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Background: The probiotic potential of Lacticacid bacteria has been studied in various medical complications, from gastrointestinal diseases to antibiotic resistance infections recently. Moreover, diabetic ulcer (DU) is known as one of the most significant global healthcare concerns, which comprehensively impacts the quality of life for these patients. Given that the conventional treatments of DUs have failed to prevent later complications completely, developing alternative therapies seems to be crucial., Methods: We designed the stable oleogel-based formulation of viable probiotic cells, including Lactobacillus rhamnosus (L. rhamnosus), Lactobacillus casei (L. casei), Lactobacillus fermentum (L. fermentum), and Lactobacillus acidophilus (L. acidophilus) individually to investigate their effect on wound healing process as an in vivo study. The wound repair process was closely monitored regarding morphology, biochemical, and histopathological changes over two weeks and compared it with the effects of topical tetracycline as an antibiotic approach. Furthermore, the antibiofilm activity of probiotic bacteria was assessed against some common pathogens., Results: The findings indicated that all tested lactobacillus groups (excluded L. casei) included in the oleogel-based formulation revealed a high potential for repairing damaged skin due to the considerably more levels of hydroxyproline content of tissue samples along with the higher numerical density of mature fibroblasts cell and volume density of hair follicles, collagen fibrils, and neovascularization in comparison with antibiotic and control groups. L. acidophilus and L. rhamnosus showed the best potential of wound healing among all lactobacillus species, groups treated by tetracycline and control groups. Besides, L. rhamnosus showed a significant biofilm inhibition activity against tested pathogens., Conclusions: This experiment demonstrated that the designed formulations containing probiotics, particularly L. acidophilus and L. rhamnosus, play a central role in manipulating diabetic wound healing. It could be suggested as an encouraging nominee for diabetic wound-healing alternative approaches, though further studies in detailed clinical trials are needed., (© 2024. The Author(s).)

Objectives: This study aimed to reveal the anti-fibrotic effects of Botrychium ternatum (Thunb.) Sw. (BT) against idiopathic pulmonary fibrosis (IPF) and to preliminarily analyze its potential mechanism on bleomycin-induced IPF rats., Methods: The inhibition of fibrosis progression in vivo was assessed by histopathology combined with biochemical indicators. In addition, the metabolic regulatory mechanism was investigated using 1H-nuclear magnetic resonance-based metabolomics combined with multivariate statistical analysis., Key Findings: Firstly, biochemical analysis revealed that BT notably suppressed the expression of hydroxyproline and transforming growth factor-β1 in the pulmonary tissue. Secondly, Masson's trichrome staining and hematoxylin and eosin showed that BT substantially improved the structure of the damaged lung and significantly inhibited the proliferation of collagen fibers and the deposition of extracellular matrix. Finally, serum metabolomic analysis suggested that BT may exert anti-fibrotic effects by synergistically regulating tyrosine metabolism; phenylalanine, tyrosine and tryptophan biosynthesis; and synthesis and degradation of ketone bodies., Conclusions: Our study not only clarifies the potential anti-fibrotic mechanism of BT against IPF at the metabolic level but also provides a theoretical basis for developing BT as an effective anti-fibrotic agent., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Dorema aucheri gum (DAG) is a bitter flavonoid gum widely used for numerous medicinal purposes including wound recovery. The present work investigates the acute toxicity and wound-healing effects of DAG in excisional skin injury in rats., Materials and Methods: Sprague Dawley rats (24) were clustered into four groups, each rat had a full-thickness excisional dorsal neck injury (2.00 cm) and addressed with 0.2 mL of the following treatments for 15 days: Group A (vehicle), rats addressed with normal saline; Group B, rats received intrasite gel; C and D, rats addressed with 250 and 500 mg/kg of DAG, respectively., Results: The results revealed the absence of any toxic signs in rats who received oral dosages of 2 and 5 g/kg of DAG. Wound healing was significantly accelerated following DAG treatments indicated by smaller open areas and higher wound contraction percentages compared to vehicle rats. Histological evaluation revealed higher fibroblast formation, collagen deposition, and noticeably lower inflammatory cell infiltration in granulated skin tissues of DAG-addressed rats compared to vehicle rats. DAG treatment caused significant modulation of immunohistochemical proteins (decreased Bax and increased HSP 70) and inflammatory mediators (reduced TNF-α, IL-6, and magnified IL-10), which were significantly varied compared to vehicle rats. Moreover, topical DAG treatment led to significant upregulation of the hydroxyproline (HDX) (collagen) and antioxidant content. At the same time, decreased the lipid peroxidation (MDA) levels in healed tissues obtained from DAG-treated rats., Conclusion: The present wound contraction by DAG might be linked with the modulatory effect of its phytochemicals (polysaccharides, flavonoids, and phenolic) on the cellular mechanisms, which justify their folkloric use and provokes further investigation as therapeutic drug additives for wound contraction., (© 2024 The Author(s). Skin Research and Technology published by John Wiley & Sons Ltd.)

Key Message: E1 holoenzyme was extensively Hyp-O-glycosylated at the proline rich linker region in plants, which substantially increased the molecular size and improved the enzymatic digestibility of the biomass of transgenic plants. Thermophilic E1 endo-1,4-β-glucanase derived from Acidothermus cellulolyticus has been frequently expressed in planta to reconstruct the plant cell wall to overcome biomass recalcitrance. However, the expressed holoenzyme exhibited a larger molecular size (~ 100 kDa) than the theoretical one (57 kDa), possibly due to posttranslational modifications in the recombinant enzyme within plant cells. This study investigates the glycosylation of the E1 holoenzyme expressed in tobacco plants and determines its impact on enzyme activity and biomass digestibility. The E1 holoenzyme, E1 catalytic domain (E1cd) and E1 linker (E1Lk) were each expressed in tobacco plants and suspension cells. The accumulation of holoenzyme was 2.0- to 2.3- times higher than that of E1cd. The proline-rich E1Lk region was extensively hydroxyproline-O-glycosylated with arabinogalactan polysaccharides. Compared with E1cd, the holoenzyme displayed a broader optimal temperature range (70 to 85 ºC). When grown in greenhouse, the expression of E1 holoenzyme induced notable phenotypic changes in plants, including delayed flowering and leaf variegation post-flowering. However, the final yield of plant biomass was not significantly affected. Finally, plant biomass engineering with E1 holoenzyme showed 1.7- to 1.8-fold higher saccharification efficiency than the E1cd lines and 2.4- to 2.7-fold higher than the wild-type lines, which was ascribed to the synergetic action of the E1Lk and cellulose binding module in reducing cell wall recalcitrance., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Wound healing involves several cellular and molecular pathways. Tridax procumbens activates genetic pathways with antibacterial, antioxidant, anticancer, and anti-inflammatory properties, aiding wound healing. This study purified Procumbenase, a serine protease from T. procumbens extract, using gel filtration (Sephadex G-75) and ion exchange (CM-Sephadex C-50) chromatography. Characterization involved analyses of protease activity, RP-HPLC, SDS-PAGE, gelatin zymogram, PAS staining, mass spectrometry, and circular dichroism. Optimal pH and temperature were determined. Protease type was identified using inhibitors. Wound-healing potential was evaluated through tensile strength, wound models, hydroxyproline estimation, and NIH 3T3 cell scratch analysis. In incision wound rat models, Procumbenase increased tensile strength on day 14 more than saline and Povidone‑iodine. It increased wound contraction by 89 % after 10 days in excision wound models, attaining full contraction by day 15 and closure by day 21. Scarless wound healing was enhanced by 18 days of epithelialization against 22 and 21 days for saline and povidone‑iodine. Procumbenase increased hydroxyproline concentration 2.53-fold (59.93 ± 2.89 mg/g) compared to saline (23.67 ± 1.86 mg/g). In NIH 3 T3 cell scratch assay, Procumbenase increased migration by 60.93 % (50 μg) and 60.57 % (150 μg) after 48 h. Thus, Procumbenase is the primary bioactive molecule in T. procumbens, demonstrates scar-free wound healing properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Sea buckthorn (Hippophae rhamnoides L.) has been designated a "medicine food homology" fruit by the National Health Commission of China due to its nutritional value. In traditional Chinese ethnomedicine, Hippophae rhamnoides L. is commonly used to treat nonhealing wounds such as burns, sores, and gastric ulcers. The aim of this study was to explore the healing effects of the ethyl acetate extract of sea buckthorn seeds (SBS-EF) on burn wounds., Aim of the Study: The primary objectives of this research were to determine the most effective medicinal site of action for treating burns with sea buckthorn seeds (SBS) and to investigate the underlying material basis and mechanisms of their therapeutic effects., Materials and Methods: The effects of different components of SBS-EF on the proliferation and migration of human skin fibroblasts (HSFs) were evaluated via MTT assays, scratch assays, transwell assays, and hydroxyproline secretion analysis. SBS-EF displayed the greatest activity amongst the extracts. Subsequent analyses included network pharmacology methodology, molecular docking studies, ultraperformance liquid chromatography UPLC-Orbitrap-Exploris-120-MS and a severe second-degree burn rat model to investigate the chemical constituents and potential therapeutic mechanisms of the SBS-EF., Results: In vitro studies demonstrated the efficacy of SBS-EF in promoting HSF growth and migration. UPLC-Orbitrap-Exploris-120-MS analysis revealed that SBS-EF had ten major constituents, with flavonoids being the predominant compounds, especially catechin, quercetin, and kaempferol derivatives. Network pharmacology and molecular docking analyses indicated that SBS-EF may exert its healing effects by modulating the Wnt/β-catenin signalling pathway. Subsequent in vivo experiments demonstrated that SBS-EF accelerated burn wound healing in rats, increased hydroxyproline expression in skin tissue, facilitated skin structure repair, and enhanced collagen production and organisation over a 21 d period. Additionally, exposure to SBS-EF upregulated WNT3a and β-catenin while downregulating GSK-3β levels in rat skin tissue., Conclusions: The wound healing properties of SBS-EF were attributed to its ability to enhance HSF growth and migration, increase hydroxyproline levels in the skin, promote collagen accumulation, reduce scarring, and decrease the skin water content. SBS-EF may also provide therapeutic benefits for burns by modulating the Wnt/β-catenin signalling pathway, as evidenced by its effective site and likely mechanism of action in the treatment of burned rats., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest relevant to the publication of this document., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: To evaluate changes of hydroxyproline concentration and its influencing factors of small incision lenticule extraction (SMILE)-derived corneal stromal lenticules with different preservation methods., Methods: A total of 390 corneal stromal lenticules of 195 patients were derived from SMILE surgeries. Thirty of the lenticules were classified as the fresh (control) group, and the rest were randomly and evenly divided and stored in anhydrous glycerol, silicone oil, Optisol, and cryopreservation for 1 day, 1 week, or 1 month. A hydroxyproline assay kit (ab222941, Abcam) was used to measure the hydroxyproline concentration in each preservation method. Concentrations of MMP-2, TIMP-2, TNFα, TGFβ2, and reactive oxygen species were also evaluated., Results: In the anhydrous glycerol group, the concentration of hydroxyproline decreased within 1 week (fresh: 1 dΔ = 0.229, P < 0.001*; 1 d - 1 wΔ = 0.055, P < 0.001*) while that in the silicone oil group remained stable in 1 week (1 d - 1 wΔ = -0.005, P = 0.929) and decreased significantly in 1 m (1 m - 1 wΔ = -0.041, P = 0.003*). The sequence of hydroxyproline concentration in the Optisol group was 1 m > 1 day > 1 week. Hydroxyproline concentration in the cryopreservation group decreased within 1 m. Hydroxyproline concentration was highest in the Optisol group and lowest in the anhydrous glycerol group under the same preservation time. Hydroxyproline concentration was negatively correlated with MMP-2 (r = -0.16, P = 0.421) and TIMP-2 (r = -0.56, P = 0.002*) while MMP-2 and TNFα (r = 0.17, P = 0.242), TIMP-2 and TGFβ2 (r = 0.21, P = 0.207), and TNFα and reactive oxygen species (r = 0.52, P = 0.007*) were positively correlated., Conclusions: More collagen was retained in SMILE lenticules preserved in Optisol under the same preservation time. The mechanism of the changes of collagen in preserved SMILE-derived lenticules and oxidative stress requires additional investigation., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Piglets receive far less hydroxyproline (Hyp) from a diet after weaning than they obtained from sow's milk prior to weaning, suggesting that Hyp may play a protective role in preserving intestinal mucosal homeostasis. This study aimed to evaluate the effect of Hyp on intestinal barrier function and its associated gut microbiota and metabolites in early-weaned piglets. Eighty weaned piglets were divided into four groups and fed diets containing different Hyp levels (0 %, 0.5 %, 1 %, or 2 %) for 21 days. Samples, including intestinal contents, tissues, and blood, were collected on day 7 for analysis of microbial composition, intestinal barrier function, and metabolites. We demonstrated that dietary supplementation with 2 % Hyp improved the feed conversion ratio and reduced the incidence of diarrhea in early-weaned piglets compared to the control group. Concurrently, Hyp enhanced intestinal barrier function by facilitating tight junction protein (zonula occludens (ZO)-1 and occludin) expression and mucin production in the jejunal, ileal, and colonic mucosas. It also improved mucosal immunity (by increasing the amount of secretory IgA (sIgA) and the ratio of CD4 + /CD8 + T lymphocytes and decreasing NF-κB phosphorylation) and increased antioxidant capacity (by raising total antioxidant capacity (T-AOC) and glutathione levels) in the intestinal mucosa. In addition, Hyp supplementation resulted in an increase in the levels of glycine, glutathione, and glycine-conjugated bile acids, while decreasing the concentrations of cortisol and methionine sulfoxide in plasma. Intriguingly, piglets fed diet containing Hyp exhibited a remarkable increase in the abundance of probiotic Enterococcus faecium within their colonic contents. This elevation occurred alongside an attenuation of pro-inflammatory responses and an enhancement in intestinal barrier integrity. Further, these changes were accompanied by a rise in anti-inflammatory metabolites, specifically glycochenodeoxycholic acid and guanosine, along with a suppression of pro-inflammatory lipid peroxidation products, including (12Z)-9,10-dihydroxyoctadec-12-enoic acid (9,10-DHOME) and 13-L-hydroperoxylinoleic acid (13(S)-HPODE). In summary, Hyp holds the capacity to enhance the intestinal barrier function in weaned piglets; this effect is correlated with changes in the gut microbiota and metabolites. Our findings provide novel insights into the role of Hyp in maintaining gut homeostasis, highlighting its potential as a dietary supplement for promoting intestinal health in early-weaned piglets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: We administered Bushen Huoxue Huazhuo Formula (BSHXHZF) and transplanted bone marrow mesenchymal stem cells (BMSCs) into mice with Wilson's disease (WD)-related liver fibrosis to evaluate the liver-protecting mechanism of this prescription., Methods: Mice, randomly divided into different treatment groups, showed histopathological changes and degree of hepatocyte apoptosis. For hepatic hydroxyproline (Hyp) determination, transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7) mRNA and protein were measured. Chemical profiling of the extract of BSHXHZF using The liquid chromatography-mass spectrometry (LC-MS/MS) and revealing its antifibrosis mechanism using metabolomics., Results: TCM+BMSC group livers exhibited few inflammatory cells. TUNEL revealed abundant brown apoptotic cells in model control groups, while the TCM+BMSC groups showed a significant increase in blue negative expression of liver cells. Hyp in toxic milk (TX) mice groups was significantly lower than that in model control groups (MG). Compared with MG, TGF-β1 expression was significantly lower than all other groups, while BMP-7 expression was significantly higher. Metabolic analysis identified 20 potential biomarkers and 10 key pathways, indicating that BSHXHZF+BMSC intervention has a significant regulatory effect on metabolic disorders of these small molecule substances., Conclusion: BSHXHZF combined with BMSCs can inhibit liver fibrosis and hepatocyte apoptosis by improving related metabolic disorders, and achieving therapeutic effects in WD-related liver fibrosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Hydrogels have emerged as a promising option for treating local scald wounds due to their unique physical and chemical properties. This study aims to evaluate the efficacy of ovalbumin/gelatin composite hydrogels in repairing deep II-degree scald wounds using a mouse dorsal skin model. Trauma tissues collected at various time points are analyzed for total protein content, hydroxyproline content, histological features, and expression of relevant markers. The results reveal that the hydrogel accelerates the healing process of scalded wounds, which is 17.27% higher than the control group. The hydrogel treatment also effectively prevents wound enlargement and redness of the edges caused by infection during the initial stage of scalding. The total protein and hydroxyproline content of the treated wounds are significantly elevated. Additionally, the hydrogel up-regulates the expression of VEGF (a crucial angiogenic factor) and down-regulates CD68 (a macrophage marker). In summary, this study provides valuable insights into the potential of multifunctional protein-based hydrogels in wound healing., (© 2024 Wiley‐VCH GmbH.)

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic form of non-alcoholic fatty liver disease. Liver fibrosis leads to liver cancer and cirrhosis, and drug therapy for NASH remains lacking. Ninjin'yoeito (NYT) has shown antifibrotic effects in a model of liver fibrosis without steatosis but has not been studied for NASH. Therefore, we evaluated the efficacy of NYT in mice fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a NASH model. Compared with the normal diet group, mice fed CDAHFD showed decreased body weight and increased white adipose tissue, liver weight, and triglyceride content in the liver. Furthermore, a substantial increase in the hepatic concentration of hydroxyproline, expression of α-smooth muscle actin (α-SMA), and transforming growth factor-β was observed in CDAHFD-fed mice. Masson's trichrome and Picro-Sirius red staining revealed a remarkable increase in collagen fiber compared with the normal diet group. Compared with mice that received CDAHFD alone, those supplemented with NYT exhibited reduced hepatic triglyceride and hydroxyproline levels and α-SMA expression. Additionally, compared with the group fed CDAHFD alone, the stained liver tissues of NYT-treated mice exhibited a reduction in Masson's trichrome- and Picro-Sirius red-positive areas. Locomotor activity was significantly reduced in the CDAHFD-fed group compared with the normal diet group. In the NYT-treated group, the CDAHFD-induced decrease in locomotor activity was significantly suppressed. The findings indicate that NYT inhibited fatty and fibrotic changes in the livers of NASH mice and alleviated the decrease in locomotor activity. Therefore, NYT may serve as a novel therapeutic approach for NASH., (© 2024. The Author(s).)

Although fibroblast growth factor 7 (FGF7) is known to promote wound healing, its mass production poses several challenges and very few studies have assessed the feasibility of producing FGF7 in cell lines such as Chinese hamster ovary (CHO) cells. Therefore, this study sought to produce recombinant FGF7 in large quantities and evaluate its wound healing effect. To this end, the FGF7 gene was transfected into CHO cells and FGF7 production was optimized. The wound healing efficacy of N-glycosylated FGF7 was evaluated in animals on days 7 and 14 post-treatment using collagen patches (CPs), FGF7-only, and CP with FGF7 (CP+FGF7), whereas an untreated group was used as the control. Wound healing was most effective in the CP+FGF7 group. Particularly, on day 7 post-exposure, the CP+FGF7 and FGF7-only groups exhibited the highest expression of hydroxyproline, fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor. Epidermalization in H&E staining showed the same order of healing as hydroxyproline content. Additionally, the CP+FGF7 and FGF7-only group exhibited more notable blood vessel formation on days 7 and 14. In conclusion, the prepared FGF7 was effective in promoting wound healing and CHO cells can be a reliable platform for the mass production of FGF7., (© 2024 Wiley‐VCH GmbH.)

Idiopathic pulmonary fibrosis (IPF) is a life-threatening disease that leads to dyspnea and progressive loss of lung function. This study aimed to investigate the protective effect of betanin (BET), the major pigment in red beetroot, on pulmonary fibrosis induced by bleomycin (BLM) in rats and to assess the underlying mechanisms. In this view, total and differential cell counts and LDH activity in bronchoalveolar lavage fluid were estimated. Furthermore, MDA and GSH contents in the lungs were colorimetrically measured, while hydroxyproline, NLRP3, ASC, caspase-1, TGF-β1, and vimentin levels in lung tissue were evaluated using the ELISA technique. Moreover, IL-1β, E-cadherin, and α-SMA expressions were analyzed by immunostaining of lung specimens. BET treatment protects against pulmonary fibrosis as indicated by the reduction in total and differential cell counts, LDH activity, hydroxyproline, NLRP3, ASC, caspase-1, IL-1β, and TGF-β1 levels. MDA content was also decreased following BET administration, while GSH content was elevated. Additionally, BET suppressed the EMT process as evidenced by an increase in E-cadherin expression besides the reduction in vimentin and α-SMA expressions. To conclude, these results revealed the protective effect of BET against pulmonary fibrosis that might be attributed to the attenuation of the NLRP3/IL-1β/TGF-β1 signaling pathway and EMT process.

Objectives: Tuberostemonine has several biological activity, the aim of study examined the impact of tuberostemonine on the proliferation of TGF-β1 induced cell model, and its ability to alleviate pulmonary fibrosis stimulated by bleomycin in mice., Methods: In vitro , we assessed the effect of tuberostemonine (350, 550 and 750 µM) on the proliferation of cells stimulated by TGF-β1 (10 μg/L), as well as on parameters such as α-SMA vitality, human fibronectin, collagen, and hydroxyproline levels in cells. In vivo , we analyzed inflammation, hydroxyproline, collagen activity and metabolomics in the lungs of mice. Additionally, a comprehensive investigation into the TGF-β/smad signaling pathway was undertaken, targeting lung tissue as well as HFL cells., Results: Within the confines of an in vitro setup, the tuberostemonine manifested a discerned IC50 of 1.9 mM. Furthermore, a significant reduction of over fifty percent was ascertained in the secretion levels of hydroxyproline, fibronectin, collagen type I, collagen type III and α-SMA. In vivo , tuberostemonine obviously improved the respiratory function percentage over 50% of animal model and decreased the hydroxyproline, lung inflammation and collagen deposition. A prominent decline in TGF-β/smad pathway functioning was identified within both the internal and external cellular contexts., Conclusions: Tuberostemonine is considered as a modulator to alleviate fibrosis and may become a new renovation for pulmonary fibrosis., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The study's objective is to clarify the probable mechanisms underlying the wound-healing properties of Helianthemum canum L. (Cistaceae), a traditional anti-inflammatory and wound-healing medicine. LC/MS-MS was used to perform phytochemical analyses on a 70 % methanol extract of the plant's aerial parts. In vivo, linear incision and circular excision models were used to evaluate the wound healing activity. For anti-inflammatory effect, in vivo acetic acid capillary permeability assay and in vitro Interleukin 1, Interleukin 6, and Interferon ɣ levels in LPS-induced FR skin fibroblast cell line were also evaluated. The extract significantly improved wound healing in experimental models, with tensile strength values of 27.8 % and a contraction value of 35.09 %. Histopathological examinations, hydroxyproline estimation, hyaluronidase, collagenase, and elastase enzyme inhibitory assays confirmed wound healing potential. Inflammatory cytokines were significantly inhibited in the LPS-induced FR cell line, with the highest effect seen on IL-6 (34.5±2.12 pg/mL). This study offered the first concrete proof that H. canum can be used to treat wounds by suggesting that the myricetin and quinic acid content identified by LCMS-MS analysis may be accountable for the effect of H. canum on wound contraction and hydroxyproline production., (© 2023 Wiley-VHCA AG, Zurich, Switzerland.)

The objective of this study was to investigate the effect of low-molecular-weight fish collagen (valine-glycine-proline-hydroxyproline-glycine-proline-alanine-glycine; LMWCP) on H 2 O 2 - or LPS-treated primary chondrocytes and monoiodoacetate (MIA)-induced osteoarthritis rat models. Our findings indicated that LMWCP treatment exhibited protective effects by preventing chondrocyte death and reducing matrix degradation in both H 2 O 2 -treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. This was achieved by increasing the levels of aggrecan, collagen type I, collagen type II, TIMP-1, and TIMP-3, while simultaneously decreasing catabolic factors such as phosphorylation of Smad, MMP-3, and MMP-13. Additionally, LMWCP treatment effectively suppressed the activation of inflammation and apoptosis pathways in both LPS-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. These results suggest that LMWCP supplementation ameliorates the progression of osteoarthritis through its direct impact on inflammation and apoptosis in chondrocytes.

This work describes the development of a flow injection method to determine hydroxyproline (HYP), one of collagen's most abundant amino acids. Collagen is a protein with several applications and high nutritional value. Evaluating the feasibility of using collagen from fish skin over its mammalian source is essential. The determination of HYP requires the pre-treatment and hydrolysis of the fish skin to break down collagen into its amino acids, and the HYP value quantified relates to the collagen content. The determination was based on the HYP oxidation with permanganate in an alkaline medium and the consequent decrease of colour intensity registered. Under optimal conditions, the developed method enables the determination of the HYP within the dynamic range of 23.8 to 500 mg L -1 , with a limit of detection (LOD) of 2.6 mg L -1 and a limit of quantification (LOQ) of 23.8 mg L -1 . Different samples were processed, and the digests were analysed by the proposed method and with the conventional procedure with good correlation (relative error < 7%). Moreover, the analyte quantification is performed faster, simpler, and more accurately, with less toxic solutions. The reproducibility of the developed method was also evaluated by calculating the relative standard deviation of the calibration curve slope (RSD < 1%).

Study Question: Is the abundance of certain biochemical compounds in human cumulus cells (CCs) related to oocyte quality?, Summary Answer: Malonate, 5-oxyproline, and erythronate were positively associated with pregnancy potential., What Is Known Already: CCs are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Mitochondrial DNA content and transcriptional analyses in CC have been shown to provide a poor predictive value of oocyte competence, but the untargeted analysis of biochemical compounds (metabolomics) has been unexplored., Study Design, Size, Duration: CCs were obtained from three groups of cumulus-oocyte complexes (COCs) of known developmental potential: oocytes not developing to blastocyst following ICSI (Bl-); oocytes developing to blastocyst but failing to establish pregnancy following embryo transfer (P-); and oocytes developing to blastocyst able to establish a pregnancy (P+). Metabolomics analyses were performed on 12 samples per group, each sample comprising the CC recovered from a single COC., Participants/materials, Setting, Methods: Human CC samples were obtained from IVF treatments. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Metabolomics analysis was performed by ultra-high performance liquid chromatography-tandem mass spectroscopy., Main Results and the Role of Chance: The analysis identified 98 compounds, five of which were differentially abundant (P < 0.05) between groups: asparagine, proline, and malonate were less abundant in P- compared to Bl-, malonate and 5-oxoproline were less abundant in P- group compared to P+, and erythronate was less abundant in Bl- group compared to P+. No significant association between the abundance of the compounds identified and donor age or BMI was noted., Limitations, Reasons for Caution: Data dispersion and the lack of coherence between developmental groups preclude the direct use of metabolic markers in clinical practice, where the uterine environment plays a major role in pregnancy outcome. The abundance of other compounds not detected by the analysis may be associated with oocyte competence. As donors were lean (only two with BMI > 30 kg/m2) and young (<34 years old), a possible effect of obesity or advanced age on the CC metabolome could not be determined., Wider Implications of the Findings: The abundance of malonate, 5-oxyproline, and erythronate in CC was significantly higher in COCs ultimately establishing pregnancy, providing clues on the pathways required for oocyte competence. The untargeted analysis uncovered the presence of compounds that were not expected in CC, such as β-citrylglutamate and the neurotransmitter N-acetyl-aspartyl-glutamate, which may play roles in chromatin remodeling and signaling, respectively., Study Funding/competing Interest(s): Research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by Madrid Region Government. The authors declare no competing interest., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

This study tested the hypothesis that the synthesis of glycine from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) was impaired in tissues of piglets with intrauterine growth restriction (IUGR), thereby contributing to a severe glycine deficiency in these compromised neonates. At 0, 7, 14, and 21 days of age, IUGR piglets were euthanized, and tissues (liver, small intestine, kidney, pancreas, stomach, skeletal muscle, and heart) were obtained for metabolic studies, as well as the determination of enzymatic activities, cell-specific localization, and expression of mRNAs for glycine-synthetic enzymes. The results indicated relatively low enzymatic activities for 4-hydroxyproline oxidase (OH-POX), proline oxidase, serine hydroxymethyltransferase, threonine dehydrogenase (TDH), alanine: glyoxylate transaminase, and 4-hydroxy-2-oxoglutarate aldolase in the kidneys and liver from 0- to 21-day-old IUGR pigs, in the pancreas of 7- to 21-day-old IUGR pigs, and in the small intestine and skeletal muscle (except TDH) of 21-day-old IUGR pigs. Accordingly, the rates of conversion of 4-hydroxyproline into glycine were relatively low in tissues of IUGR piglets. The expression of mRNAs for glycine-synthetic enzymes followed the patterns of enzymatic activities and was also low. Immunohistochemical analyses revealed the relatively low abundance of OH-POX protein in the liver, kidney, and small intestine of IUGR piglets, and the lack of OH-POX zonation in their livers. These novel results provide a metabolic basis to explain why the endogenous synthesis of glycine is insufficient for optimum growth of IUGR piglets and have important implications for improving the nutrition and health of other mammalian neonates including humans with IUGR., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ulcerative colitis is a type of inflammatory bowel disease responsible for the inflammation of the innermost lining of the colon and rectum. The present study's objective is to determine the potential synergistic impact of quercetin (QR) and lycopene (LP) in ulcerative colitis (UC) induced in rats by ochratoxin A (OTA) by biochemical and morphological alterations. QR and LP were administered alone and in combination with the OTA for 7 days. OTA administration caused UC generation, resulting in significant changes in body weight percentage, disease activity index (DAI), macroscopic evaluation, colon weight/length ratio, and histological score. In addition to the above parameters, it also leads to elevated oxidative stress, i.e. increased malondialdehyde (MDA), nitric oxide (NO), myeloperoxidase (MPO), and hydroxyproline levels and decreased superoxide dismutase (SOD) and reduced glutathione (GSH) levels. Histological changes in the colon architecture were also observed suggestive of extensive mucosal damage. In addition, a high level of matrix metalloproteinase 7 (MMP7) was observed in immunohistochemistry, and a high level of gene expression of osteopontin (OPN), runt-related transcription factor 2 (RUNX2), MMP-7, and interleukin-6 (IL-6) was observed in OTA administered animals. The combination of QR and LP significantly restored the per cent body weight loss and DAI score and improved macroscopic and histological changes, colon weight/length ratio, and macroscopic damages. It also improved the biochemical parameters to near-normal levels, i.e. reduced MDA, NO, MPO, and hydroxyproline levels and increased SOD and GSH levels. In addition, OPN, Runx2, MMP-7, and IL-6 gene expression decreased compared to the OTA-induced UC group. Outcomes of the present study indicate the potential of QR + LP as anti-inflammatory and immunomodulatory agents against OTA-induced UC in rats., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vanadium is available as a dietary supplement and also is known to be toxic if inhaled, yet little information is available concerning the effects of vanadium on mammalian metabolism when concentrations found in food and water. Vanadium pentoxide (V +5 ) is representative of the most common dietary and environmental exposures, and prior research shows that low-dose V +5 exposure causes oxidative stress measured by glutathione oxidation and protein S-glutathionylation. We examined the metabolic impact of V +5 at relevant dietary and environmental doses (0.01, 0.1, and 1 ppm for 24 h) in human lung fibroblasts (HLFs) and male C57BL/6J mice (0.02, 0.2, and 2 ppm in drinking water for 7 mo). Untargeted metabolomics using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) showed that V +5 induced significant metabolic perturbations in both HLF cells and mouse lungs. We noted 30% of the significantly altered pathways in HLF cells, including pyrimidines and aminosugars, fatty acids, mitochondrial and redox pathways, showed similar dose-dependent patterns in mouse lung tissues. Alterations in lipid metabolism included leukotrienes and prostaglandins involved in inflammatory signaling, which have been associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF) and other disease processes. Elevated hydroxyproline levels and excessive collagen deposition were also present in lungs from V +5 -treated mice. Taken together, these results show that oxidative stress from environmental V +5 , ingested at low levels, could alter metabolism to contribute to common human lung diseases. NEW & NOTEWORTHY We used relevant dietary and environmental doses of Vanadium pentoxide (V +5 ) to examine its metabolic impact in vitro and in vivo. Using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), we found significant metabolic perturbations, with similar dose-dependent patterns observed in human lung fibroblasts and male mouse lungs. Alterations in lipid metabolism included inflammatory signaling, elevated hydroxyproline levels, and excessive collagen deposition were present in V +5 -treated lungs. Our findings suggest that low levels of V +5 could trigger pulmonary fibrotic signaling.

Objective: JWH133, a cannabinoid type 2 receptor agonist, was tested for its ability to protect mice from bleomycin-induced pulmonary fibrosis. Methods: By using a random number generator, 24 C57BL/6J male mice were randomly divided into the control group, model group, JWH133 intervention group, and JWH133+a cannabinoid type-2 receptor antagonist (AM630) inhibitor group, with 6 mice in each group. A mouse pulmonary fibrosis model was established by tracheal instillation of bleomycin (5 mg/kg). Starting from the first day after modeling, the control group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution, and the model group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution. The JWH133 intervention group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg, dissolved in physiological saline), and the JWH133+AM630 antagonistic group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg) and AM630 (2.5 mg/kg). After 28 days, all mice were killed; the lung tissue was obtained, pathological changes were observed, and alveolar inflammation scores and Ashcroft scores were calculated. The content of type Ⅰ collagen in the lung tissue of the four groups of mice was measured using immunohistochemistry. The levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in the serum of the four groups of mice were measured using enzyme-linked immunosorbent assay (ELISA), and the content of hydroxyproline (HYP) in the lung tissue of the four groups of mice was measured. Western blotting was used to measure the protein expression levels of type Ⅲ collagen, α-smooth muscle actin (α-SMA), extracellular signal regulated kinase (ERK1/2), phosphorylated P-ERK1/2 (P-ERK1/2), and phosphorylated ribosome S6 kinase type 1 (P-p90RSK) in the lung tissue of mice in the four groups. Real-time quantitative polymerase chain reaction was used to measure the expression levels of collagen Ⅰ, collagen Ⅲ, and α-SMA mRNA in the lung tissue of the four groups of mice. Results: Compared with the control group, the pathological changes in the lung tissue of the model group mice worsened, with an increase in alveolar inflammation score (3.833±0.408 vs. 0.833±0.408, P <0.05), an increase in Ashcroft score (7.333±0.516 vs. 2.000±0.633, P <0.05), an increase in type Ⅰ collagen absorbance value (0.065±0.008 vs. 0.018±0.006, P <0.05), an increase in inflammatory cell infiltration, and an increase in hydroxyproline levels [(1.551±0.051) μg/mg vs. (0.974±0.060) μg/mg, P <0.05]. Compared with the model group, the JWH133 intervention group showed reduced pathological changes in lung tissue, decreased alveolar inflammation score (1.833±0.408, P <0.05), decreased Ashcroft score (4.167±0.753, P <0.05), decreased type Ⅰ collagen absorbance value (0.032±0.004, P <0.05), reduced inflammatory cell infiltration, and decreased hydroxyproline levels [(1.148±0.055) μg/mg, P <0.05]. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group showed more severe pathological changes in the lung tissue of mice, increased alveolar inflammation score and Ashcroft score, increased type Ⅰ collagen absorbance value, increased inflammatory cell infiltration, and increased hydroxyproline levels. Compared with the control group, the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK proteins in the lung tissue of the model group mice increased, while the expression of type Ⅰ collagen, type Ⅲ collagen, and α-SMA mRNA increased. Compared with the model group, the protein expression of α-SMA (relative expression 0.60±0.17 vs. 1.34±0.19, P <0.05), type Ⅲ collagen (relative expression 0.52±0.09 vs. 1.35±0.14, P <0.05), P-ERK1/2 (relative expression 0.32±0.11 vs. 1.14±0.14, P <0.05), and P-p90RSK (relative expression 0.43±0.14 vs. 1.15±0.07, P <0.05) decreased in the JWH133 intervention group. The type Ⅰ collagen mRNA (2.190±0.362 vs. 5.078±0.792, P <0.05), type Ⅲ collagen mRNA (1.750±0.290 vs. 4.935±0.456, P <0.05), and α-SMA mRNA (1.588±0.060 vs. 5.192±0.506, P <0.05) decreased. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group increased the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK protein in the lung tissue of mice, and increased the expression of type Ⅲ collagen and α-SMA mRNA. Conclusion: In mice with bleomycin-induced pulmonary fibrosis, the cannabinoid type-2 receptor agonist JWH133 inhibited inflammation and improved extracellular matrix deposition, which alleviated lung fibrosis. The underlying mechanism of action may be related to the activation of the ERK1/2-RSK1 signaling pathway.

The pollen grain cell wall is a highly specialized structure composed of distinct layers formed through complex developmental pathways. The production of the innermost intine layer, composed of cellulose, pectin and other polymers, is particularly poorly understood. Here we demonstrate an important and specific role for the hydroxyproline O-arabinosyltransferase (HPAT) FIN4 in tomato intine development. HPATs are plant-specific enzymes which initiate glycosylation of certain cell wall structural proteins and signaling peptides. FIN4 was expressed throughout pollen development in both the developing pollen and surrounding tapetal cells. A fin4 mutant with a partial deletion of the catalytic domain displayed significantly reduced male fertility in vivo and compromised pollen hydration and germination in vitro. However, fin4 pollen that successfully germinated formed morphologically normal pollen tubes with the same growth rate as the wild-type pollen. When we examined mature fin4 pollen, we found they were cytologically normal, and formed morphologically normal exine, but produced significantly thinner intine. During intine deposition at the late stages of pollen development we found fin4 pollen had altered polymer deposition, including reduced cellulose and increased detection of pectin, specifically homogalacturonan with both low and high degrees of methylesterification. Therefore, FIN4 plays an important role in intine formation and, in turn pollen hydration and germination and the process of intine formation involves dynamic changes in the developing pollen cell wall., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study aimed to investigate the mechanism of lung tissue YKL-40 promoting the interstitial transformation of alveolar epithelial cells in mice with idiopathic pulmonary fibrosis and its effect on the level of TGF-β1. For this purpose, Forty SPF SD mice were randomly divided into 4 groups. They were the blank control group (CK group), virus-negative control group (YKL-40-NC group), YKL-40 knockdown group (YKL-40-inhibitor group) and YKL-40 overexpression group (YKL-40-mimics group), respectively. The mRNA expressions of alveolar epithelial cell mesenchymal transformation-related proteins, pulmonary fibrosis-related factors and TGF-β1-related pathway proteins in the above four groups of mice were compared to determine the mechanism of the promotion of alveolar epithelial cell mesenchymal transformation by YKL-40 in the lung tissues of mice with idiopathic pulmonary fibrosis and the effect of YKL-40 on the level of TGF-β1. The results showed that in terms of lung wet/dry weight ratio, the YKL-40-NC group, YKL-40-inhibitor group and YKL-40-mimics group were significantly increased compared with the CK group (P<0.05). About YKL-40 protein expression, compared with the CK group, AOD value and YKL-40 protein expression in the YKL-40-NC group, YKL-40-inhibitor group and YKL-40-mimics group were significantly increased (P<0.05), and compared with YKL-40-NC group, The AOD value and YKL-40 protein expression in YKL-40-inhibitor group were significantly decreased, while the AOD value and YKL-40 protein expression in YKL-40-mimics group were significantly increased (P<0.05), suggesting successful lentivirus transfection. Compared with the CK group, β-catenin and E-cadherin in the alveolar epithelial cells were significantly increased, while Pro-SPC was significantly decreased (P<0.05). The mRNA expression of pulmonary fibrosis-related factors showed that compared with the CK group, the mRNA expression of vimimin and hydroxyproline was significantly increased, while the mRNA expression of E-cadherin was decreased (P<0.05). However, the mRNA expressions of vimimin and hydroxyproline in the YKL-40-inhibitor group were significantly decreased, but the mRNA expression of E-cadherin was significantly increased. Compared with CK group, the protein expressions of TGF-β1, Smad3, Smad7 and α-Sma in the CK group were significantly increased (P<0.05). The protein expressions of TGF-β1, Smad3, Smad7 and α-SMA in the YKL-40-mimics group were significantly increased, but the protein expressions of TGF-β1, Smad3, Smad7 and α-SMA in YKL-40-inhibitor group were significantly decreased (P<0.05). In general, overexpression of YKL-40 can promote the progression of pulmonary fibrosis and the interstitial transformation of alveolar epithelial cells in mice with idiopathic fibrosis.

A number of food components, such as polyphenols and phytonutrients, have immunomodulatory effects. Collagen has various bioactivities, such as antioxidative effects, the promotion of wound healing, and relieving symptoms of bone/joint disease. Collagen is digested into dipeptides and amino acids in the gastrointestinal tract and subsequently absorbed. However, the difference in immunomodulatory effects between collagen-derived dipeptides and amino acids is unknown. To investigate such differences, we incubated M1 macrophages or peripheral blood mononuclear cells (PBMC) with collagen-derived dipeptides (hydroxyproline-glycine (Hyp-Gly) and proline-hydroxyproline (Pro-Hyp)) and amino acids (proline (Pro), hydroxyproline (Hyp), and glycine (Gly)). We first investigated the dose dependency of Hyp-Gly on cytokine secretion. Hyp-Gly modulates cytokine secretion from M1 macrophages at 100 µM, but not at 10 µM and 1 µM. We then compared immunomodulatory effects between dipeptides and mixtures of amino acids on M1 macrophages and PBMC. There was, however, no difference in cytokine secretion between dipeptides and their respective amino acids. We conclude that collagen-derived dipeptides and amino acids have immunomodulatory effects on M1-differentiated RAW264.7 cells and PBMC and that there is no difference in the immunomodulatory effects between dipeptides and amino acids.

Arabinogalactan-proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high sugar content and are widely distributed in the plant kingdom. AGPs have long been suggested to play important roles in sexual plant reproduction. The synthesis of their complex carbohydrates is initiated by a family of hydroxyproline galactosyltransferase (Hyp-GALT) enzymes which add the first galactose to Hyp residues in the protein backbone. Eight Hyp-GALT enzymes have been identified so far, and in the present work a mutant affecting five of these enzymes (galt2galt5galt7galt8galt9) was analyzed regarding the reproductive process. The galt25789 mutant presented a low seed set, and reciprocal crosses indicated a significant female gametophytic contribution to this mutant phenotype. Mutant ovules revealed abnormal callose accumulation inside the embryo sac and integument defects at the micropylar region culminating in defects in pollen tube reception. In addition, immunolocalization and biochemical analyses allowed the detection of a reduction in the amount of glucuronic acid in mutant ovary AGPs. Dramatically low amounts of high-molecular-weight Hyp-O-glycosides obtained following size exclusion chromatography of base-hydrolyzed mutant AGPs compared to the wild type indicated the presence of underglycosylated AGPs in the galt25789 mutant, while the monosaccharide composition of these Hyp-O-glycosides displayed no significant changes compared to the wild-type Hyp-O-glycosides. The present work demonstrates the functional importance of the carbohydrate moieties of AGPs in ovule development and pollen-pistil interactions., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Collagen has been postulated to be the most abundant protein in our body, making up one-third of the total protein content in mammals. However, a direct assessment of the total collagen levels of an entire mammal to confirm this estimate is missing. Here we measured hydroxyproline levels as a proxy for collagen content together with total protein levels of entire mice or of individual tissues. Collagen content normalized to the total protein is approximately 0.1% in the brain and liver, 1% in the heart and kidney, 4% in the muscle and lung, 6% in the colon, 20-40% in the skin, 25-35% in bones, and 40-50% in tendons of wild-type (CD1 and CB57BL/6) mice, consistent with previous reports. To our surprise, we find that collagen is approximately 12% in females and 17% in males of the total protein content of entire wild-type (CD1 and CB57BL/6) mice. Although collagen type I is the most abundant collagen, the most abundant proteins are albumin, hemoglobulin, histones, actin, serpina, and then collagen type I. Analyzing amino acid compositions of mice revealed glycine as the most abundant amino acid. Thus, we provide reference points for collagen, matrisome, protein, and amino acid composition of healthy wild-type mice., (© 2023. The Author(s).)

Context: Oxidative stress leads to deleterious processes in the liver that resulted in liver diseases. Objective: To evaluate antioxidant activity and hepatoprotective potential of ethanolic leaves extract of Citrus reticulate against hepatic dysfunction induced by thioacetamide (TAA). Materials and Methods: Flavonoid constituents were isolated from the ethanol extract by chromatographic techniques and identified by the spectroscopic analyses. Antioxidant activity was determined using DPPH assay. Hepatotoxicity was induced in rats via intraperitoneal injection of TAA and the ethanol extract was orally administrated at a dose of 100 mg/kg/day for four weeks. Serum biomarkers, hepatic antioxidant enzymes, tumour necrosis factor-alpha (TNF-α), hepatic hydroxyproline levels, and histopathology were examined. Results: Ten known flavonoids were identified, among of them, 6,3`-dimethoxyluteolin and 8,3`-dimethoxyluteolin possessed the highest antioxidant activity. The substantially elevated serum enzymatic levels of ALT, ALP, and bilirubin were found to be restored towards normalisation significantly by the plant extract. Furthermore, the markers including MDA, GSH, SOD, NO, and protein carbonyl which were close to oxidative damage, were restored. Meanwhile, the extract treatment decreased TNF-α level and also was able to reverse the induced fibrosis by significantly reducing the hydroxyproline content. Moreover, histopathological studies further substantiate the protective effect of the extract. Conclusion: C. reticulate leaves extract is a rich source of phytochemicals with in vitro and in vivo protective effects.

Paraquat (PQ) is an herbicide which has brought some health problems through the production of reactive oxygen species. The increasing interest in the novel formulation of agrochemicals has been aiming to provide safety for non-target organisms. Chitosan is a well-known non-toxic polymer, commonly used in preparing particles via ionotropic gelation. In this study, we prepared PQ nanoparticles (PQNPs) and evaluated their toxicity in vivo and in vitro. PQNPs were prepared and characterized in two forms, with and without the utilization of chitosan. Relative cell survival of PQNPs were studied against bulk PQ in HEK-293. Also, the acute lung injury of PQNP was assessed against treatment with acetylcysteine. Total antioxidant capacity (TAC), lipid peroxidation (LPO), total thiol groups (TTG), and hydroxyproline, along with histological changes were assessed in the lungs. The size, zeta potential, and polydispersity index of the optimum particles were about 157.7 ± 7.03, 22.25 ± 4.52, and 0.701, respectively. The encapsulation efficiency was 65.11 ± 10.45, and the loading percent of PQ was 58.57 ± 2.37. PQNPs showed an initial burst of PQ release followed by a zero-degree pattern. PQNPs displayed lower cell cytotoxicity compared to bulk PQ. LPO, TAC, TTG, and hydroxyproline levels in lungs generally showed more satisfying status in PQNPQs as well. The levels of oxidative status markers indicate lower oxidative damage in lungs and a more desirable response to acetylcysteine treatment, in line with histological changes. PQ loaded in chitosan-alginate particles offers safer characteristics compared with bulk PQ., Competing Interests: Declaration of Competing Interest The authors state that they have no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Despite the fact that liver fibrosis is an intractable disease with a poor prognosis, effective therapeutic agents are not available. In this study, we focused on bone morphogenetic factor 7 (BMP7) that inhibits transforming growth factor (TGF)-β signaling, which is involved in liver fibrosis. We prepared an albumin-fused BMP7 (HSA-BMP7) that is retained in the blood and evaluated its inhibitory effect on liver fibrosis. Bile duct ligated mice were used as an acute liver fibrosis model, and carbon tetrachloride-induced liver fibrosis mice were used as a chronic model. All mice were administered HSA-BMP7 once per week. In the mice with bile duct ligation, the administration of HSA-BMP7 significantly suppressed the infiltration of inflammatory cells, the area of fibrosis around the bile duct, and decreased in the level of hydroxyproline as compared with saline administration. The mRNA expression of TGF-β and its downstream fibrosis-associated genes (α-SMA and Col1a2) were also suppressed by the administration of HSA-BMP7. In the carbon tetrachloride-induced liver fibrosis mice, the HSA-BMP7 administration significantly decreased the hepatic fibrosis area and the level of hydroxyproline. Based on these results, it appears that HSA-BMP7 has the potential for serving as a therapeutic agent for the treatment of liver fibrosis.

Introduction: The incidence of non-alcoholic fatty liver disease (NAFLD) has increased in recent years. Hepatic fibrosis (HF) is an important step in the progression of NAFLD to cirrhosis and even carcinoma and is also recognized as a possible reversal phase., Aims: We previously found that the aqueous extract of Sedum Lineare Thunb. has hepatoprotective effects. This study investigated the hepatoprotective effect and mechanism of the Sedum Lineare Thunb. n-butanol phase (SLNP) on HF in rats., Methods: Animals were intraperitoneally injected with thioacetamide solution twice a week for 8 weeks to prepare an HF model and were administered the corresponding drugs or an equal volume of normal saline by intragastric administration once a day for 8 weeks. Liver function, hydroxyproline and malondialdehyde (MDA) content, superoxide dismutase (SOD), Na + -K +- ATPase, and Ca 2+ -Mg 2+ -ATPase were analyzed using colorimetric methods. Moreover, mRNA expression and protein levels in the liver tissue were detected via quantitative polymerase chain reaction and western blotting, respectively., Results: The results showed that SLNP could effectively improve the liver function of rats with HF and significantly reduce the content of hydroxyproline; the mRNA expression and protein levels of alpha-smooth muscle actin (α-SMA), collagen I, III, and IV, transforming growth factor beta 1 (TGF-β1), Smad2/3, and Smad4 were also significantly reduced. Simultaneously, SLNP significantly increased the activities of SOD, Na + -K +- ATPase, and Ca 2+ -Mg 2+ -ATPase in the rat liver tissues, whereas it reduced the levels of MDA and SOD in the serum and liver tissues., Conclusion: This study revealed that SLNP elicits an anti-fibrotic effect by inhibiting oxidative stress and stellate cell activation, thereby reducing the formation and deposition of the extracellular matrix. The TGF-β1/Smads signaling pathway may be involved in this process., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The primary hyperoxalurias (PH), types 1-3, are disorders of glyoxylate metabolism that result in increased oxalate production and calcium oxalate stone formation. The breakdown of trans-4-hydroxy-L-proline (Hyp) from endogenous and dietary sources of collagen makes a significant contribution to the cellular glyoxylate pool. Proline dehydrogenase 2 (PRODH2), historically known as hydroxyproline oxidase, is the first step in the hydroxyproline catabolic pathway and represents a drug target to reduce the glyoxylate and oxalate burden of PH patients. This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C-terminal truncation variants indicated that residues 157-515 contain the catalytic core with one FAD molecule. The 12-fold higher kcat/Km value of 0.93 M-1·s-1 for Hyp over Pro demonstrates the preference for Hyp as substrate. Moreover, an anaerobic titration determined a Kd value of 125 µM for Hyp, a value ~1600-fold lower than the Km value. A survey of ubiquinone analogues revealed that menadione, duroquinone, and CoQ1 reacted more efficiently than oxygen as the terminal electron acceptor during catalysis. Taken together, these data and the slow reactivity with sodium sulfite support that PRODH2 functions as a dehydrogenase and most likely utilizes CoQ10 as the terminal electron acceptor in vivo. Thus, we propose that the name of PRODH2 be changed to hydroxyproline dehydrogenase (HYPDH). Three Hyp analogues were also identified to inhibit the activity of HYPDH, representing the first steps toward the development of a novel approach to treat all forms of PH. [ABSTRACT FROM AUTHOR]

Aims: Pulmonary fibrosis (PF) is considered as an end stage for many lung diseases. Mesenchymal stem cells (MSC) as regenerative therapy have become a remarkably valuable therapeutic strategy in different diseases. Hydrogen sulfide has been recently introduced into the medical field for its antifibrotic properties in addition to enhancement of MSC stemness and function. The aim of the present study was to investigate the ability of BM-MSC in combination with NaHS to attenuate Bleomycin induced pulmonary fibrosis was studied in rats. A special emphasis was given to miR-21 and GAS5 as important players in the development of PF., Main Methods: PF was induced in 32 Wistar male rats by single endotracheal injection of bleomycin, those were randomly divided into four groups (8 rats each): (untreated PF group) - (PF + MSC) treated group- (PF + NaHS treated group) - PF + combined (NAHS + MSC) treated group., Key Findings: Induction of PF was associated with increased miR-21 and decreased lncRNA-GAS5 expression. Treatment with either NaHS or BM-MSC leads to an inhibitory effect on pulmonary fibrosis as evidenced by improvement of histopathological studies, pulmonary function tests, reduction of inflammatory and fibrotic markers like Hydroxyproline, TNF α, TGF-β and caspase -3 together with downregulation miR-21 and increase lncRNA-GAS5 expression., Significance: The current work revealed the inhibitory effect of combined NaHS and BM-MSC on pulmonary fibrosis with concomitant modulation of miR-21 and lncRNA-GAS5 expression., Competing Interests: Declaration of competing interest None of the authors have potential conflicts of interest to be disclosed., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Overcoming the skin barrier properties efficiently, temporarily, and safely for successful transdermal drug delivery remains a challenge. We synthesized three series of potential skin permeation enhancers derived from natural amino acid derivatives proline, 4-hydroxyproline, and pyrrolidone carboxylic acid, which is a component of natural moisturizing factor. Permeation studies using in vitro human skin identified dodecyl prolinates with N-acetyl, propionyl, and butyryl chains (Pro2, Pro3, and Pro4, respectively) as potent enhancers for model drugs theophylline and diclofenac. The proline derivatives were generally more active than 4-hydroxyprolines and pyrrolidone carboxylic acid derivatives. Pro2-4 had acceptable in vitro toxicities on 3T3 fibroblast and HaCaT cell lines with IC 50 values in tens of µM. Infrared spectroscopy using the human stratum corneum revealed that these enhancers preferentially interacted with the skin barrier lipids and decreased the overall chain order without causing lipid extraction, while their effects on the stratum corneum protein structures were negligible. The impacts of Pro3 and Pro4 on an in vitro transepidermal water loss and skin electrical impedance were fully reversible. Thus, proline derivatives Pro3 and Pro4 have an advantageous combination of high enhancing potency, low cellular toxicity, and reversible action, which is important for their potential in vivo use as the skin barrier would quickly recover after the drug/enhancer administration is terminated., (© 2022. The Author(s).)

Current literature highlights the need for precise histological quantitative assessment of fibrosis which cannot be achieved by conventional scoring systems, inherent to their discontinuous values and reader-dependent variability. Here we used an automated image analysis software to measure fibrosis deposition in two relevant preclinical models of liver fibrosis, and established correlation with other quantitative fibrosis descriptors. Longitudinal quantification of liver fibrosis was carried out during progression of post-necrotic (CCl 4 -induced) and metabolic (HF-CDAA feeding) models of chronic liver disease in mice. Whole slide images of picrosirius red-stained liver sections were analyzed using a fully automated, unsupervised software. Fibrosis was characterized by a significant increase of collagen proportionate area (CPA) at weeks 3 (CCl 4 ) and 8 (HF-CDAA) with a progressive increase up to week 18 and 24, respectively. CPA was compared to collagen content assessed biochemically by hydroxyproline assay (HYP) and by standard histological staging systems. CPA showed a high correlation with HYP content for CCl 4 (r = 0.8268) and HF-CDAA (r = 0.6799) models. High correlations were also found with Ishak score or its modified version (r = 0.9705) for CCl 4 and HF-CDAA (r = 0.9062) as well as with NASH CRN for HF-CDAA (r = 0.7937). Such correlations support the use of automated digital analysis as a reliable tool to evaluate the dynamics of liver fibrosis and efficacy of antifibrotic drug candidates in preclinical models., (© 2022. The Author(s).)

Exposure to higher levels of arsenic is a serious threat affecting human health worldwide. We investigated the protective role of betaine (N,N,N-trimethylglycine) against sodium arsenite-induced renal dysfunction in rats. Sodium arsenite (5 mg/kg, oral) was given to rats for 4 weeks to induce nephrotoxicity. Betaine (125 and 250 mg/kg, oral) was administered in rats for 4 weeks along with sodium-arsenite feeding. Arsenic-induced renal dysfunction was demonstrated by measuring serum creatinine, creatinine clearance, urea, uric acid, potassium, fractional excretion of sodium, and microproteinuria. Oxidative stress in rat kidneys was determined by assaying thiobarbituric acid reactive substances, superoxide anion generation, and reduced glutathione levels. Furthermore, hydroxyproline assay was done to assess renal fibrosis in arsenic intoxicated rats. Hematoxylin-eosin and picrosirius red staining revealed pathological alterations in rat kidneys. Renal endothelial nitric oxide synthase (eNOS) expression was determined by immuno-histochemistry. Concurrent administration of betaine abrogated arsenic-induced renal biochemical and histological changes in rats. Betaine treatment significantly attenuated arsenic-induced decrease in renal eNOS expression. In conclusion, betaine is protective against sodium arsenite-induced renal dysfunction, which may be attributed to its anti-oxidant activity and modulation of renal eNOS expression in rat kidneys.

Objective: To investigate the effect of hydronidone on CCl 4 -induced liver fibrosis in rats and explore the possible mechanism., Methods: Sixty-six male SD rats were randomized into 5 groups, including a control group ( n =10), a liver fibrosis model group ( n =20), 2 hydronidone dose groups (100 and 250 mg/kg; n =12), and a pirfenidone (250 mg/kg) treatment group ( n = 12). Rat models of liver fibrosis were established by subcutaneous injection of CCl 4 in all but the control group. Hydronidone and pirfenidone were given daily at the indicated doses by intragastric administration for 6 weeks. After the treatments, serum samples were collected from the rats for detecting liver function parameters, and hydroxyproline content in the liver tissue was determined. Inflammation and fibrosis in the liver tissue were observed using HE staining and Sirius Red staining. In the cell experiment, human hepatic stellate cell line LX-2 was stimulated with TGF-β1 and treated with hydronidone or pirfenidone, and the expression levels of α-SMA, collagen type I and phosphorylated Smad3, phosphorylated p38, phosphorylated ERK1/2 and phosphorylated Akt were detected with Western blotting., Results: In the rat models of liver fibrosis, treatment with hydronidone obviously improved the liver functions, reduced the content of hydroxyproline in the liver tissue, and significantly alleviated liver fibrosis ( P < 0.05). In LX-2 cells, hydronidone dose-dependently decreased the expression levels of α-SMA and collagen type I. In TGF- β1-stimulated cells, the phosphorylation levels of Smad3, P38, ERK, and Akt increased progressively with the extension of the treatment time, but this effect was significantly attenuated by treatment with hydronidone ( P < 0.05)., Conclusion: Hydronidone can inhibit the phosphorylation of the proteins in the TGF-β signaling pathway, thereby preventing TGF-β1-mediated activation of hepatic stellate cells, which may be a possible mechanism by which hydronidone alleviates CCl 4 -induced liver fibrosis in rats.

Aims: Obeticholic acid (OCA) was approved for the treatment of primary biliary cholangitis (PBC) patients, as it can significantly improve the level of serum alkaline phosphatase. However, OCA-induced liver injury in PBC patients puts them at risk of acute chronic liver failure, thus limiting the clinical application of OCA. Osteopontin (OPN), an extracellular cell matrix molecule, is highly induced in many cholestatic liver diseases. Herein we explored whether liver injury exacerbation by OCA was related to OPN., Main Methods: Bile duct ligation (BDL) mice were treated with OCA (40 mg/kg) to evaluate its effect on liver injury and OPN involvement. Enzyme-linked immunosorbent assay, western blot, immunohistochemistry, and other assays were used to detect OPN levels in serum and liver. Immunohistochemistry, and immunofluorescence, among other assays, were used to evaluate the extent of ductular reaction. The extent of fibrosis was also determined using various assays, such as immunohistochemistry, quantitative real-time PCR (qPCR), and hydroxyproline assays., Key Findings: OPN was overexpressed in the liver of BDL mice treated with OCA. OCA induced overexpression of OPN exacerbated ductular reaction, fibrosis, and liver inflammation, and reduced hepatocyte proliferation., Significance: Upon liver injury, OCA upregulates the expression of OPN in the liver and accelerates disease progression. This mechanism helps explain the risk of liver damage associated with OCA., Competing Interests: Declaration of competing interest All authors disclosed no relevant relationships., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Adipose-derived mesenchymal stem cells (ASCs) have gained attention as a new treatment for systemic sclerosis (SSc). Low-molecular-weight heparin (LMWH) enhances cell function and stimulates the production of hepatocyte growth factor (HGF) in a variety of cells. This study investigated the effects of LMWH on the functions of mouse ASCs (mASCs), and the therapeutic effects of mASCs activated with LMWH (hep-mASCs) in mouse models of SSc., Methods: The cellular functions of mASCs cultured with different concentrations of LMWH were determined. Mice were divided into four groups: bleomycin (BLM)-induced SSc (BLM-alone), BLM-induced SSc administered with mASCs (BLM-mASC), and BLM-induced SSc administered with mASCs activated with 10 or 100 μg/mL LMWH (BLM-hep-mASC); there were 9 mice per group (n = 9). Skin inflammation and fibrosis were evaluated using histological and biochemical examinations and gene expression levels., Results: In vitro assays showed that migration ability and HGF production were significantly higher in hep-mASCs than in mASCs alone. The mRNA expression levels of cell migration factors were significantly upregulated in hep-mASCs compared to those in mASCs alone. The hep-mASCs accumulated in the skin tissues more than mASCs alone. The thickness of skin and hydroxyproline content in BLM-hep-mASC groups were significantly decreased, and the skin mRNA expression levels of interleukin-2, α-smooth muscle actin, transforming growth factor β1, collagen type 1 alpha 1, and tissue inhibitor of metalloproteinase 2 were significantly downregulated compared to those in the BLM-alone group., Conclusions: hep-mASCs showed higher anti-inflammatory and anti-fibrotic effects than mASCs alone and may be a promising candidate for SSc treatment., (© 2022. The Author(s).)

β-Carotene exhibits antioxidant and hepatoprotective activities via a multitude of biochemical mechanisms. However, the action mechanism involved in antioxidant and anti-inflammatory effects of this carotene in chronic liver diseases is not fully understood. In the present investigation, we have attempted to outline a plausible mechanism of β-carotene action against liver fibrosis in albino Wistar rats. To induce hepatic fibrosis, diethylnitrosamine (DEN) was administered in experimental rats for two weeks. DEN treated rats were divided into four groups, wherein each group comprised of five rats. β-Carotene supplement attenuated DEN-induced elevation in LFT markers (P < 0.05); averted depletion of glycogen (24%, P < 0.05) and, increased nitrite (P < 0.05), hydroxyproline (~67%, P < 0.05) and collagen levels (~65%, P < 0.05). Confocal microscopy of tissue sections stained with picrosirius red revealed accrued collagen in DEN-administered group, which was found to be reduced by β-carotene supplementation. Furthermore, β-carotene decreased the expression of iNOS/NOS-2 and NF-κB, as revealed by immunohistochemistry and Western immunoblotting. Collectively, these results demonstrate that β-carotene mitigates experimental liver fibrosis via inhibition of iNOS and NF-κB in-vivo . Thus, β-carotene may be suggested as a possible nutraceutical to curb experimental liver fibrosis.

Silicosis is one of the most important occupational diseases worldwide, caused by inhalation of silica particles or free crystalline silicon dioxide. As a disease with high mortality, it has no effective treatment and new therapeutic targets are urgently needed. Recent studies have identified FCER1A, encoding α-subunit of the immunoglobulin E (IgE) receptor FcεRI, as a candidate gene involved in the biological pathways leading to respiratory symptoms. FcεRI is known to be important in allergic asthma, but its role in silicosis remains unclear. In this study, serum IgE concentrations and FcεRI expression were assessed in pneumoconiosis patients and silica-exposed mice. The role of FcεRI was explored in a silica-induced mouse model using wild-type and FcεRI-deficient mice. The results showed that serum IgE concentrations were significantly elevated in both pneumoconiosis patients and mice exposed to silica compared with controls. The mRNA and protein expression of FcεRI were also significantly increased in the lung tissue of patients and silica-exposed mice. FcεRI deficiency significantly attenuated the changes in lung function caused by silica exposure. Silica-induced elevations of IL-1β, IL-6, and TNF-α were significantly attenuated in the lung tissue and bronchoalveolar lavage fluid (BALF) of FcεRI-deficient mice compared with wild-type controls. Additionally, FcεRI-deficient mice showed a significantly lower score of pulmonary fibrosis than wild-type mice following exposure to silica, with significantly lower hydroxyproline content and expression of fibrotic genes Col1a1 and Fn1. Immunofluorescent staining suggested FcεRI mainly on mast cells. Mast cell degranulation took place after silica exposure, as shown by increased serum histamine levels and β-hexosaminidase activity, which were significantly reduced in FcεRI-deficient mice compared with wild-type controls. Together, these data showed that FcεRI deficiency had a significant protective effect against silica-induced pulmonary inflammation and fibrosis. Our findings provide new insights into the pathophysiological mechanisms of silica-induced pulmonary fibrosis and a potential target for the treatment of silicosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Myostatin (MSTN), a member of the TGF-β superfamily, negatively regulates muscle growth. MSTN inhibition has been known to cause a double-muscled phenotype in skeletal muscle and fibrosis reduction in the heart. However, the role of MSTN in the cardiac extracellular matrix (ECM) needs more studies in various species of animal models to draw more objective conclusions. The main objective of the present study was to investigate whether loss of MSTN affects the cardiac extracellular matrix in pigs. Three MSTN knockouts (MSTN-/-) and three wild type (WT) male pigs were generated by crossing MSTN ± heterozygous gilts and boars. Cardiac ECM and underlying mechanisms were determined post-mortem. The role of MSTN on collagen expression was investigated by treating cardiac fibroblasts with active MSTN protein in vitro. MSTN protein was detected in WT hearts, while no expression was detected in MSTN-/- hearts. The heart-to-body weight ratio was significantly decreased in MSTN-/- pigs. The morphometric analyses, including picrosirius red staining, immunofluorescent staining, and ultra-structural thickness examination of the endomysium, revealed a significant reduction of connective tissue content in MSTN-/- hearts compared to WT. Hydroxyproline, type I collagen (Col1A), and p-Smad3/Smad3 levels were significantly lower in MSTN-/- hearts in vivo. On the contrary, cardiac fibroblasts treated with exogenous MSTN protein overexpressed Col1A and activated Smad and AKT signaling pathways in vitro. The present study suggests that inhibition of MSTN decreases cardiac extracellular matrix., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Aim: This study assessed the effects of topical application of the plant Globularia arabica extract on the diabetic rate of wound closure., Methods: The effect of methanol extracts of G. arabica leaf extract on pro-inflammatory and anti-inflammatory cytokines, as well as the rate of wound contraction, hydroxyproline, and collagen content, was examined using an excision model of wound repair in male rats., Results: The healing pattern was also highly related to using 10% plant extract and significantly, (p-values <0.025) increased wound contraction and increased hydroxyproline and collagen expression in both non-diabetic and diabetic rats, demonstrating that G. arabica extract possesses potent wound healing capacity. During the early wound healing phase, IL-6 levels were found to be upregulated by G. arabica treatment. Increased wound contraction augmented hydroxyproline and collagen content, supporting the early wound healing exhibited by G. arabica. One of the ways for speeding wound healing may be the induction of cytokine production. The result of our study shows that the fibroblast cell line treated with 20 µg/ml methanolic extract of G. arabica significantly (p-values <0.035) increases the cell migration that indicated the high wound healing activity., Conclusion: Results suggest that G. arabica may be useful in the topical management of wound healing., (© 2022 Wiley Periodicals LLC.)

Aim: Periodontitis (PD) is the sixth most prevalent disease around the world and is involved in the development and progression of multiple systemic diseases. Previous studies have reported that PD may aggravate liver injuries. The objective of this study was to investigate whether and how PD affects liver fibrosis., Materials and Methods: Ligature-induced PD (LIP) was induced in male C57/B6J mice, and sub-gingival plaques (PL) from patients with PD were applied to mouse teeth. Liver fibrosis was induced by carbon tetrachloride (CCl 4 ) injection. The mice were randomly divided into six groups: Oil, Oil+LIP, Oil+LIP+PL, CCl 4 , CCl 4 +LIP, and CCl 4 +LIP+PL. Alveolar bone resorption was evaluated by methylene blue staining. Hepatic function was analysed by serum alanine aminotransferase and hepatic hydroxyproline. Picrosirius red and α-smooth muscle actin (SMA) staining were used to evaluate the fibrotic area. RNA sequencing and quantitative RT-PCR were used to measure gene expression. Western blotting was used to measure protein levels. Flow cytometry was used to analyse the accumulation of immune cells. Mouse microbiota were analysed using 16S rRNA gene sequencing., Results: Mice in the CCl 4 +LIP+PL group displayed higher serum alanine aminotransferase and hepatic hydroxyproline as well as more Picrosirius red-positive and α-SMA-positive areas in liver samples than those of the CCl 4 group, suggesting that PD (LIP+PL) aggravated CCl 4 -induced hepatic dysfunction and liver fibrosis. Consistently, the expression of fibro-genic genes and the protein levels of transforming growth factor β were much higher in the CCl 4 +LIP+PL group than in the CCl 4 group. Flow cytometry revealed that PD increased the accumulation of immune cells, including Kupffer cells, B cells, and Th17 cells, in the liver of mice with CCl 4 treatment. PD also increased the expression of inflammatory genes and activated pro-inflammatory nuclear factor-kappa B pathway in the livers of CCl 4 -injected mice. Moreover, PD altered both oral and liver microbiota in CCl 4 -injected mice., Conclusions: PD aggravates CCl 4 -induced hepatic dysfunction and fibrosis in mice, likely through the increase of inflammation and alteration of microbiota in the liver., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Present study was conducted to undermine the wound healing potential of mangiferin vis a vis its molecular dynamics in immunocompromised excisional rat model. 120 rats were randomly and equally divided into five groups viz. group I (Healthy control), group II (Immunocompromised control), group III (Immunocompromised group treated with silver sulphadiazine), group IV (Immunocompromised group treated with 2.5 %Mangiferin) and group V (Immunocompromised group treated with 5 %Mangiferin). Immuno compromised state was achieved following intramuscular injection of Hydrocortisone @ 80 mg/kg body weight. Study was conducted for a period of 28 days. Six animals from each group were humanely sacrificed at weekly interval till day 28th of study. Planimetric analysis, biochemical studies viz. hydroxyproline assay, total protein and DNA content, antioxidative potential through LPO assay was done along with molecular studies involving expression profiling of IL1β, TNFα and COX-2 and Immunohistochemistry of angiogenic marker i.e. VEGF was performed to undermine the pharmacodynamics of mangiferin. Histopathological studies including H&E and Masson's Trichome was also performed to study histoarchitectural changes in wound healing and reparative process following application of mangiferin ointment. Study revealed significant (P ≤ 0.05) reduction in wound area measurement and significant (P ≤ 0.05) increase in wound contraction (%) following mangiferin administration in immunocompromised rats. Hydroxyproline, DNA and total protein showed significant (P ≤ 0.05) increase in skin tissues of mangiferin treated immunocompromised rats. LPO assay revealed significant (P ≤ 0.05) reduction in mangiferin treated animals. Histopathological studies of skin tissues revealed complete restoration advocating grade III of healing in 2.5% mangiferin treated group. Higher expression and strong signal intensity of VEGF was noticed in 2.5% mangiferin treatment group along with significant (P ≤ 0.05) upregulation IL1β and TNFα on day 7 in 2.5% mangiferin treatment group with significant (P ≤ 0.05) down regulation of COX-2 in mangiferin treatment group as compared to other groups i.e. group II and III. It is concluded from our study that mangiferin facilitates wound healing through improved wound closure, organized deposition of collagen deposition and granulation matrix formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The importance of insects in our ecosystems is undeniable. The indiscriminate use of broad-spectrum insecticides is a factor in the decline in insect biomass. We identify and sequence a prominent neuropeptide hormone in insects with an overarching goal to elucidate relatedness and create a database of bioactive peptides that could inform possible cross-activity in biological assays for the identification of a biorational lead compound. The major task of an adipokinetic hormone (AKH) in an insect is the regulation of metabolic events, such as carbohydrate and lipid breakdown in storage tissue during intense muscular work. From genomic and/or transcriptomic information one may predict the genes encoding neuropeptides such as the AKHs of insects. Definite elucidation of the primary structure of the mature peptide with putative post-translational modifications needs analytical chemical methods. Here we use high-resolution mass spectrometry coupled with liquid chromatography to identify unequivocally the AKHs of five insect species (one cockroach, two moths, and two flies) of which either genomic/transcriptomic information was available or sequences from related species. We confirm predicted sequences and discover novel AKH sequences, including one with a post-translational hydroxyproline modification. The additional sequences affirm an evolutionary pattern of dipteran AKHs and a conserved pattern in crambid moths.

Nonalcoholic fatty liver disease (NAFLD), a hepatic characteristic of metabolic syndrome, received significant attention in clinical settings. The multiple-hit theory is one of the proposed mechanisms of NAFLD, and gut dysbiosis is considered a hit. Thus, controlling gut microbiota is a potential target in the management of NAFLD, and probiotics can be used as a treatment agent for NAFLD. The current study aimed to investigate the efficacy of probiotics against nonalcoholic steatohepatitis in a hepatocyte-specific PTEN knockout mouse model that mimics the characteristics of human NAFLD. Probiotics were administered to male knockout mice for 8 or 40 weeks. Next, we assessed hepatic inflammation, fibrosis, carcinogenesis, and oxidative stress. Probiotics were found to reduce serum transaminase levels, NAFLD activity score, and the gene expression of pro-inflammatory cytokines. In addition, they decreased liver fibrosis grade, which was examined via Sirius red staining, gene expression of fibrotic markers, and hydroxyproline. Furthermore, probiotics suppressed the number of liver tumors, particular in HCC. Probiotics reduced oxidative stresses, including glutathione levels, and anti-oxidative stress marker, which may be an underlying mechanism for their beneficial effects. In conclusion, probiotics treatment had beneficial effects against NAFLD and carcinogenesis in hepatocyte-specific PTEN knockout mice., (© 2022. The Author(s).)

Purpose: Immobilization of skeletal muscles causes muscle atrophy, muscle contracture, and muscle pain, the mechanisms of which are related to macrophage accumulation. However, muscle contractile exercise through a belt electrode device may mitigate macrophage accumulation. We hypothesized that such exercise would be effective in preventing myofiber atrophy, muscle contracture, and muscular pain. This study tested this hypothesis in immobilized rat gastrocnemius muscle., Materials and Methods: A total of 32 rats were divided into the following control and experimental groups: immobilization (immobilized treatment only), low-frequency (LF; immobilized treatment and muscle contractile exercise with a 2 s (do) /6 s (rest) duty cycle), and high-frequency (HF; immobilized treatment and muscle contractile exercise with a 2 s (do)/2 s (rest) duty cycle). Electrical stimulation was performed at 50 Hz and 4.7 mA, and muscle contractile exercise was applied to the lower limb muscles for 15 or 20 min/session (once daily) for 2 weeks (6 times/week). After the behavioral tests, the bilateral gastrocnemius muscles were collected for analysis., Results: The number of macrophages, the Atrogin-1 and MuRF-1 mRNA expression, and the hydroxyproline content in the HF group were lower than those in the immobilization and LF groups. The cross-sectional area (CSA) of type IIb myofibers in the superficial region, the PGC-1α mRNA expression, and the range of motion of dorsiflexion in the HF group were significantly higher than those in the immobilization and LF groups. The pressure pain thresholds in the LF and HF groups were significantly higher than that in the immobilization group, and the nerve growth factor (NGF) content in the LF and HF groups was significantly lower than that in the immobilization group., Conclusion: Muscle contractile exercise through the belt electrode device may be effective in preventing immobilization-induced myofiber atrophy, muscle contracture, and muscular pain in the immobilized rat gastrocnemius muscle., Competing Interests: Financial competing interests ● Ownership of stocks or shares: No ● Paid employment or consultancy: No ● Board membership: No ● Patent applications (pending or actual), including individual applications or those belonging to the institution to which the authors are affiliated and from which the authors may benefit: No ● Research grants (from any source, restricted or unrestricted): Yes. 1. KAKENHI from Japan Society for the Promotion of Science (grant No. 19K19795, 20K20668, and 21H03291). ◇ Description of funder’s role in the study design; collection, analysis, and interpretation of data; writing of the paper; and/or decision to submit for publication: No ◇ Whether they have served or currently serve on the editorial board of the journal to which they are submitting: No ◇ Whether they have sat or currently sit on a committee for an organization that may benefit from publication of the paper: No 2. research funding from ALCARE Co., Ltd. ◇ Description of funder’s role in the study design; collection, analysis, and interpretation of data; writing of the paper; and/or decision to submit for publication: No ◇ Whether they have served or currently serve on the editorial board of the journal to which they are submitting: No ◇ Whether they have sat or currently sit on a committee for an organization that may benefit from publication of the paper: No ● Travel grants and honoraria for speaking or participation at meetings: No ● Gifts: No Non-financial competing interests Authors do not have Non-financial competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objectives: To evaluate the inhibitory activity of an ion-releasing filler (S-PRG) eluate on dentin collagen-bound metalloproteinases (MMPs) and dentin matrix degradation., Methods: Dentin beams (5 × 2 × 0.5 mm) from human molars were completely demineralized to produce dentin matrix specimens. The dry mass was measured, and a colorimetric assay (Sensolyte) determined the initial total MMP activity to allocate the beams into four treatment groups (n = 10/group): 1) water for 1 min (negative control); 2) 2% chlorhexidine digluconate (CHX - inhibitor control) for 1 min; 3) S-PRG eluate for 1 min; 4) S-PRG eluate for 30 min. After the treatments, the total MMP activity was reassessed. The specimens were stored in simulated body fluid (SBF) at 37 °C for up to 21 days. The dry mass was reassessed weekly. On day 7, the dentin matrix degradation was analyzed for the presence of collagen fragments (CF; Sirius Red) and hydroxyproline (Hyp) in the SBF. Statistical analyses were performed with ANOVA/Tukey, paired t-tests, and RM-ANOVA/Sidak (α = 5%)., Results: S-PRG eluate exposure for 1 and 30 min reduced (p < 0.0001) MMP activity. S-PRG exposure for 30 min presented MMP activity inhibition equivalent to CHX (p = 0.061). S-PRG and CHX decreased CF (p ≤ 0.007) and Hyp (p < 0.046) release. After 21 days of storage, S-PRG-treated beams, regardless of exposure time, presented a reduced (p ≤ 0.017) mass loss, intermediate between CHX and control., Conclusion: Treating demineralized dentin with S-PRG eluate for 1 or 30 min reduced matrix-bound MMP activity and dentin matrix degradation for up to 21 days., Clinical Significance: S-PRG filler may hinder the progression of dentin carious/erosive lesions and enhance the stabilization of dentin bonding interfaces., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterised by irreversible fibrosis and destruction of the alveolar structure. Receptor for advanced glycation end products (RAGE) has been identified as one of the key molecules involved in IPF pathogenesis. A RAGE-antagonist peptide (RAP) was developed based on the RAGE-binding domain of high mobility group box-1 (HMGB-1). Anti-IPF effects of RAP were evaluated in a bleomycin-induced mouse model of IPF. Bleomycin was administered intratracheally, and then RAP was administrated twice by intratracheal instillation, 1 and 3 d after bleomycin challenge. Seven days after the bleomycin challenge, the mice were sacrificed and the lungs were harvested. The results showed that pulmonary hydroxyproline was reduced in mice administered RAP compared with the control group. Tumour growth factor-β (TGF-β), α-smooth muscle actin (α-SMA) and collagen were also reduced by RAP administration in a dose-dependent manner. Longer-term effects of RAP were investigated in mice challenged with bleomycin. RAP was administered intratracheally every 7 d for 28 d, after which lung samples were harvested and analysed. The results showed that hydroxyproline, TGF-β, α-SMA and collagen were reduced by repeated RAP administration. Taken together, the results suggest that RAP is useful for treatment of IPF.