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Objective: To observe whether metformin (MET) inhibits transforming growth factor-β 1 (TGF-β 1 )/Smad3 signaling pathway by activating adenosine activated protein kinase (AMPK), so as to alleviate the pulmonary fibrosis caused by paraquat (PQ) poisoning in mice., Methods: Male C57BL/6J mice were randomly divided into the Control group, PQ poisoning model group (PQ group), MET intervention group (PQ+MET group), AMPK agonist group (PQ+AICAR group), and AMPK inhibitor group (PQ+MET+CC group), according to a random number table method. A mouse model of PQ poisoning was established by one-time peritoneal injection of 1 mL PQ solution (20 mg/kg). The Control group was injected with the same volume of normal saline. After 2 hours of modeling, the PQ+MET group was given 2 mL of 200 mg/kg MET solution by gavage, the PQ+AICAR group was given 2 mL of 200 mg/kg AICAR solution by intraperitoneal injection, the PQ+MET+CC group was given 2 mL of 200 mg/kg MET solution by gavage and then 1 mL complex C (CC) solution (20 mg/kg) was intraperitoneally injected, the Control group and PQ group were given 2 mL of normal saline by gavage. The intervention was given once a day for 21 consecutive days. The 21-day survival rate of ten mice in each group was calculated, and the lung tissues of remaining mice were collected at 21 days after modeling. The pathological changes of lung tissues were observed under light microscope after hematoxylin-eosin (HE) staining and Masson staining, and the degree of pulmonary fibrosis was evaluated by Ashcroft score. The content of hydroxyproline in lung tissue and oxidative stress indicators such as malondialdehyde (MDA) and superoxide dismutase (SOD) were detected. The protein expressions of E-cadherin, α-smooth muscle actin (α-SMA), phosphorylated AMPK (p-AMPK), TGF-β 1 and phosphorylated Smad3 (p-Smad3) in lung tissue were detected by Western blotting., Results: Compared with the Control group, the 21 days survival rate was significantly reduced, lung fibrosis and Ashcroft score were significantly increased in PQ group. In addition, the content of hydroxyproline, MDA and the protein expressions of α-SMA, TGF-β 1 and p-Smad3 in lung tissue were significantly increased, while the activity of SOD and the protein expressions of E-cadherin and p-AMPK were significantly decreased in PQ group. Compared with the PQ group, the 21 days survival rates of mice were significantly improved in the PQ+MET group and PQ+AICAR group (70%, 60% vs. 20%, both P < 0.05). The degree of pulmonary fibrosis and the Ashcroft score were significantly reduced (1.50±0.55, 2.00±0.63 vs. 6.67±0.52, both P < 0.05). The content of hydroxyproline and MDA in lung tissue, as well as α-SMA, TGF-β 1 and p-Smad3 protein expressions were significantly reduced [hydroxyproline (mg/L): 2.03±0.11, 3.00±0.85 vs. 4.92±0.65, MDA (kU/g): 2.06±1.48, 2.10±1.80 vs. 4.06±1.33, α-SMA/GAPDH: 0.23±0.06, 0.16±0.06 vs. 1.00±0.09, TGF-β 1 /GAPDH: 0.28±0.03, 0.53±0.05 vs. 0.92±0.06 p-Smad3/GAPDH: 0.52±0.04, 0.69±0.06 vs. 1.11±0.10, all P < 0.05], SOD activity and the protein expressions of E-cadherin and p-AMPK were significantly increased [SOD (μmol/g): 39.76±1.35, 33.03±1.28 vs. 20.08±1.79, E-cadherin/GAPDH: 0.91±0.08, 0.72±0.08 vs. 0.26±0.04, p-AMPK/GAPDH: 0.62±0.04, 0.60±0.01 vs. 0.20±0.04, all P < 0.05]. However, these protective effects of MET were inhibited by the addition of AMPK inhibitor CC solution., Conclusions: MET can effectively alleviate the degree of pulmonary fibrosis in mice poisoned with PQ, and its mechanism may be related to the activation of AMPK and inhibition of TGF-β 1 /Smad3 signaling pathway, which can be inhibited by AMPK inhibitor CC.

Although many efforts have been made to improve management strategies and diagnostic methods in the past several decades, the prevention of anastomotic complications, such as anastomotic leaks and strictures, remain a major clinical challenge. Therefore, new molecular pathways need to be identified that regulate anastomotic healing, and to design new treatments for patients after anastomosis to reduce the occurrence of complications. Rabbits were treated with a MST1/2 inhibitor XMU-XP-1, a Chinese medicine formula Shenhuang plaster (SHP) or a control vehicle immediately after surgery. The anastomotic burst pressure, collagen deposition, and hydroxyproline concentration were evaluated at 3 and 7 days after the surgery, and qRT-PCR and western-blot analyses were used to characterize mRNA and protein expression levels. Both XMU-XP-1 and SHP significantly increased anastomotic burst pressure, collagen deposition, and the concentration of hydroxyproline in intestinal anastomotic tissue at postoperative day 7 (POD 7). Importantly, SHP could induce TGF-β1 expression, which activated its downstream target Smad-2 to activate the TGF-β1 signaling pathway. Moreover, SHP reduced the phosphorylation level of YAP and increased its active form, and treatment with verteporfin, a YAP-TEAD complex inhibitor, significantly suppressed the effects induced by SHP during anastomotic tissue healing. This study demonstrated that activation of the Hippo-YAP pathway enhances anastomotic healing, and that SHP enhances both the TGF-β1/Smad and YAP signaling pathways to promote rabbit anastomotic healing after surgery. These results suggest that SHP could be used to treat patients who underwent anastomosis to prevent the occurrence of anastomotic complications., Competing Interests: The authors report no conflicts of interest in this work.

Antimicrobial peptides (AMPs) often display guanidinium functionalities, and hence robust synthetic procedures are needed to facilitate access to analogues with unnatural homologues of arginine (Arg = R). Initially, a resin-bound Arg/Pro-rich fluoren-9-yl-methyloxycarbonyl-protected fragment (Fmoc-RPRPPR) of the AMP oncocin (i.e., VDKPPYLPRPRPPRRIYNR-NH 2 ) was employed in a comparative on-resin assessment of commercial guanidinylation reagents head-to-head with the recently studied bis-Boc-protected triazole-based reagent, 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]-carboxamidine, which was synthesized by a chromatography-free procedure. This reagent was found to enable quantitative conversion in solid-phase peptide synthesis (SPPS) of peptides displaying homoarginine (Har) residues and/or an N-terminal guanidinium group. SPPS was used to obtain analogues of the 18-mer oncocin with single as well as multiple Arg → Har modifications. In addition, the effect of replacement of proline (Pro) residues in oncocin was explored by incorporating single or multiple trans-4-hydroxy-l-proline (Hyp) or 4,4-difluoro-l-proline (Dfp) residues, which both affected hydrophobicity. The resulting peptide library was tested against both Gram-negative and Gram-positive bacteria. Analysis of the minimal inhibitory concentrations (MICs) showed that analogues, displaying modifications at positions 4, 5 and 12 (originally Pro residues), had retained or slightly improved antimicrobial activity. Next, an oncocin analogue with two stabilizing l-Arg → d-Arg replacements in the C-terminal part was further modified by triple-replacement of Pro by either Dfp or Hyp in positions 4, 5, and 12. The resulting analogue displaying three Pro → Dfp modifications proved to possess the best activity profile: MICs of 1-2 µg/mL against E. coli and Klebsiella pneumoniae, less than 1% hemolysis at 800 µg/mL, and an IC 50 above 1280 µg/mL in HepG2 cells. Thus, incorporation of bis-fluorinated Pro residues appears to constitute a novel tool in structure-activity studies aimed at optimization of Pro-rich AMPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that have influenced the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

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.

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.

Objective: Ready to eat fried liver meat balls (LMB) were developed to fight anaemia and vitamin A deficiency and promote cognitive function., Design: Randomised controlled trial consisting of two arms: control group with no supplement and LMB group receiving LMB supplement three times a week for 90 d. Criteria of evaluations included dietary assessment, anthropometric measurements, laboratory investigations and cognitive function by Wechsler test., Setting: Kinder Garten and primary school in Urban Giza., Participants: Sixty boys and girls aging 3-9 years., Results: The LMB supplement contributed to significant increases in the intakes of high bioavailable Fe and vitamin A in the diets of all children. Initial overall prevalence of mild and moderate anaemia was 43 %, which disappeared completely from all children aging < 72 months and from 88 % of children ≥ 72 months after the 90 d dietary intervention with the LMB. Faecal systemic immune globulin A, urinary hydroxyproline index and urinary iodine excretion increased significantly ( P < 0·05) only after the dietary intervention with the LMB supplement for 90 d. The standard scores of verbal and non-verbal cognitive function tests (Δ day 90-day 0) increased significantly ( P < 0·05) among the LMB group compared with the respective changes observed among the control group. The increase in height-for-age Z score and blood Hb were good predictors for improvement in cognitive function., Conclusion: LMB supplement is effective sustainable nutritious biotherapeutic food in fighting hidden hunger and promoting the cognitive function.

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.

Propolis is rich in natural bioactive compounds, and considering its importance for many skin therapies, emulgel was prepared. This study examines how a propolis extract (PE) and Passiflora edulis seed (PS) oil emulgel affect rat deep skin wound healing. Based on preset criteria of maximum drug content and optimum drug permeation through the stratum corneum along with drug retention in the skin layers, an optimized emulgel formula based on Box-Behnken factorial design was prepared and used for subsequent in vitro and in vivo evaluations. In vivo wound-healing activities of emulgel and control treatments were investigated in a rat model. The optimized emulgel formula exhibited superior healing activity compared with plain PE suspension-treated rats on day 14 of wounding. Histopathological investigations of hematoxylin and eosin and Masson's Trichrome-stained skin sections supported this effect. Emulgel promotes cutaneous wound healing through a variety of mechanisms, including anti-inflammatory through modulation of cytokines tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 production, and promotion of collagen fiber formation, all of which contribute to tissue remodeling. Furthermore, when compared with propolis suspension, emulgel showed significant antioxidant and anti-inflammatory effects. Emulgel significantly increased the skin's hydroxyproline level, antioxidant potential, wound contraction, increased penetration, and localized propolis deposition across the skin. Incorporation of PS oil into the emulgel accelerates the tissue regeneration process. The findings suggest that 5% propolis emulgel could be used as an alternative to treat wounds.

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.)

Macrophages play an important role in causing silicosis eventually becoming an irreversible fibrotic disease, and there are no specific drugs for silicosis in the clinic so far. Pirfenidone has consistently been shown to have anti-inflammatory and anti-fibrotic effects, but the specific mechanism by which it ameliorates fibrosis in silicosis is unclear. A rat silicosis model was established in this study, and lung tissues and serum were collected by batch execution at 14, 28, and 56 days. Also, the effects of Pirfenidone on macrophage polarization and pulmonary fibrosis were evaluated in silicosis with early intervention and late treatment by histological examination, Enzyme-linked immunosorbent assay, Hydroxyproline assay, Western blot and Quantitative reverse transcription polymerase chain reaction. The results showed that Pirfenidone significantly reduced pulmonary fibrosis in rats with silicosis, and both early intervention and late treatment effectively inhibited the expression of α-SMA, Col-I, Vimentin, Hydroxyproline, IL-1β, IL-18, and the M2 macrophage marker CD206 and Arg-1, while only early intervention effectively inhibited E-cad, TGF-β1, TNF-α, and the M1 macrophage marker iNOS, CD86. Furthermore, Pirfenidone dramatically reduced the mRNA expression of the JAK2/STAT3. These findings imply that Pirfenidone may reduce pulmonary fibrosis in silicosis rats by inhibiting macrophage polarization via the JAK2/STAT3 signaling pathway., 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.)

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.)

Objective: For a long time, natural compounds have been used to accelerate wound healing. In this study, the topical effects of ammoniacum gum extract on wound healing were investigated in white male rats., Method: Following skin wound induction in aseptic conditions, 48 Wistar rats were divided into six equal groups; phenytoin cream 1% (standard), untreated (control), Eucerin (control), and 5%, 10% and 20% ointments of Dorema ammoniacum gum extract (treatment groups). All experimental groups received topical drugs daily for 14 days. The percentage of wound healing, hydroxyproline content, histological parameters, and growth factors (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-α) were measured in experimental groups., Results: The areas of the wounds in the treatment groups were significantly decreased compared with the wound areas of control groups at 5, 7 and 10 days after wounding. On the 12th day, the wounds in the treatment groups were completely healed. Hydroxyproline contents were significantly increased in the treatment groups compared with the control groups (p<0.001). In histological evaluation, the re-epithelialisation, increasing thickness of the epithelial layer, granulation tissue and neovascularisation parameters in the treatment groups showed significant increases compared with the control groups. Also, serum levels of TGF-β, PDGF, EGF and VEGF in the treatment groups were significantly increased compared to the control groups., Conclusion: The topical application of ammoniacum gum extract significantly increases the percentage of wound healing in rats and reduces the time of wound closure.

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.)

Aims/objectives: Wound healing in people with diabetes is delayed secondary to impaired nitric oxide generation, advanced glycation end products (AGE), and poor migration of epithelial cells. We developed a novel topical esmolol hydrochloride (Galnobax) and assessed its efficacy for wound healing in streptozocin-induced diabetic hairless rat., Methods: All experiments were performed at an animal laboratory and tertiary-care research facility. Ex vivo aldose reductase inhibition was assessed from enzymes obtained from a bacterial culture (spectrophotometer), sorbitol content in homogenized red blood cells, and AGE in glucose and bovine serum by fluorometry following the addition of esmolol in varying concentrations. A scratch assay of human fibroblasts, endothelial cells, and keratinocytes was assessed under a high-glucose environment and after esmolol by phase-contrast microscopy. The efficacy evaluation of the topical application of Galnobax (14 and 20%) or vehicle was conducted in streptozotocin-induced diabetic hairless rats, and endogenous nitrite and hydroxyproline from homogenized wound tissue were measured along with pharmacokinetic and dermal toxicity in Hanford miniature swine., Results: Esmolol inhibited the formation of sorbitol by 59% in erythrocytes in comparison to glucose-induced sorbitol levels. AGE generation in bovine serum albumin was reduced at 1 mM esmolol concentrations (2.6 ± 1.7) compared with control ( p < 0.05) and similar to that of diclofenac (2.5 ± 1.3). Esmolol at 1 and 10 µM enhanced the migration of fibroblasts, epithelial cells, and keratinocytes compared with control. The nitric oxide levels (day 7) were 44 and 112% higher with Galnobax (14%) than those of the diabetic group ( p < 0.05) and the vehicle control group ( p < 0.05), respectively. The days 7 and 14 hydroxyproline in the wound was higher by 22 and 44% following Galnobax (14%) compared with the diabetic and vehicle control groups. The wound area exhibited better reduction with Galnobax at 14% up to day 10 follow-up compared with the controls. The pharmacokinetic and dermal toxicity in miniature swine suggested no significant adverse event with Galnobax., Conclusions: Topical esmolol hydrochloride is a novel, safe, and effective treatment modality that acts through pleotropic mechanisms to hasten wound healing in diabetes., Competing Interests: Authors SK and SD were employed by NovaLead Pharma Pvt. Ltd., Pune, India. The authors declare that this study received funding from NovaLead Pharma Pvt. Ltd. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication., (Copyright © 2022 Kulkarni, Deshpande and Rastogi.)

Aim: This study assessed the effects of topical application of R. coriaria extract on the rate of wound closure. The rate of wound contraction was used to assess the wound healing efficacy of the R. coriaria fruit methanolic extract., Methods: Using excision and burn model of wound repair in diabetic male Wistar rats. Also, hydroxyproline, collagen content, and proinflammatory and anti-inflammatory cytokines levels were determined in this study., Results: During the early wound healing phase, interleukin 6 (IL-6) levels were found to be decreased by R. coriaria treatment and increased the level of interleukin 10 (IL-10). Increased wound contraction augmented with hydroxyproline and collagen content, supporting the early wound healing exhibited by R. coriaria. The epithelialization, neovascularization and enhanced hydroxyproline and collagen expression were strongly associated with the healing pattern., Conclusion: This study indicating that R. coriaria methanolic fruit extract has a potent wound healing capacity. And may be effective in the topical therapy of wound healing., (© 2021 Wiley Periodicals LLC.)

Objective: To assess dentin collagen denaturation from phosphoric acid and enzyme treatments using collagen hybridizing peptide (CHP) and to investigate the effect of collagen denaturation on bio-stabilization promoted by proanthocyanidins (PA)., Methods: Human molars were sectioned into 7-µm-thick dentin films, demineralized, and assigned to six groups: control with/without PA modification, H 3 PO 4 -treated collagen with/without PA modification, enzyme-treated collagen with/without PA modification. PA modification involved immersing collagen films in 0.65% PA for 30 s. H 3 PO 4 and enzyme treatments were used to experimentally induce collagen denaturation, which was quantitated by fluorescence intensity (FI) from the fluorescently-conjugated-CHP (F-CHP) staining (n = 4). FTIR was used to characterize collagen structures. All groups were subject to collagenase digestion to test the bio-stabilization effect of PA on denatured collagen using weight loss analysis and hydroxyproline assay (n = 6). Data were analyzed using two-factor ANOVA and Games-Howell post hoc tests (α = 0.05)., Results: FTIR showed collagen secondary structural changes after denaturation treatments and confirmed the incorporation and cross-linking of PA in control and treated collagen. F-CHP staining indicated high-degree, medium-degree, and low-degree collagen denaturation from H 3 PO 4 -treatment (FI = 83.22), enzyme-treatment (FI = 36.54), and control (FI = 6.01) respectively. PA modification significantly reduced the weight loss and hydroxyproline release of all groups after digestion (p < 0.0001), with the results correlated with FI values at r = 0.96-0.98., Significance: A molecular method CHP is introduced as a sensitive technique to quantitate dentin collagen denaturation for the first time. PA modification is shown to effectively stabilize denatured collagen against collagenase digestion, with the stabilization effect negatively associated with the collagen denaturation degree., (Copyright © 2022 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

The ubiquitination of the hypoxia-inducible factor-1α (HIF-1α) is mediated by interacting with the von Hippel-Lindau protein (VHL), and is associated with cancer, chronic anemia, and ischemia. VHL, an E3 ligase, has been reported to degrade HIF-1 for decades, however, there are few successful inhibitors currently. Poor understanding of the binding pocket and a lack of in-depth exploration of the interactions between two proteins are the main reasons. Hence, we developed an effective strategy to identify and design new inhibitors for protein-protein interaction targets. The hydroxyproline (Hyp564) of HIF-1α contributed the key interaction between HIF-1α and VHL. In this study, detailed information of the binding pocket were explored by alanine scanning, site-directed mutagenesis and molecular dynamics simulations. Interestingly, we found the interaction(s) between Y565 and H110 played a key role in the binding of VHL/HIF-1α. Based on the interactions, 8 derivates of VH032, 16a-h, were synthesized by introducing various groups bounded to H110. Further assay on protein and cellular level exhibited that 16a-h accessed higher binding affinity to VHL and markable or modest improvement in stabilization of HIF-1α or HIF-1α-OH in HeLa cells. Our work provides a new orientation for the modification or design of VHL/HIF-1α protein-protein interaction inhibitors., 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 © 2021 Elsevier Masson SAS. All rights reserved.)

Despite the success of imatinib in CML therapy through Bcr-Abl inhibition, acquired drug resistance occurs over time in patients. In particular, the resistance caused by T315I mutation remains a challenge in clinic. Herein, we embarked on a structural optimization campaign aiming at discovery of novel Bcr-Abl inhibitors toward T315I mutant based on previously reported dibenzoylpiperazin derivatives. We proposed that incorporation of flexible linker could achieve potent inhibition of Bcr-Abl T315I by avoiding steric clash with bulky sidechain of Ile315. A library of 28 compounds with amino acids as linker has been developed and evaluated. Among them, compound AA2 displayed the most potent activity against Bcr-Abl WT and Bcr-Abl T315I , as well as toward Bcr-Abl driven K562 and K562R cells. Further investigations indicated that AA2 could induce apoptosis of K562 cells and down regulate phosphorylation of Bcr-Abl. In summary, the compounds with amino acid as novel flexible linker exhibited certain antitumor activities, providing valuable hints for the discovery of novel Bcr-Abl inhibitors to overcome T315I mutant resistance, and AA2 could be considered as a candidate for further optimization., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Commonly used aquatic feed naturally contains low-level or no hydroxyproline (Hyp). This study was conducted to evaluate the effects of dietary Hyp inclusion on growth performance, body composition, amino acid profiles, blood biochemistry, and the expression of target of rapamycin (TOR) pathway-related genes in juvenile Nibea diacanthus. Fish with similar size (initial body weight, 133.00 ± 2.14 g) were fed six isonitrogenous and isolipidic practical diets supplemented with graded levels of Hyp (0, 5, 10, 15, 20, and 25 g kg -1 of dry matter) for 8 weeks. The results indicated that growth performance and feed utilization were improved with increased levels of dietary Hyp (P < 0.05), and the optimum amount of dietary Hyp estimated from SGR as 16.6 g kg -1 . The crude protein of whole body and swim bladder and the amino acid composition of muscle and swim bladder were significantly (P < 0.05) affected by the addition of dietary Hyp, which reflects the important role of feed composition in animal body composition. In addition, the expression levels of mammalian target of rapamycin (TOR) and ribosomal protein S6 kinase1 (S6K1) genes in the liver, muscle, and swim bladder increased with increasing Hyp content of diets, while the mRNA expression level of eukaryotic translation initiation factor 4E-binding protein (4E-BP) gene in these tissues decreased. These results indicated that Hyp improved fish growth and the ability to synthesize proteins, most likely through the TOR pathway. It is suggested that dietary Hyp supplementation is particularly necessary for application in aquatic feed.

The N -methyl-(2S,4R)-trans-4-hydroxy-l-proline-enriched fraction (NMP) from Sideroxylon obtusifolium was evaluated as a neuroprotective agent in the intracerebroventricular (icv) pilocarpine (Pilo) model. To this aim, male mice were subdivided into sham (SO, vehicle), Pilo (300 µg/1 µL icv, followed by the vehicle per os , po ) and NMP-treated groups (Pilo 300 µg/1 µL icv, followed by 100 or 200 mg/kg po ). The treatments started one day after the Pilo injection and continued for 15 days. The effects of NMP were assessed by characterizing the preservation of cognitive function in both the Y-maze and object recognition tests. The hippocampal cell viability was evaluated by Nissl staining. Additional markers of damage were studied-the glial fibrillary acidic protein (GFAP) and the ionized calcium-binding adaptor molecule 1 (Iba-1) expression using, respectively, immunofluorescence and western blot analyses. We also performed molecular docking experiments revealing that NMP binds to the γ-aminobutyric acid (GABA) transporter 1 (GAT1). GAT1 expression in the hippocampus was also characterized. Pilo induced cognitive deficits, cell damage, increased GFAP, Iba-1, and GAT1 expression in the hippocampus. These alterations were prevented, especially by the higher NMP dose. These data highlight NMP as a promising candidate for the protection of the hippocampus, as shown by the icv Pilo model.

Oxaceprol is well-defined therapeutic agent as an atypical inhibitor of inflammation in osteoarthritis. In the present study, we aimed to develop and characterize oxaceprol-loaded poly-lactide-co-glycolide (PLGA) nanoparticles for intra-articular administration in osteoarthritis. PLGA nanoparticles were prepared by double-emulsion solvent evaporation method. Meanwhile, a straightforward and generally applicable high performance liquid chromatography method was developed, and validated for the first time for the quantification of oxaceprol. To examine the drug carrying capacity of nanoparticles, varying amount of oxaceprol was entrapped into a constant amount of polymer matrix. Moreover, the efficacy of drug amount on nanoparticle characteristics such as particle size, zeta potential, morphology, drug entrapment, and in vitro drug release was investigated. Nanoparticle sizes were between 229 and 509 nm for different amount of oxaceprol with spherical smooth morphology. Encapsulation efficiency ranged between 39.73 and 63.83% by decreasing oxaceprol amount. The results of Fourier transform infrared and DSC showed absence of interaction between oxaceprol and PLGA. The in vitro drug release from these nanoparticles showed a sustained release of oxaceprol over 30 days. According to cell culture studies, oxaceprol-loaded nanoparticles had no cytotoxicity with high biocompatibility. This study was the first step of developing an intra-articular system in the treatment of osteoarthritis for the controlled release of oxaceprol. Our findings showed that these nanoparticles can be beneficial for an effective treatment of osteoarthritis avoiding side effects associated with oral administration., (© 2020 Wiley Periodicals, Inc.)

Introduction:  Impaired plasminogen activation (PA) is causally related to the development of lung fibrosis. Prior studies demonstrate that enhanced PA in the lung limits the severity of scarring following injury and in vitro studies indicate that PA promotes matrix degradation and fibroblast apoptosis. These findings led us to hypothesize that increased PA in an in vivo model would enhance the resolution of established lung fibrosis in conjunction with increased myofibroblast apoptosis., Methods:  Transgenic C57BL/6 mice with doxycycline inducible lung-specific urokinase plasminogen activator (uPA) expression or littermate controls were treated (day 0) with bleomycin or saline. Doxycycline was initiated on days 1, 9, 14, or 21. Lung fibrosis, stiffness, apoptosis, epithelial barrier integrity, and inflammation were assessed., Results:  Protection from fibrosis with uPA upregulation from day 1 through day 28 was associated with reduced parenchymal stiffness as determined by atomic force microscopy. Initiation of uPA expression beginning in the late inflammatory or the early fibrotic phase reduced stiffness and fibrosis at day 28. Induction of uPA activity in mice with established fibrosis decreased lung collagen and lung stiffness while increasing myofibroblast apoptosis. Upregulation of uPA did not alter lung inflammation but was associated with improved epithelial cell homeostasis., Conclusion:  Restoring intrapulmonary PA activity diminishes lung fibrogenesis and enhances the resolution of established lung fibrosis. This PA-mediated resolution is associated with increased myofibroblast apoptosis and improved epithelial cell homeostasis. These studies support the potential capacity of the lung to resolve existing scar in murine models., Competing Interests: J.C.H., T.H.S., K.K.K., and D.J.T. report grants from National Institutes of Health, during the conduct of the study., (Georg Thieme Verlag KG Stuttgart · New York.)

Scope: Inflammatory bowel disease (IBD) is a chronic disease of gastrointestinal tract in which oxidative stress and overactivation of inflammatory response are implicated. The aim of the present study is to test the hypothesis that hydroxyproline (Hyp), an amino acid with an antioxidative property, attenuates dextran sulfate sodium (DSS)-induced colitis in mice., Methods and Results: Male C57BL/6 mice supplemented with or without 1% Hyp are subjected to 2.5% DSS in drinking water to induce colitis. Hyp attenuates the severity of colitis as evidenced by reduced disease activity index scores, decreased myeloperoxidase activity, histological damage, and apoptosis. Furthermore, DSS-induced increases in reactive oxygen species accumulation, TNF-α and IL-6 secretion, and malonyldialdehyde activity and a decrease in reduced glutathione in the colon are ameliorated by Hyp. The enhanced phosphorylation of STAT3 and NF-κB following DSS administration is mitigated by Hyp, which is also observed in LPS-treated RAW264.7 macrophages. Moreover, the inhibitory effect of Hyp on IL-6 expression is mainly mediated by the NF-κB signaling, because the induction of STAT3 and IL-6 by LPS is markedly reversed by Bay11-7085, a specific inhibitor NF-κB., Conclusion: In summary, Hyp is a critical nutrient with an ability to attenuate DSS-induced colonic damage in mice. This beneficial effect of Hyp is partially mediated by inhibiting the NF-κB/IL-6 signaling and the restoration of redox homeostasis., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Proline (Pro) accumulation protects plant cell under abiotic stress. Hydroxyproline (Hyp) as selection agent is a toxic analog of proline and promotes Pro overaccumulation. In this study, Chardonnay calli were firstly irradiated with different dosages of 60 Co and then cultured on a Hyp-added medium. Finally, some stable hydroxyproline-resistant (HR) calli were obtained. When calli were cultured on 4 mM Hyp medium for 7 days, intracellular Pro content of the HR calli was five times higher than that detected in the normal calli. The regeneration of HR calli into plantlets was much slower than that of normal ones. When cultured on woody plant medium (WPM) containing 10 mM NaCl for 14 days, HR plantlets still grew well with lower Pro than withered normal plantlets. qRT-PCR results of Pro biosynthesis-related genes in HR plantlets showed that three genes VvP5CS, VvOAT, and VvP5CDH were conducive for Pro accumulation. These results confirmed that HR plantlets acquired salt tolerance ability. We prospect that this procedure to obtain salt-tolerant plants may be valuable to breed programs and improve grapevine genotypes with increased tolerance to salt and other abiotic stresses.

Aim: This study was conducted to estimate the aminoacid levels in the vitreous of patients with proliferative diabetic retinopathy, and to correlate it with the adiponectin levels. Secondly to test if these amino acids can alter or induce adiponectin levels and its related factors in retinal cells like pericyte as an in vitro model., Methods: All human studies were done as per declaration of Helsinki with institutional approval and after obtaining consent from participating individuals. The vitreous amino acids were estimated in PDR (Proliferative diabetic retinopathy) and MH (Macular Hole) as disease control using HPLC. Bovine retinal pericytes (BRP) were cultured in DMEM/F12 medium and treated with 0.5 mM of any one of the individual amino acids (proline, hydroxyproline, phenylalanine, alanine, serine, glycine, lysine, isoleucine or valine) along with 100 nM insulin for 14 days in high glucose (25 mM) condition. The mRNA expression profile of adipogenic markers (such as Pref1, APN, ZAG and PPARγ), angiogenic markers (VEGF, MMP-2 and MMP-9, TGF-β) and antioxidant markers (Nrf2 and UCP-2) were evaluated by qPCR. Adipogenesis was further confirmed by adipogenesis assay, secretion of adiponectin in medium and triglyceride accumulation by Oil red O staining in Bovine retinal pericytes., Results: Amino acids valine (p < 0.004), isoleucine (p < 0.0007), leucine (p < 0.022), serine (p < 0.0007), glycine (p < 0.001), alanine (p < 0.017), phenylalanine (p < 0.013), and lysine (p < 0.001) were significantly elevated in the vitreous of PDR group (n = 30) when compared to macular hole (n = 20). There was a significant positive correlation between serine (p < 0.021), alanine (p < 0.00016), phenylalanine (p < 0.04), isoleucine (p < 0.023), leucine (p < 0.043), and lysine (p < 0.026) with adiponectin level in the vitreous. The amino acids hydroxyproline, proline, lysine, glycine and alanine induced the triglyceride accumulation and expression of Adiponectin. VEGF and MMP-9 expression was decreased with all the amino acids treated and PEDF was significantly increased with phenylalanine treatment. TGFβ mRNA expression showed a significant decrease with proline, alanine, glycine, lysine and isoleucine. The Nrf2 expression was significantly increased in alanine and serine when compared to control. The UCP-2 gene showed a significant increase in proline and lysine treatment., Discussion and Conclusion: Our results suggest that amino acids hydroxyproline, proline, lysine, glycine and alanine which are elevated in the PDR vitreous show a tendency to induce adipogenic effects in retinal pericytes by triggering the accumulation of triglycerides and adiponectin. Hence we hypothesize that these aminoacids when elevated along with insulin and glucose can induce metabolic changes in pericytes. The functional implications of these changes tend to be protective as it increases the antioxidant potential and decreases the angiogenesis markers which are potentially pathogenic., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Substrate reduction therapy with analogs reduces the accumulation of substrates by inhibiting the metabolic pathways involved in their biosynthesis, providing new treatment options for patients with primary hyperoxalurias (PHs) that often progress to end-stage renal disease (ESRD). This research aims to evaluate the inhibition efficacy of Hydroxy-L-proline (HYP) analogs against calcium oxalate (CaOx) crystal formation in the Drosophila Melanogaster (D. Melanogaster) by comparing them with Pyridoxine (Vitamin B6)., Methods: Three stocks of Drosophila Melanogaster (W 118 , CG3926 RNAi, and Act5C-GAL4/CyO) were utilized. Two stocks (CG3926 RNAi and Act5C-GAL4 /CyO) were crossed to generate the Act5C > dAGXT RNAi recombinant line (F 1 generation) of D. Melanogaster which was used to compare the efficacy of Hydroxy-L-proline (HYP) analogs inhibiting CaOx crystal formation with Vitamin B 6 as the traditional therapy for primary hyperoxaluria., Results: Nephrolithiasis model was successfully constructed by downregulating the function of the dAGXT gene in D. Melanogaster (P-Value = 0.0045). Furthermore, the efficacy of Hydroxy-L-proline (HYP) analogs against CaOx crystal formation was demonstrated in vivo using D. Melanogaster model; the results showed that these L-Proline analogs were better in inhibiting stone formation at very low concentrations than Vitamin B 6 (IC 50  = 0.6 and 1.8% for standard and dietary salt growth medium respectively) compared to N-acetyl-L-Hydroxyproline (IC 50  = 0.1% for both standard and dietary salt growth medium) and Baclofen (IC 50  = 0.06 and 0.1% for standard and dietary salt growth medium respectively). Analysis of variance (ANOVA) also showed that Hydroxy-L-proline (HYP) analogs were better alternatives for CaOx inhibition at very low concentration especially when both genetics and environmental factors are intertwined (p < 0.0008) for the dietary salt growth medium and (P < 0.063) for standard growth medium., Conclusion: Addition of Hydroxy-L-Proline analogs to growth medium resulted in the reduction of CaOx crystals formation. These analogs show promise as potential inhibitors for oxalate reduction in Primary Hyperoxaluria.

Chemical cell-surface engineering is a tool for modifying and altering cellular functions. Herein, we report the introduction of an antibiotic phenotype to the green alga Chlamydomonas reinhardtii by chemically modifying its cell surface. Flow cytometry and confocal microscopy studies demonstrated that a hybrid of the antibiotic vancomycin and a 4-hydroxyproline oligomer binds reversibly to the cell wall without affecting the viability or motility of the cells. The modified cells were used to inhibit bacterial growth of Gram-positive Bacillus subtilis cultures. Delivery of the antibiotic from the microalgae to the bacterial cells was verified by microscopy. Our studies provide compelling evidence that 1) chemical surface engineering constitutes a useful tool for the introduction of new, previously unknown functionality, and 2) living microalgae can serve as new platforms for drug delivery., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: This study investigated the effects of an amino acid mixture containing arginine, glutamine, and β-hydroxy-β-methyl butyrate on secondary healing of ischemic wounds in a rat model (N = 18)., Methods: After the formation of a bipediculated flap on each rat, 2 full-thickness excisional skin wounds (2 × 2 cm) were created on every flap. The rats were then randomized into the control and treatment groups. Every rat received standardized rat food throughout the study. The rats in the treatment group were administered an extra 200 mg/kg of L-arginine, 200 mg/kg of L-glutamine, and 40 mg/kg of β-hydroxy-β-methyl butyrate per day. Wound sizes were measured on days 0, 4, 10, and 14. The rats were sacrificed, and the wounds were excised for biochemical and histologic examination on the 14th day., Results: As compared with the control group, the treatment group's wound sizes were significantly smaller on days 10 and 14 ( P < .001), as was its inflammatory cell accumulation score ( P = .008). There was no significant difference between the 2 groups in collagen accumulation ( P = .340), granulation tissue maturation ( P = .161), angiogenesis ( P = .387), or reepithelialization ( P = .190) and no significant difference between hydroxyproline concentrations in wounds ( P = .287)., Discussion: This amino acid combination seems to have a positive impact on the secondary healing of experimental ischemic wounds when introduced as a supplement to the standard diet, and the reduction in the inflammatory process appears to play a role in this effect.

Functionalized collagen-mimetic peptides (CMPs) have been widely used in the preparation of collagen-related biomaterials. Among the reported results, the induced noncovalent interactions between the implanted functional groups or moieties were frequently the key elements to promote the self-assembly of small CMPs. In this work, we designed and synthesized a series of glycosylated CMPs in which 4-O-[β-D-galactopyranosyl]-(2S,4R)-4-hydroxyproline (Hyp(Gal)) was incorporated to explore the effects of glycosylation on the stability and assembly of collagen triple helices. Circular dichroism measurements showed that glycosylation of hydroxyproline slightly destabilized the collagen triple helices, but did not reduce their refolding rate. Compared to non-glycosylated CMPs, the incorporation of Hyp(Gal) speeded up the assembly of CMPs, indicating that this modification could assist the self-assembly of CMPs into higher-order structures, such as fibrils. O-Galactosylation of hydroxyproline imposes contrary effects on the triple helix stability and the self-assembly rate of collagen triple helices, exhibiting a piece of important and useful information for designing collagen-related biomaterials. Our finding also suggests that instead of stabilizing the triple helical conformation of CMPs, installing additional forces between CMPs could be a crucial factor to promote the assembly of CMPs into large-scale constructs.

Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d₃-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise.

Conantokins are ~20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp(10) disrupts only a small region of the α-helix of the Mn(2+)·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp(10) with N(8)Q results in a Mg(2+)-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp(10) with Pro(10) allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM) as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP). After being extracted from mature seeds (cv. Huayu 20), the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation). The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS) and nine individuals with higher protein content (>30%) and 21 individuals with higher oil content (>58%) were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut.

It has been considered that concentrations of certain amino acids in the egg are not sufficient to fully support embryonic development of modern broilers. In this study we evaluated embryo growth and development with particular emphasis on one of the major components of connective tissue, collagen. Experiments were performed on Ross 308 chicken embryos from 160 fertilised eggs. Experimental solutions of silver nanoparticles (Ag), hydroxyproline solution (Hyp) and a complex of silver nanoparticles with hydroxyproline (AgHyp) were injected into albumen, and embryos were incubated until day 20. An assessment of the mass of embryo and selected organs was carried out followed by measurements of the expression of the key signalling factors' fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A). Finally, an evaluation of collagen microstructure using scanning electron microscopy was performed. Our results clearly indicate that Hyp, Ag and AgHyp administered in ovo to chicken embryos did not harm embryos. Comparing to the control group, Hyp, Ag and the AgHyp complex significantly upregulated expression of the FGF-2 at the mRNA and protein levels. Moreover, Hyp, Ag and, in particular, the complex of AgHyp significantly increased blood vessel size, cartilage collagen fibre lattice size and bundle thickness. The general conclusion from this study is that AgHyp treatment may help to build a stronger and longer lasting form of collagen fibres.

Hydroxyproline-rich glycoproteins (HRGPs) are plant cell wall proteins related to plant growth and development, and extensins (EXTs) are a subfamily of HRGPs. In this study, the function of HRGPs, especially EXTs, was investigated in the pollen tube and style cell growth of Nicotiana tabacum L. By using the techniques of protein blot and immunohistochemistry, the JIM20-recognized epitopes of EXTs were abundantly expressed in vivo for pollen tubes and transmitting tissue. A hydroxyproline synthesis inhibitor, 3,4-dehydro-l-proline (3,4-DHP), was used to investigate the functions of HRGPs. The addition of 3,4-DHP decreased the speed of pollen tube growth and shortened the length of style. Moreover, the hydroxyproline assay and JIM20 immunolocalization confirmed that 3,4-DHP treatment reduced the level of hydroxyproline and EXTs in the treated styles, respectively. These results indicate that HRGPs, most likely EXTs, may play important roles in the pollen tube and style cell growth., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

We used an unbiased approach of gene expression profiling to determine differential gene expression of all the macromolecular modulators (MMs) considered to be involved in stone formation, in hyperoxaluric rats, with and without treatment with the NADPH oxidase inhibitor apocynin. Male rats were fed rat chow or chow supplemented with 5% wt/wt hydroxy-l-proline (HLP) with or without apocynin-supplemented water. After 28 days, rats were euthanized and their kidneys explanted. Total RNA was isolated and microarray analysis was conducted using the Illumina bead array reader. Gene ontology analysis and the pathway analyses of the genes were done using Database for Annotation, Visualization of Integrated Discovery enrichment analysis tool. Quantitative RT-PCR of selected genes was carried out to verify the microarray results. Expression of selected gene products was confirmed using immunohistochemistry. Administration of HLP led to crystal deposition. Genes encoding for fibronectin, CD 44, fetuin B, osteopontin, and matrix-gla protein were upregulated while those encoding for heavy chains of inter-alpha-inhibitor 1, 3, and 4, calgranulin B, prothrombin, and Tamm-Horsfall protein were downregulated. HLP-fed rats receiving apocynin had a significant reversal in gene expression profiles: those that were upregulated came down while those that were downregulated stepped up. Apocynin treatment resulted in near complete absence of crystals. Clearly, there are two types of MMs; one is downregulated while the other is upregulated during hyperoxaluria and crystal deposition. Apparently gene and protein expressions of known macromolecular modulators of CaOx crystallization are likely regulated by ROS produced in part through the activation of NADPH oxidase., (Copyright © 2014 the American Physiological Society.)

In contrast to accumulated knowledge about gastroduodenal injury associated with nonsteroidal antiinflammatory drugs (NSAIDs) such as indomethacin, small intestinal mucosal injuries have been noticed only recently, and the precise mechanism remains to be elucidated. To clarify the mechanism, we performed 2-DE on IEC-6 rat normal intestinal cells that were treated with indomethacin (200μΜ, 24h) or a vehicle control and identified 18 up-regulated and 8 down-regulated proteins through MALDI-TOF/TOF mass spectrometry. Among these proteins, collagen I and proteins involved in collagen I biosynthesis and maturation, including prolyl 4-hydroxylase subunit α1, protein disulfide isomerase A3 (PDIA3), calreticulin, and endoplasmin, were all down-regulated by indomethacin. Immunohistochemical staining of the intestinal mucosa of indomethacin-administered rats showed a decrease of collagen I on the apical surface of intestinal cells. Cell death induced by indomethacin was prominently suppressed when IEC-6 cells were grown on collagen I-coated plates. cis-4-Hydroxy-l-proline, a proline analog that inhibits collagen synthesis, depressed IEC-6 cell viability in a concentration-dependent manner. Cell death was also induced by short interfering RNA knockdown of endogenous collagen I in IEC-6 cells. In conclusion, by comparative proteome analysis, we identified down-regulation of collagen I as an important mechanism in NSAID-induced intestinal injury., Biological Significance: Small intestinal lesions induced by NSAIDs are of great concern in clinical settings. Various hypotheses have been proposed for the origin of these inflammatory responses, such as reduction in the blood flow, intestinal hypermotility, abnormal intestinal mucosal permeability, mitochondrial dysfunction, and reactive oxygen species, many of which are related to the inhibition of prostaglandin synthesis. However, the precise mechanism is yet to be known. The cellular process of the lesions must involve up- and down-regulations of a large number of proteins and complex interactions between them. To elucidate it, global and systematic identification of the proteins in intestinal cells affected by NSAIDs is essential. We found that the proteins exhibiting reduced expression by indomethacin treatment are collagen I and the proteins involved in collagen I synthesis and maturation. Consistent with this, immunohistochemical analysis showed that the indomethacin-treated rat intestinal mucosal cells exhibits decreased collagen I expression on its apical surface. Furthermore, the cell-protective effect of collagen on intestinal mucosal cells was demonstrated by the use of a collagen-synthesis inhibitor, short interfering RNA (siRNA) knockdown of endogenous collagen I, and cell cultivation on collagen I-coated plates versus uncoated plates. These results give important information on the role of the collagen synthesis in intestinal mucosa in the mechanism of NSAID-induced small intestinal lesions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In humans and rodents, dietary hydroxyproline (hyp) and oxalate intake affect urinary oxalate (Uox) excretion. Whether Uox excretion occurs in cats was tested by feeding diets containing low oxalate (13 mg/100 g DM) with high (Hhyp-Lox), moderate (Mhyp-Lox), and low hyp (Lhyp-Lox) concentrations (3.8, 2.0, and 0.2 g/100 g DM, respectively) and low hyp with high oxalate (93 mg/100 g DM; Lhyp-Hox) to 8 adult female cats in a 48-d study using a Latin square design. Cats were randomly allocated to one of the four 12-d treatment periods and fed according to individual energy needs. Feces and urine were collected quantitatively using modified litter boxes during the final 5 d of each period. Feces were analyzed for oxalate and Ca, and urine was analyzed for specific density, pH, oxalate, Ca, P, Mg, Na, K, ammonia, citrate, urate, sulfate, and creatinine. Increasing hyp intake (0.2, 2.0, and 3.8 g/100 g DM) resulted in increased Uox excretion (Lhyp-Lox vs. Mhyp-Lox vs. Hhyp-Lox; P < 0.05), and the linear dose-response equation was Uox (mg/d) = 5.62 + 2.10 × g hyp intake/d (r(2) = 0.56; P < 0.001). Increasing oxalate intake from 13 to 93 mg/100 g DM did not affect Uox excretion but resulted in an increase in fecal oxalate output (P < 0.001) and positive oxalate balance (32.20 ± 2.06 mg/d). The results indicate that the intestinal absorption of the supplemental oxalate, and thereby its contribution to Uox, was low (5.90% ± 5.24%). Relevant increases in endogenous Uox excretion were achieved by increasing dietary hyp intake. The hyp-containing protein sources should be minimized in Ca oxalate urolith preventative diets until their effect on Uox excretion is tested. The oxalate content (up to 93 mg/100 g DM) in a diet with moderate Ca content does not contribute to Uox content.

To take advantage of the therapeutic potential of embryonic stem cells (ESCs), it is necessary to regulate their differentiation in response to defined factors. In this study, in order to explore novel molecules that regulate the differentiation of ESCs, we investigated whether collagen hydrolysate, collagen-characteristic amino acids, glycine (Gly), l-proline and trans-4-hydroxy-l-proline (l-Hyp); or dipeptides, proline-hydroxyproline and hydroxyproline-glycine regulate the differentiation of mouse embryoid bodies (EBs). We identified that treatment with collagen hydrolysate or Gly repressed the expression of the mesendodermal markers, Brachyury and Foxa2 in EBs and maintained the undifferentiated state of mESCs in a feeder-free monolayer culture. In contrast, l-Hyp promoted the expression of Brachyury, Mixl1, Gsc and Foxa2 in EBs. And the treatment with l-Hyp promoted cardiac differentiation within EBs, which was proven by the spontaneous contraction of cardiomyocytes and the expression of the cardiac markers, α-MHC, MLC-2v and Nkx2.5. Results suggest that l-Hyp is a promising new inducer for reproducible and efficient differentiation of mesendoderm lineages., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Teucrium chamaedrys, one of the most common and investigated species of the genus Teucrium, has been used for centuries in traditional medicine for many purposes. Its phytochemical components comprise, among others, phenylethanoid glycosides (PGs) and neo-clerodane diterpenoids. Several reports have demonstrated a wide range of beneficial biological and pharmacological activities of the phenylethanoid components, while the diterpenes were shown to be strongly hepatotoxic. In this work, in vitro cultures were established from leaf explants of T. chamaedrys. Both solid (callus) and liquid (cell suspension) cultures maintained the capacity to produce PGs, with teucrioside (TS) representing the most abundant one. Cell suspensions had a lower TS content than that found in leaf extracts, but higher than that of calli. An NMR-based metabolomics approach was used to compare the product profile of intact plants vs. cell suspension cultures, and results showed that neo-clerodane diterpenes, present in the intact plant, were not detected in cell cultures. Several elicitors were supplied to cell cultures with the aim of increasing TS production, and elicitation was tested at different growth phases and by exposing cells for different periods. Methyl jasmonate and fungal mycelia from Trichoderma viridae and Fusarium moniliforme were able to significantly increase TS production if supplied at the early-exponential growth phase for 24h. Based on the proposed link between proline and the phenylpropanoid pathways, proline accumulation in cell cultures was followed throughout a 14-day culture period, showing that it strictly reflected that of TS production. Moreover, exogenously supplied proline, and its analogue hydroxyproline, turned out to be very effective in increasing teucrioside production., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aim: To investigate the molecular mechanism of Total Flavonoids of Fructus Chorspondiatis (TFFC) on preventing cardiac fibroblasts collagen synthesis induced by angiotensin II., Methods: Collagen synthesis was determined by measuring (3)H-proline incorporation cardiac fibroblasts and hydroxyproline content in the culture mediums. The expression of collagen types I and III mRNA and protein was measured by RT-PCR and western blot, respectively. NO level in the culture medium was measured by the Griess reagent. NOS level in the culture medium was measured by chemical colorimetric method. The cellular concentration of cyclic GMP (cGMP) was measured by radioimmunoassay., Results: TFFC (25, 50, and 100mg/L) inhibited collagen synthesis in cardiac fibroblasts in a dose-dependent manner compared with angiotensin II group (P<0.01), and the inhibitory effects were blocked by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) and 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ). TFFC increased nitric oxide (NO) and nitric oxide synthase (NOS) levels in the culture medium, increased intracellular cGMP level in cardiac fibroblasts, decreased collagen types I and III protein level in cardiac fibroblasts. The mRNA expression of collagen type I and III was suppressed by TFFC., Conclusions: These results suggested that TFFC inhibited collagen synthesis induced by angiotensin II in cardiac fibroblasts, and the inhibitory effect might associate with the activation of the NO/cGMP signaling pathway., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We examined the effects of oral L-hydroxyproline (Hyp) on collagen in the body. After 2 weeks' administration of Hyp (0.5 or 1 g/kg) to F344 male rats, the soluble collagen content of the skin had increased, and the serum concentration of collagen peptides was correlated with the skin content of soluble collagen. This result suggests that oral Hyp augmented collagen metabolism.

Current clinically used chemotherapeutic platinum drugs can trigger severe toxic effects. To develop a model system for the evaluation of the therapeutic efficacy and the toxic effects of new platinum agents, we have synthesized a new compound N-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxylhex-1-yl]-L-hydroxyproline dichloroplatinum(ii) (PHDP), compared its in vitro anti-proliferation activity, in vivo anti-tumor activity and safety to those of oxaliplatin, and correlated all these biological actions with the platinum occurring in the spleen, kidney, heart, brain, blood, tumor tissue, urine and faeces of the treated mice. We explored the atomic absorption based determinations of the platinum which occurred in the spleen, kidney, heart, brain, blood, tumor tissue, urine and faeces and constitute a model system that can be generally used in the investigation of the novel platinum agents.

A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different hydroxyprolinamide P2 ligands were designed and synthesized. Variation of substitutions at the P2 significantly affected the enzyme inhibitory potency of the inhibitors. Compounds 2a and 2d showed excellent enzyme inhibitory activity with IC(50) values in the nanomolar range. An active site binding model for inhibitors 2a and 2d was suggested based upon the computational-docking results of the ligand with HIV-1 protease. This model offers molecular insights regarding ligand-binding site interactions of the hydroxyprolinamide-derived novel P2-ligand., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effects and mechanism of the extract of Radix Scrophulariae (ERS), a traditional Chinese herb, on experimental ventricular remodeling in rats was studied. Rats were separated randomly into 5 groups: sham, model, captopril (40 mg x kg(-1)) and ERS (8, 16 g x kg(-1)). The experimental ventricular remodeling was induced with ligating the left anterior descending branch of the coronary artery of the rats. The sham group was conducted the same procedure without ligation. After 4 weeks treatment with intragastric administration of the corresponding drugs, the left ventricular weight index (LVWI) and heart weight index (HWI) were determined. The concentrations of angiotensin II (Ang II) and hydroxyproline (Hyp) in myocardium were detected. Myocardium tissue was stained with HE and picric acid/Sirius red for cardiocyte cross-section area and collagen content measurements. Real-time RT-PCR was used to detect the gene expressions of AT1R, TNF-alpha and TGF-beta1 mRNA. ERS could significantly reduce the LVWI, HWI, decrease the content of Ang II, Hyp, diminish cardiocyte cross-section area and ameliorate collagen deposition. In addition, ERS could down regulate the gene expressions of AT1R, TNF-alpha and TGF-beta1 mRNA in myocardium. ERS has beneficial effect against ventricular remodeling. The mechanism may be related to decreasing the level of Ang II and cardiac fibrosis, modulating some gene expressions associated with cardiac hypertrophy.

Ethylene glycol (EG) exposure is a common model for kidney stones, because animals accumulate calcium oxalate monohydrate (COM) in kidneys. Wistar rats are more sensitive to EG than Fischer 344 (F344) rats, with greater COM deposition in kidneys. The mechanisms by which COM accumulates differently among strains are poorly understood. Urinary proteins inhibit COM adhesion to renal cells, which could alter COM deposition in kidneys. We hypothesize that COM accumulates more in Wistar rat kidneys because of lower levels of inhibitory proteins in urine. Wistar and F344 rats were treated with 0.75% EG in drinking water for 8 wk. Twenty-four-hour urine was collected every 2 wk for analysis of urinary proteins. Similar studies were conducted for 2 wk using 2% hydroxyproline (HP) as an alternative oxalate source. Total urinary protein was higher in F344 than Wistar rats at all times. Tamm-Horsfall protein was not different between strains. Osteopontin (OPN) levels in Wistar urine and kidney tissue were higher and were further increased by EG treatment. This increase in OPN occurred before renal COM accumulation. Untreated F344 rats showed greater CD45 and ED-1 staining in kidneys than untreated Wistars; in contrast, EG treatment increased CD45 and ED-1 staining in Wistars more than in F344 rats, indicating macrophage infiltration. This increase occurred in parallel with the increase in OPN and before COM accumulation. Like EG, HP induced markedly greater oxalate concentrations in the plasma and urine of Wistar rats compared with F344 rats. These results suggest that OPN upregulation and macrophage infiltration do not completely protect against COM accumulation and may be a response to crystal retention. Because the two oxalate precursors, EG and HP, produced similar elevations of oxalate, the strain difference in COM accumulation may result more so from metabolic differences between strains than from differences in urinary proteins or inflammatory responses.

A bacterial strain, designated DR-834 and producing immunostimulatory activities to carp (Cyprinus carpio), was isolated from Qinghai-Tibetan Plateau permafrost soil. Cultural characteristic studies suggested that this strain belongs to the genus Bacillus. The nucleotide sequence of the 16S rRNA gene of strain DR-834 exhibited close similarity (99%) with the 16S rRNA gene of Bacillus simplex. Two compounds showing potent activity were isolated from secondary metabolites of the strain through bioassay-guided isolation techniques and identified by spectral data (infrared, nuclear magnetic resonance and mass spectrometry) as: (1) 4-trans-hydroxy-l-proline and (2) cyclo-(l-Pro-Gly)(2). They were found to be significantly increased the selected innate immune function parameters, serum SOD activity, serum lysozyme activity, serum bactericidal activity, superoxide anion production and phagocytic activity by isolated blood leucocytes. The effects of two compounds on immune-related genes expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 1beta (IL-1beta) and inducible nitric oxide synthase (iNOS) mRNA in the blood have been augmented by 4-trans-hydroxy-l-proline and cyclo-(l-Pro-Gly)(2). Compounds 1 and 2 administration the challenge with live Aeromonas hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in relative percent survival (RPS) values. Compound 2 produced the highest protection with the RPS values of 87.50, 77.78, 55.56 and 55.56 after 1, 2, 3 and 4 weeks, respectively. The study indicates that the isolated compounds could be positively influence the immune response and protect the heath status of carp against A. hydrophila infection., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)

Purpose: This study addressed the question of whether the collagen metabolism modulator cis-4-Hydroxy-L-proline (CHP) is applicable for potential use as a therapeutic inhibitor of pancreatic carcinoma cell growth., Methods: Cell proliferation, as well as quantification of apoptosis of murine Panc02 cells, was assessed after CHP treatment. Supplementary, the effect of CHP on tumor growth was examined in the subcutaneous Panc02 model in vivo. Mice received daily intraperitoneal injections of CHP (300, 400, and 500 mg/kg bw). In addition to the assessment of systemic parameters (blood count, enzyme activities), histology (HE) and immunohistochemistry (Ki-67) were performed from resected tumor specimens., Results: Like reduction of metabolic activity, CHP also induced inhibition of cell growth in a dose-dependent manner, with however only slight increases in apoptosis. In vivo treatment of Panc02 tumors with CHP resulted in pronounced delay of tumor growth and decreases in tumor cell proliferation. Moreover, these effects were accompanied by a massive systemic leukocytosis as well increased leukocyte infiltration into the tumors subsequent to CHP therapy., Conclusions: CHP inhibits the proliferation of Panc02 tumor cells in a dose-dependent manner both in vitro and in vivo. Our presented data show that modulation of the collagen metabolism is an interesting strategy for treatment of pancreatic carcinoma.