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Abstract Background Emerging evidence suggested the association between gut dysbiosis and Alzheimer’s disease (AD) progression. However, it remained unclear how the gut microbiome and neuroinflammation in the brain mutually interact or how these interactions affect brain functioning and cognition. Here we hypothesized that “gut-brain” axis mediated by microbial derived metabolites was expected to novel breakthroughs in the fields of AD research and development. Methods Multiple technologies, such as immunofluorescence, 16s rDNA sequencing, mass spectrometry-based metabolomics (LC-QQQ-MS and GC-MS), were used to reveal potential link between gut microbiota and the metabolism and cognition of the host. Results Microbial depletion induced by the antibiotics mix (ABX) verified that “gut-brain” can transmit information bidirectionally. Short-chain fatty acid-producing (SCFAs-producing) bacteria and amino acid-producing bacteria fluctuated greatly in 5×FAD mice, especially the reduction sharply of the Bifidobacteriaceae and the increase of the Lachnospiraceae family. Concentrations of several Tryptophan-kynurenine intermediates, lactic acid, CD4+ cell, and CD8+ cells were higher in serum of 5×FAD mice, whilst TCA cycle intermediates and Th1/Th2 were lower. In addition, the levels of iso-butyric acid (IBA) in feces, serum, and brain of 5×FAD mice were increased compared with WT-M mice, especially in serum. And IBA in the brain was positively correlated with Aβ and proinflammatory factors. Conclusion Together, our finding highlighted that the alternation in gut microbiota affected the effective communication between the “gut-brain” axis in 5×FAD mice by regulating the immune system, carbohydrate, and energy metabolism.

The Nuclear receptor subfamily 5 group A member 2 (NR5A2) gene plays a pivotal role in ovarian development, ovulation, and reproductive traits. There is a lack of studies on its impact on ovarian traits and reproductive traits in cattle. This study aimed to explore NR5A2 gene polymorphisms associations with reproductive traits and investigate the distribution of NR5A2 gene polymorphisms across diverse bovine breeds worldwide. We identified a novel 17-bp deletion within the NR5A2 gene specifically in Chinese Holstein cows (n = 1033) leading to the observation of two genotypes DD and ID. Subsequent association analysis revealed a significant correlation between the ‘ID’ genotype at this locus and a larger number of corpus albicans (p = 0.042) in diestrus, as well as a higher number of mature follicles (p = 0.038) in estrus. In addition, we also found that the distribution of this deletion exhibits strong regionality across different cattle breeds globally. These findings indicate that the 17-bp deletion mutation within the NR5A2 gene is significantly associated with an increased corpus luteum diameter and a greater number of mature follicles, suggesting its potential utility as a valuable DNA marker for enhancing cow fertility.

Abstract Background Glycative stress, characterized by the formation and accumulation of advanced glycation end products (AGEs) associated with protein glycation reactions, has been implicated in inducing a decline of muscle function. Although the inverse correlation between glycative stress and muscle mass and strength has been demonstrated, the underlying molecular mechanisms are not fully understood. This study aimed to elucidate how glycative stress affects the skeletal muscle, particularly the adaptive muscle response to hypertrophic stimuli and its molecular mechanism. Methods Male C57BL/6NCr mice were randomly divided into the following two groups: the bovine serum albumin (BSA)‐treated and AGE‐treated groups. Mice in the AGE‐treated group were intraperitoneally administered AGEs (0.5 mg/g) once daily, whereas those in the BSA‐treated group received an equal amount of BSA (0.5 mg/g) as the vehicle control. After 7 days of continuous administration, the right leg plantaris muscle of mice in each group underwent functional overload treatment by synergist ablation for 7 days to induce muscle hypertrophy. In in vitro studies, cultured C2C12 myocytes were treated with AGEs (1 mg/mL) to examine cell adhesion and cell membrane permeability. Results Continuous AGE administration increased the levels of fluorescent AGEs, Nε‐(carboxymethyl) lysine, and methylglyoxal‐derived hydroimidazolone‐1 in both plasma and skeletal muscle. Plantaris muscle weight, muscle fibre cross‐sectional area, protein synthesis rate, and the number of myonuclei increased with functional overload in both groups; however, the increase was significantly reduced by AGE treatment. Some muscles of AGE‐treated mice were destroyed by functional overload. Proteomic analysis was performed to explore the mechanisms of muscle hypertrophy suppression and myofibre destruction by AGEs. When principal component analysis was performed on 4659 data obtained by proteomic analysis, AGE treatment was observed to affect protein expression only in functionally overloaded muscles. Enrichment analysis of the 436 proteins extracted using the K‐means method further identified a group of proteins involved in cell adhesion. Consistent with this finding, dystrophin–glycoprotein complex proteins and cell adhesion‐related proteins were confirmed to increase with functional overload; however, this was attenuated by AGE treatment. Additionally, the treatment of C2C12 muscle cells with AGEs inhibited their ability to adhere and increased cell membrane permeability. Conclusions This study indicates that glycative stress may be a novel pathogenic factor in skeletal muscle dysfunctions by causing loss of membrane integrity and preventing muscle mass gain.

Abstract Advanced glycation end products (AGEs) have been implicated in several skeletal muscle dysfunctions. However, whether the adverse effects of AGEs on skeletal muscle are because of their direct action on the skeletal muscle tissue is unclear. Therefore, this study aimed to investigate the direct and acute effects of AGEs on skeletal muscle using an isolated mouse skeletal muscle to eliminate several confounders derived from other organs. The results showed that the incubation of isolated mouse skeletal muscle with AGEs (1 mg/mL) for 2–6 h suppressed protein synthesis and the mechanistic target of rapamycin signaling pathway. Furthermore, AGEs showed potential inhibitory effects on protein degradation pathways, including autophagy and the ubiquitin–proteasome system. Additionally, AGEs stimulated endoplasmic reticulum (ER) stress by modulating the activating transcription factor 6, PKR‐like ER kinase, C/EBP homologous protein, and altered inflammatory cytokine expression. AGEs also stimulated receptor for AGEs (RAGE)‐associated signaling molecules, including mitogen‐activated protein kinases. These findings suggest that AGEs have direct and acute effect on skeletal muscle and disturb proteostasis by modulating intracellular pathways such as RAGE signaling, protein synthesis, proteolysis, ER stress, and inflammatory cytokines.

ADIPOQ plays a crucial role in regulating the reproductive system, but there are few reports on the effects of ADIPOQ on ovarian in dairy cows. Previous studies have verified the presence of a 67-bp mutation in the promoter region of the ADIPOQ gene. Hence, we employed ovarian tissues (n = 2111) and blood samples (n = 108) from Chinese Holstein cows as experimental samples to examine the association between ADIPOQ promoter variants and ovarian traits. We extracted DNA from these samples and conducted genetic typing identification on each sample using advanced techniques like PCR and agarose gel electrophoresis. Consequently, the DD, ID, and II genotypes were discovered. and it has been observed that the mutation frequency of this locus is low in the Chinese Holstein cow. Importantly, the correlational analysis unveiled a significant relationship (p < 0.05) between the weight of ovaries in late estrus and the width of ovaries during the estrus interval with the mutation. Result of the RT-PCR revealed that the ID genotype partially diminished the expression of the ADIPOQ gene. The results of this study suggest that the identified variable duplication could serve as a potential genetic marker for enhancing the ovarian traits of Chinese Holstein cows.

Background: The pathogenesis of metabolic syndrome was strongly associated with compromised metabolism homeostasis and gut microbiota imbalance. NAFLD is a progressive metabolic liver disease for which effective interventions are lacking. Bile acids exhibited appreciable metabolic regulatory effects and selective antimicrobial activity. Aim of the study: This study was designed to investigate the effect of BBBP, which mainly contained bile acids, on NAFLD from the perspectives of gut microbiota and metabolomics. Materials and methods: The present study was initiated on the anti-NAFLD effect of BBBP in HFD-fed mice. The efficacy of BBBP was evaluated by mice phenotypes, liver histopathological analysis and serum lipid and glucose levels. The activation of bile acid receptors such as Nr1h4, Nr1i2 and S1pr2 were detected by qRT-PCR analysis. Subsequently, untargeted metabolomics coupled with microbiomics was used to explore the mechanism of BBBP against NAFLD. Human L02 hepatocytes induced by OA and PA were used to investigate the effect of GABA on reducing lipid accumulation in vitro. Results: BBBP significantly and dose-dependently alleviated the obese phenotype, lipid accumulation and liver injury in mice subjected to 18 weeks HFD diet. Untargeted metabolomics and microbiomics analysis revealed that BBBP could alleviate the disturbance of lipid and amino acid metabolism and the imbalance of gut microbiota. Furthermore, BBBP oral gavage activated liver bile acid receptors, as indicated by elevated mRNA levels of Nr1h4, Nr1i2 and S1pr2. Surprisingly, we determined that BBBP, which mainly contained bile acids that possessed antimicrobial activity, could promote the growth of Lactobacillus. Correlation analysis showed a remarkable correlation between Lactobacillus and endogenous metabolites such as valine, serine, glutamine, et al. Among them, GABA which could be produced by Lactobacillus significantly reduced the lipid accumulation in L02 cells. Conclusions: The role of BBBP in regulating lipid metabolism might be achieved by activating bile acid receptors, or partially by promoting the levels of Lactobacillus and its metabolites such as GABA. Our study provided evidence that BBBP could be a novel therapeutic candidate for the treatment of NAFLD.

Abstract Background To adapt to life on land, Asiatic toads (Bufo gargarizans) must remodel their bodies and refine their chemical defenses in water. The full scope of the mechanisms underlying these processes has yet to be revealed. Bufadienolides (BDs) are chemical defense substances secreted by toads when they are in danger, and they have high medicinal value in treating heart failure, cancer, and hepatitis. However, the artificial breeding of toads to increase BDs has been unsuccessful due to the high mortality of toad larvae during metamorphosis. Method Toad larvae at different growth stages were selected to study the changes in the metamorphosis process under the same growth conditions. The differences of tadpoles were explored, including body remodeling, energy metabolism, synthesis and regulation of BDs, through lipidomic technology, transcriptomic technology, and mass spectrometry imaging technology during metamorphosis. Results During metamorphosis, tadpoles underwent significant changes in lipid metabolism due to body remodeling to adapt to terrestrial life, which involved ketosis, lipogenesis, cholesterol metabolism, and fatty acid oxidation. The accumulation trend of BDs was observed. “Pentose phosphate pathway” and “Aromatase activity” may be the critical pathway and GO term in BD synthesis, involving 16 genes predominantly expressed in the liver. The involved genes were mainly expressed in the liver, consistent with the synthetic site observed by mass spectrometry imaging. Conclusion Together, our findings presented the changes in the toad larvae during metamorphosis and highlighted the accumulation process of BDs as well as the regulatory pathways and synthetic site, providing research and theoretical basis for future development of the toad resources.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic hepatic disease characterized by excessive fat accumulation, and is the major risk for cardiovascular and metabolic disorders. We explored the therapeutic effect of gypenosides (Gyps) in combination with capsaicinoids (Caps) against high-fat-diet (HFD)-induced rat hyperlipidemia and oxidized low-density lipoprotein (ox-LDL)-treated Raw264.7 cells. Network pharmacology analysis predicted that the potential molecular mechanism of Gyps/Caps combination against hyperlipidemia was mainly associated with steroid metabolism, lipid synthesis and inflammatory response. Animal experiment demonstrated that Gyps/Caps combination lowered the liver weight, liver index, serum lipid profile and hepatic cholesterol content, improved liver function by reducing the serum markers of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), thereby relieving the hepatic injury. Moreover, Gyps/Caps combination inhibited oxidative stress by decreasing the lactic dehydrogenase (LDH) activity and increasing the superoxide dismutase (SOD) activity. Western blotting result suggested that Gyps/Caps combination could upregulate the expressions of peroxisome proliferator-activated receptors γ (PPARγ), liver X receptor α (LXRα), ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) in the liver tissue. Cellular experiment revealed that the combination treatment protected RAW264.7 cells from ox-LDL induction through inhibiting apoptosis, lipid-decreasing and anti-inflammation, and caused the dramatic enhancement on PPARγ expression. Notably, the combined use of Gyps and Caps did not influence the pharmacological outcomes of each other, but significantly enhanced the overall therapeutic effect in ameliorating HFD-induced hyperlipidemia. Our research provided an experimental basis for advanced research and clinical application of Gyps/Caps combination. Importantly, our cumulative data indicated that combination use of Gyps and Caps may be an effective therapeutic strategy for alleviating hyperlipidemia-induced NAFLD.

IntroductionThe mechanism of the immediate adverse drug reactions (ADRs) induced by ShenMai injection (SMI) has not been completely elucidated. Within 30 minutes, the ears and lungs of mice injected with SMI for the first time showed edema and exudation reactions. These reactions were different from the IV hypersensitivity. The theory of pharmacological interaction with immune receptor (p-i) offered a new insight into the mechanisms of immediate ADRs induced by SMI.MethodsIn this study, we determined that the ADRs were mediated by thymus-derived T cells through the different reactions of BALB/c mice (thymus-derived T cell normal) and BALB/c nude mice (thymus-derived T cell deficient) after injecting SMI. The flow cytometric analysis, cytokine bead array (CBA) assay and untargeted metabolomics were used to explain the mechanisms of the immediate ADRs. Moreover, the activation of the RhoA/ROCK signaling pathway was detected by western blot analysis.ResultsIn BALB/c mice, the vascular leakage and histopathology results showed the occurrence of the immediate ADRs induced by SMI. The flow cytometric analysis revealed that CD4+ T cell subsets (Th1/Th2, Th17/Treg) were imbalanced. And the levels of cytokines such as IL-2, IL-4, IL12P70 and INF-γ increased significantly. However, in BALB/c nude mice, all the indicators mentioned above have not changed significantly. The metabolic profile of both BALB/c mice and BALB/c nude mice was significantly changed after injecting SMI, and the notable increase in lysolecithin level might have a greater association with the immediate ADRs induced by SMI. The Spearman correlation analysis revealed that LysoPC (18:3(6Z,9Z,12Z)/0:0) showed a significant positive correlation with cytokines. After injecting SMI, the levels of RhoA/ROCK signaling pathway-related protein increased significantly in BALB/c mice. Protein-protein interaction (PPI) showed that the increased lysolecithin levels might be related to the activation of the RhoA/ROCK signaling pathway.DiscussionTogether, the results of our study revealed that the immediate ADRs induced by SMI were mediated by thymus-derived T cells, and elucidated the mechanisms of such ADRs. This study provided new insights into the underlying mechanism of immediate ADRs induced by SMI.

Different studies on the effects of high-fat diet (HFD) on Alzheimer’s disease (AD) pathology have reported conflicting findings. Our previous studies showed HFD could moderate neuroinflammation and had no significant effect on amyloid-β levels or contextual memory on AD mice. To gain more insights into the involvement of HFD, we performed the whole-transcriptome sequencing and ribosome footprints profiling. Combined with competitive endogenous RNA analysis, the transcriptional regulation mechanism of HFD on AD mice was systematically revealed from RNA level. Mmu-miR-450b-3p and mmu-miR-6540-3p might be involved in regulating the expression of Th and Ddc expression. MiR-551b-5p regulated the expression of a variety of genes including Slc18a2 and Igfbp3. The upregulation of Pcsk9 expression in HFD intervention on AD mice might be closely related to the increase of cholesterol in brain tissues, while Huanglian Jiedu Decoction significantly downregulated the expression of Pcsk9. Our data showed the close connection between the alterations of transcriptome and translatome under the effect of HFD, which emphasized the roles of translational and transcriptional regulation were relatively independent. The profiled molecular responses in current study might be valuable resources for advanced understanding of the mechanisms underlying the effect of HFD on AD.

Background: A previous genome-wide association study (GWAS) identified the kinesin family member 16B (KIF16B) as a candidate gene related to sheep wool production. In this work, DNA pool sequencing and SNPscanTM high-throughput genotyping methods were used to detect single-nucleotide polymorphisms (SNPs) in the sheep KIF16B gene. The correlations between the SNPs and wool length and greasy wool yield were systematically assessed. Results: Forty-five SNPs were identified and 37 of them were genotyped, including 10 exon mutations, 26 intron mutations, and 1 promoter region mutation. Most of the SNPs were of medium genetic diversity and at Hardy-Weinberg equilibrium (HWE). Among them, 10 SNPs were associated with greasy wool yield and 28 SNPs impact the wool length. Five specific SNPs were found to exert significant effects on the wool length in all body parts analyzed in this study. Furthermore, linkage disequilibrium (LD) analysis was conducted among SNP loci and they were found to be significantly associated with economically important traits. Two strongly linked SNP blocks were identified within these SNPs and they might exert significant impacts on the greasy wool yield and wool length. Conclusions: The identified SNPs exert significant effects on wool production and could be considered as potential DNA markers for selecting the individuals with superior phenotypes. How to cite: Zhao H, Hu R, Li F, et al. Two strongly linked blocks within the KIF16B gene significantly influence wool length and greasy yield in fine wool sheep (Ovis aries). Electron J Biotechnol 2021:52. https://doi.org/10.1016/j.ejbt.2021.05.003

Abstract The active sulphur compounds in transformer oil would react with copper wires and produce cuprous sulphides, which then attach themselves to the surface of copper wires and insulating paper, thus reducing the electrical performance of oil–paper insulation and causing insulation failure. This article aims to make a comprehensive summary on the field of sulphur corrosion of oil–paper insulation in the mineral oil transformer, including the corrosion mechanism of sulphide on copper sheets, the deterioration mechanism of sulphide on oil–paper, the factors influencing sulphur corrosion reaction, the method of characterising the degree of sulphur corrosion, the protection technology of sulphur corrosion and the field operation measures for sulphur corrosion. Moreover, four key problems that need to be further solved were sorted out: the synergistic deterioration mechanism of sulphide and its transformation mechanism, the micro‐process and forming path of sulphur corrosion reaction, the characteristic parameters and quantitative characterisation methods of sulphur corrosion degree, and the effective protection technology of sulphide corrosion. To solve these problems, efforts will be made for deeper understanding of the sulphur corrosion phenomenon and its deterioration mechanism based on theoretical background, the assessment methods and protection technology, endeavouring to put forward solid theoretical basis and feasible technical means for preventing sulphur corrosion insulation failure.

Abstract Background In recent years, excellent results have suggested an association between the “brain-gut” axis and Alzheimer’s disease (AD) progression, yet the role of the “brain-gut” axis in AD pathogenesis still remains obscure. Herein, we provided a potential link between the central and peripheral neuroinflammatory disorders in AD progression. Methods The Morris water maze (MWM) test, immunohistochemistry, ELISA, ProcartaPlex Multiplex immunoassay, multiple LC-MS/MS methods, and the V3-V4 regions of 16S rRNA genes were applied to explore potential biomarkers. Results In Tg-APP/PS1 mice, gut dysbiosis and lipid metabolism were highly associated with AD-like neuroinflammation. The combination of inflammatory factors (IL-6 and INF-γ), phosphatidylcholines (PCs) and SCFA-producing bacteria were expected to be early diagnostic biomarkers for AD. Huanglian Jiedu decoction (HLJDD) suppressed gut dysbiosis and the associated Aβ accumulation, harnessed neuroinflammation and reversed cognitive impairment. Conclusion Together, our findings highlighted the roles of neuroinflammation induced by gut dysbiosis and lipid metabolism disorder in AD progression. This integrated metabolomics approach showed its potential to understand the complex mechanisms of HLJDD in the treatment of AD.

Most zoonotic pathogens originate from mammals and avians, but viral diversity and related biosafety risk assessment in lower vertebrates also need to be explored. Amphibians are an important group of lower vertebrates that played a momentous role in animal evolution. To elucidate the diversity of RNA viruses in one important species of amphibians, the Asiatic toad (Bufo gargarizans), we obtained 44 samples including lung, gut, liver, and kidney tissues from Asiatic toads in Sichuan and Jilin provinces, China, for viral metagenomics sequencing. More than 20 novel RNA viruses derived from the order Bunyavirales and 7 families of Astroviridae, Dicistroviridae, Leviviridae, Partitiviridae, Picornaviridae, Rhabdoviridae, and Virgaviridae were discovered, which were distinct from previously described viruses and formed new clusters, as revealed by phylogenetic analyses. Notably, a novel bastrovirus, AtBastV/GCCDC11/2022, of the family Astroviridae was identified from the gut library, the genome of which contains three open reading frames, with the RNA-dependent RNA polymerase (RdRp) coded by ORF1 closely related to that of hepeviruses, and ORF2 encoding an astrovirus-related capsid protein. Notably, phenuiviruses were discovered for the first time in amphibians. AtPhenV1/GCCDC12/2022 and AtPhenV2/GCCDC13/2022 clustered together and formed a clade with the group of phenuiviruses identified from rodents. Picornaviruses and several invertebrate RNA viruses were also detected. These findings improve our understanding of the high RNA viral diversity in the Asiatic toad and provide new insights in the evolution of RNA viruses in amphibians.

Arenobufagin (ArBu), one of the main active bufadienolides of toad venom with cardiotonic effect, analgesic effect, and outstanding anti-tumor potentiality, is also a potential cardiotoxic component. In the present study, the cardiac effect of ArBu and its underlying mechanism were explored by integrating data such as heart rates, toxicokinetics, myocardial enzyme and brain natriuretic peptide (BNP) activity, pathological sections, lipidomics and proteomics. Under different doses, the cardiac effects turned out to be different. The oral dose of 60 mg/kg of ArBu sped up the heart rate. However, 120 mg/kg ArBu mainly reduced the heart rate. Over time, they all returned to normal, consisting of the trend of ArBu concentration-time curve. High concentrations of myocardial enzymes and BNP indicated that ArBu inhibited or impaired the cardiac function of rats. Pathological sections of hearts also showed that ArBu caused myocardial fiber disorder and rupture, in which the high-dose group was more serious. At the same time, serum and heart tissue lipidomics were used to explore the changes in body lipid metabolism under different doses. The data indicated a larger difference in the high-dose ArBu group. There were likewise many significant differences in the proteomics of the heart. Furthermore, a multi-layered network was used to integrate the above information to explore the potential mechanism. Finally, 4 proteins that were shown to be significantly and differentially expressed were validated by targeted proteomics using parallel reaction monitoring (PRM) analysis. Our findings indicated that ArBu behaved as a bidirectional regulation of the heart. The potential mechanism of cardiac action was revealed with the increased dose, which provided a useful reference for the safety of clinical application of ArBu.

Fibroblast growth factor 5 (FGF5) gene, a member of fibroblast growth factor superfamily, plays significant roles in the regulation of the hair growth cycle during the development of mammalian hair follicles as well as the skeletal muscle development. In this study, DNA sequencing was used to scan the putative SNPs within the full-length of FGF5 gene, and SNPscan high-throughput technique was applied in the individual genotyping of 604 crossbred sheep. 10 SNPs were identified within FGF5 gene while five of them located in intron 1 could be genotyped, namely SNP1 (g. 105914953 G > A), SNP2 (g. 105922232 T > C), SNP3 (g. 105922244 A > G), SNP4 (g. 105922334 A > T) and SNP5 (g. 105922340 G > T). All these SNPs were in accord with the Hardy-Weinberg equilibrium (P > 0.05), and displayed the moderate polymorphism with PIC values ranging from 0.302 to 0.374. Thereafter, the correlation analysis between each SNP locus and economic traits including wool length, greasy wool weight and growth performance of sheep was systematically implemented. In our results, SNP1, SNP3, SNP4 and SNP5 were significantly associated with wool length, greasy wool weight and growth traits of SG sheep (P < 0.05); SNP1, SNP2, SNP3, and SNP4 were significantly correlated with wool length and growth traits of SSG sheep (P < 0.05). Meanwhile, our study revealed a strong linkage disequilibrium (LD) relationship among these SNPs (r2 > 0.33), except for SNP3 and SNP4 sites (r2 = 0.30). Combination genotype analysis showed that combination genotypes were significantly associated with mean fiber diameter of SG (P < 0.05), and body weight trait of SSG (P < 0.01). The above findings suggested that these SNP loci might affect economic traits synergistically and could be regarded as potential molecular markers for improving both wool production and growth performance of fine-wool sheep, which lay a molecular foundation for the breeding of fine dual-purpose sheep thereby accelerating the pace of sheep breeding.

Development of new therapeutic strategies for breast cancer is urgently needed due to the sustained emergence of drug resistance, tumor recurrence and metastasis. To gain a novel insight into therapeutic approaches to fight against breast cancer, the cytocidal effects of hellebrigenin (Helle) and arenobufagin (Areno) were investigated in human estrogen receptor (ER)-positive breast cancer cell line MCF-7 and triple-negative breast cancer cell line MDA-MB-231. Helle exhibited more potent cytotoxicity than Areno in both cancer cells, and MCF-7 cells were more susceptible to both drugs in comparison with MDA-MB-231 cells. Apoptotic-like morphological characteristics, along with the downregulation of the expression level of Bcl-2 and Bcl-xL and the upregulation of the expression level of Bad, were observed in Helle-treated MCF-7 cells. Helle also caused the activation of caspase-8, caspase-9, along with the cleavage of poly(ADP-ribose) polymerase in MCF-7 cells. Helle-mediated necrosis-like phenotype, as evidenced by the increased propidium iodide (PI)-positive cells was further observed. G2/M cell cycle arrest was also induced by Helle in the cells. Upregulation of the expression level of p21 and downregulation of the expression level of cyclin D1, cyclin E1, cdc25C and survivin were observed in MCF-7 cells treated with Helle and occurred in parallel with G2/M arrest. Autophagy was triggered in MCF-7 cells and the addition of wortmannin or 3-MA, two well-known autophagy inhibitors, slightly but significantly rescued the cells. Furthermore, similar alterations of some key molecules associated with the aforementioned biological phenomena were observed in MDA-MB-231 cells. Intriguingly, the numbers of PI-positive cells in Helle-treated MCF-7 cells were significantly reduced by wortmannin and 3-MA, respectively. In addition, Helle-triggered G2/M arrest was significantly corrected by wortmannin, suggesting autophagy induction contributed to Helle-induced cytotoxicity of breast cancer cells by modulating necrosis and cell cycle arrest. Collectively, our results suggested potential usefulness of both Helle and Areno in developing therapeutic strategies to treat patients with different types of breast cancer, especially ER-positive breast cancer.

Summary: Day length in conjunction with seasonal cycles affects many aspects of animal biology. We have studied photoperiod-dependent alterations of complex behavior in the teleost, medaka (Oryzias latipes), a photoperiodic breeder, in a learning paradigm whereby fish have to activate a sensor to obtain a food reward. Medaka were tested under a long (14:10 LD) and short (10:14 LD) photoperiod in three different groups: mixed-sex, all-males, and all-females. Under long photoperiod, medaka mixed-sex groups learned rapidly with a stable response. Unexpectedly, males-only groups showed a strong learning deficit, whereas females-only groups performed efficiently. In mixed-sex groups, female individuals drove group learning, whereas males apparently prioritized mating over feeding behavior resulting in strongly reduced learning performance. Under short photoperiod, where medaka do not mate, male performance improved to a level similar to that of females. Thus, photoperiod has sex-specific effects on the learning performance of a seasonal vertebrate.

Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by the complex interaction of multiple mechanisms. Recent studies examining the effect of high-fat diet (HFD) on the AD phenotype have demonstrated a significant influence on both inflammation and cognition. However, different studies on the effect of high-fat diet on AD pathology have reported conflicting conclusions. To explore the involvement of HFD in AD, we investigated phenotypic and metabolic changes in an AD mouse model in response to HFD. The results indicated there was no significant effect on Aβ levels or contextual memory due to HFD treatment. Of note, HFD did moderate neuroinflammation, despite spurring inflammation and increasing cholesterol levels in the periphery. In addition, diet affected gut microbiota symbiosis, altering the production of bacterial metabolites. HFD created a favorable microenvironment for bile acid alteration and arachidonic acid metabolism in APP/PS1 mice, which may be related to the observed improvement in LXR/PPAR expression. Our previous research demonstrated that Huanglian Jiedu decoction (HLJDD) significantly ameliorated impaired learning and memory. Furthermore, HLJDD may globally suppress inflammation and lipid accumulation to relieve cognitive impairment after HFD intervention. It was difficult to define the effect of HFD on AD progression because the results were influenced by confounding factors and biases. Although there was still obvious damage in AD mice treated with HFD, there was no deterioration and there was even a slight remission of neuroinflammation. Moreover, HLJDD represents a potential AD drug based on its anti-inflammatory and lipid-lowering effects.

A-kinase anchoring protein 12 (AKAP12) plays key roles in male germ cells and female ovarian granulosa cells, whereas its influence on livestock litter size remains unclear. Herein we detected the genetic variants of AKAP12 gene and their effects on litter size as well as alternative splicing variants expression in Shaanbei white cashmere (SBWC) goats, aiming at exploring theoretical basis for goat molecular breeding. We identified two Insertion/deletions (Indels) (7- and 13-bp) within the AKAP12 gene. Statistical analyses demonstrated that the 13-bp indel mutation in the 3′ UTR was significantly associated with litter size (n = 1,019), and the carriers with DD genotypes presented lower litter sizes compared with other carriers (P < 0.01). Bioinformatics analysis predicted that this 13-bp deletion sequence could bind to the seed region of miR-181, which has been documented to suppress porcine reproductive and respiratory syndrome virus (PRRSV) infection by targeting PRRSV receptor CD163 and affect the pig litter size. Therefore, luciferase assay for this 13-bp indel binding with miRNA-181 was performed, and the luciferase activity of pcDNA-miR-181-13bp-Deletion-allele vector was significantly lower than that of the pcDNA-miR-181-13bp-Insertion-allele vector (P < 0.05), suggesting the reduced binding capability with miR-181 in DD genotype. Given that alternative spliced variants and their expression considerably account for the Indel genetic effects on phenotypic traits, we therefore detected the expression of the alternative spliced variants in different tissues and identified that AKAP12-AS2 exhibited the highest expression levels in testis tissues. Interestingly, the AKAP12-AS2 expression levels of homozygote DD carriers were significantly lower than that of individuals with heterozygote ID, in both testis and ovarian tissues (P < 0.05), which is consistent with the effect of the 13-bp deletion on the reduced litter size. Taken together, our results here suggest that this 13-bp indel mutation within goat AKAP12 might be utilized as a novel molecular marker for improving litter size in goat breeding.

“Shanghuo” (“excessive internal heat”) is caused by exuberant endogenous fire, which does not have a comprehensive and systematic traditional Chinese medicine theory. In previous study, we had evaluated the therapeutic effect of Huanglian Jiedu Decoction (HLJDD) (granule) on patients with “Shanghuo”, however, the specific mechanism was not clear, which need further exploration. To explain its intervention mechanism, we select 57 patients with oral diseases caused by “Shanghuo” and 20 health volunteers to divide into oral disease group, HLJDD intervention group and healthy control group. Firstly, biochemical indicators before and after HLJDD intervention are detected, such as inflammatory factors, oxidative stress factors and energy metabolism factors. The results exhibit that HLJDD significantly decreases indicators succinic acid (p < 0.001); tumor necrosis factor-alpha, adenosine triphosphate, citric acid (p < 0.01); interleukin-8 (IL-8), 4-hydroxynonenal, pyruvic acid, lactate dehydrogenase (p < 0.05). The levels of glucocorticoid, adrenocorticotropic hormone (p < 0.01); lactic acid, IL-4, IL-10 (p < 0.05) significantly increase after HLJDD intervention. In addition, we adopt multi-omics analysis approach to investigate the potential biomarkers. Nontargeted metabolomics demonstrate that the levels of 7 differential metabolites approach that in the healthy control group after HLJDD intervention, which are correlated with histidine metabolism, beta-alanine metabolism and sphingolipid metabolism through metabolic pathway analysis. Targeted lipidomics results and receiver operating characteristic curve analysis show that 13 differential lipids are identified in the three groups mainly focuse on lysophosphatidylcholines, lysophosphatidylethanolamines. Finally, the network associations of those differential biomarkers reveal the regulation of adenosine triphosphate and tricarboxylic acid cycle play essential role in the therapeutic effect mechanism of HLJDD in “Shanghuo”. The study has laid the foundation for further revealing the mechanism and finding clinical biomarkers related to “Shanghuo”.

We have gained considerable insight into the mechanisms which recognize and repair DNA damage, but how they adapt to extreme environmental challenges remains poorly understood. Cavefish have proven to be fascinating models for exploring the evolution of DNA repair in the complete absence of UV-induced DNA damage and light. We have previously revealed that the Somalian cavefish Phreatichthys andruzzii, lacks photoreactivation repair via the loss of light, UV and ROS-induced photolyase gene transcription mediated by D-box enhancer elements. Here, we explore whether other systems repairing UV-induced DNA damage have been similarly affected in this cavefish model. By performing a comparative study using P. andruzzii and the surface-dwelling zebrafish, we provide evidence for a conservation of sunlight-regulated Nucleotide Excision Repair (NER). Specifically, the expression of the ddb2 gene which encodes a key NER recognition factor is robustly induced following exposure to light, UV and oxidative stress in both species. As in the case of the photolyase genes, D-boxes in the ddb2 promoter are sufficient to induce transcription in zebrafish. Interestingly, despite the loss of D-box-regulated photolyase gene expression in P. andruzzii, the D-box is required for ddb2 induction by visible light and oxidative stress in cavefish. However, in the cavefish ddb2 gene this D-box-mediated induction requires cooperation with an adjacent, highly conserved E2F element. Furthermore, while in zebrafish UV-induced ddb2 expression results from transcriptional activation accompanied by stabilization of the ddb2 mRNA, in P. andruzzii UV induces ddb2 expression exclusively via an increase in mRNA stability. Thus, we reveal plasticity in the transcriptional and post transcriptional mechanisms regulating the repair of sunlight-induced DNA damage under long-term environmental challenges.

Venenum Bufonis (VB), also called Chan Su in China, has been extensively used as a traditional Chinese medicine (TCM) for treating heart failure (HF) since ancient time. However, the active components and the potential anti-HF mechanism of VB remain unclear. In the current study, the major absorbed components and metabolites of VB after oral administration in rats were first collected from literatures. A total of 17 prototypes and 25 metabolites were gathered. Next, a feasible network-based pharmacological approach was developed and employed to explore the therapeutic mechanism of VB on HF based on the collected constituents. In total, 158 main targets were screened out and considered as effective players in ameliorating HF. Then, the VB components–main HF putative targets–main pathways network was established, clarifying the underlying biological process of VB on HF. More importantly, the main hubs were found to be highly enriched in adrenergic signalling in cardio-myocytes. After verified by molecular docking studies, four key targets (ATP1A1, GNAS, MAPK1 and PRKCA) and three potential active leading compounds (bufotalin, cinobufaginol and 19-oxo-bufalin) were identified, which may play critical roles in cardiac muscle contraction. This study demonstrated that the integrated strategy based on network pharmacology and molecular docking was helpful to uncover the synergistic mechanism of multiple constituents in TCM.

Hellebrigenin is a natural product found in the toad skin secretions and plants of Urginea, including Hellebores and Kalanchoe genera. It has been shown to be active against Leishmania chagasi promastigotes and Trypanosoma cruzi trypomastigotes and also reported to play an anti-tumor effect on several cancer cell lines in vitro, including pancreatic cancer. This study is aimed to investigate the effects of Hellebrigenin on pancreatic carcinoma cells, SW1990 and BxPC-3 in vitro and its molecular mechanism involved in antitumor activities. Our results showed that Hellebrigenin effectively inhibited the proliferation of SW1990 and BxPC-3 cells in dose- and time-dependent manner. Flow cytometry results showed that Hellebrigenin induced the G0/G1 arrest in both of SW1990 and BxPC-3 cells and promoted cell early apoptosis and autophagy according to morphological observation. Immunofluorescence staining results further confirmed that cell apoptosis and autophagy also increased upon the Hellebrigenin treatment. Moreover, higher dose of Hellebrigenin further increased the cell apoptosis rate while decrease the mitochondrial membrane potential 24 h after treatment. The autophagy rate increased 48 h after treatment with significant difference (P < 0.05). Western blot analysis showed that the expression of caspase 3, 7, cleaved caspase 7, Atg 12, LC3 proteins were increased in SW1990 cell after treatment with Hellebrigenin. In addition, increasing expression of caspase 3, 7, 9, PARP, cleaved caspase 3, 7, 9, PARP, the sub basic protein of the PI3K family, Beclin-1, LC 3, Atg 3, 5, 12, 16 L were also observed after BxPC-3 cells treated with Hellebrigenin. In summary, this study reported for the first time that Hellebrigenin effectively induced autophagy and apoptosis especially the early apoptosis in SW1990 and BxPC-3 cells.

Melanocortin-3 receptor (MC3R) plays an important role in the energy homeostasis of animals under different nutritional conditions. Onychostoma macrolepis is a hibernating cavefish found in the northern part of the Yangtze River, and its adaptation to a nutrient-poor environment has attracted growing interest. In this study, we characterized the protein structure of Onychostoma macrolepis Mc3r (omMc3r), examined its tissue distribution, and investigated its function in mediating cellular signaling. We showed that the CDS of omMc3r is 978 bp, encoding a putative protein of 325 amino acids. Homology and phylogenetic analyses indicated that omMc3r is evolutionary close to cyprinids. Real-time quantitative PCR (RT-qPCR) revealed that omMc3r was highly expressed in the liver and brain. The functions of omMc3r to mediate ligands activating downstream signaling have also been confirmed by using signal pathway-specific reporters. The four agonists α-MSH, β-MSH, NDP-MSH, and ACTH (1–24) can all activate the cAMP and MAPK/ERK signaling pathway, albeit with different potency orders. The “primitive” ligand ACTH (1–24) had the highest potency on the cAMP signaling pathway, while the synthetic ligand NDP-MSH had the highest activation effect on the MAPK/ERK signaling pathway. This research will lay the foundation for studying the energy regulation mechanism of cavefish in an oligotrophic environment.

Huanglian Jiedu decoction (HLJDD) has been used in the clinical treatment of inflammatory conditions. To clarify the mechanism of its comprehensive anti-inflammatory activities, the correlation between lipid homeostasis and the TLR4/MyD88 signaling pathway in zebrafish was established in the present study. In the lipopolysaccharide (LPS)-induced inflammation in zebrafish model, RT-PCR assays of five inflammatory cytokines and six targeted proteins were measured. Lipidomics analysis was conducted to identify potential lipid markers. HLJDD displayed strong efficacies, with a 61% anti-inflammatory rate at a concentration of 50 μg/mL. The activation of TLR4/MyD88 played an essential role in the inflammatory process. All protein indexes in the HLJDD group exhibited a tendency to reverse back to normal levels. Moreover, 79 potential pathological lipid biomarkers were identified. Compared with the model group, 61 therapeutic lipid biomarkers were detected in HLJDD group. Most perturbations of lipids were ameliorated by HLJDD, mainly through the glycerophospholipid metabolic pathway. In the visual network study, the corresponding lipoproteins such as PLA2, SGMS, and SMDP were observed as important intermediates between lipid homeostasis and the TLR4/MyD88 signaling pathway.

There is an urgent need to improve bovine fertility, and molecular marker-assisted selection (MAS) can accelerate this process. Genome-wide association studies suggest that Integrin β5 (ITGβ5) might affect fertility in bovines. As a member of the integrins family, ITGβ5 can bind to the extracellular matrix and mediate various cellular processes. In our study, primers spanning six potential insertion/deletion (indel) polymorphisms within the ITGβ5 gene were designed and 696 ovary samples from different individuals, the vast majority not in oestrum were collected for genetic variation detection. A deletion locus, rs522759246, namely P1-D13-bp, was found to be polymorphic. The allele D frequency was 0.152 and the polymorphism information content (PIC) value was 0.224, indicating a low-degree PIC. This locus did not follow the Hardy–Weinberg equilibrium (p = 1.200E-23). Importantly, associations between P1-D13-bp and ovarian morphological traits were established. Polymorphisms of this locus had significant correlations with ovarian width (p = 0.015). The corpus luteum is also linked to fertility and P1-D13-bp was significantly correlated with corpus luteum diameter (p = 0.005). In conclusion, an indel mutation within the bovine ITGβ5 gene was identified, which was significantly associated with several ovarian and luteal traits.

Cinobufacin injection, also known as huachansu, is a preparation form of Cinobufacini made from Cinobufacin extract liquid. Despite that Cinobufacin injection is shown to shrink liver and gastric tumors, improving patient survival and life quality, the effective components in Cinobufacin remain elusive. In this study, we aim to screen antitumor components from Cinobufacin injection to elucidate the most effective antitumor components for treatment of liver and gastric cancers.High performance liquid chromatography (HPLC) and LC-MS/MS analysis were used to separate and determine the components in Cinobufacin injection. Inhibition rates of various components in Cinobufacin injection on liver and gastric cancer cells were determined with MTT assay; Hepatocellular carcinoma and gastric cancer models were used to assess the antitumor effect of the compounds in vivo.The major constituents in Cinobufacin injection include peptides, nucleic acids, tryptamines and bufotalins. MTT assay revealed that bufadienolides had the best antitumor activity, with peptides being the second most effective components. Bufadienolides showed significant inhibition rates on gastric and hepatocellular tumour growth in vivo.Bufadienolides are the most effective components in Cinobufacini injection for the treatment of liver and gastric cancers. This discovery can greatly facilitate further research in improving the therapeutic effects of Cinobufacin injection, meanwhile reducing its adverse reaction.

Aristolochic acid I (AAI) was regarded as the major toxic component of aristolochic acid (AA). In addition to aristolochic acid nephropathy (AAN), liver cancers induced by AAI has aroused increasing attention recently. In this paper, the discovery of diagnostic biomarkers for AAI-induced liver injury has been studied, especially for the lipid markers. From the histopathological characteristics, the injury was observed clearly in the liver apart from the kidney after 30 mg/kg of AAΙ treatment for one week, while the lesion alleviated after AAΙ discontinuance. The serum biochemical indexes were manifested to the normal tendency after AAΙ discontinuance for two weeks. According to the evaluation of pathology slices and serum biochemical indexes, they indicated that the hepatotoxicity induced by AAΙ was reversible to some extent. A total of 44 lipid markers were identified in the liver, as well as 59 in the serum. Twenty-six common lipid markers were observed in both serum and liver. Furthermore, nine out of 26 lipids exhibited the excellent diagnostic ability to differentiate the control group from the AAΙ group and AAΙ discontinuance group with high sensitivity and specificity. The changed lipid markers might serve as characteristics to explain the mechanisms of pathogenesis and progression in hepatotoxicity induced by AAΙ.

Rhein is used as an active ingredient in laxatives in medicinal herbal products and is a chemical marker for quality control purposes. Thus, a simple and effective method for the optimized extraction of a high amount of rhein from the fruit pulp of Cassia fistula was investigated using ultrasonic-assisted extraction (UAE). The response surface methodology was applied to find the most suitable parameters for optimizing the extraction process and to study the factors’ relationships with each other. The best conditions for ultrasonic extraction were the application of 1:40 g/mL solid-to-liquid ratio and 10% EtOH−H2O as a solvent at 75 °C for 40 min. This method was compared to a conventional decoction in two variations. In these experiments, it was confirmed that the UAE was more favorable than the decoction methods. The resulting crude extract was further purified by liquid−liquid extraction with a basic pH adjustment, followed by recrystallization. High-purity rhein was obtained by using chromatographic techniques and nuclear magnetic resonance spectroscopy. Therefore, this study suggests that UAE is an efficient alternative method for the extraction of rhein from C. fistula pod pulp. The resulting optimized conditions can be applied as a useful tool for the large-scale industrial production of a rhein-rich plant extract.

Toad skin and toad venom, as two kinds of Chinese medicine, are prepared from Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. However, they display distinct properties in traditional application, and the hydrophilic ingredients are the possible distinguishing chemicals between them. In this work, 36 and 22 hydrophilic components were characterized from toad skin and venom, respectively, by UHPLC-HR-MS/MS, including amino acids, nucleosides, polypeptides, and indolealkylamines (IAAs). Among them, 15 compounds were unambiguously confirmed by comparison with standards. The CID-MS/MS fragmentation behaviors of seven indolealkylamine references were investigated to ascertain three types of structures. Subsequently, 11 high abundance contents of hydrophilic ingredients were determined from 11 batches of toad skin and 4 batches of toad venom by UPLC-QqQ-MS/MS. The quantitative results showed that the content of main IAAs in toad venom was much higher than in skin. In addition, the N-methyl serotonin (free IAA), bufothionine (combined IAA), and total IAAs sample were selected for anti-inflammatory evaluation in lipopolysaccharide (LPS) stimulated zebrafish embryo models. The obvious anti-inflammatory activities of IAAs were observed, especially for the free IAAs. This study illustrated IAAs were the main distinct hydrophilic components that probably lead to the difference between toad skin and toad venom in traditional applications.

Macamides are very important secondary metabolites produced by Lepidium meyenii Walp, which possess multiple bioactivities, especially in the neuronal system. In a previous study, we observed that macamides exhibited excellent effects in the recovery of injured nerves after 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neuronal damage in zebrafish. However, the mechanism underlying this effect remains unclear. In the present study, we observed that N-benzylhexadecanamide (XA), which is a typical constituent of macamides, improved the survival rate of neurons in vitro. We determined the concentration of neurotransmitters in MN9D cells and used it in conjunction with an integrated proteomics and lipidomics approach to investigate the mechanism underlying the neuroprotective effects of XA in an MPP+-induced neurodegeneration cell model using QqQ MS, Q-TOF MS, and Orbitrap MS. The statistical analysis of the results led to the identification of differentially-expressed biomarkers, including 11 proteins and 22 lipids, which may be responsible for the neuron-related activities of XA. All these potential biomarkers were closely related to the pathogenesis of neurodegenerative diseases, and their levels approached those in the normal group after treatment with XA. Furthermore, seven lipids, including five phosphatidylcholines, one lysophosphatidylcholine, and one phosphatidylethanolamine, were verified by a relative quantitative approach. Moreover, four proteins (Scarb2, Csnk2a2, Vti1b, and Bnip2) were validated by ELISA. The neurotransmitters taurine and norepinephrine, and the cholinergic constituents, correlated closely with the neuroprotective effects of XA. Finally, the protein⁻lipid interaction network was analyzed. Based on our results, the regulation of sphingolipid metabolism and mitochondrial function were determined to be the main mechanisms underlying the neuroprotective effect of XA. The present study should help us to better understand the multiple effects of macamides and their use in neurodegenerative diseases.

Metabolic study of bioactive compounds that undergo a dynamic and sequential process of metabolism is still a great challenge. Salidroside, one of the most active ingredients of Rhodiola crenulata, can be metabolized in different sites before being absorbed into the systemic blood stream. This study proposed an approach for describing the sequential biotransformation process of salidroside based on comparative analysis. In vitro incubation, in situ closed-loop and in vivo blood sampling were used to determine the relative contribution of each site to the total metabolism of salidroside. The results showed that salidroside was stable in digestive juice, and it was metabolized primarily by the liver and the intestinal flora and to a lesser extent by the gut wall. The sequential metabolism method described in this study could be a general approach to characterizing the metabolic routes in the digestive system for natural products.