TMT quantitative proteomics and network pharmacology reveal the mechanism by which asiaticoside regulates the JAK2/STAT3 signaling pathway to inhibit peritoneal fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine, Centella asiatica (L.) Urb., has been extensively utilized in clinics to treat a variety of fibrotic disorders. Asiaticoside (ASI), as an important active ingredient, has attracted much attention in this field. However, the effect of ASI on peritoneal fibrosis (PF) is still unclear. Therefore, we evaluated the benefits of ASI for PF and mesothelial-mesenchymal transition (MMT) and revealed the underlying mechanisms. AIM OF STUDY: The objective of this investigation was to anticipate the potential molecular mechanism of ASI against peritoneal mesothelial cells (PMCs) MMT employing proteomics and network pharmacology, and to confirm it using in vivo and in vitro studies. MATERIALS AND METHODS: The mesentery of peritoneal fibrosis mice and normal mice were analyzed quantitatively for proteins that were differentially expressed using a technique tandem mass tag (TMT). Next, the core target genes of ASI against PF were screened through network pharmacology analysis, and PPI and C-P‒T networks were constructed by Cytoscape Version 3.7.2. According to the findings of a GO and KEGG enrichment analysis of differential proteins and core target genes, the signaling pathway with a high correlation degree was selected as the key signaling pathway of ASI inhibiting the PMCs MMT for further molecular docking analysis and experimental verification. RESULTS: TMT-based quantitative proteome analysis revealed the identification of 5727 proteins, of which 70 were downregulated and 178 were upregulated. Among them, the levels of STAT1, STAT2, and STAT3 in the mesentery of mice with peritoneal fibrosis were considerably lower than in the control group, indicating a role for the STAT family in the pathogenesis of peritoneal fibrosis. Then, a total of 98 ASI-PF-related targets were identified by network pharmacology analysis. JAK2 is one of the top 10 core target genes representing a potential therapeutic target. JAK/STAT signaling may represent a core pathway mediating PF effects by ASI. Molecular docking studies showed that ASI had the potential to interact favorably with target genes involved in the JAK/STAT signaling pathway, such as JAK2 and STAT3. The experimental results showed that ASI could significantly alleviate Chlorhexidine Gluconate (CG)-induced peritoneal histopathological changes and increase JAK2 and STAT3 phosphorylation levels. In TGF-ß1-stimulated HMrSV5 cells, E-cadherin expression levels were dramatically reduced whereas Vimentin, p-JAK2, α-SMA, and p-STAT3 expression levels were considerably increased. ASI inhibited the TGF-ß1-induced HMrSV5 cell MMT, decreased the activation of JAK2/STAT3 signaling, and increased the nuclear translocation of p-STAT3, which was consistent with the effect of the JAK2/STAT3 pathway inhibitor AG490. CONCLUSION: ASI can inhibit PMCs MMT and alleviate PF by regulating the JAK2/STAT3 signaling pathway.

Total extract of Abelmoschus manihot L. alleviates uric acid-induced renal tubular epithelial injury via inhibition of caspase-8/caspase-3/NLRP3/GSDME signaling.

Purpose: The incidence of uric acid (UA)-induced kidney injury is increasing owing to the high incidence of hyperuricemia in recent years. The flower of Abelmoschus manihot (Linneus) Medik is a traditional Chinese medicinal herb widely used in the treatment of some kidney diseases. In our previous study, we reported that the total extract of A. manihot L. flower (TEA) attenuated adriamycin-induced renal tubular cell injury. In this study, we aimed to evaluate the role of TEA in UA-induced tubular cell injury. Methods: Normal rat proximal epithelial NRK-52E cells were incubated with UA to mimic hyperuricemia conditions. The role of TEA in the renal tubular cells was also assessed. The cellular morphology was observed using phase-contrast microscopy, and cell viability was analyzed using the Cell Counting kit-8. Living and dead cells were stained using a Calcein-AM/PI double stain kit. The release of lactate dehydrogenase (LDH) was analyzed by LDH cytotoxicity Assay Kit. The expression of target proteins was analyzed using western blot analysis. Results: UA triggered NRK-52E cell injury, as evidenced by morphological changes, detachment of cells from the bottom, cell swelling, large bubbles blowing from cell membrane and loss of cell viability. UA increased release of LDH. UA induced the expression of p-ERK1/2 and the subsequent activation of caspase-8, caspase-3, and NLRP3 inflammasomes. Pyroptosis was elicited by UA after gasdermin E N-terminal (GSDME-NT) was cleaved from gasdermin E (GSDME). Z-DEVD-FMK, a caspase-3 inhibitor, suppressed the expression of both NLRP3 and GSDME-NT, but not that of caspase-8. INF39, an NLRP3 inhibitor, altered the expression of GSDME-NT expression, but not that caspase-3 and caspase-8. TEA alleviated UA-induced cell injury by suppressing ERK1/2/caspase-8/caspase-3/NLRP3/GSDME signaling. Conclusion: GSDME-mediated pyroptosis was involved in UA-induced renal tubular cell injury. This is the first study to report that TEA protects renal tubular epithelial cells against UA by inhibiting the ERK/1/2/caspase-8/caspase-3/NLRP3/GSDME pathway.

Astragalus Total Saponins Ameliorate Peritoneal Fibrosis by Promoting Mitochondrial Synthesis and Inhibiting Apoptosis.

Peritoneal fibrosis (PF) is a disease caused by prolonged exposure of the peritoneum to high levels of dialysis fluid. Astragalus total saponins (ATS) is a phytochemical naturally occurring in Radix Astragali that has anti-inflammatory and anti-oxidant properties. In this study, we constructed an in vivo model of PF using 4.25% glucose-containing administered intraperitoneally to rats and incubated peritoneal mesothelial cells (PMCs) with 4.25% glucose-containing peritoneal dialysis fluid to construct an in vitro model of PF. Furthermore, siRNA of PGC-1[Formula: see text] was used to inhibit the expression of PGC-1[Formula: see text] to further investigate the mechanism of the protective effect of ATS on PF. In both in vivo and in vitro models, ATS treatment showed a protective effect against PF, with ATS reducing the thickness of peritoneal tissues in PF rats, increasing the viability of PMCs, increasing the mitochondrial membrane potential and reducing apoptosis ratio. ATS treatment also reduced the expressions of peritoneal fibrosis markers (Smad2, p-Smad2 and [Formula: see text]-SMA) and apoptosis markers (Caspase3, cleaved-Caspase3 and Bax) and restored the expressions of mitochondrial synthesis proteins (PGC-1[Formula: see text], NRF1 and TFAM) in ATS-treated peritoneal tissues or PMCs. Furthermore, in the presence of PGC-1[Formula: see text] inhibition, the protective effect of ATS on PF was blocked. In conclusion, ATS treatment may be an effective therapeutic agent to inhibit high glucose-induced in peritoneal fibrosis through PGC-1[Formula: see text]-mediated apoptosis.

Downregulation of let-7 by Electrical Acupuncture Increases Protein Synthesis in Mice.

BACKGROUND: Our previous study found that acupuncture with low frequency electrical stimulation (Acu/LFES) prevents muscle atrophy by attenuation of protein degradation in mice. The current study examines the impact of Acu/LFES on protein synthesis. METHOD: C57/BL6 mice received Acu/LFES treatment on hindlimb for 30 min once. Acu/LFES points were selected by WHO Standard Acupuncture Nomenclature and electric stimulation applied using an SDZ-II Electronic acupuncture instrument. Muscle protein synthesis was measured by the surface-sensing of translation (SUnSET) assay. Exosomes were isolated using serial centrifugation and concentration and size of the collected exosomes were measured using a NanoSight instrument. The mature microRNA library in serum exosomes was validated using a High Sensitivity DNA chip. RESULTS: Protein synthesis was enhanced in the both hindlimb and forelimb muscles. Blocking exosome secretion with GW4869 decreased the Acu/LFES-induced increases in protein synthesis. MicroRNA-deep sequencing demonstrated that four members of the Let-7 miRNA family were significantly decreased in serum exosomes. Real time qPCR further verified Acu/LFES-mediated decreases of let-7c-5p in serum exosomes and skeletal muscles. In cultured C2C12 myotubes, inhibition of let-7c not only increased protein synthesis, but also enhanced protein abundance of Igf1 and Igf1 receptors. Using a luciferase reporter assay, we demonstrated that let-7 directly inhibits Igf1. CONCLUSION: Acu/LFES on hindlimb decreases let-7-5p leading to upregulation of the Igf1 signaling and increasing protein synthesis in both hindlimb and forelimb skeletal muscles. This provides a new understanding of how the electrical acupuncture treatment can positively influence muscle health.

Astragalus Inhibits Epithelial-to-Mesenchymal Transition of Peritoneal Mesothelial Cells by Down-Regulating ß-Catenin.

BACKGROUND/AIMS: The epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a crucial event in the induction of peritoneal fibrosis (PF), in which canonical Wnt/ß-catenin signaling participates. Smads signaling is reported to interact with ß-catenin and synergistically regulates EMT. This study was aimed to reveal the effect of Astragalus on ß-catenin in EMT of PMCs. METHODS: To obtain the role of ß-catenin in EMT, gene transfer into HMrSV5 cell line and rats has been achieved. After Astragalus treatment, EMT markers and signaling pathway-related indicators were detected by western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation and real time-PCR. RESULTS: ß-catenin knockdown suppressed EMT of HMrSV5 cells. Astragalus alleviated EMT of PMCs characterized by increased E-cadherin and decreased α-SMA and Vimentin. In rat model of peritoneal dialysis (PD), Astragalus attenuated peritoneal thickening and fibrosis. Astragalus down-regulated ß-catenin by stabilizing the Glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin complex and further inhibited the nuclear translocation of ß-catenin. Meanwhile, Astragalus down-regulated ß-catenin by enhancing Smad7 expression. Silencing Smad7 antagonized the EMT-inhibitory effect of Astragalus. CONCLUSION: Astragalus inhibits EMT of PMCs by down-regulating ß-catenin. The modulation of ß-catenin in peritoneum can be a novel tool to prevent PF.