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

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. 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. 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. 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-β1-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-β1-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. ASI can inhibit PMCs MMT and alleviate PF by regulating the JAK2/STAT3 signaling pathway.

Sun J ，Tang L ，Shan Y ，Yu M ，Sheng L ，Huang L ，Cao H ，Dai H ，Wang F ，Zhao J ，Sheng M ... -  《-》

Network pharmacology, molecular docking and experimental verification of the mechanism of huangqi-jixuecao herb pair in treatment of peritoneal fibrosis.

The Huangqi-Jixuecao herb pair (HQJXCHP) is a traditional herbal formula composed of two widely applied TCM prescriptions, Huangqi (Astragalus membranaceus (Fisch.) Bunge) and Jixuecao (Centella asiatica (L.) Urb.), used for hundreds of years to replenish qi and clear away heat. However, the therapeutic effects of HQJXCHP against peritoneal fibrosis (PF) and potential targets are currently unclear. The main objective of this study was preliminary prediction and validation of the effects and molecular mechanisms of action of HQJXCHP against PF based on network pharmacology analysis and experimental verification. The ingredients of HQJXCHP were analyzed via HPLC-Q-TOF/MS. Bioactive compounds of HQJXCHP used for network pharmacology analysis were obtained from the TCMSP database. HQJXCHP-related therapeutic targets in PF were obtained from the GeneCards, OMIM, Therapeutic Targets and PharmGkb databases. Therapeutic target-related signaling pathways were predicted via GO and KEGG pathway enrichment analyses. The targets of HQJXCHO were further validated in a PDS-induced PF mouse model in vivo and PMCs MMT model in vitro. A total of 23 bioactive compounds of HQJXCHP related 188 target genes were retrieved. The HQJXCHP compound-target and PF-related target networks identified 131 common target genes. Subsequent protein-protein interaction (PPI) network analysis results disclosed Akt1, TP53, TNF, VEGFA and CASP3 as the top five key targets of HQJXCHP. Further molecular docking data revealed strong affinity of the two key compounds of HQJXCHP, quercetin and kaempferol, for these key targets. GO and KEGG pathway enrichment analyses further showed that PI3K/Akt, IL-17, TNF and TLR pathways contribute to the therapeutic effects of HQJXCHP on PF. An in vivo PDS-induced PF mouse model and in vitro PMCs mesothelial-to-mesenchymal transition (MMT) model with or without HQJXCHP intervention were used to confirm the effects and mechanisms of action of HQJXCHP. Western blot and qRT-PCR results showed that HQ, JXC and HQJXCHP reduced PDS-induced inflammatory cell aggregation and peritoneal thickening through suppressing the MMT process, among which HQJXCHP exerted the greatest therapeutic effect. Moreover, HQJXCHP inhibited activation of the PI3K/Akt, IL-17, TNF and TLR signaling pathways induced by PDS. This is the first study to employ network pharmacology and molecular docking analyses to predict the targets of HQJXCHP with therapeutic effects on PDS-related PF. Data from in vivo and in vitro validation experiments collectively showed that HQJXCHP delays the PF process through inhibiting PI3K/Akt, IL-17, TNF and TLR signaling pathways. Overall, our findings highlight the successful application of network pharmacology theory to provide a scientific basis for clinical utility of HQJXCHP against PF.

Dai H ，Shan Y ，Yu M ，Wang F ，Zhou Z ，Sun J ，Sheng L ，Huang L ，Sheng M ... -  《-》

Asiaticoside inhibits TGF-β1-induced mesothelial-mesenchymal transition and oxidative stress via the Nrf2/HO-1 signaling pathway in the human peritoneal mesothelial cell line HMrSV5.

Peritoneal fibrosis (PF) is a frequent complication caused by peritoneal dialysis (PD). Peritoneal mesothelial cells (PMCs), the first barrier of the peritoneum, play an important role in maintaining structure and function in the peritoneum during PD. Mesothelial-mesenchymal transition (MMT) and oxidative stress of PMCs are two key processes of PF. To elucidate the efficacy and possible mechanism of asiaticoside inhibition of MMT and ROS generation in TGF-β1-induced PF in human peritoneal mesothelial cells (HPMCs). MMT and ROS generation of HPMCs were induced by TGF-β1. To explain the anti-MMT and antioxidant role of asiaticoside, varied doses of asiaticoside, oxygen radical scavenger (NAC), TGF-β receptor kinase inhibitor (LY2109761) and Nrf2 inhibitor (ML385) were used separately. Immunoblots were used to detect the expression of signaling associated proteins. DCFH-DA was used to detect the generation of ROS. Transwell migration assay and wound healing assay were used to verify the capacity of asiaticoside to inhibit MMT. Immunofluorescence assay was performed to observe the subcellular translocation of Nrf2 and expression of HO-1. Asiaticoside inhibited TGF-β1-induced MMT and suppressed Smad signaling in a dose-dependent manner. Migration and invasion activities of HPMCs were decreased by asiaticoside. Asiaticoside decreased TGF-β1-induced ROS, especially in a high dose (150 μM) for 6 h. Furthermore, ML385 partly abolished the inhibitory effect of asiaticoside on MMT, ROS and p-Smad2/3. Asiaticoside inhibited the TGF-β1-induced MMT and ROS via Nrf2 activation, thus protecting the peritoneal membrane and preventing PF.

Zhao J ，Shi J ，Shan Y ，Yu M ，Zhu X ，Zhu Y ，Liu L ，Sheng M ... -  《-》

N-methylpiperazine-diepoxyovatodiolide ameliorates peritoneal fibrosis via suppressing TGF-β/Smad and JAK/STAT signaling pathway.

Peritoneal fibrosis (PF) is the main cause of peritoneal ultrafiltration failure in patients undergoing long-term peritoneal dialysis (PD). Epithelial-mesenchymal transition (EMT) is the key pathogenesis of PF. However, currently, no specific treatments are available to suppress PF. N-methylpiperazine-diepoxyovatodiolide (NMPDOva) is a newly synthesized compound that involves a chemical modification of ovatodiolide. In this study, we aimed to explore the antifibrotic effects of NMPDOva in PD-related PF and underlying mechanisms. A mouse model of PD-related PF was established via daily intraperitoneal injection of 4.25% glucose PD fluid. In vitro studies were performed using the transforming growth factor-beta1 (TGF-β1)-stimulated HMrSV5 cell line. Pathological changes were observed, and fibrotic markers were significantly elevated in the peritoneal membrane in mice model of PD-related PF. However, NMPDOva treatment significantly alleviated PD-related PF by decreasing the extracellular matrix accumulation. NMPDOva treatment decreased the expression of fibronectin, collagen Ⅰ, and alpha-smooth muscle actin (α-SMA) in mice with PD-related PF. Moreover, NMPDOva could alleviate TGF-β1-induced EMT in HMrSV5 cells, inhibited phosphorylation and nuclear translocation of Smad2/3, and increased the expression of Smad7. Meanwhile, NMPDOva inhibited phosphorylation of JAK2 and STAT3. Collectively, these results indicated that NMPDOva prevents PD-related PF by inhibiting the TGF-β1/Smad and JAK/STAT signaling pathway. Therefore, because of these antifibrotic effects, NMPDOva may be a promising therapeutic agent for PD-related PF.

Mo M ，Zeng Y ，Zeng Y ，Li S ，He X ，Chen X ，Luo Q ，Liu M ，Luo C ，Dou X ，Peng F ，Long H ... -  《-》

Astragalus membranaceus and its monomers treat peritoneal fibrosis and related muscle atrophy through the AR/TGF-β1 pathway.

Background: To anticipate the potential molecular mechanism of Astragalus membranaceus (AM) and its monomer, Calycosin, against peritoneal fibrosis (PF) and related muscle atrophy using mRNA-seq, network pharmacology, and serum pharmacochemistry. Methods: Animal tissues were examined to evaluate a CKD-PF mice model construction. mRNA sequencing was performed to find differential targets. The core target genes of AM against PF were screened through network pharmacology analysis, and CKD-PF mice models were given high- and low-dose AM to verify common genes. Serum pharmacochemistry was conducted to clarify which components of AM can enter the blood circulation, and the selected monomer was further validated through cell experiments for the effect on PF and mesothelial mesenchymal transition (MMT) of peritoneal mesothelial cells (PMCs). Results: The CKD-PF mice models were successfully constructed. A total of 31,184 genes were detected in the blank and CKD-PF groups, and 228 transcription factors had significant differences between the groups. Combined with network pharmacology analysis, a total of 228 AM-PF-related targets were identified. Androgen receptor (AR) was the remarkable transcription factor involved in regulating transforming growth factor-β1 (TGF-β1). AM may be involved in regulating the AR/TGF-β1 signaling pathway and may alleviate peritoneal dialysis-related fibrosis and muscle atrophy in CKD-PF mice. In 3% peritoneal dialysis solution-stimulated HMrSV5 cells, AR expression levels were dramatically reduced, whereas TGF-β1/p-smads expression levels were considerably increased. Conclusion: AM could ameliorate PF and related muscle atrophy via the co-target AR and modulated AR/TGF-β1 pathway. Calycosin, a monomer of AM, could partially reverse PMC MMT via the AR/TGF-β1/smads pathway. This study explored the traditional Chinese medicine theory of "same treatment for different diseases," and supplied the pharmacological evidence of "AM can treat flaccidity syndrome."

Sheng L ，Sun J ，Huang L ，Yu M ，Meng X ，Shan Y ，Dai H ，Wang F ，Shi J ，Sheng M ... -  《Frontiers in Pharmacology》