#3005 Asiaticoside alleviates renal fibrosis by regulating Th17/Treg cell homeostasis through binding to STAT3

Abstract Background and Aims Renal fibrosis (RF) is a significant pathological change in chronic kidney disease (CKD) that leads to kidney function loss and ultimately results in end-stage kidney disease (ESRD). CD4+ T cells play a role in RF development. Th17 cells promote RF, whereas Treg cells help alleviate it. Centella asiatica was a traditional herb with a variety of pharmacological activities, which was used in traditional Chinese medicine for anti-inflammation and anti-scar formation. Asiaticoside (AS), the primary active ingredient in Centella asiatica, is known for its therapeutic properties. It has been reported in many animal experiments that AS could alleviate renal fibrosis, but its mechanism have not been well defined. Thus, we employed network pharmacological analysis and mouse renal tissue transcriptome sequencing to systematically elucidate how AS alleviates RF. Method We used network pharmacological analysis to predict the potential targets and signaling pathways of RF treatment by AS. To verify and evaluate these mechanisms, we constructed a UUO (10 d) mouse model, which was divided into normal control group, UUO model group, and AS drug group (divided into 3 dose groups, 100 mg/ (kg*d), 50 mg/ (kg*d), and 10 mg/ (kg*d), with 10 mice in each group. DdH2O was used to dissolve AS to the appropriate concentration. AS was administered intragastrically starting on the first day after surgery for 10 days, and renal fibrosis was assessed via Masson staining and immunohistochemistry (IF). Transcriptome sequencing and bioinformatics were conducted on kidney tissues, while Luminex and qPCR measured inflammatory factors. Th17 and Treg cells were quantitatively analyzed using flow cytometry and IF. The DARTS+LC-MS/MS technique screened asiaticoside's therapeutic targets, which were validated by molecular docking, dynamics simulation, and surface plasmon resonance (SPR). STAT3 phosphorylation was analyzed by Western blot and IF. Results The network pharmacological analysis identified 281 therapeutic targets for RF mitigation by AS, with 196 signaling pathway interactions, notably the Th17 cell differentiation pathway. PPI analysis highlighted STAT3, SRC, and AKT1 as central network nodes. In vivo, AS at 100 mg/(kg*d) alleviated RF in UUO mice, revealing 107 differentially expressed genes via transcriptomic sequencing. KEGG analysis confirmed the role of these genes, with AS affecting Th17 cell differentiation. Flow cytometry showed increased Treg and decreased Th17 cells post-treatment, alongside reduced IL-17A and increased IL-10 levels in kidney tissue. AS also lowered TNF-α, IL-2, IL-4, IL-6, IL-17, and IFN-r in serum without affecting IL-10. DARTS+LC-MS/MS indicated increased hydrolysis of STAT3 in renal tissue after AS treatment. Then, we used molecular docking and molecular dynamics to predict that AS could bind to Phosphorylated Tyr705 binding pocket (pY pocket) in SH2 domain of STAT3 with good affinity (−8.323 kcal/mol) and very low MM-GBSA binding free energy (−53.07 kcal/mol). SPR found that the binding of STAT3 and AS show fast binding and fast dissociation, the binding dissociation parameter was KD = 4.736E−6 M. IF and western blot indicated that after treatment with AS, renal pSTAT3 level decreased, while total STAT3 level did not change. Conclusion AS may bind to the pY pocket in the SH2 domain of STAT3, which could inhibit the phosphorylation of STAT3 Tyr705. This inhibition affects the signaling pathway responsible for Th17 cell differentiation. By regulating the homeostasis of Th17 and Treg cells, AS may alleviate kidney inflammation and fibrosis in mice. STAT3 was considered as a key therapeutic target for AS treatment of RF.