Astragalus membranaceus and Salvia miltiorrhiza Inhibit Tryptase/PAR2-Mediated Mast Cells – Peritoneal Mesothelial Cells Crosstalk to Ameliorate Peritoneal Fibrosis

Yanlin Chen,1,2 Yuqing Wu,1,2 Chenghua Zhang,3 Yuxin Tang,1,2 Weiwei Zhu,1 Wei Li,1,2 Kun Gao,1,4 Enchao Zhou,1,2,4 Xufang Wang1,4 1Department of Nephrology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China; 2Department of Clinical Medicine, The First School of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China; 3Department of Nephrology, Changzhou Hospital of Chinese Medicine, Changzhou, Jiangsu, People’s Republic of China; 4Department of Nephrology, Jiangsu University Key Laboratory of Tonifying Kidney and Anti-Senescence, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of ChinaCorrespondence: Xufang Wang, Department of Nephrology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China, Email [email protected] Enchao Zhou, Department of Nephrology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China, Email [email protected]: Mast cells (MCs) act as sentinels of the immune system and are involved in peritoneal fibrosis (PF), but the precise mechanism remains unclear. To investigate this, we focused on Astragalus membranaceus and Salvia miltiorrhiza (AS), a combination known for its anti-inflammatory properties. This study aimed to elucidate the effect of AS on MCs and its potential to alleviate PF.Methods: The major chemical components of AS were identified by UPLC-Q-TOF-MS/MS. Subsequently, we employed network pharmacology, molecular docking, and molecular dynamics simulations to investigate the key targets and mechanisms of AS in the treatment of PF. In vivo, a mouse PF model was established by daily intraperitoneal injection of high-glucose (4.25%) peritoneal dialysis fluid (PDF) for 4 weeks. Following AS treatment, the mechanism was explored through transcriptomics analysis, immunohistochemistry (IHC), immunofluorescence (IF), and Western blotting (WB). In vitro, an activated MC model was established by stimulating murine P815 mast cells with LPS. The activation of P815 cells was assessed using transcriptomics analysis, ELISA, and transmission electron microscopy (TEM). Finally, a fibrosis model was further generated by stimulating human peritoneal mesothelial cells (HMrSV5) with tryptase, and the effects of AS were assessed by WB.Results: UPLC-Q-TOF-MS/MS identified 40 active components in AS. Network pharmacology and molecular docking indicated favorable predicted binding affinities of the key constituents calycosin and tanshinone IIA to MAPK1 and NF-κB1, with binding energies ranging from − 9.1 to − 6.8 kcal/mol. In vivo experiments demonstrated that high-glucose PDF increased peritoneal tryptase levels by approximately 3.2-fold, whereas AS treatment reduced tryptase release by 71.5%, respectively (baseline-corrected). Correspondingly, AS significantly inhibited MC degranulation and suppressed activation of the PAR2/MAPK/NF-κB pathway. In vitro, AS decreased LPS-induced tryptase and TNF-α secretion in P815 cells by 71.6% and 58.2%, respectively, and attenuated tryptase-induced fibrosis in HMrSV5 cells, accompanied by reduced phosphorylation of MAPK/NF-κB signaling proteins.Conclusion: This study demonstrates that the MC tryptase/PAR2 signaling axis contributes to PDF-induced peritoneal fibrotic responses. AS alleviates PF at least in part by inhibiting tryptase-mediated MC-PMC crosstalk and downstream PAR2/MAPK/NF-κB activation. Mast cell activation causes peritoneal fibrosis, affected by Astragalus and Salvia.The diagram illustrates the process of mast cell activation leading to peritoneal fibrosis. At the top, Astragalus membranaceus and Salvia miltiorrhiza are shown as influencing factors. Below, an activated mast cell releases substances affecting peritoneal mesothelial cells. The central section depicts tryptase interacting with PAR2, leading to activation of pathways involving p-JNK, p-p38 and p-ERK1/2. This results in NF-kappa B activation, producing TNF-alpha and IL-1 beta, which contribute to peritoneal fibrosis. The sequence shows the progression from mesothelial cell interaction to fibrosis development.Keywords: Astragalus membranaceus and Salvia miltiorrhiza, mast cells, peritoneal mesothelial cells, peritoneal fibrosis, protease-activated receptor 2, tryptase