Ping Na Zhang,1,* Jing Yi Tang,1,* Ke Zhen Yang,2,* Qi Yan Zheng,1 Zhao Cheng Dong,1 Yun Ling Geng,1 Yu Ning Liu,1 Wei Jing Liu1 1Renal Research Institution of Beijing University of Chinese Medicine, and Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing, Peopleâs Republic of China; 2School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing, Peopleâs Republic of China*These authors contributed equally to this workCorrespondence: Yu Ning Liu; Wei Jing Liu, Renal Research Institution of Beijing University of Chinese Medicine, and Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing, Peopleâs Republic of China, Tel/Fax +86-10-84013190, Email liuyn2021@126.com; liuweijing-1977@hotmail.comBackground: Triptolide, a major active ingredient isolated from Tripterygium wilfordii Hook f., is effective in the treatment of membranous nephropathy (MN); however, its pharmacological mechanism of action has not yet been clarified. We applied an approach that integrated network pharmacology and experimental validation to systemically reveal the molecular mechanism of triptolide in the treatment of MN.Methods: First, potential targets of triptolide and the MN-related targets were collected from publicly available database. Then, based on a proteinâprotein interaction network as well as GO and KEGG pathway enrichment analyses, we constructed target-pathway networks to unravel therapeutic targets and pathways. Moreover, molecular docking was applied to validate the interactions between the triptolide and hub targets. Finally, we induced passive Heymann nephritis (PHN) rat models and validated the possible molecular mechanisms of triptolide against MN.Results: The network pharmacology results showed that 118 intersected targets were identified for triptolide against MN, including mTOR, STAT3, CASP3, EGFR and AKT1. Based on enrichment analysis, signaling pathways such as PI3K/AKT, MAKP, Ras and Rap1 were involved in triptolide treatment of MN. Furthermore, molecular docking confirmed that triptolide could bind with high affinity to the PIK3R1, AKT1 and mTOR, respectively. Then, in vivo experiments indicated that triptolide can reduce 24 h urine protein (P < 0.01) and protect against renal damage in PHN. Serum albumin level was significantly increased and total cholesterol, triglycerides, and low-density lipoprotein levels were decreased by triptolide (P < 0.05). Compared with PHN group, triptolide treatment regulated the PI3K/AKT/mTOR pathway according to Western blot analyses.Conclusion: Triptolide could exert antiproteinuric and renoprotective effects in PHN. The therapeutic mechanism of triptolide may be associated with the regulation of PI3K/AKT/mTOR signaling pathway. This study demonstrates the pharmacological mechanism of triptolide in the treatment of MN and provides scientific evidence for basic and clinical research.Graphical Abstract: Keywords: membranous nephropathy, network pharmacology, triptolide, molecular docking, experiment verification