Guang-Yao Chen,1,2 Xiang-Yu Ji,3 Ying Li,4 Si-Si Zheng,5 Qi Jin,6 Qing-Wen Tao1,2 

1Department of TCM Rheumatology, China-Japan Friendship Hospital, Beijing, 100029, People’s Republic of China; 2Beijing Key Laboratory for Immune-Mediated Inflammatory Diseases, China-Japan Friendship Hospital, Beijing, 100029, People’s Republic of China; 3College of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan, 250355, People’s Republic of China; 4Graduate School, Beijing University of Chinese Medicine, Beijing, 100029, People’s Republic of China; 5Beijing Key Laboratory of Mental Disorders, National Clinical Research Center for Mental Disorders & National Center for Mental Disorders, Beijing Anding Hospital, Capital Medical University, Beijing, 100088, People’s Republic of China; 6Department of Dermatology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, 100010, People’s Republic of China

Correspondence: Qing-Wen Tao, Department of TCM Rheumatology, China-Japan Friendship Hospital, Beijing, People’s Republic of China, Tel +8613910528490, Email taoqg1@outlook.com

Objective: Total glycoside of peony (TGP) enhances methotrexate efficacy and attenuates its hepatotoxicity in rheumatoid arthritis, but the mechanisms remain unclear. This study investigates the mechanisms of TGP against methotrexate-induced liver injury through a network pharmacology-based approach.
Methods: A liver injury model was established in CD-1 mice by intraperitoneal injection of 20 mg/kg methotrexate. TGP and the positive control drug silybin were used to intervene in the methotrexate-induced liver injury model in mice. Serum ALT and AST activities, liver index test and histopathology was detected to evaluate the effects of the treatment on methotrexate-induced liver injury. Additionally, network pharmacology and serum metabolomics were employed to predict the mechanisms of TGP in treating methotrexate-induced liver injury. Experimental validation was conducted by RT-PCR, ELISA and Western blot.
Results: TGP effectively alleviated the liver index and pathological liver damage induced by methotrexate and reduced the liver injury markers, serum ALT and AST, showing effects comparable to those of the positive control drug silybin. Network pharmacology predicted that the key targets and key signaling pathways of TGP in treating methotrexate-induced liver injury are closely associated with inflammatory response. Furthermore, serum metabolomics and network pharmacology analysis indicated a close association between effects of TGP on methotrexate-induced liver injury and arachidonic acid pathway. Experimental validation results confirmed that the expression levels of IL-6, TNF and COX-2 in liver tissues were significantly elevated, with the activation of the PI3K/AKT, MAPK, and NFκB pathways. TGP intervention can reverse these changes to a certain extent.
Conclusion: TGP treatment effectively mitigates methotrexate-induced liver injury, and its mechanism is closely associated with the inhibition of hepatic inflammatory responses.

Keywords: total glucosides of paeony, methotrexate, liver injury, network pharmacology, serum metabolomics