Weixin Hou,1,2 Peng Fang,3 Jiajun Liang,4 Xiaoyi Wei,5 Chongyang Ma,6,7 Yanbin Gao,8,9 Qiuyun Zhang,6,7,* Jingnan Li1,2,* 

1Department of Gastroenterology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, People’s Republic of China; 2Key Laboratory of Gut Microbiota Translational Medicine Research, Chinese Academy of Medical Sciences, Beijing, People’s Republic of China; 3Department of Infectious Diseases, First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang Province, People’s Republic of China; 4Department of Hepatology, Southern Medical University Hospital of Integrated Traditional Chinese and Western Medicine, Guangzhou, Guangdong province, People’s Republic of China; 5Office of Science and Technology Administration, Beijing Friendship Hospital, Capital Medical University, Beijing, People’s Republic of China; 6Department of Hepatology, School of Traditional Chinese Medicine, Capital Medical University, Beijing, People’s Republic of China; 7Department of Hepatology, Beijing Key Laboratory of Traditional Chinese Medicine Collateral Disease Theory Research, Capital Medical University, Beijing, People’s Republic of China; 8Department of Endocrinology, School of Traditional Chinese Medicine, Capital Medical University, Beijing, People’s Republic of China; 9Department of Endocrinology, Beijing Key Laboratory of Traditional Chinese Medicine Collateral Disease Theory Research, Capital Medical University, Beijing, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Qiuyun Zhang, Department of Hepatology, School of Traditional Chinese Medicine, Capital Medical University, No. 10 Youanmenwai, Xitoutiao, Fengtai District, Beijing, 100069, People’s Republic of China, Tel +86 10 8391 1638, Email 19970059@ccmu.edu.cn Jingnan Li, Department of Gastroenterology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, No. 1 Shuaifuyuan, Dongcheng District, Beijing, 100730, People’s Republic of China, Tel +86 10 6915 5017, Email 13240937099@163.com

Background: Acute-on-chronic liver failure (ACLF) is a global intractable disease. HMGB1-induced hepatocyte pyroptosis expanding inflammatory responses contributes to the pathogenesis of ACLF. The JDNW formula (JDNWF) has a significant clinical effect on ACLF, but its hepatoprotective mechanisms remain elusive.
Purpose: To explore the potential molecular mechanisms of the JDNWF in ACLF by HMGB1-induced hepatocyte pyroptosis.
Methods: Rats were divided into normal, ACLF, Caspase-1 inhibitor, HMGB1 inhibitor, JDNW, JDNW+Caspase-1 inhibitor and JDNW+HMGB1 inhibitor groups. The ACLF rat model was established by 40% carbon tetrachloride-induced liver fibrosis, followed by intraperitoneal injection of D-galactosamine and lipopolysaccharide. The liver function, coagulation function, liver pathological damage and ultrastructural changes of hepatocytes were evaluated. Triple-immunostaining of active Caspase-1, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and albumin were performed to evaluate the percentage of pyroptotic hepatocytes. Western blot, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (RT-qPCR) were used to analyze the expressions of key genes and proteins in HMGB1-induced pyroptosis pathways and the level of inflammatory factors.
Results: The JDNWF improved liver function, coagulation function and liver pathological damage, reduced the percentage of pyroptotic hepatocytes and inflammatory responses, and down-regulated the expressions of key genes and proteins in the HMGB1-induced pyroptosis pathways in ACLF rats. The effect of the JDNWF was better than those of HMGB1 inhibitor (glycyrrhizin) and Caspase-1 inhibitor (VX-765). Compared with glycyrrhizin or VX-765, there were no significant differences in the above indicators after the JDNWF in combination with glycyrrhizin or VX-765. These results indicated that the JDNWF inhibited hepatocyte pyroptosis and liver inflammation in ACLF rats through the HMGB1-induced pyroptosis pathways.
Conclusion: The JDNWF protects the livers of ACLF rats by inhibiting HMGB1-induced hepatocyte pyroptosis reducing inflammatory responses, suggesting that HMGB1-induced hepatocyte pyroptosis may be a potential therapeutic target of ACLF.

Keywords: traditional Chinese medicine, ACLF treatment, HMGB1, pyroptosis, inflammatory response, pharmacological effect