Linlin Pan,1 Juhai Liu,1 Guowei Pang,2 Guirong Liu,1 Yuan Tian3,4 

1College of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan City, Shandong Province, People’s Republic of China; 2Acupuncture and Moxibustion Massage College, Shandong University of Traditional Chinese Medicine, Jinan City, Shandong Province, People’s Republic of China; 3Foreign Languages College, Shandong University of Traditional Chinese Medicine, Jinan City, Shandong Province, People’s Republic of China; 4First Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan City, Shandong Province, People’s Republic of China

Correspondence: Yuan Tian, Foreign Languages College, Shandong University of Traditional Chinese Medicine, No. 4655, Daxue Road, Changqing District, Jinan City, Shandong Province, 250355, People’s Republic of China, Tel +86-053189628253, Email tianyuan8612@163.com

Background: Metabolic dysfunction-associated steatohepatitis (MASH) is characterized primarily by hepatocyte lipoapoptosis and hepatic inflammation, frequently developing from overweight/obesity. To date, no specific therapeutics exist to reverse MASH. Although resmetirom has been approved in some regions, patients in many Asian countries, including China, still lack access to approved pharmacotherapy for MASH. Lipi Jiangzhuo decoction (LPJZD) is a promising traditional Chinese medicine formula for MASH. However, to date, there have been no comprehensive studies clarifying its potential mechanism of action. This study aims to elucidate the underlying mechanism of action of LPJZD in the treatment of MASH.
Materials and Methods: A MASH mouse model was established by feeding a high-fat diet and subjecting them to fatigue protocols and cold stress for 12 weeks. After treating MASH mice with LPJZD, biochemical assays were conducted to assess the efficacy of LPJZD in alleviating the MASH symptoms. In addition, the in vitro effects of LPJZD on MASH were evaluated using L-02 cells. Specifically, we analyzed the effect of LPJZD on endoplasmic reticulum (ER) stress, mitophagy, and ferroptosis by Western blot analysis, flow cytometry, immunofluorescence staining, and enzyme-linked immunosorbent assay.
Results: In vivo, LPJZD effectively improved the inflammatory response, reduced body weight and blood lipid levels, improved liver function, reduced liver lipid droplet accumulation, and ameliorated the pathological status of MASH mice. In vitro, LPJZD effectively inhibited ferroptosis by reducing ferrous ions and reactive oxygen species levels, increasing GPx4 protein expression, elevating glutathione levels, and ameliorating mitochondrial swelling and matrix thinning. Simultaneously, LPJZD activated mitophagy by increasing PINK1 and Parkin protein expression, augmenting mitophagosome number, and restoring mitochondrial membrane potential. Additionally, LPJZD suppressed ER stress by decreasing PERK protein expression. Notably, activation of ER stress using a PERK activator attenuated LPJZD’s effects on mitophagy activation and ferroptosis inhibition, inhibition of mitophagy via a PINK1 inhibitor diminished LPJZD’s anti-ferroptotic effect, and administration of a GPx4 inhibitor reduced LPJZD’s suppression of ferroptosis. Therefore, these results demonstrate that LPJZD ameliorates MASH by regulating the PERK/PINK1/GPx4 pathway.
Conclusion: LPJZD can improve MASH by regulating ER stress-mitophagy -ferroptosis axis in liver cells. The role of LPJZD in anti-inflammatory therapy provides new insights for the clinical prevention and treatment of MASH.

Keywords: Lipi Jiangzhuo decoction, metabolic dysfunction-associated steatohepatitis, endoplasmic reticulum stress, mitophagy, ferroptosis