Injinoryeong-San attenuates metabolic dysfunction-associated steatohepatitis via regulation of YAP/TAZ-signaling pathway.

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Citation abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Injinoryeong-san (IJO), known as Yinchen-Wuling Powder in Chinese medicine, is a traditional herbal formula comprising six herbal medicinal plants, including Atractylodes macrocephala Koidz., Polyporus umbellatus (Pers.) Fries, Poria cocos (Schw.) Wolf, Cinnamomum cassia Presl, Alisma orientale (Sam.) Juz., and Artemisia capillaris Thunb. This formulation has been used in East Asian countries for centuries to treat liver-related diseases; however, there is currently no direct evidence elucidating its pharmacological effects against metabolic dysfunction-associated steatohepatitis (MASH).

AIM OF THE STUDY: This study aimed to investigate the pharmacological effects and underlying mechanisms of IJO in Western diet (WD)-induced MASH mouse model and in an in vitro human hepatocyte cell line model, with particular focus on the Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ)-signaling pathway.

MATERIAL AND METHODS: The MASH condition was established by administering a WD diet for 8 weeks. To assess the pharmacological properties of IJO, comprehensive indicators for lipid metabolism, oxidative stress, inflammation, hepatic fibrosis, and apoptosis signals were determined in the liver tissue and serum samples. To further validate the underlying mechanism, in vitro experiments were conducted using the human hepatocyte cell line, Huh7 cells.

RESULTS: IJO administration significantly improved serum levels of alanine aminotransferase and lactate dehydrogenase (P < 0.05 or 0.01), as well as histopathological analysis. Lipid accumulation by Oil Red O staining, and hepatic triglyceride and total cholesterol were markedly reduced (P < 0.05 or 0.01). IJO also normalized key regulators of lipid metabolism proteins, phospho-Adenosine monophosphate-activated protein kinase-α activity, Fatty acid synthase, and mature/precursor Sterol regulatory protein 1c ratio (p < 0.05 to 0.001). Oxidative stress markers, including 4-hydroxyneal signals, nitric oxide, reactive oxygen species, and lipid peroxidation, were enhanced in WD-fed liver tissue (P < 0.05 or 0.01). However, IJO significantly decreased these markers by recovering total glutathione, superoxide dismutase activities, and catalase activities (P < 0.05 to 0.001). IJO remarkably decreased pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6, but increased IL-10 in hepatic protein levels (P < 0.05 or 0.01). Furthermore, Toll-like receptor-4 and Nuclear factor kappa B activation were also notably attenuated by IJO by increases of NF-κB inhibitor α (P < 0.05 to 0.001). Fibrotic alterations were alleviated by reducing hepatic collagen deposition, extracellular matrix accumulation, and TGF-β1 expression (P < 0.05). Hepatic apoptosis was also significantly decreased as indicated by reduced TUNEL-positive cells, cytochrome c release, and caspase-3 activity (P < 0.05 or 0.01). Mechanistically, IJO suppressed nuclear translocation of YAP and TAZ (WWTR1) in both mouse liver tissues and Huh7 cells under MASH conditions. In addition, IJO significantly inhibited human YAP1 gene promoter activity (P < 0.01). Notably, major chemical constituents of IJO, including Alisol B, Cinnamic acid, and Scoparone, exhibited inhibitory effects on YAP/TAZ nuclear translocation at the cellular level.

CONCLUSION: IJO exhibits significant therapeutic potential against MASH by simultaneously targeting key pathological processes, including lipid dysregulation, oxidative stress, inflammation, fibrosis, and apoptosis. These multi-targeted effects are mechanistically linked to suppression of the YAP/TAZ signaling pathway, suggesting IJO as a promising candidate for further development as a hepatoprotective agent.