Rebalancing TGF-β/Smad7 signaling via Compound kushen injection in hepatic stellate cells protects against liver fibrosis and hepatocarcinogenesis

Background: Liver fibrosis and fibrosis-related hepatocarcinogenesis are a rising cause for morbidity and death worldwide. Although transforming growth factor-beta (TGF-beta) is a critical mediator of chronic liver fibrosis, targeting TGF-beta isoforms and receptors lead to unacceptable side effect. This study was designed to explore the antifibrotic effect of Compound kushen injection (CKI), an approved traditional Chinese medicine formula, via a therapeutic strategy of rebalancing TGF-beta/Smad7 signaling. Methods: A meta-analysis was performed to evaluate CKI intervention on viral hepatitis-induced fibrosis or cirrhosis in clinical randomized controlled trials (RCTs). Mice were given carbon tetrachloride (CCl4) injection or methionine-choline deficient (MCD) diet to induce liver fibrosis, followed by CKI treatment. We examined the expression of TGF-beta/Smad signaling and typical fibrosis-related genes in hepatic stellate cells (HSCs) and fibrotic liver tissues by qRT-PCR, Western blotting, RNA-seq, immunofluorescence, and immunohistochemistry. Results: Based on meta-analysis results, CKI improved the liver function and relieved liver fibrosis among patients. In our preclinical studies by using two mouse models, CKI treatment demonstrated promising antifibrotic effects and postponed hepatocarcinogenesis with improved liver function and histopathologic features. Mechanistically, we found that CKI inhibited HSCs activation by stabilizing the interaction of Smad7/TGF-beta R1 to rebalance Smad2/Smad3 signaling, and subsequently decreased the extracellular matrix formation. Importantly, Smad7 depletion abolished the antifibrotic effect of CKI in vivo and in vitro. Moreover, matrine, oxymatrine, sophocarpine, and oxysophocarpine were identified as material basis responsible for the antifibrosis effect of CKI. Conclusions: Our results unveil the approach of CKI in rebalancing TGF-beta/Smad7 signaling in HSCs to protect against hepatic fibrosis and hepatocarcinogenesis in both preclinical and clinical studies. Our study suggests that CKI can be a candidate for treatment of hepatic fibrosis and related oncogenesis.