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Abstract
Background Biliary atresia (BA) is a severe neonatal hepatobiliary disease that leads to progressive liver fibrosis and liver failure. Hepatic stellate cells (HSCs) have been identified as the main contributors to liver fibrogenesis, with their activation playing a crucial role in fibrosis development. Yiqihuoxue prescription (YQHX), a traditional herbal formula, has demonstrated effective anti-fibrotic properties in multiple hepatic diseases. This study aims to investigate the function and underlying mechanisms of YQHX in HSCs activation and liver fibrogenesis in BA.
Methods We conducted a series of experiments using rhesus-rotavirus (RRV)-induced BA mice model and cultured HSCs. Neonatal mice were intraperitoneally injected with RRV within 12 hours of birth. Starting from day five, YQHX group received YQHX by oral gavage daily for 9 days. Pups were sacrificed on day 14 for tissue collection and analysis, and serum was collected to measure liver function. H&E and Masson’s trichrome staining were performed for the examination of histological changes. A fibrosis model was established by inducing HSCs activation with tgfβ, and the effects of YQHX on cellular ferroptosis and fibrosis were investigated. UPLC-HRMS was utilized to identify the chemical components in YQHX, and molecular docking analysis was performed between them and NCOA4.
Results We found that YQHX ameliorates hepatic injury and fibrosis in the RRV-induced BA mice model (IDDF2024-ABS-0308 Figure 1). In vitro experiments demonstrate that YQHX inhibits HSCs activation by suppressing their proliferation/migration and promoting apoptosis (IDDF2024-ABS-0308 Figure 2), which also occurs in the tgfβ-induced HSCs activation model (IDDF2024-ABS-0308 Figure 3, IDDF2024-ABS-0308 Figure 4). Mechanistically, we found that seven of the top ten compounds in YQHX exhibited molecular docking with NCOA4, a major regulator of ferritinophagy, thereby reducing the release of ferrous iron. We observed a decrease in NCOA4 expression with increasing YQHX concentration.YQHX enhanced the inhibition of activated HSCs after the knockdown of NCOA4 with lentivirus. Consequently, YQHX improved cellular iron overload and oxidative stress by inhibiting the expression of NCOA4, thereby suppressing the activation of HSCs (IDDF2024-ABS-0308 Figure 5).
Conclusions YQHX can ameliorate hepatic fibrosis by targeting NCOA4 to induce the ferritinophagy of activated HSCs, providing a promising therapeutic strategy for hepatic fibrosis in BA.