Experimental Study of YaJieShaBa Antialcoholic Hepatic Fibrosis Through TGF-β1/Smad Signaling Pathway

This study aimed to clarify the pharmacodynamic effects of YaJieShaBa (YJSB) against alcoholic hepatic fibrosis (HF) and elucidate its mechanism in regulating the transforming growth factor-β1 (TGF-β1)/Smad pathway.

Induce the alcoholic HF model in rats using 56% ethanol (10 mL/kg). The pharmacological efficacy of YJSB in combating liver fibrosis was evaluated through comprehensive assessments of key indicators: body weight, liver mass and index, biochemical liver function parameters (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]), liver fibrosis biomarkers (type Ⅲ procollagen amino-terminal propeptide [PⅢNP], type-Ⅳ collagen (COL-Ⅳ), laminin (LN), and hyaluronic acid [HA]), serum hydroxyproline (Hyp) and TGF-β1 levels, hepatocyte homogenate levels of COL-I, COL-Ⅲ, and α-smooth muscle actin (α-SMA), along with histopathological changes observed in liver tissue via hematoxylin and eosin (H&E) staining, Ag staining, and Masson staining. Pathway-focused qPCR array analysis was used to detect the expression of 72 genes related to signaling pathways such as TGF-β1, Keap1-Nrf2, and TLR4/MyD88 in liver tissue from the control group, model group, and YJSB group, identifying differentially expressed genes (DEGs) and key signaling pathways between the model group and the YJSB treatment group. Finally, based on the results of the pathway-focused qPCR array, the mechanism of action of YJSB against HF was validated using ELISA, WB, and immunofluorescence methods. Concurrently, TGF-β1 receptor inhibitors were employed in vitro experiments to determine whether YJSB could still provide additional protective effects when the TGF-β1/Smad pathway was maximally blocked. Furthermore, after confirming that YJSB could inhibit TGF-β1-induced activation, a rescue experiment was conducted by adding exogenous TGF-β1 to observe whether it could reverse the inhibitory effects of YJSB.

YJSB administration significantly increased body mass and decreased liver index in alcoholic HF rats. Serum levels of AST, ALT, PⅢNP, COL-Ⅳ, LN, HA, Hyp, and TGF-β1 were significantly reduced, as were the levels of COL-I, Ⅲ, and α-SMA in liver homogenates. Histological analyses, including H&E, Ag, and Masson staining, revealed a significant reduction in liver damage. Pathway-focused qPCR array results showed that, compared with the blank group, 65 genes were upregulated and seven genes were downregulated in the model group, among which the relative expression levels of 40 genes were statistically significant (expression change factor ≥ 1 and p  < 0.05). Compared with the model group, 68 genes were downregulated, and four genes were upregulated in the YJSB group, with 34 genes showing statistically significant relative expression levels (fold change [FC] ≥ 2 and p  < 0.05). The DEGs were primarily enriched in the TGF-β1 signaling pathway. Additionally, YJSB reduced the levels of inflammatory factors IL-1β, TNF-α, IL-6, and IL-8 in liver tissue homogenates while increasing SOD, GSH-Px, and catalase (CAT) levels and decreasing MDA and ROS levels. Western blotting results showed that YJSB downregulated the expression levels of TGF-β1, Smad2, Smad3, P-Smad2, and P-Smad3 in the liver. Immunofluorescence results indicated that YJSB downregulated the expression levels of Smad4 in hepatocyte nuclei. In vitro experiments demonstrated that the mechanism of YJSB involves primarily inhibiting TGF-βR1 receptor activation, effectively downregulating P-Smad2/3 protein levels, as validated by the TGF-βR1 inhibitor LY2157299. Concurrently, in rescue experiments, the inhibitory effect of YJSB on P-Smad2/3 protein was partially reversed by exogenous TGF-β1, indicating that YJSB's antifibrotic action is highly correlated with the TGF-β1/Smad pathway.

YJSB effectively inhibits the inflammatory and oxidative stress-related cascade by regulating the TGF-β1/Smad signaling pathway (TSSP), thus suppressing the progression of alcoholic HF. This demonstrates that YJSB possesses potential for combating alcoholic HF in animal models, providing experimental evidence for its subsequent research and clinical application.