Puerarin, isolated from Pueraria lobata (Willd.), protects against hepatotoxicity via specific inhibition of the TGF-β1/Smad signaling pathway, thereby leading to anti-fibrotic effect.
Recently, the TGF-β1/Smad signaling pathway has been investigated in the pathogenesis of hepatofibrosis, and pharmacological treatment of liver fibrosis targeted this pathway to determine its contribution to the inhibition of fibrotic development. Importantly, ethnopharmacology-derived Pueraria lobata has been reported to effectively reverse the fibrotic process in the liver. In the present study, we performed dimethylnitrosamine (DMN)-induced liver fibrosis in rats to assess the benefits of puerarin (PR), which was isolated from Pueraria lobata (Willd.), on ECM-derived hepatocytes associated with the TGF-β1/Smad pathway. Our results showed that the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN), type III precollagen (PCIII) and type IV collagen (CIV) were significantly reduced by PR treatment, while hepatic homogenates showed decreased levels of hydroxyproline (Hyp) and collagen I (Col I). Masson's trichrome staining indicated that the DMN-induced liver fibrosis was alleviated. In addition, the protein expression levels of transforming growth factor-β l (TGF-β l), smad2, smad3, α-SMA and TIMP-1 were downregulated specifically by PR treatment, whereas the protein expression levels of smad7 and MMP-1 were upregulated. Furthermore, we evaluated the PR-mediated inhibitory effect on TGF-β1-treated proliferation and activation in a rat liver stellate cell line (HSC-T6). These data resulted in inhibition of the cell growth of HSC-T6 in a dose-dependent manner and a reduction in TβRI, smad2 and smad3 expressed proteins in the presence of PR on TGF-β1-treated HSC-T6 cells, while smad7 levels were downregulated. Taken together, these findings identify a unique effect for PR-regulation of the TGF-β1/Smad pathway in blocking fibrotic development and provide a promising strategy for hepatofibrosis treatment.
Xu L
，Zheng N
，He Q
，Li R
，Zhang K
，Liang T
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Experimental study on H(2)O(2) activation of HSC-T6 and hepatic fibrosis in cholestatic mice by "Yajieshaba".
Yajieshaba (YJSB), approved by the Yunnan Provincial Food and Drug Administration in 2008, are known for their anti-inflammatory, antiviral, and pro-apoptotic properties, effectively treating Hepatic fibrosis (HF). However, its mechanism of action remains unclear.
The objective of this investigation is to explore how YJSB influences the TGF-β1/Smad signaling pathway as a strategy for reducing HF.
The establishment of a HF model in mice involved ligation of the common bile duct, followed by administration of YJSB. Body and liver weights were measured, and the liver index calculated. Serum levels of ALT, AST, ALP, TBA, and TBIL were assessed using colorimetric methods. Additionally, liver homogenates were analyzed for PIIINP, Col-IV, LN, HA, and Hyp, as well as TGF-β1 activity, using ELISA. Histological analyses of liver sections, stained with H&E, Ag, and Masson's trichrome, were performed to examine inflammation and the accumulation of collagen and reticular fibers. These studies aimed to elucidate the pharmacodynamic effects of YJSB on HF in mice with bile duct obstruction. The target pathways of YJSB were preliminarily identified through immunofluorescence detection of TGF-β1, P-Smad2L, P-Smad2C, P-Smad3L, P-Smad3C, and Smad4 proteins. In vitro experiments included the induction of hepatic stellate cell (HSC-T6) activation by H2O2. A cell injury model was established for HSC-T6, and the CCK-8 assay was used to determine the optimal YJSB concentration and treatment duration. After pirfenidone (PFD) administration, which inhibits the TGF-β1/Smad pathway, the effects of YJSB on HSC-T6 cell proliferation were observed. ELISA assays quantified Col-III, α-SMA, and Col-I in cell lysates to assess YJSB's impact on collagen synthesis in HSC-T6 cells. Western blot analysis was performed to assess the protein levels within the TGF-β1/Smad signaling cascade.
In the HF mouse model, administration of YJSB notably augmented the body weight and reduced the liver index. Concurrently, there was an elevation in serum concentrations of ALP, AST, ALT, TBA, and TBIL. Similarly, in the liver homogenates of HF mice, increases were observed in the levels of HA, PIIINP, Col-IV, LN, Hyp, and TGF-β1. Histological assessments using H&E, Ag, and Masson stains indicated a substantial diminution in liver tissue damage. Through immunofluorescence analysis, it was discerned that YJSB modulated the expression of TGF-β1, P-Smad2L, P-Smad2C, and P-Smad3L downwards, while elevating P-Smad3C and Smad4 protein expressions. Additional investigations revealed a significant reduction in α-SMA, Col-I, and Col-III levels in cell culture fluids, suggesting a decrease in collagen synthesis and a protective role against cellular damage. Western blot analyses demonstrated that the TGF-β1/Smad pathway inhibitor, PFD, acted in synergy with YJSB, enhancing its regulatory effects on this pathway, decreasing levels of TGF-β1, P-Smad2L, P-Smad2C, P-Smad3L, and promoting the expression of P-Smad3C.
YJSB demonstrates a pharmacodynamic effect against HF, enhancing liver functionality and effectively mitigating the damage associated with bile duct obstruction. The proposed action mechanism of YJSB involves modulation of the TGF-β1/Smad signaling pathway. Research indicates that YJSB might play a role in suppressing the movement, programmed cell death, and activation of HSC-T6, potentially decelerating the advancement of hepatic fibrosis.
Bai Y
，Liu F
，Luo S
，Wan Y
，Zhang L
，Wu X
，Chen Q
，Xie Y
，Guo P
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Methyl ferulic acid attenuates liver fibrosis and hepatic stellate cell activation through the TGF-β1/Smad and NOX4/ROS pathways.
Liver fibrosis is a pathological wound-healing response caused by chronic liver damage due to a virus, autoimmune disorder, or drugs. Hepatic stellate cells (HSCs) play an essential role in the pathogenesis of liver fibrosis. Methyl ferulic acid (MFA), a biologically active monomer, has a protective effect on liver injury. However, the effects and roles of MFA in liver fibrosis remain unknown. The purpose of the current study was to investigate the effect of MFA on hepatic fibrosis and the underlying mechanisms. Human hepatic stellate LX-2 cells were exposed to 5 μg/L TGF-β1 for 48 h to stimulate liver fibrosis in vitro. Using MTT, RT-PCR and Western blot analysis, we revealed that MFA significantly inhibited the proliferation of LX-2 cells as well as decreased the expressions of α-SMA and type I collagen in LX-2 cells. SD rats were fed with ethanol, and this combined with the intraperitoneal injection of CCl4 induced liver fibrosis in vivo. We found that the administration of MFA markedly decreased the levels of hyaluronic acid (HA), procollagen type III (PC-III), type IV collagen (CIV) and laminin (LN) in the serum, inhibited the expression of α-smooth muscle actin (α-SMA) as well as type I and type III collagen, and up-regulated the ratio of MMP-2/TIMP-1 in rats. The antifibrotic effects of MFA were also evaluated by H&E staining and Masson's trichrome staining. In addition, further studies suggested that this protection by MFA from liver fibrosis was possibly related to the inhibition of TGF-β1/Smad and NOX4/ROS signalling. In conclusion, our results demonstrate that MFA attenuated liver fibrosis and hepatic stellate cell activation by inhibiting the TGF-β1/Smad and NOX4/ROS signalling pathways.
Cheng Q
，Li C
，Yang CF
，Zhong YJ
，Wu D
，Shi L
，Chen L
，Li YW
，Li L
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