背景:Yajeshaba(YJSB),2008年由云南省食品药品监督管理局批准,以其抗炎作用而闻名,抗病毒,和促凋亡特性,有效治疗肝纤维化(HF)。然而,其作用机制尚不清楚。
目的:本研究的目的是探讨YJSB如何影响TGF-β1/Smad信号通路作为减少HF的策略。
方法:建立胆总管结扎小鼠HF模型,其次是YJSB的管理。测量体重和肝脏重量,计算肝脏指数。血清ALT水平,AST,ALP,TBA,和TBIL使用比色法进行评估。此外,分析肝脏匀浆的PIIINP,Col-IV,LN,HA,Hyp,以及TGF-β1活性,使用ELISA。肝脏切片的组织学分析,染色H&E,Ag,和马森的三色,进行检查炎症和胶原和网状纤维的积累。这些研究旨在阐明YJSB对胆管阻塞小鼠HF的药效学作用。通过免疫荧光检测TGF-β1、P-Smad2L、P-Smad2C,P-Smad3L,P-Smad3C,和Smad4蛋白。体外实验包括H2O2诱导肝星状细胞(HSC-T6)活化。建立HSC-T6的细胞损伤模型,并使用CCK-8测定法确定最佳YJSB浓度和治疗持续时间。吡非尼酮(PFD)给药后,抑制TGF-β1/Smad通路,观察YJSB对HSC-T6细胞增殖的影响。ELISA定量检测Col-III,α-SMA,和Col-I在细胞裂解物中评估YJSB对HSC-T6细胞胶原合成的影响。进行蛋白质印迹分析以评估TGF-β1/Smad信号传导级联内的蛋白质水平。
结果:在HF小鼠模型中,YJSB的给药显著增加了体重并降低了肝脏指数。同时,血清ALP浓度升高,AST,ALT,TBA,TBIL。同样,在HF小鼠的肝脏匀浆中,观察到HA水平增加,PIIINP,Col-IV,LN,Hyp,和TGF-β1。使用H&E进行组织学评估,Ag,和Masson染色表明肝组织损伤显著减少。通过免疫荧光分析,发现YJSB调节TGF-β1,P-Smad2L的表达,P-Smad2C,P-Smad3L向下,同时升高P-Smad3C和Smad4蛋白表达。其他调查显示,α-SMA显著减少,Col-I,和细胞培养液中的Col-III水平,表明胶原蛋白合成的减少和对细胞损伤的保护作用。Westernblot分析表明TGF-β1/Smad通路抑制剂,PFD,与YJSB协同行动,增强其对这一途径的调节作用,降低TGF-β1,P-Smad2L,P-Smad2C,P-Smad3L,并促进P-Smad3C的表达。
结论:YJSB具有抗HF的药效学作用,增强肝功能并有效减轻与胆管阻塞相关的损害。YJSB的作用机制涉及TGF-β1/Smad信号通路的调节。研究表明,YJSB可能在抑制运动中发挥作用,程序性细胞死亡,和HSC-T6的激活,可能减缓肝纤维化的进展。
BACKGROUND: 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.
OBJECTIVE: The objective of this investigation is to explore how YJSB influences the TGF-β1/Smad signaling pathway as a strategy for reducing HF.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.