College of Ethnic Medicine, Yunnan University of Chinese Medicine, Yunnan, Kunming, 650500, China.
College of Chinese Medicine, Yunnan University of Chinese Medicine, Yunnan, Kunming, 650500, China.
J Ethnopharmacol. 2024 Dec 5;335:118712. doi: 10.1016/j.jep.2024.118712. Epub 2024 Aug 22.
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 HO. 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.
雅积沙巴（YJSB）于 2008 年获得云南省食品药品监督管理局批准，以其抗炎、抗病毒和促凋亡特性而闻名，可有效治疗肝纤维化（HF）。然而，其作用机制尚不清楚。
本研究旨在探讨 YJSB 如何通过 TGF-β1/Smad 信号通路影响 HF，以作为减少 HF 的策略。
通过结扎胆总管建立 HF 小鼠模型，然后给予 YJSB 治疗。测量体质量和肝质量，并计算肝指数。使用比色法评估 ALT、AST、ALP、TBA 和 TBIL 血清水平。此外，通过 ELISA 分析肝匀浆中 PIIINP、Col-IV、LN、HA 和 Hyp 的水平，以及 TGF-β1 活性。用 H&E、Ag 和 Masson 三色染色的肝组织切片进行炎症和胶原及网状纤维积聚的组织学分析。这些研究旨在阐明 YJSB 对胆源性梗阻性 HF 小鼠的药效作用。通过 TGF-β1、P-Smad2L、P-Smad2C、P-Smad3L、P-Smad3C 和 Smad4 蛋白的免疫荧光检测，初步确定了 YJSB 的作用途径。体外实验包括 HO 诱导肝星状细胞（HSC-T6）激活。建立 HSC-T6 细胞损伤模型，用 CCK-8 测定确定 YJSB 的最佳浓度和治疗时间。用 TGF-β1/Smad 通路抑制剂 PFD 给药后，观察 YJSB 对 HSC-T6 细胞增殖的影响。用 ELISA 试剂盒测定细胞裂解物中 Col-III、α-SMA 和 Col-I 的含量，以评估 YJSB 对 HSC-T6 细胞胶原合成的影响。通过 Western blot 分析评估 TGF-β1/Smad 信号级联中的蛋白水平。
在 HF 小鼠模型中，YJSB 给药显著增加了体质量并降低了肝指数。同时，血清中 ALP、AST、ALT、TBA 和 TBIL 的浓度也升高。同样，在 HF 小鼠的肝匀浆中，HA、PIIINP、Col-IV、LN、Hyp 和 TGF-β1 的水平也升高。用 H&E、Ag 和 Masson 染色的组织学评估表明，肝组织损伤明显减轻。通过免疫荧光分析，发现 YJSB 下调了 TGF-β1、P-Smad2L、P-Smad2C 和 P-Smad3L 的表达，同时上调了 P-Smad3C 和 Smad4 蛋白的表达。进一步的研究表明，细胞培养液中α-SMA、Col-I 和 Col-III 的水平显著降低，提示胶原合成减少，对细胞损伤有保护作用。Western blot 分析表明，TGF-β1/Smad 通路抑制剂 PFD 与 YJSB 协同作用，增强了其对该通路的调节作用，降低了 TGF-β1、P-Smad2L、P-Smad2C、P-Smad3L 的水平，促进了 P-Smad3C 的表达。
YJSB 对 HF 具有药效作用，可增强肝功能，有效减轻胆管梗阻引起的损伤。YJSB 的作用机制可能涉及 TGF-β1/Smad 信号通路的调节。研究表明，YJSB 可能通过抑制 HSC-T6 的迁移、程序性细胞死亡和激活，从而抑制肝纤维化的进展。
