A characterized saponin extract of Panax japonicus suppresses hepatocyte EMT and HSC activation in vitro and CCl4-provoked liver fibrosis in mice: Roles of its modulatory effects on the Akt/GSK3β/Nrf2 cascade,Phytomedicine

当前位置： X-MOL 学术 › Phytomedicine › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

A characterized saponin extract of Panax japonicus suppresses hepatocyte EMT and HSC activation in vitro and CCl4-provoked liver fibrosis in mice: Roles of its modulatory effects on the Akt/GSK3β/Nrf2 cascade
Phytomedicine ( IF 7.9 ) Pub Date : 2021-09-11 , DOI: 10.1016/j.phymed.2021.153746
Chenxi Dai ₁ , Arslan Yusuf ₁ , Hui Sun ₁ , Guangwen Shu ₁ , Xukun Deng ₁

Affiliation

Background and purpose

Liver fibrosis constitutes a pathologic condition resulting in a series of advanced liver diseases. Oleanane-type saponins are distinctive active constituents in the medicinal plant Panax japonicus C. A. Mey (P. japonicus). Herein, we assessed protective effects of a characterized saponin extract of rhizomes of P. japonicus (SEPJ) on hepatocyte EMT and HSC activation in vitro and liver fibrosis in mice. We also investigated molecular mechanisms underlying the hepatoprotective activity of SEPJ.

Methods

EMT of AML-12 hepatocytes was evaluated by observing morphology of cells and quantifying EMT marker proteins. Activation of LX-2 HSCs was assessed via scratch assay, transwell assay, and EdU-incorporation assay, and by quantifying activation marker proteins. Liver fibrosis in mice was evaluated by HE, SR, and Masson staining, and by measuring related serum indicators. Immunoblotting and RT-PCR were performed to study mechanisms underlying the action of SEPJ.

Results

SEPJ inhibited TGF-β-induced EMT in AML-12 hepatocytes and activation of LX-2 HSCs. SEPJ elevated Akt phosphorylation at Ser473 and GSK3β phosphorylation at Ser9 in these cells, giving rise to a descent of the catalytic activity of GSK3β. These events increased levels of both total and nuclear Nrf2 protein and upregulated expressions of Nrf2-responsive antioxidative genes. In addition, enhanced phosphorylation of Akt and GSK3β acted upstream of SEPJ-mediated activation of Nrf2. Knockdown of Nrf2 or inhibition of Akt diminished the protective activity of SEPJ against TGF-β in both AML-12 and LX-2 cells. Our further in vivo experiments revealed that SEPJ imposed a considerable alleviation on CCl₄-provoked mouse liver fibrosis. Moreover, hepatic Akt/GSK3β/Nrf2 cascade were potentiated by SEPJ. Taken together, our results unveiled that SEPJ exerted protective effects against fibrogenic cytokine TGF-β in vitro and ameliorated liver fibrosis in mice. Mechanistically, SEPJ regulated the Akt/GSK3β/Nrf2 signaling which subsequently enhanced intracellular antioxidative capacity.

Conclusions

SEPJ inhibits hepatocyte EMT and HSC activation in vitro and alleviates liver fibrosis in mice. Modulation of the Akt/GSK3β/Nrf2 cascade attributes to its hepatoprotective effects. Our findings support a possible application of SEPJ in the control of liver fibrosis.

中文翻译：

人参皂苷提取物抑制体外肝细胞 EMT 和 HSC 活化以及 CCl4 诱发的小鼠肝纤维化：其对 Akt/GSK3β/Nrf2 级联反应的调节作用

背景和目的

肝纤维化是导致一系列晚期肝病的病理状态。齐墩果型皂苷是药用植物Panax japonicus CA Mey ( P. japonicus )中独特的活性成分。在此，我们评估了具有特征的P. japonicus (SEPJ)根茎皂苷提取物对体外肝细胞 EMT 和 HSC 活化以及小鼠肝纤维化的保护作用。我们还研究了 SEPJ 保肝活性的分子机制。

方法

通过观察细胞形态和量化 EMT 标记蛋白来评估 AML-12 肝细胞的 EMT。LX-2 HSCs 的激活通过划痕试验、transwell 试验和 EdU 掺入试验进行评估，并通过量化激活标记蛋白。小鼠肝纤维化通过HE、SR、Masson染色及相关血清指标的测定进行评价。进行免疫印迹和 RT-PCR 以研究 SEPJ 作用的潜在机制。

结果

SEPJ 抑制 AML-12 肝细胞中 TGF-β 诱导的 EMT 和 LX-2 HSC 的激活。SEPJ 提高了这些细胞中 Ser473 的 Akt 磷酸化和 Ser9 的 GSK3β 磷酸化，导致 GSK3β 的催化活性下降。这些事件增加了总 Nrf2 蛋白和核 Nrf2 蛋白的水平，并上调了 Nrf2 反应性抗氧化基因的表达。此外，Akt 和 GSK3β 的增强磷酸化作用于 SEPJ 介导的 Nrf2 激活的上游。Nrf2 的敲低或 Akt 的抑制降低了 SEPJ 对 AML-12 和 LX-2 细胞中 TGF-β 的保护活性。我们进一步的体内实验表明，SEPJ 对 CCl ₄施加了相当大的缓解- 诱发小鼠肝纤维化。此外，SEPJ 增强了肝脏 Akt/GSK3β/Nrf2 级联反应。总之，我们的结果表明，SEPJ在体外对纤维化细胞因子 TGF-β 发挥保护作用，并改善小鼠的肝纤维化。从机制上讲，SEPJ 调节 Akt/GSK3β/Nrf2 信号，从而增强细胞内抗氧化能力。