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三种温阳健脾汤对非酒精性脂肪肝细胞增殖及凋亡的影响

[Effects of three Wenyang Jianpi Tang on cell proliferation and apoptosis of nonalcoholic fatty liver cells].

作者信息

Yang Jia-Yao, Tao Dong-Qing, Liu Song, Zhang Shu, Ma Wei, Shi Zhao-Hong

机构信息

Wuhan Integrated Traditional Chinese Medicine & Western Medicine Hospital(Wuhan No.1 Hospital), Wuhan 430022, China.

Zhongshan Hospital, Medical College of Wuhan University, Wuhan 430026, China.

出版信息

Zhongguo Zhong Yao Za Zhi. 2017 Apr;42(8):1591-1596. doi: 10.19540/j.cnki.cjcmm.20170222.010.

Abstract

To investigate the effects of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang on the cell proliferation and apoptosis of nonalcoholic fatty liver cells through the nonalcoholic fatty liver cell model established by inducing L02 cells with oleic acid. Different concentrations of oleic acid were added into L02 cells to induce the nonalcoholic fatty liver cell model. Oil red O staining was used to observe fatty droplets of fatty liver cells. Automatic biochemical analyzer was used to detect the levels of aspartic transaminase(AST), alanine aminotransferase(ALT), total cholesterol(TC), and triglyceride(TG) in the cell supernatants. There were five groups, namely normal group, model group, model and Sijunzi Tang group, model and Lizhong Tang group, and model and Fuzi Lizhong Tang group. The cell proliferation and apoptosis of the five groups were detected by MTT colorimetry test and flow cytometer. The expressions of PCNA, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax and Bcl-2 proteins of the five groups were detected by Western blot. The oil red O staining results showed that the optimum concentration of oleic acid that was used to induce nonalcoholic fatty liver cell models was 80 mg•L-1. The levels of AST, ALT, TC and TG in the nonalcoholic fatty liver cell supernatants were higher than that in normal liver cell supernatants(P<0.01). MTT colorimetry test and flow cytometer results showed that all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells(P<0.01). And Fuzi Lizhong Tang showed the best effect. Western blot results showed that Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could down-regulate the expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax proteins, and up-regulate the expressions of PCNA and Bcl-2 proteins of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. In conclusion, all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. The pharmacodynamic mechanism may be related to the expressions of key factors in pathways related with proliferation and apoptosis mediated by the three decoctions.

摘要

通过油酸诱导L02细胞建立非酒精性脂肪肝细胞模型,探讨四君子汤、理中汤和附子理中汤对非酒精性脂肪肝细胞增殖和凋亡的影响。向L02细胞中加入不同浓度的油酸以诱导非酒精性脂肪肝细胞模型。采用油红O染色观察脂肪肝细胞中的脂滴。使用自动生化分析仪检测细胞上清液中天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、总胆固醇(TC)和甘油三酯(TG)的水平。分为五组,即正常组、模型组、模型加四君子汤组、模型加理中汤组和模型加附子理中汤组。通过MTT比色法检测和流式细胞仪检测五组细胞的增殖和凋亡情况。通过蛋白质印迹法检测五组PCNA、裂解的caspase-3、裂解的caspase-8、裂解的caspase-9、Bax和Bcl-2蛋白的表达。油红O染色结果显示,用于诱导非酒精性脂肪肝细胞模型的油酸最佳浓度为80mg•L-1。非酒精性脂肪肝细胞上清液中AST、ALT、TC和TG的水平高于正常肝细胞上清液(P<0.01)。MTT比色法检测和流式细胞仪检测结果显示,四君子汤、理中汤和附子理中汤均能有效促进非酒精性脂肪肝细胞的增殖,并抑制其细胞凋亡(P<0.01)。其中附子理中汤效果最佳。蛋白质印迹法结果显示,四君子汤、理中汤和附子理中汤均可下调非酒精性脂肪肝细胞中裂解的caspase-3、裂解的caspase-8、裂解的caspase-9和Bax蛋白的表达,并上调PCNA和Bcl-2蛋白的表达。其中附子理中汤效果最佳。综上所述,四君子汤、理中汤和附子理中汤均能有效促进非酒精性脂肪肝细胞的增殖,并抑制其细胞凋亡。其中附子理中汤效果最佳。其药效机制可能与三方剂介导的增殖和凋亡相关通路中关键因子的表达有关。

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