连翘酯苷A对肝星状细胞增殖及凋亡的影响

Effects of Forsythiaside A on Proliferation and Apoptosis of Hepatic Stellate Cells

目的 研究连翘酯苷A(FA)对肝星状细胞增殖活化及凋亡的影响。方法 以人肝星状细胞(LX-2)作为实验对象,采用血小板衍生生长因子(PDGF)-BB模拟肝纤维化微环境,实验分为对照组、PDGF-BB组、FA组、PDGF-BB+FA组。采用细胞计数试剂盒(CCK-8)检测LX-2细胞活力;采用油红O检测细胞内脂滴的情况;采用免疫荧光染色检测细胞内α平滑肌肌动蛋白(α-SMA)的表达;采用蛋白质印迹技术(Western Blot)检测纤维化相关标志蛋白[α-SMA、α1-I型胶原蛋白(COL1A1)]和凋亡相关蛋白[B淋巴细胞瘤-2蛋白(BCL-2)、剪切的-腺苷二磷酸核糖聚合酶(cleaved-PARP)、BCL-2相关X蛋白(BAX)、剪切的-半胱氨酸天冬氨酸蛋白酶3(cleaved-CASPASE-3)]的表达;采用流式细胞仪检测细胞凋亡。结果 FA处理后LX-2细胞活力明显下降,且呈剂量依赖性;油红O实验结果显示,与PDGF-BB组比较,FA组细胞内脂滴数量较多;免疫荧光染色结果显示,与PDGF-BB组相比,FA组细胞内α-SMA荧光斑点的数量减少;WesternBlot实验结果显示,FA组α-SMA、COL1A1、BCL-2蛋白表达减少,cleaved-PARP、BAX、cleaved-CASPASE-3蛋白表达增加。结论 FA可抑制肝星状细胞的增殖、活化,促进凋亡,改善PDGF-BB诱导的体外肝纤维化。

Objective To study the effects of forsythiaside A(FA)on the proliferation,activation and apoptosis of hepatic stellate cells(HSCs).Methods Human HSCs(LX-2)were used as the model.And platelet derived growth factor(PDGF)-BB was used to simulate the microenvironment of liver fibrosis.LX-2 cells were divided into control group,PDGF-BB group,FA group and PDGF-BB+FA group and received corresponding treatment.The activity of LX-2 cells was detected by cell counting kit-8(CCK-8).The lipid droplets in cells were detected by oil red O.The protein expression ofαsmooth muscle actin(α-SMA)in cells was detected by immunofluorescence staining.The expressions of fibrosis-related marker proteins[α-SMA,and collagen type I alpha1(COL1A1)]and apoptosis-related proteins[B-cell lymphoma-2(BCL-2),cleaved poly ADP-ribose polymerase(cleaved-PARP),BCL-2-associated X protein(BAX),and cleaved cysteine aspartate proteinase 3(cleaved-Caspase-3)]were detected by Western Blot.The cell apoptosis was detected by flow cytometry.Results The activity of FA treated LX-2 cells decreased in a dose-dependent manner.Oil red O experiment showed that compared with PDGF-BB treatment,FA treatment up-regulated the number of lipid droplets in cells.Immunofluorescence results showed that compared with PDGF-BB treatment,FA treatment down-regulated the number ofα-SMA fluorescence spots in cells.Western Blot results showed that FA treatment down-regulated the protein expression ofα-SMA,COL1A1 and BCL-2,and up-regulated the protein expression of cleaved-PARP,BAX,and cleaved-Caspase-3.Conclusion FA can inhibit the proliferation and activation of HSCs,promote their apoptosis,and improve the in vitro liver fibrosis induced by PDGF-BB.