松萝酸对增生性瘢痕成纤维细胞生物学行为及JNK/MAPK信号通路的影响

Effect of usnic acid on biological behaviors of hypertrophic scar fibroblasts and JNK/MAPK signaling pathway

目的:探讨松萝酸对增生性瘢痕成纤维细胞(HSFBs)增殖、凋亡、迁移和侵袭的影响,并阐明其对c-Jun氨基末端激酶(JNK)/丝裂原活化蛋白激酶(MAPK)信号通路的调节作用。方法:取对数生长期HSFBs分为对照组、10μmol·L^(-1)松萝酸组、20μmol·L^(-1)松萝酸组和50μmol·L^(-1)松萝酸组,除对照组外,其他各组细胞给予相应浓度松萝酸处理。噻唑蓝(MTT)法检测各组细胞存活率,流式细胞术检测各组细胞凋亡率,Transwell小室实验检测各组细胞侵袭和迁移能力,Western blotting法检测各组细胞中磷酸化JNK(p-JNK)、JNK、B细胞淋巴瘤2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达水平。结果:MTT法和流式细胞术检测,与对照组比较,10、20和50μmol·L^(-1)松萝酸组细胞存活率均降低(P<0.05),细胞凋亡率均升高(P<0.05);与10μmol·L^(-1)松萝酸组比较,20和50μmol·L^(-1)松萝酸组细胞存活率均降低(P<0.05),细胞凋亡率均升高(P<0.05);与20μmol·L^(-1)松萝酸组比较,50μmol·L^(-1)松萝酸组细胞存活率降低(P<0.05),细胞凋亡率升高(P<0.05)。Transwell小室实验检测,与对照组比较,10、20和50μmol·L^(-1)松萝酸组侵袭细胞数和迁移细胞数均减少(P<0.05);与10μmol·L^(-1)松萝酸组比较,20和50μmol·L^(-1)松萝酸组侵袭细胞数和迁移细胞数均减少(P<0.05);与20μmol·L^(-1)松萝酸组比较,50μmol·L^(-1)松萝酸组侵袭细胞数和迁移细胞数均减少(P<0.05)。Westernblotting法检测,与对照组比较,10、20和50μmol·L^(-1)松萝酸组细胞中Bcl-2蛋白表达水平均降低(P<0.05),Bax和p-JNK蛋白表达水平均升高(P<0.05);与10μmol·L^(-1)松萝酸组比较,20和50μmol·L^(-1)松萝酸组细胞中Bcl-2蛋白表达水平均降低(P<0.05),Bax和p-JNK蛋白表达水平均升高(P<0.05);与20μmol·L^(-1)松萝酸组比较,50μmol·L^(-1)松萝酸组细胞中Bcl-2蛋白表达水平降低(P<0.05),Bax和p-JNK蛋白表达水平均升高(P<0.05)。结论:松萝酸可抑制HSFBs增殖、侵袭和迁移,诱导HSFBs凋亡,并激活JNK/MAPK信号通路。

Objective:To discuss the effect of usnic acid on the proliferation,apoptosis,migration,and invasion of the hypertrophic scar fibroblasts(HSFBs),and to clarify its regulatory effect on the c-Jun N-terminal kinase(JNK)/mitogen-activated protein kinase(MAPK)signaling pathway.Methods:The HSFBs at logarithmic growth phase were divided into control group,10μmol·L^(-1)usnic acid group,20μmol·L^(-1)usnic acid group,and 50μmol·L^(-1)usnic acid group.Except for control group,the cells in the other groups were treated with the corresponding concentrations of usnic acid.The survival rates of the cells in various groups were detected by methylthiazolyidiphenyl-tetrazolium bromide(MTT)method;the apoptotic rates of the cells in various groups were detected by flow cytometry;the invasion and migration abilities of the cells in various groups were detected by Transwell chamber experiment;the expression levels of phosphorylated JNK(p-JNK),JNK,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)proteins in the cells in various groups were detected by Western blotting method.Results:The MTT and flow cytometry results showed that compared with control group,the survival rates of the cells in 10,20,and 50μmol·L^(-1)usnic acid groups were decreased(P<0.05),and the apoptotic rates were increased(P<0.05).Compared with 50μmol·L^(-1)usnic acid group,the survival rates of the cells in 10 and 20μmol·L^(-1)usnic acid groups were decreased(P<0.05),and the apoptotic rates were increased(P<0.05).The Transwell chamber experiment results showed that compared with control group,the numbers of invasion and migration cells in 10,20 and 50μmol·L^(-1)usnic acid groups were decreased(P<0.05);compared with 10μmol·L^(-1)usnic acid group,the numbers of invasion and migration cells in 20 and 50μmol·L^(-1)usnic acid groups were decreased(P<0.05);compared with 20μmol·L^(-1)usnic acid group,the numbers of invasion and migration cells in 50μmol·L^(-1)usnic acid groups were decreased(P<0.05).The Western blotting results showed that compared with control group,the expression levels of Bcl-2 protein in the cells in 10,20,and 50μmol·L^(-1)usnic acid groups were decreased(P<0.05),and the expression levels of Bax and p-JNK proteins were increased(P<0.05);compared with 10μmol·L^(-1)usnic acid group,the expression levels of Bcl-2 protein in the cells in 20 and 50μmol·L^(-1)usnic acid groups were decreased,and the expression levels of Bax and p-JNK proteins in the cells were increased(P<0.05);compared with 20μmol·L^(-1)usnic acid group,the expression level of Bcl-2 protein in the cells in 50μmol·L^(-1)usnic acid group was decreased(P<0.05),and the expression levels of Bax and p-JNK proteins were increased(P<0.05).Conclusion:Usnic acid can inhibit the proliferation,invasion,and migration of the HSFBs,induce the apoptosis of the HSFBs,and activate the JNK/MAPK signaling pathway.