青蒿琥酯对人Tenon囊成纤维细胞自噬的影响

Effect of artesunate on autophagy in human tenon′s capsule fibroblasts

目的观察青蒿琥酯(Art)对人Tenon囊成纤维细胞(HTFs)自噬的影响,探讨Art阻止HTFs增生机制。方法体外培养HTFs,实验分为空白对照组和不同浓度(50、100、150、200μg/mL)Art实验组,给药24 h和48 h后噻唑蓝(MTT)法检测HTFs活性。48 h后采用逆转录定量PCR(qRT⁃PCR)和蛋白免疫印迹(Western Blot)法检测微管相关蛋白1轻链3(LC3)⁃II、自噬相关蛋白Beclin⁃l mRNA和蛋白的相对表达水平。选用150μg/mL Art处理HTFs 48 h,采用Cyto⁃ID染色法检测HTFs自噬水平,对Cyto⁃ID染色阳性的细胞数量进行检测,并与空白对照组进行比较。结果MTT法结果显示:Art抑制HTFs增殖(均P<0.05),且存在一定的浓度和时间依赖性,随着浓度升高和时间延长,其抑制率由4.7%增至68.6%。qRT⁃PCR法结果显示:不同浓度Art作用48 h后,LC3⁃II和Beclin⁃l mRNA的相对表达量增加,分别从(1.367±0.214)升至(4.765±0.371),(1.235±0.164)升至(3.487±0.356),各组间差异均有统计学意义(FL=20.920,P<0.01;FB=26.370,P<0.01)。Western Blot结果显示:不同浓度Art作用48 h后,LC3⁃II和Beclin⁃l蛋白的相对表达量增加,分别从(0.101±0.025)升至(0.808±0.102),(0.098±0.036)升至(0.784±0.064),各组间差异均有统计学意义(FL=32.852,P<0.01;FB=29.874,P<0.01)。Cyto⁃ID染色结果显示:150μg/mL Art作用48 h后,HTFs自噬阳性细胞的比例由空白对照组的(13.302±2.301)%上升至(53.274±7.851)%,差异有统计学意义(t=8.804,P<0.01)。结论Art有效促进HTFs中LC3⁃II、Beclin⁃l mRNA和蛋白的表达,提高细胞自噬水平,抑制细胞增殖。

Objective To observe autophagy of human tenon′s capsule fibroblasts(HTFs)induced by arte⁃sunate(Art)and explore the possible mechanism.Methods The cultured in vitro HTFs were divided into control group and Art(50,100,150,200μg/mL)groups.After treatment for 24 and 48 h,cell activity was determined by methyl thiazol tetrazolium(MTT)assay.48 h later,quantitative reverse transcriptase PCR(qRT⁃PCR)and Western Blot analysis were employed to assay the relative expression of microtubule⁃associated protein light chain 3(LC3)⁃Ⅱand Becline⁃1 in HTFs.The autophagy activity of HTFs was evaluated by a Cyto⁃ID autophagy detection kit,and then the autophagic flux was evaluated by counting the Cyto⁃ID positive cells.Results The results of MTT showed that Art could significantly inhibit the growth of fibroblasts in vitro in a concentration and time dependent manner(P<0.05),and the inhibition rate increased from 4.7%to 68.6%with increase of concentration and prolongation of time.qRT⁃PCR results showed that LC3⁃Ⅱand beclin⁃l mRNA relative expression increased after Art treatment,from(1.367±0.214)to(4.765±0.371),and(1.235±0.164)to(3.487±0.356),respectively.The difference between the groups had statistical significance(FL=20.920,P<0.01;FB=26.370,P<0.01).Western Blot results showed that LC3⁃Ⅱand beclin⁃l protein relative expression increased after Art treatment,from(0.101±0.025)to(0.808±0.102),and(0.098±0.036)to(0.784±0.064),respectively.The difference between the groups had statistical significance(FL=32.852,P<0.01;FB=29.874,P<0.01).Cyto⁃ID staining showed that the percentage of autophagic cell increased dramatically from(13.302±2.301)%to(53.274±7.851)%upon the exposure of HTFs to 150μg/mL Art(t=8.804,P<0.01).Conclusion Artesunate can effectively promote the production of LC3⁃Ⅱand Beclin⁃l,increase the Ievel of autophagy in HTFs,and inhibit the growth of fibroblasts.