天麻素对高糖条件下RF/6A细胞SIRT1/TLR4信号通路及细胞增殖与凋亡的影响

Effects of gastrodin on SIRT1/TLR4 signaling pathway and cell proliferation and apoptosis of RF/6A cells under high glucose condition

目的探究天麻素对高糖诱导的RF/6A细胞增殖、迁移、凋亡及SIRT1/TLR4表达水平的影响。方法将RF/6A细胞随机分5组:正常对照组(NC组),正常培养,不做任何处理;高糖对照组(HG组),在高糖环境下诱导、培养;低浓度组(LC组),在高糖环境下培养,并用5μmol/L天麻素预处理;中浓度组(MC组),在高糖环境下培养,并用10μmol/L天麻素预处理;高浓度组(HC组),在高糖环境下培养,并用20μmol/L天麻素预处理。通过CCK-8法、TUNEL法、细胞划痕法、Western blot法、qRT-PCR法等方法分析检测5组细胞增殖情况、迁移能力、凋亡、SIRT1/TLR4的蛋白含量及mRNA的表达含量的差异性。结果(1)细胞增殖:与NC组比较,HG组细胞OD值降低(t=5.394,P=0.000);与HG组比较,LC组、MC组、HC组细胞OD值依次增高(t LC=4.638,t MC=4.938,t HC=8.389,均P=0.000),差异均有统计学意义。(2)细胞凋亡:与NC组比较,HG组细胞凋亡数量升高(t=12.690,P=0.000);与HG组比较,LC组、MC组、HC组细胞凋亡数量依次降低(t LC=4.713,t MC=7.588,t HC=10.050,均P=0.000),差异均有统计学意义。(3)细胞迁移:与NC组比较,HG组细胞迁移数量降低(t=23.100,P=0.000);与HG组比较,LC组、MC组、HC组细胞迁移数量依次升高(t LC=5.040,t MC=11.280,t HC=15.550,均P=0.000),差异均有统计学意义。(4)SIRT1蛋白含量:与NC组比较,HG组细胞含量降低(t=6.606,P=0.000);与HG组比较,LC组、MC组、HC组细胞SIRT1蛋白含量依次升高(t LC=2.195,P=0.043;t MC=3.808,P=0.002;t HC=5.556,P=0.000),差异均有统计学意义。(5)TLR4蛋白含量:与NC组比较,HG组细胞含量升高(t=6.746,P=0.000);与HG组比较,LC组、MC组、HC组细胞含量依次降低(t LC=2.141,P=0.048;t MC=4.427,P=0.000;t HC=5.321,P=0.000),差异均有统计学意义。结论高糖条件下可诱导RF/6A细胞凋亡,天麻素能够抑制高糖诱导的RF/6A细胞凋亡,其机制可能与上调SIRT1表达、抑制TLR4信号通路等有关。

OBJECTIVE To investigate the effects of gastrodin on proliferation,migration,apoptosis and SIRT1/TLR4 expression of RF/6A cells induced by high glucose.METHODS The RF/6A cells were randomly divided into 5 groups:normal control group(NC group),cells were normally cultured without any treatment;high glucose control group(HG group),cells were induced and cultured in a high glucose environment;low concentration group(LC group),cells were cultured in a high glucose environment and pretreated with 5μmol/L gastrodin;medium concentration group(MC group),cells were intervened in a high glucose environment and pretreated with 10μmol/L gastrodin;and high concentration group(HC group),cells were cultured in a high glucose environment and pretreated with 20μmol/L gastrodin.Cell proliferation,migration and apoptosis in the 5 groups were detected by CCK-8 method,TUNEL method,cell scratch method,Western blot method,qRT-PCR method and other methods,the difference of SIRT1/TLR4 protein content and mRNA expression content.RESULTS(1)Cell proliferation:Compared with the NC group,the cell OD value of the HG group decreased(t=5.394,P=0.000);compared with the HG group,the cell OD value of the LC group,MC group,and HC group increased in succession(t LC=4.638,t MC=4.938,t HC=8.389,all P=0.000),the difference was statistically significant.(2)Apoptosis:Compared with the NC group,the number of apoptotic retinal vascular endothelial cells in the HG group increased(t=12.690,P=0.000);compared with the HG group,the number of apoptotic cells in the LC group,MC group and HC group decreased successively(t LC=4.713,t MC=7.588,t HC=10.050,all P=0.000),and the difference was statistically significant.(3)Cell migration:Compared with the NC group,the number of migration cell in the HG group decreased(t=23.100,P=0.000);compared with the HG group,the number of cell migration in the LC group,MC group,and HC group increased in succession(t LC=5.040,t MC=11.280,t HC=15.550,all P=0.000),the difference was statistically significant.(4)SIRT1 protein content:Compared with the NC group,the cell content of the HG group was decreased(t=6.606,P=0.000);compared with the HG group,the SIRT1 protein content of the cells in the LC,MC,and HC groups increased in succession(t LC=2.195,P=0.043;t MC=3.808,P=0.002;t HC=5.556,P=0.000),the difference was statistically significant.(5)TLR4 protein content:Compared with the NC group,the cell content of the HG group increased(t=6.746,P=0.000);compared with the HG group,the cell content of the LC group,MC group,and HC group decreased(t LC=2.141,P=0.048;t MC=4.427,P=0.000;t HC=5.321,P=0.000),the difference was statistically significant.CONCLUSIONS The apoptosis of RF/6A cells could be induced by high glucose.Gastrodin could significantly inhibit the apoptosis of RF/6A cells induced by high glucose.The mechanism may be related to the up-regulation of SIRT1 expression and the inhibition of TLR4 signaling pathway.