摘要目的 探讨左旋肉碱通过内质网应激途径对人晶状体上皮细胞凋亡产生的影响.方法 实验研究.选取人晶状体细胞株(HLE-B3),利用H2O2处理12 h建立氧化应激模型,诱导内质网应激产生.实验分为4组:H2O2组、左旋肉碱(100 μmol/L)+H2O2组、磷酸盐缓冲液(PBS)组和左旋肉碱(100 μmol/L)组,其中PBS组和左旋肉碱组为对照组.采用细胞计数试剂盒8(CCK-8)法检测不同浓度(200、400、600、800 μmol/L)H2O2作用下各组HLE-B3细胞活力.根据CCK-8结果选择适宜的H2O2浓度(400 μmol/L)处理细胞,采用流式细胞仪检测各组细胞凋亡率;实时定量(RT)-PCR和Western印迹分别检测各组细胞内半胱氨酸蛋白酶3(caspase-3)及葡萄糖调节蛋白78(GRP78)在mRNA及蛋白水平的表达.4组数据的比较采用单因素方差分析,组间两两比较采用LSD-t检验.结果 CCK-8法检测结果显示,当H2O2浓度分别为200、400、600、800 μmol/L时,HLE-B3细胞存活率H2O2组(77.6%±0.8%,58.1%±3.1%,39.2%±1.5%,28.1%±2.2%)、左旋肉碱+H2O2组(83.3%±4.2%, 74.5%±3.1%,46.4%±1.7%,32.4%±1.2%)、PBS组(97.6%±2.1%,98.3%±0.2%,96.3%±2.2%,98.5%± 1.1%)、左旋肉碱组(98.5%±1.3%,96.1%±2.1%,98.1%±0.2%,97.3%±1.4%)4组比较差异均有统计学意义(均P＜0.05),PBS组和左旋肉碱组两组间比较差异均无统计学意义(均P>0.05);当H2O2浓度为400 μmol/L时,左旋肉碱+H2O2组细胞存活率高于H2O2组,差异有统计学意义(t=18.14,P=0.020),且H2O2组细胞存活率接近50%,但随着H2O2浓度的进一步增加,左旋肉碱+H2O2组与H2O2组相比差异无统计学意义(均P>0.05).选取H2O2浓度为400 μmol/L,流式细胞仪检测结果显示H2O2组、左旋肉碱+H2O2组、PBS组和左旋肉碱组细胞凋亡率依次为31.4%±4.5%、16.5%±2.8%、2.1%±0.2%、1.9%±1.8%,4组比较差异有统计学意义(F=126.784,P=0.024),左旋肉碱+H2O2组凋亡率低于H2O2组,差异有统计学意义(t=24.67,P=0.013),PBS组和左旋肉碱组两组间差异无统计学意义(P>0.05);RT-PCR结果显示,左旋肉碱+H2O2组caspase-3、GRP78的mRNA表达量均低于H2O2组(0.424±0.041比0.752±0.203,0.521±0.223比0.821±0.103),差异均有统计学意义(t=27.92,P=0.018;t=16.31,P=0.019);Western印迹结果显示,左旋肉碱+H2O2组caspase-3、GRP78的蛋白表达量均低于H2O2组(0.712±0.212比1.126±0.251,0.512± 0.012比0.735±0.051),差异均有统计学意义(t=15.43,P=0.010;t=20.62,P=0.018).结论 左旋肉碱能够通过抑制内质网应激途径降低人晶状体细胞的凋亡率.

abstractsObjective To investigate the effect of L-carnitine on the apoptosis of human lens epithelial cells through endoplasmic reticulum (ER) stress pathway. Methods HLE-B3 cell lines were used to set up an oxidative stress model with H2O2 treatment for 12 h, and lead to ER stress. Cells were divided into four groups: H2O2 group, L-carnitine (100 μmol/L) with H2O2 group,phosphate buffered saline (PBS) group and L-carnitine (100 μmol/L) group. Cell counting kit-8 was used to detect the cell viability under the treatment of different concentrations(200,400,600 and 800 μmol/L)of H2O2.The apoptosis ratio of HLE-B3 treated by 400 μmol/L H2O2 was tested by flow cytometry. When HLE-B3 was treated by 400 μmol/L H2O2, the expression levels of cysteinyl aspartate specific proteinase 3 (caspase-3) gene and glucose-regulated protein 78 (GRP78) gene were measured by RT-PCR, and the expression levels of caspase-3 protein and GRP78 protein were assayed by Western blotting.Data from groups was analyzed by the one-way analysis of variance,and the LSD-t test was used for the comparison of groups. Results Cell counting kit-8 assay showed that when H2O2 concentration was 200, 400, 600 and 800 μmol/L, there was significant difference in the H2O2 group(77.6%±0.8%,58.1%±3.1%,39.2%±1.5%,28.1%±2.2%),L-carnitine with H2O2 group(83.3%±4.2%,74.5%±3.1%,46.4%±1.7%,32.4%±1.2%),PBS group(97.6%±2.1%,98.3%± 0.2%,96.3%±2.2%,98.5%±1.1%)and L-carnitine group(98.5%±1.3%,96.1%±2.1%,98.1%±0.2%,97.3%± 1.4%)(all P＜0.05).There was no significant difference between groups(PBS group compared to L-carnitine group,all P>0.05).When the concentration of H2O2 was 400 μmol/L,the survival rate of the L-carnitine with H2O2 group was higher than the H2O2 group.The difference was statistically significant(t=18.14,P=0.020). With increasing of the H2O2 concentration, cell necrosis was increased. The cell survival rate had no significant difference between the L-carnitine with H2O2 group and H2O2 group (both P>0.05). Flow cytometry results of the H2O2 group, L-carnitine with H2O2 group, PBS group and L-carnitine group were 31.4%±4.5%,16.5%±2.8%,2.1%±0.2% and 1.9%±1.8%,respectively(F=126.784,P=0.024).The rate of apoptosis in the L-carnitine with H2O2 group was lower than that in the H2O2 group(t=24.67,P=0.013).There was no significant difference between the PBS group and L-carnitine group(P>0.05).The results of RT-PCR showed that the expression of caspase-3 mRNA in the L-carnitine with H2O2 group was lower than the H2O2 group (0.424 ± 0.041 vs. 0.752 ± 0.203), and the expression of GRP78 mRNA in the L-carnitine with H2O2 group was lower than the H2O2 group (0.521 ± 0.223 vs. 0.821 ± 0.103). The difference was statistically significant (caspase-3: t=27.92,P=0.018;GRP78: t=16.31,P=0.019). Western blotting showed that the protein expression of caspase-3 in the L-carnitine with H2O2 group was lower than the H2O2 group(0.712± 0.212 vs.1.126±0.251),and the GRP78 protein expression in the L-carnitine with H2O2 group was lower than the H2O2 group (0.512 ± 0.012 vs. 0.735 ± 0.051). The difference was statistically significant (caspase-3: t=15.43, P=0.010;GRP78: t=20.62, P=0.018). Conclusion L-carnitine can reduce the apoptosis rate of HLE-B3 during oxidative stress through ER stress pathway.