The effect of L-carnitine on the apoptosis of human lens epithelial cells through endoplasmic reticulum stress

Abstract

Objective: 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 H(2)O(2) treatment for 12 h, and lead to ER stress. Cells were divided into four groups: H(2)O(2) group, L-carnitine (100 μmol/L) with H(2)O(2) 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 H(2)O(2). The apoptosis ratio of HLE-B3 treated by 400 μmol/L H(2)O(2) was tested by flow cytometry. When HLE-B3 was treated by 400 μmol/L H(2)O(2), 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 H(2)O(2) concentration was 200, 400, 600 and 800 μmol/L, there was significant difference in the H(2)O(2) group(77.6%±0.8%,58.1%±3.1%,39.2%±1.5%,28.1%±2.2%), L-carnitine with H(2)O(2) 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 H(2)O(2) was 400 μmol/L, the survival rate of the L-carnitine with H(2)O(2) group was higher than the H(2)O(2) group. The difference was statistically significant (t=18.14, P=0.020). With increasing of the H(2)O(2) concentration, cell necrosis was increased. The cell survival rate had no significant difference between the L-carnitine with H(2)O(2) group and H(2)O(2) group (both P>0.05). Flow cytometry results of the H(2)O(2) group, L-carnitine with H(2)O(2) 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 H(2)O(2) group was lower than that in the H(2)O(2) 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 H(2)O(2) group was lower than the H(2)O(2) group (0.424±0.041 vs. 0.752±0.203), and the expression of GRP78 mRNA in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) 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 H(2)O(2) group was lower than the H(2)O(2) group (0.712±0.212 vs. 1.126±0.251), and the GRP78 protein expression in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) 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. (Chin J Ophthalmol, 2018, 54: 363-368).