Effect And Mechanism of Endoplasmic Reticulum Stress Induced By Tunicamycin In Trabecular Meshwork Cells
Authors: Wu Yuyu , Wang Xiaorui , Huang Guangqian , Ou Tingting 
Published: July 2021

Abstract
Objective: To explore the role of endoplasmic reticulum stress in apoptosis of trabecular meshwork cellsMethods: Human trabecular meshwork cells(HTMCs) were cultured in vitro. When the cells grew to logarithmic phase, they were digested and resuspended, and then cultured in 96-well plate. When the cells are close to fusion, the culture is continued for 24h in a serum-free medium, The HTMCs were treated with different concentrations of tunicamycin (0mg/L, 1.25mg/L, 2.5mg/L, 5mg/L, 10mg/L, and 20mg/L) for different durations (0h, 6h, 12h, 24h, 36h, and 48h). The cytoactive was detected by CCK-8, and the optimal intervention concentration of tunicamycin was determined. Then we use the 5mg/L tunicamycin to interfere with HTMCs for different durations (0h, 6h, 12h, and 24h). The apoptotic rate was detected by flow cytometry, and the expression levels of GRP78, CHOP, Bcl-2 and Caspase-3 were detected by RT-PCR and Western blot. Results: The number of RGCs in IRI group was significantly lower than that in NC group (P < 0.05), demonstrated by HE staining. Western blotting results indicated that the protein expression of LC3 and Beclin-1 in the IRI group were significantly elevated compared with those in the NC group (P < 0.05). However, with 3-MA treatment, the number of RCGs in 3-MA treated-IRI group was elevated and protein levels of LC3, Beclin-1 were downregultated, compared with those in the IRI group (P < 0.05). Further immunohistochemistry staining and Western blot showed that 3-MA treated-IRI group presented downregulated Caspase-3 and upregulated Bcl-2 protein expression with comparison of IRI group (P < 0.05). CCK-8 results showed that the inhibition rate of HTMCs increased with the increase of tunicamycin concentration and duration of intervention. When the concentration of tunicamycin was 1.25mg / L and 2.5mg / L, there was no significant difference in cell inhibition rate compared with the control group (P> 0.05). When the concentration of tunicamycin was 5mg / L and the duration of action was 12h or more, the cells showed significant inhibition, of which the cell inhibition rates at 12h, 24h, 36h and 48h were (21.11 ± 1.23)% and (31.34 ± 0.86, respectively) )%, (44.37 ± 1.01)%, (51.47 ± 0.36)%, compared with the 0h group, the differences were statistically significant (P <0.05).The results of flow cytometry suggest that with the increase of the time of action of tunicamycin, the early apoptosis rate and the late apoptosis rate of the cells increase significantly. Compared with the control group, the experimental group with different intervention durations had significantly higher early cell apoptosis rate and late cell apoptosis rate, and the difference was statistically significant (P <0.05). RT-PCR test results indicated that the expression levels of GRP-78, CHOP, and Caspase-3 mRNA in the experimental groups (6h, 12h, and 24h) with different intervention durations were all increased compared with the control group, and the difference was statistically significant ( P <0.05); BCL-2 mRNA expression in the experimental groups (6h, 12h, 24h) with different intervention durations was reduced compared with the control group, and the difference was statistically significant (P<0.05).Western blot test results showed that compared with the control group, GRP-78 and CHOP protein expressions of the experimental groups (6h, 12h, 24h) with different intervention durations increased, the difference was statistically significant (P <0.05). Compared with the control group, the expression level of Caspase-3 protein increased in the 12h group and the difference was statistically significant (P <0.05). The increase in the expression level of the remaining experimental group was not obvious, and the difference was not statistically significant (P> 0.05); 24h Compared with the control group, the expression level of BCL-2 protein was significantly reduced, the difference was statistically significant (P <0.05), the expression level of BCL-2 protein in the remaining experimental group was not significantly reduced, and the difference was not statistically significant (P> 0.05). Conclusion: Tunicamycin can increase the expression of endoplasmic reticulum stress proteins in trabecular meshwork cells and induce apoptosis. Our study demonstrated that endoplasmic reticulum stress played an important role in the apoptosis of trabecular meshwork cells.