The Preliminary Study Of Endoplasmic Reticulum Stress Influence On Lens Epithelial Cell Apoptosis And Cataract Formation

Back groundCataract is the biggest cause for visual impairment in the world and also a main cause for diminution of vision in China. With the increase in the average lifespan and the aging of the Chinese population, cataract not only affects patients' living quality, but also imposes tremendous pressure on the society and the medical insurance system. Currently, surgery is the only means of curing this disease. There is an urgent need for us to look deep into the pathogenesis of cataract and find more methods of treatment. In recent years, endoplasmic reticulum stress?ERS? has become a hot research topic in the medical world. Some literatures have reported that ERS plays a part in cataract formation. However, there has been lack of systematic and complete in vitro and in vivo experiments. This paper will, from two perspectives, analyze the development of ERS during the apoptosis of human lens epithelial cells. An in-depth study will be conducted on the mechanism of ERS, which will provide a reliable basis for subsequent experiments. Objective1. To study if ERS is involved in the occurrence of senile cataract and diabetic cataract2. To study under the condition of oxidative stress, the activation of ERS to human lens epithelial cells is a protective effect or promoting apoptosis.3. To study the protective effect of Salubrinal on human lens epithelial cells?HLEC? and its mechanism under endoplasmic reticulum stress?ERS?. Methods1. Adopt the method of HE staining and immunohistochemical to detect GRP78, CHOP, Caspase-12 expression in anterior lens capsules of 30 cases senile cataract,30 cases diabetic cataract and 30 cases normal lens.2. Hydrogen peroxide?H₂O₂ 200?mol/L? was used to intervene in the cultured HLE-B3 so as to create an oxidative stress model and induce ERS in the model. Different concentrations of Salubrinal?10?mol/L, 15?mol/L, 20?mol/L, 25?mol/L, 30?mol/L, and 35?mol/L? were added to the cultured HLE-B3 with H₂O₂ intervention and without H₂O₂ intervention, respectively. The mixtures were left to stand for 24 hours. Then, the cell counting kit?CCK-8? assay was used to test the viability of HLE-B3.3. The cells were divided into three groups, namely, Group A?normal control group?, Group B?H₂O₂ 200?mol/L group?, and Group C?H₂O₂ 200?mol/L+ Salubrinal 25?mol/L group?. After 48 h, TUNEL and flow cytometry assay?FCM? were used to examine the effect of Salubrinal on HLE-B3 apoptosis.4. Under different time points extraction group B protein, Western blotting was used to test the expression of GRP78, CHOP, Caspase- 12, p- eIF2?.5. Western blotting was used to detect the expression of GRP78, CHOP, Caspase-12, p-eIF2? in each group at different points in time. Results1. HE and immunohistochemical results showed that all organizations dyeing, fixed well, organization structure complete.Three kinds of protein in the NC group were negative or weak positive, and expressed in the ARC and DC group are all positive, ARC group of positive rate were 78%, 82%, 79%, DC group positive rate were 83%, 75% and 81% respectively.2. CCK-8 results showed that without the intervention of H₂O₂, different concentrations of Salubrinal had no inhibitive effect on HLE-B3 viability, and that survival rates were 98.56±3.29%, 98.73±2.58%, 99.39±3.23%, 98.57±1.89%, 98.81±2.46%, 99.34±3.23%, and 99.50±2.36%. There was no statistically significant difference between them?F=0.087, P=0.997?. As the Salubrinal concentration grew, survival rates of HLE-B3 in the presence of H₂O₂ intervention were 52.86±4.67%, 65.01±3.56%, 72.89±3.82%, 84.45±3.57%, 91.64±2.14%, 93.12±2.92%, and 92.02±3.33%, There was statistically significant difference from the control group?all P=0.000?. When the Salubrinal concentration was greater than 25 ?mol/L, the increase of survival rates was not significant?#P = 0.555?0.878?.3. FCM results indicated that apoptosis rates of Group A, B and C were 1.93%±0.70%, 8.80%±0.51%, and 4.29%±0.30%, respectively. The differences between them were statistically significant?F = 396.263, P =0.000, compare with Group A all P = 0.000?. TUNEL results showed that apoptosis indexes of Group A, B, and C were 7.67%±0.99%, 36.90%±0.95%, and 16.69%±2.16%, respectively?F=618.393,P=0.000,compare with Group A all P=0.000?. The differences between them were statistically significant.4. Results of western blotting in group B at different points in time?0h, 12 h, 24 h, 36 h, 48h? showed that p-eIF2? had increased by 2.16±0.384 times by 6h; GRP78 had increased by 2.56±0.147 times by 12h; CHOP started to rise after 12 h until it dropped after 24 h, and its amount had increased by 2.49±0.231 times by 48h; Caspase-12 had increased significantly by 3.53±0.300 times by 48 h. The expression of GRP78, CHOP and p-eIF2? in group C was greater than that in Group B, but the expression of Caspase-12 in Group C was lower than that in Group B?GRP78: F = 37.849, p = 0.000; CHOP: F = 61.087, p = 0.000; Caspse-12: F = 22.265, p = 0.000; p-eIF2?: F=15.105, P=0.000?. Conclusions1. ERS participate in the occurrence of senile cataract and diabetic cataract.2. In the early stage of oxidative stress,UPR activated to play the role of cell protection.as the oxidative stress continued, the apoptosis pathway activated.3. Salubrinal can protect HLE-B3 against H₂O₂-induced apoptosis by inhibiting ERS related apoptosis pathways.