Downregulation Of Smac Effects On Apoptosis Of Human Lens Epithelial Cells By Endoplasmic Reticulum Stress

Back groundCataract is the main reason of blindness in the world.With the older population continuous increaseing,the prevalence of cataract also have been increasing.At present,cataract is mainly treated by surgery.The popularity of cataract surgery and intraocular lens constantly updated and improved bring millions of cataract patients the light,and the country annually support the medical team to free surgery for cataract patients with poor,which will increase the economic burden on society.Because of the large population base in china,poor medical conditions in some remote areas and high cost of operation,there are still some cataract patients who can not get effective surgical treatment.Therefore,the study of the pathogenesis of cataract is of great significance to prevent and delay the occurrence and development of cataract.Endoplasmic reticulum plays an important role in regulating the metabolism of the human body,when the body is stimulated by the outside,endoplasmic reticulum stress?ERS?is activated for restore homeostasis,but the strong ERS will cause cell apoptosis.In recent years,many scholars have discovered that ERS have associated with the pathogenesis of age-related cataract.In the earlier experiment we have verified that High expression of Smac is associated with lens opacity,however,we are the lack of further research and exploration,.Thisexperiment consists of two parts.On the one hand,we study the relationship between Smac and age-related cataract lens opacity,on the other hand,we study downregulation the expression of Smac protein effect on human lens epithelial cells via ER stress pathway.Objective1.To study whether Smac is related to the occurrence of cataract.2.To study effects of Smac on human lens epithelial cells by endoplasmic reticulum stress pathway.Methods1.Anterior lens capsules with age-related cataract patients were collected in our hospital.There are a total of 152 subjects of anterior lens capsules?A total of 169 eyes?and normal tissue in 5 subjects?A total of 10 eyes?.According to Emery classification,the expression of Smac in human anterior lens capsules were detected by Immunohisochemistry semiquantitative analysis that analyzd relationship between Smac and turbid degree of cataract.2.Choosing cells in logarithmic phase.Knockdown of Smac expression was declined by small interfering RNA?siRNA?.Cells were treated with H₂O₂ to induce endoplasmic tericulum stress-induced apoptosis.The experiment is divided into four groups: PBS group,H₂O₂ group,siRNA-Smac group,siRNA-Smac+H₂O₂ group.3.After transfection of 24 h,Western blot was used to select the optimal plasmid4.CCK-8?Cell Counting Kit-8?was used to detect the concentration of H₂O₂ induced cell apoptosis ratio close to 50%,and Cell proliferation was evaluated using were used by CCK-8.5.After transfection of 24-36 h.PBS group,H₂O₂ group,siRNA-NC+H₂O₂group,siRNA-Smac + H₂O₂ group apoptosis rate was detected by Annexin V-APC/PI method.6.The expression of GRP78,CHOP,caspase-3,BAX and Bcl-2 in mRNA were detected by RT-PCR in group.7.Western blot was used to detect the expression of GRP78,CHOP,cleaved caspase-3,Bax and Bcl-2.Results1.Immunohistochemistry results showed that expression of Smac anterior capsule in ARC patients was higher than control group,and the expression of Smac crystal in mild and moderate cataract patients had no significant difference,and when lens opacity degree was serious,the expression of Smac positive rate was high.2.siRNA-Smac3 could effectively downregulate the expression of Smac.3.CCK-8 showed that the ratio of apoptosis close to 50% was 200?mol/L H₂O₂,siRNA-Smac could increase cell viability.4.Flow cytometry showed that apoptosis rate The results was?1.7±1.2?%,?40.1±3.9?%,?45.3±4.4?%,?26.5±2.8?%,respectively.Compared to the H₂O₂ group,the rate of apoptosis was decline?P<0.05?.5.RT-PCR showed that the expression of GRP78,CHOP,caspase-3 and BAX mRNA in the siRNA-Smac+H₂O₂ group was decreased,and the expression of Bcl-2in the H₂O₂ group was increased.6.Western blot tested cells to show that Smac could increase the expression of GRP78,CHOP,cleaved caspase-3,Bax protein,and decreased the expression of Bcl-2.ConclusionSmac is closely related to age-related cataract,and downregulation the expression of Smac can inhibit the apoptosis of human lens epithelial cells by endoplasmic reticulum stress pathway.