| Objective and purposeThis study intends to explore the effects of ERS on the biological behaviors and processes of lens epithelial cells.A series of experiments in vitro and vivo were conducted to observe the proliferation,apoptosis,epithelial-mesenchymal transition,collagen deposition and inflammation of lens epithelial cells to determine the relationship between ERS and cataractSingle nucleotide polymorphism is the most common form of genetic polymorphism in the human genome.Polymorphism detection and analysis of the metalloproteinase 2(MMP-2)gene rs243865 enriches the experimental data of gene polymorphisms related to cataract,and may also provide a new means for the clinical treatment of cataract,which has an important clinical and social valueOur findings help to enrich the theoretical and experimental data on the pathogenesis of cataract,providing theoretical and experimental support for finding potential biological therapeutic targets for cataracts.The polymorphism analysis of candidate genes may also provide new ideas for the development of therapeutic drugs and treatments,and has important clinical and social valueResults1.Twenty 8-day-old SPF-free rats were randomly divided into model group and control group.Rats in the model group were injected intraperitoneally with sodium selenite solution at 20 μmol/kg once every other day and 5 injections in total to induce the selenium cataract model.The model was verified by gross observation and pathological examination.Rats in the control group were injected with normal saline,and the other operations were the same as the model group2.The human lens epithelial cells were cultured in vitro and randomly divided into endoplasmic reticulum stress(ERS)activator group,ERS inhibitor group and control group,and the cells were treated accordingly.Western Blot was used to detect the expression of ERS marker proteins including glucose regulatory protein 78/Binding Protein 1(GRP78/Bip),Protein kinase R-like endoplasmic reticulum kinase(PERK),Activating transcription factor 4(ATF4)and C/EBP-homologous protein(CHOP),as well as lens epithelial-mesenchymal transition(EMT)-related proteins including a-smooth muscle actin(α-SMA),E-cadherin and MMP-2.MTT assay was to detect cell proliferation,flow cytometry was for cell apoptosis,immunohistochemistry was to detect the IV collagen deposition in lens epithelium and ELISA was performed to assay the expression of inflammatory factors.3.The TaqMan-MGB probe was used to detect and analyze the polymorphism of MMP-2 gene rs243865,which was closely related to lens epithelial-mesenchymal transition,and to explore the relationship between MMP-2 gene polymorphism and cataract genetic susceptibility.Results1.Selenium cataract rat models were induced successfully in 10 days by 20 μmol/kg sodium selenite injection through the abdominal cavity for 5 times every other day The modeling success rate was 100%.Among them,2 cases had a degree II turbidity(20%),3 cases had a grade III turbidity(30%),3 cases had a grade IV turbidity(30%),1 case had a grade V turbidity(10%),and 1 case had a grade VI turbidity(10%)2.After HE staining,normal epithelium and abnormal epithelium were observed in the lens of the model group.The abnormal epithelium cells showed edema,different shapes and sizes,light and loose cytoplasm,polymorphism nuclear and a lot of vacuoles,and the gaps between cells increased.The fiber thickness was uneven and the arrangement was irregular.There are vesicles and water cracks between the fibers.and distortion and breakage could also be seen3.The expression levels of GRP78,p-PERK,ATF4 and CHOP in lens tissues of cataract rats were(3.89 ± 0.22)(p<0.01),(2.97 ± 0.09)(p<0.01),(2.15 ± 0.17)(p<0.01)and(3.11 ± 0.19)(p<0.01)times conpared with that of the control group respectively.4.The expression levels of GRP78,p-PERK,ATF4 and CHOP of SRA01/04 cells in the ERS activator group were(1.76 ± 0.11)(p<0.01),(3.62 ± 0.17)(p<0.01),(3.35±0.13)(p<0.01)and(4.39 ± 0.20)(p<0.01)times compared with that of the control group respectively.The expression levels of GRP78,p-PERK,ATF4 and CHOP in the ERS inhibitor group were(0.43 ± 0.05)(p<0.01),(0.52 ± 0.08)(p<0.01),(0.32± 0.06)(p<0.01)and(0.29 ± 0.04)(p<0.01)times than that of the control cells,respectively.5.The survival rates of SRA01/04 cells in the ERS activator group at Ohr,12 hr,24 hr,48 hr,72 hr and 96 hr were(102 ± 5)%,(108 ± 9)%,(119 ± 6)%,(134 ± 5)%,(152 ±8)%,(168 ± 8)%,respectively.The survival rate of SRA01/04 cells in the ERS inhibitor group at 0 hr,12 hr,24 hr,48 hr,72 hr and 96 hr They were(102 ± 4)%,(117 ± 7)%,(130 ± 6)%,(162 ± 7)%,(186 ± 8)%,(209 ± 8)%,respectively.The growth curve of lens epithelial cells treated with ERS activator was under the control group,and the survival rates at 48 hr,72 hr and 96 hr were statistically different from the control group(p<0.05).The growth curve of lens epithelial cells treated with ERS inhibitor was above the control group,and the survival rates at 48 hr and 72 hr were statistically different from the control group(p<0.05).6.Apoptosis rate of SRA01/04 cells in ERS activator group was(15.83 ± 1.07)%,which was significantly higher than that of control group(7.94 ± 0.56)%(p<0.01).The apoptotic rate of SRA01/04 cells in ERS inhibitor group was(5.05 ± 0.71)%,which was lower than that of the control group(p<0.05).7.The expression levels of a-SMA,E-cadherin and MMP-2 of SRA01/04 cells in the ERS activator group were(2.78±0.13),(0.29 ± 0.09)and(1.70 ± 0.08)times than that of the control group,respectively(p all<0.01).The expression levels of a-SMA,E-cadherin and MMP-2 in SRA01/04 cells of ERS inhibitor group were(0.17 ± 0.06),(2.89 ± 0.12)and(0.20 ± 0.07)times than that of the control group,respectively(p all<0.01).8.There was a large amount of Ⅳ collagen deposition in SRA01/04 cells of the ERS activator group,with the average optical density of(0.65 ± 0.08),which was significantly higher than that of the control group(0.23 ± 0.03)(p<0.01).The average optical density of Ⅳ collagen in the ERS inhibitor group was(0.11±0.06),which was lower than that of the control group(p<0.05).9.The expression levels of IL-1β,IL-6,CRP and TNF-la in SRA01/04 cells of ERS activator group were(42.53 ± 4.14)ng/ml,(6.17 ± 2.51)ng/ml,(9.27 ± 0.80)mg/L and(25.22±3.28)ng/ml,respectively;which were(28.52±5.00)ng/ml,(3.23±2.01)ng/ml,(2.01 ± 0.35)mg/L and(11.17 ± 2.01)ng/ml,respectively in SRA01/04 cells of ERS inhibitor group.The expression levels of IL-1β,IL-6,CRP and TNF-1α in SRA01/04 cells of the control group were(30.47±4.83)ng/ml,(4.41 ± 1.74)ng/ml,(2.23 ± 0.29)mg/L and(15.57 ± 3.08)ng/ml,respectively.The levels of IL-1β,IL-6,CRP and TNF-1α in ERS activator group were higher than those of the control group(p<0.05),and the levels of IL-6 and TNF-1α in inhibitor group were lower than those of the control group(p<0.05).10.The detection frequency and expected frequency of CC,CT and TT genotypes in the cataract group were 62%and 57.01%,30%and 39.90%,12%and 7.01%.respectively;The detection frequency and expected frequency of CC,CT and TT genotypes in the control group were 72%and 70.04%,25%and 26.20%,3%and 2.40%,respectively.According to the Hardy-Weinberg equilibrium law,the frequency distribution of rs243865 genotypes of MMP-2 gene in both groups were accorded with Hardy-Weinberg equilibrium law(p>0.05),which was comparable.11.The number of GG,GA,and AA genotypes of MMP-2 gene rs243865 in the cataract group were 62,30,and 12 cases,respectively,accounting for 57.01%,28.85%,and 11.54%.The number of GG,GA and AA genotypes of MMP-2 gene rs243865 was 72,25 and 3 cases,respectively,accounting for 72%,25%and 3%.The three genotypes were statistically different between the two groups(p<0.05)12.The frequencies of C and T alleles in the cataract group were 74.04%and 25.96%,respectively.The frequencies of C and T alleles in the control group were 84.50%and 15.50%,respectively.The distribution frequency of C/T alleles in the two groups was different(p<0.05)13.For the genetic model analysis of MMP-2 gene rs243865,there were differences between the cataract group and the control group in the additive model(p<0.05),but there was no significant difference between the recessive model and the dominant model(p>0.05).Conclusions1.ERS reaction was over-activated in the lens tissue of cataract rats,and the expression level of ERS-related protein wers significantly higher than that of normal rats.2.Over-activated ERS can inhibit the proliferation,promote apoptosis,epithelial-mesenchymal transition,collagen deposition and inflammatory reactions and other biological behaviors and processes of lens epithelial cells,which can promote the development of cataract.3.The rs243865 polymorphism of the MMP-2 gene is associated with susceptibility to human cataract. |
