Oxidative Stress-Induced E2F1 Entry Into The Nucleus Mediates The Increase Of THBS1 Expression In High Glucose Environment And Its Correlation With Apoptosis Of Lens Epithelial Cells And Diabetic Cataract

Background:Diabetes is very harmful.Chronic hyperglycemia can affect the visual system,especially the lens.Most diabetic patients have accelerated cataract development compared with non-diabetic patients.The lens epithelial cells of rats exposed to high blood glucose for a long time undergo apoptosis,and the progression and maturation of diabetic cataract can be delayed by feeding diabetic rats with antioxidants.THBS1 is an extracellular matrix protein,and its multi-binding domain allows it to interact with a large number of other proteins.Therefore,THBS1 and its major receptors are considered as potential therapeutic targets for metabolic diseases.Transgenic mice overexpressing THBS1 have been reported to have an increased incidence of cataracts.However,the role of THBS1 in Human lens epithelial cells(HLEpi CS)remains unclear.Objective:To investigate the changes of THBS1 and its receptors CD36 and CD47 in HLEpi CS of diabetic patients,and to explore whether ROS affects the expression and function of THBS1 protein in high glucose environment,and whether THBS1 and its receptors CD36 and CD47 induce the occurrence of diabetic cataract by promoting the apoptosis of lens epithelial cells.Fully understanding the pathogenesis of THBS1 in diabetic cataract will provide a new target for prevention and treatment.Methods:(1)Immunohistochemistry(IHC)was used to detect the expression of THBS1 and its receptors CD36 and CD47 in lens capsule epithelial tissues of diabetic cataract patients and senile cataract patients.(2)Western blotting(WB)was used to detect the differences in the expression levels of THBS1 and its receptors CD36 and CD47 in HLEpi CS cultured in normal(NG,5.6m M)and high glucose(HG 25.6 m M)environments.(3)Enzyme-linked immunosorbent assay(Elisa)was used to detect the difference in THBS1 protein content in the cell supernatant of HLEpi Cs cultured in NG and HG environments for 7 days.(4)WB was used to detect the differences in the expression levels of apoptosis-related proteins in HLEpi Cs transfected with THBS1 si RNA,CD47 si RNA and treated with CD36 inhibitor SSO in HG environment.(5)TUNEL assay was used to detect and compare the proportion of apoptotic cells in each group.(6)The differences of reactive oxygen species(ROS)and mitochondrial membrane potential among groups were detected by fluorescence microplate reader.(7)WB and immunofluorescence(IF)were used to detect the effect of ROS on THBS1 expression.Results:(1)Compared with senile cataract,the expression levels of THBS1 and CD47 proteins in lens capsule epithelial tissues of diabetic cataract are higher,while the expression level of CD36 protein is lower.(2)Compared with NG culture,the expression levels of THBS1 and CD47 proteins were significantly increased,while CD36 was significantly decreased in HLEpi Cs cultured in HG environment on day 7.(3)HLEpi Cs cultured in HG increased THBS1 secretion.(4)THBS1 si RNA,CD47 si RNA transfection and CD36 inhibitor SSO treatment in HG environment;The ratio of Bcl-2/ Bax was increased,while the levels of Cleaved Caspase-9/Caspase-9 and Cleaved Caspase-3/Caspase-3 were decreased.(5)TUNEL analysis showed that the proportion of apoptotic cells in the experimental group was lower than that in the control group.(6)Mitochondrial membrane potential was restored in HG-treated cells with THBS1 si RNA,CD47 si RNA and CD36 inhibitor SSO,but ROS level was not changed.(7)The expression level of THBS1 protein in HLEpi Cs was significantly decreased after treatment with the E2F1 inhibitor HLM006474.HLEpi Cs were cultured in NG or HG medium for 3 days and treated with the antioxidant Tempol.Compared with NG group,the expression levels of THBS1 and its transcription factor E2F1 protein in HLEpi Cs were increased in HG group.Compared with the control group,the expression levels of THBS1 and its transcription factor E2F1 protein were decreased in HLEpi Cs treated with the antioxidant Tempol.IF showed that HG induced E2F1 into the nucleus,and ROS was involved in this process.Conclusions:This study demonstrates for the first time that high glucose induces E2F1 nuclear entry in HLEpi Cs to mediate increased THBS1 expression through oxidative stress,and that inhibition of THBS1 and its receptors CD36 and CD47 can attenuate HLEpi Cs apoptosis under high glucose conditions.Our findings suggest that THBS1 and its receptors CD36 and CD47 play an important role in HLEpi Cs apoptosis induced by high glucose and may be key potential targets for the prevention or treatment of diabetic cataract.