| Objective:The International Commission on Non-Ionising Radiation Protection(ICNIRP)defines the 280-315 nm wavelength portion of UV radiation as Ultraviolet Radiation B(UVB),based on wavelength and radiation level.For everyday human health,moderate exposure to UVB can promote calcium absorption and health,but excessive UVB exposure can induce the development of some eye diseases such as age-related cataracts.Cataracts,as one of the major blinding eye diseases,have become one of the most important public health issues to be addressed.Studies have shown that oxidative damage caused by UVB exposure and apoptosis of lens epithelial cells are the main causes of cataracts.When UVB acts on the lens,it can increase the amount of Reactive Oxygen Species(ROS)in the lens,leading to oxidative damage to the lens.However,the exact mechanism of action has not been demonstrated in detail.Sirtuin 1(Sirt1),a deacetylase,has been shown in previous studies to regulate cell function,mitigate apoptosis and delay cellular senescence.Previous studies have shown a decrease in Sirt1levels in the lens and an increase in apoptosis after UVB exposure.The transcription factor NF-E2-related factor(Nrf2)and its cytoplasmic junction protein(Kelch like ECH associated protein 1,Keap1)are important regulators of resistance to oxidative stress and play an important role in the response to damage in the oxidative-oxidative imbalance of the body.It plays an important role in the response to oxidative-oxidative damage.However,it is not known whether UVB regulates Nrf2 through Sirt1,thereby affecting oxidative damage and apoptosis in lens epithelial cells.Resveratrol(Rsv),a natural antioxidant,has been shown to act as an activator of Sirt1,and EX527 is widely used as an inhibitor of Sirt1.In this study,we investigated the molecular mechanism of lens oxidative damage and apoptosis during UVB-induced cataract by analysing the expression of Sirt1 and its downstream signalling pathway-related molecules after UVB exposure,and provided molecular targets and theoretical and experimental basis for the treatment and prevention of UVB-induced cataract.Methods:1.The UVB exposure model of female Sprague-Dawley rats was established.According to body weight,the rats were random Ly divided into control group(normal light),Rsv control group(Rsv gavage 25 mg/kg),EX527 control group(EX527intraperitoneal injection of 5 mg/kg),UVB treatment group(UVB wavelength 312 nm,irradiation time 15 min,UV irradiation time 15 min,UV irradiation time 15 min,UV irradiation time 15 min,UV irradiation time 15 min.Radiation intensity of 900μW/cm~2),Rsv pretreatment group(Rsv 25 mg/kg,2 h later UVB exposure,wavelength of 312 nm,irradiation time of 15 min,irradiation intensity of 900μW/cm~2),EX527 pretreatment group(EX527 intraperitoneal injection of 5 mg/kg,radiation intensity of 900μW/cm~2)Two hours later,they were exposed to UVB at a wavelength of 312 nm,irradiation time of 15 min,and irradiation intensity of 900μW/cm~2).After 7 days of continuous exposure,the cumulative radiation was 8.1 k J/m~2.The degree of lens damage caused by UVB exposure was evaluated by observing and calculating the opacification area under slit lamp microscope.The apoptosis rate of lens epithelial cells was evaluated by flow cytometry.The mRNA and protein expression levels of apoptosis-related proteins Bcl-2,Bax and Caspase-3 were detected by RT-qPCR and Western Blot.The protein expression level of CRYAA was detected by Western Blot.The ROS level of lens epithelial cells was evaluated by flow cytometry.The levels of MDA,SOD,GSH,GSH-Px and CAT in the lens of rats were detected by the kit.The amount of Sirt1 protein was detected by ELISA.The mRNA and protein expression levels of Sirt1,Keap1,Nrf2,Nqo1,Sod-1,Gpx-1 and Ho-1 were detected by Real time-qPCR and Western Blot.2.In this study,UVB exposure model of human lens epithelial cells(SRA01/04)were established.Cells in logarithmic growth phase were cultured with 20μM Rsv and10μM EX527 for 0.5 h,1 h,2 h and 4 h,respectively.Cell viability was detected by CCK-8(Cell Kounting Kit-8)kit,and the optimal intervention time of Rsv and EX527was determined.According to the previous experiments of the research group(UVB exposure wavelength of 312 nm,irradiation intensity of 150μW/cm~2,irradiation time of120 s,cumulative radiation amount of 0.18 k J/m~2),the experimental groups were finally determined as control group(culture medium)and Rsv control group(20μm pretreatment for 2 h).EX527 control group(10μm pretreated for 1 h),UVB treatment group(wavelength 312 nm,irradiation intensity 150μW/cm~2,irradiation time 120 s),Rsv pretreatment group(20μmRsv pretreated for 2 h,UVB exposure wavelength 312nm,irradiation intensity 150μW/cm~2,the irradiation time was 120 s),the UVB exposure wavelength was 312 nm,the irradiation intensity was 150μW/cm~2,and the irradiation time was 120 s after pretreatment with 10μm EX527 for 1 h.The cumulative radiation dose was 0.18 k J/m~2.The morphology of cells in each group was observed under bright field under microscope.Co-immunoprecipitation was used to detect the interaction between Keap1 and Nrf2,and Sirt1 and Keap1.The interactions between Keap1 and Nrf2,Sirt1 and Keap1 were detected by fluorescence colocalization.Results:1.In vivo experiments of rats:compared with the control group,the lens of UVB treatment group showed white fog opacity.Compared with the UVB treatment group,the opacities in the Rsv pretreatment group were smaller than those in the UVB treatment group,and the opacities in the EX527 pretreatment group were larger than those in the UVB treatment group,indicating that the UVB-induced cataract exposure model was successfully established.Compared with the control group,the early apoptosis rate,protein expression and mRNA content of Bax/Bcl-2 and Cleaved Caspase3 were increased in the UVB treatment group.The protein expression level of CRYAA was significantly decreased.The levels of ROS and MDA increased,while the levels of GSH,GSH-Px,SOD and CAT decreased.With UVB exposure,the enzyme activity,protein expression and mRNA level of Sirt1 decreased,the protein expression and mRNA content of Keap1 increased,and the protein expression and mRNA content of Nrf2 and its downstream related factors decreased.These results suggest that UVB can induce oxidative stress and apoptosis of lens epithelial cells in rat lens.Compared with UVB treatment group,the early apoptosis rate,protein expression and mRNA content of Bax/Bcl-2 and Cleaved Caspase3 were decreased,and the protein expression level of CRYAA was increased in Rsv pretreatment group.The levels of ROS and MDA decreased,while the expressions of GSH,GSH-Px,SOD and CAT increased.The enzyme activity,protein expression and mRNA level of Sirt1 increased,the protein expression level and mRNA content of Keap1 decreased,and the protein expression and mRNA content of Nrf2 and its downstream related factors increased.The results of the EX527 pretreatment group were opposite.These results suggest that UVB can cause the abnormalities of Sirt1/Keap1/Nrf2 pathway,and Sirt1 plays an important role in UVB-induced oxidative damage and apoptosis of lens epithelial cells.2.In vitro cell experiments:compared with the control group,the cell viability of UVB treatment group was decreased,and the viability of Rsv control group and EX527control group was slightly increased,but the difference was not statistically significant.Compared with the UVB-treated group,cell viability was increased in the resveratrol pretreatment group and decreased in the EX527 pretreatment group,suggesting that UVB exposure caused damage to human lens epithelial cells.Compared with the control group,Sirt1 deacetylase activity was decreased and the acetylation levels of Keap1 and Nrf2were increased in the UVB-treated group;compared with the UVB group treatment group,Sirt1 deacetylase activity was increased and the acetylation levels of Keap1 and Nrf2 were decreased in the resveratrol pretreatment group,and Sirt1 deacetylase activity was decreased and the acetylation levels of Keap1 and Nrf2,suggesting that UVB exposure reduced Sirt1 deacetylase activity and negatively regulated the acetylation levels of Keap1 and Nrf2.According to the results of immunoprecipitation and fluorescence co-localization,compared with the control group,the protein interaction ability between Sirt1/Keap1 and Nrf2/Keap1 was decreased in the UVB treatment group.Compared with UVB treatment group,the protein interaction ability between Sirt1/Keap1 and Nrf2/Keap1 in Rsv pretreatment group was increased,while the protein interaction ability between Sirt1/Keap1 and Nrf2/Keap1 in EX527 pretreatment group was decreased.These results suggest that the regulation of Keap1/Nrf2 pathway by Sirt1plays an important role in UVB-induced lens epithelial cell injury.Conclusion:UVB exposure causes early signs of cataract in rats with lens clouding and induces oxidative stress and apoptosis in lens epithelial cells by interfering with the Sirt1-regulated Keap1/Nrf2 pathway. |
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