Background:This study compared the role of autophagy regulators Rapamycin and 3-MA in oxidative damage and apoptosis of human lens epithelial cells(HLECs)caused by two doses of Ultraviolet Radiation B(UVB).Methods:HLE...Background:This study compared the role of autophagy regulators Rapamycin and 3-MA in oxidative damage and apoptosis of human lens epithelial cells(HLECs)caused by two doses of Ultraviolet Radiation B(UVB).Methods:HLECs were irradiated with UVB,and two doses of UVB damage models were constructed.After treatment with autophagy regulators,cell damage tests such as CCK-8,LDH activity,and Ros detection were performed.Western blotting was used to detect the levels of autophagy-related proteins and apoptosis-related proteins.Quantitative real-time PCR(RT-qPCR)was used to detect the mRNA leve of secondary antioxidant enzymes.Flow cytometry was used to examine cell viability and apoptosis.Finally,the proportion of autophagy and apoptosis was observed by electron microscope.Results:Autophagy inhibitor 3-MA promoted oxidative damage and apoptosis of HLECs at low doses of UVB(5 mJ/cm2),which corresponds to 1.3 h of exposure to sunlight in human eyes.Under the high dose of UVB(50mJ/cm2),which is equivalent to 13 h of exposure to sunlight in human eyes,the autophagy inducer Rapamycin caused more extensive oxidative damage and apoptosis of HLECs.3-MA was able to reduce this damage,indicating that moderate autophagy is necessary for HLECs to cope with mild oxidative stress.For high dose UVBinduced oxidative stress,the use of 3-MA inhibiting autophagy is more beneficial to reduce cell damage and apoptosis.The mechanisms include degradation of damaged organelles,regulation of the expression of antioxidant enzymes HO-1,NQO1,GCS and regulation of apoptosis-related proteins.Conclusions:Autophagy played different roles in HLECs oxidative stress induced by two doses of UVB.It provides new ideas for reducing oxidative damage and apoptosis of HLECs to prevent or delay the progression of agerelated cataract(ARC).展开更多
Objective： Ultraviolet （UV） radiation is one of the important cataract risk factors, However, the pathogenesis is still poorly understood. The migration of human lens epithelial cells（HLECs） plays a Clucial role ...Objective： Ultraviolet （UV） radiation is one of the important cataract risk factors, However, the pathogenesis is still poorly understood. The migration of human lens epithelial cells（HLECs） plays a Clucial role in the remodeling of lens capsule and cataract formation. The purpose of this study is to investigate the mechanism of UV inducing cataractogenesis. Methods：The toxicity of UV-irradiation on HLECs was assessed by Methyl thiazolyl tetrazolium（MTT） assay. The activity of matrix metalloproteinase-2（MMP-2） was observed by Gelatin zymography. The migration of HLECs was examined by Cell Track Motility. Results：UV-irradiation does great harm to HLECs, and may induce apoptosis in the cells when UV higher than 15 mj/cm2. UV significantly increased MMP-2 activity in a timedependent manner. In addition, the irradiation could induce the migration of HLECs. Conclusion：UV-irradiation could induce the migration of HLECs by increasing the activity of MMP-2.展开更多
Posterior capsule opacification(PCO)remains the predominant complication following cataract surgery,significantly impairing visual function restoration.In this study,we developed a PCO model that closely mimics the an...Posterior capsule opacification(PCO)remains the predominant complication following cataract surgery,significantly impairing visual function restoration.In this study,we developed a PCO model that closely mimics the anatomical structure of the crystalline lens capsule post-surgery.The model incorporated a threaded structure for accurate positioning and observation,allowing for opening and closing.Utilizing 3D printing technology,a stable external support system was created using resin material consisting of a rigid,hollow base and cover.To replicate the lens capsule structure,a thin hydrogel coating was applied to the resin scaffold.The biocompatibility and impact on cellular functionality of various hydrogel compositions were assessed through an array of staining techniques,including calcein-AM/PI staining,rhodamine staining,BODIPY-C11 staining and EdU staining in conjunction with transwell assays.Additionally,the PCO model was utilized to investigate the effects of eight drugs with anti-inflammatory and anti-proliferative properties,including 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR),THZ1,sorbinil,4-octyl itaconate(4-OI),xanthohumol,zebularine,rapamycin and caffeic acid phenethyl ester,on human lens epithelial cells(HLECs).Confocal microscopy facilitated comprehensive imaging of the PCO model.The results demonstrated that the GelMA 605%þPLMA 2%composite hydrogel exhibited superior biocompatibility and minimal lipid peroxidation levels among the tested hydrogels.Moreover,compared to using hydrogel as the material for 3D printing the entire model,applying surface hydrogel spin coating with parameters of 2000 rpm�2 on the resin-based 3D printed base yielded a more uniform cell distribution and reduced apoptosis.Furthermore,rapamycin,4-OI and AICAR demonstrated potent antiproliferative effects in the drug intervention study.Confocal microscopy imaging revealed a uniform distribution of HLECs along the anatomical structure of the crystalline lens capsule within the PCO model,showcasing robust cell viability and regular morphology.In conclusion,the PCO model provides a valuable experimental platform for studying PCO pathogenesis and exploring potential therapeutic interventions.展开更多
基金the National Natural Science Foundation of China(Grant no.81970781)the Natural Science Foundation of Zhejiang Province(no.LY17H090004)the National Natural Science Foundation of China(Grant no.81800807).
文摘Background:This study compared the role of autophagy regulators Rapamycin and 3-MA in oxidative damage and apoptosis of human lens epithelial cells(HLECs)caused by two doses of Ultraviolet Radiation B(UVB).Methods:HLECs were irradiated with UVB,and two doses of UVB damage models were constructed.After treatment with autophagy regulators,cell damage tests such as CCK-8,LDH activity,and Ros detection were performed.Western blotting was used to detect the levels of autophagy-related proteins and apoptosis-related proteins.Quantitative real-time PCR(RT-qPCR)was used to detect the mRNA leve of secondary antioxidant enzymes.Flow cytometry was used to examine cell viability and apoptosis.Finally,the proportion of autophagy and apoptosis was observed by electron microscope.Results:Autophagy inhibitor 3-MA promoted oxidative damage and apoptosis of HLECs at low doses of UVB(5 mJ/cm2),which corresponds to 1.3 h of exposure to sunlight in human eyes.Under the high dose of UVB(50mJ/cm2),which is equivalent to 13 h of exposure to sunlight in human eyes,the autophagy inducer Rapamycin caused more extensive oxidative damage and apoptosis of HLECs.3-MA was able to reduce this damage,indicating that moderate autophagy is necessary for HLECs to cope with mild oxidative stress.For high dose UVBinduced oxidative stress,the use of 3-MA inhibiting autophagy is more beneficial to reduce cell damage and apoptosis.The mechanisms include degradation of damaged organelles,regulation of the expression of antioxidant enzymes HO-1,NQO1,GCS and regulation of apoptosis-related proteins.Conclusions:Autophagy played different roles in HLECs oxidative stress induced by two doses of UVB.It provides new ideas for reducing oxidative damage and apoptosis of HLECs to prevent or delay the progression of agerelated cataract(ARC).
基金This work was supported by the project of the Natural Science Foun- dation of Jiangsu province(No.BK2007234) the Institution Natural Science Foundation of Jiangsu province(No.07KJD320140)
文摘Objective： Ultraviolet （UV） radiation is one of the important cataract risk factors, However, the pathogenesis is still poorly understood. The migration of human lens epithelial cells（HLECs） plays a Clucial role in the remodeling of lens capsule and cataract formation. The purpose of this study is to investigate the mechanism of UV inducing cataractogenesis. Methods：The toxicity of UV-irradiation on HLECs was assessed by Methyl thiazolyl tetrazolium（MTT） assay. The activity of matrix metalloproteinase-2（MMP-2） was observed by Gelatin zymography. The migration of HLECs was examined by Cell Track Motility. Results：UV-irradiation does great harm to HLECs, and may induce apoptosis in the cells when UV higher than 15 mj/cm2. UV significantly increased MMP-2 activity in a timedependent manner. In addition, the irradiation could induce the migration of HLECs. Conclusion：UV-irradiation could induce the migration of HLECs by increasing the activity of MMP-2.
基金supported by research grants from the National Natural Science Foundation of China(82122017,82271069 and 81900839)Shanghai Science and Technology Commission(22xtcx00103)Clinical Science and Technology Innovation Project of Shanghai Shenkang Hospital Development Center(SHDC12019X08,SHDC12020111 and SHDC2020CR4078).
文摘Posterior capsule opacification(PCO)remains the predominant complication following cataract surgery,significantly impairing visual function restoration.In this study,we developed a PCO model that closely mimics the anatomical structure of the crystalline lens capsule post-surgery.The model incorporated a threaded structure for accurate positioning and observation,allowing for opening and closing.Utilizing 3D printing technology,a stable external support system was created using resin material consisting of a rigid,hollow base and cover.To replicate the lens capsule structure,a thin hydrogel coating was applied to the resin scaffold.The biocompatibility and impact on cellular functionality of various hydrogel compositions were assessed through an array of staining techniques,including calcein-AM/PI staining,rhodamine staining,BODIPY-C11 staining and EdU staining in conjunction with transwell assays.Additionally,the PCO model was utilized to investigate the effects of eight drugs with anti-inflammatory and anti-proliferative properties,including 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR),THZ1,sorbinil,4-octyl itaconate(4-OI),xanthohumol,zebularine,rapamycin and caffeic acid phenethyl ester,on human lens epithelial cells(HLECs).Confocal microscopy facilitated comprehensive imaging of the PCO model.The results demonstrated that the GelMA 605%þPLMA 2%composite hydrogel exhibited superior biocompatibility and minimal lipid peroxidation levels among the tested hydrogels.Moreover,compared to using hydrogel as the material for 3D printing the entire model,applying surface hydrogel spin coating with parameters of 2000 rpm�2 on the resin-based 3D printed base yielded a more uniform cell distribution and reduced apoptosis.Furthermore,rapamycin,4-OI and AICAR demonstrated potent antiproliferative effects in the drug intervention study.Confocal microscopy imaging revealed a uniform distribution of HLECs along the anatomical structure of the crystalline lens capsule within the PCO model,showcasing robust cell viability and regular morphology.In conclusion,the PCO model provides a valuable experimental platform for studying PCO pathogenesis and exploring potential therapeutic interventions.
