Effect Of Chloride Channel ClC-3 In Apoptosis Of Human Lens Epithelial Cells

Background and Objective:Cataract is a familiar ophthalmologic disease in clinic and the world's leading cause of blindness,which directly affects people's quality of life.Apoptosis of lens epithelial cells is an important pathological basis for the occurrence and development of cataract.Therefore,it is of great significance to study the mechanism of lens epithelial cells apoptosis for the prevention and treatment of cataract.A large number of studies have found that apoptosis of lens epithelial cells is regulated by a variety of pathways,among which oxidative stress and endoplasmic reticulum stress play an important role in apoptosis of lens epithelial cells.Chloride ion is the most abundant anion in organism,which exists in all tissues and cells of the body.Voltage gated chloride channel(CLC)is a member of chloride channel family,and ClC-3 is one of its subtypes.ClC-3 is widely distributed in mammalian tissues,organs and various cells,and plays an important role in maintaining the balance of cell volume,regulating cell electrical activity,cell proliferation,cell apoptosis and other physiological and pathological processes.Some studies have confirmed that ClC-3 is involved in the proliferation and migration of lens epithelial cells,which is closely related to posterior capsule opacification.However,its role in apoptosis of lens epithelial cells has not been fully elucidated.In this study,human lens epithelial cells(HLECs)were used as the research object.The effects of chloride channel blocker 5-nitro-2-(3-phenylpropaamino)-benzoic acid(NPPB)were used to investigate the effect of chloride channel on apoptosis of HLECs.The apoptosis model of HLECs was established by hydrogen peroxide and light.The role and mechanism of ClC-3 in apoptosis of HLECs were further studied.The study provided a new theoretical basis for prevention and treatment of cataract.Method:1.The effect and mechanism of chloride channel on apoptosis of HLECs.We treated HLECs with different concentrations of NPPB.CCK-8 was used to detect the effect of different concentrations of NPPB on the cell viability of HLECs,and the appropriate concentration was selected for subsequent experiments.The suitable concentration of NPPB was applied to HLECs.The fluorescence intensity of mitochondrial membrane potential(JC-1)and reactive oxygen species(ROS)were detected by fluorescence staining.The expression levels of BAX,Bcl-2 and cleaved caspase 3 were detected by Western blot;The fluorescence intensity of intracellular calcium was detected by fluorescence staining.The expression levels of endoplasmic reticulum stress related proteins ATF6,JNK,CHOP,PERK,p-PERK and caspase 12were detected by Western Blot to evaluate the occurrence of endoplasmic reticulum stress in HLECs.ROS scavenger NAC and endoplasmic reticulum stress inhibitor 4-PBA combined with NPPB were used to treat HLECs.The effects of different concentrations of NAC and 4-PBA on the cell viability of HLECs were detected by CCK-8,and the appropriate concentration was selected for subsequent experiments.Appropriate concentration of NAC and 4-PBA were combined with 50?M NPPB to detect the changes of oxidative stress,apoptosis and endoplasmic reticulum stress.2.Light injury model and oxidative stress damage model were established in HLECs.The changes of oxidative stress,apoptosis,endoplasmic reticulum stress and the expression of ClC-3 were detected in these two models.HLECs were exposed to 1500 lux visible light and different concentrations of hydrogen peroxide for different time,respectly.CCK-8 was used to detect the effects of light and hydrogen peroxide on cell viability of HLECs,and the appropriate concentration and time were selected for subsequent experiments.HLECs were exposed to suitable light and hydrogen peroxide.The fluorescence intensity of mitochondrial membrane potential(JC-1)and ROS were detected by fluorescence staining.The expression levels of BAX,Bcl-2 and cleaved caspase 3 were detected by Western blot;The fluorescence intensity of intracellular calcium was detected by fluorescence staining.The expression levels of endoplasmic reticulum stress related proteins ATF6,JNK,CHOP,PERK,p-PERK and caspase 12 were detected by Western Blot to evaluate the occurrence of endoplasmic reticulum stress in HLECs.The expression and localization of ClC-3 in HLECs were detected by immunofluorescence.The changes of the expression of ClC-3 in protein levels under different time of hydrogen peroxide and light were detected by Western blot method.3.To establish ClC-3 overexpression HLECs and explore the role and mechanism of ClC-3 in oxidative stress injury model and light injury model of HLECs.Hydrogen peroxide damage model and light damage model were constructed in overexpressing ClC-3 HLECs.The infection efficiency of lentivirus was determined by fluorescence.The overexpression of ClC-3 in HLECs was verified by q-PCR and Western blot.The fluorescence intensity of mitochondrial membrane potential(JC-1)and ROS were detected by fluorescence staining.The expression levels of cleaved caspase 3 were detected by Western blot;The fluorescence intensity of intracellular calcium was detected by fluorescence staining.The expression levels of endoplasmic reticulum stress related proteins ATF6,JNK,PERK,and p-PERK were detected by Western Blot to evaluate the occurrence of endoplasmic reticulum stress in HLECs.To observe the relationship between ClC-3 and oxidative stress,endoplasmic reticulum stress and mitochondrial apoptosis in HLECs.Result:1.Chloride channel blocker NPPB inhibited the cell viability of HLECs in a concentration dependent manner.50?M NPPB could decrease the mitochondrial membrane potential,up regulate the expression of BAX and cleaved caspase 3,and down regulate the expression of Bcl-2 in HLECs.These results suggested that NPPB can induce apoptosis in mitochondrial pathway of HLECs.NPPB can induce the increase of ROS and calcium in cells,and up regulate the expression of ATF6,JNK,chop,p-perk and caspase 12.The apoptosis,oxidative stress and endoplasmic reticulum stress induced by NPPB can be reduced by ROS scavenger NAC and endoplasmic reticulum stress inhibitor 4 PBA.It is suggested that chloride channel may protect HLECs through oxidative stress and endoplasmic reticulum stress.2.The models of HLECs damaged by light and oxidative stress were established.The cell viability of HLECs decreased gradually under 1500 lux visible light in a time dependent manner,and cell viability of HLECs decreased gradually under hydrogen peroxide in a concentration and time dependent manner.After exposure to 200?M hydrogen peroxide and 1500 lux visible light for 24 hours,the mitochondrial membrane potential of HLECs decreased,the expression of BAX and cleaved caspase 3 was up-regulated,the expression of Bcl-2 was down regulated,the intracellular ROS and Ca²⁺were increased,and the expression of endoplasmic reticulum stress-related proteins ATF6,JNK,chop,p-perk and caspase 12 were up-regulated.It is suggested that both light injury model and oxidative stress damage model are related to mitochondrial apoptosis induced by oxidative stress and endoplasmic reticulum stress in HLECs.The expression of ClC-3 protein in HLECs was confirmed by immunofluorescence staining,and the expression of ClC-3 was located in the endoplasmic reticulum and other cytoplasm.The expression of ClC-3 protein in HLECs was detected after exposure to 200?m hydrogen peroxide and 1500 lux visible light for 24 hours,48 hours and 72 hours.Western blot analysis showed that the expression of ClC-3 was up-regulated in HLECs exposed to 200?m hydrogen peroxide and 1500lux visible light for 24 and 48 hours.At 72 hours,the expression of ClC-3 in HLECs decreased and was lower than the normal level.These results suggest that ClC-3 may be involved in the process of oxidative stress injury and light injury of HLECs,ClC-3played a protective role in HLECs.3.Lentivirus containing ClC-3 c DNA was used to infect HLECs,and stable overexpression of ClC-3 HLECs were constructed.The overexpression of ClC-3 in HLECs at m RNA and protein levels was verified by q-PCR and Western blot.Compared with HLECs infected with empty vector,overexpression of ClC-3 HLECs can reduce the production of ROS,the accumulation of calcium ions,inhibit the expression of p-PERK,ATF6 and JNK in endoplasmic reticulum stress pathway,reduce the activation of caspase 3,and reduce the expression of cleaved caspase 3,thus reducing the apoptosis of mitochondrial pathway.These results suggest that ClC-3 can inhibit the apoptosis of mitochondrial pathway in HLECs by reducing the oxidative stress and endoplasmic reticulum stress induced by hydrogen peroxide and light.Conclusion:1.NPPB,a chloride channel blocker,can promote oxidative stress,endoplasmic reticulum stress and mitochondrial pathway apoptosis in HLECs.Chloride channel has protective effect on lens epithelial cells.2.Hydrogen peroxide damage and light injury can induce the production of ROS and endoplasmic reticulum stress and promote cell apoptosis in HLECs.We successfully constructed the HLECs model of light damage and oxidative stress damage.3.In HLECs,the expression of ClC-3 was localized in endoplasmic reticulum and other cytoplasm;in HLECs with oxidative stress injury and light injury,the expression of ClC-3 changed.4.ClC-3 can inhibit oxidative stress,endoplasmic reticulum stress and apoptosis in mitochondrial pathway induced by hydrogen peroxide and light.ClC-3 has protective effect on lens epithelial cells.