The Role And Mechanism Of Chloride Channel 3 In Oxidative Stress-induced Apoptosis And Extracellular Matrix Deposition In Human Trabecular Meshwork Cells

Background:Primary open-angle glaucoma(POAG)is a common blinding eye disease.Pathological increase of intraocular pressure(IOP)is the most important risk factor for POAG.The trabecular meshwork is the main pathway for drainage of aqueous humor out of the eye,and it's dysfunction will increase aqueous outflow resistance,leading to increased IOP.Studies have shown that oxidative stress is involved in the pathological damage of POAG trabecular meshwork,promoting the apoptosis of trabecular meshwork cells,and increasing the abnormal synthesis of extracellular matrix.Therefore,exploring the molecular mechanism of oxidative stress-induced injury in the trabecular meshwork and seeking effective antioxidant targets are crucial for the prevention and treatment of POAG.The voltage-gated chloride channel(CLC)is abundantly expressed in mammals.CLC3 is closely related to the transepithelial transport of fluid and ion.It plays an important role in various physiological and pathological processes such as cell volume regulation,proliferation,apoptosis,and phagocytosis.Our previous studies showed that CLC3 affects trabecular meshwork filtration function in cell phagocytosis,cell proliferation,etc.Next,we will explore the role and mechanism of CLC3 in oxidative stress injury of the trabecular meshwork.H₂O₂ is a classic oxidative stress inducer,which is often used in researches related to cellular oxidative stress.However,the oxidative stress response induced by H₂O₂ is sharp,which is different from the physiological conditions in vivo.We are committed to finding an oxidative stress-inducing factor and building a oxidative stress model that is closer to physiological state.Angiotensin II(Ang II)is a stress-inducing factor that exists in the body.It can activate reduced nicotinamide adenine dinucleotide phosphate oxidase(NOX),under emotional stress and cold stimulation,inducing cellular oxidative stress.The renin-angiotensin system is widely expressed in the trabecular meshwork tissue and is significantly elevated in the aqueous humor of POAG patients.Therefore,we chose H₂O₂ and Ang II to construct two oxidative stress models to explore the role and mechanism of CLC3 in the two oxidative stress models,in order to seek new targets for the treatment of glaucoma.Objective:To explore the role and mechanism of CLC3 in H₂O₂ and Ang II-induced oxidative stress damage in human trabecular meshwork cells(HTMCs).Methods:1.After approval by the Ethics Committee of the Second Hospital of Jilin University,we collected the trabecular meshwork tissue discarded by POAG patients for surgery,and performed frozen section and immunofluorescence staining to detect the expression of CLC3.2.The H₂O₂-induced oxidative stress model of HTMCs was established,and CLC3 protein expression,cell viability,cell morphology,intracellular oxidative stress level and cell apoptosis were detected by CCK-8,immunofluorescence,Western Blot and other experimental methods.The oxidative stress model of HTMCs was intervened with the oxidative stress inhibitor NAC,CLC3 overexpression lentivirus and CLC3si RNA,respectively,and the changes of cell viability,cellular oxidative stress level and apoptosis were detected.To explore the effect of CLC3 overexpression lentivirus and CLC3 si RNA intervention on the activation of the antioxidant signaling pathway Nrf2/HO-1.HTMCs were co-treated with CLC3-overexpressing lentivirus and Nrf2si RNA to clarify the signaling pathway of CLC3 regulating cellular oxidative stress and apoptosis.3.The Ang II-induced HTMCs oxidative stress model was established,and CLC3protein expression,cell viability,intracellular oxidative stress level and extracellular matrix expression were detected by CCK-8,q PCR,immunofluorescence,Western Blot and other experimental methods.The HTMCs oxidative stress model was intervened with the oxidative stress inhibitor NAC and CLC3 overexpressing lentivirus,respectively,and the changes of cellular oxidative stress and extracellular matrix were detected.Next,we explore the effect of CLC3 overexpression lentivirus on the activation of the antioxidant signaling pathway Nrf2/HO-1.Results:1.Frozen sections and immunofluorescence staining were performed on the trabecular meshwork tissue of 3 POAG patients collected.The results showed that CLC3 was strongly positive expressed in the trabecular meshwork tissue of POAG patients.2.The HTMCs oxidative stress model induced by H₂O₂ was successfully constructed.The 200?M H₂O₂ was used for 24 hours,the cell viability was significantly decreased,the intracellular ROS content and MDA content were significantly increased,and the mitochondrial membrane potential decreased.Western Blot results showed that the ratio of Bcl-2/BAX was decreased,and the cleavage and activation of caspase3 was enhanced.5m M NAC pretreatment significantly enhanced HTMCs cell viability(%)and reduced H₂O₂-induced oxidative stress and mitochondrial pathway apoptosis.These results suggest that H₂O₂ induces mitochondrial pathway apoptosis in HTMCs through oxidative stress response.H₂O₂ induced CLC3 protein expression in HTMCs,suggesting that CLC3 is involved in H₂O₂-induced oxidative stress.The results of q PCR and Western Blot showed that CLC3 overexpression and knockdown HTMCs cells were successfully constructed.CLC3 knockdown inhibited Nrf2/HO-1 protein expression and promoted oxidative stress and mitochondrial pathway apoptosis in HTMCs cells.Overexpression of CLC3 promotes Nrf2 nuclear transfer,promotes Nrf2/HO-1 protein expression,and inhibits oxidative stress and mitochondrial pathway apoptosis in HTMCs.Nrf2knockdown disrupted the anti-oxidative and anti-apoptotic functions of CLC3overexpression.3.An Ang II-induced oxidative stress model was successfully constructed.After 1?M Ang II was used for 24 hours,the intracellular ROS content increased.In addition,the levels of NOX1 and NOX2 m RNA increased.Ang II promotes the expression of Fibronectin and Collagen1.NAC and Irbesartan block Ang II-induced oxidative stress and increased extracellular matrix expression.Ang II promotes CLC3 protein expression.CLC3 overexpression promotes Nrf2 nuclear transfer,promotes HO-1protein expression,and inhibits Ang II-induced oxidative stress and extracellular matrix deposition.conclusion:1.CLC3 was expressed in the trabecular meshwork tissue of POAG patients.2.The H₂O₂-induced oxidative stress model of HTMCs was successfully constructed.H₂O₂ induces the mitochondrial-pathway apoptosis of HTMCs through oxidative stress.H₂O₂ promotes CLC3 expression of HTMCs.CLC3 inhibits H₂O₂-induced oxidative stress injury and apoptosis of HTMCs by promoting the Nrf2/HO-1signaling pathway.3.The Ang II-induced oxidative stress model of HTMCs was successfully constructed.Ang II induces extracellular matrix deposition through oxidative stress,which points that Ang II plays an important role in POAG development.CLC3 also inhibits Ang II-induced oxidative stress and extracellular matrix deposition in HTMCs by promoting Nrf2/HO-1 signaling pathway.4.The chloride channel CLC3 has a protective effect on oxidative stress in the trabecular meshwork induced by H₂O₂ and Ang II,and CLC3 may become a new target for POAG therapy.