Study On The Effect And Mechanism Of Lack Of Hsf4 Upregulating ROS Induced Cataract

BackgroundCataract is the main cause of blindness in the world,and many factors cause the disorder of lens structure,which leads to cataract,such as genetic mutation,aging,ultraviolet radiation,diabetes,long-term exposure to some chemical drugs,decreased antioxidant capacity and so on.According to the etiology,cataract can be divided into congenital cataract,age-related cataract,posterior cataract and cataract secondary to other causes.At present,the main treatment of cataract is phacoemulsification,the effect of drug treatment is not obvious.Therefore,revealing the pathogenesis of cataract and providing a new theoretical basis and research target for the clinical treatment of cataract is of great significance to solve this major public health problem.At present,more than 40 genes encoding crystallins and transcription factors have been found to be the pathogenic genes of cataract.Heat shock transcription factor 4（heatshocktranscriptionfactor4,Hsf4）,a member of the heat shock transcription factor family,is a known cataract pathogenic gene.This family mediates heat shock response by binding to heat shock element（Heat Shock Response Element,HSE）transcription to regulate the expression of heat shock proteins.Studies have shown that mutations in R74H and L115P of Hsf4 will cause congenital cataract,while mutations in Q62R and R117H will lead to senile cataract.In previous laboratory studies,it was found that the C57/BL6 mouse model with amino acid deletion in exon 5of Hsf4 gene showed obvious cataract,and Hsf4 directly regulated the expression of Hsp25 and B-crystallin,while Hsp25 and B-crystallin were involved in the cellular oxidation defense system.Many studies have found that Hsf4 plays an important role in the differentiation and development of lens.However,the mechanism of cataract caused by Hsf4 dysfunction is not clear.Oxidative stress is considered to be the beginning of cataract.When the redox state of lens is out of balance,that is,excessive production of reactive oxygen species,the accumulation of damage caused by oxidative stress accelerates the aging of lens,resulting in abnormal cell proliferation and cell cycle,and eventually develops into cataract.Studies have shown that Hsf4can directly regulate the expression of heme oxygenase-1（hemeoxygenase,HO-1）.HO-1 is an intracellular antioxidant and protective enzyme,which is regulated by the Keap1-NRF2 pathway and plays an important role in the cellular antioxidant system.This study suggests that Hsf4 may be involved in the regulation of lens oxidative stress defense system,but the mechanism of Hsf4involved in lens antioxidant stress is not completely clear.Further exploration is still needed.ObjectiveHsf4^del42mice of different ages and Hsf4 knockout lens epithelial cells were used as models to explore the role of Hsf4 in lens oxidative stress biological defense system and its molecular mechanism,so as to provide new ideas for further elucidating the mechanism of cataract and developing its targeted drugs.Methods1.Wild-type and Hsf4 mutant mice of different ages were selected and killed under anaesthesia.The lens of eyeball was taken and the content of GSH/GSSG was detected by homogenate lysis kit.The expression levels ofγH2AX and 4-HNE were detected by immunoblotting.2.The lens of wild type and Hsf4 mutant mice of different ages were taken out after anaesthesia.The total protein and total RNA of lens were extracted after homogenate lysis.The expression levels of oxidative stress related proteins and genes were detected by Western blotting and real-time fluorescence quantitative PCR.3.Total reactive oxygen species DCFH-DA probe and mitochondrial reactive oxygen species probe mito SOX were incubated in cultured mouse lens epithelial cells.The expression level of reactive oxygen species was detected by flow cytometry and the expression level of mitochondrial reactive oxygen species was detected by immunofluorescence.4.Western blotting and real-time fluorescence quantitative PCR were used to detect the levels of oxidative stress-related proteins and genes in mouse lens epithelial cells.5.In vitro,Annexin V-FITC/PI apoptosis kit was used to detect apoptosis by flow cytometry,and immunoblotting and immunofluorescence were used to detect the expression level ofγH2AX.6.The expression level of reactive oxygen species（Ros）was detected by flow cytometry after over-expression of HO-1 protein in m LEC/Hsf4^-/-cells.7.Western blotting was used to detect the expression of Keap1-NRF2 pathway in lens tissues and lens epithelial cells.8.CHX,an inhibitor of protein synthesis,was added to m LEC and Hsf4 cells,and the degradation of Keap1 was detected by immunoblotting,and the ubiquitin modification level of NRF2 in normal cellls and Hsf4 knockout cells was detected by immunoprecipitation.9.Keap1 interference RNA was transfected into m LEC and goat cells,and the expression of reactive oxygen species（Ros）was detected by flow cytometry.10.Western blotting and immunofluorescence were used to detect the entry of NRF2into the nucleus.The cells were treated with sulforaphane to detect the entry of NRF2 into the nucleus and the expression of downstream proteins of NRF2.Results1.In the lens of Hsf4 mutant mice,the expression of GSH/GSSG was down-regulated,while the expression of 4-HNE andγH2AX was up-regulated,which was more obvious with the increase of age.The expressions of ROS-producing enzymes NOX2,NOX4 and NOX5 were up-regulated,while the expressions of antioxidant enzymes HO-1,SOD1 and CAT were down-regulated.The above results showed that the level of oxidative stress in lens of Hsf4mutant mice was increased and the antioxidant function was impaired.2.The total ROS and mitochondrial ROS of lens epithelialcells in Hsf4 knockout mice were increased,the expression of ROS producing enzymes NOX2 and NOX4 was up-regulated,and the expression of antioxidant enzymes HO-1 and NQO1 was down-regulated.The apoptosis of lens epithelial cells in Hsf4 knockout mice increased and the expression ofγH2AX was up-regulated.The results showed that the level of oxidative stress in lens of Hsf4 knockout mice increased,the function of antioxidation was impaired,and lens epithelial cells of mice suffered endogenous damage.Supplementation of HO-1 can partially reduce the up-regulation of ROS caused by Hsf4 knockout.It is suggested that the down-regulation of HO1 caused by Hsf4knockout leads to the increase of endogenous ROS in some cells.3.The expression of Keap1 was up-regulated and the expression of NRF2 was inhibited in lens epitheoiao ceoos of Hsf4 mutant mice and Hsf4 knockout mice.The degradation rate of Keap1 became slower and the ubiquitin modification level of NRF2 increased.4.After transfection of si Keap1 into normal and Hsf4 knockout mouse lens epithelial cells,only normal mouse lens epithelial cells decreased ROS and Hsf4 knockout mouse lens epithelial cells decreased NRF2 entry into the nucleus,indicating that NRF2 entry into the nucleus was blocked after Hsf4 knockout.Conclusions1.During lens development,HSF4 can up-regulate the signal pathway of antioxidant stress.HSF4 up-regulates the antioxidant stress ability of lens tissue by inhibiting the expression of oxidase NOX2 and up-regulating the expression of HO-1.2.Hsf4 can directly or indirectly regulate the degradation of KEAP protein,promote the nuclear transport of NRF2,and up-regulate the expression of antioxidant HO-1.Innovation points1.The deletion of Hsf4 resulted in the production of ROS and the decrease of antioxidant function of mouse lens and lens cells.2.The absence of 2.Hsf4 leads to cataract to modulate the production of ROS is related to HO-1.3.Hsf4 regulates the expression of NRF2 by regulating the metabolism of Keap1,while Hsf4 also regulates the entry of NRF2 into the nucleus.