| Objective: With the increasing prevalence of diabetes in the world,more and more attention has been paid to the complications of diabetes.Many ocular diseases can be caused by diabetes because eye is one of the most easily affected organs of diabetes,among which cataract is one of the main causes of vision loss in diabetic patients.However,the pathogenesis of diabetic cataract has not been fully elucidated,so it is of great clinical significance to further study the pathogenesis and delay and prevent its occurrence and development.Excessive apoptosis of human lens epithelial cells(HLECs)is considered to be one of the main pathogenic mechanisms of cataract.Apoptosis is one of the widely recognized forms of programmed cell death,but in recent years,we have a more diversified understanding of the various forms of programmed cell death,including apoptosis,pyroptosis,necroptosis,autophagy and so on.Pyroptosis,which is also known as Caspase-1-dependent apoptotic process,is mediated by activated inflammatory bodies and ultimately depends on the effects of Gasdermin D(GSDMD),accompanied by the release of pro-inflammatory factors.Pyroptosis can be induced by many pathological stimuli and plays an important role in ischemic brain injury,heart disease and many kinds of tumor diseases.Cell pyroptosis has also received increasing attention in a variety of eye diseases,including cataract.The expression of NLRP3,active Caspase-1,GSDMD-N and IL-1β in cataract patients has been shown to be significantly increased,suggesting that pyroptosis may play an important role in the pathogenesis of cataract,as well as apoptosis.Oxidative stress injury is one of the most important factors in the pathogenesis of cataract.The damage of lens epithelial cells induced by ROS(Reactive oxygen species)may lead to protein oxidation,DNA damage,lipid peroxidation,and eventually lead to cataract.DJ-1 protein,as an important antioxidant protein,is encoded by PARK7 gene on human chromosome 1 and widely distributed in many tissues and cells.Studies have shown that the overexpression of DJ-1 can effectively produce antioxidant stress response and reduce apoptosis and pyroptosis of cells,thus reducing cell damage in oxidative stress response.At present,the role of DJ-1 gene on diabetic cataract is still unclear.This study aims to further reveal the pathogenesis of diabetic cataract by studying the occurrence of pyroptosis and apoptosis in the pathogenesis of diabetic cataract,as well as the inhibitory effect and mechanism of DJ-1 gene on pyroptosis and apoptosis.Meanwhile,we also discussed whether DJ-1 overexpression can delay the progression of diabetic cataract,hoping to provide a new idea for the treatment of diabetic cataract.Methods: 1.Diabetic SD rat model was established by intraperitoneal injection of Streptozotocin(STZ),and the control group was established by intraperitoneal injection of solvent.2.Gross anatomy and HE staining were used to observe the lens opacity degree between experimental group and control group at 8 and 12 weeks.3.Real-time PCR and Western blot were used to detect the expression of DJ-1 in the LECs of SD rats in experimental group and control group.4.Western blot was used to detect the expression of NLRP3,cleaved Caspase1 and IL-1β in the LECs of SD rats in the experimental group and control group.5.Culture of human lens epithelial cells with lentivirus infection: Human lens epithelial cells HLE-B3 were separated into 5 groups including: uninfected group,empty vector group,DJ-1 overexpression group,NC group and DJ-1 silenced group,respectively.Real-time PCR and Western blot were used to verify the overexpression/silencing infection efficiency of DJ-1.6.Cell treatment: After 48 h of infection,experimental groups were separated into A: blank control group,B: hyperosmotic control group,C: high glucose group,D:high glucose + empty vector group,E: high glucose +DJ-1 overexpression group,F:high glucose +NC group,G: high glucose +DJ-1 silenced group.The hyperglycemic group was treated with medium containing 55.5 m M glucose for 48 h,and the hypertonic group was treated with medium containing 5.5 m M glucose +50 m M mannitol for 48 h.7.Real-time PCR and Western blot were used to detect the mRNA and protein expressions of pyroptosis and apoptosis-related markers in each group.8.Cell proliferation ability of each group was detected by CCK-8 assay.9.ROS,MDA,SOD,LDH kits and immunofluorescence techniques were used to evaluate the oxidative damage and antioxidant capacity of cells in each group.10.Annexin V-FITC/PI double-staining cells,JC-1 mitochondrial membrane potential detection,Acridine Orange(AO)/Ethidium Bromide(EB)staining and scanning electron microscopy were used to detect the apoptosis and pyroptosis of cells in each group.11.The datas were presented as mean ± standard deviation(SD)and All experiments were performed independently at least three times.Data analysis was performed using Graph Pad Prism Version 9.The differences between two groups were analyzed using independent samples t-test.The differences among multiple groups were analyzed using one-way ANOVA.P<0.05 was considered statistically significant.Results: 1.Diabetic SD rat model construction: The results showed that the blood glucose levels of the experimental group were 20.7±2.27mmol/ L and23.35±2.9mmol/ L respectively at 8 and 12 weeks,which were significantly higher than the control group,with statistical significance(*P<0.05).2.Lens opacity evaluation of SD rats in the experimental group and the control group: After STZ injection,the lens of SD rats showed severe opacity at 8 weeks and the lens was completely opacity at 12 weeks,while the lens of SD rats in the control group was completely transparent.HE staining results in two groups: in the control group,the lens was uniformly dyed red,and the epithelial cells were orderly and evenly distributed with regular morphology.In the experimental group,at 8 weeks the epithelial cells of the lens showed multi-level phenomenon,the cells were disordered,in different sizes,and a small number of inflammatory cells were infiltrated.At 12 weeks,the epithelial cells of the lens were exfoliated,the cells were disordered,and a large number of inflammatory cells were infiltrated.3.The expression of DJ-1 in the LECs of SD rats in the experimental and control group was detected: Real-time PCR and Western blot showed that the expression of DJ-1 in the lenses of experimental rats was down-regulated compared with that in the control group,and the difference was statistically significant(**P<0.001).4.Pyroptosis in lens cells of SD rats in experimental group and control group was detected: Western blot analysis showed that the expression levels of NLRP3,Cleaved Caspase1 and IL-1β in the LECs tissues of SD rats at 8 and 12 weeks in the experimental group were up-regulated compared with that in the control group,and the differences were statistically significant(**P<0.001).5.Lentivirus infection and high glucose treatment of cells: The cells were divided into uninfected group,empty vector group,DJ-1 overexpression group,NC group and DJ-1 silenced group.After infection with lentivirus,Real-time PCR and Western blot were used to verify the overexpression/silencing efficiency of DJ-1.The results showed that lentivirus infection was successful,and the expression of DJ-1 in each group was significantly different(**P<0.001).6.Real-time PCR and Western blot were used to detect the expression of apoptosis related genes and proteins in each group: In groups of A(control group)and C(high glucose group),the expression levels of Bax,cleaved Caspase3(Caspase3)and p53(p53 acetyl K379)in high glucose group were up-regulated,while the expression levels of Bcl-2 and SIRT1 were down-regulated,with statistical significance(**P<0.001).In groups of D(high glucose + empty vector group)and E(high glucose +DJ-1 overexpression group),the expression levels of Bax,cleaved Caspase3(Caspase3)and p53(p53 acetyl K379)were decreased,while the expression levels of Bcl-2 and SIRT1 were increased in the group E,with statistical significance(**P<0.001).In groups of G(high glucose +DJ-1 silenced group)and F(high glucose+NC group),the changes of above parameters were more obvious in the group G,and with statistical significance(**P<0.001).7.Real-time PCR and Western blot were applied to detect the expression differences of pyroptosis related genes and proteins in each group: In groups of A(control group)and C(high glucose group),the expression levels of NLRP3 、IL-1β(mature IL-1β)and Caspase1(cleaved-Caspase1)were up-regulated in high glucose group,and the expression levels of Nrf2,HO-1 and NQO1 were down-regulated,and with statistical significance(**P<0.001).In groups of D(high glucose + empty vector group)and E(high glucose +DJ-1 overexpression group),the expression levels of NLRP3、IL-1β(mature IL-1β)and Caspase1(cleaved-Caspase1) were decreased in the group E,while the protein expressions of Nrf2,HO-1 and NQO1 were increased,and with statistical significance(**P<0.001).In groups of G(high glucose +DJ-1 silenced group)and F(high glucose +NC group),the protein expression levels of NLRP3、IL-1β(mature IL-1β)and Caspase1(cleaved-Caspase1)were significantly increased,and the protein expressions of Nrf2,HO-1 and NQO1 were significantly down-regulated in the group G,with statistical significance(**P<0.001).8.CCK-8 assay was applied to detect the proliferation ability of cells in each group: compared with the control group,the proliferation ability of lens epithelial cells was significantly decreased after high glucose treatment,and the cells viabilities of DJ-1 overexpression group was significantly increased compared with the empty vector group,while the cells viabilities of DJ-silencing group was significantly decreased compared with the NC group.(*P<0.05).9.Flow cytometry was used to detect cell apoptosis in aforementioned groups:Annexin V-FITC/PI double staining cells and JC-1 mitochondrial membrane potential detection method were used to detect the apoptosis ratio of cells in each group.The results showed that the apoptosis of lens epithelial cells in the high glucose treatment group was significantly higher than that in the control group,and the difference was statistically significant.Moreover,the apoptosis of DJ-1 overexpression group was significantly decreased,and the difference was statistically significant(**P<0.001).10.Immunofluorescence: Immunofluorescence detection results showed that the protein levels of Nrf2 in the high glucose group was lower than that in the control group in the nuclei.In the DJ-1 silenced group,the protein levels of Nrf2 in the nuclei was further decreased compared with that in the NC group,but when DJ-1 was overexpressed,the level of Nrf2 in the nuclei was significantly increased.11.Evaluating the oxidative damage of cells in each group: Compared with the control group,ROS,MDA and LDH in the group induced by high glucose were increased,while SOD content was significantly decreased.Compared with the empty vector group,ROS,MDA and LDH of DJ-1 overexpression group were significantly decreased,while SOD content was significantly increased,with statistical significance(**P<0.001).12.AO/EB staining was used to evaluate the level of pyroptosis cells in each group: in the high glucose group,a small amount of normal cells showed green fluorescence,while a large number of cells showed orange fluorescence,and some nuclei showed pyretic phenomenon,indicating that the number of broken membrane cells increased.These results indicated that the proportion of pyroptosis and apoptotic cells in lens epithelial cells increased;However,the above situation was significantly improved after DJ-1 overexpression treatment,and the opposite result was obtained in the DJ-1 silenced group.13.Scanning electron microscopy(Sem)observation of cell pyroptosis: in the high glucose group,the cells were swollen and expanded with numerous pores,which led to cells pyroptosis.After DJ-1 overexpression treatment,cell pores were reduced,indicating that the occurrence of cell pyroptosis was reduced,while obvious pyroptosis occurred in the DJ-1 silenced group.Conclusions: Both pyroptosis and apoptosis play important roles in the pathogenesis of diabetic cataract.DJ-1 can inhibit the apoptosis of lens epithelial cells induced by high glucose through SIRT1 signaling.Meanwhile,DJ-1 is related to oxidative stress inhibition and reduce NLRP3-mediated pyroptosis,thus delaying the occurrence and development of diabetic cataract. |
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