| Objective:On the basis of the successful establishment of rat model of age-related cataract(ARC)induced by sodium selenite(Na2SeO3)in vivo and human lens epithelial B3 cell(HLEB3)injury model induced by hydrogen peroxide(H2O2)in vitro,firstly,to be clear that Sagittaria sagittifolia polysaccharide(SSP),the main active substance in Sagittaria sagittifolia L.,has an inhibition effect on lens opacity and HLEB3 damage,and SSP’s best protect dose.Then,the relevant mechanism of SSP’s inhibitory effect was discussed from three aspects:apoptosis,endoplasmic reticulum stress(ERS)and nuclear factor E2-related factor 2(Nrf2)regulation.Finally,siRNA transfection was used to confirm that the protective effect of SSP was related to the targeted regulation of Nrf2.In this way,to provide experimental basis for the development of traditional Chinese medicine dietary therapy and health products based on Sagittaria sagittifolia L.for the prevention and treatment of ARC.Method:1 In vivo animal experiments1.1 SSP alleviated lens opacity in Na2SeO3-induced rats cataractsHealthy 8-day-old Sprague-Dawley suckling rats were randomly divided into 6 groups(n=6):control group,model group(Na2SeO3,20 μmol/kg),positive control group(calcium dobesilate,75 mg/kg+Na2SeO3 20 μmol/kg)and SSP treatment groups(SSP+Na2SeO3 20μmol/kg,high dose of 800 mg/kg,medium dose of 400 mg/kg,low dose of 200 mg/kg).On postpartum days 12 and 14,rat pups of model group,positive control group and SSP treatment groups underwent subcutaneous injection of Na2SeO3(20 μmol/kg body weight)to generate a cataract model.In addition,positive control group and SSP treatment groups received administration of corresponding dose of drugs by gavage from day 10 to day 37 postpartum.Rat pups in the positive control group and SSP treatment groups were administered 1 hour prior to Na2SeO3 injection.After opening the eyes,images of bilateral lenses opacity in rats were obtained by using hand-held slit lamp microscope every other day and scored.On postpartum day 38,all animals were cervical dislocation under pentobarbital sodium anesthesia.Lenses samples were isolated and collected.The changes of lens epithelial cells were observed by HE staining,fiber cells changes were observed by Masson staining,and ultrastructural changes of lens epithelial cells were observed by transmission electron microscopy(TEM).1.2 Mechanism research of SSP on alleviating lens opacity in Na2SeO3-induced rats cataractsRats lenses were divided into 3 groups:control group,model group and SSP optimal dose treatment group(SSP 800 mg/kg).Apoptosis was observed by TUNEL fluorescence staining.The expression levels of apoptosis-related proteins(B-cell lymphoma-2 associated X,Bax;Bcell lymphoma-2,Bcl-2;cleaved-cysteinyl aspartate specific proteinase-3,cleaved-caspase-3),ERS-mediated-apoptosis-related proteins(C/EBP homology protein,CHOP;c-Jun N terminal kinase,JNK;caspase-12)and Nrf2/HO-1 signaling pathway related proteins(Nrf2,Kelch Like ECH Associated Protein 1,Keap1;Heme Oxygenase-1,HO-1)in lens tissues were detected by western blotting.The expression levels of ERS-related proteins(Binding immunoglobulin heavy chain protein,BiP;protein kinase RNA(PKR)-like kinase,PERK)were detected by immunohistochemical staining.2 In vitro cell experiments2.1 The effect of SSP on apoptosis in H2O2-induced HLEB3 cellsAccording to the previous experimental results,the HLEB3 cells were divided into 7 groups:normal control group,injury model group(H2O2 400 μM),positive control group(glutathione(GSH),H2O2 400 μM+GSH 1 mM)and SSP treatment groups(H2O2 400 μM+SSP 0.25,0.5,1,2 mg/mL).The cells in SSP groups and GSH group were cultured with SSP or GSH for 24 hours first.Then the injury model group,GSH group and SSP groups were added with 400 μM H2O2 for 24 hours.JC-1 staining combined with flow cytometry were used to detect the apoptosis rate,and the best intervention concentration of SSP was selected for subsequent experiments.Mitochondrial membrane potential was detected by JC-1 staining combined with flow cytometry and DAPI staining combined with confocal microscopy to observe cell apoptosis.2.2 The effect of SSP on ERS in H2O2-induced HLEB3 cellsThe HLEB3 cells were divided into 3 groups:normal control group,injury model group(H2O2 400 μM)and SSP treatment group(H2O2 400μM+SSP 1 mg/mL).Fluo-4 AM fluorescence probe combined with confocal microscopy were used to observe intracellular Ca2+levels.Western blotting was used to test the expression levels of ERS-related proteins of BiP,PERK,inositol-requiring enzyme 1(IRE1α),activating transcription factor 6(ATF6),CHOP,JNK,caspase-12,calpain-2 and sarco/endoplasmic reticic-type Ca2+ transport ATPase 2(SERCA2).2.3 The effect of SSP on Nrf2 signal in H2O2-induced HLEB3 cellsCell grouping and administration were the same as 2.2.Immunofluorescence was used to observe the expression site and nuclear translocation of Nrf2.Western blotting was used to detect the expression level of Nrf2 both in cytoplasm and nucleus protein.2.4 Nrf2 gene knockdown verifies the protective effect of SSP on HLEB3 cellsThe cells were divided into negative control group and siRNA-Nrf2 group,and each group was further divided into control group,model group(400 μM H2O2)and SSP treatment group(400 μM H2O2+SSP 1 mg/mL).siRNA was used to interfere the expression of Nrf2 in HLEB3 cells,and the silencing effect of Nrf2 was detected by western blotting.Then western blotting was used to detect the expression level of BiP,PERK,CHOP,Bax,Bcl-2 and caspase-3 to verify that the regulation of endoplasmic reticulum stress and apoptosis in H2O2-induced HLEB3 cells by SSP was closely related to the targeted regulation of Nrf2.Results:1 In vivo animal experiments1.1 SSP alleviated lens opacity in Na2SeO3-induced rats cataractsThis study found that the lenses in control group were completely transparent,all the Na2SeO3-induced rats showed different degrees of cataracts,and the lens opacity score of more than 80%of the model group rats reached grade V.Compared with the model group,the scores of positive control group and SSP treatment groups were decreased,and the scores of high-dose SSP group were significantly lower than the model group and close to the positive control group.The histomorphological observation showed that compared with the control group,the lens epithelial cells in the model group were swollen,the lens fibers were deformed,swollen and ruptured,and a large number of vacuoles appeared near the posterior pole of the lens.The fibrocyte damage in the lens of the SSP treatment groups and the positive control group were significantly improved,and the epithelial cells were in a relatively normal state.The improvement effect of the high dose group was the best,which was close to the control group,and the high dose of SSP was determined to be the best protective dose.1.2 Mechanism research of SSP on alleviating lens opacity in Na2SeO3-induced rats cataractsTUNEL fluorescence staining showed that compared with the control group,the relative fluorescence intensity of the model group was increased,but significantly decreased after SSP intervention.The results of western blotting and immunohistochemical staining showed that compared with the model group,the protein expressions of Bax,cleaved-caspase-3,BiP,PERK,CHOP,JNK,caspase-12 and Keapl in SSP intervention group were significantly decreased,and the protein expressions of Bcl-2,Nrf2 and HO-1 were up-regulated.It is suggested that the delay effect of SSP on lens opacity in Na2SeO3-induced cataract rats is closely related to the regulation of Nrf2/HO-l and the improvement of apoptosis induced by endoplasmic reticulum stress.2 In vitro cell experiments2.1 The effect of SSP on apoptosis in H2O2-induced HLEB3 cellsJC-1 staining combined with flow cytometry showed that the apoptosis rate in the model group was significantly higher than that in the control group.Compared with the model group,the apoptosis rate of the SSP treatment groups and the GSH group were significantly decreased,and SSP concentration of 1 mg/mL shown the best protective effect,which was close to that of the GSH group.Therefore,SSP concentration of 1 mg/mL was selected as the SSP treatment group for the subsequent mechanism research.Compared with the model group,the mitochondrial membrane potential was significantly increased in the SSP treatment group,and the nuclear observed by DAPI staining showed less nuclear chromatin condensation and nucleus fragmentation,and the apoptosis was significantly improved.2.2 The effect of SSP on ERS in H2O2-induced HLEB3 cellsWestern blotting showed that compared with the control group,the expressions of BiP,PERK,IRE1α,ATF6,CHOP,JNK,caspase-12 and calpain-2 in the model group were significantly increased,while the expression of SERCA2 was significantly decreased.Compared with the model group,SERCA2 expression was significantly increased in the SSP treatment group,while the expression of other above proteins was significantly decreased.Fluorescence detection showed that Ca2+ level in the model group was significantly higher than the control group,and Ca2+level was significantly reduced in the SSP treatment group compared with the model group.It is suggested that the protective effect of SSP in H2O2induced HLEB3 cells is closely related to improvement of endoplasmic reticulum stress.2.3 The effect of SSP on Nrf2 signal in H2O2-induced HLEB3 cellsImmunofluorescence observation and western blotting showed that Nrf2 expression and nuclear translocation were decreased in the model group compared with the control group.Compared with the model group,the nuclear expression level of Nrf2 was increased and the nuclear translocation level of Nrf2 was enhanced in SSP treatment group.It is suggested that the protective effect of SSP in H2O2-induced HLEB3 cells is closely related to the activation of Nrf2.2.4 Nrf2 gene knockdown verifies the protective effect of SSP on HLEB3 cellsAfter siRNA intervention,Nrf2 expression was reduced by more than 40%,and the silencing intervention was successful.Further tests showed that SSP could effectively reduce the expression levels of ERS-related and apoptosis-related proteins of BiP,PERK,CHOP,Bax and caspase-3 in Nrf2 gene knockdown HLEB3 cells,and increase the expression of Bcl-2 protein.Therefore,it was confirmed that the improvement of ERS-mediated apoptosis in HLEB3 cells by SSP was closely related to the targeted regulation of Nrf2 protein expression.Conclusion:SSP,the major bioactive component of medicine-food Sagittaria sagittifolia L.,can effectively delay the progression of lens opacity in rats and protect the damaged HLEB3 cells.The protective mechanism may be related to the targeted regulation of Nrf2,activation of Nrf2/HO-1 signaling pathway,alleviation of Ca2+metabolic imbalance,and thus alleviating the ERS-mediated apoptosis of lens epithelial cells. |
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