The Mechanism Of Sulforaphane Through The Keapl/Nrf2 Pathway To Alleviate Oxidative Stress In High Glucose Cultured Mouse Podocytes

BackgroundDiabetic kidney disease(DKD)has become the leading cause of end-stage renal disease(ESRD)and one of the main causes of death in diabetic patients.More and more evidences show the important role of oxidative stress in diabetes and its complications.Since DKD was reported,renal mesangial cell damage has been considered to be the main cause of glomerular disease.In addition,researchers have realized that the thinning of glomerular basement membrane(GBM)and the damage of podocyte also have important pathophysiological significance.The mechanism of DKD is complicated,and the oxidative stress damage caused by high glucose is at the center of the pathogenesis:polyol bypass activation,Glycosylation end products(AGEs)accumulation,hexosamine,protein kinase C(Protein kinase C)C,PKC)activation,etc.,are closely related to the excessive accumulation of reactive oxygen species(ROS),leading to proteinuria and renal function damage.It has been proven to improve the level of oxidative stress in the diabetic kidney and maintain mitochondrial homeostasis,which can effectively reduce the structural damage and dysfunction of podocytes and mesangial cells,and slow down the deterioration of kidney function.Cells can maintain a stable redox internal environment through a variety of mechanisms under stress,involving multiple signaling pathways.Among them,Kelch-like ECH-associated protein 1(Keap1)-Nuclear factor E2-related factor 2,Nrf2)-Antioxidant response element(ARE)signaling pathway,namely Keap1/Nrf2/ARE,is the most important antioxidant stress pathway in the body one.Among them,Keap1 is the transcription factor Nrf2 repressor protein.Under normal circumstances,Keapl binds to Nrf2 through the Cullin3/Ringboxl E3 ubiquitin ligase complex and makes it ubiquitinated,thereby being degraded by the 26S proteasome;under stress conditions or application In the case of Nrf2 activator,Nrf2 activated by phosphorylation translocates into the nucleus,and after binding to Musculoaponeurotic fibrosarcoma(MAF),it promotes the binding of Nrf2 to the cell Antioxidant response element(ARE)binding,To initiate its powerful anti-oxidative stress and anti-inflammatory effects.Sulforaphane(SFN)is an isothiocyanate compound widely found in cabbage,cauliflower,broccoli and other cruciferous plants.SFN is considered to be a promising drug after in vivo and in vitro experimental evaluation.SFN can stimulate Nrf2 to exert a powerful cytoprotective effect in in vitro and in vivo experiments,thereby enhancing the resistance to carcinogenicity and other diseases mediated by exposure to electrophiles and oxidants,and preventing the excessive generation and release of endogenous ROS into cells.Previous studies on the effects of Nrf2 agonists have mostly focused on Nrf2 and its downstream pathways,and seldom focused on the direct action target of drugs Keapl protein,and its interaction with Nrf2,and there is currently no sulforaphane on diabetic kidneys.Research on the protective mechanism of podocytes,therefore,this topic will conduct a preliminary exploration of the mechanism of sulforaphane against oxidative stress in a mouse podocyte model in a high-glucose environment.ObjectiveExplore the protective effect and mechanism of sulforaphane on mouse podocytes against oxidative stress damage under high glucose conditions.MethodsCulturing differentiated conditionally immortalized mouse podocyte cell lines(CIMPs)in a high-glucose medium(glucose concentration 25.0mmol/L)for 1.5h,3h,6h,12h,24h,48h,96h,The mouse podocytes cultured with normal sugar medium(glucose concentration 5.6mmol/L)after differentiation were set as the control NG group,and the time of high-glycemic state that had the most obvious impact on podocytes was selected as the high-glycemic group(HG).The optimal concentration of sulforaphane on podocytes was 1 μM and the action time was 24h to interfere with podocytes under high glucose state,which was designed as the HG+SFN group.qRT-PCR was used to detect the expression of Keapl and Nrf2 mRNA;Westernblot was used to detect the anti-oxidative stress related proteins Keap1,Nrf2,p-Nrf2,Nqo1,the expression of the podocyte marker Nephrin,and the expression of the mesenchymal marker Desmin;Focusing microscope combined with DCHF-DA probe to detect intracellular ROS.Construct Keapl overexpression lentiviral vector(LV-Keapl)and Keap1-shRNA lentiviral expression vector(LV-shRNA-Keapl),empty lentiviral vector(LV-NC)as a blank control group,transfect CIMPs,after differentiation is stable Grouping treatment:normal glucose control group(NG,5.6mmol/L),high glucose group(HG,25.0mmol/L),high glucose+SFN group(HG+SFN),high glucose+Keap1 downregulation group(HG+LV-sh-Keapl),high glucose+Keapl downregulation+SFN group(HG+LV-sh-Keap1+SFN),high glucose+Keapl upregulation group(HG+LV-Keap1),high glucose+Keapl upregulation group+SFN group(HG+LV-Keap1+SFN),high glucose+negative control virus group(HG+LV-NC).qPCR was used to detect the changes in the expression of Keap1 mRNA in podocytes after transfection of LV-Keapl and LV-shRNA-Keapl;after processing in groups,the expression levels of Keap1 and Nrf2 mRNA were detected by qRT-PCR,and Keap1 was detected by western blotting.Nrf2,p-Nrf2,Nqo1,Nephrin,Desmin protein;laser confocal microscope combined with DCFH-DA fluorescent probe method to detect ROS.Results1.Compared with the NG group,the expression level of the podocyte marker protein Nephrin in the HG group was down-regulated,and the expression level of the damage marker protein Desmin was up-regulated(P<0.05).Compared with the HG group,the Nephrin protein level increased after the intervention of sulforaphane,and the Desmin protein level decreased(P<0.05).Confocal laser microscope combined with DCHF-DA probe to detect the ROS level of mouse podocytes in each group,the results showed that:compared with NG group,HG group ROS level was significantly increased,and compared with HG group,HG+SFN group ROS level Significantly reduced.2.Compared with the NG group,in the podocytes cultured with high glucose,the Keap1 protein expression reached the highest at 48h(P<0.05),and the Keap1 protein expression in the HG96h group had no significant change compared with 48h(P>0.05),so take 48h as the best intervention time for Keapl expression in the HG group for the next experiment.Keap1 and Nrf2 mRNA expression in HG group were significantly increased compared with NG group(P<0.05),while Keap1 and Nrf2 mRNA expression in HG+SFN group had no significant changes compared with HG+SFN group(P>0.05).The expression of Keap1,Nrf2,p-Nrf2,and Nqo1 protein in the HG group were increased compared with the NG group(P<0.05),and compared with the HG group,Nrf2,p-Nrf2,and Nqo1 proetin in the HG+SFN group increased(P<0.05),but Keap1 protein expression did not change significantly(P>0.05).3.Under the same normal sugar medium culture condition,compared with the NG and LV-NC group,the Keap1 knockdown group(LV-sh-Keap1)Keap1 expression was significantly down-regulated(P<0.05),while the Keapl overexpression group(LV-Keap1)Keapl expression was significantly increased(P<0.05).Compared with the NG group,the ROS level in the HG group was significantly increased;compared with the HG group,the ROS in the HG+SFN group and the HG+LV-sh-Keapl group were significantly decreased;compared with the HG+SFN group,HG+SFN+LV-Keap1 group ROS level was significantly increased.4.Compared with the HG group,the expression of Keapl and Nrf2 mRNA in the HG+Keap1 knockdown group and the HG+Keap1 knockdown+sulforaphane group were significantly decreased(P<0.05),but there was no significant changsxe in the two groups in the above-mentioned mRNA expression(P>0.05).Compared with the HG group,the HG+Keap1 knockdown group and the HG+Keap1 knockdown+sulforaphane group were down-regulated Keap1 and Desmin protein expression levels,and Nrf2 and p-Nrf2 protein levels were up-regulated,but there was no significant change in the two groups in the expression of the above proteins during the period.5.Compared with the HG group,Keap1 overexpression group and Keap1 overexpression+sulforaphane group Keapl mRNA expression were significantly up-regulated(P<0.05),but Nrf2 mRNA expression did not change significantly,And there was no significant change in the above-mentioned mRNA expression between the two groups(P>0.05).Compared with the HG group,Keapl overexpression group and Keapl overexpression+sulforaphane group,the Keapl protein expression levels were significantly increased,Nrf2 and p-Nrf2 protein levels were significantly decreased,but Desmin protein expression levels did not change significantly,And there was no significant change in the above-mentioned protein expression between the two groups.6.The co-IP result suggest that Keapl and Nrf2 interact in the cytoplasm of podocytes.Conclusions1.Sulforaphane can alleviate the oxidative stress damage of mouse podocytes under high glucose conditions;2.Sulforaphane may play an anti-oxidative stress effect by regulating the Keap1/Nrf2 pathway.