Effects And Mechanisms Of Rutaecarpine On Podocyte Injury On Diabetic Kidney Disease

Background and objective:The number of people with diabetes mellitus（DM）is increasing at an alarming rate worldwide.In 2021,537 million adults worldwide were estimated to be living with the disease.Ninety percent of diabetics have type 2 diabetes.Diabetic kidney disease（DKD）is a disease caused by diabetes involving the kidney.The incidence of DKD is on the rise in China and has become the leading cause of ESRD in the world today.DKD clinic is characterized by microproteinuria to macroproteinuria with renal impairment,and the glomerular podocyte is one of the important components of the glomerular charge barrier and plays an important role in regulating glomerular filtration function.Exploring the mechanism of podocyte damage is of great significance for the treatment of DKD,and finding key therapeutic targets has a large research space for the prevention and treatment of the disease.Traditional Chinese medicine（TCM）has a history of thousands of years in C hina,and Chinese herbal medicine is widely used in the treatment of diabetes and its complications.With further study on the curative effect and mechanism of TCM in DKD treatment,TCM may play an important role in alleviating proteinuria.Rutaecarpine is an important active component of Chinese medicine Euodia rutaecarpa,which has been widely reported in anti-inflammatory treatment.Therefore,Rutaecarpine can be used as a potential therapeutic agent for a variety of diseases.The results of previous studies showed that Rutaecarpine（Rut）has a good protective effect in kidney diseases.The purpose of this study is to explore whether Rutaecarpine can improve renal injury in diabetic kidney disease and its specific mechanism of action,to provide new ideas for drug development in the treatment of diabetic kidney disease.Methods:In vivo experiment: db/db mice（C57BLKS/J db/db）were used to simulate type 2diabetic kidney disease,and the model treatment group was given the intragastric treatment of Rutaecarpine respectively for 12 weeks.The changes in body weight and blood glucose of the mice were detected and recorded every two weeks.The experiment was divided into six groups（n=6-8）: db/m control group,single perfusion group（Rut）,db/db model group,and model treatment group（db/db +Rut5,10,20mg/kg）.After 12 weeks,the general and pathological changes in the mice were detected,and the urine protein was quantified at 24 h.The podocyte injury indexes were observed.The morphological changes of glomerulus were observed by HE,PAS,and transmission electron microscopy staining.The expression of podocyte injury marker protein（nephrin/podocin/WT-1）in the glomerulus of diabetic mice was detected by immunohistochemistry and Western blot,and the expression and activation of inflammatory indicators（NLRP3/ASC/IL-18/IL-1β）were detected.The m RNA expression levels of inflammatory factors（IL-18/IL-1β）were detected by real-time PCR.Western blot was used to detect the content changes of the drug target protein VEGFR2.Western blot,immunohistochemistry,and real-time PCR were used to detect the activation and expression of pyroptosis-associatedd protein（caspase-1/GSDMD）.In vitro experiment: Mouse podocyte（MPC5）was selected as the experimental object.MTT assay was used to screen the drug toxicity and the optimal concentration of Rutaecarpine.The experiment was divided into seven groups: normal group,mannitol group,normal + Rutaecarpine group,high glucose group,and high glucose +Rutaecarpine group（0.25μM,0.5μM,1μM）.Western blot and immunofluorescence were used to detect cell damage（nephrin/podocin）.Western blot and real-time PCR were used to detect the expression of inflammatory factors（NLRP3/ASC/IL-18/IL-1β）.Western blot and real-time PCR were used to detect the activation and content of caspase-1 and GSDMD.TUNEL kit was used to detect programmed cell death.Discovery Studio software was used to predict drug targets,and the most promising drug target for VEGFR2 was selected.Molecular docking,thermal displacement（CESTA）,and surface plasmon resonance（SPR）were used to verify whether the dru g could be combined with the target,the mode of action,and the stability of the combination.Meanwhile,we used immunoprecipitation（CO-IP）to inflame the binding of the drug target protein to the downstream signaling pathway protein NLRP3.The expression of VEGFR2 was detected by Western blot assay.After successfully silencing MPC5 cells,Western blot and real-time PCR were used to detect the effects of the drug on cell damage,inflammation and pyroptosis response of MPC5 cells.Results:In vivo experiment: the results of 24 h urinary albumin showed that the renal injury of db/db2 type diabetic mice could be significantly reversed after treatment with Rutaecarpine.Samples of HE and PAS staining and transmission electron microscopy showed that the pathological features of the glomerulus,such as mesangial hyperplasia,mesangial area expansion,foot process width,and basement membrane thickness,were improved in db/db diabetic mice after treatment with Rutaecarpine.Western blot and immunohistochemistry results showed that the expression of nephrin/podocin /WT-1 in the kidney of db/db diabetic mice was decreased,and Rutaecarpine could up-regulate these indexes.Similarly,Western blot and/or realtime PCR and immunohistochemistry showed that the high expression of NLRP3/ASC/IL-18/IL-1β in the kidney of db/db diabetic mice was alleviated after treatment with brucine.Western blot and/or real-time PCR and immunohistochemistry also showed that the expression of caspase-1 /GSDMD in diabetic mice was significantly inhibited after treatment with Rutaecarpine.All the above indexes were up-regulated or down-regulated in drug dependence.In vitro experiment: MTT method showed that the effective concentrations of Rutaecarpine were 0.25 u M,0.5u M,and 1u M,respectively.Western blot and immunofluorescence showed that Rutaecarpine up-regulated the expression of nephrin/ Podocin protein,a podocin marker,induced by high glucose.Western blot and real-time PCR showed that the expression level and activation degree of NLRP3/ASC/IL-18/IL-1β in the high glucose group were significantly higher than those in the normal group,and the expression of these inflammatory factors could be inhibited by the intervention of Rutaecarpine.Western blot and real-time PCR results also indicated that the content and activation level of Caspase-1 /GSDMD were inhibited by Rutaecarpine in the high glucose group.The results of TUNEL staining suggested that Rutaecarpine could alleviate the high rate of programmed cell necrosis in the high glucose group.Bioinformatics,molecular docking assay,CESTA,and SPR assay confirmed that VEGFR2 was a potential target of Rutaecarpine.Western blot results also suggested that Rutaecarpine could down-regulate VEGFR2 expression in the high glucose group.After successful silencing of VEGFR2,Western blot and real-time PCR assay results indicated that MPC5 damage induced by high glucose was alleviated,but the drug could not further exert its protective effect on MPC5 cells.Meanwhile,immunoprecipitation（CO-IP）results showed that VEGFR2 can directly bind to NLRP3.Thus,we demonstrated that Rutaecarpine acts through the VEGFR2/NLRP3 dependent pathway.Conclusion:（1）In vivo experiments showed that Rutaecarpine effectively alleviated renal damage in db/db type 2 diabetic mice,and inhibited renal inflammation and pyroptosis reaction.（2）In vitro studies have shown that Rutaecarpine can effectively prevent podocyte damage,cell inflammation,and pyroptosis induced by high glucose.（3）A variety of pharmacological analyses have shown that Rutaecarpine can play a role by targeting VEGFR2.Co-ip suggested that evodogine may protect against podocyte injury in db/db2 type diabetic mice through VEGFR2/NLRP3 dependent pathway.