The Effect Of Zhen-wu-tang On Chronic Glomerulonephritis Through Regulating Mitophagy And NLRP3 Inflammasome

Objectives:Chronic glomerulonephritis(CGN),kinds of immune-inflammatory diseases,is the main reason to lead end-stage renal failure and regarded as a threat of human health.Therefore,it is crucial to find some safe and effective drugs for CGN treatment.Zhen-wu-tang is a representative formula of warming yang and excerting water in Febrile Disease,Clinical and experimental studies confirmed that Zhen-wu-tang(ZWT)has exactly curative effect on CGN.NLRP3 inflammasome,a research hotspot in immune-inflammation,is closely related to the pathogenesis of CGN.Recent researches proved that mitophagy inhibits the activation of NLRP3 inflammasome by regulating mitochondrial function.Therefore,exploring the regulation of mitophagy on NLRP3 inflammasome in CGN would help to reveal the pathogenesis of CGN,as well as demonstrate the mechanism of ZWT on CGN.Methods:1.The protective effect ZWT in CGN model ratsSD rats were subcutaneously injected with cationic bovine serum albumin(C-BSA)on the first week,then the rats were injected with C-BSA in tail vein three times a week in three weeks.Afterwards,the CGN model rats were daily administrated with ZWT in different dose(16.8 g/kg,8.4 g/kg,4.2 g/kg)or prednisone acetate(2 mg/kg)for 4 weeks.The weights of all the rats were measured weekly,while urine samples were collected in 24 h for urine protein detection weekly.After finishing the administration,the blood of all the rats were obtained to detect serum creatinine(Scr),blood urea nitrogen(BUN),total cholesterol(TC)and triglyceride(TG).HE and PAS staining of renal tissues were used to observe the renal pathological morphology.Besides,IgG and complement C(C3)in glomeruli were detected by immunofluorescence.The expressions of desmin(a podocyte injury marker)and podocin(a constituent protein of podocyte structure)in glomeruli were measured by immunohistochemistry.The ultrastructure of foot process was observed with transmission electron microscope.2.The effects of ZWT on NLRP3 inflammasome and podocyte injuryCultured mouse podocytes were stimulated with 5-80 ng/mL TNF-?,and then the cell apoptosis was measured by flow cytometry,the protein expressions of NLRP3 and desmin were detected by western blot,the colocalization of NLRP3 and ASC were examined by immunofluorescence.The mRNA level of NLRP3 of podocytes transfected with siNLRP3 was detected by RTPCR,as the protein expressions and the co-localization of NLRP3 and desmin was detected simultaneously.Under the administration of NLRP3 inhibitor MCC950 and Caspase-1 inhibitor VX765,podocytes were used to detect the activation of NLRP3 inflammasome,as well as the expression of desmin.Podocytes were stimulated with TNF-? and ZWT-containing serum,then the cell vitality,apoptosis,and expressions of desmin and podocin were tested.Moreover,the activation of NLRP3 inflammasome in renal tissue of CGN model rats and podocytes with the were detected.3.The effects of ZWT on mitophagy and NLRP3 inflammasomePodocytes stimulated with mitophagy inducer CCCP or inhibitor Mdivi-1 were used to determine the expressions of Hsp60,TOMM20,LC3 ?/? and P62,as well as the co-location of LC3 and mitochondrial marker COX ?.Besides,the expressions of NLRP3 inflammasome proteins and mitochondrial ROS(mtROS)were detected.The effects of ZWT on mitophagy in vivo and vitro were measured.Furthermore,ATP content,mitochondrial membrane potential(MMP),and mtROS in podocytes,as the mRNA levels of antioxidant phosphorylated proteases superoxide dismutase-2(SOD2),catalase(CAT)and peroxiredoxin3(PRDX3)in the CGN model rats were detected.4.The regulation of ZWT on PI3K/AKT/mTOR pathwayThe expressions of PI3K,p-AKT,p-mTOR under ZWT treatment in vivo and vitro were detected.Furthermore,PI3K inhibitor LY294002 or PI3K agonist IGF-1 were administrated in podocytes alone or combined with ZWT.The expression of proteins involved in PI3K/AKT/mTOR pathway and NLRP3 inflammasome were detected.Results:1.The protective effect ZWT in CGN model ratsThe contents of 24 h urine protein,and the levels of Scr,BUN,TC,TG in serum were all decreased after ZWT treatment.ZWT obviously ameliorated the pathological changes of renal tissue and restored the morphology of glomeruli and renal tubules.ZWT also inhibited the deposition of IgG and C3 in glomeruli.Besides,ZWT protected foot process fusion with a certain degree of recovery,reduced the expression of desmin and promoted podocin expression in CGN model rats.2.The effects of ZWT on NLRP3 inflammasome and podocyte injuryTNF-? increased the apoptosis rate of podocyte,as well as the expressions of desmin and NLRP3.TNF-? also induced the co-location of NLRP3 and ASC in podocytes.What's more,siNLRP3 significantly reduced the mRNA and protein expression of NLRP3,and down-regulated the protein expression of desmin,reduced the combination of NLRP3 with desmin.MCC950 and VX765 inhibited the expressions of NLRP3,Caspase-1 and desmin.ZWT-containing serum improved the cellular activity,inhibited apoptosis,reduced protein expression of desmin but increased podocin in podocytes.ZWT significantly decreased the protein levels of NLRP3,Caspase-1,and IL1? in the renal tissue of CGN model rats and podocytes.ZWT also inhibited the co-localization of NLRP3 and ASC in glomeruli,reduced the mRNA levels of IL-1? and IL-18 in renal tissue.3.The adjustment of ZWT on mitophagy and NLRP3 inflammasomeCCCP promoted mitophagy with the increased expression of LC3 ?/?,and decreased the protein levels of Hsp60,TOMM20,and P62,as well as induced the co-location between LC3 and COX ?.CCCP also suppressed the expressions of NLRP3 inflammasome related proteins and the accumulation of mtROS.On the contrary,Mdivi-1 suppressed mitophagy and inhibited NLRP3 inflammasome activation.ZWT reduced mitophagy in CGN model rats and podocytes.In addition,ZWT showed an improvement of mitochondrial function by increasing the ATP content,increasing MMP,inhibiting the accumulation of mtROS in podocytes,and reducing the mRNA levels of SOD2,CAT and PRDX3 in CGN model rats.4.The regulation of ZWT on PI3K/AKT/mTOR pathwayZWT significantly inhibited the expressions of PI3K,p-AKT,p-mTOR in vivo and vitro.In addition,ZWT showed suppressive effect on PI3K/AKT/mTOR pathway which is similar to LY294002,while ZWT inhibited the activation of PI3K pathway induced by IGF-1.LY294002 suppressed the activation of NLRP3 inflammasome,which was further weakened by the combination with ZWT.ZWT also prevented the activation of NLRP3 inflammasome caused by IGF-1.Conclusion:1.ZWT ameliorated kidney damage in CGN model rats induced with C-BSA.2.The inhibition of NLRP3 inflammasome improved podocyte injury caused by TNF-?.In addition,ZWT ameliorated podocyte injury in vitro and inhibited the activation of NLRP3 inflammasome.3.Mitophagy inhibited the activation of NLRP3 inflammasome and the accumulation of mtROS.ZWT induced mitophagy in vitro and vivo,meanwhile improved mitochondrial function,and inhibited the generation of mtROS.4.The mechanism of ZWT protecting against CGN podocyte injury through inactivation of NLRP3 inflammasome is related to the inhibition of PI3K/AKT/mTOR signaling pathway.