Zhen-wu-tang Protects IgA Nephropathy By Inhibiting NF-κB/NLRP3 Pathway Via Exosomes

ObjectiveIgA nephropathy(IgAN)is a refractory kidney disease mediated by immune inflammation.NLRP3 inflammasome is closely a.ssociated with the pathogenesis of IgAN,inhibition of NF-κB/NLRP3 pathway plays a protective role on IgAN.There are certain effects of Zhen-wu-tang(ZWT)on IgAN according to clinical and experimental studies.However,its pharmacological mechanism remains unclear.Exosomes participate in the physiological and pathological processes of diverse renal diseases by mediating intercellular communication.The purpose of this study is to explore whether ZWT improving IgAN is related to the regulation of exosomes,which affects the NFκB/NLRP3 pathway.It will provide a new notion for the treatment of IgAN.Methods1.ZWT regulated secretion of exosomes,whcih affected NF-κB/NLRP3 signaling pathway in HMC proliferation model(1)Effect of ZWT on exosomal secretion in HK-2 cellsAn injury model was established of human renal tubular epithelial cells(HK-2)by LPS(100 ng/mL).After that,ZWT-containing serum was prepared for intervention,and the cell viability was detected via MTT.Structure and particle size of the exosomes from HK-2 cells were identified by transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA),respectively.Western blot was used to analyze the expression levels of exosomal makers CD9,CD63 and CD81 in the HK-2 cells.Therefore,the effect of ZWT-containing serum on exosomes secreted by HK-2 cells was evaluated.(2)Effect of exosomes on NF-κB/NLRP3 signaling pathway in HMC proliferation modelHuman mesangial cells(HMC)were cultured that a proliferation model was induced by LPS of 20 ug/mL.The cell viability was also detected by MTT,The collected exosomes from HK-2 cells treated by ZWT-containing serum(16 ug/mL)were applied to the HMC proliferation model.Furthermore,coculture d HK-2 cells and HMC by transwell system that one was cultured in the upper chamber the other was cultured in the lower layer.Exosomes secretion inhibitor GW4869(40 uM)was provided to interfere with the HK-2 cells at the same time.The exosomes were labeled with DIO,immunofluorescence method was able to detect the uptake of exosomes by HMC as well as co-localization with CD63.Western blot was used to detect the levels of NLRP3,p-p65,IL-1β and caspase-1 that expressed in the HMC.The effect of ZWT-containing serum on regulating the exosomal liberation from HK-2 cells which would have an influence on the inflammatory response in the HMC proliferation model was investigated.2.Effect of ZWT on IgAN model rats by regulating exosomes to inhibit NFκB/NLRP3 pathway(1)Effect of ZWT-EXO on IgAN model ratsThere were forty-eight male Sprague-Dawley rats that were fed adaptively for a week,then 8 rats were selected randomly as a.control group.The remaining rats were modeled for 12 weeks as following:administered orally of BSA(600 mg/kg)every other day,and 0.6 mL of CCL4 mixed solution was injected subcutaneously to each rat once a week.At the same time,0.3 mL of LPS solution was injected to each rat in caudal vein on the 6th,8th,10th week for once.Urine protein level for 24 h and microscopic hematuria were used as indicators of success in this model.When the model was constructed,the rats were randomly divided into model group,ZWT low-dose group(8.4 g/kg),ZWT high-dose group(16.8 g/kg),ZWTEXO group(7.5 mg/kg),and prednisone group(2 mg/kg),there were 8 rats in each group.The exosomes were extracted from HK-2 cells after being treated with 10%ZWT-containing serum,then the exosomes were injected to the rats by tail vein injection once a week as ZWT-EXO group.The others were administered orally at the rat weight of 10 mL/kg,the control and the model groups were provided for equal volume of saline once a day lasted for 4 weeks.At the end of the experimental period,24 h urine protein,urinary erythrocyte,organ index,serum creatinine,urea nitrogen,albumin and total protein content were determined.Besides,pathological morphology of renal tissues were observed by H&E and PAS staining.ELISA was applied to measure IL-18 and IL-1β content.Immunofluorescence was used to examine deposition of IgA,C3 and IgG.Consequences of ZWT-EXO on IgAN rats were comprehensively assessed.(2)ZWT regulated the expression of exosomes in kidney tissues and its effect on NF-κB/NLRP3 signaling pathwayImmunofluorescence was carried out to research co-localization of ASC and NLRP3,as well as the expression of CD63 in the renal tissues.The expression levels of CD63,CD9 and CD81 and relative proteins of the NFκB/NLRP3 pathway including NLRP3,p-p65,IL-1β and caspase-1 were detected by western blot.Thus,regulation of exosomes on the NF-κB/NLRP3 signaling pathway in the IgAN model rats and the intervention of ZWT were evaluated comprehensively.Results1.ZWT regulated secretion of exosomes,whcih affected NF-κB/NLRP3 signaling pathway in HMC proliferation model(1)Effect of ZWT on exosomal secretion in HK-2 cellsThe results shown that the main peaks of exosomal particle size in the control,LPS and ZWT group were 109.0 nm,136.3 nm and 95.6 nm respectively,which were in the range of 30 to 150 nm in accordance with the NTA standard.The concentration of HK-2-derived exosomes was increased after LPS stimulation,but there was no significant difference compared with the control group.While it was enhanced significantly after treatment of ZWTcontaining serum(P<0.01).In addition,ZWT-containing serum could boost the expressions of CD9,CD63,and CD81 from HK-2 cells(P<0.01 or P<0.05).These results suggested that ZWT-containing serum could reinforce HK-2 cells to release exosomes.(2)Effect of exosomes on NF-κB/NLRP3 signaling pathway in HMC proliferation modelThe expressions of NLRP3,p-p65,IL-1β and caspase-1 in the HMC proliferation model were down-regulated by exosomes which derived from HK2 cells treated with ZWT-containing serum(P<0.01 or P<0.05).After being co-cultured by transwell system,HK-2-derived exosomes were actively taken up by HMC that ZWT-containing serum could reinforce this uptake effect,and the expressions of NLRP3,p-p65,IL-1β and caspase-1 were also restrained(P<0.01).The uptake effect.was weakened after undergoing GW4869 treatment,which had an influence on the NF-κB/NLRP3 signaling pathway.These data demonstrated that ZWT-containing serum was able to augment the secretion of exosomes from HK-2 cells.Exosomes could be taken in by HMC,which impeded the activation of NF-κB/NLRP3 signaling pathway to exert anti-inflammatory effects.2.Effect of ZWT on IgAN model rats by regulating exosomes to inhibit NFκB/NLRP3 pathway(1)Effect of ZWT-EXO on IgAN model ratsIn vivo,the results suggested that compared with the model group,ZWTEXO were able to reduce the 24-hour urine protein level(P<0.01 or P<0.05)and the urinary erythrocyte numbers(P<0.01).What’s more,the content of SCR and BUN was declined(P<0.01)while the TP and ALB levels were up-regulated(P<0.01).The levels of IL-18 and IL-1β expressed in the renal tissues were lessen by ZWT-EXO(P<0.01).ZWT-EXO ameliorated the IgA,C3 and IgG deposition in the mesangial region,inhibited expansion of glomerular basement membrane and mesangial cell proliferation which improved renal damage.These data.showed that ZWT-EXO had a good protective effect on IgAN rats.(2)ZWT regulated the expression of exosomes in kidney tissues and its effect on NF-K B/NLRP3 signaling pathwayIn comparison with model group,ZWT facilitated CD63,CD81 and CD9 expressions(P<0.01 or P<0.05)while suppressed NLRP3,p-p65,IL-1β and caspase-1 levels of the renal tissues(P<0.01 or P<0.05).Besides,the co-localization of ASC and NLRP3 was aslo hindered after intervention of ZWT.There were similar findings in the exosomes group.These results indicated that ZWT improved the inflammatory response of IgAN through exosomes.Conclusion1.ZWT-containing serum is able to stimulate HK-2 cells to secret exosomes,enhance the ability of HMC to uptake exosomes actively which restricts the activation of NLRP3 inflammasome in the HMC proliferation model so that relieve the inflammation.2.ZWT-EXO has a good protective effect on IgAN rats.ZWT could stimulate the liberation of exosomes in the renal tissues,which inhibits NF-κB/NLRP3 pathway to restrain kidney damage and protect renal function.