Title Protective Effects Of Artemisinin And Hydroxychloroquine Combination Mediating Exosomes On IgA Nephropathy

ObjectiveIgA nephropathy(IgAN),with mesangial area IgA deposition accompanied by mesangial cell proliferation and mesangial matrix expansion as the main pathological changes,is a kind of primary immune glomerulonephritis.There are main clinical symptoms of hematuria,accompanied by proteinuria,hypertension and renal dsyfunction,which causes end-stage renal failure(ESRD).Immunoinflammatory injury is a primary factor in the development of IgAN.Studies have shown that NLRP3 inflammasome is involved in the pathogenesis of IgAN.It is effective that inhibiting the activation of NF-κB/NLRP3 pathway will improve IgAN.Exosomes participate in the treatment of kidney diseases as a messenger during cell communication,which plays an important role in regulating the occurrence and development of chronic kidney disease.Artemisinin(ART)is an effective substance of Artemisia annua L.Hydroxychloroquine(HCQ)is a hydroxychloroquine sulfate composed of 4-aminoquinoline compound.Both of them are highly effective antimalarial drugs with anti-inflammatory and immunosuppressive or immunoregulatory properties.Previous study has found that the combination of artemisinin and hydroxychloroquine(AH)has the advantage of synergism and detoxification and exert the better therapeutic effect on IgAN.However,the mechanism of AH on IgAN is still unclear.Thus,the exosome-mediated NF-κB/NLRP3 pathway is a new entry point.It is necessary to discuss the intrinsic relationship between exosomes and NF-κB/NLRP3 pathway on the trnt of AH in IgAN.In vivo,we have observed the therapeutic effect and investigated potential mechanism of AH on IgAN rats.In vitro,the effect of AH has been demonstrated on secretion of exosomes in human renal tubular epithelial cells(HK-2);co-culturing HK-2 and human mesangial cells(HMCs)is used to observe the regulation of exosomes for proliferation of mesangial cells.Additionally,it is crucial to explore the role of AH in the treatment of IgAN via exosomes-mediated NF-κB/NLRP3 pathway.Methods1 Therapeutic effect of AH on IgA nephropathy rats and the regulation of NF-κB/NLRP3 by exosomesNinty SPF male Sprague-Dawley rats were feeded one week.Then randomly selected ten rats as a normal control group,the others administrated with 600 mg/kg bovine serum albumin(BSA),lipopolysaccharide(LPS)and tetrachloromethane(CCl4)to establish the IgAN model;while the normal control rats drank the same volume of distilled water.The 24 h urine protein and hematuria were regarded as evaluated indicators for the successful model.After modeling,the rats were stochasticly divided into model group,dexamethasone group(0.078 mg/kg),artemisinin group(33.33 mg/kg),hydroxychloroquine group(33.33 mg/kg),AH low dose group(16.66 mg/kg),AH middle dose group(33.33 mg/kg),AH high dose group(66.66 mg/kg)and exosomes group(7.5 mg/kg).Normal control group and model group rats were gaven the same volume of distilled water,others were administrated with corresponding drugs for 4 weeks.①24 h urine protein,urinary erythrocyte;serum biochemical indicators:the levels of serum creatinine,urea nitrogen,total cholesterol,triglyceride,albumin,total protein,alanine aminotransferase and aspartate aminotransferase;the inflammatory factors of IL-4,IL-17 in kidney tissues and organ indexs were tested respectively.Renal histopathological morphology was detected to evaluate the efficacy of AH by H&E staining and PAS staining.Transmission electron microscopy was used to detect the deposition of electron dense matter in the mesangial area and basement membrane thickness.The depositions of IgG,IgA,and C3 in glomeruli were detected by immunofluorescence.Expression of exosomes CD63 in glomeruli was used to evaluate the therapeutic effect of AH on IgAN.②Western blot was used to test the expressions of exosomes CD81 and the NF-κB/NLRP3 inflammasome-associated proteins including p65,p-p65,ASC,IκB-α,NLRP3,caspase-1 and IL-1β.2 Intervention of AH on renal tubular epithelial cells and the regulation of exosome on NF-κB/NLRP3 inflammasome in mesangial cells2.1 Effect of AH on exosomes of renal tubular epithelial cellsRenal tubular epithelial cells(HK-2)were stimulated with 1.25 μg/mL LPS for 24 h to establish the inflammatory injury model in vitro.There were six groups as:control group(complete medium DEME F12),model group(1.25μg/mL LPS),artemisinin group(0.5 μM ART and 1.25 μg/mL LPS),hydroxychloroquine group(1.5 μM HCQ and 1.25 μg/mL LPS),AH low concentration group(0.5:1.5μM AH and 1.25 μg/mL LPS),AH high concentration group(1:3 μM AH and 1.25μg/mL LPS).The intervention was performed for 24 h.①Cell viability and the optimal concentration of the drugs were detected by MTT.②Immunofluorescence was used to detect the expressions of exosoms CD63.③The expressions of exosome marker proteins CD63,CD9 or CD81 in supernatant and lysate of HK-2 cells by western blot analysis.④Transmission electron microscopy(TEM)technique was used to identify the exosomal morphology in the supernatant of HK-2 cells.The size and concentration of exosomes were detected by using NanoFCM Flow Nano Analyzer to accurately investigate the effect of AH on the secretion of exosomes in HK-2 cells.2.2 AH mediated the regulation of exosomes on NF-κB/NLRP3 inflammasome in mesangial cellsHK-2 cells and HMCs were co-cultured in vitro by Transwell.HK-2 cells were cultured in the upper chamber,and HMCs were cultured in the lower layer.HK-2 cells were stimulated with 1.25 μg/mL LPS for 24 h to establish an inflammatory injury model.The established groups were:control group(complete medium DEME F12),model group(1.25 μg/mL LPS),ATR group(0.5 μM ART and 1.25 μg/mL LPS),HCQ group(1.5μM HCQ and 1.25 μg/mL LPS),AH low group(0.5:1.5 μM AH and 1.25μg/mL LPS),AH high group(1:3μM AH and 1.25μg/mL LPS).In addition,HMCs were treated with 20μg/mL LPS for 24 h to establish a model with NLRP3 inf lammasome activation.The aim of the experiment is to investigate the effect of exosomes on the activation of NF-κB/NLRP3 inflammasome.①The exosomes taken up HMCs of HK-2 cells were labeled with DiO dye and detected by immunofluorescence.Besides,detecting the expression of CD63 by co-localization in HMCs.②Western blot was used to detect the expressions of NF-κB/NLRP3 inflammasome-associated proteins,such as p65,p-p65,ASC,I K B-a,NLRP3,caspase-1 and IL-1 β in HMCs.2.3 Inhibition of AH on exosomal secretion in HK-2 cells,in order to study the regulation of AH on NF-κB/NLRP3 inflammasome in HMCsExosomes inhibitor GW4869 with different concentrations were used to impact HK-2 cells in vitro.The established groups were:control group(complete medium DEME F12),model group(1.25μg/mL LPS),2.5 μM GW4869(2.5μM GW and 1.25μg/mL LPS),5 μM GW4869(5 μM GW and 1.25 μg/mL LPS),10μM GW4869(10 μM GW and 1.25 μg/mL LPS),20 μM GW4869(20 μM GW and 1.25μg/mL LPS),40 μM GW4869(40 μM GW and 1.25 μg/mL LPS),80μM GW4869(80μM GW and 1.25μg/mL LPS),The intervention was performed for 24 h.In addition,HK-2 cells were co-cultured with HMCs by Transwell.HK-2 cells were cultured in the upper chamber;HMCs were cultured in the lower layer.Secretion of exosomes was inhibited by 40μM GW4869.The established groups were:control group(complete medium DEME F12),model group(1.25 μg/mL LPS),ART group(0.5 μM ART and 1.25μg/mL LPS),HCQ group(1.5 μM HCQ and 1.25μg/mL LPS),AH low group(0.5:1.5 μM AH and 1.25μg/mL LPS),AH high group(1:3 μM AH and 1.25 μg/mL LPS),GW4869 group(40 μM GW and 1.25 μg/mL LPS),GW4869+AH group(40 μM GW and 0.5:1.5 μM AH and 1.25 μg/mL LPS).Meanwhile,HMCs were stimulated with 20 μg/mL LPS for 24 h to establish the model of activated NLRP3 inflammasome.① Western blot was used to detect the expressions of exosomes CD9 and CD63 in supernatant and lysate of HK-2 cells,and select the optimal concentration of exosome inhibitor GW4869 on HK-2 cells.②The expressions of NF-κB/NLRP3 inflammasome-associated proteins including p65,p-p65,ASC,IκB-α,NLRP3,caspase-1 and IL-1β were detected by western blot in HMCs.Results1 Therapeutic effect of AH on IgA nephropathy rats and the regulation of NF-κB/NLRP3 by exosomes①In vivo,results show that AH effectively improved renal pathological damage,reduced 24 h urine protein and urinary erythrocyte,decreased the levels of serum creatinine,urea nitrogen,triglyceride,total cholesterol,albumin and total protein,inhibited the expressions of inflammatory factors IL-4 and IL-17 in renal tissues.Besides,renal pathology revealed that AH could alleviate renal damage,reduce expansion of glomerular basement membrane and decrease mesangial area hyperplasia and immune complexs deposition,compared to the IgAN model group.Immunofluorescence results exhibited that AH significantly mitigated the deposition of IgA,IgG and C3 in glomeruli and enhanced the expression of’ exosome CD63 in glomeruli compared with the model group.Transmission electron microscopy showed that AH significantly inhibited the deposition of electron dense material in the glomerular mesangial area and thickened the mesangial basement membrane.Taken together,these results demonstrated that the therapeutic effect of AH on IgAN was more significant than ART or HCQ alone.②Western blot analysis showed that the expression of renal exosomes CD81 was increased in the IgAN model group.The NF-κB/NLRP3 pathway was activated,the expressions of NF-κB-related proteins p-p65,ASC,IκB-α were enhanced,as well as NLRP3,caspase-1 and IL-1β However,AH significantly heightened secretion of exosomes and inhibited NF-κB/NLRP3 inflammasome activation.2 Intervention of AH on renal tubular epithelial cells and the regulation of exosomes on NF-κB/NLRP3 inflammasome in mesangial cells①Immunofluorescence results showed that LPS promoted the expression of exosomes CD63 in HK-2 cells and AH further enhanced the secretion of exosomes.Transmission electron microscopy(TEM)combined with NanoFCM Analyzer were used to demonstrat that secretion of exosomes was also increased after LPS stimulation,compared with the control group.AH further promoted secretion of exosomes and changed the appearance of exosomes,compared with the LPS group.It was likely to reveal that AH possiblely regulated inflammatory by altering certain component of the exosomes.②In the co-cultured test of HK-2 cells and HMCs,immunofluorescence results illustrated that the secretion of HK-2 cells derived exosomes taken up by HMCs was significantly heightened after AH treatment.Western blot analysis also showed that AH promoted the secretion of exosomes,which significantly inhibited the expressions of NF-κB-related proteins p-p65,IκB and ASC in HMCs.The activation of NLRP3 inflammasome and the expressions of IL-1β and caspase-1 were inhibited by AH.③ Western blot analysis showed that GW4869 significantly inhibited secretion of exosomes and obvious prevented the inhibitory effect of AH on expressions of p-p65,NLRP3,caspase-1 and IL-1β.Hovever,AH still have the promotional capacity on exosomes.In conclusion,all of results demonstrated that AH could exert anti-inflammatory effect via mediating secretion of exosomes to inhibit NF-κB/NLRP3 inflammasome activation in IgAN rats.ConclusionsThere was a significantly therapeutic effect of AH on IgAN.AH could prominently reduce the production of 24 h urine protein and urinary erythrocyte in IgAN rats,and decrease the levels of creatinine,urea nitrogen,total cholesterol and triglyceride in serum,while increase the levels of albumin and total protein.In addition,the generation of IL-4 and IL-17 of inflammatory factors in kidney tissue was obviously inhibited and renal dysfunction was prevented by AH.Moreover,AH obviously alleviated renal pathological damage and inhibited the expansion of mesangial matrix and proliferation of mesangial cell.Further underlying mechanisms was that there was an inhibitory effect of AH on the activation of NF-κB/NLRP3 inflammasome by promoting the secretion of exosomes,which could prevent the renal injury and improve the kidney function.