Pharmacological Research Of Artemisinin Plus Hydroxychloroquine Therapy Forchronic Glomerulonephritis

Background:Primary immunoglobulin A nephropathy(Ig AN),always has immune complex deposition of Ig A in glomerular mesangial area,which was accompanied by primary glomerulonephritis of glomerular mesangial cell proliferation and mesangial matrix expansion.Ig AN has a high morbidity including light-to-severe proteinuria,blood urine,renal insufficiency,high blood pressure with edema,nephrotic syndrome and kidney failure.ACEI,ARB,glucocorticoids,immune inhibitors,fish oil,anticoagulation and antiplatelet are used in Ig AN clinical therapy,which are controversial at the same time for side effects and inconsistent conclusion in clinical treatment.The pathogenesis of Ig AN is possibly related to body immune dysfunction,cytokines and inflammatory mediator,abnormal renal hemodynamics,blood coagulation dysfunction,and genetic factors.Immune dysfunction is considered as the major factor of Ig AN.Nephritis model rats induced by cationic bovine serum albumin(C-BSA)is a common model for chronic glomerulonephritis research.The main symptoms of C-BSA nephropathy are proteinuria and the pathological changes of kidney,which is similar to human membranous nephropathy.This model is a good animal model to study human membranous nephritis as good repeatability and high incidence.Artemisinin(ART),the main medicinal ingredients extracted from artemisia annua and artemisia annua leaves,with high antimalarial effect,is the first-line antimalarias in clinical.In recent years,many researches demonstrate that artemisinin has effects of anti-inflammation and immunosuppression.Hydroxychloroquine(HCQ),widely used as anti-malarial and lupus nephritis therapy,has anti-inflammation,anti-tumor,immune regulation,skin rashes,anticoagulation and hematic fat reduction through anti-infection,immunosuppression and immune regulation.Both ART and HCQ has good anti-inflammatory and immune-suppression effect,which can alleviate the kidney damage in Ig AN and C-BSA model by immune regulation,as well as inhibition of inflammatory mediator and proliferation of glomerular mesangial cell.Therefore,the research on Ig AN or C-BSA model by combination AH can provide a new insight and scientific basis for clinical therapy.Purpose:The results of pharmacological efficacy of combination ART with HCQ on Ig AN and C-BSA glomerulonephritis rats can provide a new insight and scientific basis for clinical therapy.Methods:1.Preparing animal models: Ig AN rats model was established by oral bovine serum albumin,intravenous fat polysaccharide and subcutaneous injection of carbon tetrachloride.Blood in the urine,urinary protein increasing,kidney with Ig A strong fluorescence are successful criteria for this model.C-BSA glomerulonephritis rats model was established by subcutaneous injection,intravenous injection cationic bovine serum albumin.Urine protein increasing and pathological changes in kidney are successful criteria for this model.2.Groups and treatment: Except for the control group of ful 10 normal rats,40 successful SD model rats,male and female half,were randomly divided into model control group(model group),dexamethasone group,ART with HCQ(1:1)group,ART with HCQ(1:3)group.Dexamethasone group,ART with HCQ(1:1)group,ART with HCQ(1:3)group were given corresponding drugs one time per day,respectively,90 days in a row;Normal group and model group were given the amount of water.3.Sample collection and index determination: During the treatment,body weight and urine protein were recorded weekly.End of the experiment,the rats were fasting to collect 24 hours of urine after placed in metabolism cage.Blood in the urine was detected by microscopy,and urine protein was detected by fully automatic biochemical instrument.After anesthesia,the rats were dissected for blood and kidney collection.Blood biochemical indexes and blood coagulation four were measured in blood by corresponding instruments;Blood immunoglobulin A(Ig A),C3,nitric oxide(NO),superoxide dismutase(SOD)and malondialdehyde(MDA)content were detected in serum by enzyme immunoassay assay(ELISA).Renal pathology in Ig AN rat were detected by PAS staining and immunofluorescence method and transmission electron microscopy.Renal pathology in the C-BSA glomerulonephritis rats were detected by HE staining.Results:1?General situation in ratsAs the building time extended,activity decrease,body weight loss,listlessness,withened,reluster and shed easily were appeared in model group rats compared with normal group.Feeding situation and activity hair loss were improved in dose groups compared with model group and control group.2?Urine protein and blood in urineUrine protein,blood in urine increased during building time,and gradually reduced after administration.Urine protein and urine red blood cell counts was significantly decreased at 24 h after administration in AH groups compared with model group.3?Blood biochemical testsIn Ig AN rats: Compared with control group,the contents of creatinine(CRE),urea(UREA),cholesterol(CHOL)and triglycerides(TG)were increased,while the levels of the total protein(TP)and albumin(ALB)were decreased in moel group.Compared with model group,the contents of CRE,UREA,CHOL and TG were decreased,while the levels of TP and ALB were increased in AH group.In C-BSA glomerulonephritis rats: Compared with control group,the contents of CRE,UREA,CHOL and TG were increased,while the levels of TP,ALB and GLB were decreased in moel group.Compared with model group,the contents of CRE,UREA,CHOL and TG decreasing,while the levels of TP and GLB increasing in AH group.4?Four coagulation testsIn Ig AN rats,the times of prothrombin time(PT),international normalized ratio(INR)and activated partial thromboplastin time(APTT)significantly shortened.the activity of prothrombin activity level(PTA),the level of fibrinogen(Fbg)and the thrombin time(TT)significantly prolonged compared with control group.The times of PT,INR and APTT significantly prolonged,PTA,Fbg and TT significantly shortened in AH(1:3)group rats compared with model group.APTT significantly elevated,PTA and TT significantly shortened in AH(1:1)group rats compared with model group.PT?INR and Fbg had no no statistical significance in two group.5?The determination of Ig A,C3 in the serumIn Ig AN rats,the expression of Ig A in the model group was obviously elevated compared with normal group,but the expression of C3 not was significantly statistical;Compared with model group,Ig A in two AH groups obviously reduced.6?The determination of NO in the serumThe expression of NO in the serum was obviously decreased in the model group compared with normal group;The expression of NO of two AH groups were obviously increased compared with model group.7?The determination of SOD?MDA in the plasmaThe level of MDA was significantly increased in model group compared with control group,but the expression of SOD in the plasma wasn't statistical significantly.The level of MDA was significantly decreased in the AH(1:1)group compared with model group,meanwhile the level of SOD in the AH(1:3)group was obviously increased compared with model group,and the level of MDA was significantly decreased.8?The detection of renal pathological changeIn Ig AN rat: For the results of Ig A immunofluorescence test in rats kidney,intensity expression was significantly decreased in AH group compared with the model group.For the results of PAS staining in renal pathology,dyed purple sediment in renal glomerular mesangial area was decreased significantly in AH group compared with model group;As the results of kidney electron microscopy detection,thickening of glomerular mesangial cell and mesangial matrix was obviously improved in AH group.There was no obvious sediment in basement membrane in AH group,and epithelial cell foot process fusion was improved significantly compared with model group.In C-BSA rat:The results of C-BSA glomerulonephritis rats showed that there were rat glomerular enlargement,balloon lumen narrowed and irregular thickening of glomerular basement membrane were shown in model group,but there was not obvious difference between control and model group in the number of glomerular cells;The glomerular size of basicly returned to normal level,glomerular morphology began to recover,balloon open and mesangial cells decreased,uniform matrix,mesangial area gradually narrow compared with model group.The glomerular number of AH groups decreased significantly,the lesion rate decreased significantly,the extent of disease significantly reducing,glomerular cells was significantly reduced(P<0.01).The results with statistical differences,the kidney pathological damage degree in AH(1:3)was better than AH(1:1)group.The above results showed that the AH could improve the C-BSA glomerulonephritis rats in renal pathological damage,and reduce the risk of kidney failure.Conclusion:1.AH could significantly reduce the levels of urine protein,blood in urine,reduce the CRE,UREA level in the Ig AN rats and C-BSA glomerulonephritis rats,elevated serum level of TP and ALB,lower levels of CHOL.AH could restore glomerular filtration function and improve kidney damage,the glomeruli defects caused by loss of serum protein,lipid metabolic disorder and renal function.2.AH could significantly reduce the level of Ig A in serum,increase the level of SOD,reduce the level of MDA,which suggestes that AH could confrontate and block the damage on cells caused by free radicals,and repair the damaged kidney cells.AH could reduce the Ig A immune complex deposition in renal tissue of Ig AN model rats,and repair kidney pathological injury in Ig AN rats.3.AH could improve the kidney pathological damage in C-BSA glomerulonephritis rats,reduce the risk of kidney failure.