The Study On The Effect And Mechanism Of Exacerbating Lupus Nephritis Following BPA Exposure

Objective:Systemic lupus erythematosus(SLE)is an autoimmune disease characterized by multiple organs involvement,multiple autoantibodies production and immunologic dysfunction,among which lupus nephritis(LN)is one of the common and serious complications.SLE mainly occurs in women in their reproductive years with quick progresses and poor prognosis.The pathogenesis of LN is still not clear.Inflammation,oxidative injury of the kidney and immune cells disorders are important reasons for the aggravation of lupus nephritis.Autophagy is an adaptive response of cells to metabolic stress and environmental changes,which is involved in the pathogenesis of autoimmune diseases.Studies have found that autophagy has a regulatory effect on inflammation and oxidative stress,as well as T-cell-mediated autoimmunity,which plays an important role in maintaining T lymphocyte homeostasis.Abnormal autophagy was found in blood and kidney tissues of lupus patients and mice.Another type of atypical autophagy,LC3-associated phagocytosis(LAP)is an important pathway for degradation and clearance of apoptotic or necrotic cells.Studies found that mice with LAP defect showed elevated levels of anti-dsDNA and ANA antibodies,renal immune complex deposition and immune dysfunction,suggesting that abnormal autoantibody clearance may be one of the important reasons for the aggravation of lupus.At the same time,abnormal T cell function plays an important role in the pathogenesis of LN,especially Th17/Treg balance has attracted extensive attention in recent years.The PI3K/AKT/m TOR signaling pathway is involved in T cell differentiation and the progression of autoimmune disease.The occurrence of SLE is the result of the combination of genetic and environmental factors.On the premise of relatively stable genetic background,the incidence of SLE is still increasing year by year,suggesting that the role of environmental factors should not be ignored.Therefore,more and more researches focus on the role of environmental factors in the development of diseases through epigenetic modification or causing immune cells dysfunction.It was found thatexposure to environmental pollutants aggravated renal structure and pathological damage in human and animals.Bisphenol A(BPA),A ubiquitous pollutant that mimics the effects of estrogen,is a major ingredient in the production of plastics and canned goods,and can be detected in blood,urine and milk.Studies have found that BPA exposure can increase the level of microalbuminuria,increase the ratio of urine protein to creatinine,and is closely related to renal insufficiency stage.However,the risk and mechanism of kidney injury in patients with self-dysfunction is unclear,which needs further study.The studies on the immunotoxic mechanism of BPA are still in the initial stage,and there is a lack of data on whether BPA exposure affects SLE patients in particular.In this study,the MRL/lpr mouse model was used to determine whether BPA aggravates LN,and we analyzed the effect of BPA on kidney tissue of lupus-prone mice from the perspectives of inflammation,antioxidant and autophagy.In addition,the effect of BPA on Th17/Treg balance in MRL/lpr mice and its possible mechanism were discussed.This study provides daily life guidance for patients with lupus,and provides scientific basis for revising the minimum inactive dose of BPA for normal and immunodysfunction patients.Methods:Part Ⅰ: 18 female MRL/lpr mice aged 7 weeks were randomly divided into 3groups: control group(1% anhydrous ethanol solution,n=6);low-dose BPA group(0.1 μg/m L BPA solution,n=6);high dose BPA group(0.2 μg/mL BPA solution,n=6).12 female C57BL/6 mice aged 7 weeks were randomly divided into 2 groups: control group(1% anhydrous ethanol solution,n=6);high dose BPA group(0.2 μg/mL BPA solution,n=6).During the experiment,the mice were free drinking and feeding for 6weeks.The volume of remaining water was measured daily to obtain the capacity of drinking water,and the weight of remained food and body weight were weighed weekly.Since two weeks of exposure,urine samples of mice were collected once a week to detect the ratio of urine protein to creatinine.After the experiment,the size and weight of spleen,thymus and kidney were measured.Serum samples of mice were collected and the concentration of anti-dsDNA was detected by ELISA.Kidney samples of mice were collected,the glomerulonephritis activity index was calculatedby HE staining,and the deposits of IgG and C3 were observed by immunohistochemical staining.Part Ⅱ: The kidneys were collected and Western Blot was used to determine the expression levels of NF-κB,Nrf2,mTOR,LC3,Beclin-1,P62,Rubicon,ERα,AhR and TLR4 protein in each group.Part Ⅲ: Fresh spleen samples were milled and filtered to prepare splenic cell suspension.Treg cells were detected by flow cytometry after staining with FITC-CD4,PE-CD25 and Alexa Fluor? 647-Foxp3 antibodies.The cell suspension was cultured for 6 hours with stimulants,and Th17 cells were detected after staining with FITC-CD4 and PE-IL17 A antibodies.The levels of RORγt,Foxp3,PI3 K,p-PI3 K,p-AKT,m TOR,p-mTOR,ULK,Rubicon,P62,Beclin-1,LC3,ERα,AhR and TLR4 protein were determined by Western Blot.Results:Part Ⅰ: 1.BPA exposure had no significant effect on body weight,food intake,water consumption and organ coefficient of MRL/lpr and C57 mice.2.0.2μg/mL BPA exposure increased serum level of anti-ds DNA antibody in MRL/lpr mice,while no effect of the anti-dsDNA level was observed in C57 mice.3.0.2μg/mL BPA exposure increased the urine protein/creatinine ratio in MRL/lpr mice,which had no significant effect on C57 mice.4.In the unexposed MRL/lpr group,the pathological manifestations of renal HE staining were mainly mesangial cell hyperplasia and interstitial inflammatory cells infiltration.In the MRL/lpr + 0.1μg/ml BPA group,there were increased capillary cells,increased interstitial inflammatory cells infiltration,microthrombus formation,and focal glomerular sclerosis.In the MRL/lpr+ 0.2μg/ml BPA group,cells in the capillaries increased,with multiple crescent formation,interstitial inflammatory cells infiltration,partial glomerular sclerosis,renal tubular epithelial cells enlargement,and urinary protein tubular type.At the same time,the glomerulonephritis activity index score of the high dose BPA(0.2μg/ml)group was significantly higher than that of the control group and the low dose BPA(0.1 μg/ml)group.However,BPA exposure did not significantly change kidney pathology in C57 mice.5.BPA exposure increased renal IgG deposition in MRL/lpr mice.MRL/lpr mice in the three groups showed complement C3 deposition,and therewere no significant statistical differences between the three groups.Part Ⅱ: 1.0.2μg/ml BPA exposure increased the expression of NF-κB in the kidney of MRL/lpr and C57 mice.0.2μg/ml BPA exposure increased the expression of Nrf2 in the kidney of C57 mice,while decreased the expression of Nrf2 in MRL/lpr mice.2.Compared with the MRL/lpr control group,BPA exposure decreased the expression of mTOR and Rubicon protein in renal tissues and increased the expression of LC3,while no significant effect on Beclin-1 and P62 protein was observed.BPA exposure to C57 mice increased LC3 and Rubicon expression in the kidney compared to control.3.In MRL/lpr mice,0.2μg/ml BPA exposure significantly increased the expression of ERα and AhR protein in renal tissue,but had no effect on the expression of TLR4 protein.However,in kidney tissues of C57 mice,0.2μg/ml BPA exposure significantly increased the expression of AhR protein,while ERα and TLR4 protein expression levels were not significant changed.Part Ⅲ: 1.BPA exposure increased the proportion of Th17 cells and the expression of RORγt protein in MRL/lpr mice,but had no significant effect on the proportion of Treg cells and the expression of Foxp3 protein.2.Compared with the MRL/lpr control group,the relative expression levels of PI3 K,p-PI3 K,p-AKT(Ser473),p-AKT(T308),mTOR and p-mTOR in spleen cells of MRL/lpr+0.1μg/ml group were significantly increased.The relative expression levels of p-PI3 K,p-AKT(Ser473),p-AKT(T308)and p-mTOR proteins in spleen cells of MRL/lpr+0.2μg/ml group were significantly increased.Compared with C57 control group,0.2μg/ml BPA significantly increased the expression level of p-PI3 K protein.3.Compared with the MRL/lpr control group,the relative expression levels of ULK,Rubicon,P62 and Beclin-1 protein in spleen cells of MRL/lpr+0.1μg/ml group were significantly increased.The relative expression levels of ULK,Beclin-1 and LC3 proteins in spleen cells of MRL/lpr+0.2μg/ml group were significantly increased.Compared with C57 control group,0.2μg/ml BPA significantly increased the expression levels of Rubicon,P62 and Beclin-1.4.0.2μg/ml BPA exposure increased the expression levels of ERαand AhR proteins in the spleen tissues of MRL/lpr mice,and increased the expression levels of AhR proteins in the spleen tissues of C57 mice.Conclusion:1.BPA exposure aggravated disease activity of lupus nephritis in MRL/lpr mice,and aggravated the pathological damage of renal tissue and the deposition of immune complex IgG.2.BPA increase the inflammatory response and aggravate LN by increasing the expression of NF-κB protein in the renal tissues,as well as down-regulating mTOR protein and activating autophagy in MRL/lpr mice.However,the autophagy lacks Beclin-1-involved regulation and P62-involved degradation.BPA down-regulate the expression of Nrf2 and Rubicon proteins in the renal tissues of MRL/lpr mice,weaken the antioxidant capacity and scavenging activity,which may be one of the reasons for the aggravation of LN after BPA exposure.BPA play a toxic role in renal tissues of MRL/lpr mice through both ERα and AhR pathways.3.BPA exposure increased the proportion of Th17 cells in spleen of MRL/lpr mice,causing Th17/Treg imbalance.BPA exposure up-regulated the PI3K/AKT/mTOR signaling pathway in the spleen tissues of MRL/lpr mice,activating autophagy related proteins.BPA play a role in spleen tissue of MRL/lpr mice through ERα and AhR pathways,while through AhR pathway in spleen tissue of C57 mice.