The Effects And Mechanisms Of RAS Activation On The Inflammation Of CKD ApoE^-/- Mice

ObjectiveTo explore the effects and mechanisms of RAS activation on inflammation in CKD apolipoprotein E knockout(ApoE-/-)mice.MethodAnimal experiment1.The model of CKD was induced by a 5/6 nephrectomy(SNx)in male ApoE-/-mice.2.ApoE-/-mice were randomly allocated into 3 subgroups: the control group,SNx group and losartan group.The fifth week after building model the mice in losartan group were taken losartan at a dose of 30 mg/kg/d by intragastric administration for 12 weeks.While the other mice were treated with the same volume of 0.5% sodium carboxymethylcellulose.3.Sixteen weeks after nephrectomy,serum levels of urea,creatinine and AngⅡwere determined.4.HE staining were used to observe the general morphology changes of atherosclerotic plaque.5.Immunohistochemical method was used to detect the content of macrophages in aortic root atherosclerotic plaque,and the macrophages were labeled with CD68.Flow Cytometry was performed to detect the proportion of inflammatory monocytes in blood.qPCR was performed to detect the mRNA expression of proinflammatory cytokines(IL-6 and TNF-α)and chemokines(MCP-1 and CX3CL1)in aorta.6.Immunohistochemical method was used to detect the protein expression of P-IRE1α in aortic root atherosclerotic plaque;western blot analysis was performed to detect the protein expression of GRP78 and the phosphorylation of IRE1α in aorta.The cell experiment in vitro1.MTT cell proliferation assay kit was used to analyze cell proliferation under AngⅡstimulation.2.Western blot analysis was used to detect the protein expression of GRP78 and the phosphorylation of IRE1α in Raw264.7 macrophages treated with AngⅡin different concentrations and different times.3.After losartan intervened the Ang Ⅱ-stimulated Raw264.7macrophages,western blot analysis was used to detect the protein expression of GRP78,P-IRE1α,P-IKKα/β,IκB and nuclear NF-κB p65.Fluorescence microscope was performed to observe the NF-κBp65 nuclear translocation.4.LV-IRE1αsiRNA and empty lentivirus(LV-empty)were transfectedinto Raw264.7 macrophages respectively.qPCR and western blot analysis were used to detect the mRNA and protein expression of IRE1α.5.After Ang Ⅱ stimulated the transfected Raw264.7 macrophages,MTT cell proliferation assay kit was used to analyze cell proliferation.Western blot analysis was used to detect the protein expression of P-IKKα/β,IκB and nuclear NF-κB p65.qPCR was used to detect the mRNA expression of proinflammatory cytokines and chemokines.Transwell was used to observe the changes of migration ability.ResultsAnimal experiment(一)、Serum biochemical analysisSixteen weeks after nephrectomy,compared with the control group,the serum levels of urea,creatinine and AngⅡin SNx group ApoE-/-mice markedly increased(P<0.01).Compared with SNx group,the serum levels of urea,creatinine and AngⅡin losartan group ApoE-/-mice significantly decreased(P<0.05).(二)、The results of aortic root tissue with HE stainingSixteen weeks after nephrectomy,the ApoE-/-mice in the SNx group showed a significant increase in the size of aortic root plaques compared to the aortic root plaques in the control mice(P<0.01).Compared to the SNx group,the plaque sizes in aortic root of the ApoE-/-mice in losartan group significantly decreased(P<0.05).(三)、The changes of inflammatory reactionCompared with control mice,macrophage content within the aortic root plaques,the proportion of CD11b+Ly6C+ inflammatory monocytes in blood and the mRNA expression of proinflammatory cytokines and chemokines in aorta of the SNx group ApoE-/-mice significantly increased(P<0.01);Such effects could be significantly decrease by losartan(P<0.05).(四)、To evaluate the ERSThe protein expression of P-IRE1α in the aortic root plaques,the protein expression of GRP78 and the phosphorylation of IRE1α in the aorta in SNx group ApoE-/-mice were significant higher than that of control group mice(P<0.01).Such effects could be significantly suppressed by losartan(P<0.05).The cell experiments in vitro(一)、The Raw264.7 macrophages proliferation was promoted when stimulated by 1 μg/ml AngⅡ,and the most significant effect was 8h group than the others.(二)、The changes of ERS markers in AngⅡ-stimulated Raw264.7macrophagesAng Ⅱinduced the expression of GRP78 and the phosphorylation of IRE1α in Raw264.7 macrophages in time-and concentration-dependent manners,and the differences were significant(P<0.05).(三)、The effect of losartan on AngⅡ induced ERS in Raw264.7macrophagesThe results of western blot analysis showed that the protein expression of GRP78 and phosphorylation of IRE1α were significantly attenuated by losartan in Ang Ⅱ-stimulated Raw264.7 macrophages(P<0.01).(四)、The effect of losartan on the NF-κB signal pathway in AngⅡ-stimulated Raw264.7 macrophagesCompared to the control group,Ang Ⅱ could increase the protein expression of P-IKKα/β,nuclear NF-κBp65,and fluorescence intensity in nuclear NF-κBp65,and the differencese were significant(P<0.01).Losartan significantly markedly suppressed such changes(P<0.01).Compared to the control group,AngⅡ could markedly decrease the protein expression of IκB(P<0.01).Losartan could significantly increase the expression of IκB in AngⅡ-stimulated Raw264.7 macrophages(P<0.01).(五)、 The effect of ERS inhibition on the inflammation of Ang Ⅱ-stimulated Raw264.7 macrophages1.The results of qPCR and Western blot analysis showed that the mRNA and protein expression of IRElα were markedly decreased(P<0.01)in IRElα siRNA lentivirus transfected Raw264.7 macrophages compared to control group.But there was no difference between LV-empty and control group.2.MTT results showed that LV-IRE1αsiRNA significantly suppressed AngⅡinduced Raw264.7 macrophages proliferation(P<0.01).3.The effect of ERS inhibition on the AngⅡactivated NF-κB signal pathwayIn Ang Ⅱ stimulated Raw264.7 macrophages,IRElαsiRNA could markedly(P<0.01)decrease the expression of P-IKKα/β and nuclear NF-κB p65 and significantly up-regulated the expression of IκB compared with the AngⅡand AngⅡ+LV-empty group.4.In Ang Ⅱ stimulated Raw264.7 macrophages,IRElαsiRNA could markedly decrease(P<0.01)the mRNA expression of proinflammatory cytokines,chemokines and the number of migratory cells compared with the AngⅡand AngⅡ+LV-empty group.Conclusion1.These findings suggest that RAS activation could accelerate the development of AS and up-regulated the endoplasmic reticulum stress and inflammation in CKD AopE-/-mice.The mechanism of acclerated atherosclerosis by RAS activation may associate with the up-regulated endoplasmic reticulum stress.2.AngⅡinduced ERS in macrophages in time-and concentrationdependent manners.3.AngⅡinduced inflammation via AT1R/IRE1α/NF-κB pathway in macrophages.4.Renin-angiotensin system activation accelerates atherosclerosis in CKD AopE-/-mice by up-regulating endoplasmic reticulum stress-related inflammation.