The Role Of TLR4 And Relative Inflammatory Factors In Hypertensive Renal Injury

Part 1 Experiment in vitro——Cell CultureSection 1 The Expressions of TLR4,NF-κB,IL-6 and TNF-αin Renal Tubular Epithelia Cells stimulated by AngiotensinⅡObjectiveTo investigate the expressions of Toll like Receptor 4(TLR4), NF-κB,IL-6 and TNF-αstimulated by angiotensinⅡ(AngⅡ),in a bid to evaluate whether AngⅡregulated TLR4 and related inflammatory factors in renal tubular epithelia cells(NRK-52E).Methods①NRK-52E were incubated with different concentration of AngⅡ[0(normal control),10^-6M,10^-7M,10^-8M,10^-9M,10^-10M]for 24 hours. The expression of TLR4 mRNA was tested by Reverse transcriptionpolymerase chain reaction(RT-PCR).②According to the above results,The optimal concentration of AngⅡ(10^-7mol/L)was selected to incubated with NRK-52E for 0(normal control),6h,12h,24h,48h.The expression of TLR4 mRNA was tested by RT-PCR to choose the best cultivate time.③According to the above results,The optimal concentration of AngⅡwas selected to incubated NRK-52E for the best cultivate time, NF-κB nuclear translocation was tested by immunocytochemistry,IL-6 and TNF-αsupernatant levels were tested by enzyme linked immunosorbent assay(ELISA). Results①In NRK-52E,AngⅡcan up-regulate TLR4 mRNA expression in a concentration-dependent manner.The mRNA expression of TLR4 increased significantly after 6h(P＜0.05),with a peak at 12h～24h,then there is inhibition and decrease at 48h.②10^-7M AngⅡobviously increased the supematant excretive levels of IL-6 and TNF-α,and induced nuclear translocation of NF-κB(P＜0.01).ConclusionsAngⅡinduced TLR4 expression in NRK-52E and participated in the up-regulation of inflammatory mediators and cytokines such as NF-κB, IL-6 and TNF-a. Section 2 TLR4-specific RNA interferenceObjectiveTo investigate the expressions of NF-κB,IL-6 and TNF-a by using the method of RNA interference to knock down the TLR4 gene in NRK-52E and try to find whether there are direct relations between TLR4 and the downstream cytokines.Methods①TLR4-specific RNAi plasmids(Si525,Si526)and negative control plasmid(Si461)were stably transfected into NRK-52E.TLR4 mRNA and protein levels were tested by RT-PCR and Western Blot to verify the plasmids' silence efficiency.②According to the above results,the best plasmid was chosen to construct stable transfected NRK-52E cell line.Then four groups were divided as follows:NRK-52E(normal control group),NRK-52E+AngⅡ(10^-7M),NRK-52E which transfected TLR4-specific RNAi plasmid +AngⅡ(10^-7M),NRK-52E which transfected negative control plasmid +AngⅡ(10^-7M).IL-6 and TNF-a mRNAs and supernatant excretive levels were quantified by RT-PCR and ELISA,the nuclear protein and nuclear translocation of NF-κB were investigated by Western Blot and immuno- cytochemistry.Results①TLR4-specific RNAi plasmids(Si525 and Si526)can obviously inhibit TLR mRNA and protein expressions(P＜0.05 or P＜0.01),the silence efficiency of Si526 is better than Si525(P＜0.05).The negative control plasmid(Si461)has no obvious effect on TLR4 as compared to the normal control(P＞0.05).②After stimulation by 10^-7M AngⅡfor 24h,IL-6 and TNF-a mRNAs and supernatant excretive levels in NRK-52E which stably transfected TLR4-specific RNAi plasmid were decreased,in contrast with to the levels that stimulated the normal NRK-52E(P＜0.05).But no significant difference appeared regarding the nuclear activation of NF-κB (P＞0.05).ConclusionsTLR4-specific RNA interference can depress the expressions of IL-6 and TNF-αwhen incubated with AngⅡ,indicating that TLR4 may induce the expressions of inflammatory factors in renal hypertensive injury. There was no obvious influence on NF-κB,the reason being that there are many pathways enabling AngⅡto induce its nuclear activation. Section 3 Role of Fosinopril and Losartan on the Signalling Pathway of AngⅡ-TLR4-NF-κB-IL6/TNF-αin NRK-52EObjectiveTo investigate the role of Fosinopril(Fos)and Losartan(Los)on the signalling pathway of AngⅡ-TLR4-NF-κB-IL6/TNF-αin NRK-52E, and study the renal protective mechanisms of ACEI/ARB from the angle of microinflammatory.MothedsNRK-52E were incubated with medias such as AngⅡ,Fos and Los, then divided into five groups:NRK-52E(normal control),NRK-52E+ AngⅡ(10^-7M),NRK-52E+AngⅡ(10^-7M)+Fos(10^-5M),AngⅡ(10^-7M)+ Los(10^-5M),NRK-52E+AngⅡ(10^-7M)+Fos(10^-5M)+Los(10^-5M).The expressions of TLR4,IL6,TNF-a mRNAs were tested by RT-PCR.The total protein of TLR4 and nuclear protein of NF-κB were quantified by Western Blot.The nuclear translocation of NF-κB was investigated by immunocytochemistry.IL-6 and TNF-a supernatant excretive levels were tested by ELISA.Results①Fosinopril or/and Losartan can down-regulate TLR4 mRNA and protein levels in NRK-52E induced with AngⅡ(P＜0.05).The IL-6 and TNF-a mRNA and supernatant excretive levels significantly decreased(P＜0.05).The nuclear protein and translocation of NF-κB also depressed(P＜0.05).②No cooperation effects were observed in the combined treatment group as compared to the Fos group and Los group(P＞0.05).ConclusionsFosinopril and Losartan antagonized the effect of AngⅡinduction of the expression of TLR4 and depressed the nuclear activation of NF-κB, thus blocking the expressions of downstream cytokines such as IL-6 and TNF-a.This may contribute to anti inflammatory reaction in hypertensive renal injury. Part 2 Experiment in Vivo——Animal ExperimentThe Expression of Toll like Receptor 4 in Spontaneously Hypertensive Rat and the Role of Fosinopril and Losartan interventionObjectiveTo observe the impact of Fosinopril(Fos)and Losartan(Los)on the expressions of TLR4,NF-κB,IL-6 and TNF-αin spontaneously hypertensive rats(SHR),in order to investigate the role of renin angiotensin system(RAS)on TLR4 regulation and study its mechanism of action in hypertensive renal injury.MethodsTwenty 22 weeks-old male SHRs were randomly divided into four groups for each of five:hypertensive group(SHR),Fos treated group (10mg·kg^-1·d^-1),Los treated group(50mg·kg^-1·d^-1),and associated treated group(Fos 10mg·kg^-1·d^-1add Los 50mg·kg^-1·d^-1).Five 22 weeks -old male WKY rats were used as the normal control group.After 8 weeks,the body weigh,cuff-tail blood pressure,24 hours urinary protein (Upro),urinary N-acetyl-β-D-glucosaminidase(NAGase),blood urea nitrogen(Bun),serum creatinine(Scr)and left renal weigh/body weigh ratio were measured.The pathological changes were viewed under light microscopy by HE and Masson.TLR4,IL-6 and TNF-αmRNAs expressions were detected by RT-PCR.The expression of TLR4 protein was quantified by Western Blot.The serum levels of IL-6 and TNF-αwere tested by ELISA. Results①The general data prior to therapy showed that the blood pressure, Upro and NAGase of SHR rats were significantly higher than WKY rats(P＜0.05),the body weigh,levels of Scr and Bun were not obviously different(P＞0.05).②After treatment with Fos or/and Los for 8 weeks,the blood pressure,Upro and NAGase of SHR rats were decreased(P＜0.05),but there are no statistic variance among the three treated groups(P＞0.05).③The typical pathological characteristics and infiltrative inflammatory cells in hypertensive renal injury were demonstrated in the kidney of SHR as compared to the control,and the hypertensive renal injury was ameliorated after treatment with Fos or/and Los.④In SHR,the expressions of TLR4,IL-6,TNF-a mRNAs and proteins were notably increased(P＜0.05).Fosinopril or/and Losartan can down-regulated the expressions of TLR4 and the above cytokines in SHR(P＜0.05).There was no statistical difference between the combined treatment group as compared to the Fos group and Los group(P＞0.05).ConclusionsFosinopril and Lorsartan can decreased the levels of TLR4 and the correlated inflammatory factors in SHR,this may constitute one of the therapeutic mechanisms against hypertensive renal injury.