| BackgroundEssential hypertension was a widespread cardiovascular disease.Kidney was the most important target organ of hypertension.Elevated levels of Angll in lte development of hypertension could stimulate vascular endothelial,increasing expression of NADPH oxidase and ROS,promoting oxidative stress aiKUnflammatory by various inflammatory signaling pathway. Therefore, oxidative stress and inflammation which effecting blood pressure promoting renal arteriolar vascular endothehai cell damage, remodeling,hyperplasia and deposition of extracellular matrix glomerular mesangial cells were a vicious spiral. As mentioned above the non hemodynamic factors in the pathogenesis was significant in our Study-According to nlt experiment and literature research,we found ltat inflammatory facto。 were increased by NOX4 mediated ROS via the activation of NF-kB signaling pathway in renal damage,which was the main aspect of our research. On the other hand,TLR2/4 signal pathway,a hot research topic,was COnsiderably extensive and deep in recent years, which expressed in multiple regions of the kidney, especially glomerular mesangial cells.So both were important significance in hypertensive renal damage of important signiincanoe.RASS blocker (ACEI,ARB) weK recormnencied to treat hypertensive renal damage in the new guidelines These medcine not only had a clear effect,but some boundedness, so needed some beneficial supplement.The cmrent treatment of hypertensive renal damage not only emphasized the hypertension effect,but should be paid U) the renal proactive effect of drug specific. Qian Yang Yu Yin granules were preparated in Jiangsu Province Traditional Chinese Medicine Hospital, mainly used for lte treatment of hypertension and renal damage of "Vmdeficiency of liver and kidney". The obvious curative effect was confirmed by preliminary research, which improving the symptoms of renal damage. The main target was not clear,so related molecular mechanism still needed 1:0 fiirther studied according to the preliminary research.AimTo investigate the effect of CJian Yang Yu in n granules (QYYYG) on the levels of Angicrtensin II in renal tissues of Spontaneously Hypertensive Rats(SHRs) and discussed the effect of lte medicated serum of QYYYG(GYGS) on Angiotensin n-induced proliferation and inflammatory reaction in Human mesangial cells(HMCs). Elaborated the influence and poinltle prcrtective mechanism of QYYYG by Kgulating Oxidative stress and inflammation.especially NOX4 mediated NF-κB signaling pathway and TLR2/4/MyD88 signal pathway. To seach for a intervented method of Traditional Chinese medicine to provide experimental basis of hypertensive renal damage.Methods1.In vivoSHRs were randomly divided into model group(SHR),group of benazepril (SHR+B),group of QYYYG(SHR+Q),group ofQYYYG and benszepril (SHR+Q+B),8 only each group. And Choosed Wisltr rats as control groupRats were nlied into the stomach with different medicines according to the different groups, once daily for 8 weeks.The kidneys were taken out I day later after the last administration in rats of each group.Effects of QYYYG on prot;ein expression was assessed by "Westem blot.①in the preliminary experimental. All indicators have been completed as followrEffects of QYYYG 0n expression in SHR was assessed by eLISA;Effects of QYYYG on rat renal damage related indicators such as: urine m-ALB, urine beta 2- microglobulin, urinary NAG were asseined by full automatic biochemical analyzer and radioimmunoassay; Effects of QYYYG about the morphology of kidneys was assessed by HE and Masson;②Effects of QYYYG on NOX4 protein expression was assessed by Western blot;③Effects of QYYYG on Myd88,TLR4,TLR2,NF-kB P65,TNF-a and IL-6 protein cxprcssion were asltsltd by Western blot;2.In VitroWistar rats were randomly divided into normal group, group of Valsartan, group of QYYYG, 10 only each group. Rats were filled into the stomach with different medicines according to the different groups. Then the medicated serum was prepared. HMCs wei*e divided into 5 groups. Except the normal group,1;he Other HMCs were induced by 10"Wnol/L Angiotensin II. And each medicated serum was intervented.①Effects of QYGS on the proliferation of HMCs was assessed by MTT method;②Using Flow cytometry instrument to detect the contents of ROS;③QYGS counteracted Ang Il-induced up-regulation of NOX4, p22,and GTP-Racl in HMCs. RT-PCR and Western blot were carried out lt detect mRNA and protein levels;④QYGS counteracted Ang Il-induced up-regulation of NF-kB P65,IL6 and TNF-a in HMCs. Western blot was carried out to detect prertein levels;⑤RT-PCR and Western blot were carried out 1;o detect mRNA and prertem levels of TLR2JLR4 and MyD88;⑥RT-PCR and Western blot were carried out lt detect mRNA and protein levels of NOX4, p22 and GTP-Racl in NOX4 siRNA-transfected HMCs;⑦ RT-PCR and Western blot were carried out to detect mRNA and protein levels of TLR2、 TLR4 and MyD88 in TLR2 siRNA-transfected HMCs;⑧ Western blot was carried out to detect protein levels of NF-κB P65、IL-6 and TNF-a in TLR2 or NOX4 siRNA-transfected HMCs.Results1.In vivo① The protein expression of TLR2/4,Myd88 were significantly increased in SHR vs WKY(P<0.01).SHR+Q,SHR+B,especially SHR+Q+B suppressed the protein expression of TLR2/4,Myd88(P<0.01),which were assessed by Western blot vs SHR;② The protein expression of NF-κB P65 was significantly increased in SHR vs WKY(F< 0.01).SHR+Q,SHR+B,especially SHR+Q+B suppressed the protein expression of NF-κB P65 (P<0.01),which was assessed by Western blot vs SHR;③ The protein expression of TNF-a,IL-6 were significantly increased in SHR vs WKY(P <0.01).SHR+Q,SHR+B,especially SHR+Q+B suppressed the protein expression of TNF-α,IL-6 (P<0.01),which were assessed by Western blot vs SHR;④ The protein expression of NOX4 was significantly increased in SHR vs WKY(P< 0.01).SHR+Q,SHR+B,especially SHR+Q+B suppressed the protein expression of NOX4 (P<0.01),which was assessed by Western blot vs SHR;2.In Vitro① QYGS inhibited AngⅡ-induced cell proliferation at different extent, and especially high and moderate dose (P<0.01);② The expression of ROS was significantly increased in Ang Ⅱ-induced HMCs vs normal group(P<0.01).QYGS counteracted AngⅡ-induced up-regulation of ROS in HMCs significantly(P<0.01).And there was a dose-dependent effect.③ The protein and mRNA expression of GTP-Racl,p22phox and NOX4 were significantly increased in AngⅡ-induced HMCs vs normal group(P<0.01).QYGS counteracted AngⅡ-induced up-regulation of GTP-Racl,p22phox and NOX4 in HMCs significantly(P <0.01). And there was a dose-dependent effect.④ The protein expression of NF-κB P65 and inflammatory factors were significantly increased in AngⅡ-induced HMCs vs normal group(P<0.01).QYGS counteracted Angll-induced up-regulation of NF-κB P65 and inflammatory factors in HMCs significantly(P<0.01).And there was a dose-dependent effect.⑤ The protein and mRNA expression of TLR2,TLR4 and MyD88 were significantly increased in Angll-induced HMCs vs normal group(P<0.01).QYGS counteracted Angll-induced up-regulation of TLR2,TLR4 and MyD88 in HMCs significantly(P< 0.01).And there was a dose-dependent effect.⑥ After transfection with NOX4 siRNA for 24 h, RT-PCR showed that the mRNA expression of NOX4, p22phox, and GTP-Racl in HMCs had decreased by 72.7%,69.3%, and 61.8%, respectively, which was statistically significant as compared with the RNAcon group(P<0.01) When cells were pre-incubated with QYGS followed by AngⅡ stimulation, the expression levels of ROS,p22pox and GTP-Racl were decreased mildly(P<0.05).Similar results showing protein levels in QYGS-pretreated HMCs were also obtained by Western blot analysis.⑦ After transfection with TLR2 siRNA for 24 h, RT-PCR showed that the mRNA expression of TLR2 in HMCs had decreased. When cells were pre-incubated with QYGS followed by AngⅡ stimulation, the expression levels of TLR4 and MyD88 were significantly decreased (P<0.01).Similar results showing decreased in QYGS-pretreated HMCs were also obtained by Western blot analysis.⑧ After transfection with NOX4 siRNA for 24 h,Western blot also showed that the protein expression of IL-6、TNF-α and NF-κB P65 were decreased mildly(P<0.05)or had no change(P>0.05),When cells were pre-incubated with QYGS followed by AngⅡ stimulation;But after transfection with TLR2 siRNA for 24 h, Western blot showed that the protein expression of IL-6、TNF-α and NF-κB P65 were decreased significantly (P<0.01).Conclusion1.QYYYG might attenuate 1;he expression of NOX4, GTP-Racl and p22Ph°x via Angll/NAD (P)H oxidase4/ROS and NF-kB pathway, effectively restrained the ECM deposition which caused by inflammation factor increases, thus improvea the renal damage as an antioxidant.2.QYYYG also attenuated the expression of TLR2,TLR4,MyD88, NF-kB P65 and numerous of inflammatory factor via TLR2/4/MyD88 or NF-kB pathway as an antiinflammatory.3.QYYYG attenuated hypertensive renal nbrosis and damage in multiple targetis.4.QYYYG and Benazepril main mechanism was (different in many aspects,combing each other could proltct the kidney as a synergistic effect5.QYYYG showed a dose-dependent effect. Oxidative stress and inflammation would be inhibited in lte high dose. |
PDF Full Text Request