辣蓼黄酮通过调控核因子E2相关因子2信号通路及组蛋白乙酰化缓解伪狂犬病毒感染小鼠诱导的脾脏和肺脏氧化应激

Flavonoids of Polygonum hydropiper L.Alleviates Oxidative Stress in Spleen and Lung Tissues of Pseudorabies Virus Infected Mice by Regulating Nuclear Factor E2-Related Factor 2 Signaling Pathway and Histone Acetylation

本研究通过探索辣蓼黄酮对核因子E2相关因子2(Nrf2)信号通路及组蛋白乙酰化的调控效果,以阐明辣蓼黄酮干预伪狂犬病毒(PRV)感染小鼠诱导氧化应激的分子机制。将60只无特定病原体(SPF)级昆明种小鼠,随机分为6个组(每组10只小鼠),即空白对照组、PRV组、维生素C(VC)组(灌服100 mg/kg BW VC)及辣蓼黄酮高、中、低剂量组(灌服200、100和50 mg/kg BW辣蓼黄酮混悬液),各组灌服量均为0.2 mL。PRV感染小鼠7 d后,通过荧光定量PCR测定小鼠脾脏和肺脏组织中诱导型一氧化氮合酶(iNOS)、Nrf2、血红素加氧酶-1(HO-1)、依赖还原型辅酶Ⅰ(Ⅱ)醌氧化还原酶1(NQO1)、乙酰转移酶(HAT)和去乙酰转移酶(HDAC)的mRNA表达水平;通过免疫组化和蛋白免疫印迹法(Western Blot)测定iNOS、Nrf2、HO-1、NQO1、乙酰化组蛋白H3(AcH3)和乙酰化组蛋白H4(AcH4)的蛋白表达水平。结果表明:与空白对照组相比,PRV组脾脏组织中iNOS、HAT的mRNA表达水平和AcH3的蛋白表达水平显著或极显著升高(P<0.05或P<0.01),脾脏组织中HO-1、HDAC的mRNA表达水平极显著降低(P<0.01);PRV组肺脏组织中iNOS、Nrf2、HO-1、NQO1、HAT的mRNA表达水平和iNOS、Nrf2、AcH3的蛋白表达水平显著或极显著升高(P<0.05或P<0.01),肺脏组织中AcH4的蛋白表达水平极显著降低(P<0.01)。与PRV组相比,辣蓼黄酮高、中、低剂量组脾脏组织中Nrf2、HO-1和HDAC的mRNA表达水平和AcH4的蛋白表达水平显著或极显著升高(P<0.05或P<0.01),脾脏组织中HAT的mRNA表达水平和AcH3的蛋白表达水平显著或极显著下降(P<0.05或P<0.01);辣蓼黄酮高、中、低剂量组肺脏组织中iNOS的mRNA表达水平和AcH3的蛋白表达水平显著或极显著下降(P<0.05或P<0.01),肺脏组织中AcH4的蛋白表达水平极显著升高(P<0.01)。由此可见,辣蓼黄酮对Nrf2信号通路和组蛋白乙酰化修饰具有调控作用,且其通过该调控作用缓解PRV感染小鼠脾脏和肺脏诱导的氧化应激。

The objective of this study was to investigate the regulatory effect of flavonoids of Polygonum hydropiper L.on the nuclear factor E2-related factor 2(Nrf2)signaling pathway and histone acetylation,and to clarify the molecular mechanism of its intervention on the oxidative stress induced by pseudorabies virus(PRV)infected mice.Sixty specific pathogen free(SPF)level Kunming breeding mice were randomly divided into 6 groups(10 mice in each group),which were blank control group,PRV group,vitamin C(VC)group(100 mg/kg BW VC by gavage)and flavonoids of Polygonum hydropiper L.high,medium and low dose groups(200,100 and 50 mg/kg BW flavonoids of Polygonum hydropiper L.suspension by gavage),the gavage volume in each group was 0.2 mL.After 7 days of PRV infection in mice,the mRNA expression levels of inducible nitric oxide synthase(iNOS),Nrf2,heme oxygenase-1(HO-1),NAD(P)H:quinone oxidoreductase 1(NQO1),acetyltransferase(HAT)and deacetyltransferase(HDAC)in spleen and lung tissues of mice were determined by fluorescence quantitative PCR,and the protein expression levels of iNOS,Nrf2,HO-1,NQO1,acetylated histone H3(AcH3)and acetylated histone H4(AcH4)were determined by immunohistochemistry and Western Blot.The results showed that compared the blank control group,the mRNA expression levels of iNOS and HAT and protein expression level of AcH3 in spleen tissue of PRV group were significantly increased(P<0.05 or P<0.01),and the mRNA expression levels of HO-1 and HDAC in spleen tissue were significantly decreased(P<0.01);the mRNA expression levels of iNOS,Nrf2,HO-1,NQO1 and HAT and protein expression levels of iNOS,Nrf2 and AcH3 in lung tissue of PRV group were significantly increased(P<0.05 or P<0.01),and the protein expression level of AcH4 in lung tissue was significantly decreased(P<0.01).Compared with the PRV group,the mRNA expression levels of Nrf2,HO-1 and HDAC and protein expression level of AcH4 in spleen tissue of flavonoids of Polygonum hydropiper L.high,medium and low dose groups were significantly increased(P<0.05 or P<0.01),and the mRNA expression level of HAT and protein expression level of AcH3 in spleen tissue were significantly decreased(P<0.05 or P<0.01);the mRNA expression level of iNOS and protein expression level of AcH3 in lung tissue of flavonoids of Polygonum hydropiper L.high,medium and low dose groups were significantly decreased(P<0.05 or P<0.01),and the protein expression level of AcH4 in lung tissue was significantly increased(P<0.01).In conclusion,the flavonoids of Polygonum hydropiper L.can regulate the Nrf2 signaling pathway and histone acetylation modification,and it alleviated the oxidative stress in spleen and lung tissues of RPV infected mice through the regulation.