湿热中阻方对脾胃湿热证小鼠氧化应激和炎症因子影响研究

Study on the eff ect of Shire Zhongzha Fang on the oxidative stress and infl ammatory factors in mice with the spleen and stomach dampness-heat syndrome

目的观察湿热中阻方对脾胃湿热证小鼠氧化应激和炎症因子的调节作用,探讨湿热中阻方治疗脾胃湿热证的作用机制。方法采用高脂高糖饮食建立脾胃湿热证模型,以湿热中阻方(6.2、12.4、24.8 g/kg)进行干预。采用Western blotting法检测细胞磷酸化p38丝裂原活化蛋白酶(P-p38MAPK)、磷酸化核因子-κB(P-NF-κB)、磷酸化人核因子κB抑制蛋白α(P-IKBα)表达水平;ELISA法检测细胞上清液中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)的释放和检测超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性;流式细胞术检测细胞活性氧(ROS)含量;实时荧光定量PCR(qPCR)检测COX2 mRNA、HO-1 mRNA、iNOS mRNA表达。观察不同浓度的湿热中阻方和阳性药对脾胃湿热证小鼠炎症因子诱导的氧化应激影响。结果与正常组比较,湿热模型组小鼠SOD、CAT、GSH-Px含量显著降低(P<0.001),LDH、COX2 mRNA、HO-1 mRNA、iNOS mRNA含量显著升高(P<0.001),MDA含量明显升高(P<0.05),TNF-α、IL-6含量显著升高(P<0.01);P-p38MAPK、P-NF-κB、P-IKBα含量显著升高(P<0.001)。与湿热模型组比较,湿热中阻方组LDH释放水平显著降低(P<0.05、P<0.01、P<0.001);减少了炎症因子COX2 mRNA、HO-1 mRNA、iNOS mRNA表达;ROS生成受到显著抑制(P<0.01);TNF-α、IL-6释放受到显著抑制(P<0.001);丙二醛(MDA)释放显著降低;SOD、CAT、GSH-Px释放均显著升高(P<0.05、P<0.01、P<0.001);湿热中阻方高剂量组P-p38MAPK、P-NF-κB、P-IKBα蛋白表达显著降低(P<0.01)。结论湿热中阻方可以通过抑制氧化应激,增加细胞内SOD、CAT、GSH-Px的含量,降低ROS、MDA、LDH的含量,改善脾胃湿热型小鼠炎症反应。

Objective To observe the regulatory effect of Shire Zhongzhu Fang on the oxidative stress and infl ammatory factors in mice with spleen and stomach dampness-heat syndrome,and to explore its activity mechanism.Methods A high-fat and high-sugar diet was used to establish a model of dampness-heat syndrome of the spleen and stomach,and the Shire Zhongzhu Fang(6.2,12.4,24.8 g/kg)was used for intervention.Western blotting was used to detect the expression levels of phosphorylated p38 mitogen-activated protease(P-p38MAPK),phosphorylated nuclear factor-κB(P-NF-κB),phosphorylated human nuclear factor-κB inhibitory proteinα(P-IKBα).The ELISA method was used to detect the release of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in the cell supernatant,to detect the activity of superoxide dismutase(SOD),malondialdehyde(MDA),lactate Hydrogenase(LDH),catalase(CAT),glutathione peroxidase(GSH-Px).The content of reactive oxygen species(ROS)was detected by flow cytometry.The expression of COX 2 mRNA,HO-1 mRNA and iNOS mRNA were detected by real-time fluorescent quantitative PCR(qPCR).The eff ects of diff erent concentrations of Shire Zhongzhu Fang and positive medicines on the oxidative stress induced by infl ammatory factors in mice with the spleen and stomach dampness-heat syndrome were observed.Results Compared with the normal group,the contents of SOD,CAT,and GSH-Px in the model group were extremely signifi cantly reduced(P<0.001),and the contents of LDH,COX2 mRNA,HO-1 mRNA,and iNOS mRNA were dramatically significantly increased(P<0.001),and the content of MDA was significantly increased(P<0.05),while the TNF-α,IL-6 content was more significantly increased(P<0.01),and the P-p38MAPK,P-NF-κB,P-IKBαcontents were most signifi cantly increased(P<0.001).Compared with the model group,the LDH release level of the Shire Zhongzu Fang group was signifi cantly reduced(P<0.05,P<0.01,P<0.001),and the expression of infl ammatory factors COX2 mRNA,HO-1 mRNA,and iNOS mRNA was reduced,and the ROS production was more signifi cantly inhibited(P<0.01),the release of TNF-αand IL-6 was most signifi cantly inhibited(P<0.001).The release of malondialdehyde(MDA)was signifi cantly reduced,and the release of SOD,CAT,and GSH-Px were signifi cantly increased(P<0.05,P<0.01,P<0.001),and the P-p38MAPK,P-NF-κB,P-IKBαprotein expression in the high-dose group was more significantly reduced(P<0.01).Conclusion Shire Zhongzhu Fang can inhibit the oxidative stress,increase the content of SOD,CAT,and GSH-Px in cells,and reduce the content of ROS,MDA,and LDH,thereby improve the infl ammatory response of mice with the spleen and stomach dampness-heat syndrome.