针刺对溃疡性结肠炎大鼠氧化应激和内质网应激的影响

Acupuncture reduce colonic inflammation by suppressing oxidative stress and endoplasmic reticulum stress in rats with ulcerative colitis

目的:观察针刺治疗2,4,6-三硝基苯磺酸(TNBS)诱导的溃疡性结肠炎(UC)大鼠的效应机制。方法:雄性SD大鼠随机分为空白组、模型组、手针组和电针组,每组7只。采用TNBS结肠灌注法建立UC模型。手针组和电针组分别予针刺或电针刺激"曲池""足三里"穴,20min/次,隔日1次,连续14d。干预14d后,HE染色观察大鼠结肠组织外观及病理变化,ELISA法检测结肠组织中氧化应激相关因子含量,Western blot法检测结肠组织中核转录因子κB通路中关键蛋白和内质网应激(ERS)关键蛋白的表达水平。结果:与空白组比较,模型组大鼠结肠组织明显肿胀,大量炎性细胞浸润,肠绒毛缺失,结肠组织形态学评分较空白组显著升高(P<0.01),结肠组织内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、还原型谷胱甘肽(GSH)含量和总抗氧化能力(T-AOC)均显著降低(P<0.01),丙二醛(MDA)含量及磷酸化核因子κB抑制蛋白(p-IκBα)、p-p65、葡萄糖调节蛋白(GRP78)、磷酸化蛋白激酶样内质网激酶(p-PERK)、磷酸化真核转录起始因子2α(p-eIF2α)表达水平均显著升高(P<0.01)。与模型组比较,电针组大鼠结肠病变明显减轻,肠绒毛较完整,结肠组织形态学评分显著降低(P<0.01),SOD、CAT、GSH含量及T-AOC显著升高(P<0.01,P<0.05),MDA含量及p-IκBα、p-p65、GRP78、p-PERK、p-eIF2α表达水平显著下降(P<0.01,P<0.05);手针组大鼠结肠病变改善显著,结肠组织形态学评分显著降低(P<0.01),SOD、CAT、GSH含量显著升高(P<0.01,P<0.05),MDA含量及p-IκBα、p-p65、GRP78、p-PERK、p-eIF2α表达水平显著降低(P<0.05,P<0.01)。结论:手针或电针刺激"曲池""足三里"均能通过抑制TNBS诱导的氧化应激和内质网应激反应,减轻大鼠结肠炎性病变。

Objective To investigate the effect of manual acupuncture(MA)and electroacupuncture(EA)on histopathological changes,and levels of oxidative-stress related cytokines and key proteins of the endoplasmic reticulum stress(ERS)pathway in ulcerative colitis(UC)rats,so as to reveal their mechanisms underlying improvement of UC.Methods Twenty-eight male SD rats were randomly divided into control,model,EA and MA groups(n=7 rats per group).The UC model was established by enema of mixture solution of 5%2,4,6-trinitrobenzene sulfonic acid(TNBS,100 mg/kg).Rats of the control group received intra-rectal perfusion of normal saline.After modeling,the left"Quchi"(LI11)and"Zusanli"(ST36)were stimulated with EA(2-4 mA,8 Hz/25 Hz)or MA for 20 min,once every other day for consecutive 2 weeks.The rats in the control and model group were just anesthetized and fixed.At the end of experiments,the colon tissue was collected for observing histopathological changes with H.E.staining.The contents of oxidative stress-related factors as superoxide dismutase(SOD),catalase(CAT),malondialdehyde(MDA),reduced glutathione(GSH)and total antioxidant capacity(T-AOC)were detected by ELISA,and the expression levels of key proteins of ERS as phosphorylated inhibitor of nuclear factor kappa B kinaseα(p-IκBα),phosphorylated p65(p-p65),glucose regulated protein 78(GRP78),phosphorylated protein kinase R-like endoplasmic reticulum kinase(p-PERK)and phosphorylated eukaryotic translation initiation factor 2α(p-eIF2α)by using Western blot.Results After modeling,the colon tissues showed severe swelling,disordered arrangement of intestinal mucosal cells,hemorrhage with infiltration of inflammatory cell and partial loss of colon villus,which was relatively milder in the EA and MA groups.The colonic lesion score was remarkably increased in the model group in contrast to the control group(P<0.01),and obviously reduced in both EA and MA groups relevant to the model group(P<0.01).The levels of SOD,CAT,GSH and T-AOC were all significantly decreased(P<0.01),and the content of MDA,and expression levels of p-IκBα,p-p65 and GRP78,p-PERK and p-eIF2αproteins were all significantly increased in the model group relevant to the control group(P<0.01).After the treatment,modeling-induced down-regulation of SOD,CAT and GSH in both EA and MA groups,and T-AOC in the EA group,and up-regulation of levels of MDA,p-IκBα,p-p65,GRP78,p-PERK and p-eIF2α in both groups were reversed(P<0.01,P<0.05).Conclusion Both EA and MA treatment can obviously alleviate colonic inflammation in UC rats via inhibiting oxidative stress and ERS.