基于Akt/NF-κB信号通路探讨牛蒡根水提物对盐酸/乙醇诱导的急性胃溃疡大鼠的保护作用

Protective Effect of Aqueous Extract of Arctium lappa Roots on HCl/EtOH-induced Acute Gastric Ulcer in Rats Based on Akt/NF-κB Signaling Pathway

目的:基于蛋白激酶B(Akt)/核转录因子-κB(NF-κB)信号通路研究牛蒡根水提物(ALR-AE)对盐酸/乙醇(HCl/EtOH)诱导的大鼠急性胃溃疡的作用机制。方法:雄性大鼠随机分为5组,分别为正常组、模型组、雷尼替丁组(35 mg·kg^(-1))和ALR-AE低、高剂量组(50、100 mg·kg^(-1)),各给药组连续灌胃给药3 d,每天2次;末次给药30 min后,模型组及各给药组给予HCl/EtOH(以60%EtOH为溶剂,使HCl浓度为150 mmol·L^(-1))诱导大鼠急性胃溃疡。取各组胃组织样品,采用电子成像技术扫描溃疡面,ImageJ 1.8.0软件计算溃疡抑制率;分别采用苏木素-伊红(HE)和高碘酸-希夫(PAS)染色观察病理变化及糖蛋白的分布;比色法测定组织中氧化应激因子丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的水平;酶联免疫吸附试验(ELISA)检测组织样品中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-1β的水平;通过蛋白质免疫印迹法(Western blot)检测各组组织中磷酸化(p)-Akt/Akt、p-NF-κB p65/NF-κB p65、p-NF-κB抑制蛋白α(IκBα)/IκBα、p-IκB激酶α(IKKα)/IKKα和蛋白水平。结果:与正常组比较,模型组胃组织损伤严重,胃溃疡面积显著增大(P<0.01);MDA、TNF-α、IL-6和IL-1β水平显著升高(P<0.01),GSH-Px和SOD水平显著降低(P<0.01),Akt、NF-κB p65、IKKα和IκBα的磷酸化水平显著升高(P<0.01);与模型组比较,ALR-AE可显著降低HCl/EtOH诱导的胃组织损伤程度、提高溃疡抑制率(P<0.01),剂量依赖性地降低MDA、TNF-α、IL-6和IL-1β的水平(P<0.05,P<0.01),显著升高GSH-Px和SOD的水平(P<0.01),ALR-AE低剂量组可明显抑制Akt的磷酸化水平(P<0.05),ALR-AE高剂量组可明显抑制Akt、NF-κB p65、IKKα、IκBα的磷酸化水平(P<0.05,P<0.01)。结论:ALR-AE对HCl/EtOH诱导的大鼠胃溃疡有显著的保护作用,其作用机制可能与抑制Akt/NF-κB信号通路介导的炎症介质表达及降低氧化应激水平有关。

Objective:To investigate protective effect of Arctium lappa root aqueous extract(ALR-AE on hydrochloric acid/ethanol(HCl/EtOH)-induced acute gastric ulcer in rats based on protein kinase B/nuclear transcription factor-κB(Akt/NF-κB)signaling pathway.Method:Rats were randomly divided into 5 groups,namely normal group,model group,ranitidine group(35 mg·kg^(-1)),ALR-AE low dose group(50 mg·kg^(-1),ALR-AE-L group)and ALR-AE high dose group(100 mg·kg^(-1),ALR-AE-H group).Different doses of ALR-AE were orally administered twice daily for three consecutive days before the animals were subjected to HCl/EtOH(60%ethanol in 150 mmol·L^(-1)HCl)to induce acute gastric ulcer.For the gastric tissue samples,the ulcer surface was recorded by electronic imaging technique,and then the ulcer inhibition rate was calculated using ImageJ 1.8.0,hematoxylin-eosin(HE)and periodic acid-Schiff(PAS)staining were used to observe the pathological changes and mucoprotein distribution,respectively.The levels of oxidative stress factors of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in rat gastric tissues were determined by colorimetric method,the levels of pro-inflammatory mediators of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and IL-1β were determined by enzyme linked immunosorbent assay(ELISA),protein levels of phosphorylation and non-phosphorylation of Akt,NF-κB p65,NF-κB inhibitor proteinα(IκBα)and IκB kinaseα(IKKα)were evaluated by Western blot.Result:Compared with the normal group,the gastric tissue of the model group was severely damaged,and the area of gastric ulcer were significantly enlarged(P<0.01),the levels of MDA,TNF-α,IL-6 and IL-1β in gastric tissue were significantly increased(P<0.01),levels of GSH-Px and SOD were significantly decreased(P<0.01),and phosphorylation levels of Akt,NF-κB p65,IKKαand IκBαin gastric tissue were significantly increased(P<0.01).Compared with the model group,ALR-AE significantly attenuated HCl/EtOH-induced gastric tissue damage,significantly increased ulcer inhibition rate(P<0.01),and dose-dependently reduced the levels of MDA,TNF-α,IL-6 and IL-1β(P<0.05,P<0.01),and elevated GSH-Px and SOD levels(P<0.01),ALR-AE-L group could significantly inhibit the phosphorylation levels of Akt(P<0.05),and ALR-AE-H group could significantly inhibit phosphorylation levels of Akt,NF-κB p65,IKKαand IκBα(P<0.05,P<0.01).Conclusion:ALR-AE has a significant protective effect on HCl/EtOH-induced acute gastric ulcers in rats,and its mechanism may be related to the inhibition of inflammatory mediator expression and reduction of oxidative stress levels mediated by Akt/NF-κB signaling pathway.