基于p38 MAPK/NF-κB信号通路探讨中华雪胆醇提物对盐酸/乙醇诱导的大鼠急性胃溃疡的保护作用

Protective Effect of Ethanolic Extract of Hemsleya chinensis on HCl/Ethanol-induced Acute Gastric Ulcer in Rats Based on p38 MAPK/NF-κB Signaling Pathway

目的:探讨中华雪胆醇提物(HC-EE)对盐酸/乙醇(HCl/EtOH)诱导的大鼠急性胃溃疡保护作用机制。方法:采用脂多糖(LPS)诱导的RAW264.7细胞体外评价HC-EE对炎症介质生成的抑制作用;采用HCl/EtOH(以60%EtOH为溶剂,使HCl浓度为150 mmol·L^(-1))诱导的大鼠急性胃溃疡模型评价HC-EE对急性胃溃疡的保护作用,雄性SD大鼠随机等分为5组,即正常组、模型组、HC-EE低剂量(30 mg·kg^(-1),HC-EE 30)组、HC-EE高剂量(60 mg·kg^(-1),HC-EE 60)组及雷尼替丁(35 mg·kg^(-1))组,各组连续灌胃给药7 d,每天2次,末次给药30 min后,模型组及各给药组给予HCl/EtOH诱导大鼠急性胃溃疡。对各组动物进行麻醉,分离胃组织,采用电子成像技术记录大鼠胃内壁溃疡面,运用ImageJ 1.8.0计算药物的溃疡抑制率,通过苏木素-伊红(HE)和高碘酸-希夫(PAS)染色观察大鼠病理组织学变化,利用Griess法测定细胞培养液中的一氧化氮(NO)含量,酶联免疫吸附试验(ELISA)测定大鼠血清(或细胞培养液)中白细胞介素(IL)-1β、肿瘤坏死因子-α(TNF-α)、IL-6、血管细胞黏附分子-1(VCAM-1)、前列腺素E2(PGE2)的水平,通过蛋白质免疫印迹法(Western blot)检测各组大鼠胃组织磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)/p38 MAPK、p-核转录因子-κB(NF-κB)p65/NF-κB p65蛋白表达水平。结果:体外实验结果显示,HC-EE能明显下调模型细胞NF-κB信号介导的炎症介质NO,以及TNF-α、IL-1β、IL-6、VCAM-1的表达(P<0.05,P<0.01)。体内实验结果显示,与正常组比较,模型组大鼠胃组织损伤明显,溃疡面积显著增大,TNF-α、IL-6水平显著升高(P<0.01),PGE2含量显著降低(P<0.01),p38 MAPK、NF-κB p65的磷酸化水平显著升高(P<0.01);与模型组比较,HC-EE呈剂量依赖性地改善HCl/EtOH诱导的胃组织损伤和炎细胞浸润,提高溃疡抑制率,显著减少TNF-α、IL-6的释放量(P<0.01),HC-EE 60组可以增加PGE2的含量(P<0.05)、显著抑制胃组织蛋白p38 MAPK、NF-κB p65的磷酸化水平(P<0.05,P<0.01)。结论:HC-EE可改善HCl/EtOH诱导的大鼠急性胃溃疡,其作用机制可能与抑制p38 MAPK/NF-κB信号通路介导的炎症介质表达有关。

Objective:To investigate the mechanism of protective effect of ethanol extracts of Hemsleya chinensis(HC-EE)on hydrochloric acid/ethanol(HCl/EtOH)-induced acute gastric ulcer in rats.Method:Lipopolysaccharide(LPS)-induced RAW264.7 cells were used to evaluate inhibitory effect of HC-EE on the production of inflammatory mediators in vitro.A rat acute gastric ulcer model induced by HCl/EtOH(60%ethanol in 150 mmol·L^(-1)HCl)was used to evaluate protective effect of HC-EE on acute gastric ulcer.Rats were divided into five groups,including normal group,model group,HC-EE low dose(HC-EE 30,30 mg·kg^(-1))group,HC-EE high dose(HC-EE 60,60 mg·kg^(-1))group and ranitidine(35 mg·kg^(-1))group.For model and drug-treated groups,vehicle solvent or drugs were orally administered twice daily for 7 consecutive days before the rats were subjected to HCl/EtOH to induce acute gastric ulcer.After being anesthetized,ulcer surface of each rat was obtained and recorded using electronic imaging technology,and the ulcer inhibition rate was calculated by ImageJ 1.8.0.Hematoxylin-eosin(HE)and periodic acid-Schiff(PAS)staining were used to observe the pathological histological changes in rats.Content of nitric oxide(NO)in cell culture medium was measured by the Griess method.The levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),IL-6,vascular cell adhesion molecule-1(VCAM-1)and prostaglandin E2(PGE2)in rat serum(or cell culture medium)were determined by enzyme linked immunosorbent assay(ELISA).The protein expressions of phosphorylation(p)-p38 mitogen activated protein kinase(MAPK)/p38 MAPK and p-nuclear transcription factor-κB(NF-κB)p65/NF-κB p65 in rat gastric tissue were detected by Western blot.Result:In vitro assay showed HC-EE could significantly down-regulate the expressions of NO,TNF-α,IL-1β,IL-6 and VCAM-1 in LPS-induced cells(P<0.05,P<0.01).In vivo experimental results showed that,compared with the normal group,gastric tissue of the model group was severely damaged,and the area of gastric ulcer was significantly enlarged,levels of TNF-α,IL-6 were significantly increased(P<0.01),and the level of PGE2 was significantly decreased(P<0.01),the phosphorylation levels of of p38 MAPK,NF-κB p65 in gastric tissue were significantly increased(P<0.01).Compared with the model group,HC-EE dose-dependently improved HCl/EtOH-induced gastric tissue injury and inflammatory cell infiltration,and it could increase ulcer inhibition rate,significantly decreased the release of TNF-α and IL-6(P<0.01),HC-EE 60 group could increase the content of PGE2(P<0.05),and significantly inhibit the phosphorylation levels of p38 MAPK and NF-κB p65(P<0.05,P<0.01).Conclusion:HC-EE can exert protective effect on HCl/EtOH-induced acute gastric ulcer in rats,and its mechanism may be related to the inhibition of expression of inflammatory mediators mediated by p38 MAPK/NF-κB signaling pathway.