黄芪甲苷Ⅳ治疗溃疡性结肠炎模型大鼠的机制研究

Mechanism of astragaloside Ⅳ in the treatment of ulcerative colitis in rats

目的探讨黄芪甲苷Ⅳ(astragalosideⅣ, AsⅣ)对溃疡性结肠炎(ulcerative colitis, UC)大鼠血清肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α),结肠组织p-p38丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)蛋白、p38 MAPK mRNA表达的影响。方法 32只SD大鼠,随机分为AsⅣ组、柳氮磺吡啶(sulfasalazine, SASP)组、模型组、对照组各8只。AsⅣ组、SASP组、模型组饮用质量分数4%葡聚糖硫酸钠溶液7 d制备UC大鼠模型,对照组自由饮用纯水。造模成功后,AsⅣ组给予AsⅣ溶液20 mL/(kg·d)灌胃,SASP组给予SASP溶液25 mL/(kg·d)灌肠,模型组给予生理盐水20 mL/(kg·d)灌肠,均连续7 d;对照组不作任何处理。干预7 d后,评估4组大鼠疾病活动指数评分(disease activity index, DAI);麻醉后取腹主动脉血,采用ELISA法检测血清TNF-α水平;取血后处死大鼠,截取距离肛门2 cm以上结肠组织,HE染色后行病理检查评定组织学损伤评分,采用Western blot法检测p-p38 MAPK蛋白表达,实时荧光定量PCR法检测p38 MAPK mRNA表达。结果干预7 d后,模型组、AsⅣ组、SASP组DAI评分[(5.50±1.38)、(3.17±1.21)、(2.83±1.67)分]、血清TNF-α水平[(248.17±7.85)、(186.33±11.90)、(208.67±8.38)μg/L]均高于对照组[0,(158.37±6.13)μg/L](P<0.05),模型组高于AsⅣ组、SASP组(P<0.05),AsⅣ组与SASP组比较差异无统计学意义(P>0.05);对照组大鼠结肠黏膜完整,腺体存在,排列规律,可见隐窝结构,黏膜及黏膜下层无炎症细胞浸润;模型组大鼠结肠黏膜不完整,腺体紊乱,排列不规则或消失,黏膜及黏膜下层大量炎症细胞浸润;AsⅣ组与SASP组大鼠结肠黏膜修复,腺体存在,黏膜及黏膜下层炎症细胞明显减少;干预7 d后,模型组、AsⅣ组、SASP组组织学损伤评分[(3.86±0.64)、(2.29±0.88)、(1.86±0.83)分]均高于对照组(0)(P<0.05),模型组高于AsⅣ组、SASP组(P<0.05),AsⅣ组与SASP组比较差异无统计学意义(P>0.05);模型组p-p38 MAPK蛋白相对表达量(17.29±3.72)高于AsⅣ组(9.52±1.27)、SASP组(10.02±0.22)、对照组(7.25±1.09)(P<0.05),SASP组高于对照组(P<0.05),AsⅣ组、对照组比较差异无统计学意义(P>0.05);模型组p38 MAPK mRNA相对表达量(2.42±0.09)高于AsⅣ组(1.08±0.08)、SASP组(0.55±0.19)、对照组(1)(P<0.05),AsⅣ组、SASP组、对照组比较差异无统计学意义(P>0.05)。结论对葡萄糖硫酸钠诱导的UC大鼠模型,AsⅣ可通过抑制p38 MAPK信号通路活化,减少TNF-α释放,缓解大鼠肠黏膜损伤,与SASP具有相同的效果。

Objective To investigate the effects of astragalosideⅣ(AsⅣ)on serum tumor necrosis factor-α(TNF-α),p-p38 mitogen-activated protein kinase(MAPK)protein in colon tissue and p38 MARK mRNA expression in rat models with ulcerative colitis(UC).Methods Thirty-two SD rats were randomly divided into AsⅣgroup,sulfasalazine(SASP)group,model group and control group,with 8 rats in each group.AsⅣgroup,SASP group and model group were orally administrated with 40%dextran sodium sulfate solution for 7 days to prepare UC rats models,and control group was orally administrated with purified water freely.After modeling,AsⅣgroup was given lavage with AsⅣsolution 20 mL/(kg·d),SASP group was given enema with SASP solution 25 mL/(kg·d),model group was given enema with normal saline 20 mL/(kg·d)for 7 days,and control group received no treatment.The disease activity index(DAI)was evaluated after intervention in 4 groups,and the serum level of tumor necrosis factor-α(TNF-α)was detected in abdominal aorta by ELISA after anesthesia.After the rats were sacrificed,the colon tissue 2 cm away from the anus was acquired to evaluate histological injury score by HE staining.The expressions of p-p38 MAPK protein and p38 MARK mRNA were detected by Western blot and real-time quantitative PCR technique.Results After intervention,the DAI scores(5.50±1.38,3.17±1.21,2.83±1.67)and serum TNF-αlevels((248.17±7.85),(186.33±11.90),(208.67±8.38)μg/L)were significantly higher in model group,AsⅣgroup and SASP group than those in control group(0,(158.37±6.13)μg/L)(P<0.05),higher in model group than those in AsⅣgroup and SASP group(P<0.05),and showed no significant differences between AsⅣgroup and SASP group(P>0.05).Control group was found intact colonic mucosa,normal and regularly arranged glands,visible crypt structure,and no inflammatory cell infiltration in mucosal and submucosal tissue.Model group was found incomplete colonic mucosa,irregularly arranged or absent glands,and a large number of inflammatory cells infiltration in mucosal and submucosal tissue.AsⅣgroup and SASP group were found repaired mucosa,normal gland,and decreased inflammatory cells infiltration in mucosal and submucosal tissue.After intervention,the injury score was significantly higher in model group(3.86±0.64),AsⅣgroup(2.29±0.88)and SASP group(1.86±0.83)than that in control group(0)(P<0.05),higher in model group than that in AsⅣgroup and SASP group(P<0.05),and showed no significant difference between AsⅣgroup and SASP group(P>0.05).The relative expression of p-p38 MAPK protein was significantly higher in model group(17.29±3.72)than that in AsⅣgroup(9.52±1.27),SASP group(10.02±0.22)and control group(7.25±1.09)(P<0.05),higher in SASP group than that in control group,and showed no difference in AsⅣgroup compared with that in SASP group and control group(P>0.05).The relative expression of p38 MAPK mRNA was significantly higher in model group(2.42±0.09)than that in AsⅣgroup(1.08±0.08),SASP group(0.55±0.19)and control group(1),and showed no differences in multiple comparison among AsⅣgroup,SASP group and control group(P>0.05).Conclusion AsⅣcan alleviate colonic mucosal injury by inhibiting the activation of p38 MAPK signaling pathway and reduce the release of TNF-αin DSS-induced UC rat models,and has the same therapeutic effect as SASP.