黄芪多糖通过miR-193a-3p/STMN1轴对溃疡性结肠炎大鼠结肠细胞凋亡和氧化应激的影响

Astragalus polysaccharides affect colon cell apoptosis and oxidative stress in rats with ulcerative colitis through miR-193a-3p/STMN1 axis

目的 研究黄芪多糖是否通过miR-193a-3p/STMN1轴对溃疡性结肠炎大鼠结肠细胞凋亡和氧化应激的影响。方法 将60只SD大鼠随机分为对照组和模型组,每组30只。模型组采用三硝基苯磺酸(TNBS)/乙醇复合法构建大鼠溃疡型结肠炎模型,对照组给予等量的50%乙醇,之后分离培养对照组和模型组大鼠结肠上皮细胞。溃疡型结肠炎大鼠结肠上皮细胞分别给予100、200、400μg/mL黄芪多糖,流式细胞术、免疫印迹实验(Western blot)和实时定量PCR(qRT-PCR)分别检测细胞凋亡、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、miR-193a-3p表达。试剂盒检测丙二醛(MDA)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平。在溃疡性结肠炎大鼠结肠上皮细胞中转染anti-miR-con、anti-miR-193a-3p模拟物、si-NC、si-STMN1过表达质粒,并给予400μg/mL黄芪多糖,分别记为APS+anti-miR-con组、APS+anti-miR-193a-3p组、APS+anti-miR-193a-3p+si-NC组、APS+anti-miR-193a-3p+si-STMN1组,采用上述方法检测溃疡性结肠炎大鼠结肠上皮细胞凋亡、氧化应激变化。生物信息学starbase预测和双荧光素酶报告实验验证miR-193a-3p与STMN1靶向作用。结果 与对照组比较,模型组大鼠结肠上皮细胞的凋亡率、Bax、STMN1蛋白表达量、MDA、LDH水平增加,Bcl-2蛋白、miR-193a-3p表达量、SOD、GSH-Px水平减少,差异有统计学意义(P<0.05)。与模型组比较,100、200、400μg/mL黄芪多糖减少溃疡性结肠炎大鼠结肠上皮细胞的凋亡率、Bax、STMN1蛋白表达量、MDA、LDH水平,提高Bcl-2蛋白、miR-193a-3p表达量、SOD、GSH-Px水平,均呈剂量依赖性,且差异有统计学意义(P<0.05)。与APS+anti-miR-con组比较,APS+anti-miR-193a-3p组溃疡性结肠炎大鼠结肠上皮细胞的miR-193a-3p表达量、凋亡率、Bax蛋白表达量、MDA、LDH水平升高,Bcl-2蛋白表达量、SOD、GSH-Px水平降低,差异有统计学意义(P<0.05)。miR-193a-3p直接靶向STMN1。与APS+anti-miR-193a-3p+si-NC组比较,APS+anti-miR-193a-3p+si-STMN1组溃疡性结肠炎大鼠结肠上皮细胞的STMN1、Bcl-2蛋白表达量、SOD、GSH-Px水平增加,凋亡率、Bax蛋白表达量、MDA、LDH水平减少,差异有统计学意义(P<0.05)。结论 黄芪多糖通过上调miR-193a-3p靶向STMN1,减轻溃疡性结肠炎大鼠结肠上皮细胞的凋亡和氧化应激。

Objective To investigate whether astragalus polysaccharides(APS) affect colon cell apoptosis and oxidative stress through miR-193 a-3 p/STMN1 axis in rats with ulcerative colitis.Methods Sixty SD rats were randomly divided into control group and model group,with 30 rats in each group.The model group was treated with trinitrobenzene sulfonic acid(TNBS) and ethanol to construct rat ulcerative colitis.The control group was given the same amount of 50% ethanol,and then the colonic epithelial cells were separated,cultured,and treated with APS 100,200,400 μg/ml.Flow cytometry,Western blot and qRT-PCR were used to detect apoptosis,Bcl-2,Bax,and miR-193 a-3 p expression.The levels of malondialdehyde(MDA),lactate dehydrogenase(LDH),superoxide dismutase(SOD),and glutathione peroxidase(GSH-Px)were examined.Rat colonic epithelial cells with ulcerative colitis were transfected with anti-miR-con,anti-miR-193 a-3 p mimics,si-NC,si-STMN1 overexpression plasmids,and treated with APS 400 μg/ml.Bioinformatics starbase prediction and dual luciferase report experimentswere used to verify the targeting effect of miR-193 a-3 p and STMN1.Results Compared with the control group,the apoptosis rate,protein expression levels of Bax and STMN1,MDA and LDH levels of colonic epithelial cells in the model group were increased,and the protein expression of Bcl-2 and miR-193 a-3 p,SOD and GSH-Px levels were decreased(P<0.05).Treatment with APS 100,200,400 μg/ml reduced apoptosis,levels of Bax and STMN1,MDA and LDH ofcolonic epithelial cells with ulcerative colitis,and increased the expression of Bcl-2 protein and miR-193 a-3 p,SOD and GSH-Px levels(P<0.05).Anti-miR-193 a-3 p reversed APS effects.miR-193 a-3 p directly targets STMN1,si-STMN1 reversed APS+anti-miR-193 a-3 p effects.Conclusion APS can reduce the apoptosis and oxidative stress of colonic epithelial cells in rats with ulcerative colitis by up-regulating miR-193 a-3 p target STMN1.