黄芩汤对溃疡性结肠炎小鼠氧化应激及铁死亡相关指标GSH-Px4,P53,SLC7A11的影响

Effect of Huangqintang on Oxidative Stress and Ferroptosis-related Indexes GSH-Px4,P53,SLC7A11 in Ulcerative Colitis Mice

目的:探讨黄芩汤治疗溃疡性结肠炎(UC)的可能作用机制。方法:采用葡聚糖硫酸钠进行造模,实验分正常组,模型组,黄芩汤低、中、高剂量组(4.55,9.1,18.2 g·kg^(-1))5个组。造模同时给予灌胃给药,连续7 d,第8天实验结束时采集标本测量结肠长度、质量,计算结肠质量指数,苏木素-伊红(HE)染色观察病理变化,生化试剂检测小鼠血清铁离子,超氧化物歧化酶(SOD),还原型谷胱甘肽(GSH),过氧化氢酶(CAT),髓过氧化物酶(MPO)等含量,蛋白免疫印迹法(Western blot)检测结肠中长链脂酰辅酶A合成酶4(ACSL4),谷胱甘肽过氧化物酶4(GSH-Px4),铁蛋白重链(FTH1)蛋白的表达。实时荧光定量聚合酶链式反应(Real-time PCR)检测结肠组织中肿瘤蛋白53(P53),胱氨酸/谷氨酸逆向转运蛋白溶质载体家族7成员11(SLC7A11)mRNA表达水平。结果:与正常组比较,模型组小鼠血清MPO,铁离子含量,ACSL4蛋白阳性表达及P53 mRNA表达均明显升高(P<0.05),血清SOD,CAT,GSH含量,GSH-Px4,FTH1蛋白表达及SLC7A11 mRNA表达均显著降低(P<0.01);与模型组比较,黄芩汤干预后,小鼠血清MPO,铁离子含量,ACSL4蛋白阳性表达及P53 mRNA表达均明显降低,血清SOD,CAT,GSH水平,GSH-Px4,FTH1蛋白表达及SLC7A11 mRNA表达均明显升高,且以高剂量组差异明显(P<0.05,P<0.01)。小鼠一般状态、结肠长度、肠重指数及HE染色结果表明,黄芩汤可在不同程度上改善小鼠临床症状及病理组织学变化。结论:以上结果表明黄芩汤对溃疡性结肠炎小鼠有治疗作用,其作用机制可能与抑制氧化应激及细胞铁死亡相关。

Objective: To explore the possible mechanism of Huangqintang in treating ulcerative colitis(UC). Method: The animal model of UC was induced by dextran sodium sulfate(DSS). The experimental animals were divided into control group,model group,Huangqintang low dose(4.55 g·kg^(-1)),medium dose(9.1 g·kg^(-1)),and high dose(18.2 g·kg^(-1))groups. Intragastric administration was also given in the modeling process for 7 consecutive days. At the end of the 8 th day,colon tissues were collected to measure colon length and mass,and calculate the colon mass index. Pathological changes were observed by hematoxylin-eosin(HE)staining. Serum iron content,superoxide dismutase(SOD),glutathione(GSH),catalase(CAT)and myeloperoxidase(MPO)were determined by biochemical assay. Western blot was used to detect the protein expression of glutathione peroxidase 4(GSH-Px4),long-chain acyl-CoA synthetase 4(ACSL4)and ferritin heavy chain 1(FTH1). The mRNA expression levels of tumor trotein 53(P53)and solute carrier family 7 member 11(SLC7A11)in colon tissues were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR). Result: The experimental studies showed that compared with normal group,serum MPO and iron content,ACSL4 protein level and relative P53 mRNA expression in the model group significantly increased(P<0.05),while serum SOD,CAT,GSH content,GSH-Px4,FTH1 relative protein expression level and relative SLC7A11 mRNA expression in the model group significantly decreased(P<0.01). Compared with model group, serum MPO and iron content, ACSL4 protein level and relative P53 mRNA expression significantly decreased(P<0.05),while serum SOD,CAT,GSH content,GSH-Px4,FTH1 relative protein expression level and relative SLC7A11 mRNA expression significantly increased(P<0.05)after the intervention of Huangqintang,and the effect was most significant in the high-dose group(P<0.05). The results of general condition,colon length,colon mass index and HE staining showed that Huangqintang could relieve clinical symptoms and histopathological changes in UC mice. Conclusion: These results indicated that Huangqintang had therapeutic effect on ulcerative colitis mice,and its mechanism might be related to inhibiting the oxidative stress and ferroptosis.