atRA对大鼠结肠炎中TGF-β1/Smad3通路的作用研究

Effect of atRA on TGF-β1/Smad3 pathway in colitis rat models

目的探讨全反式维甲酸(atRA)对TNBS诱导的大鼠结肠炎模型中的TGF-β1/Smad3信号通路的影响。方法大鼠随机分为5组,分别为对照组(C组),造模组(TNBS组),atRA灌胃组(A组),美沙拉嗪组(M组),美沙拉嗪+atRA组(MA组);使用TNBS/乙醇灌肠液诱导结肠炎;之后对照组、造模组予以生理盐水灌胃,其余各组分别予以相应药物灌胃,记录大鼠的疾病活动指数(DAI)、组织学评分(TDI)。QT-PCR检测结肠组织中TGF-β1 mRNA、Smad7 mRNA的水平,免疫组化测定结肠组织中Smad7蛋白及磷酸化Smad3(p Smad3)水平。ELISA法检测血清中IL-17、TNF-α水平。结果与TNBS组比较,各治疗组的DAI、TDI评分较TNBS组下降,差异有统计学意义(P<0.05),MA组下降最明显(P<0.05)。C组TGF-β1 mRNA水平降低最明显,TNBS组升高,各治疗组升高更明显,与TNBS组比较差异有统计学意义(P<0.05)。治疗后,与TNBS组比较,各组Smad7 mRNA水平均下降(P<0.05);TNBS组Smad7蛋白水平最高,治疗后各组Smad7蛋白水平均明显降低(P<0.05),但A组与M组比较差异无统计学意义(P=0.586),MA组较A组或M组下降更明显(P<0.05)。TNBS组p Smad3水平明显下调,而各治疗组均升高,与TNBS组比较差异有统计学意义(P<0.01),其中MA组升高最明显(P<0.05)。各治疗组IL-17、TNF-α水平较TNBS组均明显降低,MA组降低最明显(P<0.05)。结论炎症性肠病中存在着TGF-β1/Smad3通路受损,导致过度的免疫炎症反应。atRA可通过下调Smad7蛋白表达,增加p Smad3水平,修复该通路,恢复TGF-β1的效应,缓解症状。

Objective To investigate the effect of all-trans retinoic acid（ atRA） on TGF-β1/Smad3 signaling pathway in 2,4,6-trinitrobenzene sulfonic acid（ TNBS）-induced colitis in rat models. Methods The rats were randomly divided into 5 groups： control group（ group C）,TNBS group,atRA gavage group（ group A）,masalazine gavage group（ group M）,and masalazine combined with atRA group（ group MA）. Rats were instilled TNBS/ethanol enema to induce colitis. After the administration,group C and TNBS group were gavaged with physiological saline solution,and the other groups were gavaged with corresponding drugs. The DAI and TDI score were accessed. The expression of TGF-β1 mRNA and Smad7 mRNA in colorectal tissues was detected by RT-PCR. The expression of Smad7 protein and p Smad3 protein in colorectal tissues was detected by immunohistochemistry. The plasma level of IL-17 and TNF-α was detected by ELISA.Results The DAI and TDI scores of all therapeutic groups were decreased significantly compared with TNBS group,the difference being statistically significant（ P〈0. 05）,and the decrease in group MA was the most significant（ P〈0. 05）.The expression of TGF-β1 mRNA was decreased most in group C,but increased in TNBS group. After drug intervention,all therapeutic groups had a marked increase in TGF-β1 mRNA compared with TNBS group（ P〈0. 05）. Smad7 mRNA was lower in TNBS group than in therapeutic groups（ P〈0. 05）. The Smad7 protein of TNBS group was the highest,and after treatment,the Smad7 of all therapeutic groups was decreased significant compared with TNBS group（ P〈0. 05）,but there was no significant difference between group A and group M（ P = 0. 586）. The lower expression of Smad7 was observed in group MA compared with group A and group M（ P〈0. 05）. The expression of p Smad3 was decreased markedly in TNBS group,but significantly increased in each therapeutic group（ P〈0. 01）,with group MA increasing most among them（ P〈0. 05）. The plasma level of IL-17 and TNF-α of all therapeutic groups was significantly lower than that in TNBS group,and the lowest level was observed in group MA（ P〈0. 05）. Conclusion The TGF-β1/Smad3 signal pathway is defective in colitis rats,which results in the excessive immune response. The atRA can relieve ulcerative colitis symptoms by down-regulating Smad7 protein expression while up-regulating p Smad3 expression and restoring TGFβ1/Smad3 signaling,which enables TGFβ1 to down-regulate the excessive immune response.