清肠温中方通过miR-675-5p/VDR信号通路调控溃疡性结肠炎Th17/Treg免疫平衡及肠黏膜屏障的机制研究
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摘要
目的研究清肠温中方调控DSS诱导溃疡性结肠炎(UC)大鼠Th17/Treg免疫平衡及肠黏膜屏障的作用机制。方法 24只SPF级雄性SD大鼠,按体质量依照随机数字表将其随机分为空白组、模型组、清肠温中方组。空白组自由饮用去离子水,同时予去离子水灌胃;模型组和清肠温中方组自由饮用4.5%DSS溶液制备UC模型,同时模型组给予去离子水,清肠温中方组予清肠温中方灌胃。7 d后麻醉大鼠后取血及结肠组织,应用Real time-PCR法检测结肠miR-675-5p、VDR m RNA、RORγt mRNA、Foxp3 m RNA的表达,ELISA法检测血清IL-10、IL-17的表达,免疫组织化学法检测ZO-1、Occludin的蛋白表达及分布。结果与空白组比较,DSS诱导的UC大鼠结肠mi R-675-5p、RORγt mRNA及血清IL-17的表达较空白组明显升高(P <0.05或P <0.01),VDR mRNA、Foxp3 mRNA、ZO-1、Occludin、血清IL-10的表达较空白组明显降低(P <0.05或P <0.01);清肠温中方干预后,大鼠结肠mi R-675-5p、RORγt mRNA、血清IL-17的表达较模型组明显降低(P <0.05),VDR mRNA、Foxp3 mRNA、ZO-1、Occludin、血清IL-10的表达较模型组明显升高(P <0.05或P <0.01)。结论清肠温中方可能通过降低mi R-675-5p的表达,靶向调控VDR信号通路,从而调控溃疡性结肠炎Th17/Treg免疫平衡、修复肠黏膜屏障损伤,达到治疗UC的目的。
Objective: To explore the mechanism of regulating the immune balance of Th17/Treg and intestinal mucosal barrier in rats with DSS induced ulcerative colitis. Methods: 24 SPF male SD rats were randomly divided into three groups according to their body weight: control group,model group and Qingchang Wenzhong Decoction group. Control group: drinking free deionized water,while deionized water was administered. The model group and the Qingchang Wenzhong Decoction group: free drinking 4.5% DSS solution to make UC model,while deionized water was administered in model group and Qingchang Wenzhong Decoction was administered in model group. After 7 days Intervention,blood and colonic tissue were taken from the anesthetized rats. Real time-PCR was used to detect the expression of miR-675-5 p,VDR m RNA,RORγt mRNA,Foxp3 mRNA in colon,Elisa to detect the expression of serum IL-10,IL-17,and immunohistochemical method to detect the expression and distribution of ZO-1,Occludin protein. Results: Compared with the control group,the expression of miR-675-5 p,RORγt mRNA and serum IL-17 in the colon of UC rats induced by DSS were significantly higher than those in the control group(P < 0.05 or P < 0.01) and the expression of VDR m RNA,Foxp3 mRNA,ZO-1,Occludin,serum IL-10 was significantly lower than that in the control group(P < 0.05 or P < 0.01). After the intervention of Qingchang Wenzhong Decoction,The expression of miR-675-5 p,RORγt mRNA in colon and serum IL-17 of rats was significantly lower than those in model group(P < 0.05) and the expression of VDR mRNA,Foxp3 mRNA,ZO-1,Occludin,serum IL-10 was significantly higher than those in the model group(P < 0.05 or P < 0.01). Conclusion: Qingchang Wenzhong Decoction may regulate the VDR signaling pathway by decreasing the expression of miR-675-5 p,thereby regulating the immune balance of Th17/Treg in ulcerative colitis,repairing the intestinal mucosal barrier injury,and achieving the purpose of treating UC.
Objective: To explore the mechanism of regulating the immune balance of Th17/Treg and intestinal mucosal barrier in rats with DSS induced ulcerative colitis. Methods: 24 SPF male SD rats were randomly divided into three groups according to their body weight: control group,model group and Qingchang Wenzhong Decoction group. Control group: drinking free deionized water,while deionized water was administered. The model group and the Qingchang Wenzhong Decoction group: free drinking 4.5% DSS solution to make UC model,while deionized water was administered in model group and Qingchang Wenzhong Decoction was administered in model group. After 7 days Intervention,blood and colonic tissue were taken from the anesthetized rats. Real time-PCR was used to detect the expression of miR-675-5 p,VDR m RNA,RORγt mRNA,Foxp3 mRNA in colon,Elisa to detect the expression of serum IL-10,IL-17,and immunohistochemical method to detect the expression and distribution of ZO-1,Occludin protein. Results: Compared with the control group,the expression of miR-675-5 p,RORγt mRNA and serum IL-17 in the colon of UC rats induced by DSS were significantly higher than those in the control group(P < 0.05 or P < 0.01) and the expression of VDR m RNA,Foxp3 mRNA,ZO-1,Occludin,serum IL-10 was significantly lower than that in the control group(P < 0.05 or P < 0.01). After the intervention of Qingchang Wenzhong Decoction,The expression of miR-675-5 p,RORγt mRNA in colon and serum IL-17 of rats was significantly lower than those in model group(P < 0.05) and the expression of VDR mRNA,Foxp3 mRNA,ZO-1,Occludin,serum IL-10 was significantly higher than those in the model group(P < 0.05 or P < 0.01). Conclusion: Qingchang Wenzhong Decoction may regulate the VDR signaling pathway by decreasing the expression of miR-675-5 p,thereby regulating the immune balance of Th17/Treg in ulcerative colitis,repairing the intestinal mucosal barrier injury,and achieving the purpose of treating UC.
引文
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[11] Beaugerie,Laurent. Use of Immunosuppressants and biologicals in patients with previous cancer[J]. Digestive Disease,2013,31(2):254-259.
[12] Zou T,Jaladanki SK,Liu L,et al. H19 Long Noncoding RNA Regulates Intestinal Epithelial Barrier Function via MicroRNA 675 by Interacting with RNA-Binding Protein HuR[J].Molecular&Cellular Biology,2016,36(9):1332-1341.
[13] Mousa A,Misso M,Teede H,et al. Effect of vitamin D supplementation on inflammation:protocol for a systematic review[J]. Bmj Open,2016,6(4):e010804.
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[15] Reich KM,Fedorak RN,Madsen K,et al. Vitamin D improves inflammatory bowel disease outcomes:Basic science and clinical review[J]. World Journal of Gastroenterology,2014,20(17):4934-4947.
[16] Lu D,Lan B,Din Z,et al. A vitamin D receptor agonist converts CD4+T cells to Foxp3+regulatory T cells in patients with ulcerative colitis[J]. Oncotarget,2017,8(32):53552-53562.
[17]张博,熊壮,姜鑫.自拟扶正平溃汤联合针灸治疗慢性溃疡性结肠炎的疗效及对肠道菌群失调和Th17/Treg细胞免疫平衡的影响[J].现代中西医结合杂志,2018,27(11):1164-1167,1171.
[18]张静,付妤,邹开芳,等.雷公藤甲素对小鼠实验性结肠炎Th17/Treg的调节作用[J].胃肠病学和肝病学杂志,2011,20(12):1118-1121.
[19]庞艳华,吴东方,马亚会,等.白细胞介素-6在溃疡性结肠炎模型小鼠Th17/Treg细胞失衡中的作用[J].胃肠病学和肝病学杂志,2018,27(5):488-491.
[20] Cui H,Cai Y,Wang L,et al. Berberine Regulates Treg/Th17Balance to Treat Ulcerative Colitis Through Modulating the Gut Microbiota in the Colon[J]. Frontiers in Pharmacology,2018,9:571.
[21] You P,Chen N,Su L,et al. Local level of TGF-β1 determines the effectiveness of dexamethasone through regulating the balance of Treg/Th17 cells in TNBS-induced mouse colitis[J]. Experimental and Therapeutic Medicine,2018,15(4):3639-3649.
[2] Mayer L,Sandborn WJ,Stepanov Y,et al. Anti-IP-10 antibody(BMS-936557)for ulcerativecolitis:a phaseⅡrandomised study[J]. Gut,2014,63(3):442-450.
[3]毛堂友,程佳伟,魏仕兵,等.清肠温中方治疗溃疡性结肠炎84例[J].环球中医药,2016,9(4):479-481.
[4] Mao TY,Li JX,Liu LJ,et al. Qingchang wenzhong decoction attenuates DSS-Induced colitis in rats by reducing inflammation and improving intestinal barrier function via upregulating the MSP/RON signalling pathway[J]. Evidence-Based Complementary and Alternative Medicine,2017,2017:4846876.
[5] Mao TY,Shi R,Zhao WH,et al. Qingchang Wenzhong Decoction Ameliorates Dextran Sulphate Sodium-Induced Ulcerative Colitis in Rats by Downregulating the IP10/CXCR3 AxisMediated Inflammatory Response[J]. Evidence-Based Complementary and Alternative Medicine,2016,2016:4312538.
[6] Da SB,Lyra AC,Rocha R,et al. Epidemiology, demographic characteristics and prognostic predictors of ulcerative colitis[J]. World Journal of Gastroenterology,2014,20(28):9458-9467.
[7] Coward S,Heitman SJ,Clement F,et al. Ulcerative colitis-associated hospitalization costs:A population-based study[J].Canadian Journal of Gastroenterology&Hepatology,2015,29(7):357-362.
[8] Curkovic I,Egbring M,Kullakublick GA. Risks of inflammatory bowel disease treatment with glucocorticos teroids and aminosalicylates[J]. Digestive Diseases,2013,31(3-4):368-373.
[9] Gabryel M,Skrzypczakzielinska M,Kucharski MA,et al. The impact of genetic factors on response to glucocorticoids therapy in IBD[J]. Scand J Gastroenterol,2016,51(6):654-665.
[10]张瑞芳,陈朝晖,刘漪沦,等.溃疡性结肠炎的发病机制及其治疗进展[J].生命的化学,2018,38(2):241-249.
[11] Beaugerie,Laurent. Use of Immunosuppressants and biologicals in patients with previous cancer[J]. Digestive Disease,2013,31(2):254-259.
[12] Zou T,Jaladanki SK,Liu L,et al. H19 Long Noncoding RNA Regulates Intestinal Epithelial Barrier Function via MicroRNA 675 by Interacting with RNA-Binding Protein HuR[J].Molecular&Cellular Biology,2016,36(9):1332-1341.
[13] Mousa A,Misso M,Teede H,et al. Effect of vitamin D supplementation on inflammation:protocol for a systematic review[J]. Bmj Open,2016,6(4):e010804.
[14] Wang L,Wang ZT,Hu JJ,et al. Polymorphisms of the vitamin D receptor gene and the risk of inflammatory bowel disease:a meta-analysis[J]. Genetics&Molecular Research Gmr,2014,13(2):2598-2610.
[15] Reich KM,Fedorak RN,Madsen K,et al. Vitamin D improves inflammatory bowel disease outcomes:Basic science and clinical review[J]. World Journal of Gastroenterology,2014,20(17):4934-4947.
[16] Lu D,Lan B,Din Z,et al. A vitamin D receptor agonist converts CD4+T cells to Foxp3+regulatory T cells in patients with ulcerative colitis[J]. Oncotarget,2017,8(32):53552-53562.
[17]张博,熊壮,姜鑫.自拟扶正平溃汤联合针灸治疗慢性溃疡性结肠炎的疗效及对肠道菌群失调和Th17/Treg细胞免疫平衡的影响[J].现代中西医结合杂志,2018,27(11):1164-1167,1171.
[18]张静,付妤,邹开芳,等.雷公藤甲素对小鼠实验性结肠炎Th17/Treg的调节作用[J].胃肠病学和肝病学杂志,2011,20(12):1118-1121.
[19]庞艳华,吴东方,马亚会,等.白细胞介素-6在溃疡性结肠炎模型小鼠Th17/Treg细胞失衡中的作用[J].胃肠病学和肝病学杂志,2018,27(5):488-491.
[20] Cui H,Cai Y,Wang L,et al. Berberine Regulates Treg/Th17Balance to Treat Ulcerative Colitis Through Modulating the Gut Microbiota in the Colon[J]. Frontiers in Pharmacology,2018,9:571.
[21] You P,Chen N,Su L,et al. Local level of TGF-β1 determines the effectiveness of dexamethasone through regulating the balance of Treg/Th17 cells in TNBS-induced mouse colitis[J]. Experimental and Therapeutic Medicine,2018,15(4):3639-3649.
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