清肠栓脐贴调控TLR4/MyD88/NF-κB信号通路减轻小鼠溃疡性结肠炎
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摘要
目的:研究清肠栓脐贴对葡聚糖硫酸钠(DSS)诱导的溃疡性结肠炎(UC)小鼠的作用,并基于Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核因子-κB(NF-κB)信号通路探讨其作用机制。方法:取40只雄性Balb/c小鼠,采用3.5%DSS自由饮用连续7 d的方法制备UC模型,另取10只小鼠作为正常组。造模7 d后,造模小鼠随机分为模型组、美沙拉嗪组、清肠栓方组和清肠栓脐贴组,每组10只。美沙拉嗪组灌胃给予0.1 g/kg美沙拉嗪溶液;清肠栓方组和清肠栓脐贴组分别采用灌胃和脐贴方式给予0.118 g/kg清肠栓生药;正常组和模型组灌胃给予等量0.9%NaCl溶液。均每日1次,连续9 d。造模期间,观察小鼠的一般活动和排便情况,计算疾病活动指数(DAI)。末次给药后采集血液和结肠样本,HE染色后光镜下观察各组结肠组织的病理学变化;ELISA检测血清TNF-α和IL-6水平,PCR检测结肠组织TNF-α、IL-6、IL-1β基因表达,Western blot检测结肠组织TLR4、MyD88、NF-κB p65蛋白表达。结果:①造模第7天,造模小鼠均出现明显血便,且精神萎靡、体质量明显下降;DAI评分较正常组显著升高(P<0.01)。②结肠组织病理观察显示,与正常组比较,模型组小鼠结肠上皮组织部分缺损,腺管萎缩或消失,结构紊乱,杯状细胞减少,炎症细胞浸润严重。美沙拉嗪组、清肠栓方组、清肠栓脐贴组小鼠结肠黏膜及黏膜下层可见少量炎症细胞浸润,病变程度较模型组明显减轻。③与正常组比较,模型组小鼠血清IL-6和TNF-α水平显著升高(P<0.01);与模型组比较,美沙拉嗪组、清肠栓方组、清肠栓脐贴组血清IL-6和TNF-α水平显著降低(P<0.05,P<0.01)。不同药物组间比较,差异无统计学意义(P>0.05)。④与正常组比较,模型组小鼠结肠TNF-α、IL-6、IL-1βmRNA表达水平及TLR4、MyD88、NF-κB p65蛋白表达水平显著升高(P<0.01);与模型组比较,美沙拉嗪组、清肠栓方组、清肠栓脐贴组结肠TNF-α、IL-6、IL-1βmRNA表达水平及TLR4、MyD88、NF-κB p65蛋白表达水平显著下调(P<0.01)。不同药物组间比较,差异无统计学意义(P>0.05)。结论:清肠栓脐贴可明显改善UC小鼠的一般活动及排便情况,减轻肠道黏膜上皮损伤和炎症浸润,其作用机制与抑制TLR4/MyD88/NF-κB信号通路有关。
Objective: To study the effects of Qingchang suppository umbilical paste on dextran sulfate sodium(DSS)-induced ulcerative colitis(UC) mice, and explore its mechanism based on Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Methods: Forty male Balb/c mice were taken, and treated with 3.5% DSS by freely drinking for continuous 7 days to induce UC model. Another 10 mice were used as the normal group. After modeling for 7 days, the modeling mice were randomly divided into the model group, mesalazine group, Qingchang suppository decoction group and Qingchang suppository umbilical paste group, 10 mice in each group. The mesalazine group was treated with 0.1 g/kg mesalazine solution by intragastric administration, the Qingchang suppository decoction group and the Qingchang suppository umbilical paste group were treated with 0.118 g/kg Qingchang suppository crude drug by intragastric administration or umbilical compress respectively, the normal group and model group were treated with 0.9% NaCl solution at equal volume, with a course of 9 days. During the modeling, the general activity and defecation of mice were observed, and the disease activity index(DAI) was calculated. After last administration, the serum and colon tissue were collected. The histopathological changes of colon tissue were observed by light microscope after HE staining. The serum levels of TNF-α and IL-6 were measured by ELISA. The mRNA expressions of TNF-α, IL-6 and IL-1β in colon tissue were detected by PCR, and the protein expressions of TLR4, MyD88 and NF-κB p65 in colon tissue were detected by Western blot. Results: ① On the 7 th day of modeling, the UC model mice showed obvious bloody stool, fatigued spirit and decreased body weight. The DAI score of the model group was significantly higher than that of the normal group(P<0.01). ② Histopathological observation on colon tissue showed that, compared with the normal group, the partial defect of epithelium was observed in the model group, as well as atrophy or disappearance of glands with disordered arrangement, less goblet cells, and serious inflammatory cell infiltration. The mesalazine group, Qingchang suppository decoction group and Qingchang suppository umbilical paste group showed a small amount of inflammatory cells infiltration in the mucosa and submucosa, and the degree of lesion was significantly improved compared with the model group.③Compared with the normal group, the serum levels of IL-6 and TNF-α were significantly increased in the model group(P<0.01); compared with the model group, the serum levels of IL-6 and TNF-α were significantly decreased in the mesalazine group, Qingchang suppository decoction group and Qingchang suppository umbilical paste group(P<0.05, P<0.01). There were no statistically significant differences among the different drug treatment groups(P>0.05). ④Compared with the normal group, the mRNA expression levels of TNF-α, IL-6 and IL-1β and the protein expression levels of TLR4, MyD88 and NF-κB p65 were significantly increased in the model group(P<0.01); compared with the model group, the mRNA expression levels of TNF-α, IL-6 and IL-1β and the protein expression levels of TLR4, MyD88 and NF-κB p65 were significantly down-regulated in the mesalazine group, Qingchang suppository decoction group and Qingchang suppository umbilical paste group(P<0.01). There were no statistically significant differences among the different drug treatment groups(P>0.05). Conclusion: Qingchang suppository umbilical paste can significantly improve the general activity and defecation of UC mice, and alleviate the colon mucosal epithelial damage and inflammatory infiltration. Its mechanism is related to inhibiting TLR4/MyD88/NF-κB signaling pathway.
Objective: To study the effects of Qingchang suppository umbilical paste on dextran sulfate sodium(DSS)-induced ulcerative colitis(UC) mice, and explore its mechanism based on Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Methods: Forty male Balb/c mice were taken, and treated with 3.5% DSS by freely drinking for continuous 7 days to induce UC model. Another 10 mice were used as the normal group. After modeling for 7 days, the modeling mice were randomly divided into the model group, mesalazine group, Qingchang suppository decoction group and Qingchang suppository umbilical paste group, 10 mice in each group. The mesalazine group was treated with 0.1 g/kg mesalazine solution by intragastric administration, the Qingchang suppository decoction group and the Qingchang suppository umbilical paste group were treated with 0.118 g/kg Qingchang suppository crude drug by intragastric administration or umbilical compress respectively, the normal group and model group were treated with 0.9% NaCl solution at equal volume, with a course of 9 days. During the modeling, the general activity and defecation of mice were observed, and the disease activity index(DAI) was calculated. After last administration, the serum and colon tissue were collected. The histopathological changes of colon tissue were observed by light microscope after HE staining. The serum levels of TNF-α and IL-6 were measured by ELISA. The mRNA expressions of TNF-α, IL-6 and IL-1β in colon tissue were detected by PCR, and the protein expressions of TLR4, MyD88 and NF-κB p65 in colon tissue were detected by Western blot. Results: ① On the 7 th day of modeling, the UC model mice showed obvious bloody stool, fatigued spirit and decreased body weight. The DAI score of the model group was significantly higher than that of the normal group(P<0.01). ② Histopathological observation on colon tissue showed that, compared with the normal group, the partial defect of epithelium was observed in the model group, as well as atrophy or disappearance of glands with disordered arrangement, less goblet cells, and serious inflammatory cell infiltration. The mesalazine group, Qingchang suppository decoction group and Qingchang suppository umbilical paste group showed a small amount of inflammatory cells infiltration in the mucosa and submucosa, and the degree of lesion was significantly improved compared with the model group.③Compared with the normal group, the serum levels of IL-6 and TNF-α were significantly increased in the model group(P<0.01); compared with the model group, the serum levels of IL-6 and TNF-α were significantly decreased in the mesalazine group, Qingchang suppository decoction group and Qingchang suppository umbilical paste group(P<0.05, P<0.01). There were no statistically significant differences among the different drug treatment groups(P>0.05). ④Compared with the normal group, the mRNA expression levels of TNF-α, IL-6 and IL-1β and the protein expression levels of TLR4, MyD88 and NF-κB p65 were significantly increased in the model group(P<0.01); compared with the model group, the mRNA expression levels of TNF-α, IL-6 and IL-1β and the protein expression levels of TLR4, MyD88 and NF-κB p65 were significantly down-regulated in the mesalazine group, Qingchang suppository decoction group and Qingchang suppository umbilical paste group(P<0.01). There were no statistically significant differences among the different drug treatment groups(P>0.05). Conclusion: Qingchang suppository umbilical paste can significantly improve the general activity and defecation of UC mice, and alleviate the colon mucosal epithelial damage and inflammatory infiltration. Its mechanism is related to inhibiting TLR4/MyD88/NF-κB signaling pathway.
引文
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[11] 李戈,董宇翔,金晶.神阙穴对消化系统疾病的治疗作用[J].长春中医药大学学报,2007,23(1):86.
[12] 王福隆.神阙灸——敷脐疗法及其机理的探讨[J].针灸学报,1989,5(4):7- 8.
[13] FUKATA M,ABREU M T.TLR4 signaling in the intestine in health and disease[J].Biochem Soc Trans,2007,35(Pt 6):1473- 1478.
[14] HISAMATSU T,KANAI T,MIKAMI Y,et al.Immune aspects of the pathogenesis of inflammatory bowel disease[J].Pharmacol Ther,2013,137(3):283- 297.
[15] NAGPAL K,PLANTINGA T S,WONG J,et al.A TIR Domain Variant of MyD88 Adapter- like (Mal)/TIRAP Results in Loss of MyD88 Binding and Reduced TLR2/TLR4 Signaling[J].J Biol Chem,2009,284(38):25742- 25748.
[2] LIN X,LIU J.TLR4/MyD88/NF- κB Signaling Pathway and Ulcerative Colitis[J].Chin J Gastroenterol,2013,18(4):244- 246.
[3] WU N,LEE Y C,SHAH N,et al.Cost of biologics per treated patient across immune- mediated inflammatory disease indications in a pharmacy benefit management setting:a retrospective cohort study[J].Clin Ther,2014,36(8):1231- 1241.
[4] 贾立伟,孙辉,李欣,等.溃疡性结肠炎药物治疗的研究进展[J].北京医学,2018,40(1):63- 65.
[5] 马贵同,龚雨萍,胡鸿毅,等.清肠栓治疗溃疡性结肠炎53例回顾性分析[J].中国中西医结合消化杂志,2003,11(4):231- 232.
[6] 王立娟,刘旭,唐志鹏,等.清肠栓对DSS诱导结肠炎小鼠脾CD4+T细胞CD44、CD62L表达的影响[J].上海中医药杂志,2014,48(5):104- 108.
[7] 李军祥,陈誩.溃疡性结肠炎中西医结合诊疗共识意见(2017年)[J].中国中西医结合消化杂志,2018,26(2):105- 111.
[8] 龚雨萍,柳文,马贵同,等.清肠栓治疗溃疡性结肠炎的随机对照研究[J].上海中医药大学学报,2007,21(6):33- 36.
[9] 李超男,龚雨萍.清肠栓治疗溃疡性结肠炎的临床和实验研究进展[J].临床医药文献电子杂志,2018,5(9):181- 183.
[10] 钟兰,诸毅晖,王苏娜,等.“强身保健药贴”神阙穴透皮给药对老年人衰老症状和免疫功能的影响[J].成都中医药大学学报,2010,33(4):15- 17.
[11] 李戈,董宇翔,金晶.神阙穴对消化系统疾病的治疗作用[J].长春中医药大学学报,2007,23(1):86.
[12] 王福隆.神阙灸——敷脐疗法及其机理的探讨[J].针灸学报,1989,5(4):7- 8.
[13] FUKATA M,ABREU M T.TLR4 signaling in the intestine in health and disease[J].Biochem Soc Trans,2007,35(Pt 6):1473- 1478.
[14] HISAMATSU T,KANAI T,MIKAMI Y,et al.Immune aspects of the pathogenesis of inflammatory bowel disease[J].Pharmacol Ther,2013,137(3):283- 297.
[15] NAGPAL K,PLANTINGA T S,WONG J,et al.A TIR Domain Variant of MyD88 Adapter- like (Mal)/TIRAP Results in Loss of MyD88 Binding and Reduced TLR2/TLR4 Signaling[J].J Biol Chem,2009,284(38):25742- 25748.