调肝理脾方对原发性胆汁性肝硬化小鼠调节性T细胞及肝组织病理的影响
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摘要
目的探讨调肝理脾方治疗原发性胆汁性肝硬化的可能作用机制。方法 120只C57BL/6小鼠随机分为正常组、模型组、阳性组和治疗组,每组30只。除正常组外其余各组应用聚肌胞苷酸腹腔注射(5 mg/kg,2次/周,共24周)建立原发性胆汁性肝硬化动物模型,造模同时阳性组给予熊去氧胆酸胶囊0.1 g/(kg·d),治疗组给予调肝理脾方27 g/(kg·d),正常组及模型组给予等体积生理盐水,每日灌胃1次,共24周。于实验第8、16、24周时检测各组小鼠血清抗线粒体抗体M_2亚型(AMA-M2)阳性率、外周血CD4~+CD25~+Foxp3~+调节性T细胞(Treg)细胞计数、肝组织病理学检查及肝组织细胞角蛋白19(CK19)表达。结果除正常组外,第8、16、24周时各组血清AMA-M_2阳性率比较差异均无统计学意义(P>0.05)。与正常组比较,模型组小鼠第8、16、24周时外周血CD4~+CD25~+Foxp3~+Treg细胞计数明显下降(P<0.05);在第24周时,阳性组和治疗组外周血CD4~+CD25~+Foxp3~+Treg细胞计数均较模型组升高(P<0.05),且治疗组升高较阳性组明显(P<0.05)。与模型组比较,治疗组在第8、16、24周时肝组织病理分期差异有统计学意义(P<0.05)。与正常组比较,模型组第8、16、24周时CK19表达均明显升高(P<0.05);与模型组比较,治疗组第24周时CK19表达降低(P<0.05)。结论调肝理脾方能够上调原发性胆汁性肝硬化模型小鼠外周血CD4~+CD25~+Foxp3~+Treg细胞计数、降低肝组织CK19蛋白表达,从而改善胆小管损伤和肝组织纤维化。
Objective To study the possible mechanism of Tiaogan Lipi Fang( 调肝理脾方) in the treatment of primary biliary cirrhosis. Methods Totally 120 C57 BL /6 rats were randomly divided into normal group,model group,positive group and treatment group,with 30 rats in each group. Except the normal group,the rats in the other groups received intraperitoneal injection of poly I-poly( C) 5 mg / kg,twice a week,for 24 weeks to establish the animal model of primary biliary cirrhosis. Meanwhile,the positive group was given Ursodeoxycholic Acid Capules 0. 1 g /( kg·d),the treatment group was given Tiaogan Lipi Fang 27g /( kg·d),the normal group and the model group were given equal volume of normal saline,all by gavage once a day for 24 weeks. At the 8th,16 th,and 24 th week of the experiment,the positive rate of anti mitochondrial antibody M_2 subtype( AMA-M_2),peripheral blood CD4~+CD25~+Foxp3~+regulatory T cell( Treg) count,and liver tissue pathological examination and expression of liver tissue and cell keratin 19( CK19) were detected in all rats. Results There was no significant difference in the positive rate of AMA-M2 between the model group,positive group and treatment group at the 8th,16 th and 24 th week( P >0. 05). Compared with the normal group,the number of CD4~+CD25~+Foxp3~+Treg cells in peripheral blood in the model group was significantly decreased at 8th,16 th and 24 th week( P < 0. 05); At the 24 th week,the number of CD4~+CD25~+Foxp3~+Treg cells in peripheral blood in the positive group and the treatment group was higher than that in the model group,but the treatment group was significantly higher than that in the positive group( P < 0. 05).There was significant difference in the pathological staging of liver tissues between the treatment group and the model group at the 8th,16 th and 24 th week( P < 0. 05). Compared with the normal group,the expression of CK19 in the model group was much higher than that in normal group at the 8th,16 th and 24 th week( P < 0. 05); Compared with the model group,CK19 expression was decreased in the treatment group at the 24 th week( P < 0. 05). Conclusion Tiaogan Lipi Fang can up-regulate the activity of CD4~+CD25~+Foxp3~+Treg in peripheral blood and reduce the expression of CK19 protein in liver tissue of rats model with primary liver cirrhosis,so as to improve the bile duct injury and hepatic fibrosis.
Objective To study the possible mechanism of Tiaogan Lipi Fang( 调肝理脾方) in the treatment of primary biliary cirrhosis. Methods Totally 120 C57 BL /6 rats were randomly divided into normal group,model group,positive group and treatment group,with 30 rats in each group. Except the normal group,the rats in the other groups received intraperitoneal injection of poly I-poly( C) 5 mg / kg,twice a week,for 24 weeks to establish the animal model of primary biliary cirrhosis. Meanwhile,the positive group was given Ursodeoxycholic Acid Capules 0. 1 g /( kg·d),the treatment group was given Tiaogan Lipi Fang 27g /( kg·d),the normal group and the model group were given equal volume of normal saline,all by gavage once a day for 24 weeks. At the 8th,16 th,and 24 th week of the experiment,the positive rate of anti mitochondrial antibody M_2 subtype( AMA-M_2),peripheral blood CD4~+CD25~+Foxp3~+regulatory T cell( Treg) count,and liver tissue pathological examination and expression of liver tissue and cell keratin 19( CK19) were detected in all rats. Results There was no significant difference in the positive rate of AMA-M2 between the model group,positive group and treatment group at the 8th,16 th and 24 th week( P >0. 05). Compared with the normal group,the number of CD4~+CD25~+Foxp3~+Treg cells in peripheral blood in the model group was significantly decreased at 8th,16 th and 24 th week( P < 0. 05); At the 24 th week,the number of CD4~+CD25~+Foxp3~+Treg cells in peripheral blood in the positive group and the treatment group was higher than that in the model group,but the treatment group was significantly higher than that in the positive group( P < 0. 05).There was significant difference in the pathological staging of liver tissues between the treatment group and the model group at the 8th,16 th and 24 th week( P < 0. 05). Compared with the normal group,the expression of CK19 in the model group was much higher than that in normal group at the 8th,16 th and 24 th week( P < 0. 05); Compared with the model group,CK19 expression was decreased in the treatment group at the 24 th week( P < 0. 05). Conclusion Tiaogan Lipi Fang can up-regulate the activity of CD4~+CD25~+Foxp3~+Treg in peripheral blood and reduce the expression of CK19 protein in liver tissue of rats model with primary liver cirrhosis,so as to improve the bile duct injury and hepatic fibrosis.
引文
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[5]中华医学会风湿病学分会.自身免疫性肝病诊断和治疗指南[J].中华风湿病学杂志,2011,15(8):556-558.
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[11]BERNUZZI F,FENOGLI D,BATTAGLIA F,et al.Phenotypical and functional alterations of CD8 regulatory T cells in primary biliary cirrhosis[J].Autoimmune,2010,35(3):176-180.
[12]刘娟,曹雪涛.2012年度免疫学研究重要进展[J].中国免疫学杂志,2013,29(1):3-13.
[13]RAMSDELL F.Foxp3 and natural regulatory T cells:key to a cell linage?[J].Immunity,2003,19(2):165-168.
[14]许茵,杨小娟,蒯守刚.原发性胆汁性肝硬化患者外周血CD4+CD25+Foxp3+调节性T细胞检测及其临床意义[J].中国实验诊断学,2014,18(4):592-594.
[2]KUIPER EM,HANSEN BE,DE VRIES RA,et al.Improved prognosis of patients with primary biliary cirrhosis that have a biochemical response to ursodeoxycholic acid[J].Gastroenterology,2009,136(4):1281-1287.
[3]郭晓霞,李良学,武玉鹏,等.调肝理脾方干预原发性胆汁性肝硬化小鼠肝损伤的组织学观察[J].中西医结合肝病杂志,2015,25(1):36-38.
[4]马欢,王邦茂.原发性胆汁性肝硬化动物模型研究进展[J].国际消化病杂志,2012,32(2):93-95.
[5]中华医学会风湿病学分会.自身免疫性肝病诊断和治疗指南[J].中华风湿病学杂志,2011,15(8):556-558.
[6]许良中,杨文涛.免疫组织化学反应结果的判断标准[J].中国癌症杂志,1996,6(4):229-231.
[7]Working Subgroup(English version)for Clinical Practice Guidelines for Primary Biliary Cirrhosis.Guidelines for the management of primary biliary cirrhosis:the intractable hepatobiliary disease study group supported by the Ministry of Health,Labour and Welfare of Japan[J].Hepatol Res,2014,44(Suppl S1):71-90.
[8]KOHJIMA M,ENJOJI M,YADA R,et al.Pathophysiological analysis of primary biliary cirrhosis focusing on choline/phospholipid metabolism[J].Liver Int,2015,35(3):1095-1102.
[9]INVERNIZZI P,SELMI C,GERSHWIN ME.Update on primary biliary cirrhosis[J].Dig Liver Dis,2010,42(6):401-408.
[10]刘然,陆伦根.抗线粒体抗体对原发性胆汁性肝硬化诊断价值研究进展[J].实用肝脏病杂志,2014,17(2):218-221.
[11]BERNUZZI F,FENOGLI D,BATTAGLIA F,et al.Phenotypical and functional alterations of CD8 regulatory T cells in primary biliary cirrhosis[J].Autoimmune,2010,35(3):176-180.
[12]刘娟,曹雪涛.2012年度免疫学研究重要进展[J].中国免疫学杂志,2013,29(1):3-13.
[13]RAMSDELL F.Foxp3 and natural regulatory T cells:key to a cell linage?[J].Immunity,2003,19(2):165-168.
[14]许茵,杨小娟,蒯守刚.原发性胆汁性肝硬化患者外周血CD4+CD25+Foxp3+调节性T细胞检测及其临床意义[J].中国实验诊断学,2014,18(4):592-594.