软坚消瘿颗粒对肝郁脾虚型桥本甲状腺炎模型大鼠Treg和Th17细胞因子表达的影响
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摘要
目的:观察软坚消瘿颗粒对肝郁脾虚型桥本甲状腺炎(HT)大鼠Treg和Th17特异性转录因子及细胞因子表达的影响,探讨软坚消瘿颗粒对HT的治疗作用及其免疫学作用机制。方法:采用随机数字法将48只SD大鼠分为正常组(12只)和造模组(36只)。采用高碘饮水联合甲状腺球蛋白皮下注射方法建立HT大鼠模型,再结合慢性束缚应激、过度疲劳、饮食失节等复合方法制备肝郁脾虚模型。造模后,造模组大鼠随机分为模型组、雷公藤组和软坚消瘿组,每组12只,连续给药8周。去除实验期间死亡大鼠和不合格标本,每组留取10只检测结果,观察各组大鼠一般情况;末次给药后采集大鼠腹主动脉血和甲状腺组织,采用ELISA法检测大鼠血清甲状腺球蛋白抗体(TGAb)、甲状腺过氧化酶抗体(TPOAb)、血清游离三碘甲腺原氨酸(FT3)、游离甲状腺素(FT4)、促甲状腺激素释放激素(TRH)和超敏促甲状腺激素(TSH)水平及血清白细胞介素10(IL-10)、IL-17和IL-23水平;HE染色法检测各组大鼠甲状腺组织病理形态表现;Western blotting法检测各组大鼠甲状腺组织中Foxp3和RORγt蛋白表达水平。结果:与正常组比较,模型组、雷公藤组和软坚消瘿组大鼠血清TGAb和TPOAb水平升高(P<0.05);血清FT3、FT4和TRH水平降低(P<0.05),TSH水平升高(P<0.05);甲状腺组织中Foxp3蛋白表达水平降低(P<0.05),RORγt蛋白表达水平升高(P<0.05);血清IL-10水平降低(P<0.05),IL-17和IL-23水平升高(P<0.05)。与模型组比较,雷公藤组和软坚消瘿组大鼠血清TGAb和TPOAb水平降低(P<0.05);大鼠血清FT3、FT4和TRH水平升高(P<0.05),TSH水平降低(P<0.05);甲状腺组织中Foxp3蛋白表达水平升高(P<0.05),RORγt蛋白表达水平降低(P<0.05);血清IL-10水平升高(P<0.05),IL-17和IL-23水平降低(P<0.05)。与雷公藤组比较,软坚消瘿组大鼠血清TGAb和TPOAb水平降低(P<0.05);血清FT3、FT4和TRH水平升高(P<0.05),TSH水平降低(P<0.05);甲状腺组织中Foxp3蛋白表达水平升高,RORγt蛋白表达水平降低,但差异无统计学意义(P>0.05);血清IL-10水平升高(P<0.05),IL-17和IL-23水平降低(P<0.05)。正常组大鼠甲状腺滤泡形态规则,结构完整,无淋巴细胞浸润;模型组大鼠甲状腺滤泡腔扩大,滤泡结构大量破坏,滤泡腔内及周围可见大量淋巴细胞浸润;雷公藤组甲状腺滤泡腔扩大,部分滤泡结构破坏,滤泡腔内及周围有少量淋巴细胞浸润,浸润程度轻于模型组;软坚消瘿组甲状腺病理形态学改变与雷公藤组类似,滤泡腔内及周围亦有少量淋巴细胞浸润。结论:软坚消瘿颗粒可以通过调节Treg和Th17特异性转录因子Foxp3和RORγt蛋白及其相关细胞因子的表达,对肝郁脾虚型HT大鼠起治疗作用。
Objective:To observe the effects of RuanJianXiaoYing Granule on the expressions of specific transcription factors and cytokines of Treg and Th17 in the Hashimoto thyroiditis(HT)rats with liver depression and spleen deficiency,and to investigate the therapeutic effect of RuanJianXiaoYing Granule on HT and its immunological mechanism.Methods:A total of 48 SD rats were randomly divided into normal group(12 rats)and modeling group(36 rats).The HT model was established by subcutaneous injection of excessive iodine drinking water and thyroglobulin,and then combined with chronic restraint stress,excessive fatigue and dietary disorders to prepare the rat models with liver depression and spleen deficiency.After modeling,the rats were randomly divided into model group(n=12),Tripterygium wilfordii group(n=12),and RuanJianXiaoYing group(n=12),and the rats were treated for 8 weeks.In addition to the dead rats and unqualified specimens during the experiment,the test results of 10 rats in each group were retained.The general situation of the rats in various groups was observed;the abdominal aortic blood and thyroid gland tissue of the rats in various groups were collected after the last administration;the levels of thyroglobulin antibody(TGAb),thyroid peroxidase antibody(TPOAb),free triiodothyronine(FT3),free thyroxine(FT4),thyrotropin-releasing hormone(TRH)and thyrotropin(TSH)in serum and the levels of IL-17,IL-23 and IL-10 were detected by ELISA method;the pathomorphology of thyroid gland tissue of the rats in various groups were detected by HE staining;the expression levels of Foxp3 and RORγt proteins in thyroid gland tissue of the rats in various groups were detected by Western blotting method.Results:Compared with normal group,the levels of serum TGAb and TPOAb of the rats in model group,Tripterygium wilfordii group and RuanJianXiaoYing～group were increased(P< 0.05);the levels of FT3,FT4 and TRH were decreased(P<0.05),the levels of TSH were increased(P<0.05);the expression levels of Foxp3 protein in thyroid gland tissue were decreased(P<0.05),and the expression levels of RORγt protein were increased(P<0.05);the levels of serum IL-10 were decreased(P<0.05),and the levels of serum IL-17 and IL-23 were increased(P<0.05).Compared with model group,the levels of TGAb and TPOAb of the rats in Tripterygium wilfordii group and RuanJianXiaoYing group were decreased(P<0.05);the levels of FT3,FT4 and TRH were increased(P< 0.05),and the levels of TSH were decreased(P< 0.05);the expression levels of Foxp3 protein in thyroid gland tissue were increased(P<0.05),and the expression levels of RORγt protein were decreased(P< 0.05);the levels of serum IL-10 were increased(P< 0.05),and the levels of IL-17 and IL-23 were decreased(P< 0.05).Compared with Tripterygium wilfordii group,the levels of TGAb,TPOAb,FT3,FT4,and TRH of the rats in RuanJianXiaoYing group were increased(P< 0.05),and the levels of TSH of the rats in RuanJianXiaoYing group was decreased(P<0.05);the expression level of Foxp3 protein in thyroid gland tissue of the rats was increased,and the expression level of RORγt protein in thyroid gland tissue was decreased,but there no were significant differences(P>0.05);the level of serum IL-10 was increased(P<0.05),and the levels of serum IL-17 and IL-23 were decreased(P<0.05).The follicular cavity of thyroid gland in normal group had regular morphology,complete structure and no lymphocyte infiltration.In model group,the follicular cavity of thyroid gland was enlarged,the follicular structure was destroyed,and a large number of lymphocyte infiltration was observed in and around the follicular cavity.In Tripterygium wilfordii group,the follicular cavity of thyroid gland was enlarged,part of the follicular structure was destroyed,and there was a small amount of lymphocyte infiltration in and around the follicular cavity,which was less than that in model group.The morphological changes of thyroid gland in RuanJianXiaoYing group were similar to those in Tripterygium wilfordii group.Conclusion:RuanJianXiaoYing Granule has the therapeutic effect in the HT rats with liver depression and spleen deficiency by regulating the expressions of the specific transcription factors Foxp3 and RORγt protein and related cytokines of Treg and Th17.
Objective:To observe the effects of RuanJianXiaoYing Granule on the expressions of specific transcription factors and cytokines of Treg and Th17 in the Hashimoto thyroiditis(HT)rats with liver depression and spleen deficiency,and to investigate the therapeutic effect of RuanJianXiaoYing Granule on HT and its immunological mechanism.Methods:A total of 48 SD rats were randomly divided into normal group(12 rats)and modeling group(36 rats).The HT model was established by subcutaneous injection of excessive iodine drinking water and thyroglobulin,and then combined with chronic restraint stress,excessive fatigue and dietary disorders to prepare the rat models with liver depression and spleen deficiency.After modeling,the rats were randomly divided into model group(n=12),Tripterygium wilfordii group(n=12),and RuanJianXiaoYing group(n=12),and the rats were treated for 8 weeks.In addition to the dead rats and unqualified specimens during the experiment,the test results of 10 rats in each group were retained.The general situation of the rats in various groups was observed;the abdominal aortic blood and thyroid gland tissue of the rats in various groups were collected after the last administration;the levels of thyroglobulin antibody(TGAb),thyroid peroxidase antibody(TPOAb),free triiodothyronine(FT3),free thyroxine(FT4),thyrotropin-releasing hormone(TRH)and thyrotropin(TSH)in serum and the levels of IL-17,IL-23 and IL-10 were detected by ELISA method;the pathomorphology of thyroid gland tissue of the rats in various groups were detected by HE staining;the expression levels of Foxp3 and RORγt proteins in thyroid gland tissue of the rats in various groups were detected by Western blotting method.Results:Compared with normal group,the levels of serum TGAb and TPOAb of the rats in model group,Tripterygium wilfordii group and RuanJianXiaoYing～group were increased(P< 0.05);the levels of FT3,FT4 and TRH were decreased(P<0.05),the levels of TSH were increased(P<0.05);the expression levels of Foxp3 protein in thyroid gland tissue were decreased(P<0.05),and the expression levels of RORγt protein were increased(P<0.05);the levels of serum IL-10 were decreased(P<0.05),and the levels of serum IL-17 and IL-23 were increased(P<0.05).Compared with model group,the levels of TGAb and TPOAb of the rats in Tripterygium wilfordii group and RuanJianXiaoYing group were decreased(P<0.05);the levels of FT3,FT4 and TRH were increased(P< 0.05),and the levels of TSH were decreased(P< 0.05);the expression levels of Foxp3 protein in thyroid gland tissue were increased(P<0.05),and the expression levels of RORγt protein were decreased(P< 0.05);the levels of serum IL-10 were increased(P< 0.05),and the levels of IL-17 and IL-23 were decreased(P< 0.05).Compared with Tripterygium wilfordii group,the levels of TGAb,TPOAb,FT3,FT4,and TRH of the rats in RuanJianXiaoYing group were increased(P< 0.05),and the levels of TSH of the rats in RuanJianXiaoYing group was decreased(P<0.05);the expression level of Foxp3 protein in thyroid gland tissue of the rats was increased,and the expression level of RORγt protein in thyroid gland tissue was decreased,but there no were significant differences(P>0.05);the level of serum IL-10 was increased(P<0.05),and the levels of serum IL-17 and IL-23 were decreased(P<0.05).The follicular cavity of thyroid gland in normal group had regular morphology,complete structure and no lymphocyte infiltration.In model group,the follicular cavity of thyroid gland was enlarged,the follicular structure was destroyed,and a large number of lymphocyte infiltration was observed in and around the follicular cavity.In Tripterygium wilfordii group,the follicular cavity of thyroid gland was enlarged,part of the follicular structure was destroyed,and there was a small amount of lymphocyte infiltration in and around the follicular cavity,which was less than that in model group.The morphological changes of thyroid gland in RuanJianXiaoYing group were similar to those in Tripterygium wilfordii group.Conclusion:RuanJianXiaoYing Granule has the therapeutic effect in the HT rats with liver depression and spleen deficiency by regulating the expressions of the specific transcription factors Foxp3 and RORγt protein and related cytokines of Treg and Th17.
引文
[1]王家红.软坚消瘿颗粒治疗自身免疫性甲状腺炎大鼠的实验研究[D].沈阳:辽宁中医药大学,2015.
[2]王家红,张兰.软坚消瘿颗粒对AIT大鼠细胞因子Th1/Th2及凋亡蛋白Fas/FasL、Bcl-2/Bax的影响[J].辽宁医学院学报,2015,36(2):9-12.
[3]KRISTENSEN B,HEGEDUS L,MADSEN H O,et al.Altered balance between self-reactive T helper(Th)17cells and Th10cells and between full-length forkhead box protein 3(FoxP3)and FoxP3 splice variants in Hashimoto’s thyroiditis[J].Clin Exp Immunol,2015,180(1):58-69.
[4]XUE H,YU X,MA L,et al.The possible role of CD4+CD25(high)Foxp3+/CD4+IL-17A+cell imbalance in the autoimmunity of patients with Hashimoto thyroiditis[J].Endocrine,2015,50(3):665-673.
[5]LIU Y Z,TANG X Y,TIAN J,et al.Th17/Treg cells imbalance and GITRL profile in patients with Hashimoto’s thyroiditis[J].Int J Mol Sci,2014,15(12):21674-21686.
[6]BOSSOWSKI A,MONIUSZKO M,IDZKOWSKA E,et al.Decreased proportions of CD4+IL17+/CD4+CD25+CD127and CD4+IL17+/CD4+CD25+CD127-FoxP3+T cells in children with autoimmune thyroid diseases[J].Autoimmunity,2016,49(5):320-328.
[7]徐叔云,卞如濂,陈修.药理实验方法学[M].2版.北京:人民卫生出版社,1982:1535.
[8]马清钧,王淑玲.临床实用中药学[M].南昌:江西科学技术出版社,2002.
[9]杨成,金剑虹,唐成坤,等.自身免疫性甲状腺炎中西医研究进展[J].浙江中西医结合杂志,2018,28(4):335-338.
[10]Kristensen B.Regulatory B and T cell responses in patients with autoimmune thyroid disease and healthy controls[J].Dan Med J,2016,63(2):B5177.
[11]关溪,赵亮,刘力,等.消瘿合剂对自身免疫性甲状腺炎大鼠调节性T细胞/Th17细胞轴的影响[J].医药导报,2017,36(2):136-140.
[12]曾蓉.桥本氏甲状腺炎-甲状腺乳头状癌的免疫微环境及基因关联性初步研究[D].昆明:昆明理工大学,2017.
[13]梅沉成,张云璧,陈曦,等.Treg/Th17平衡偏移与寻常型银屑病中医三证型的相关性研究[J].中华中医药杂志,2017,32(6):2717-2720.
[14]苏斯佳.桥本甲状腺炎患者血清25-(OH)D3水平与Th17/Treg细胞因子失衡的相关性研究[D].石家庄:河北医科大学,2016.
[15]杜建超,冯淬灵,葛东宇,等.清金化痰汤对慢性阻塞性肺疾病急性加重期模型大鼠肺组织Foxp3和RORγt表达的影响[J].北京中医药大学学报,2016,39(12):1006-1012.
[16]SAFDARI V,ALIJANI E,NEMATI M,et al.Imbalances in T cell-related transcription factors among patients with Hashimoto’s thyroiditis[J].Sultan Qaboos Univ Med J,2017,17(2):e174-e180.
[17]毕建华,郝兰香,黄飞,等.桥本甲状腺炎患者外周血CD4+CD25+Foxp3+调节性T细胞及IL-10的检测及临床意义[J].临床和实验医学杂志,2017,16(1):38-40.
[18]LI C,YUAN J H,ZHU Y F,et al.Imbalance of Th17/Treg in different subtypes of autoimmune thyroid diseases[J].Cell Physiol Biochem,2016,40(1/2):245-252.
[19]任志华,王婵,邓俊良,等.中药组方对腹腔注射大肠杆菌小鼠血清Th17、Treg型细胞因子的影响[J].中国兽医学报,2017,37(12):2378-2383.
[20]刘岳花,卢凤艳,陆东庆,等.白细胞介素-17和23在湿疹患者血清中的表达及其临床意义[J].临床皮肤科杂志,2018,47(3):157-159.
[21]冯勤,李忠武,刘卉,等.免疫组化检测63例原发甲状腺癌中BRAF(V600E)突变及其价值研究[J].中国实用内科杂志,2018,38(12):1188-1190.
[2]王家红,张兰.软坚消瘿颗粒对AIT大鼠细胞因子Th1/Th2及凋亡蛋白Fas/FasL、Bcl-2/Bax的影响[J].辽宁医学院学报,2015,36(2):9-12.
[3]KRISTENSEN B,HEGEDUS L,MADSEN H O,et al.Altered balance between self-reactive T helper(Th)17cells and Th10cells and between full-length forkhead box protein 3(FoxP3)and FoxP3 splice variants in Hashimoto’s thyroiditis[J].Clin Exp Immunol,2015,180(1):58-69.
[4]XUE H,YU X,MA L,et al.The possible role of CD4+CD25(high)Foxp3+/CD4+IL-17A+cell imbalance in the autoimmunity of patients with Hashimoto thyroiditis[J].Endocrine,2015,50(3):665-673.
[5]LIU Y Z,TANG X Y,TIAN J,et al.Th17/Treg cells imbalance and GITRL profile in patients with Hashimoto’s thyroiditis[J].Int J Mol Sci,2014,15(12):21674-21686.
[6]BOSSOWSKI A,MONIUSZKO M,IDZKOWSKA E,et al.Decreased proportions of CD4+IL17+/CD4+CD25+CD127and CD4+IL17+/CD4+CD25+CD127-FoxP3+T cells in children with autoimmune thyroid diseases[J].Autoimmunity,2016,49(5):320-328.
[7]徐叔云,卞如濂,陈修.药理实验方法学[M].2版.北京:人民卫生出版社,1982:1535.
[8]马清钧,王淑玲.临床实用中药学[M].南昌:江西科学技术出版社,2002.
[9]杨成,金剑虹,唐成坤,等.自身免疫性甲状腺炎中西医研究进展[J].浙江中西医结合杂志,2018,28(4):335-338.
[10]Kristensen B.Regulatory B and T cell responses in patients with autoimmune thyroid disease and healthy controls[J].Dan Med J,2016,63(2):B5177.
[11]关溪,赵亮,刘力,等.消瘿合剂对自身免疫性甲状腺炎大鼠调节性T细胞/Th17细胞轴的影响[J].医药导报,2017,36(2):136-140.
[12]曾蓉.桥本氏甲状腺炎-甲状腺乳头状癌的免疫微环境及基因关联性初步研究[D].昆明:昆明理工大学,2017.
[13]梅沉成,张云璧,陈曦,等.Treg/Th17平衡偏移与寻常型银屑病中医三证型的相关性研究[J].中华中医药杂志,2017,32(6):2717-2720.
[14]苏斯佳.桥本甲状腺炎患者血清25-(OH)D3水平与Th17/Treg细胞因子失衡的相关性研究[D].石家庄:河北医科大学,2016.
[15]杜建超,冯淬灵,葛东宇,等.清金化痰汤对慢性阻塞性肺疾病急性加重期模型大鼠肺组织Foxp3和RORγt表达的影响[J].北京中医药大学学报,2016,39(12):1006-1012.
[16]SAFDARI V,ALIJANI E,NEMATI M,et al.Imbalances in T cell-related transcription factors among patients with Hashimoto’s thyroiditis[J].Sultan Qaboos Univ Med J,2017,17(2):e174-e180.
[17]毕建华,郝兰香,黄飞,等.桥本甲状腺炎患者外周血CD4+CD25+Foxp3+调节性T细胞及IL-10的检测及临床意义[J].临床和实验医学杂志,2017,16(1):38-40.
[18]LI C,YUAN J H,ZHU Y F,et al.Imbalance of Th17/Treg in different subtypes of autoimmune thyroid diseases[J].Cell Physiol Biochem,2016,40(1/2):245-252.
[19]任志华,王婵,邓俊良,等.中药组方对腹腔注射大肠杆菌小鼠血清Th17、Treg型细胞因子的影响[J].中国兽医学报,2017,37(12):2378-2383.
[20]刘岳花,卢凤艳,陆东庆,等.白细胞介素-17和23在湿疹患者血清中的表达及其临床意义[J].临床皮肤科杂志,2018,47(3):157-159.
[21]冯勤,李忠武,刘卉,等.免疫组化检测63例原发甲状腺癌中BRAF(V600E)突变及其价值研究[J].中国实用内科杂志,2018,38(12):1188-1190.