补中益气颗粒对EAT大鼠Treg/Th17细胞因子表达的影响
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摘要
目的探讨补中益气颗粒对实验性自身免疫性甲状腺炎(EAT)大鼠调节性T细胞(Treg)/辅助性T细胞17(Th17)细胞因子表达的影响,从Treg/Th17细胞因子失衡角度探讨其治疗桥本氏甲状腺炎的机制。方法运用猪甲状腺球蛋白与完全弗氏佐剂和不完全弗氏佐剂混合免疫注射法及高碘喂养法复制EAT大鼠模型,随机分为空白组、模型组、补中益气组、优甲乐组、补中益气+优甲乐组,药物干预2个月后,取大鼠脾脏组织,采用荧光定量PCR、蛋白质印迹(Western blot)法分别检测大鼠脾脏转化生长因子-β(TGF-β)、叉头翼状螺旋转录因子(Foxp3)、维甲酸相关孤儿受体γT(RORγT)、白细胞介素-17(IL-17)基因和蛋白表达水平。结果与空白组比较,模型组RORγT、IL-17基因和蛋白表达上调(P<0.05),Foxp3、TGF-β基因表达减少(P<0.05),蛋白表达无统计学意义(P>0.05)。与模型组比较,补中益气组、优甲乐组、补中益气+优甲乐组RORγT、IL-17基因表达下调(P<0.05),蛋白表达减少,RORγT改变具有统计学意义(P<0.05);Foxp3、TGF-β基因表达上调(P<0.05),蛋白表达无统计学意义(P>0.05)。药物各组组间比较,仅优甲乐组Foxp3蛋白表达高于补中益气组(P<0.05),RORγT、IL-17、TGF-β蛋白和基因表达3组间无统计学意义。结论补中益气颗粒能够抑制甲状腺自身免疫性炎症,其作用机制可能与调节Treg/Th17相关细胞因子的平衡状态有关。
Objective To investigate the influence of BuzhongYiqi(Middle-tonifying Qi-replenishing) Granule(BZYQ Granule) on expressions of Treg/Th17 cytokines in rats with experimental autoimmune thyroiditis(EAT), and discuss the mechanism of BZYQ Granule in treatment of Hashimoto's thyroiditis from the aspect of unbalance of Treg/Th17 cytokines. Methods EAT rat model was established by using mixed immune injection of porcine thyroglobulin and complete and incomplete Freund's adjuvant and high iodine feeding, and then the rats were randomly divided into blank group, model group, BZYQ Granule group, Euthyrox group and BZYQ Granule+Euthyrox group. After medicinal intervention for 2 months, the samples of rat spleen tissue were collected for detecting the protein and mRNA expressions of transforming growth factor-β(TGF-β), Foxp3 transcription factor(Foxp3), retinoic acid-related orphan receptor γT(RORγT) and interleukin-17(IL-17) by using RT-qPCR and Western blotting assay. Results Compared with blank group, the protein and mRNA expressions of RORγT and IL-17 were up-regulated(P<0.05), and mRNA expressions of Foxp3 and TGF-β were down-regulated(P<0.05) but protein expressions of them had no statistical significance(P>0.05) in model group. Compared with model group, the mRNA expressions of RORγT and IL-17 were down-regulated(P<0.05) and protein expressions of them decreased, and the expression of RORγT had statistical significance(P<0.05) but expression of IL-17 had no statistical significance in all medicinal groups. The mRNA expressions of Foxp3 and TGF-β were up-regulated(P<0.05) and protein expressions of them had statistical significance(P>0.05) in all medicinal groups compared with model group. The comparison among medicinal groups showed that the mRNA expressions of RORγT, IL-17, Foxp3 and TGF-β had no significant difference among these groups, while protein expression of Foxp3 was higher in Euthyrox group than that in BZYQ Granule group(P<0.05). The protein expressions of RORγT, IL-17 and TGF-β had no significant difference among BZYQ Granule group, Euthyrox group and BZYQ Granule+Euthyrox group. Conclusion BZYQ Granule can inhibit thyroid autoimmune inflammatory, and the mechanism may be related to the regulation of balance status of Treg/Th17 cytokines.
Objective To investigate the influence of BuzhongYiqi(Middle-tonifying Qi-replenishing) Granule(BZYQ Granule) on expressions of Treg/Th17 cytokines in rats with experimental autoimmune thyroiditis(EAT), and discuss the mechanism of BZYQ Granule in treatment of Hashimoto's thyroiditis from the aspect of unbalance of Treg/Th17 cytokines. Methods EAT rat model was established by using mixed immune injection of porcine thyroglobulin and complete and incomplete Freund's adjuvant and high iodine feeding, and then the rats were randomly divided into blank group, model group, BZYQ Granule group, Euthyrox group and BZYQ Granule+Euthyrox group. After medicinal intervention for 2 months, the samples of rat spleen tissue were collected for detecting the protein and mRNA expressions of transforming growth factor-β(TGF-β), Foxp3 transcription factor(Foxp3), retinoic acid-related orphan receptor γT(RORγT) and interleukin-17(IL-17) by using RT-qPCR and Western blotting assay. Results Compared with blank group, the protein and mRNA expressions of RORγT and IL-17 were up-regulated(P<0.05), and mRNA expressions of Foxp3 and TGF-β were down-regulated(P<0.05) but protein expressions of them had no statistical significance(P>0.05) in model group. Compared with model group, the mRNA expressions of RORγT and IL-17 were down-regulated(P<0.05) and protein expressions of them decreased, and the expression of RORγT had statistical significance(P<0.05) but expression of IL-17 had no statistical significance in all medicinal groups. The mRNA expressions of Foxp3 and TGF-β were up-regulated(P<0.05) and protein expressions of them had statistical significance(P>0.05) in all medicinal groups compared with model group. The comparison among medicinal groups showed that the mRNA expressions of RORγT, IL-17, Foxp3 and TGF-β had no significant difference among these groups, while protein expression of Foxp3 was higher in Euthyrox group than that in BZYQ Granule group(P<0.05). The protein expressions of RORγT, IL-17 and TGF-β had no significant difference among BZYQ Granule group, Euthyrox group and BZYQ Granule+Euthyrox group. Conclusion BZYQ Granule can inhibit thyroid autoimmune inflammatory, and the mechanism may be related to the regulation of balance status of Treg/Th17 cytokines.
引文
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[11] 韩静,袁泉,刘昕怡,等.补中益气颗粒对EAT大鼠甲状腺功能、甲状腺抗体的影响[J].北京中医药大学学报,2018,41(12):1007-1011.Han J,Yuan Q,Liu XY,et al.Effects of BuzhongYiqi granule on thyroid function and thyroid antibodies in rats with experirental autoimmune thyroiditis[J].Journal of Beijing University of Traditional Chinese Medicine,2018,41(12):1007-1011.
[12] Harrington LE,Hatton RD,Mangan PR,et al.Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages[J].Nat Immunol,2005,6(11):1123-1132.
[13] Araujo-Pires AC,Francisconi CF,Biguetti CC,et al.Simultaneous analysis of T helper subsets (Th1,Th2,Th9,Th17,Th22,Tfh,Tr1 and Tregs) markers expression in periapical lesions reveals multiple cytokine clusters accountable for lesions activity and inactivity status[J].J Appl Oral Sci,2014,22(4):336-346.
[14] Ghoreschi K,Laurence A,Yang XP,et al.Generation of pathogenic Th17 cells in the absence of TGF-beta signalling[J].Nature,2010,467(7318):967-971.
[15] Zhou L,Lopes JE,Chong MM,et al.TGF-beta-induced Foxp3 inhibits Th17 cell differentiation by antagonizing RORyt function[J].Nature,2008,453(7192):236-240.
[2] 周桂荣,徐萍芝,崔鹏,等.补中益气汤加味治疗桥本甲状腺炎60例[J].实用中医内科杂志,2007,21(2):66-67.Zhou GR,Xu PZ,Cui P,et al.Treatment of 60 cases of Hashimoto’s thyroiditis with BuzhongYiqi Decoction[J].Journal of Practical Traditional Chinese Internal Medicine,2007,21(2):66-67.
[3] 卢桂玲,李群燕.益气养阴活血方治疗系统性红斑狼疮的探讨[J].中国皮肤性病学杂志,2012,26(6):544-547.Lu GL,Li QY.Research on strengtnening qi,nourishing yin and acting blood principle for systemic lupus erythematosus[J].Chin J Derm Venereol,2012,26(6):544-547.
[4] 关溪,赵亮,刘力,等.消瘿合剂对自身免疫性甲状腺炎大鼠甲状腺Fas/FasL的影响[J].上海中医药杂志,2016,50(10):73-76.Guan X,Zhao L,Liu L,et al.Effect of Xiaoying Mixture on Fas and FasL in the thyroid of autoimmune thyroiditis rats[J].Shanghai Journal of Traditional Chinese Medicine,2016,50(10):73-76.
[5] 刘淑清,陈湘君,刘家昌.益气补肾活血方对佐剂关节炎大鼠CD4+CD25+Tr和Foxp3的影响[J].中医药信息,2015,32(2):75-77.Liu SQ,Chen XJ,Liu JC.Effect of Yiqi Bushen Huoxue Decoction on levels of CD4+CD25+Tr and Foxp3 in rats with adjuvant arthritis[J].Information on Traditional Chinese Medicine[J].2015,32(2):75-77.
[6] Xia N,Chen G,Liu M,et al.Anti-inflammatory effects of luteolin on experimental autoimmune thyroiditis in mice[J].Experimental and Therapeutic Medicine,2016,12(6):4049-4054.
[7] Naufel AO,Aguiar MCF,Madeira FM,et al.Treg and Th17 cells in inflammatory periapical disease:a systematic review[J].Braz Oral Res,2017,31:1-14.
[8] Omenetti S,Pizarro TT.The Treg/Th17 Axis:A Dynamic Balance Regulated By The Gut Microbiome[J].Front Immunol,2015,6(639):1-8.
[9] 宋进展,吴汉妮,钱伟.慢性淋巴细胞性甲状腺炎患者外周血Th17的检测及意义[J].细胞与分子免疫学杂志,2009,25(10):927-931.Song JZ,Wu HN,Qian W.Significance of the alteration of Th17 cells in patients with chronic lymphocytic thyroiditis[J].Chin J Cell Mol Immunol,2009,25(10):927-931.
[10] Campos K,Franscisconi CF,Okehie V,et al.FOXP3 DNA methylation levels as a potential biomarker in the development of periapical lesions[J].J Endod,2015,41(2):212-218.
[11] 韩静,袁泉,刘昕怡,等.补中益气颗粒对EAT大鼠甲状腺功能、甲状腺抗体的影响[J].北京中医药大学学报,2018,41(12):1007-1011.Han J,Yuan Q,Liu XY,et al.Effects of BuzhongYiqi granule on thyroid function and thyroid antibodies in rats with experirental autoimmune thyroiditis[J].Journal of Beijing University of Traditional Chinese Medicine,2018,41(12):1007-1011.
[12] Harrington LE,Hatton RD,Mangan PR,et al.Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages[J].Nat Immunol,2005,6(11):1123-1132.
[13] Araujo-Pires AC,Francisconi CF,Biguetti CC,et al.Simultaneous analysis of T helper subsets (Th1,Th2,Th9,Th17,Th22,Tfh,Tr1 and Tregs) markers expression in periapical lesions reveals multiple cytokine clusters accountable for lesions activity and inactivity status[J].J Appl Oral Sci,2014,22(4):336-346.
[14] Ghoreschi K,Laurence A,Yang XP,et al.Generation of pathogenic Th17 cells in the absence of TGF-beta signalling[J].Nature,2010,467(7318):967-971.
[15] Zhou L,Lopes JE,Chong MM,et al.TGF-beta-induced Foxp3 inhibits Th17 cell differentiation by antagonizing RORyt function[J].Nature,2008,453(7192):236-240.