基于PI3K/Akt通路探究迷迭香酸对自身免疫性甲状腺炎大鼠的作用及机制

Exploring the Effect and Underlying Mechanism of Rosmarinic Acid on Autoimmune Thyroiditis in Rats Based on the PI3K/Akt Pathway

目的基于磷酸肌醇3激酶(PI3K)/蛋白激酶B(AKT)通路探究迷迭香酸对自身免疫性甲状腺炎(AIT)大鼠的作用及机制。方法通过高碘水喂养联合皮下注射甲状腺球蛋白及弗氏佐剂来诱导建立AIT大鼠模型,随机分为4组:模型组、迷迭香酸低剂量(20mg/kg)组、迷迭香酸高剂量(40mg/kg)组、迷迭香酸高剂量(40mg/kg)+IGF-1(PI3K/Akt激活剂,0.75μg/kg)组,每组10只,另选10只SD大鼠,正常水喂养并皮下注射等剂量生理盐水作为对照组,以迷迭香酸和IGF-1分组处理后,ELISA测量大鼠血清甲状腺过氧化物酶抗体(TPOAb)、抗甲状腺球蛋白抗体(TGAb)水平;HE染色检测各组大鼠甲状腺组织病理损伤;免疫组织化学染色检测各组大鼠甲状腺组织CD45R表达;TUNEL染色检测各组大鼠甲状腺细胞凋亡情况;ELISA测量大鼠血清中Th细胞分泌的炎症相关因子γ干扰素(IFN-γ)、白细胞介素(IL)-4、IL-17、IL-10水平;免疫印迹实验检测各组大鼠甲状腺组织PI3K/Akt通路相关蛋白表达。结果与对照组相比,模型组大鼠TGAb、TPOAb、IFN-γ及IL-17水平、病理损伤评分、CD45R相对阳性表达、甲状腺细胞凋亡率、p-PI3K/PI3K、p-AKT/Akt明显升高(P<0.05),IL-4、IL-10水平明显降低(P<0.05);与模型组相比,迷迭香酸低剂量组、迷迭香酸高剂量组大鼠TGAb、TPOAb、IFN-γ及IL-17水平、病理损伤评分、CD45R相对阳性表达、甲状腺细胞凋亡率、p-PI3K/PI3K、p-AKT/Akt均降低(P<0.05),IL-4、IL-10水平均升高(P<0.05),且高剂量迷迭香酸作用更强;IGF-1可减弱迷迭香酸对AIT大鼠的作用。结论迷迭香酸可通过阻断PI3K/Akt通路激活来调控Th细胞因子分泌,从而抑制免疫炎症反应,减轻甲状腺组织淋巴细胞浸润及甲状腺细胞凋亡,改善AIT大鼠症状。

Objective To investigate the effect and underlying mechanism of rosmarinic acid on autoimmune thyroiditis(AIT)in rats based on the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)pathway.Methods A rat model of AIT was established by feeding high iodine water combined with subcutaneous injection of thyroglobulin and Freund’s adjuvant.The study rats were randomly grouped into four groups:model group,low-dose rosmarinic acid(20mg/kg)group,high-dose rosmarinic acid(40mg/kg)group,and high-dose rosmarinic acid(40mg/kg)+IGF-1(PI3K/Akt activator,0.75μg/kg)group,with 10 rats in each group.Another 10 SD rats were selected as the control group:fed with normal water and subcutaneously injected with equivalent doses of physiological saline.After treated with rosmarinic acid and IGF-1,ELISA were applied to measure levels of serum thyroid peroxidase antibody(TPOAb)and anti-thyroid autoantibodies(TGAb)in rats;HE staining was applied to detect pathological damage in the thyroid tissue of rats in each group;Immunohistochemical staining was applied to detect the expression of CD45R in the thyroid tissue of rats in each group;TUNEL staining was applied to detect the apoptosis of thyroid cells in rats of each group;ELISA was applied to measure levels of inflammatory related factors,such asγ-interferon(IFN-γ),interleukin-4(IL-4),IL-17,and IL-10 secreted by Th cells in rat serum;Immunoblotting experiments were applied to detect the expression of PI3K/Akt pathway related proteins in the thyroid tissue of rats in each group.Results Compared with the control group,levels of TGAb,TPOAb,IFN-γ,and IL-17,pathological injury score,CD45R relative positive expression,thyroid cell apoptosis rate,p-PI3K/PI3K,and p-AKT/Akt in the model group were significantly increased(P<0.05),while levels of IL-4 and IL-10 were significantly decreased(P<0.05);Compared with the model group,levels of TGAb,TPOAb,IFN-γ,and IL-17,pathological injury score,CD45R relative positive expression,thyroid cell apoptosis rate,p-PI3K/PI3K,p-AKT/Akt in the low-dose and high-dose rosmarinic acid groups were all reduced(P<0.05),while levels of IL-4 and IL-10 were increased(P<0.05);Compared with the low-dose rosmarinic acid group,levels of TGAb,TPOAb,IFN-γ,and IL-17,pathological injury score,CD45R relative positive expression,thyroid cell apoptosis rate,p-PI3K/PI3K,p-AKT/Akt in the high-dose rosmarinic acid group were all reduced(P<0.05),while levels of IL-4 and IL-10 were increased(P<0.05),and high-dose rosmarinic acid had a stronger effect;IGF-1 could weaken the effect of rosmarinic acid on AIT rats.Conclusion Rosmarinic acid can regulate Th cytokine secretion by blocking the activation of the PI3K/Akt pathway,thereby inhibiting immune inflammatory response,reducing lymphocyte infiltration and apoptosis in thyroid tissue,and improving symptoms in AIT rats.