基于NLRP3/Caspase-1/GSDMD通路探讨补中益气汤干预NOD.H-2^(h4)小鼠AIT细胞焦亡的机制

Mechanism of Buzhong Yiqitang Intervening in Pyroptosis of AIT in NOD.H-2h4 Mice Based on NLRP3/Caspase-1/GSDMD Pathway

目的:基于NOD样受体热蛋白结构域相关蛋白3(NLRP3)/胱天蛋白酶-1(Caspase-1)/消皮素D(GSDMD)通路探讨补中益气汤对自身免疫性甲状腺炎(AIT)小鼠细胞焦亡的作用机制。方法:60只NOD.H-2h4小鼠分为正常组、模型组、补中益气汤低、中、高剂量(4.10、8.19、16.38 g·kg^(-1))组和硒酵母片(0.26 mg·kg^(-1))组,每组10只。除正常组外,其余各组均给予0.05%碘化钠(NaI)灌胃8周造模,继续给药干预8周。酶联免疫吸附测定法(ELISA)检测血清甲状腺过氧化物酶抗体(TPO-Ab)、甲状腺球蛋白抗体(Tg Ab)水平;苏木素-伊红(HE)染色观察小鼠甲状腺组织病理学改变;免疫组化法检测甲状腺组织中NLRP3、Caspase-1、白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测甲状腺组织中NLRP3、Caspase-1、IL-1β、IL-18 mRNA表达;蛋白免疫印迹法(Western blot)检测甲状腺组织中细胞焦亡相关蛋白水平。结果:与正常组比较,模型组小鼠血清TPO-Ab、Tg Ab水平显著升高(P<0.01),甲状腺滤泡或增大呈立方状,或破坏萎缩,滤泡周围可见大量淋巴细胞浸润;与模型组比较,给药组TPO-Ab、Tg Ab水平显著降低(P<0.01),滤泡形态结构有所改善,淋巴细胞浸润程度有所减轻,其中补中益气汤中剂量组降低和改善效果最为明显。与正常组比较,模型组小鼠甲状腺组织中NLRP3、Caspase-1、IL-1β、IL-18蛋白阳性产物和mRNA表达显著增加(P<0.01),NLRP3、剪切的(cleaved) Caspase-1、IL-1β、IL-18、GSDMD-N蛋白表达水平明显升高(P<0.05,P<0.01);与模型组比较,给药组NLRP3、Caspase-1、IL-1β、IL-18蛋白阳性产物和mRNA表达明显降低(P<0.05,P<0.01),其中补中益气汤中剂量组降低效果最为明显,给药组NLRP3、cleaved Caspase-1、IL-1β、IL-18、GSDMD-N蛋白表达水平明显降低(P<0.05,P<0.01)。结论:补中益气汤可改善AIT,其机制可能是通过调控NLRP3/Caspase-1/GSDMD信号通路抑制细胞焦亡实现的。

Objective:To explore the mechanism of Buzhong Yiqitang on pyroptosis in autoimmune thyroiditis(AIT)mice based on the NOD-like receptor hot protein domain related protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(Caspase-1)/Gasdermin D(GSDMD)pathway.Method:Sixty NOD.H-2h4 mice were divided into normal group,model group,low,medium,and high dose groups(4.10,8.19,16.38 g·kg^(-1))of Buzhong Yiqitang,and selenium yeast tablet group(0.26 mg·kg^(-1)),with 10 mice in each group.Except for the normal group,all other groups were given 0.05%NaI by gavage for eight weeks to establish a model and then received the drug treatment for eight weeks.The serum levels of thyroid peroxidase antibody(TPO-Ab)and thyroglobulin antibody(TgAb)were detected using enzyme-linked immunosorbent assay(ELISA)method.Hematoxylin-eosin(HE)staining was used to observe the pathological changes in mouse thyroid tissue.The immunohistochemical method was used to detect the protein expression of NLRP3,Caspase-1,interleukin-1β(IL-1β),and interleukin-18(IL-18).Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of NLRP3,Caspase-1,IL-1β,and IL-18.Western blot was used to detect the levels of pyroptosis-related proteins in thyroid tissue.Result:Compared with the normal group,the serum levels of TPO-Ab and TgAb in the model group were significantly increased(P<0.01).Thyroid follicles either increased in a cubic shape or were damaged and atrophied,with a large number of lymphocytes infiltrating around the follicles.Compared with the model group,the levels of TPO-Ab and TgAb in other groups were significantly reduced(P<0.01),and the morphology and structure of follicles were improved.The degree of lymphocyte infiltration was reduced.Among them,the medium dose group of Buzhong Yiqitang had the most significant reduction and improvement effect.Compared with the normal group,the positive products and mRNA expression of NLRP3,Caspase-1,IL-1β,and IL-18 proteins in the thyroid tissue of the model group significantly increased(P<0.01),and the protein expression levels of NLRP3,cleaved Caspase-1,IL-1β,IL-18,and GSDMD-N were significantly increased(P<0.05,P<0.01).Compared with the model group,the positive products and mRNA expression of NLRP3,Caspase-1,IL-1β,and IL-18 proteins in other groups were significantly reduced(P<0.05,P<0.01),with the most significant reduction effect in the medium dose group of Buzhong Yiqitang.The protein expression levels of NLRP3,cleaved Caspase-1,IL-1β,IL-18,and GSDMD-N were significantly reduced(P<0.05,P<0.01).Conclusion:Buzhong Yiqitang can improve AIT,and its mechanism may be achieved by regulating the NLRP3/Caspase-1/GSDMD signaling pathway to inhibit pyroptosis.