瘿痛饮对腺病毒致甲状腺滤泡上皮细胞感染模型细胞焦亡机制

Pyroptosis mechanism of Yingtong Decoction on the pathogenesis of thyroid follicle epithelial cells infected by adenovirus

目的:基于细胞焦亡病理表型对升阳清热法瘿痛饮干预腺病毒致甲状腺滤泡上皮Nthy-roi3-1细胞感染模型的机制进行探索。方法:Nthy-roi3-1细胞随机分为6组:空白组,模型组,强的松组,中药低、中、高剂量组,采用腺病毒感染Nthy-roi3-1细胞模拟亚急性甲状腺炎体外模型,培养14 d后分别应用免疫荧光法检测细胞中的Caspase-1蛋白含量,免疫化学发光法测定细胞上清液T3、T4水平,比色法测定细胞上清液LDH,ELISA检测各组细胞上清液IL-1β、IL-18水平,Western blot测定细胞GSDMD-N、Caspase-1、NLRP3、ELAVL-1蛋白表达,Real-time PCR测定细胞Tg、TPO、Caspase-1、NLRP3、ELAVL-1 mRNA表达,Annexin V-FITC/PI染色检测细胞焦亡水平。结果:与空白组比较,模型组Caspase-1、ELAVL-1、GSDMD-N、NLRP3转录和表达水平,IL-1β、IL-18、T3、T4表达水平,Tg、TPO转录水平,细胞死亡率,以及Annexin V(+)/PI(+)和AnnexinV(+)/PI(-)细胞比例均显著升高(P<0.01)。各药物组上述指标均有不同程度下调(P<0.01),其中,中药低、中剂量组在下调T3、T4、LDH、Caspase-1、ELAVL-1、GSDMD-N、NLRP3表达水平及Tg、TPO、Caspase-1、ELAVL-1、GSDMD-N、NLRP3转录水平,上调Annexin V(+)/PI(+)细胞比例中具有显著疗效。结论:复方瘿痛饮能够抑制腺病毒致Nthy-roi3-1细胞感染的焦亡过程,其机制主要与下调焦亡上游调控分子ELAVL-1表达和转录水平,抑制NLRP3炎症小体活化、下调Caspase-1活化水平、抑制炎症细胞因子IL-1β、IL-18和GSDMD-N的表达有关。

Objective: To explore the efficacy and mechanism of lifting yang and clearing heat therapy in inhibiting pyroptosis of adenovirus-infected Nthy-roi3-1 cells. Methods: Nthy-roi3-1 cells were randomly divided into 6 groups which were respectively named blank group, model group, prednisone group, Yingtong Decocation(YTY) low dose group, YTY medium dose group and YTY high dose group. Then we simulated subacute thyroiditis in vitro by using adenovirus to infect Nthyroi3-1 cells. After 14 days, we tested protein level of Caspase-1 by fluorescence immunoassay, T3 and T4 in cellular supernatant by immunochemiluminometric assays, LDH in cellular supernatant by colorimetry, IL-1β, IL-18 in cellular supernatant by ELISA, intracellular protein expression level of GSDMD-N, Caspase-1, NLRP3, ELAVL-1 by Western blot, intracellular mRNA transcriptional level of GSDMD-N, Caspase-1, NLRP3, ELAVL-1 by Real-time PCR, and pyroptosis level of Nthy-roi3-1 cells by Annexin V-FITC/PI. Results: Compared with the blank group, the transcription and expression level of Caspase-1, ELAVL-1,GSDMD-N, NLRP3, expression level of IL-1β, IL-18, T3, T4, transcription level of Tg, TPO, level of LDH, and the cell proportion of AnnexinV(+)/PI(+) and AnnexinV(+)/PI(-) were all rising in model group. After the intervention of each drug group, the above indexes were all downregulated to varying degrees. The YTY low-dose and medium-dose groups decreased the expression level of T3, T4, LDH, Caspase-1, EVAVL-1, GSDMD-N and NLRP3, and the transcription level of Tg, TPO,Caspase-1, ELAVL-1, GSDMD-N and NLRP3 and increased the proportion of annexin V(+)/PI(+) cells. Conclusion: YTY could inhibiting pyroptosis of adenovirus-infected Nthy-roi3-1 cells by down-regulating the expression and transcription level of ELAVL-1, inhibiting activation of NLRP3 inflammatome, down-regulating activation level of Caspase-1, and inhibiting the expression of IL-1β, IL-18 and GSDMD-N.