黄芪甲苷对新生大鼠缺氧缺血性脑损伤NLRP3炎性小体表达的影响

Effect of astragaloside IV on the expression of NOD-like receptor protein 3 inflammasome in neonatal rats with hypoxic-ischemic brain damage

摘要:

目的 探讨黄芪甲苷（AS-IV）对缺氧缺血性脑损伤（HIBD）新生大鼠Nod样受体蛋白3（NLRP3）炎性小体的影响。方法 将24只7日龄Sprague-Dawley大鼠随机分为假手术组、HIBD组、AS-IV治疗组（AS-IV组），每组8只。各组动物造模24 h后取脑组织进行苏木精-伊红染色、yo-PRO-1和EthD-2免疫荧光染色，以观察AS-IV在体内的脑保护作用。构建HT22细胞氧糖剥夺（OGD）模型，设置AS-IV浓度梯度（50~400 μmol/L）进行干预。采用CCK8法检测HT22细胞活力，RT-PCR和Western blot法检测不同浓度AS-IV对NLRP3、Gasdermin D蛋白（GSDMD）、半胱氨酸蛋白酶-1（Caspase-1）、白细胞介素1β（IL-1β）mRNA及其蛋白表达的影响。结果 Yo-PRO-1和EthD-2染色结果显示，与假手术组比较，HIBD组焦亡细胞增多，同时可见少量坏死细胞，AS-IV组焦亡细胞和坏死细胞均减少。HIBD组脑组织NLRP3、IL-1β、Caspase-1及GSDMD蛋白表达水平显著高于假手术组（P < 0.05）；与HIBD组比较，AS-IV组NLRP3、Caspase-1及GSDMD蛋白表达水平显著降低（P < 0.05）。HT22细胞实验结果显示，与OGD组比较，AS-IV可以抑制NLRP3、GSDMD、Caspase-1、IL-1β mRNA及其蛋白表达，200 μmol/L时治疗效果最佳（P < 0.05）。结论 AS-IV可能通过抑制NLRP3、GSDMD、Caspase-1、IL-1β的表达减轻新生大鼠HIBD。

Abstract:

Objective To study the effect of astragaloside IV(AS-IV) on NOD-like receptor protein 3(NLRP3) inflammasome in neonatal rats with hypoxic-ischemic brain damage(HIBD). Methods A total of 24 Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group, an HIBD group, and an AS-IV treatment group, with 8 rats in each group. After 24 hours of modeling, brain tissue was collected for hematoxylin-eosin staining, yoPRO-1 staining, and EthD-2 immunofluorescent staining in order to observe the cerebral protection effect of AS-IV in vivo. HT22 cells were used to prepare a model of oxygen-glycogen deprivation(OGD), and a concentration gradient(50-400 μmol/L) was established for AS-IV. CCK-8 assay was used to measure the viability of HT22 cells. RT-PCR and Western blot were used to observe the effect of different concentrations of AS-IV on the mRNA and protein expression of NLRP3, gasdermin D(GSDMD), caspase-1, and interleukin-1β(IL-1β). Results Yo-Pro-1 and EthD-2 staining showed that compared with the sham-operation group, the HIBD group had an increase in pyroptotic cells with a small number of necrotic cells, and the AS-IV group had reductions in both pyroptotic and necrotic cells. Compared with the sham-operation group, the HIBD group had significantly higher protein expression levels of NLRP3, IL-1β, caspase-1, and GSDMD(P<0.05). Compared with the HIBD group, the AS-IV group had significant reductions in the protein expression levels of NLRP3, caspase-1, and GSDMD(P<0.05). HT22 cell experiment showed that compared with the OGD group, the AS-IV group had inhibited mRNA and protein expression of NLRP3, GSDMD, caspase-1, and IL-1β, with the best therapeutic effect at the concentration of 200 μmol/L(P<0.05). Conclusions AS-IV may alleviate HIBD in neonatal rats by inhibiting the expression of NLRP3, GSDMD, caspase-1, and IL-1β.