shRNA干扰Nod样受体蛋白在新生大鼠脑缺血再灌注模型中的作用及机制

Protective Effect of ShRNA Interference with NLRP3 on the Myocardial injury and Immune Response of Neonatal Rats with Hypoxic-ischemic Brain Injury and Its Mechanism

目的探究shRNA(short hairpin RNA)干扰Nod样受体蛋白(NLRP3)对新生大鼠脑缺血再灌注模型(MCAO)的心肌损伤和免疫反应的作用及机制。方法构建新生大鼠MCAO,实验分4组:假手术组、模型组(MCAO组)、MCAO+阴性对照组和MCAO+sh-NLRP3组,每组10只。采用RT-PCR和蛋白质印迹技术(Western blot)检测NLRP3表达;HE染色观察心肌组织病理学改变;免疫组化检测Ki67表达;Western blot 检测Ki67和PCNA表达;TUNEL染色检测心肌细胞凋亡率;Western blot检测心肌细胞Caspase-3、Caspase-9 表达;Elisa检测Mb、 CK-MB 和cTnⅠ含量及心肌炎标记分子IL-1β、iNOS、IL-6表达;Western blot检测TGF-β1、NF-κB p65和TNF-α表达。结果 sh-NLRP3 能抑制模型大鼠NLRP3在基因和蛋白水平上的表达( P <0.01),改善心肌坏死,上调心肌组织中Ki67和PCNA表达( P < 0.01),降低心肌细胞凋亡率( P <0.01)和Caspase-3、Caspase-9 的蛋白表达( P <0.01),降低大鼠血清中Mb、cTnⅠ和CK-MB的含量( P <0.01),同时下调TGF-β1、P-P65/P65和TNF-α的蛋白表达。结论 sh-NLRP3对MCAO新生大鼠的心肌损伤和免疫反应起保护作用,其机制与抑制 TGF-β1/NF-κB p65/TNF-α炎症通路活化相关。

Objective To explore the protective effect of short hairpin RNA (shRNA) interference with NLRP3 on the myocardial injury and immune response of neonatal rats with hypoxic-ischemic brain injury and its mechanism. Methods The neonatal rat models were established by MCAO and divided into four groups including the sham-operated group, model group (MCAO group), MCAO and Scramble group and MCAO and sh-NLRP group with 10 rats in each group. The expression of NLRP3 was detected by RT-PCR and Western blotting;the pathological changes of myocardium were observed by HE staining;the expression of Ki67 was detected by immunohistochemistry and the expression of Ki67 and PCNA was detected by Western blotting;the apoptotic rate of myocardial cells was detected by TUNEL staining and the expression of Caspase-3 and Caspase-9 was detected by Western blotting;the content of Mb, CK-MB and cTn I and the expression of the Myocarditis marker molecules including IL-1β, iNOS, IL-6 and L-10 were detected by the Elisa method;the expression of TGF-β1, NF-κB p65 and TNF-α was detected by Western blotting. Results Sh-NLRP3 inhibited the expression of NLRP3 at gene and protein levels ( P <0.01), improved myocardial necrosis, up-regulated the expression of Ki67 and PCNA in myocardial tissues ( P <0.01), decreased the apoptotic rate of myocardial cells ( P <0.01) and the expression of Caspase-3 and Caspase-9 ( P <0.01), reduced the content of Mb, cTnⅠ and CK-MB in serum ( P <0.01), decreased the content of IL-1β, iNOS and IL-6, increased the content of IL-10, and down-regulated the protein expression of TGF-β1, P-P65/P65 and TNF-α in the rat model. Conclusion Sh-NLRP3 has protective effect on the myocardial injury and immune response of neonatal rats with hypoxic-ischemic brain injury, and its mechanism is related to inhibiting the activation of the inflammatory pathway of TGF-β1/NF-κB p65/TNF-α.