miR-133a调控NLRP3炎性小体活化对大鼠脊髓损伤修复的影响

Influence of miR-133a regulating NLRP3 inflammasome activation on spinal cord injury repair in rats

目的:探究miR-133a对大鼠脊髓损伤(SCI)修复的作用及机制。方法:采用改良的Allen法诱导急性SCI大鼠模型。进行Basso-Beattie-Bresnahan(BBB)评分以确定运动功能恢复;通过qRT-PCR和Western blot检测miR-133a水平与NLRP3炎性小体相关蛋白水平;HE染色和Nissl染色评估脊髓神经元形态变化情况;TUNEL染色用于评估细胞凋亡;ELISA用于检测TNF-α、IL-1β和IL-10含量;免疫荧光用于评估GFAP、CD11b和NeuN阳性细胞表达情况。结果:SCI后大鼠BBB评分降低,Nissl小体数量减少,TUNEL凋亡率升高;并且TNF-α、IL-1β含量上调,IL-10含量下调,GFAP、CD11b蛋白水平升高,NeuN蛋白水平降低;此外miR-133a表达被抑制,而NLRP3炎性小体活化(P<0.05)。通过注射miR-133a激动剂上调其表达,进而可有效改善SCI大鼠的损伤情况以及对NLRP3炎性小体的激活情况(P<0.05)。此外,NLRP3抑制剂与miR-133a激动剂效果相似(P>0.05),但NLRP3抑制剂疗效稍差于miR-133a激动剂。进一步研究结果表明,NLRP3激动剂可以部分逆转上调miR-133a对SCI损伤的保护作用(P<0.05)。结论:miR-133a的上调可能通过抑制NLRP3炎性小体活化促进大鼠SCI损伤修复。

Objective: To explore the influence and mechanism of miR-133a on the repair of spinal cord injury(SCI) in rats. Methods: The acute SCI rat model was induced by modified Allen method. Basso-Beattie-Bresnahan(BBB) score was used to determine motor recovery;the levels of miR-133a and NLRP3 inflammasome-related proteins were detected by qRT-PCR and Western blot;HE staining and Nissl staining were used to evaluate the morphological changes of spinal cord neurons;TUNEL staining was used to assess apoptosis;ELISA was used to detect the contents of TNF-α, IL-1β and IL-10;immunofluorescence was used to evaluate the expression of GFAP, CD11b and NeuN positive cells. Results: After SCI, the BBB score of the rats decreased, the number of Nissl bodies decreased, and the apoptosis rate of TUNEL increased;and the contents of TNF-α and IL-1β were up-regulated, the content of IL-10 was down-regulated, the protein levels of GFAP and CD11b were increased, and the protein level of NeuN was decreased;in addition, miR-133a expression was inhibited, while NLRP3 inflammasome was activated(P<0.05). The expression of miR-133a was up-regulated by injection of miR-133a agonist, which could effectively improve the injury and activation of NLRP3 inflammasome in SCI rats(P<0.05). In addition, the effects of NLRP3 inhibitor and miR-133a agonist were similar(P>0.05), but the efficacy of NLRP3 inhibitor was slightly less effective than that of miR-133a agonist. Further results showed that NLRP3 agonists could partially reverse the protective effect of up-regulated miR-133a on SCI injury(P<0.05). Conclusion: The up-regulation of miR-133a may promote the repair of SCI in rats by inhibiting the activation of NLRP3 inflammasome.