ATG12基因对易卒中自发性高血压大鼠大脑组织自噬的影响

Effect of ATG12 gene on autophagy in brain tissue of stroke-prone spontaneously hypertensive rats

目的探讨自噬相关蛋白12(autophagy-related protein 12,ATG12)对易卒中自发性高血压大鼠(stroke-prone spontaneously hypertensive rats,SHR/SP)大脑组织自噬的影响。方法将30只SHR/SP分为SHR/SP模型(SHR/SP)组、对照病毒(NC shRNA SHR/SP)组和ATG低表达病毒(ATG12 shRNA SHR/SP)组,其中ATG12 shRNA SHR/SP和NC shRNA SHR/SP组大鼠分别为通过尾静脉注射病毒构建ATG12低表达和对照大鼠,10只WKY(Wistar-Kyoto)大鼠作为SHR/SP的空白对照(WKY组)。荧光定量PCR检测WKY和SHR/SP大脑组织中ATG12 mRNA表达水平。通过大鼠血压计检测各组大鼠血压,分光光度法检测各组大鼠大脑组织氧化应激指标SOD和MDA水平;Western blot检测各组大鼠大脑组织自噬相关蛋白LC3A/B、ATG12和P62蛋白表达水平。结果与WKY组大鼠相比,SHR/SP模型组大鼠大脑组织ATG12 mRNA水平显著升高(P <0. 01)。血压检测结果显示,与WKY大鼠组比较,SHR/SP组大鼠血压显著升高,与NC shRNA SHR/SP组比较,ATG12 shRNA SHR/SP组大鼠血压显著降低(P <0. 01)。与WKY组比较,SHR/SP模型组大鼠大脑组织SOD活性显著降低,MDA含量显著升高(P <0. 01);与NC shRNA SHR/SP组比较,ATG12 shRNA-SHR/SP组大鼠大脑组织SOD活性显著升高,MDA含量显著降低(P <0. 01)。Western blot结果显示,与WKY组比较,SHR/SP模型组大鼠大脑组织ATG12和LC3A/B蛋白表达显著升高,P62蛋白表达显著降低;与NC shRNA SHR/SP组比较,ATG12 shRNA-SHR/SP组大鼠大脑组织ATG12和LC3A/B蛋白表达显著降低,P62蛋白表达显著升高(P <0. 01)。结论易卒中自发性高血压大鼠大脑组织ATG12高表达,抑制ATG12表达能够抑制大鼠脑组织自噬水平,降低大鼠血压,可能与抑制大鼠脑组织氧化应激水平有关。

Objective To investigate the effect of autophagy-related protein 12( ATG12) on autophagy in brain tissue of stroke-prone spontaneously hypertensive rats( SHR/SP). Methods The ATG12 low expression rat model was constructed by injecting adeno-associated virus( AAV) into the tail vein. Thirty rats were divided into SHR/SP model group,negative control virus SHR/SP model( NC shRNA SHR/SP) group and ATG low expression virus SHR/SP model( ATG12 shRNA-SHR/SP) group. The ATG12 low expression( ATG12 shRNA-SHR/SP) and control( NC shRNA SHR/SP) rats were injected with adeno-associated virus( AAV) into the tail vein.Ten WKY rats were chosen as blank controls( WKY group). The expression level of ATG12 mRNA in brain tissue was detected by real-time PCR. The blood pressure was measured. The levels of SOD and MDA in brain tissues were detected by spectrophotometry. The expression levels of autophagy-related proteins LC3 A/B,ATG12 and P62 in brain tissues were detected by Western blot. Results Compared with WKY group,the ATG12 mRNA level in the brain was significantly increased in SHR/SP model group( P < 0. 01). The blood pressure in SHR/SP group was significantly higher than that in WKY group. Compared with NC shRNA SHR/SP group,the blood pressure was significantly decreased in ATG12 shRNA SHR/SP group( P < 0. 01). Compared with WKY group,the SOD activity in the brain tissue was significantly decreased in SHR/SP model group,while the MDA content was significantly increased( P < 0. 01).Compared with NC shRNA SHR/SP group,the SOD activity in the brain tissue was significantly increased in ATG12 shRNA-SHR/SP group,while the MDA content was significantly decreased( P < 0. 01). Western blot results showed that compared with WKY group,the expression of ATG12 and LC3 A/B protein in the brain was significantly increased in SHR/SP model group,while the expression of P62 protein was significantly decreased. Compared with NC shRNA SHR/SP group,the expression of ATG12 and LC3 A/B protein in brain tissue was significantly decreased in ATG12 shRNA-SHR/SP group,while the expression of P62 protein was significantly increased( P < 0. 01). Conclusion The ATG12 expression is high in SHR/SP rats. Inhibition of ATG12 expression can inhibit autophagy levels in rat brain tissue and decrease blood pressure in rats,which may be related to inhibition of oxidative stress levels in rat brain tissue.