氢溴酸樟柳碱对抗大鼠急性脑缺血/再灌注损伤的作用机制研究

The protective mechanism of anisodine hydrobromide against cerebral ischemia-reperfusion injury in rats

目的探讨氢溴酸樟柳碱对抗大鼠急性脑缺血/再灌注损伤的作用机制。方法体内实验采用线栓法制备大鼠大脑中动脉阻塞(MCAO)致脑缺血/再灌注损伤模型,氢溴酸樟柳碱尾静脉注射进行干预。HE染色评价脑组织一般病理学情况;尼氏染色评价脑组织健存神经元情况;检测脑组织匀浆过氧化氢酶(CAT)活性、脂质过氧化物(LPO)含量、乳酸脱氢酶(LDH)活性;采用Western blot技术检测脑组织Bax、Bcl-2、caspase-3、p-Akt等蛋白的表达。体外实验采用PC12细胞氧糖剥夺再灌注损伤模型(OGD-R),采用Western blot技术检测细胞内Bax、Bcl-2、caspase-3、p-Akt等蛋白的表达情况,对氢溴酸樟柳碱作用的信号通路进行确认。结果氢溴酸樟柳碱0.15 mg·kg^(-1)能明显降低MCAO模型大鼠一般病理学评分,提高存活神经元数目;氢溴酸樟柳碱0.3、0.15 mg·kg^(-1)能明显提高脑组织CAT活性,氢溴酸樟柳碱0.3 mg·kg^(-1)能明显降低LPO含量;氢溴酸樟柳碱1.2mg·kg^(-1)明显降低LDH活性;各剂量组均能明显降低促凋亡蛋白Bax的表达,提高Bcl-2/Bax比值,促进p-Akt表达,明显提高p-Akt/Akt比值,除氢溴酸樟柳碱0.15 mg·kg^(-1)剂量外,其余剂量均能明显提高抗凋亡蛋白Bcl-2的表达。体外实验结果显示,氢溴酸樟柳碱在25~100μmol·L^(-1)时能明显提高Bcl-2蛋白的表达,提高Bcl-2/Bax比值,在50μmol·L^(-1)剂量下能明显提高p-Akt/Akt的比值。结论氢溴酸樟柳碱对抗急性脑缺血/再灌注损伤大鼠的作用机制与抗氧化损伤及提高p-Akt的表达有关。

Aim To investigate the protective mechanism of anisodine hydrobromide against cerebral ischemia-reperfusion injury in rats. Methods In vivo： the cerebral ischemia-reperfusion injury model was established by middle cerebral artery occlusion（ MCAO）via suture method in rats; the rats were injected anisodine hydrobromide（ 1. 2, 0. 6, 0. 3, 0. 15 mg ·kg-1）; the morphological changes were detected by HE staining; the Nissl staining was used to count the number of surviving neurons; the activity of CAT and LDH,the LPO contents in the brain tissue were measured; the expressions of Bax,Bcl-2,caspase-3 and pAkt in brain tissue were detected by Western blot. In vitro： Western blot assay was used to determine the expression of Bax,Bcl-2,caspase-3 and p-Akt protein expression in the OGD-R model of PC12 cells. The signal pathway of anisodine hydrobromide was identified. Results Anisodine hydrobromide with the dose of 0. 15 mg · kg-1could significantly lessen the morphological changes,and improve the number of surviving neurons; the dose of 0. 3 and 0. 15 mg · kg-1could significantly improve the activity of CAT; the dose of 0. 3 mg · kg-1could significantly reduce the contents of LPO in the rat brain tissue; the dose of 1. 2mg· kg-1could significantly decrease the activity of LDH; the dose of 0. 15 ~ 1. 2 mg·kg-1could inhibit the expression of Bax,promote the expression of p-Akt in rat brain tissue. All the doses except 0. 15 mg ·kg-1could promote the expression of Bcl-2 in rat brain tissue. In vitro, the results showed that anisodine hydrobromide in 25 ~ 100 μmol · L-1could significantly improve the expression of Bcl-2 and the ratio of Bcl-2/Bax,and the dose of 50 μmol ·L-1could significantly improve the ratio of p-Akt/Akt. Conclusion The mechanism of anisodine hydrobromide against cerebral ischemia-reperfusion injury model rats might be related to its anti-oxidative activity and the activation of Akt.