基于PI3K/Akt/eNOS信号通路探讨补阳还五汤对大鼠蛛网膜下腔出血后脑血管痉挛的影响

Effect of Buyang Huanwu Tang on Cerebral Vasospasm After Subarachnoid Hemorrhage in Rats Based on PI3K/Akt/eNOS Signaling Pathway

目的:探讨补阳还五汤改善蛛网膜下腔出血后脑血管痉挛的相关分子机制。方法:将雄性SD大鼠80只随机分为假手术组、模型组、补阳还五汤低、高剂量组(13,26 g·kg^(-1)·d^(-1)),每组20只。按照10 mL·kg^(-1)每天灌胃给药2次,连续7 d。采用二次枕大池注血法制作蛛网膜下腔出血模型,评估各组大鼠1,3,5,7 d的神经功能评分,利用苏木素-伊红(HE)染色对各组大鼠基底动脉直径进行测量,蛋白免疫印迹法(Western blot)检测基底动脉脑组织磷酸化磷脂酰肌醇3-激酶(p-PI3K),磷酸化蛋白激酶B(p-Akt),内皮型一氧化氮合酶(eNOS),神经型一氧化氮合酶(n NOS)蛋白的表达;酶联免疫吸附测定(ELISA)检测大鼠脑脊液中一氧化氮(NO),内皮素-1(ET-1)的表达。结果:与假手术组比较,模型组大鼠神经功能评分明显下降(P <0. 05),基底动脉直径明显缩小(P <0. 05),基底动脉脑组织p-PI3K,p-Akt,eNOS,n NOS蛋白的表达明显降低(P <0. 05),脑脊液中NO明显减少(P <0. 05),ET-1明显升高(P <0. 05);与模型组比较,不同剂量补阳还五汤组(26,13 g·kg^(-1)·d^(-1))在治疗后3～5 d后大鼠神经功能评分均有不同程度升高,基底动脉直径明显增宽(P <0. 05),p-PI3K,p-Akt,eNOS蛋白的表达明显升高(P <0. 05),NO明显升高(P <0. 05),ET-1明显下降(P <0. 05);与低剂量组比较,经过7 d的补阳还五汤治疗后高剂量组神经功能评分明显升高(P <0. 05),基底动脉直径增宽更明显(P <0. 05),p-PI3K,p-Akt,eNOS蛋白的表达更高(P <0. 05),NO升高更明显(P <0. 05),ET-1下降更明显(P <0. 05)。结论:补阳还五汤对蛛网膜下腔出血后脑血管痉挛的保护作用可能与上调PI3K/Akt/eNOS信号通路中的p-PI3K,p-Akt,eNOS的表达,从而增加NO产生有关。

Objective: To investigate the molecular mechanism of Buyang Huanwu Tang in improving cerebral vasospasm after subarachnoid hemorrhage. Method: Eighty male Sprague-Dawley rats were randomly divided into sham operation group,model group,Buyang Huanwu Tang low and high dose( 13,26 g·kg-1·d-1)group. According to 10 mL·kg-1,the drug was administered twice a day for 7 days. The subarachnoid hemorrhage model was made by double occipital pool injection method. The neurological function scores of rats in each group were evaluated at 1,3,5 and 7 days. The diameter of basilar artery was measured by hematoxylin-eosin( HE)staining. The expressions of phosphp-phosphoinositide 3-kinases( p-PI3 K),phosphp-protein kinase B( p-Akt),endothelial nitric oxide synthase( eNOS) and neuronal nitric oxide synthase( nNOS) protein in basilar artery brain tissue were detected by Western blot. The expression of nitric oxide( NO) and endothelin-1( ET-1) in rat cerebrospinal fluid was detected by enzyme-linked immunosorbent assay( ELISA). Result: Compared with sham operation group,the neurological function scores of the model group were significantly decreased( P < 0. 05),the diameter of the basilar artery was significantly reduced( P < 0. 05),and The expression of p-PI3 K,p-Akt,eNOS,nNOS proteins of the basilar artery brain tissue were significantly decreased( P < 0. 05). The level of NO in the cerebrospinal fluid was significantly decreased( P < 0. 05),and the ET-1 was significantly increased( P < 0. 05).Compared with model group,the different doses in Buyang Huanwu Tang group( 26,13 g·kg^-1·d^-1) increased the neurological function scores 3 to 5 days after treatment,and the basilar artery diameter was significant increased( P < 0. 05). The expression of p-PI3 K,p-Akt and eNOS protein was significantly increased( P < 0. 05). The level of NO was significantly increased( P < 0. 05),and ET-1 was significantly decreased( P < 0. 05). Compared with low-dose group,the neurological scores of high-dose group were significantly increased after 7 days of treatment with Buyang Huanwu Tang( P < 0. 05),and the diameter of the basilar artery was significantly increased( P < 0. 05),p-PI3 K,The expression of p-Akt and eNOS protein was higher( P < 0. 05),the increase of NO was more obvious( P < 0. 05),the decrease of ET-1 was more significant( P < 0. 05). Conclusion: The protective effect of Buyang Huanwu Tang on cerebral vasospasm after subarachnoid hemorrhage may be related to up-regulation of p-PI3 K,p-Akt and eNOS expression in PI3 K/Akt/eNOS signaling pathway,thereby increasing NO production.