高渗氯化钠羟乙基淀粉溶液对蛛网膜下腔出血后脑血管痉挛的影响及机制研究

Effects of hypertonic sodium chloride hydroxyethyl starch solution on cerebral vasospasm following subarachnoid hemorrhage and its mechanism

目的 探讨高渗氯化钠羟乙基淀粉溶液（HSH）对蛛网膜下腔出血（SAH）后脑血管痉挛（CVS）的影响及其相关机制.方法 按随机数字表法将24只雄性SD大鼠分为4组,每组6只.利用枕大池二次注血法建立SAH-CVS模型.模型组和HSH治疗组于制模后每日分别经尾静脉注射生理盐水或HSH 8 mL/kg;假手术组进行SAH手术但仅在枕大池内注射1.5 mL/kg生理盐水并每日给予生理盐水8 mL/kg治疗;正常组不予任何处理.各组大鼠均于7d后处死,苏木素-伊红（HE）染色分析检测基底动脉血管壁厚度及管腔面积.分离血管平滑肌细胞（VSMC）,采用流式细胞仪检测细胞凋亡率;荧光法检测细胞天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）活性;蛋白质免疫印迹试验（Western Blot）检测细胞Bax及Bcl-2蛋白表达;H2DCFDA荧光探针检测细胞活性氧（ROS）水平.结果 与正常组比较,模型组基底动脉血管壁厚度明显增加（μm：27.72±1.94比18.30±1.11,P＜0.05）,管腔面积明显减小（μm2：26 115±1 991比55 080±2 091,P＜0.05）,细胞凋亡率显著增加[（35.05±5.54）％比（5.93±1.53）％,P＜0.05].与模型组比较,HSH治疗组基底动脉血管壁厚度明显减小（μm：22.55±1.50比27.72±1.94,P＜ 0.05）,管腔面积明显增加（μm2：48 115±2 460比26 115±1 991,P＜0.05）,细胞凋亡率明显下降[（16.54±5.94）％比（35.05±5.54）％,P＜0.05].模型组细胞caspase-3活性、ROS水平、Bax和Bcl-2表达分别为正常组的（188.40±19.35）％、（163.50±17.02）％、（208.71±26.04）％和（44.52±9.61）％,与正常组比较差异均有统计学意义（均P＜0.05）;HSH治疗组caspase-3活性、ROS水平、Bax和Bcl-2表达分别为正常组的（135.05±19.52）％、（119.44±11.50）％、（139.20±18.04）％和（85.35±13.12）％,与模型组比较均明显改善（均P＜0.05）.假手术组各指标与正常组比较差异均无统计学意义.结论 HSH可抑制SAH后CVS程度,改善基底动脉血管壁增厚及管腔狭窄,其机制可能与抑制VSMC凋亡有关.

Objective To investigate the protective effect and potential mechanisms of hypertonic sodium chloride hydroxyethyl starch solution （ HSH ） against the cerebral vasospasm （ CVS ） following subarachnoid hemorrhage （SAH）. Methods Twenty-four male Sprague-Dawley （SD） rats were randomly assigned to four groups according to the random number table, with 6 rats in each group. The SAH-CVS model was reproduced by injection of the blood twice through the cisterna magna. Rats in both model and HSH treatment groups received 8 mL/kg normal saline （ NS ） or HSH treatment everyday via caudal vein. Rats in sham group were injected with 1.5 mL/kg NS into cisterna magna followed by 8 mL/kg NS treatment. Rats in normal group received no treatment. Rats were sacrificed to harvest basilar artery after 7 days. The thickness of vessel wall and lumen area were measured using hematoxylin-eosin （HE） staining. The rate of apoptosis of vascular smooth muscle cell （VSMC） was assessed using flow cytometry. Caspase-3 activity was measured by a fluorometric assay. The expressions of Bax and Bcl-2 were determined by Western Blot. Intracellular reactive oxygen species （ROS） was detected by H2DCFDA. Results Compared with normal group, increased thickness of vessel wall （μm： 27.72 ± 1.94 vs. 18.30 ± 1.10, P〈0.05）, decreased lumen area （μm2：26 115 ± 1 991 vs. 55 080 ± 2 091, P〈0.05）, and elevation of rate of apoptosis of VSMCs （（35.05 ± 5.54） % vs. （5.93 ± 1.53） %, P〈 0.05 ] were found in model group. Compared with model group, decreased thickness of vessel wall （μm： 22.55 ± 1.50 vs. 27.72 ± 1.94, P〈0.05）, increase of lumen area （μm2：48 115 ±2 460 vs. 26 115 ± 1 991, P〈0.05）, and depressed rate of apoptosis of VSMCs [ （ 16.54 ± 5.94 ） % vs. （35.05 ± 5.54） %, P〈0.05] were found in HSH treatment group. Caspase-3 activity, intracellular ROS level, Bax and Bcl-2 expressions in model group were （188.40 ± 19.35）%, （163.50 ± 17.02）%, （208.71 ±26.04）% and （44.52 ±9.61） % of those of normal group, and the differences of these parameters between model and normal groups were statistically significant （all P〈0.05 ）. Caspase-3 activity, intraceUular ROS level, Bax and Bcl-2 expressions in HSH treatment group were （135.05 ± 19.52）% , （119.44 ± 11.50）%, （139.20 ± 18.04）% and （85.35 ± 13.12）% of those of normal group, respectively, and the differences of these parameters between HSH treatment and model groups were statistically significant （all P〈0.05 ）. The differences of all measurements between sham and normal groups were not statistically significant. Conclusion The current results demonstrate that HSH attenuates the SAH-induced CVS, alleviates thickness of vessel wall, and increases lumen area via inhibition of VSMCs apoptosis.