硫化氢对蛛网膜下腔出血后急性脑血管痉挛的影响及其作用机制

Protective effect of hydrogen sulfide on acute cerebral vasospasm in subarachnoid hemorrhage models

目的观察硫化氢（H2S）供体硫氢化钠（NaHS）对蛛网膜下腔出血（SAH）后急性脑血管痉挛（CVS）的影响，并初步探讨其可能作用机制。方法（1）48只雄性SD大鼠按随机数字表法分为空白组、SAH组、NaHS低剂量组和NariS高剂量组，每组12只。后3组采用视交叉池注血法制作SAH模型，后2组分别在造模后0．5h腹腔注射0．7mg／kg、2．8mg／kgNaHS。造模后24h进行Garcia神经功能评分，采用HE染色、TUNEL与免疫组化双标染色分别观察大鼠大脑前动脉（ACA）A2段血管近段形态、管壁厚度、管腔面积以及内皮细胞凋亡情况。（2）取培养好的胎鼠脑微血管内皮细胞分成4组：空白组、氧化血红蛋白（OxyHb，100μmol／L）干预组、低剂量（25μmol／L）NaHS预处理组、高剂量（100μmol／L）NaHS预处理组。培养24h后观察、统计脱皿内皮细胞数，并收集细胞采用Western blotting方法检测凋亡蛋白Caspase-3表达。结果（1）SAH组大鼠神经功能评分[（8．5±2．4）分]较空白组显著下降，管壁厚度[（43．5±6．2）μm]显著增厚，管腔面积[（30488±938）μm^2]显著减小，TUNEL阳性细胞数[（36．51±11．45）％]显著增多，差异均有统计学意义（P〈0．05）；NaHS低剂量组、NaHS高剂量组神经功能评分[（11．6±1．9）分，（15．4±2．3）分]较SAH组显著升高，管壁厚度[（34．7±3．7）μm，（31．7±4．6）μm]显著减薄，管腔面积[（41463±1104）μm^2，（45244±1217）μm^2]显著增加，TUNEL阳性细胞数[（17．14±5.36）％，（8．10±4．62）％]显著下降，差异均有统计学意义（P〈0．05）；NaHS高剂量组神经功能评分较NaHS低剂量组显著升高，TUNEL阳性细胞数显著下降，差异有统计学意义（P〈0．05）。（2）OxyHb干预组脱皿内皮细胞数[（40．56±9．85）％]较空白组显著增多，凋亡蛋白Caspase-3表达（0．395±0．122）明显上调，差异均有统计学意义（P〈0．05）；低剂量NaHS预处理组、高剂量NaHS预处理组脱皿内皮细胞数[（16．65±6.35）％，（14．12±6．65）％]较OxyHb干预组显著减少，Caspase-3表达（0．223±0．083，0．208±0．104）显著下降，差异均有统计学意义（P〈0．05）。结论NaHS可有效缓解痉挛的脑血管，其保护作用可能是通过抑制脑血管内皮细胞凋亡实现的。

Objective To study the effect of hydrogen sulfide （H2S） on acute cerebral vasospasm （CVS） after subarachnoid hemorrhage （SAH） and explore its mechanism. Methods （1） A total of 48 adult male SD rats were randomly allocated into a normal group, a SAIl group, a low dose Naris group and a high dose NariS group （n=12）. Rat models of SAH were induced by injecting autologous blood into the prechiasmatic cistern. Rats in the later two groups were given 0.7 mg/kg and 2.8 mg/kg NariS, respectively, 0.5 h after modeling. Neurological scale scores were assessed 24 h after modeling; HE staining, TUNEL and immunohistochemical double-staining were employed to detect the morphology of approximated A2 blood vessel of anterior cerebral artery （ACA）, tube wall thickness and endothelial cell apoptosis, respectively. （2） Brain microvascular endothelial cells （BMECs） were chosen and divided into blank control group, 100 μmol/L OxyHb prevention group, 25 μmol/L Naris pretreatment group and 100 μmol/L NariS pretreatment group. The cells were collected and observed 24 h after treatment, and then, the number of endotheliocytes was counted, and the Caspase-3 protein expression was detected by Western blotting. Results （1） The neurological scale scores （8.5±2.4） were significantly lower, the vessel wall （[43.5±6.2] μm） was significantlythickened, the lumenarea （[30488±938） μm^2） was obviously reduced, and the number of TUNEL positive cells （[36.51 ± 11.451%） was remarkably increased in the SAH group as compared with those in the normal group （16.1±1.7, [25.8±3.5] μm, [51707±1422] μm^2 and [2.86±0.75]% in turn, P〈0.05）. The neurological scale scores （11.6±1.9 and 15.4±2.3） were significantly higher, the vessel wall （[34.7±3.7] and [31.7±4.6] μm） was significantly thinned, the lumen area （[41 463±1104] and [45 244±1217] μm^2） was obviously increased, and the number of TUNEL positive cells （[17.14±5.36] and [8.10±4.62] %） was remarkably reduced in the low dose NariS group and high dose NariS group as compared with those in the SAH group （P〈0.05）. The neurological scale scores in high dose NariS group were significantly higher than those in the low dose group and the number of TUNEL positive cells was signficantly smaller than that in the low dose group （P〈0.05）. （2） The number of apoptotic endothelial cells （[40.56±9.85] %） and the expression of Caspase-3 （0.395±0.122） in OxyHb prevention group were significantly larger/higher than those in the blank control group （P〈0.05）. The number of apoptotic endothelial cells and the expression of Caspase-3 in the low dose group （[16.65± 6.35]% and [0.223±0.083]） and high dose group（ [14.12±6.65] % and [0.208±0.104]） were obviously reduced as compared with those in the OxyHb prevention group, with significant differences （P〈0.05）. Conclusion H2S can effectively expand cerebral vasospasm, and its vasoprotective mechanism may be through inhibiting vascular endothelial cell apoptosis.