葛根素对小鼠脑缺血再灌注损伤的神经保护作用及机制研究

The Mechanism and Neuroprotection Study of Perarin on Cerebral Ischemia Reperfusion Injury in Mice

目的观察葛根素对脑缺血再灌注小鼠的神经保护作用并探讨其对雌激素受体(estrogen receptor,ER)及缺氧诱导因子-1(hypoxia inducible factor-1,HIF-1)表达及相关炎症因子释放的影响。方法 C57BL小鼠随机分为假手术组、溶剂对照组以及葛根素低剂量组(100 mg·kg^(-1))、中剂量组(250 mg·kg^(-1))和高剂量组(500 mg·kg^(-1)),采用线栓法制备小鼠大脑中动脉栓塞模型(middle cerebral artery occlusion,MCAO),缺血2 h,再灌注24 h后观察葛根素对损伤后脑梗死体积、含水量、神经功能评分等指标的影响。根据再灌注不同时间点,溶剂对照组又分为2 h、6 h、12 h、24 h共4个亚组,分别在相应时间点采用蛋白质免疫印迹技术测定脑组织中ER-α及HIF-1α的变化情况。取变化最显著的缺血2 h再灌注12 h这一时间点测定葛根素对ER-α及HIF-1α表达的影响,并通过酶联免疫吸附技术测定葛根素在这一时间点对下游炎症因子如肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)和白细胞介素6(interleukin-6,IL-6)的调节作用。结果相比溶剂对照组,低、中和高剂量组葛根素均能降低缺血2 h再灌注24 h小鼠的脑梗死体积[低剂量(29.6±3.6)%,中剂量(15.2±3.9)%,高剂量(8.2±2.1)%vs对照组(39.3±5.0)%]和脑含水量[低剂量(84.9±8.8)%,中剂量(83.7±8.2)%,高剂量(80.9±8.7)%vs对照组(85.3±10.2)%],差异有统计学意义。对于脑缺血2 h再灌注12 h小鼠,中、高剂量葛根素组较溶剂对照组ER-α水平升高,HIF-1α水平降低,差异有统计学意义;中、高剂量葛根素组TNF-α[(420.7±27.2)μg·g^(-1),(379.6±23.9)μg·g^(-1)]、IL-1β[(211.8±19.2)μg·g^(-1),(182.4±13.5)μg·g^(-1)]和IL-6[(111.2±9.1)μg·g^(-1),(104.1±12.4)μg·g^(-1)]水平较溶剂对照组[TNF-α(505.8±36.7)μg·g^(-1);IL-1β(291.6±21.8)μg·g^(-1);IL-6(138.4±11.7)μg·g^(-1)]下降,差异有统计学意义。结论葛根素在一定剂量范围内可降低脑缺血再灌注小鼠病变脑组织的含水量,减小梗死体积,其作用机制可能与激活ER-α、抑制HIF-1α表达并抑制相关炎症因子TNF-α、IL-1β和IL-6的释放有关。

Objective To observe the neuroprotection of Puerarin on cerebral ischemia reperfusion injury and investigate its effect on the expression of estrogen receptor α（ER-α）, hypoxia inducible factor-1（HIF-1α） and related inflammatory cytokines.Methods Mice were randomly divided into a sham group, a solvent-control group and three Puerarin-treated groups with different doses（100, 250, 500 mg·kg^-1）. Middle cerebral artery occlusion（MCAO） model was made and then the infarct volume, water content and neurological scores were evaluated after 2 h ischemia and 24 h reperfusion. Western blot was used to determine the expression of ER-α and HIF-1α after 2 h, 6 h, 12 h, 24 h reperfusion respectively. Both Western blot and ELISA were used to determine the effect of Puerarin on the expression of ER-α, HIF-1αand related inflammatory cytokines such as tumor necrosis factor-α（TNF-α）, interleukin-1β（IL-1β） and interleukin-6（IL-6） in 2 h ischemia and 12 h reperfusion mice.Results Compared with solvent-control group, Puerarin reduced the infarct volume [low dose group（29.6±3.6）%, medium dose group（15.2±3.9）%, high dose group（8.2±2.1）% vs control group（39.3±5.0）%] and water content [low dose group（84.9±8.8）%, medium dose group（83.7±8.2）%, high dose group（80.9±8.7）% vs control group（85.3±10.2）%], which had significant difference. It also activated ER-α and suppressed HIF-1α. In addition, medium and high dose of Puerarin further inhibited TNF-α [（420.7± 27.2） μg·g^-1,（379.6±23.9） μg·g^-1], IL-1β [（211.8±19.2） μg·g^-1,（182.4±13.5） μg·g^-1] and IL-6 [（111.2±9.1） μg·g^-1,（104.1±12.4） μg·g^-1] respectively compared to solvent control group [TNF-α（505.8±36.7） μg·g^-1; IL-1β（291.6±21.8） μg·g^-1; IL-6（138.4±11.7） μg·g^-1] in 2 h ischemia and 12 h reperfusion mice.Conclusion Puerarin within certain range of doses could reduce the water content and infarct volume of cerebral ischemia reperfusion injury. Its mechanism might be linked to activation of ER-α, inhibition the expression of HIF-1α, and the inhibition of release of related inflammatory cytokines such as TNF-α, IL-1β and IL-6.