雷公藤红素对大鼠脑缺血后血管新生和神经保护作用的机制研究

Mechanism of celastrol on angiogenesis and neuroprotection in rats after cerebral ischemia

目的观察雷公藤红素(CSL)在脑缺血后对血管新生的作用及CSL在脑缺血后的神经保护作用。方法将80只SD大鼠随机分入24 h和7 d两大组,分别再分为假手术组,试剂对照组,低剂量CSL组(0.5 mg/kg)、中剂量组(2 mg/kg)、高剂量组(5 mg/kg),每组8只大鼠。除假手术组仅进行手术而不造成缺血状态外,其余各组均建立大鼠大脑中动脉闭塞模型。在缺血90 min再灌注30 min首次灌胃给药,每日一次给予相应剂量的CSL。对大鼠行为学进行评估,脑组织切片染色综合评价大鼠脑缺血再灌注损伤程度,检测脑组织炎症因子IL-1β的含量,观察细胞凋亡率的改变,采用免疫组化染色观察大脑皮层微血管密度改变,ELISA法检测VEGF和MMP-9的含量。结果 (1)与对应假手术组比较,各干预组各指标差异均有统计学意义(P<0.05)。(2)神经功能评分、梗死体积、细胞凋亡率和VEGF、MMP-9水平:与试剂对照组同时点比较,CSL中、高剂量组差异有统计学意义(P<0.05);CSL中、高剂量组同时点比较差异有统计学意义(P<0.05)。(3)CD31:与试剂对照组同时点比较,CLS中、高剂量组CD31阳性率减少(P<0.05);CLS中、高剂量组24 h与CLS中、高剂量组7 d比较,CD31阳性率差异有统计学意义(P<0.05)。(4)IL-1β:7 d干预时与试剂对照组比较,CSL低、中、高剂量组差异均有统计学意义(P<0.05);在24 h和7 d干预时,CSL高、中、低剂量组组间差异均有统计学意义(P<0.05)。结论一定剂量范围内,CSL在脑缺血再灌注急性期存在两种相反的作用,即通过抗炎和抗细胞凋亡产生神经保护作用和抑制血管新生作用,并且其抑制血管新生的作用随着时间的推移可能更加显著。在脑缺血急性期,CSL抑制炎性因子,减轻细胞凋亡的作用是占有主导地位的,这并不受其抑制血管新生作用的影响。

Objective To observe the effects of celastrol（CSL） on the angiogenesis after cerebral ischemia,and discuss its neuroprotection effects after cerebral ischemia. Methods 80 SD rats were randomly divided into the 24 h-treatment group and 7 d-treatment group,then sub-divided into the sham operation group,reagent control group,CSL groups with low-（0.5 mg/kg）,middle-（2 mg/kg） and high-dose（5 mg/kg）,8 rats in each group. The sham operation group was performed the operation but without the ischemia state. The rat model of middle cerebral artery occlusion was established in the other groups. The first-time intragastric administration was conducted after ischemia for 90 minutes and reperfusion for 30 minutes,and the rats were treated with CSL at corresponding dose once a day. The rat behavior was evaluated and the brain tissue section staining was used to evaluate the degree of cerebral ischemia reperfusion injury in rats. The IL-1β content in brain tissue was detected,and the change on cell apoptosis rate was observed. The microvessel density of cerebral cortex was observed by immunohistochemical staining,and the contents of vascular endothelial growth factor（VEGF） and matrix metalloproteinase-9（MMP-9） were detected by ELISA.Results（1）There were statistically significant differences on all indicators between the intervention group and the corresponding sham operation group（P〈0.05）.（2）For the comparison of neurological function score,infarct volume,apoptosis rate and VEGF and MMP-9 levels,at the same timepoint,there were statistically significant differences between the reagent control group and CSL groups with middle-and high-dose（P〈0.05）,and there were statistically significant differences between the middle-and high-dose group（P〈0.05）.（3）Compared with the reagent control group at the same timepoint,the positive rate of CD31 was decreased in the CSL groups with middle-and high-dose（P〈0.05）. For the comparison between 24 h-treatment group and 7 d-treatment group,there was statistically significant difference on the positive rate of CD31 in the CSL groups with middle-and high-dose（P〈0.05）.（4）In the 7 d-treatment groups,there was statistically significant difference on IL-1β level between the reagent control group and CSL groups with different doses（P〈0.05）. In the 24 h-treatment groups and 7 d-treatment groups,there was statistically significant difference on IL-1β level among the different doses groups（P〈0.05）. Conclusion Within certain dose,there are opposite effects of CSL on cerebral ischemia-reperfusion in acute period,that is the neuroprotection and the inhibition on angiogenesis through anti-inflammation and anti-apoptosis. And its inhibitory effect on angiogenesis may be more significant over time. In the acute period of cerebral ischemia,the effects of CSL in inhibiting inflammatory factors and alleviating cell apoptosis are dominant,which is not affected by its inhibition on angiogenesis.