大鼠局灶性脑缺血再灌注后LPA1的表达对神经元凋亡的影响

目的 探讨大鼠局灶性脑缺血再灌注后溶血磷脂酸受体1(LPA1)的表达对神经元凋亡的影响及其可能机制。方法 将24只SPF级SD雄性大鼠随机分为4组,每组各6只,分别为假手术组(A组)、大脑中动脉栓塞(MCAO)组(B组)、MCAO+溶剂组(C组)、MCAO+LPA1拮抗剂(Ki16425)组(D组); 4组均于手术后48 h取标本; 利用HE染色观察大鼠脑组织细胞形态的变化; 四氮唑红(TTC)染色观察大鼠脑梗死面积; 免疫荧光技术检测LPA1在大鼠皮层半暗带神经元表达水平; 免疫印迹、免疫组化技术检测大鼠脑组织中Caspase-3蛋白及p-Akt蛋白表达水平。结果 与A组比较,B组有明显的缺血再灌注损伤,表现为细胞肿胀,细胞溶解坏死,大鼠皮层半暗带神经元的LPA1表达水平较高; 与C组比较,D组大鼠脑梗死面积显著增大(P<0.05),缺血半暗带细胞肿胀更加明显,胞浆空泡区增大,细胞核固缩更加严重,细胞间隙增宽更明显; D组较C组缺血半暗带Caspase-3蛋白表达水平明显升高(P<0.05),而p-Akt蛋白表达水平明显降低(P<0.05)。结论 抑制大鼠局灶性脑缺血再灌后LPA1的表达可使大鼠脑梗死面积增大,细胞凋亡增加,同时p-Akt蛋白表达减少,这说明在大鼠局灶性缺血再灌注过程中LPA1对神经元具有保护作用,其机制可能是通过Akt途径来发挥保护作用的。

The effects of LPA1 expression level on neuron apoptosis in rats after focal cerebral ischemia and reperfusion

ObjectiveTo explore the effects of LPA1 expression level on cortical neuron apoptosis in ischemic penumbra after focal cerebral ischemia and reperfusion in rats and its possible mechanism.Methods 24 SD rats were randomly divided into four groups,namely,the sham group(group A),the right middle cerebral artery occlusion(MCAO)group(group B),MCAO +vehicle group(group C),MCAO+LPA1 antagonist(Ki16425)group(group D).All rats in four groups were taken brain tissue at the second day after surgery,the morphological changes of rats were measured by using H&E staining; the infarction area was measured by TTC staining; LPA1 expression level in neurons was located by immunofluorescence of Nuen and LPA1; the expression levels of caspase-3 and p-Akt were detected by Western blotting and immunohistochemical technology.Results Compared with group A,nerve cells were more obviously swelling,cytoplasm vacuoles area was increased,and the gap between cells was widened obviously,the expression level of LPA₁ protein in rat cortex penumbra neurons was significantly higher in group B; compared with group C,the cerebral infarction area was significantly larger(P<0.05),nerve cells were more obviously swelling,cytoplasm vacuoles area was increased,and the gap between cells was widened obviously in the penumbra of group D; Western blotting and immunofluorescence showed that,compared with group C,the expression level of caspase-3 protein in the penumbra of group D was significantly increased(P<0.05),and the expression level of p-Akt protein was significantly decreased(P<0.05).Conclusion After administration of an LPA1 antagonist,the cerebral infarction area increased,brain tissue edema and neuronal apoptosis were more serious,the expression level of Caspase-3 protein increased in the penumbra,and the expression level of p-akt protein decreased,all above evidence conversely proved that LPA1 had a protective effect on ischemic neurons and it might play a protective role through the Akt pathway.