脑出血后血红蛋白对大鼠脑组织Shp2表达及血脑屏障黏着连接的影响

Effect of hemoglobin on Shp2 expression in brain tissues and adherens junction in blood brain barrier in rats after intracerebral hemorrhage

目的探讨脑出血后血红蛋白对脑组织蛋白酪氨酸磷酸酶Shp2表达及血脑屏障黏着连接的影响。方法将80只雄性SD大鼠按随机数字表法分为假手术组和脑出血6 h、24 h、3 d、7d组,每组16只,其中后4组大鼠于脑内注射20μL血红蛋白制备成脑出血模型。采用Longa评分法对各组大鼠进行神经功能评估,采用干湿重法检测脑组织含水量和脑系数,采用实时荧光定量逆转录-聚合酶链式反应(qRT-PCR)检测Shp2 mRNA的表达,采用免疫组化染色检测Shp2阳性表达情况,采用Western blotting检测Shp2及黏着连接蛋白α-连环蛋白、β-连环蛋白、血管内皮钙黏蛋白、磷酸化α-连环蛋白、磷酸化β-连环蛋白、磷酸化血管内皮钙黏蛋白的表达。结果与假手术组比较,脑出血6h、24h、3d、7d组大鼠的Longa评分均明显升高,差异均有统计学意义(P<0.05)。与假手术组比较,脑出血24h、3d、7d组大鼠的脑组织含水量和脑系数均明显升高,Shp2 mRNA、免疫组化染色阳性表达及蛋白表达均明显降低,磷酸化α-连环蛋白、磷酸化β-连环蛋白及磷酸化血管内皮钙黏蛋白表达均明显升高,差异均有统计学意义(P<0.05)。结论脑出血后血红蛋白可下调脑组织中Shp2的表达并促进黏着连接蛋白磷酸化,诱导黏着连接破坏,从而导致血脑屏障破坏和脑水肿形成。

Objective To investigate the effect of hemoglobin on Shp2 expression in brain tissues and adherens junction in blood brain barrier in rats after cerebral hemorrhage.Methods Eighty male SD rats were randomly allocated into sham-operated group and groups of cerebral hemorrhage for 6 h,24 h,3 d,and 7 d(n=16).Rat models in the groups of cerebral hemorrhage were established by intracerebral injection of 20μL of hemoglobin.Six h,24 h,3 d,and 7 d after injection,neurological functions were assessed by Longa scale;brain water content and brain organ coefficient in the harvested cerebral tissues were calculated by wet-dry weighting method;Shp2 mRNA expression was analyzed by real-time fluorescence quantification reverse transcription PCR(qRT-PCR);Shp2 positive neurons was detected by immunohistochemistry;the protein expressions of Shp2,α-catenin,β-catenin and vascular endothelial(VE)-cadherin,and phosphorylated(p-)α-catenin,p-β-catenin and p-VE-cadherin were explored by Western blotting.Results As compared with those in the sham-operated group,Longa scale scores in groups of intracerebral hemorrhage for 6 h,24 h,3 d and 7 d were significantly increased(P<0.05).As compared with the sham-operated group,groups of intracerebral hemorrhage for 24 h,3 d and 7 d had significantly increased brain water content and brain organ coefficient,statistically reduced Shp2 mRNA expression,statistically samller amount of Shp2 positive neurons,and statistically reduced Shp2 protein expression,and significantly increased protein expressions of p-α-catenin,p-β-catenin and p-VE-cadherin(P<0.05).Conclusion Intracerebral injection of hemoglobin can downregulate Shp2 expression and promote phosphorylation of adherens junction proteins,therefore induce the disruption of adherens junction,which might be a critical mechanism of blood-brain barrier disruption and brain edema.