小豆蔻明调节YAP/TAZ信号通路对脑梗死大鼠模型血脑屏障的影响

Effects of cardamomine on blood-brain barrier in rat model of cerebral infarction by regulating YAP/TAZ signaling pathway

目的 探讨小豆蔻明(Cardamonin, CAR)对脑梗死大鼠模型血脑屏障(Blood-brain barrier, BBB)及Hippo-p-Yes相关蛋白(Yes associated protein, YAP)/转录共激活因子(Tafazzin, TAZ)信号通路的影响。方法 建立脑梗死大鼠模型,所有大鼠分为对照组[Control(CT)组]、[模型组(Model(M)组]、CAR低剂量组(CAR L组,5 mg·kg^(-1)·d^(-1)CAR)、CAR高剂量组(CAR H组,20 mg·kg^(-1)·d^(-1)CAR)和CAR高剂量+PY60组(CAR H+PY60组,20 mg·kg^(-1)·d^(-1)CAR+10 mg/mL PY60);给药期间各组大鼠做神经功能评分;给药结束后苏木精-伊红(Hematoxylin-eosin, HE)染色观察脑组织病理变化,氯化三苯基四氮唑(Triphenyl-tetrazolium chloride, TCC)染色测定脑梗死体积,免疫组化检测脑组织闭合蛋白(Occludin)、紧密连接蛋白(Claudin-5)的表达水平,计算脑含水量和伊文思蓝(Evans blue, EB)渗透量,Western blot技术验证脑组织磷酸化(Phosphorylation, p)-哺乳动物STE20样蛋白激酶1(Mammalian sterile20-like kinase1,MST1)、MST1、p-大肿瘤抑制因子1(Large tumor suppressor factor 1,LATS1)、LATS1,p-YAP,YAP蛋白表达水平。结果 与CT组比较,M组大鼠脑组织细胞间隙变大且有大量炎症细胞浸润及少量空泡变性,神经功能评分、脑梗死体积、脑含水量、EB渗透量及p-YAP/YAP蛋白表达水平升高,Occludin, Claudin-5,p-MST1/MST1,p-LATS1/LATS1蛋白表达水平降低(P<0.05);与M组比较,CAR L,H组大鼠炎症细胞浸润、细胞空泡变性、细胞间隙减少,脑组织结构更完整,神经功能评分、脑梗死体积、脑含水量、EB渗透量及p-YAP/YAP蛋白表达水平降低,Occludin, Claudin-5,p-MST1/MST1,p-LATS1/LATS1蛋白表达水平升高(P<0.05);与CAR H组比较,CAR H+PY60组大鼠炎症细胞浸润和细胞空泡变性增加,神经功能评分、脑梗死体积、脑含水量、EB渗透量及p-YAP/YAP蛋白表达水平升高,Occludin, Claudin-5,p-MST1/MST1,p-LATS1/LATS1蛋白表达水平降低(P<0.05)。结论 CAR可能通过抑制YAP/TAZ信号通路来改善脑梗死大鼠模型BBB功能。

Objective To investigate the effects of cardamonin(CAR) on the blood-brain barrier(BBB) and the Hippo/p-Yes-associated protein(YAP)/transcription coactivator(TAZ) signaling pathway in a rat model of cerebral infarction. Methods A rat model of cerebral infarction was established, and all rats were divided into a control group(CT group), a model group(M group), a low-dose CAR group(CAR-L group, 5 mg/kg/d CAR), a high-dose CAR group(CAR-H group, 20 mg/kg/d CAR), and a high-dose CAR+PY60 group(CAR-H+PY60 group, 20 mg/kg/d CAR+10 mg/m PY60);during the administration period, neurological function scores were evaluated for each group. After administration, HE staining was applied to observe pathological changes in brain tissue, triphenyltetrazolium chloride(TCC) staining was used to determine the volume of cerebral infarction, and immunohistochemistry was used to detect the expression levels of Occludin and Claudin-5 in brain tissue. The brain water content and Evans blue(EB) penetration were calculated. Western blotting was used to verify the expression levels of the brain tissue phosphorylation(p) proteins, mammalian STE20-like protein kinase 1(MST1), MST1, p-large tumor suppressor factor 1(LATS1), LATS1, p-YAP, and YAP. Results Compared with those in the CT group, the intercellular spaces in the brain tissue of rats in the M group were greater, with a large amount of inflammatory cell infiltration and a small amount of vacuolar degeneration. The neurological function score, cerebral infarction volume, brain water content, EB permeability, and p-YAP/YAP protein expression level increased, and the protein expression levels of Occludin, Claudin-5, p-MST1/MST1, and p-LATS1/LATS1 decreased(P<0.05). Compared with those in the M group, the infiltration of inflammatory cells, cell vacuolar degeneration, and intercellular spaces in the CARL and H groups were lower, the brain tissue structure was more complete, the neurological function score, cerebral infarction volume, brain water content, EB permeability, and p-YAP/YAP protein expression levels were lower, and the protein expression levels of Occludin, Claudin-5, p-MST1/MST1, and p-LATS1/LATS1 were greater(P<0.05). Compared with those in the CAR H group, the infiltration of inflammatory cells and cellular vacuolar degeneration in the CAR H+PY60 group were greater, the neurological function score, cerebral infarction volume, brain water content, EB permeability, and p-YAP/YAP protein expression were greater, and the expression of Occludin, Claudin-5, p-MST1/MST1, and p-LATS1/LATS1 proteins was lower(P<0.05). Conclusion CAR may improve BBB function in a rat model of cerebral infarction by inhibiting the YAP/TAZ signaling pathway.