PAR1对蛛网膜下腔出血大鼠神经细胞自噬、神经肽及BDNF水平的影响

Effects of PAR-1 on the autophagy, neuropeptide and BDNF of the nerve cells in rats with subarachnoid hemorrhage

目的探讨蛋白酶激活受体1(Protease-activated receptors 1,PAR1)对蛛网膜下腔出血(Subarachnoid hemorrhage,SAH)大鼠神经细胞自噬、神经肽及脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)水平的影响。方法Sham组(假手术组)、SAH组(SAH模型大鼠10只)、BDNF组(SAH模型大鼠给予BDNF干预)、PAR1抑制剂组(SAH模型大鼠给予PAR1抑制剂干预)和联合组(SAH模型大鼠给予BDNF联合PAR1抑制剂干预)均9只;采用放射免疫法检测血液及脑脊液中神经肽γ(Neuropeptide Y,NPY)水平,苏木精-伊红(Hematoxylin eosin,HE)染色检测脑皮质组织形态,免疫印迹检测自噬蛋白表达水平,缺口末端标记技术(Terminal deoxynucleotidyl transferase-mediated nick end labeling,TUNEL)检测细胞凋亡率,免疫组化检测BDNF及酪氨酸激酶受体B(Tyrosine Kinase receptor B,TrKB)蛋白表达阳性细胞数。结果Sham组大鼠脑组织结构未见异常,染色区细胞浆及细胞核染色清晰,无神经元缺失;SAH组大鼠于结构松散的软化灶内存在大量炎性细胞,存在神经细胞坏死;BDNF组、PAR1抑制剂组和联合组脑组织病理学变化均改善,神经元排列规则及数目较多。与Sham组比较,SAH组大鼠血液及脑脊液中NPY水平、脑皮质中自噬相关基因5(Autophagy-related gene 5,Atg5)、微管相关蛋白1轻链3(Microtuble-associated protein 1 light chain 3,LC3)-Ⅱ蛋白表达水平及脑细胞凋亡率升高(P<0.05),泛素结合蛋白P62(P62)蛋白表达水平及BDNF,TrkB阳性细胞数降低(P<0.05);与SAH组比较,BDNF组、PAR1抑制剂组大鼠血液及脑脊液NPY水平、脑皮质中Atg5,LC3-Ⅱ蛋白表达水平及脑细胞凋亡率降低(P<0.05),p62蛋白表达水平及BDNF,TrkB阳性细胞数升高(P<0.05),且联合组效果更为显著,与BDNF组、PAR1抑制剂组比较差异显著(P<0.05);BDNF组、PAR1抑制剂组上述各项指标水平比较均无显著差异(P>0.05)。结论PAR1抑制剂SCH79797通过抑制血液及脑脊液中NPY表达,降低自噬蛋白Atg5及LC3-Ⅱ活性,减少蛛网膜下腔出血大鼠脑细胞凋亡,其机制可能与上调BDNF及TrkB表达水平有关。

Objective To investigate the effects of PAR-1 on autophagy, neuropeptide levels and BDNF of nerve cells in rats with subarachnoid hemorrhage. Methods Forty-five rats were randomly divided into four groups: the Sham group(sham operation group), SAH group(SAH model rats), BDNF group(SAH model rats given BDNF intervention), PAR1 inhibitor group(SAH model rats given PAR1 inhibitor intervention) and combination group( SAH model rats were treated with BDNF combined with PAR1 inhibitor intervention), 9 in each group. Radioimmunoassay was used to detect the level of NPY in blood and cerebrospinal fluid. HE staining was used to detect the morphology of cerebral cortex. Western blotting was used to detect autophagy protein. TUNEL method was used to detect the apoptosis rate of brain cells. Immunohistochemical method was used to quantify the numbers of BDNF and TrKB positive neurons. Results There was no abnormality in the structure of the brain tissue of rats in the Sham group. The cytoplasm and nucleus of the staining area were clearly stained, and there was no loss of neuronal cells. In the SAH group, there were a large number of inflammatory cells and nerve cell necrosis in loosely structured softened foci. The pathology of brain tissue in the BDNF group, PAR1 inhibitor group and the combination group was improved, and the number of neurons increased. Compared with the Sham group, the levels of NPY in the blood and cerebrospinal fluid, Atg5 and LC3-Ⅱ in the cerebral cortex of rats, and the brain cell apoptosis rate in the SAH group increased respectively(P<0.05), and the expression of p62 protein and the number of BDNF and TrkB positive neurons decreased(P<0.05). Compared with SAH group, the expression levels of NPY in blood and cerebrospinal fluid, Atg5 and LC3-Ⅱ protein in cerebral cortex and brain cell apoptosis rate decreased in the BDNF group and PAR1 inhibitor group(P<0.05), with the increased level of p62 protein and increased numbers of BDNF and TrkB positive neurons(P<0.05), meanwhile, the effect of the combination group was much more significant than that of the BDNF group and the PAR1 inhibitor group(P<0.05). There was no significant difference among the above indicators between the BDNF group and the PAR1 inhibitor group(P>0.05). Conclusion PAR1 inhibitor SCH79797 reduced the activity of autophagy protein Atg5 and LC3-Ⅱ by inhibiting the expression of NPY in blood and cerebrospinal fluid, thus improved brain cell apoptosis in subarachnoid hemorrhage, which is related to up-regulation of BDNF and TrkB expression levels.