汉黄芩素调节Notch信号通路对脑缺血/再灌注模型小鼠神经细胞凋亡的影响

Impact of wogonin on neuronal apoptosis in a cerebral ischemia/reperfusion mouse model by regulating Notch signaling pathway

目的探讨汉黄芩素(Wogonin,WOG)通过调节Notch信号通路对脑缺血/再灌注模型(Ischemia/reperfusion,I/R)小鼠神经细胞凋亡的影响。方法采用改良线栓堵塞法建立I/R小鼠模型,建模成功后将小鼠随机分为假手术组(Sham组)、模型组(I/R组)、汉黄芩素低、中、高剂量组(WOG-L组、WOG-M组、WOG-H组),汉黄芩素高剂量+Notch信号通路抑制剂组[(WOG+3,5-二氟苯乙酰基)-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯(DAPT)组],每组各15只;采用神经功能缺损评分评价小鼠神经功能损伤,苏木素-伊红(Hematoxylin and eosin,HE)染色法、原位末端标记法(TDT mediated dutp nick end labeling,Tunel)观察小鼠脑组织海马CA1区病理情况及神经细胞凋亡情况,2,3,5-氯化三苯基四氮唑(2,3,5-Triphenyltetrazolium chloride,TTC)染色法计算小鼠脑梗死体积,酶联免疫吸附测定法(Enzyme-linked immunosorbent assay,ELISA)法检测海马组织中肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素-6(Interleukin 6,IL-6)水平,Western blot法检测半胱氨酸蛋白酶-3(Cysteinyl aspartate specific proteinase-3,Caspase-3)及Notch信号通路相关蛋白(受体Notch1、配体蛋白Jagged1、下游转录因子Hes1)表达水平。结果与Sham组比较,I/R组小鼠脑组织细胞损伤严重,神经功能缺损评分、脑梗死体积、神经细胞凋亡率、TNF-α及IL-6,Caspase-3,Notch1,Jagged1,Hes1蛋白表达水平显著升高(P>0.05);与I/R组比较,WOG-L组、WOG-M组、WOG-H组小鼠脑组织细胞损伤减轻,神经功能缺损评分、脑梗死体积、神经细胞凋亡率、TNF-α及IL-6,Caspase-3蛋白表达水平显著降低,Notch1,Jagged1,Hes1表达水平显著升高(P<0.05);与WOG-H组比较,WOG+DAPT组DAPT可部分逆转汉黄芩素对I/R组小鼠神经细胞的保护作用(P<0.05)。结论汉黄芩素可以减少脑缺血/再灌注小鼠神经细胞的凋亡,改善神经功能损伤,其作用机制可能与激活Notch信号通路有关。

Objective To investigate the impact of wogonin(WOG)on neuronal apoptosis in cerebral ischemia/reperfusion(I/R)mice by regulating Notch signaling pathway.Methods The I/R rat model was established by the modified thread plug method.After successful modeling,the mice were randomly grouped into Sham surgery group(Sham group),model group(I/R group),low,medium and high dose Wogonin groups(WOG-L group,WOG-M group,WOG-H group),and high dose Wogonin+Notch signaling pathway inhibitor group(WOG+DAPT group),with 15 mice in each group.The neurological deficit score was applied to evaluate neurological impairment in mice.Hematoxylin and eosin(HE)staining and terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(Tunel)staining were applied to observe the pathological changes and neuronal apoptosis in the CA1 region of the hippocampus in mice.The 2,3,5-triphenyltetrazolium chloride(TTC)staining method was applied to calculate the volume of cerebral infarction in mice.Enzyme-linked immunosorbent assay(ELISA)was applied to detect the levels of tumor necrosis factor-α(TNF-α)and interleukin 6(IL-6)in the hippocampus.Western blot method was applied to detect the expression of cysteinyl aspartate specific proteinase-3(Caspase-3)and Notch signaling pathway related proteins(receptor Notch1,ligand protein Jagged1,downstream transcription factor Hes1).Results Compared with the Sham group,the cells of the I/R group mice were severely damaged.The neurological deficit score,cerebral infarction volume,neuronal apoptosis rate,TNF-αand IL-6 levels,Caspase-3,Notch1,Jagged1,and Hes1 protein expression were obviously increased(P>0.05).Compared with the I/R group,the damage of cells in the WOG-L,WOG-M,and WOG-H groups was alleviated.And the neurological deficit score,cerebral infarction volume,neuronal apoptosis rate,TNF-αand IL-6 levels,and Caspase-3 protein expression were obviously reduced,while the expressions of Notch1,Jagged1,and Hes1 were obviously increased(P<0.05).Compared with the WOG-H group,the experimental results in the WOG+DAPT group showed that DAPT could partially reverse the protective effect of Wogonin on neural cells in the I/R group(P<0.05).Conclusion Wogonin can reduce the neuronal apoptosis and improve the neurological damage in mice with cerebral ischemia/reperfusion,and its mechanism may be related to the activation of Notch signaling pathway.