荭草苷对脑缺血再灌大鼠神经保护作用及其机制

Neuroprotective effect and mechanism of orientin on cerebral ischemia-reperfusion injury in rats

目的探讨荭草苷对大鼠脑缺血再灌注损伤保护作用机制。方法按照体重将大鼠随机分为5组:假手术组、模型组、中药组、中药合用JNK抑制剂(SP600125)组和中药合用ERK抑制剂(AZD6244)组,每组15只。除假手术组以外,其余大鼠用线栓法制备大鼠局部性脑中动脉阻断模型。术后0,12 h,中药组、中药合用JNK抑制剂组和中药合用ERK抑制剂组均腹腔注射荭草苷溶液2. 9 mg·m L-1;在术前0. 5和24 h,中药合用JNK抑制剂组给予SP600125溶液2. 0mg·m L-1,中药合用ERK抑制剂组给予AZD6244溶液2. 0 mg·m L-1。脑缺血再灌注24h,以蛋白质免疫印迹法检测脑缺血区自噬相关蛋白Beclin1、微管相关蛋白轻链3(LC3)、磷酸化的细胞外调节蛋白激酶(p-ERK)和c-Jun氨基末端激酶(p-JNK)蛋白表达水平。结果假手术组、模型组、中药组和中药合用JNK抑制剂组的自噬相关蛋白Beclin1的相对表达量分别为1. 00±0. 08,3. 15±0. 13,1. 58±0. 11和0. 92±0. 07;这4组的自噬相关蛋白LC3蛋白相对表达量分别为1. 00±0. 05,3. 02±0. 12,1. 38±0. 10和0. 83±0. 07。假手术组、模型组、中药组和中药合用ERK抑制剂组的自噬相关蛋白Beclin1相对表达量为1. 00±0. 07,2. 98±0. 15,1. 95±0. 13和1. 35±0. 10;这4组的LC3蛋白相对表达量分别为1. 00±0. 04,3. 01±0. 14,1. 59±0. 15和1. 20±0. 12。模型组与假手术组相比,上述指标的差异均有统计学意义(均P <0. 01);中药组和中药合用抑制剂组与模型组相比,差异均有统计学意义(P <0. 05,P <0. 01);中药合用JNK抑制剂组与中药组相比,上述指标的差异均有统计学意义(均P <0. 05);中药合用ERK抑制剂组与中药组相比,Beclin1表达差异有统计学意义(P <0. 05)。假手术组、模型组、中药组和中药合用JNK抑制剂组的p-JNK相对表达量分别为1. 00±0. 02,2. 95±0. 13,1. 52±0. 07和1. 29±0. 04;假手术组、模型组、中药组和中药合用ERK抑制剂组的p-ERK相对表达量分别为1. 00±0. 03,2. 50±0. 13,1. 17±0. 11和0. 79±0. 07;模型组与假手术组相比,差异均有统计学意义(均P <0. 01);中药合用抑制剂组和中药组与模型组相比,差异均有统计学意义(均P <0. 05);中药合用ERK抑制剂组与中药组相比,差异有统计学意义(P <0. 05)。结论通过调控JNK/ERK通路,可降低自噬蛋白表达,抑制自噬水平,推测JNK/ERK通路可能参与荭草苷对大鼠脑缺血再灌注损伤保护作用机制。

Objective To explore protective effect and mechanism of orientin on cerebral ischemia-reperfusion injury in rats.Methods SD rats were randomly divided into five groups:sham group,model group,Chinese medicine(CM)group,CM combined with JNK inhibitor(SP600125)group and CM combined with ERK inhibitor(AZD6244)group,with 15 rats in each group.Middle cerebral artery occlusion model was established by suture method in each group except sham group.The 2.9 mg·m L-1 of orientin was administrated at 0 h and 12 h of reperfusion in CM group and two inhibitor groups.At 0.5,24 h before the operation,SP600125 2.0 mg·m L-1 and AZD6244 2.0 mg·m L-1 were administrated in CM combined with JNK inhibitor group and CM combined with ERK inhibitor group,respectively.The protein levels of Beclin1,microtubule-associated protein1 light chain3(LC3),phosphorylated c-Jun N-terminal kinase(p-JNK)and phosphorylated extracellular signal-regulated kinase(p-ERK)were determined by protein immunoblotting.Results The expression of autophagy protein Beclin1 using JNK inhibitor in sham group,model group,CM group,CM combined with JNK group were 1.00±0.08,3.15±0.13,1.58±0.11,0.92±0.07,respectively;the protein expression of LC3 in the four groups were 1.00±0.05,3.02±0.12,1.38±0.10,0.83±0.07,respectively.The expression of autophagy protein Beclin1 using ERK inhibitor in sham group,model group,CM group,CM combined with ERK group were 1.00±0.07,2.98±0.15,1.95±0.13,1.35±0.10,respectively;the protein expression of LC3 in the four groups were 1.00±0.04,3.01±0.14,1.59±0.15,1.20±0.12,respectively.Comparing between sham group and model group,the differences of the factors were statistically significant(all P<0.01);comparing between three drug groups and model group,the differences of the factors were statistically significant(all P<0.05);comparing between CM group and JNK inhibitor group,the differences of the factors were statistically significant(all P<0.05);comparing between CM group and ERK inhibitor group,the difference of the Beclin1 protein expression was statistically significant(all P<0.05).The expression of p-JNK in sham group,model group,CM group and CM combined with JNK inhibitor group were 1.00±0.02,2.95±0.13,1.52±0.07,1.29±0.04,respectively;the protein expression of p-ERK in sham group,model group,CM group and CM combined with ERK inhibitor group were 1.00±0.03,2.50±0.13,1.17±0.11,0.79±0.07.Comparing between model group and sham group,the differences of the factors were statistically significant(all P<0.01);comparing between three drug groups and model group,the differences of the factors were statistically significant(all P<0.05);comparing between CM group and ERK inhibitor group,the difference of the p-ERK protein expression was statistically significant(P<0.05).Conclusion By regulating JNK/ERK pathway,autophagic protein expression can be reduced and autophagic level can be inhibited.It is speculated that JNK/ERK pathway might be involved in the protective mechanism of orientin against cerebral ischemia-reperfusion injury in rats.