基于miRNA-219,NR2B,DISC1,CaMKⅡγ探讨温胆汤干预精神分裂症模型大鼠的分子机制

Molecular Mechanism of Wendantang Against Schizophrenia in Rats:An Exploration Based on miRNA-219,NR2B,DISC1 and CaMKⅡγ

目的:研究温胆汤对精神分裂症模型大鼠额叶组织miRNA-219,N-甲基-D-天冬氨酸受体2B(NR2B),精神分裂症断裂基因1(DISC1),钙离子/钙调素依赖性蛋白激酶Ⅱγ(CaMKⅡγ)表达的影响。方法:将60只大鼠随机分为6组,正常组、模型组、温胆汤高、中、低剂量组、氯氮平组,每组10只。温胆汤高、中、低剂量组分别灌胃温胆汤溶液40,20,10 g·kg^(-1),氯氮平组灌胃氯氮平0.02 g·kg^(-1),正常组和模型组灌胃等体积生理盐水,1次/d。给药21 d后,除正常组外,其余各组均进行左腹腔注射地卓西平马来酸盐(MK-801)0.6 mg·kg^(-1),建立急性精神分裂症模型。依照SAMS和HOFFMAN标准对大鼠的刻板行为和共济失调评分;苏木素-伊红(HE)染色观察大鼠前额叶皮层病理形态变化;蛋白免疫印迹法(Western blot)检测额叶组织NR2B,DISC1,CaMKⅡγ蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测额叶组织miRNA-219,NR2B,DISC1,CaMKⅡγmRNA表达。结果:与正常组比较,模型组大鼠刻板行为和共济失调评分显著升高(P<0.01);模型组前额叶皮层大部分神经元细胞核固缩或溶解;模型组额叶组织NR2B,DISC1,CaMKⅡγ蛋白表达水平显著降低(P<0.01);miRNA-219,NR2B,DISC1,CaMKⅡγmRNA表达水平显著降低(P<0.01);与模型组比较,温胆汤各组大鼠在50,60 min时的刻板行为和共济失调评分明显降低(P<0.05,P<0.01);温胆汤高、中剂量组前额叶皮层神经元细胞排列较为紧密,核固缩和溶解有不同程度减轻;温胆汤高、中剂量组额叶组织NR2B蛋白表达水平均显著升高(P<0.01),温胆汤中、低剂量组DISC1的蛋白表达水平均明显升高(P<0.05,P<0.01),温胆汤各组CaMKⅡγ蛋白表达水平明显升高(P<0.05);温胆汤各组额叶组织miRNA-219,NR2B,DISC1,CaMKⅡγmRNA表达水平明显升高(P<0.05,P<0.01)。结论:温胆汤可以通过改善精神分裂症模型大鼠的刻板行为和共济失调表现,减轻前额叶皮层神经元细胞核固缩和溶解程度,升高miRNA-219,NR2B,DISC1,CaMKⅡγ表达水平,从而达到改善精神分裂样症状和防治精神分裂症的目的。

Objective:To study the effects of Wendantang on the expression of miRNA-219,N-methylD-aspartate receptor 2 B(NR2B),disrupted in schizophrenia 1(DISC1),and Ca;/calmodulin-dependent protein kinase Ⅱγ(CaMKⅡγ)in the frontal lobe of rats with schizophrenia. Method: Sixty rats were randomly divided into six groups, namely normal group, model group, high-, medium-, and low-dose Wendantang groups,and clozapine group,with 10 rats in each group. Rats in high-,medium-,and low-dose Wendantang groups were intragastric with 40,20,and 10 g·kg^(-1) Wendantang,and the ones in clozapine group were intragastric with 0.02 g·kg^(-1) clozapine,those in normal and model group were intragastric with equal volume of normal saline,once a day. After 21 days of administration,rats in all groups except for the normal group were injected with 0.6 mg·kg^(-1) dizocilpine maleate(MK-801)into the left abdominal cavity for inducing acute schizophrenia. The stereotypic behavior and ataxia in rats were scored according to SAMS and HOFFMAN criteria. The morphological changes in the prefrontal cortex were observed by hematoxylin-eosin(HE)staining.The protein expression levels of NR2 B, DISC1 and CaMKⅡγ in the frontal lobe was detected by Western blot.The mRNA expression levels of miRNA-219 was detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR). Result: Compared with normal group,the model group exhibited significantly increased stereotypic behavior and ataxia scores(P<0.01),karyopyknosis and karyolysis in most neurons of the prefrontal cortex,and down-regulated NR2 B,DISC1,and CaMKⅡγ protein expression(P<0.01)and miRNA-219,NR2 B,DISC1,and CaMKⅡγ mRNA expression(P<0.01). Compared with model group,Wendantang high-,medium-,and low-doses group lowered the scores of stereotypic behavior and ataxia at 50,60 mmin(P<0.05,P<0.01). In high-and medium-dose Wendantang groups,the neurons in the prefrontal cortex were densely arranged. The karyopyknosis and karyolysis were alleviated to varying degrees. The NR2 B protein expression in the frontal lobe was up-regulated(P<0.01). In the medium-and low-dose Wendantang groups,the DISC1 protein expression in the frontal lobe was up-regulated(P<0.05,P<0.01). Wendantang at each dose significantly increased the CaMKⅡγ protein expression(P<0.05)and miRNA-219,NR2 B,DISC1,and CaMKⅡγ mRNA expression in the frontal lobe(P<0.05,P<0.01). Conclusion: Wendantang improves the scores of stereotypical behavior and ataxia,relieves the karyopyknosis and karyolysis of neurons in the prefrontal cortex,and increases the expression levels of miRNA-219,NR2B,DISC1,and CaMKⅡγ of rats with schizophrenia,so as to alleviate the schizophrenic-like symptoms and schizophrenia.