百事乐加味方含药血清调控CMPK2/NR信号改善卒中后抑郁前扣带皮层谷氨酸能神经元突触损伤的机制研究

Mechanism of Modified Baishile Decoction to alleviate the synaptic damage of anterior cingulate cortex glutaminergic neuron via regulating CMPK2/NR signal in post-stroke depression

关键词:  百事乐加味方  卒中后抑郁  CMPK2/NR信号  前扣带皮层谷氨酸能神经元  突触损伤

Key words:  Modified Baishile Decoction  post-stroke depression  CMPK2/NR signal  anterior cingulate cortex glutaminergic neuron  synaptic damage

摘要:

目的 揭示百事乐加味方（BSL）通过调控胞苷/尿苷单磷酸激酶2（cytidine/uridine monophosphate kinase 2，CMPK2）/谷氨酸N-甲基-D-天冬氨酸受体（N-methyl-D-aspartate receptor，NR）改善卒中后抑郁（post-stroke depression，PSD）前扣带皮层（anterior cingulate cortex，AAC）谷氨酸能神经元突触损伤的保护作用及分子机制。方法 首先原代分离、培养SD大鼠前扣带皮层小胶质细胞和神经元，然后以氧糖剥夺联合脂多糖（lipopolysaccharide，LPS）和皮质酮干预造模6h，复制模拟PSD环境下的体外细胞模型，实验设正常组、模型组、CMPK2激活剂组、CMPK2抑制剂组、阳性药(阿司匹林+氟西汀)组、百事乐加味方组、百事乐加味方联合激活剂组、百事乐加味方联合抑制剂组。采用细胞成像分析观察小胶质细胞和前扣带皮层神经元形态结构；采用CCK8法检测前扣带皮层神经元细胞存活率；采用Calcein-AM/PI染色观察前扣带皮层神经元活死细胞情况；采用尼氏染色观察前扣带皮层神经元突触损伤情况；采用酶联免疫吸附法检测细胞上清液中五羟色胺（serotonin，5-HT）、多巴胺（dopamine，DA）含量；采用免疫荧光染色观察小胶质细胞中CMPK2、离子钙结合适配器分子1（ion calcium binding adaptor molecule 1，IBA1）及前扣带皮层谷氨酸能神经元中谷氨酸受体2A（glutamate receptor 2A，GluN2A）、谷氨酸受体2B（glutamate receptor 2B，GluN2B）、突触素1（synaptophysin 1，SYN1）与突触后密度蛋白95（postsynaptic density protein 95，PSD95）蛋白表达水平；采用Western blot法检测小胶质细胞中CMPK2与前扣带皮层神经元中GluN2A、GluN2B、SYN1与PSD95蛋白表达情况。结果 百事乐加味方含药血清能有效改善小胶质细胞和前扣带皮层神经元形态结构受损，抑制小胶质细胞异常活化，增加前扣带皮层神经元细胞存活，并提升单胺神经递质5-HT、DA水平，进而缓解PSD前扣带皮层神经元突触损伤。进一步的机制研究发现，CMPK2激活剂和抑制剂分别加重和减轻上述异常改变，百事乐加味方含药血清通过下调小胶质细胞CMPK2信号从而抑制前扣带皮层谷氨酸能神经元中突触相关蛋白GluN2A、GluN2B表达和促进突触前膜蛋白SYN1、突触后膜蛋白PSD95表达。有趣的是，百事乐加味方含药血清还能显著逆转CMPK2激活剂LOS所致的上述蛋白表达异常升高或降低，最终修复PSD前扣带皮层谷氨酸能神经元突触损伤。结论 百事乐加味方含药血清能有效促进细胞存活，提升单胺神经递质5-HT、DA水平，进而改善前扣带皮层谷氨酸能神经元突触损伤，其分子机制可能与调控异常的CMPK2/NR信号有关。

Abstract:

Objective This study aims to reveal the mechanism of Modified Baishile Decoction to alleviate the synaptic damage of anterior cingulate cortex(ACC) glutaminergic neuron via regulating cytidine/uridine monophosphate kinase 2(CMPK2) /N-methyl-D-aspartate receptor(NR) signal in post-stroke depression. Methods Firstly, microglia and anterior cingulate cortex neuron from SD rats were primitively isolated and cultured. In vitro cell models simulating the damage environment of PSD was established by oxygen-glucose deprivation(OGD) combined with lipopolysaccharide and corticosterone intervention for 6 hours. The cultured cells were randomly divided into normal, model, CMPK2 agonist, CMPK2 blocker, positive(aspirin+fluoxetine),Modified Baishile Decoction, Modified Baishile Decoction combined with CMPK2 agonist and Modified Baishile Decoction combined with CMPK2 blocker group. Morphology and structure of microglia and ACC neuron were observed by cell imaging analysis. The survival rate of ACC neuron was detected by the CCK8. Calcein-AM/PI staining was used to observe the dead/alive cell ratio of ACC neuron. Nissl's staining was used to observe synaptic damage of ACC neuron. Enzyme-linked immunosorbent assay was used to measure the levels of 5-hydroxytryptamine (5-HT) and dopamine (DA) in the cell supernatant. The protein expressions of CMPK2 and ion calcium binding adaptor molecule 1 (IBA1) in microglia, and glutamate receptor 2A (GluN2A), glutamate receptor 2B (GluN2B), synaptophysin 1 (SYN1) and postsynaptic density protein 95 (PSD95) in ACC glutaminergic neurons were detected by immunofluorescence. Western blot was used to detected the protein expression of CMPK2 in microglia and GluN2A, GluN2B, SYN1 and PSD95 inACC neuron. Results Our results indicated that drug-containing serum of Modified Baishile Decoction could effectively improve the morphological and structural damage of microglia and ACC neuron, inhibit the abnormal activation of microglia, increase the cell survival of ACC neurons, and enhance the levels of monoamine neurotransmitters 5-HT and DA, and ultimately alleviate the synaptic damage of ACC neurons in PSD. Further mechanistic research reveals that CMPK2 activators and inhibitors respectively aggravate and alleviate the above-mentioned abnormal changes. Additionally, the drug-containing serum of Modified Baishile Decoction could inhibit the expression of synaptic-related proteins GluN2A and GluN2B in ACC glutaminergic neuron and promote the expression of presynaptic membrane protein SYN1 and postsynaptic membrane protein PSD95 via down-regulating the CMPK2 signal in microglia. Interestingly, the drug-containing serum of Modified Baishile Decoction could also significantly reverse the abnormal increase or decrease in the expression of the above-mentioned proteins triggered by the CMPK2 activator LOS, and ultimately repair the synaptic damage of ACC glutaminergic neuron of PSD. Conclusion This research confirms that Modified Baishile Decoction colud effectively promote cell survival, increase the levels of monoamine neurotransmitters 5-HT and DA, and alleviate the synaptic injury of ACC glutaminergic neuron, the molecular mechanism of which might be related to the regulation of abnormal CMPK2/NR signal.