小胶质细胞活化激活糖酵解PFKFB3/HMGB1通路对大鼠血管性认知障碍的影响

Effect of microglia activation of glycolytic PFKFB3/HMGB1 pathway on vascular cognitive impairment in rats

目的探讨小胶质细胞活化激活糖酵解PFKFB3/HMGB1通路对大鼠血管性认知障碍的影响。方法48只Sprague-Dawley雄性大鼠采用永久性双侧颈总动脉结扎法建立血管性认知障碍(VCI)模型,并采用完全随机方法分为假手术组(Sham组)、VCI组、VCI+PFKFB3抑制剂3PO处理组(VCI+3PO组)、VCI+糖酵解激动剂二甲氧乙二酰甘氨酸(DMOG)处理组(VCI+DMOG组)4组,每组12只。采用条件性恐惧实验评估大鼠的认知功能;采用酶联免疫吸附试验(ELISA)法检测血清神经丝轻链蛋白(NFL)、S100钙结合蛋白β(S100-β)和海马组织中乳酸、趋化因子配体2(CCL2)、白细胞介素6(IL-6)及高迁移率蛋白1(HMGB1)的表达量;采用蛋白质免疫印迹(WB)法检测离子钙结合适配器分子1(Iba1)、PFKFB3、AMP激活蛋白激酶α(AMPKα)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)蛋白的表达情况;通过苏木素-伊红(HE)染色法评估海马神经元的变性情况,TUNEL染色法评估海马神经元的凋亡情况。结果4组大鼠的认知功能,血清中NFL、S100-β及海马组织中HMGB1、乳酸、CCL2、IL-6、Iba1、PFKFB3、AMPKα、NLRP3、Caspase-3的表达量,神经元的变性、凋亡率,差异均有统计学意义(均P<0.05)。与Sham组相比,VCI组大鼠的认知功能低下,NFL、S100-β、乳酸、CCL2、HMGB1、PFKFB3、Iba1、NLRP3、Caspase-3表达升高,AMPKα表达降低,神经元变性、凋亡率增加,差异均有统计学意义(均P<0.05)。与VCI组相比,VCI+3PO组认知功能改善而VCI+DMOG组加重;VCI+3PO组NFL、S100-β、乳酸、CCL2、HMGB1表达降低,而VCI+DMOG组升高;VCI+3PO组Iba1、PFKFB3、NLRP3、Caspase-3表达降低,而VCI+DMOG组Iba1、PFKFB3、NLRP3、Caspase-3表达升高,AMPKα表达降低;VCI+3PO组神经元变性、凋亡率降低,而VCI+DMOG组增加;差异均有统计学意义(均P<0.05)。结论小胶质细胞活化激活糖酵解关键酶PFKFB3增强可上调炎性小体NLRP3的表达并诱导中枢炎性反应,导致神经元损伤、变性和凋亡,加重大鼠VCI,其机制可能与海马组织中促炎因子HMGB1的增多有关。

Objective To evaluate the effect of PFKFB3/HMGB1 signal pathway activated by glycolysis of microglia activation on vascular cognitive impairment(VCI)in rats.Methods The model of vascular cognitive impairment(VCI)was established by permanent bilateral common carotid artery occlusion in 48 Sprague-Dawley male rats.The rats were randomly divided into 4 groups:sham group,VCI group,VCI+PFKFB3 inhibitor 3PO group(VCI+3PO group)and VCI+glycolytic agonist DMOG group(VCI+DMOG group)with 12 rats in each group.The cognitive function of rats was evaluated by conditioned fear test.The expressions of neurofilament light chain protein(NFL),S100 calcium binding protein-β(S100-β),lactic acid(Lac),chemokine ligand 2(CCL2),interleukin-6(IL-6)and high mobility group box protein 1(HMGB1)in serum and hippocampus were detected by enzyme-linked immunosorbent assay(ELISA).Western blot(WB)was used to detect the expression of ionized calcium binding adapter molecule 1(Iba1),PFKFB3,AMP-activated protein kinaseα(AMPKα),NOD-like receptor thermoprotein domain associated protein 3(NLRP3)and Caspase-3 protein.The degeneration of hippocampal neurons was evaluated by hematoxylin-eosin(HE)staining,and the apoptosis of hippocampal neurons was evaluated by TUNEL staining.Results The cognitive function and the expression of NFL,S100-βin blood,the expression of HMGB1,Lac,CCL2,IL-6,Iba1,PFKFB3,AMPKα,NLRP3 and Caspase-3 in hippocampal tissues,neuronal degeneration and apoptosis among the 4 groups showed significant differences(all P<0.05).Compared with the sham group,the cognitive function decreased and the expression of NFL,S100-β,Lac,CCL2,HMGB1,PFKFB3,Iba1,NLRP3 and Caspase-3 increased in the VCI group,while the expression of AMPKαdecreased and the neuron degeneration and apoptosis increased in VCI group,and the differences were statistically significant(all P<0.05).Compared with the VCI group,the cognitive impairment of VCI+3PO group was improved,the cognitive impairment of VCI+DMOG group was aggravated,and the expression of NFL,S100-β,Lac,CCL2 and HMGB1 decreased in VCI+3PO group and increased in VCI+DMOG group.The expression of PFKFB3,Iba1,NLRP3 and Caspase-3 decreased in VCI+3PO group,while the expression of PFKFB3,Iba1,NLRP3 and Caspase-3 increased and the expression of AMPKαdecreased in VCI+DMOG group.The degeneration and apoptosis of neurons in VCI+3PO group decreased,while the degeneration and apoptosis of neurons in VCI+DMOG group increased,and the differences were statistically significant(all P<0.05).Conclusion The enhancement of key glycolytic enzyme PFKFB3 in microglia activation could induce central inflammation and upregulate the expression of NLRP3,leading to neuronal injury,degeneration and apoptosis,and aggravating vascular cognitive dysfunction in rats,which may be related to the increase of pro-inflammatory factor HMGB1 in the hippocampus.