摘要
目的:从脑源性神经营养因子(BDNF)/酪氨酸激酶受体B(TrkB)信号通路研究乌头汤对神经病理性疼痛模型小鼠的神经元保护作用。方法:40只雄性ICR小鼠随机分为假手术组(Sham),SNL模型组,乌头汤组(126 mg·kg-1),ANA-12+乌头汤组。建立神经病理性疼痛L5脊神经结扎(SNL)小鼠模型,造模成功后,灌胃给药10 d,脑立体定位注射BDNF/TrkB受体拮抗剂ANA-12(50 nmol·L^-1)7 d,免疫组化法检测小鼠海马BDNF,蛋白激酶B(Akt),环磷酸腺苷(c AMP)反应元件结合蛋白(CREB)蛋白的表达。免疫荧光法观察谷氨酸(Glu)和γ-氨基丁酸(GABA)神经元变化。小鼠海马神经元细胞分离于精准孕18 d的胎鼠脑组织,分为正常组、甘氨酸处理组,ANA-12组(0.5 mmol·L^-1),ANA-12+甘氨酸组,ANA-12+乌头汤组,ANA-12+乌头汤+甘氨酸组,细胞免疫荧光法观察原代神经元形态学变化;海马神经元细胞分为正常组、甘氨酸处理组(200 mmol·L^-1),肿瘤坏死因子(TNF)-α(5 mg·L^-1)+甘氨酸组,TNF-α+乌头汤(5 mg·L^-1)+甘氨酸组,TNF-α+乌头汤+甘氨酸+BDNF-siRNA组,TNF-α+乌头汤+甘氨酸+Akt-siRNA组,TNF-α+乌头汤+甘氨酸+CREB-siRNA组,细胞免疫荧光法检测神经元初、次级树突棘内外的突触后密度蛋白95(PSD95)的表达量。结果:与正常组比较,模型组BDNF,Akt和CREB阳性细胞数显著下降(P<0.01),Glu-GABA能神经元失衡(P<0.01);与模型组比较,乌头汤组BDNF,Akt和CREB阳性细胞数显著上升(P<0.01),Glu-GABA能神经元恢复平衡(P<0.01);与乌头汤组比较,ANA-12+乌头汤组BDNF和CREB阳性细胞数又显著下降(P<0.05)而Akt组并无显著变化,Glu-GABA能神经元失衡(P<0.01)。与正常组比较,甘氨酸处理组神经元初、次级树突棘数量显著上升(P<0.01),且突触后密度蛋白95(PSD95)表达量显著上升(P<0.01),ANA-12组,ANA-12+甘氨酸组,ANA-12+乌头汤组,ANA-12+乌头汤+甘氨酸组神经元初、次级树突棘数量均无明显变化;与甘氨酸处理组比较,TNF-α+甘氨酸组PSD95表达量显著下降(P<0.01);与TNF-α+甘氨酸组比较,TNF-α+乌头汤+甘氨酸组PSD95表达量明显上升(P<0.01);与TNF-α+乌头汤+甘氨酸组比较,TNF-α+乌头汤+甘氨酸+BDNF-siRNA组,TNF-α+乌头汤+甘氨酸+Akt-siRNA组,TNF-α+乌头汤+甘氨酸+CREB-siRNA组PSD95表达量均显著下降(P<0.01)。结论:乌头汤对SNL小鼠海马谷氨酸能神经元兴奋性及可塑性损伤具有修复作用,这一作用与其对BDNF/TrkB通路的调控有关。
Objective:To investigate the neuroprotective effect and mechanism by Wutoutang(WTT)in the spinal nerve ligation(SNL)mice by neurotrophic factor(BDNF)/tyrosine kinase receptor B(TrkB)signaling BDNF/tyrosine kinase receptor B(TrkB).Method:The 40 mice were randomly divided into Sham group,SNL group,WTT group(126 mg·kg-1),ANA-12+WTT group.The L5 spinal nerve ligation model mice were established in mice,After that,WTT was administrated from the first day to the 10 thday,then the consecutive 7-day hippocampal injection of ANA-12(0.05 nmol·L^-1)were lasted for 7 days.The levels of brainderived BDNF,cAMP-response element binding protein(CREB),and protein kinase B(Akt)and the change of hippocampal glutamatergic and GABAergic neurons in mice were detected by tissue immunofluorescence.E14 pregnant ICR mice were sacrificed and the hippocampus were dissected which were divided into control group,glycine group,tumo necrosis factor(TNF)-α(5 mg·L^-1)+glycine group,TNF-α+WTT(5 mg·L^-1)+glycine group,TNF-α+WTT+glycine+BDNF-siRNA group,TNF-α+WTT+glycine+Akt-siRNA group,TNF-α+WTT+glycine+CREB-siRNA group,the primary and secondary dendrrictes,in which the arrowheads indicate the expression od postsynapti desity protein 95(PSD95)in the shafts and arrows were tested by cellular immunofluorescence.The neurons were divided into control group,glycine group,ANA-12 group(0.5 mmol·L^-1),ANA-12+glycine group,ANA-12+WTT group,ANA-12+WTT+glycine group,the morphology of hippocampal neurons were tested by cellular immunofluorescence.Result:Compared with Sham group,BDNF,Akt and CREB positive cell of SNL group decreased significantly(P<0.01),the hippocampal glutamatergic and GABAergic neurons out of balance(P<0.01).Compared with SNL group,the BDNF,Akt and CREB positive cell of WTT group increased significantly(P<0.01),Glutamine-aminobutyric acid neurons regein balance(P<0.01).Compared with WTT group,BDNF and CREB positive cell of ANA-12+WTT group decreased significantly(P<0.05),Glutamine-aminobutyric acid neurons was disorderedd(P<0.05).Comparaed with control group,the level of PSD95 of glycine group were increase significantly(P<0.01).The number of dendritic spine density apically and basally of glycine group were increase significantly(P<0.01),but the primary and secondary dendrites of ANA-12 group,ANA-12+glycine group,ANA-12+WTT group,ANA-12+WTT+glycine group were not change.Comparaed with glycine group,the level of PSD95 of TNF-α+glycine group were decreased significantly(P<0.01).Comparaed with TNF-α+glycine group,the level of PSD95 of TNF-α+WTT+glycine group were increase significantly(P<0.01).Comparaed with TNF-α+WTT+glycine group,the level of PSD95 of TNF-α+WTT+glycine+BDNF-siRNA group,TNF-α+WTT+glycine+Akt-siRNA group,TNF-α+WTT+glycine+CREB-siRNA group were decreased significantly(P<0.01).Conclusion:In vivo and in vitro studies have shown that the WTT mediated remission of the primary hippocampal glutamatergic neurons were dependent on the BDNF/TrkB pathway.
作者
师钰琪
吴红艳
朱春燕
林娜
SHI Yu-qi;WU Hong-yan;ZHU Chun-yan;LIN Na(Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Institute of Chinese Materia Medica,China Academy of Chinese Medicine Sciences,Beijing 100700,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2020年第7期23-30,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81630107,81603322)
国家科技重大专项(2019ZX09731002)
中央级公益性科研院所基本科研业务费专项(L2017046).
