抑肝散对创伤后应激障碍小鼠海马神经元凋亡的影响与作用机制

Therapeutic Effect and Mechanism of Yigan Powder on Apoptosis of Hippocampal Neurons in Mice with Post-traumatic Stress Disorder

目的探究抑肝散对创伤后应激障碍(post-traumatic stress disorder,PTSD)小鼠行为学与神经元凋亡的影响,并从环磷腺苷效应元件结合蛋白(cyclic adenosine monophosphate response element-binding protein,CREB)/脑源性神经影响因子(brain-derived neurotrophic factor,BDNF)信号通路分析其作用机制。方法选取58只雄性C57BL/6J小鼠随机分为正常组(不造模+等体积生理盐水灌胃,n=12)、模型组(PTSD造模+等体积生理盐水灌胃,n=10),抑肝散低剂量组[PTSD造模+0.5 g/(kg·d)抑肝散灌胃,n=12]、抑肝散高剂量组[PTSD造模+1.0 g/(kg·d)抑肝散灌胃,n=12]、盐酸舍曲林组[PTSD造模+0.01 g/(kg·d)盐酸舍曲林灌胃,n=12]。采用旷场实验、创伤线索回避实验、情景恐惧实验评价小鼠的行为学变化;尼式染色法观察小鼠海马细胞病理变化;末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling,TUNEL)法检测小鼠海马组织细胞凋亡情况;Western blot法检测小鼠海马组织中BDNF、磷酸化CREB(phosphorylated CREB,p-CREB)、脑源性神经营养因子酪氨酸激酶受体B(tyrosine kinase receptor B,TrkB)的蛋白含量。结果与正常组比较,模型组小鼠旷场实验的中心区域运动距离百分比、探索物体时间明显减少,僵直时间明显增加(P均<0.05);海马组织细胞存在结构异常、坏死情况;海马神经元凋亡数量增加(P<0.05);海马组织中TrkB、BDNF、p-CREB蛋白相对表达水平明显下降(P均<0.01)。给药后,与模型组比较,抑肝散低剂量组、抑肝散高剂量组、盐酸舍曲林组小鼠旷场实验的中心区域运动距离百分比均增加(P均<0.05),抑肝散低剂量组小鼠探索物体时间增加(P<0.05),抑肝散高剂量组小鼠僵直时间减少(P<0.05);与模型组比较,抑肝散低剂量组、抑肝散高剂量组、盐酸舍曲林组小鼠海马组织细胞结构异常、坏死情况缓解;抑肝散高剂量组与盐酸舍曲林组小鼠海马神经元凋亡数量减少(P<0.01);与模型组比较,抑肝散低剂量组、抑肝散高剂量组、盐酸舍曲林组小鼠海马组织TrkB、p-CREB、BDNF蛋白相对表达水平明显升高(P均<0.01)。结论抑肝散可激活CREB/BDNF信号通路,抑制PTSD小鼠神经元凋亡,改善小鼠的焦虑、主动回避行为和再体验行为。

Objective To investigate the effects of Yigan Powder on the behavior and neuronal apoptosis of post-traumatic stress disorder(PTSD)mice,and to analyze its mechanism of action through cyclic adenosine monophosphate response element-binding protein(CREB)/brain-derived neurotrophic factor(BDNF)signaling pathway.Methods A total of 58 male C57BL/6J mice were selected and randomly divided into normal group(do not build model+giving volume normal saline lavage,n=12),model group(PTSD modeling+giving volume normal saline lavage,n=10),low-dose Yigan Powder group[PTSD modeling+giving 0.5 g/(kg·d)Yigan Powder by gavage,n=12),high-dose Yigan Powder group[PTSD modeling+giving 1 g/(kg·d)Yigan Powder by gavage]and sertoline hydrochloride group[PTSD modeling+giving 0.01 g/(kg·d)sertoline hydrochloride by gavage,n=12].The behavioral changes of the mice were evaluated by open field test,trauma cue avoidance test and contextual fear test;the pathological changes of hippocampal cells in mice were observed by Nissl staining;the apoptosis of hippocampal tissue cells in mice was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL);the protein levels of BDNF,phosphorylated CREB(p-CREB)and tyrosine kinase receptor B(TrkB)in hippocampal tissues of mice were detected by Western blot.Results Compared with the normal group,the model group showed a significant decrease in the percentage of central zone movement distance and exploration object time and an significant increase in cataleptic time(all P<0.05),the hippocampal tissue cells showed structural abnormalities and necrosis;the number of apoptotic hippocampal neurons increased(P<0.05);and the protein expression levels of TrkB,BDNF and p-CREB in the hippocampal tissues significantly decreased(P<0.01).After administration,compared with the model group,the low-dose Yigan Powder group,high-dose Yigan Powder group and the sertoline hydrochloride group showed an increase in the percentage of central zone movement distance in the open field test(all P<0.05),the low-dose Yigan Powder group showed an increase in exploration object time(P<0.05),and the high-dose Yigan Powder group showed a decrease in cataleptic time(P<0.05);compared with the model group,the low-dose Yigan Powder group,high-dose Yigan Powder group and the sertoline hydrochloride group showed alleviation of hippocampal tissue cell structural abnormalities and necrosis,the high-dose Yigan Powder group and the sertoline hydrochloride group showed a decrease in the number of apoptotic hippocampal neurons(P<0.01);compared with the model group,the low-dose Yigan Powder group,high-dose Yigan Powder group and the sertoline hydrochloride group showed an increase in protein expression levels of TrkB,p-CREB and BDNF in hippocampal tissues(all P<0.01).Conclusion Yigan Powder can activate CREB/BDNF signaling pathway,inhibit neuronal apoptosis in PTSD mice,and improve anxiety,active avoidance behavior and re-experience behavior in mice.