逍遥散乙酸乙酯部位对LPS致抑郁样模型小鼠影响的实验研究

Effect of Ethyl Acetate Fraction of Xiaoyaosan on Neuroinflammation and Neurons in LPS-induced Depression Model Mice

目的:基于炎症与抑郁症的关联及逍遥散抗抑郁作用的研究基础,本研究采用LPS诱导的抑郁样小鼠模型,探究逍遥散乙酸乙酯部位对模型小鼠神经炎症的影响及海马神经元的保护作用机制。方法:C57BL/6J雄性小鼠按体质量分层随机分为正常对照组、模型对照组、盐酸氟西汀13 mg/kg组、逍遥散水煎液40 g/kg组、逍遥散乙酸乙酯部位0.23、0.46 g/kg组,连续给药15 d,在给药第12 d~14 d,除正常对照组外,其余各组小鼠连续3 d腹腔注射脂多糖(LPS,1 mg/kg)造模,正常对照组小鼠腹腔注射等容积生理盐水,末次给药1 h后进行悬尾和强迫游泳行为学测试;ELISA法检测血清中白细胞介素-1β(IL-1β)含量;Western blot法测定小鼠海马组织NOD样蛋白结构域受体3(NLRP3)、半胱天冬氨酸酶-1(Caspase-1)、裂解半胱氨酸蛋白酶-1(Cleaved-Caspase-1)、IL-1β、白细胞介素-1β前体(Pro-IL-1β)、消皮素(GSDMD)、GSDMD的N端结构域(GSDMD-N)以及钙离子结合蛋白-1(IBA-1)的蛋白表达;RT-PCR法检测海马部位Nlrp3、Il1b mRNA表达;免疫荧光双标法检测小鼠海马CA3区NLRP3及激活小胶质细胞的分布;尼氏染色法检测小鼠海马CA3区神经元尼氏小体的变化。结果:与正常对照组比较,模型对照组小鼠悬尾及强迫游泳试验不动时间显著增加(P<0.01);小鼠血清IL-1β含量显著升高(P<0.01);炎症小体通路明显激活(P<0.05或P<0.01),海马CA3区NLRP3、IBA-1蛋白表达显著上调(P<0.01);小鼠海马CA3区神经元损伤显著(P<0.01);与模型对照组比较,逍遥散40 g/kg组、逍遥散乙酸乙酯部位0.23、0.46 g/kg组均能明显缩短模型小鼠悬尾及强迫游泳试验的不动时间(P<0.05或P<0.01);能显著降低小鼠血清IL-1β含量(P<0.01),抑制炎症小体通路的激活(P<0.05或P<0.01);降低模型小鼠海马CA3区NLRP3、IBA-1的表达、明显抑制小胶质细胞激活(P<0.05),并显著减轻神经炎症所致的神经元损伤(P<0.01)。结论:逍遥散乙酸乙酯部位能对抗因神经炎症所诱发的小鼠抑郁样行为,作用发挥与抑制NLRP3/Caspase-1/IL-1β通路激活,从而抑制小胶质细胞激活、减轻神经炎症及神经元损伤有关。

Objective:To explore the mechanism of ethyl acetate fraction of Xiaoyaosan against neuroinflammation and hippocampal neuron protection in LPS-induced depression mice based on the correlation of inflammation and depression,and the research basis of Xiaoyaosan treating depressive disorder.Methods:According to the body weight,C57 BL/6 J male mice were randomly divided into normal group,model group,fluoxertine hydrochloride(13 mg/kg)group,Xiaoyaosan decoction(40 g/kg)group,and Xiaoyaosan ethyl acetate fraction groups(0.46,0.23 g/kg).Drugs were administered for 15 consecutive days.Except for normal group,mice in the other groups were intraperitoneally injected with lipopolysaccharide(LPS,1 mg/kg)on days 12-14 to induce depression mice model,while those in normal group were given the same volume of normal saline.After 1 hour of the last administration,tail suspension test and forced swimming test were performed.Content of serum interleukin1β(IL-1β)was measured by enzyme linked immunosorbent assay(ELISA).The protein expression levels of NOD-like receptor thermal protein domain associated protein 3(NLRP3),caspase 1,cleaved caspase 1,IL-1β,pro IL-1β,gasdermin D(GSDMD),GSDMD N,and ionized calcium-binding adapter molecule 1(Iba 1)in the hippocampus were determined by Western blot.The mRNA expression levels of NLRP3 and IL1βin the hippocampus were determined by reverse transcription-polymerase chain reaction(RT-PCR).The distribution of NLRP3 and activated microglia in the CA3 region of hippocampus of mice was detected by double-label immunofluorescence,and the changes of hippocampal CA3 neurons were observed by Nissl staining.Results:Compared with the conditions in normal group,the immobility time of tail suspension test and forced swimming test(P<0.01),the level of serum IL-1β(P<0.01),and the protein expression of NLRP3 and Iba 1 in the CA3 region of hippocampus(P<0.01)in model group were increased;the NLRP3/caspase 1/IL-1βpathway was activated(P<0.05 or P<0.01),and the hippocampal CA3 neurons were damaged(P<0.01).Compared with model group,the Xiaoyaosan decoction(40 g/kg)group and Xiaoyaosan ethyl acetate fraction groups(0.46,0.23 g/kg)had shortened immobility time of tail suspension test and forced swimming test(P<0.05 or P<0.01),reduced level of serum IL-1β(P<0.01),and decreased expression of NLRP3 and iba1 in the CA3 region of hippocampus(P<0.05);additionally,the activation of NLRP3/caspase 1/IL-1βpathway and microglia was inhibited(P<0.05 or P<0.01),and the neuronal damage caused by neuroinflammation was ameliorated(P<0.01).Conclusion:The ethyl acetate fraction of Xiaoyaosan can inhibit depression-like behavior induced by neuroinflammation in mice,and its mechanism may be related to inhibiting the activation of NLRP3/caspase1/IL-1βpathway and microglia,and thereby reducing neuroinflammation and neuronal damage.