【摘要】 目的 探究柴胡-黄芩药对核心成分柴胡皂苷a-黄芩苷(saikosaponin a-baicalin,SSa-BA)的抗抑郁作用及分子机制。方法 将雄性小鼠随机分为空白对照组、模型组、阳性对照组(氟西汀,Flu)、SSa-BA低/高剂量组(SSa-BA-L/H),每组10只。慢性不可预知温和应激造模28 d,造模14 d后给药14 d;通过旷场实验、悬尾实验评价抑郁样行为;苏木精-伊红(HE)染色观察海马病理变化;酶联免疫吸附试验(ELISA)法检测海马5-羟色胺(5-HT)含量;Western blot检测海马NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体、小胶质细胞焦亡相关蛋白及钙离子信号通路相关蛋白表达;免疫组化分析IL-1β、IL-18阳性表达。RNA-Seq筛选差异表达基因(differentially expressed genes,DEGs),京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析锁定关键通路。结果 与空白对照组相比,模型组小鼠旷场步行得分、站立次数显著降低(P <0.01),悬尾不动时间显著延长(P<0.01),海马5-HT水平显著降低(P <0.01),神经元损伤明显,NLRP3炎症小体及焦亡相关蛋白表达及IL-1β、IL-18表达显著升高(P<0.01)。SSa-BA显著下调上述蛋白表达(P <0.01),且SSa-BA-H组行为学、海马病理及5-HT水平接近空白组。转录组分析显示,模型组与空白组DEGs共1 864个,SSa-BA组与模型组DEGs共1 376个,交集483个;KEGG提示钙离子信号通路、神经信号传递通路及ECM-受体交互通路与SSa-BA抗抑郁作用相关,且钙离子通路可能为NLRP3通路上游调控通路。分子验证显示,模型组海马IP3R1、PLCγ1、p-CaMKⅡδ、VDAC1蛋白表达高于空白对照组(P<0.01),而SSa-BA可剂量依赖性抑制其表达(P<0.01)。结论 SSa-BA可能通过抑制抑郁模型小鼠海马区钙离子信号通路相关蛋白表达,进而减少NLRP3炎症小体活化与小胶质细胞焦亡,恢复单胺类神经递质水平并改善海马神经元损伤,最终发挥抗抑郁作用。更多还原

【Abstract】 OBJECTIVE To explore the antidepressant effect and molecular mechanism of saikosaponin a-baicalin（SSa-BA）,the core component of the Bupleuri Radix-Scu tellariae Radix herb pair.METHODS Male ICR mice were randomly divided into 5groups（n=10 per group）:blank control group,chronic unpredictable mild stress（CUMS） model group,positive control group（fluoxetine,Flu）,SSa-BA low-dose group（SSa-BA-L）,and SSa-BA high-dose group（SSa-BA-H）.The chronic unpredictable mild stress（CUMS） model was established for 28 days,and drug administration was conducted for 14 days starting from the 14^th day of modeling.Depression-like behaviors were evaluated by open field test（OFT） and tail suspension test（TST）.Hippocampal pathological changes were observed by hematoxylin-eosin（HE） staining;hippocampal serotonin（5-HT） content in the hippocampus was detected by enzyme-linked immunosorbent assay（ELISA）;the expressions of NLRP3 inflammasome,microglial pyroptosis-related proteins,and calcium signaling pathway-related proteins were detected by western blot.Immunohistochemistry was performed to analyze the positive expressions of IL-1β and IL-18.RNA-Seq was used to screen differentially expressed genes（DEGs）,KEGG enrichment analysis was performed to identify key pathways.RESULTS Compared with control group,the model group showed a significant decrease in OFT walking score and rearing frequency（P<0.01）,a significant prolongation of immobility time in TST（P<0.01）,a significant reduction in hippocampal 5-HT level（P<0.01）,obvious hippocampal neuron damage,and a significant increase in the expressions of NLRP3 inflammasome,pyroptosis-related proteins,IL-1β,and IL-18（P<0.01）.SSa-BA significantly downregulated the expressions of the above proteins（P<0.01）,and the behavioral indicators,hippocampal pathology,and 5-HT level in the SSa-BA-H group were close to those in control group.Transcriptome analysis showed that there were 1 864 DEGs between the model group and the blank control group,1 376 DEGs between the SSa-BA group and the model group,with 483 overlapping DEGs.KEGG enrichment analysis suggested that the calcium signaling pathway,neural signal transmission pathway,and ECM-receptor interaction pathway were associated with the antidepressant effect of SSa-BA,and the calcium signaling pathway might be the upstream regulatory pathway of the NLRP3 pathway.Verification experiments showed that the expressions of IP3R1,PLCγ1,p-CaMKⅡδ,and VDAC1 proteins in the hippocampus of the model group were significantly higher than those in the blank control group（P<0.01）,while SSa-BA inhibited their expressions in a dosedependent manner（P<0.01）.CONCLUSION SSa-BA may exert its antidepressant effect by inhibiting the expression of proteins related to the calcium signaling p athway in the hippocamp us of depressed model mice,thereby suppressing the activation of NLRP3 inflammasome and microglial pyroptosis,restoring the levels of monoamine neurotransmitters,and improving hippocampal neuron damage.更多还原