基于外周血单个核细胞转录组学的柴归颗粒抗抑郁作用机制研究

Antidepressant mechanism of Chaigui Granules based on transcriptomics of peripheral blood mononuclear cells

目的通过转录组测序(RNAsequencing,RNA-seq)技术分析柴归颗粒对慢性不可预知温和应激(chronic unpredictable mild stress,CUMS)模型大鼠外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)的转录组学特征,并采用qRT-PCR法验证其作用机制。方法将大鼠随机分为对照组、模型组、盐酸文拉法辛(35 mg/kg)组和柴归颗粒低、中、高剂量(4.2、8.3、16.6 g/kg)组,造模同时给药,连续28 d。通过抑郁症传统药效学指标(体质量、旷场实验、糖水偏爱实验及强迫游泳实验)评价柴归颗粒的药效。采用RNA-seq技术对大鼠PBMCs进行转录组学分析,筛选差异基因,对其进行京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析;通过qRT-PCR法对筛选出的关键表达基因进行实验验证。结果行为学指标显示柴归颗粒能显著改善CUMS模型大鼠的抑郁行为。转录组学中KEGG通路富集分析表明抑郁发生时及柴归颗粒干预后,都显著富集到嘌呤代谢通路,因此针对嘌呤代谢相关代谢酶基因表达进行深入研究。qRT-PCR结果表明,与模型组比较,柴归颗粒组大鼠PBMCs中的胞外核苷酸焦磷酸酶/磷酸二酯酶3(ectonucleotide pyrophosphatase/phosphodiesterase 3,Enpp3)、胞质-5′-核苷酸酶IIIA(cytosolic-5′-nucleotidase IIIA,Nt5c3a)、嘌呤核苷磷酸化酶(purine nucleoside phosphorylase,Pnp)、腺苷酸脱氨酶(adenosine deaminase,Ada)m RNA表达水平显著升高(P<0.05、0.01、0.001),黄嘌呤脱氢酶/氧化酶(xanthine dehydrogenase/oxidase,Xdh)、嘌呤能受体P2X7(purinergic receptor P2X7,P2rX7)、白细胞介素1β(interleukin 1β,IL1β)m RNA表达水平显著降低(P<0.05、0.01)。结论柴归颗粒具有抗抑郁作用,其作用机制与调节嘌呤代谢途径上的基因及嘌呤能受体P2X7和炎症因子IL1βmRNA表达有关。

Objective To analyze the transcriptomic characteristics of Chaigui Granules(柴归颗粒)on peripheral blood mononuclear cells(PBMCs)of rats with chronic unpredictable mild stress(CUMS)model by RNA sequencing(RNA-seq),qRT-PCR was used to verify the mechanism.Methods Rats were randomly divided into control group,model group,venlafaxine hydrochloride(35 mg/kg)group,and Chaigui Granules low-,medium-and high-dose(4.2,8.3,16.6 g/kg)groups.Rats were given drugs for 28 d while modeling.The efficacy of Chaigui Granules was evaluated through traditional pharmacodynamic indicators for depression(body weight,open field test,sugar water preference test and forced swimming test).RNA-seq technology was used to perform transcriptome sequencing analysis on rat PBMCs,screen for differential genes,and perform Kyoto encyclopedia of genes and genomes(KEGG)analysis;The key expressed genes screened out were verified experimentally by qRT-PCR.Results Behavioral indicators showed that Chaigui Granules significantly improved the depressive behavior of CUMS model rats.Enrichment analysis of KEGG pathway in transcriptomics showed that when depression occurs after the intervention of Chaigui Granules,purine metabolism pathway was significantly enriched.Therefore,in-depth research was conducted on gene expression of metabolic enzymes related to purine metabolism.qRT-PCR results showed that compared with model group,expression levels of ectonucleotide pyrophosphatase/phosphodiesterase 3(Enpp3),cytosolic-5′-nucleotidase IIIA(Nt5c3a),purine nucleoside phosphorylase(Pnp)and adenosine deaminase(Ada)mRNA in PBMCs of Chaigui Granules group were significantly increased(P<0.05,0.01,0.001),expression levels of xanthine dehydrogenase/oxidase(Xdh),purinergic receptor P2X7(P2rX7),interleukin 1β(IL1β)mRNA were significantly decreased(P<0.05,0.01).Conclusion Chaigui Granules has antidepressant effects,its mechanism is related to the regulation of genes in purine metabolism pathway,purinergic receptor P2X7 and inflammatory factor IL1β.