基于转录组学技术探讨基底外侧杏仁核参与电针治疗癌痛及其诱发的抑郁情绪的分子机制

Molecular mechanism of basolateral amygdala involved in electroacupuncture-induced amelioration of cancer pain and concomitant depression based on transcriptomics techniques

目的:观察电针对癌痛模型小鼠疼痛及其诱发的抑郁情绪的影响,采用转录组学技术从基因层面探讨基底外侧杏仁核(BLA)参与癌痛多维度调节效应的分子机制。方法:将C57BL/6小鼠分为假手术组、模型组、电针组,每组10只。于股骨内注射含有Lewis肺癌细胞的PBS悬液建立骨癌痛模型。电针组小鼠在造模后第10天开始进行电针干预“足三里”“三阴交”,每日1次,每次20 min,连续干预12 d。采用X线和HE染色观察荷瘤股骨远端骨质损伤情况;von Frey检测小鼠机械性痛阈值变化;糖水偏好实验和强迫游泳实验观察小鼠抑郁情绪;提取小鼠BLA组织RNA建库,转录组学测序筛选差异基因并进行生物信息学分析;实时荧光定量PCR检测差异表达基因肿瘤坏死因子超家族8(Tnfsf8)、骨髓基质细胞抗原1(Bst1)、前强啡肽原(Pdyn)、电压门控钠离子通道β4(Scn4b)mRNA表达水平。结果:X线和HE染色可见小鼠股骨远端显著骨质损伤。与假手术组比较,模型组小鼠造模后第1、4、7、10、14、21天机械性痛阈值显著降低(P<0.001,P<0.0001),糖水偏好指数显著降低(P<0.001),强迫游泳不动时间显著延长(P<0.001)。与模型组比较,电针组小鼠造模后第14、21天机械痛阈值显著升高(P<0.0001),糖水偏好指数显著升高(P<0.05),强迫游泳不动时间显著缩短(P<0.01)。与假手术组比较,模型组小鼠BLA共筛选出404条差异基因,与模型组比较,电针组小鼠BLA共筛选出329条差异基因,其中在模型组表达上调而在电针组下调的基因共45条,在模型组表达异常上调而被电针干预下调的基因共28条。将筛选的差异表达基因进行基因本体(GO)注释和京都基因组百科全书(KEGG)通路分析,GO注释结果显示上述差异基因主要功能富集在与神经炎性反应相关的配体受体活性、细胞因子受体结合及细胞因子活性,以及与神经元兴奋相关的神经肽信号通路、钙离子介导的信号传导等方面;KEGG通路分析显示差异基因主要富集在与炎性反应相关的通路如白细胞介素-17通路等。模型组Tnfsf8、Bst1 mRNA的表达较假手术组显著升高(P<0.01,P<0.05),电针组较模型组显著下降(P<0.01,P<0.05);模型组Pdyn、Scn4b mRNA的表达较假手术组显著下降(P<0.01),电针组较模型组显著升高(P<0.05,P<0.01),实时荧光定量PCR结果与基因测序结果变化趋势相一致。结论:电针可以显著缓解癌痛及其诱发的抑郁样情绪,其机制可能与模型动物BLA内神经炎性反应的缓解及特定神经元的异常活动的恢复有关。

Objective To observe the effect of electroacupuncture(EA)of“Zusanli”(ST36)and“Sanyinjiao”(SP6)on cancer pain and concomitant negative emotion in cancer pain model mice,and to explore its molecular mechanisms in the basolateral amygdala(BLA)by using transcriptomics techniques.Methods C57BL/6 mice were randomized into sham operation,model and EA groups,with 10 mice in each group.The cancer pain model was established by injecting PBS suspension containing Lewis lung cancer cells into the femur.The mice in the EA group received EA stimulation(1 mA,2 Hz)on ST36 and SP6 from the 10th day after modeling,20 min per day for 12 successive days.The bone damage of the distal femur was observed with X-ray and H.E.staining,respectively.The mechanical pain threshold(MPT)was detected by using von Frey.The depression-like behavior was detected by using sucrose-preference test(sucrose preference index in 12 h),and the immobility(feeling of despair)duration of forced swimming within 4 min.The BLA tissue was extracted for RNA sequencing(RNA library construction,and screening differential gene profiling by transcriptomic sequencing)and bioinformatics analysis.The real-time PCR was used to validate the mRNA expression of differentially expressed genes:tumor necrosis factor superfamily 8(Tnfsf8),bone marrow stromal cell antigen 1(Bst1),prodynorphin(Pdyn)and voltage-gated sodium channelβ4(Scn4b).Results H.E.staining and X-ray showed significant bone damage in the distal femur in cancer pain mice.In contrast to the sham operation group,the MPT on the 1st,4th,7th,10th,14th and 21st day after modeling and sucrose preference index were significantly decreased(P<0.001,P<0.0001),and the immobility time of the forced swimming was considerably increased in the model group(P<0.001).In contrast to the model group,the MPT values on the 14th and 21st day and sucrose preference index were obviously increased(P<0.0001,P<0.05),and the immobility time was strikingly decreased in the EA group(P<0.01).RNA sequencing showed that a total of 404 differentially expressed genes(205 up-regulated,199 down-regulated)were screened in the model group compared with the sham operation group,and a total of 329 differentially expressed genes(206 up-regulated and 123 down-regulated)were screened in the EA group compared with the model group.Venn diagram analysis of the differentially expressed genes showed that 45 up-regulated and 28 down-regulated genes in the model group were completely reversed by EA.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses of the screened differentially expressed genes revealed that the above differential genes were mainly enriched in the ligand receptor activity,cytokine receptor binding,and cytokine activity related to neuro-inflammation,as well as in neuropeptide signaling pathways related to neuronal excitability,and calcium ion mediated signal transduction.The analysis of KEGG pathway showed that the differentially expressed genes were mainly enriched in the inflammation-related pathways,such as interleukin-17 pathway.Validation analysis of the differentially expressed genes showed that the expression levels of Tnfsf8 and Bst1 were significantly up-regulated in the model group compared with the sham operation group(P<0.01,P<0.05),and downregulated by EA(P<0.01,P<0.05),while the expression levels of Pdyn and Scn4b were down-regulated in the model group in comparison with the sham operation group(P<0.01),and up-regulated by EA(P<0.05,P<0.01),which was consistent with the changing trend of the gene sequencing results.Conclusion Acupuncture of ST36 and SP6 can significantly relieve cancer pain and concomitant negative emotion in cancer pain mice,which may be related to its functions in alleviating neuro-inflammation and relieving the abnormal activities of specific neurons in the BLA.