基于miR-26a-5p/EZH2/H3K27me3通路探讨"通督调神"针刺对CUMS大鼠神经炎症的影响OA

Objective To observe the effects of Tongdu Tiaoshen(unblocking the circulation of the governor vessel and regulating the mind)acupuncture on depressive-like behavior and the expression of microRNA-26a-5p(miR-26a-5p)/enhancer of zeste homolog 2(EZH2)/trimethylation of lysine 27 on histone H3(H3K27me3)proteins and inflammatory factors in chronic unpredictable mild stress(CUMS)rats,and to explore its antidepressant mechanism.Methods Thirty-two SPF-grade male SD rats were randomly divided into a normal group(8 rats)and a modeling group(24 rats).The modeling group was subjected to CUMS to establish depression model.After successful modeling,the rats were randomized into a model group,an acupuncture group and a fluoxetine group,with 8 rats in each group.In the acupuncture group,Tongdu Tiaoshen acupuncture was operated at"Baihui"(GV20),"Shenting"(GV24),"Shuigou"(GV26)and"Dazhui"(GV14),once daily for 6 consecutive days followed by one day of interval;and the completion of intervention was composed of 24 sessions of acupuncture.The gavage with fluoxetine solution(2.1 mg/kg)was administered in the fluoxetine group;and with 0.9％sodium chloride solution(10 mL/kg)in the other 3 groups,continuously for 28 days.Sucrose preference and the results of the open field test were observed.The hippocampal neuron morphology and Nissl bodies were examined using HE and Nissl staining.The protein expression of hippocampal EZH2 and H3K27me3 was detected using Western blot,and the mRNA expression of hippocampal miR-26a-5p and EZH2 was detected with RT-qPCR.ChIP-qPCR was applied to detect the enrichment levels of H3K27me3 in the promoter regions of interleukin-4(IL-4),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α).ELISA was provided to measure the contents of IL-6,TNF-α,interleukin-1β(IL-1β),and IL-4 in the hippocampus and serum.Results Compared with the normal group,the model group showed the decrease in sucrose preference(P<0.01),the total distance,the numbers of uprighting and cleaning behavior in the open field test(P<0.01);hippocampal neurons exhibited partial cell dissolution,irregular arrangement,nuclear pyknosis,reduced Nissl bodies(P<0.01),and uneven distribution.The mRNA expression of hippocampal EZH2,and the protein expression of hippocampal EZH2 and H3K27me3 increased(P<0.01),while the mRNA expression of miR-26a-5p decreased(P<0.01).The enrichment levels of H3K27me3 in the promoter regions of IL-6 and TNF-α were elevated(P<0.01),whereas that in IL-4 was reduced(P<0.01).The levels of IL-6,TNF-α and IL-1β in the hippocampus and serum increased(P<0.01),and the levels of IL-4 decreased(P<0.01).Compared with the model group,the acupuncture group and the fluoxetine group showed the increase in sucrose preference(P<0.01),the total distance,the numbers of uprighting and cleaning behavior in the open field test(P<0.01);hippocampal neurons exhibited the reduced cell dissolution,regular arrangement,declined nuclear pyknosis,higher Nissl bodies(P<0.01),and even distribution.The mRNA expression of hippocampal EZH2,the protein expression of hippocampal EZH2 and H3K27me3 decreased(P<0.01),and the mRNA expression of miR-26a-5p increased(P<0.01);and the mRNA expression of miR-26a-5p was negatively correlated with that of EZH2(P<0.001).The enrichment levels of H3K27me3 in the promoter regions of IL-6 and TNF-α were lower(P<0.01),and that of IL-4 was higher(P<0.01,P<0.05).The levels of IL-6,TNF-α and IL-1β in the hippocampus and serum decreased(P<0.01),and the levels of IL-4 increased(P<0.01).The differences of the above indexes were not statistically significant between the acupuncture group and fluoxetine group(P>0.05).Conclusion Tongdu Tiaoshen acupuncture can ameliorate depression-like behavior in CUMS rats,with the mechanism potentially related to regulating the miR-26a-5p/EZH2/H3K27me3 pathway and reducing central neuroinflammation.