基于定量蛋白质组学探讨针刺对慢性不可预测温和应激抑郁大鼠的作用机制

Study on mechanisms of acupuncture underlying improvement of CUMS-induced depression in rats based on tandem mass spectrometry proteomics technique

目的:应用串联质谱标签(TMT)定量蛋白质组学技术,探讨调气解郁法针刺治疗慢性不可预测温和应激(CUMS)诱导的抑郁模型大鼠的作用机制。方法:将36只SD大鼠随机分为正常组、模型组和针刺组,每组12只。采用CUMS诱导抑郁大鼠模型。造模成功后,针刺组于“百会”“印堂”给予针刺治疗20min,1次/d,连续21d。采用旷场实验、糖水偏好实验及强迫游泳实验评价大鼠行为学改变;以TMT蛋白质组学获取各组大鼠海马的差异蛋白并富集分析相关信号通路;以Westernblot法及免疫荧光法验证差异通路蛋白。结果:与正常组比较,模型组大鼠总穿格次数及总行走距离减少(P<0.05),糖水偏好百分比下降(P<0.05),漂浮不动时间延长(P<0.05);与模型组比较,干预后针刺组大鼠总穿格次数、总行走距离及糖水偏好百分比增加(P<0.05),漂浮不动时间缩短(P<0.05)。蛋白质组学结果显示,正常组、模型组的差异蛋白有71种;其中与模型组比较,针刺调控差异蛋白下调的有15种、上调的有5种。经过GO和KEGG富集分析发现,这些差异蛋白主要集中在凝血系统调控、丝裂原活化蛋白激酶(MAPK)信号通路等。Westernblot法验证结果显示,与正常组比较,模型组大鼠海马c-JUN、磷酸化c-JUN氨基末端激酶(p-JNK)蛋白表达水平升高(P<0.05);与模型组比较,针刺组大鼠海马c-JUN、p-JNK蛋白表达水平降低(P<0.05)。免疫荧光染色结果显示,与正常组比较,模型组大鼠海马CA1区、CA3区、DG区c-JUN、p-JNK蛋白平均荧光强度增强(P<0.05);与模型组比较,针刺组大鼠海马CA1区、CA3区、DG区c-JUN、p-JNK蛋白平均荧光强度减弱(P<0.05)。结论:调气解郁法针刺可明显改善CUMS抑郁模型大鼠抑郁样行为,其作用机制涉及多靶点、多通路,MAPK/JNK信号通路可能是其中之一。

Objective To investigate the possible mechanism of“regulating qi and relieving depression”acupuncture underlying improvement of chronic unpredictable mild stress(CUMS)-induced depression in rats by using Tandem Mass Tags(TMT)quantitative proteomics technique.Methods Thirty-six male SD rats were randomly divided into control,model and acupuncture groups,with 12 rats in each group.The depression model was induced by CUMS stress for 21 days.After the depression model was successfully established,the rats in the acupuncture group received manual acupuncture stimulation at“Baihui”(GV20)and“Yintang”(GV24+)for 20 min,once daily for 21 days.Open field test,sugar water preference test and forced swimming test(FST)were used to evaluate the behavioral changes.TMT quantitative proteomics was used to obtain differential proteins in the hippocampus tissue and related signaling pathways enrichment was analyzed,followed by verifying differential protein pathways by using Western blot and immunofluorescence methods.Results Behavior tests showed that on the 21+)for 20 min,once daily for 21 days.Open field test,sugar water preference test and forced swimming test(FST)were used to evaluate the behavioral changes.TMT quantitative proteomics was used to obtain differential proteins in the hippocampus tissue and related signaling pathways enrichment was analyzed,followed by verifying differential protein pathways by using Western blot and immunofluorescence methods.Results Behavior tests showed that on the 21(st)and 42(st)and 42(nd)days,the horizontal crossing times,walking distance and percentage of sugar water consumption were significantly decreased(P<0.05),while the immobility time of FST was obviously increased(P<0.05)in the model group relevant to the control group.After acupuncture intervention,the horizontal crossing times,walking distance and percentage of sugar water consumption were significantly increased(P<0.05),and the immobility time was apparently decreased(P<0.05)in the acupuncture group relevant to the model group.The TMT quantitative proteomics of hippocampus tissue displayed that of the 71 differential proteins(model group vs control group),32 was down-regulated and 39 up-regulated in the model group;and among the above 71 differential proteins,there were 20 differential proteins between acupuncture group and model group,15 down-regulated and 5 up-regulated in the acupuncture group(vs the model group).The expression of Mapk8ipl was up-regulated in the model group(vs the control group)and down-regulated in the acupuncture group(vs the model group).GO and KEGG enrichment analysis showed that these acupuncture-related differential proteins mainly involve the regulation of blood coagulation system,MAPK signaling pathway,etc.We selected the MAPK/JNK signaling pathway related to depression for verification.Western blot showed that the expression levels of c-JUN and phosphorylated c-JUN terminal kinase(p-JNK)proteins in the hippocampus were up-regulated in the model group relevant to the control group(P<0.05);while the expression levels of c-JUN and p-JNK proteins in the hippocampus were down-regulated in the acupuncture group relevant to the model group(P<0.05).The results of immunofluorescence showed that the mean fluorescence intensity of c-JUN and p-JNK in hippocampal CA1,CA3 and DG regions was increased in the model group relevant to the control group(P<0.05),while the mean fluorescence intensity of c-JUN and p-JNK in hippocampal CA1,CA3 and DG regions was obviously lower in the acupuncture group than in the model group(P<0.05).Conclusion Acupuncture for“regulating qi and relieving depression”can significantly improve depression-like behavior in CUMS-induced depression model rats,which involves multiple targets and multiple pathways,including MAPK/JNK signaling.