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.