The Effect Of Electroacupuncture On The Signaling Pathway Of Chronic Stress Rats

Depression is a mental disorder with high prevalence and mortality,resulting in massive socioeconomic burden.The pathophysiology mechanism of depression has been poorly understood.Nowdays it is most porpular therapeutic target to modulate monoaminergic neurotransmission.But this therapeutic method only just alleviate the symptoms of 30%patients.Furthermore,all the antidepressant drugs exist side effects and lag period for their clinical effects.Acupuncture belong to traditional Chinese medicine and can regulate the balance for human body.The datas,from the animal experiments and clinical reports,show that acupuncture is effective for depression.It has little side effect and can play roles on multiple targets.Although acupuncture is effective for depression,the mechanism for the efficiency is still not clear.The pathogenesis of depression may be relevant to molecular signaling pathway.So chronic stress animal model was chosen for the study.Two signaling pathways were chosen for the mechanism study on acupuncture antidepression.The upper stream and down stream indexes were detected.At the same time,the special inhibitor was chosen for the signaling pathway.We hope to make it clear for the mechanism of acupuncture antidepression.Methods and Results1.Chronic stress depression animal model was established,and Open field test, sucrose intake,and body weight balance were used to evaluate the model.After stress,compared with control group rats,the model group rats'crossing numbers,rearing times,the level of sacchar-consumption,body weight changes,were apparently less than those of control group rats'(P<0.05).In comparison with model group,the crossing numbers,rearing times,body weight changes,the level of sacchar-consumption,being apparently increasing in electroacupuncture group and Prozac group(P<0.05),those indexes were not apparently differences between electroacupuncture group and Prozac group(P>0.05).2.Annexin V-FITC/PI double staining and PI single staining flow cytometric method were used to detect the hippocampal apoptotic rates.Annexin V-FITC/PI double staining result indicated that hippocampal apoptotic rates in model group was higher than control group(P<0.05),apoptotic rates of EA group and Pozac group was lower than model group(P<0.05),EA group was more lower than Pozac group(P<0.05).PI single staining result indicated that there was no difference between model group and control group(P>0.05),apoptotic rates of EA group was signifant different from model group(P<0.05),there was no difference between Pozac group and EA group(P>0.05).3.Real-time PCR method was used to detect Bcl-2mRNA and BadmRNA expression.The results showed that hippocampal Bcl-2mRNA expression for EA group was more higher than model group(P<0.05),there was no difference between EA group and Pozac group(P>0.05).BadmRNA expression in model group was higher than EA group and Pozac group,but the difference was not significant.4.cAMP-PKA signaling pathway is more relative to depression.So the signaling was chosen to detect.cAMP in hippocampus was detected by radioimmunoassay method.Immunohistochemistry method was used to detect PKA expression,and Western blotting method was used to detect CREB protein.The results showed that the level of cAMP in hippocampus for model group is lower than control group(P<0.05), cAMP level for EA group and Pozac group were more higher than model group(P <0.05),there was no difference between EA group and Pozac group(P>0.05).PKA expressin in cortex was almost similar in each groups,Hippocampal PKA expression in Pozac group was stronger than model group(P<0.05).CREB protein level in cortex for EA group was higher than model group,CREB protein level hippocampus for Prozac group was higher than model group.5.ERK is another important signaling pathway for depression.Western blotting method was used to detect ERK signaling pathway.The data showed that Ras,c-raf,p-c-raf,ERK protein level was almost similar in each groups.p-ERK level in cortex for EA group and Pozac group were higher than model group(P<0.05).p-ERK level in cortex for model group was lower than control group(P<0.05).p-ERK level in hippocampus for model group was lower than control group(P<0.05),p-ERK level for EA group was higher than model group(P<0.05).RSK protein level in cortex for EA group was higher than model group(P<0.05),and RSK level in cortex for model group was lower than control group(P<0.05),there was no difference for cortex RSK level between Pozac group and model group(P>0.05).Although RSK level in hippocampus increased for EA group and Pozac group,there was no different from model group (P>0.05).6.Inhibitor was used to detect the ERK signaling pathway function.Chronic forced swimming stress model was established.Open field test and body weight balance were used to evaluate the model.The results showed that,compared with control group, the crossing,rearing times,body weight changes for model group was significant different on day 7(P<0.01) and day13(P<0.05).The open field test and body weight changes for EA group was more apparent.EA group and EA+PD98059group was significant different for crossing times(P<0.05).ERK level in cortex was almost similar for each group.p-ERK level in cortex of EA group was higher than model group (P<0.05) and EA+PD98059 group(P<0.05).p-ERK level in hippocampus for each group was almost similar.There were no differences for BDNF level in cortex of each group.BDNF level in hippocampus of EA group was higher than model group (P<0.05) and EA+PD98059 group(P<0.05).Conclusions1.The data from open field test,sucrose intake,body weight changes showed that chronic stress could cause the rats to move less,suppress body weight gain of rats, decreasing of sucrose intake,whereas electroacupuncture could improve them.2.Flow cytometric results indicated that chronic stress could cause hippoampal apoptotic rates increasing,but electroacupuncture could decrease it.3.Real-time PCR results indicated that chronic stress could cause Bcl-2mRNA decreasing and BadmRNA increasing in hippocampus,but electroacupuncture could reverse it.4.Chronic stress could cause cAMP level decreasing in hippocampus,but electroacupuncutre and Pozac could reverse it.Pozac could cause PKA expression increasing under chronic stress.We thought that Pozac was more effective than electroacupuncture on cAMP-PKA signaling pathway.5.Chronic stress could inhibit the ERK signaling pathway,but electroacupuncture could active ERK signaling pathway under stress by phosphorate ERK and play some roles on antiapoptosis and antidepression.6.Chronic forced swimming model could be used for inhibitor study.The results indicated that electroacupuncture could effect ERK pathway expression,and PD98059 could inhibit its function.