Study On Effective Target Screening Of Electroacupuncture For Antidepressant Based On ITRAQ Technique

ObjectiveDepression, addiction and neurology diseases are main problems in global public health. Comorbid disorder among the three kinds of diseases which is gradually become the attention spo.However,the physiological mechanism of depression is not clear, but the clinical effect on depression by electroacupuncture(EA) has been widely recognized, EA can adjust the combination of multiple targets.Meanwhile,EA can decrease toxic and improving the quality life of people. Proteins play crucial part in human physiological activity process, disease, the body’s response and disease outcomes.In our study,We choose chronic restraint stress rats pre-treated by EA,based on Immunohistochemical technique(IHC), ITRAQ, Western blot to detect the proteins expressed on hippocampus.Methods1. Choosing chronic restraint stress combine solitary raise made depressive-like model.2. Using open-field test and bodyweight to evaluate whether the model was successful.3. Using IHC technique show the expression of IL-1β,IL-6,TGF-β and syntaxin-1 on hippocampal CA3 region.4. Using iTRAQ to mark protein peptides of hippocampus, after separating by strong cation exchange(SCX), quantitative identification of the protein peptide by Triple TOF 5600. Then select Mascot for analyse, describing differential protein spectrum. To analyse the differential proteins by GO ontology, Cluster of Orthologous Groups of proteins (COG) database and KEGG pathway.5. Using Western blot verify a part of differential proteins.Results1. On days 28, the crossing number, rearing number and bodyweight in model group were significantly lower than those in control group(P< 0.01), it indicated that the model is successful. Comparing to model group, the crossing number, rearing number and bodyweight were higher than model group(P< 0.01).2. After a 28-day procedure, It showed that the expression of IL-1βin model group was significantly increased compared with that in control group and EA group based on IOD value (P< 0.01).The expression of IL-6 in model group was significantly increased compared with that in control group(P<0.01). whereas there were no significantly difference of IL-1β and ILA-6 between EA group and control group (P> 0.05).3. On days 28, according to the IOD value of TGF-βin model group was significantly increased compared with that in control group, indicating that restraint stress can stimulate the expression of TGF-beta, whereas EA reduced its expression, but the effect was not of statistical significance(P> 0.05).4. After a 28-day procedure, IOD value of syntaxin-1 in model group was significantly decreased compared with that in control group (P<0.01) and EA group (P<0.01).5. Using iTRAQ, thirty three differential proteins were selected between model group and EA group. Through COG database, we predicted that these proteins may function to classify statistics. Among the differential proteins, eighteen proteins in model group were decreased which including four uncharacterized proteins; fifteen proteins in model group were increased which including two uncharacterized proteins. After GO enrichment, terms from the process ontology mainly includes response to nutrient levels, locomotory behavior, response to extracellular stimulus, catechol-containing compound biosynthetic and dopamine biosynthetic process, etc. Terms from the function ontology mainly includes ferric iron binding, dopamine binding, catecholamine binding, etc. Terms from the component ontology mainly includes haptoglobin-hemoglobin complex and cell projection. COG mainly includes function unknown, general function prediction only, amino acid transport ans metabolism, etc.6. Using western blot,compared with control group, the express of DAT in rat’s hippocampus was significantly higher(P< 0.01).After 28 procedure,DAT express in EA group was lower than model gourp,but there was no significant difference (P > 0.05).Th expression of Th among the three groups have no significant difference(P> 0.05). Compared with control group,rats Krpc expression in model group increased significantly(P< 0.01).After 28 days pre-treatment, Krpc expression in EA group was lower than that in model group(P< 0.05). Compared with control group and EA group, Mapt expression in model group was lower(P< 0.05).Conclusions1. The results of behavior test indicated that chronic restraint stress combine solitary made the depressive-like model success.EA has a great adventage on improving the bodyweight, activities and exploration of rats.2. EA can improve the damage of neurons by chronic restraint stress and solitary, reduce the relase of inflammatory mediators, in addition, EA can improve the expression of syntaxin.So as to play a protection role in hippocampal neurons, especially in CA3 region.3. Based on Itraq, twenty seven differential proteins were selected from model group and EA group.Then anlaysed the biological processes,gene function and cell position of the differential proteins, combining to HAMD seven factors do further discussion. To provide a scientific basis for EA pre-treatment.4. There is a high comorbidity existence among depression, addiction and neurology diseases.5. DAT, Th, Krpc and Mapt were tested by western blot, the results indicated that they are all the key targets in antidepression.The expression of Krpc and Mapt are consistent with Itraq results.Although DAT and Th were not vertified due to some reasons, based on the catecholamine hypothesis, EA may play antidepressant effect: by regulating the Dopaminergic synapse pathway.6. iTRAQ is a kind of stable and reliable proteomic detection method.