| Depression is one of the most common mental illnesses at present.The obvious persistent and incompatible state of mood is the main clinical features of the disease,accompanied by corresponding cognitive and behavioral changes.The disease has a high incidence,suicide rate and so on.At present,acupuncture has significant curative effect and no side effects in the treatment of depression.Therefore,it is of great clinical and social significance to strengthen the mechanism research of acupuncture on depression.ObjectiveIn this experiment,the rat model of chronic mild and unpredictable stress(CUMS)was established,and continues the research on the anti-apoptosis effect of acupuncture on depression in the previous study of this research group.The SP600125 blocking agent was used to specifically block the c-Jun amino terminal kinase(JNK)signaling pathway,and by detecting the apoptosis-associated protein B lymphoma tumor-2 gene(bcl-2)and cysteine-containing aspartate proteolytic enzyme(caspase-12)expression on the JNK signaling pathway during stress,observing the morphological changes of key factor JNK in hippocampal brain regions,to explore the antidepressant mechanism of acupuncture.Methods1.Experimental animals and grouping:72 clean grade SD rats were randomly divided into normal group,model group,electroacupuncture group,fluoxetine group,model + dimethyl sulfoxide(model + DMSO)group,model + blocker group(model+SP group),electroacupuncture + blocker group(electroacupuncture + SP group),and fluoxetine + blocker group(fluoxetine + SP group),8 groups in total,9 rats in each group.2.Processing method:after one week of adaptive feeding,lateral ventricular cannula was implanted in rats requiring injection of solvents and blockers.The 7d was restored after operation.The normal group reared in groups,free to feed water,and did not give any intervention.Normal groups live in groups and freely ingest water without any intervention.The electroacupuncture group received acupuncture 30 minutes before model establishment,and the fluoxetine group received intragastric administration 30 minutes before model establishment.The model + DMSO group was administered intracerebroventricular injection of DMSO solution 1 hour before model establishment.In the ventricles of the model+ SP group,electroacupuncture+SP and the fluoxetine + SP group,lOul/d diluted SP600125 blocker was injected.3.Modeling methods:in addition to the normal group after the cannula embedding,the rest of the rats adopt the internationally recognized CUMS modeling method:single cage rearing,7 kinds of unpredictable stimulation methods(24h fasting;24h water cut;2h restraint;shaking for 30 min;clipping tail 3 min;moist padding 24 h;night light 12 h)stimulating continuously for 21 days,with an average of 3 times per stimulus.4.Cannula embedment method:refer to previous studies for the following operations.First,the rats were intraperitoneally anesthetized with 10%chloral chloral(0.3ml/100g),and then fixed to the stereotaxis.The anterior fontanelle was fully exposed at the level of the skull.Location of lateral ventricle:first locate the anterior fontanelle,perforate the skull with 0,9mm and 1.5mm,and penetrate the skull.Apply the denture base resin(denture powder and denture water mixture)to the injection sleeve and fix it around.In addition to injection into the lateral ventricle,the casing was covered by casing caps for the rest of the time.The postoperative recovery time was 7d.Penicillin was needed three days before the recovery phase to avoid postoperative infection.The lateral ventricle was then injected with a blocker.5.Intervention method:select the bilateral "Neiguan" and "Sanyinjiao" points,and use size 30 1-inch needle to perform a vertical penetration with a depth of 0.3-0.5 cm.Connect the HANSLH-202 electro-acupuncture instrument to the left and right sides of "Sanyinjiao" at a frequency of 2Hz,current intensity of 0.6mA,acupuncture when the rat limb extremity tremble is appropriate,last for 20 min each time;fluoxetine dubbed with distilled water into a 0.2mg/ml suspension,was administered at 1.8mg/kg concentration.;The above interventions were performed before the daily modelling,once a day for a total of 21 days.Intracerebroventricular injection of DMSO solvent and SP600125 solution(dissolved in DMSO solution at a concentration of 10 mg/ml)was performed at a dose of 10 ul/d and was injected every other day from the beginning of the experiment.6.Detection methods:Behavioral tests:We used the weight gain and sugar water consumption test before and on the 21st day after modeling to evaluate the behavioral changes of depression model rats to determine if the model was successful.Detection of key indicators:ELISA was used to detect the expression of caspase-12 and bcl-2 in hippocampus and serum of rats.Immunohistochemistry was used to detect the expression of JNK in CA1 and CA3 regions of hippocampus in rats.Results1.Effect of acupuncture on behavior of depression model ratsBefore modeling:There was no significant difference in the consumption of sugar and body weight between the groups,indicating that there was no statistical significance(P>0.05)and experiments could be performed.On the 21st day of modeling,compared with the normal group,the difference of weight loss in the model group was statistically significant(P<0.01).Compared with the model group,there was no significant difference between the model +DMSO group and the fluoxetine group+SP group(P>0.05),and the difference between the model +SP group,fluoxetine group,the electroacupuncture group and the electroacupuncture group +SP group was statistically significant(P<0.01).Compared with the fluoxetine group,the body weight increased significantly in the EA group,but the difference was not statistically significant(P>0.05).Compared with the normal group,the difference of sugar water consumption in the model group was statistically significant(P<0.05).Compared with the model group,the decrease of sugar water consumption in the model +DMSO group,the model +SP group and the electroacupuncture group was statistically significant(P<0.05),the glucose water consumption in the fluoxetine +SP group decreased significantly(P<0.01),the fluoxetine group and the electroacupuncture group.There was no significant difference in the consumption of sugar and water(P>0.05);there was no significant difference in the consumption of sugar between fluoxetine group and EA group(P>0.05).2.Effect of acupuncture on key molecules of JNK signaling pathway in depression model ratsCompared with the normal group,the caspase-12 protein expression in the hippocampus of the model rats was significantly higher than that in the model group(P<0.01).Compared with the model group,the expression of caspase-12 protein in the hippocampus of the hippocampus of model +DMSO group,model +SP group,fluoxetine group,fluoxetine +SP group,electroacupuncture group and electroacupuncture group +SP group decreased significantly.There was no significant difference in learning meaning(P<0.01)between fluoxetine group and EA group(P>0.05).Compared with the normal group,the expression of serum caspase-12 protein in the model group was decreased.Compared with the model group,the expression of caspase-12 protein in the serum of model +DMSO group,model +SP group,fluoxetine group and electroacupuncture group was decreased,but the expression of caspase-12 in the group of fluoxetine +SP group and Electroacupuncture group increased,but the difference was not statistically significant(P>0.05).Compared with the normal group,the expression of Bcl-2 protein in the hippocampus of the model rats was significantly decreased(P<0.01).Compared with the model group,the expression of the model +DMSO group increased(P<0.05).The serum Bcl-2 protein expression in the model +SP group,fluoxetine group,fluoxetine +SP group,electroacupuncture group and Electroacupuncture +SP group increased significantly(P<0.01);fluorine West was significant.There was no significant difference between Ting Group and EA group(P>0.05).Compared with the normal group,the expression of Bcl-2 protein in the model group was significantly decreased(P<0.01).Compared with the model group,the difference between the model+DMSO group and the model +SP group was not statistically significant(P>0.05).The expression of Bcl-2 protein in the hippocampal Bcl-2 in the fluoxetine group and the electroacupuncture group of +SP group were all significantly higher than that in the group of fluoxetine +SP(P<0.05)and the fluoxetine +SP group.The significant difference in the expression of electroacupuncture group was significant(P<0.01),but there was no significant difference between the fluoxetine group and the electroacupuncture group(P>0.05).Compared with normal group,the expression of JNK in hippocampal CA1 area of model+DMSO group increased significantly,and the difference was statistically significant(P<0.01).Compared with the model group,the expression of JNK in the CA1 region of the model+DMSO group was significantly higher(P<0.01).Compared with the model +DMSO group,the expression of fluoxetine group,fluoxetine +SP group and electroacupuncture group decreased significantly(P<0.01),and the JNK table of fluoxetine group decreased but the difference was not statistically significant(P>0.).05).Compared with normal group,the expression of JNK in hippocampal CA3 area of model +DMSO group increased significantly,and the difference was statistically significant(P<0.01).Compared with the model group,the expression of JNK in the CA3 region of the model +DMSO group was significantly increased(P<0.01).Compared with the model +DMSO group,the expression of the model +SP group,fluoxetine group,fluoxetine +SP group,the electroacupuncture group and the electroacupuncture +SP group decreased significantly,and the difference was statistically significant(P<0.01).Compared with the electroacupuncture group,the expression of JNK in the fluoxetine group was decreased but the difference was different.There was no statistical significance(P>0.05).Conclusions1.Behavioural results show that chronic unpredictable stress leads to a decrease in weight gain in rats,which is improved after electroacupuncture and fluoxetine intervention.2.The effect of electroacupuncture on the decrease of body weight gain in rats may be that electroacupuncture and fluoxetine can be achieved by inhibiting the activity of JNK,caspase-12,and bcl-2 activation,and electroacupuncture and fluoxetine intervention may be effective in the depression model.The central role of rats is more pronounced than peripheral blood serum.3.The effect of electroacupuncture and fluoxetine intervention on behavioral improvement in depressive model rats was not significantly different. |
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