The Effect Of Acupuncture On The JNK Signal Transduction Pathway In CUMS Rats Was Blocked By SP600125

BackgroundDepression has seriously affected the quality of human life.February 23,2017,the World Health Organization(WHO)website published:the global incidence of depression accounted for 4.3%of the total population,has reached 322 million;2005 to 2015,the number of patients increased by 18.4%;depression in China Rate of 4.2%,total disability loss of healthy life(YLD)of 8,981,401 person-years.The pathogenesis of depression is more complex,involving multiple functional disorders,and social-psychological-physiology is closely related to its pathogenesis still exists in many unknown areas.JNK(cN-Jun N terminal kinase,JNK)signal pathway can be activated by external stress stimulation and/or cytokines,the activation of JNK Pathway can lead to apoptosis of hippocampal neurons.And,its specific blocker SP600125 can selectively inhibit JNK,JNK blockade with a high degree of specificity by this blocker.Studies have shown that SP600125 has a synergistic effect of electroacupuncture and fluoxetine on the acute stress status of forced swimming model rats.With the function of blocker,the mechanism of acupuncture treatment will be further explored in this study.Purpose1.From the clinical and experimental research status of depression,this study tries to analyze the mechanism of depression and the relationship between acupuncture and fluoxetine on JNK signal transduction pathway of CUMS rats;2.Using the method of lateral ventricle injection,this study compares and observes the differences between the 2 modes of route of administration of blocking agent SP600125.3.Comparing with the former studies,this study observes the morphological changes of key factors in protein level,gene level and key factor distribution in different brain regions,and to explore the effect of acupuncture on JNK signal transduction pathway,in order to provide clinical Valuable experimental data.Methods1.Animal grouping:clean SD rats were randomly divided into 8 groups after adaptive feeding:control group,model group,model+acupuncture group,model+ fluoxetine group,model+DMSO group,model+SP600125 group,model+acupuncture+SP600125 group,model+fluoxetine+SP600125 group(n=9)2.Modeling methods:isolation combined with recognized CUMS model rats---7 kinds of unpredictable stimulation methods(ambrosia for 12h,removing water for 24h,horizontal vibrating for 30min,ambrosia for 24h,reversing day and night for 24h,clipping tails for 3min,constrainting for 2h),continuous stimulation of 21 days,the average application of each method used in the experiment was 3 times.3.Intervention methods:exclude control group,model group,model + acupuncture group and model + fluoxetine group,the other four groups were used lateral ventricle surgery,and embedded casing pipes,postoperative recovery 1 week(3 Days intraperitoneal injection of penicillin against infection).The control group did not give any intervention,free diet and water;model group:randomly selected 1 stress stimulation method used each day,1 time/day;model + DMSO group:1/2h before modeling,lateral ventricle injection of solvent DMSO 10ul/Day;model+SP600125 group:1 hour before stress stimulation,lateral ventricle injection of solution SP600125 10ul/day;model + acupuncture group:1h before stress stimulation,point through point acupuncture of Bai hui and In tang,simple sting of Zu san li,20min/day;model +acupuncture + SP600125 group:1 hour before stress stimulation,giving acupuncture,and 1/2h before stress stimulation,lateral ventricle injection of solution SP600125 10ul/day;model + fluoxetine group:1 hour before stress stimulation,intragastric administration at a does of 10ml/kg,1 time/day;model + fluoxetine + SP600125 group:1hour before stress stimulation,giving fluoxetine gavage,and 1/2hour before the stress stimulation,lateral ventricle injection of solution SP600125 lOul/day.4.Datas and methods of testing4.1 weight and behavioral changes,a total of 4 times(Before modeling,modeling day 7,day 14,day 21);4.2 ELISA:Detecting the protein expression changes of Caspase-3,HSP70,JNK,p-MKK4 and p-MKK7 in the hippocampus and Caspase-3 and HSP70 in the serum.4.3 Western Blot:Detecting the protein expression changes of JNK and p-JNK in the hippocampus.4.4 Rt-PCR:Detecting the gene level changes of MKK4,MKK7 and JNK in hippocampus,frontal lobe and hypothalamus.4.5 Immunohistochemistry:Detecting the morphological changes of JNK and c-Jun in hippocampus.Results1.Behavioral test:There were no significant differences in behavioral testing among the groups before the experiment.The gradually differences appeared after modeling showed that the model rats were successfully established.After intervention of treatments,according to the differences among the intervention groups,it can be seen that the conditions of blockers and different treatments have different effects on behavior.2.ELISA test results2.1 Caspase-3 can induce apoptosisHippocampus Compared with normal group,A + SP group has highly significant difference(P<0.01);compared with model group,model + F + SP group has significantly different(P<0.05),model + A+ SP group has highly significant difference(P<0.01);compared with model+ A group,model + A+ SP group has significant difference(P<0.05);compared with model + F + SP group has significant difference(P<0.05).Serum Only model + F + SP + acupuncture group and model group have significantly differences(P<0.05),the other groups had no significant difference2.2 HSP70 is a stress-stimulating response factorHippocampus Compared with normal group,the model + A group,model + A + SP group have highly significantly different(P<0.01);compared with model group,the model + A group,model + A + SP group have highly significantly different(P<0.01);compared with model + A group,there are highly significant differences among normal group,model group,model + SP and model + F group(P<0.01);compared with model + F group,there are highly significant differences between acupuncture group and model + A + SP group(P<0.01).Serum Compared with normal group,model + F group has significant differences(P<0.05);compared with model + F group,normal group and model + SP group have significantly differences(P<0.05)2.3 JNK is a key factor in the signal transduction pathwayHippocampus Compared with model + A group,there are highly significant differences among model + SP,model + F,model + F + SP groups(P<0.01);compared with model + F group,There are highly significant differences from model + A group(P<0.01).2.4 p-MKK4 is an actived factor of upstream in the JNK signaling pathwayHippocampus The significance data among groups are 0.699<0.05,which indicats no significance differences among groups.2.5 p-MKK7 is an actived factor of upstream in the JNK signaling pathwayHippocampus Compared with control group,there are significant difference among model group,and model + A + SP group(P<0.05);compared with model group,there are significant differences between control group(P<0.05).3.Western Blot test result3.1 JNK is a key factor in the signaling pathway Hippocampus There was no significant difference among the groups(P>0.05).3.2 p-JNK is the JNK factor that is activated in the signaling pathwayHippocampus Compared with control group,there are highly significant differences among model group,model + D Group was a significant differences(P<0.01),there is significant differences between model + F group(P<0.05);compared with model group,there are highly significant differences between control group(P<0.01),there are significant differences model + F + SP and model + A + SP(P<0.05);compared with model + F group,there are significant differences between contral group(P<0.05).4.Rt-PCR test results4.1 MKK4 is the upstream key factor of JNK signaling pathwayHippocampus Compared with the control group,there are highly significantly differences among the model group,model + SP,model + D,model + F group(P<0.01);compared with the model group,there are highly significantly differences among the control group,model+F + SP,mode 1+ A,model + A + SP group(P<0.01),there are significant differences among the model + SP,model + F group(P<0.05);compared with the model + A group,there are highly significantly differences among model group and model + D group(P<0.01);compared with model + F group,there are highly significant differences between with control group(P<0.01),there are significant differences between with model group(P<0.05).4.2 MKK7 is the upstream key factor of JNK signaling pathwayHippocampus Compared with control group,there are highly significant differences among model group,model + D group(P<0.01);compared with model group,there are highly significant differences among control group,model + SP,model + F,model + F + SP model,model + A,model + A+ SP group(P<0.01);compared with the model + A group,there are highly significant differences between with model group(P<0.01);compared with the model+ F group The model group is highly significantly different(P<0.01).4.3 JNK is a key factor in the signaling pathwayHippocampus Compared with control group,model group,there are highly significant differences among model group,model + D,model + F,model + A group(P<0.01),model +SP group is significantly different(P<0.05);compared with the model group,there are highly significant differences among control group,model + F + SP,model + A + SP group(P<0.01);compared with model + A group,there are highly significant differences between with control group(P<0.01),there was significant difference between model + F + SP,model + A + SP group(P<0.05);compared with model + F group,control group is highly significantly different(P<0.01).5.Immunohistochemical test results5.1 JNK is a key factor in the signaling pathwayHippocampus CA1 Compared with model + F group,there are significant differences between with model + D group(P<0.05),highly significant differences between with model+ A+SP(P<0.01).Hippocampus CA3 Compared with model + A group,there are significant differences between with model + D group(P<0.05);compared with model + F group,highly significant differences can be seen in model + D group(P<0.01).5.2 C-Jun is the key downstream factor in the JNK signaling pathwayHippocampus CA1 Compared with control group,there are highly significant differences between with model + F group(P<0.01).Hippocampus CA3 There is no significant difference among the groups(P>0.05).Conclusions1.Behavioral test shows that CUMS model rats are successfully established;2.SP600125 can block the key protein and mRNA(JMK?MKK4?MKK7?Caspase-3)of JNK signal transduction pathway by acupuncture;3.Acupuncture can inhibit JNK signaling pathways,and decrease neurons apoptosis of CA1,CA3 area in hippocampal to be antidepressant role;4.Acupuncture can achieve the effect of antidepressant through peripheral blood and JNK signal transduction pathway in hippocampus.