| Objective:We aim to study the effect of different stimulation duration on the expression of BDNF-CREB signal pathway protein in hippocampus of rats with autism and explore the time-dependent pattern of electroacupuncture response.Methods:Twenty pairs of SPF Wistar rats were caged overnight according to the ratio of male and female 1:1.The female vaginal suppository was found on next day that was designated as the first day of gestation,which was raised separately in separate cages.On the 12.5th day of pregnancy,sixteen pregnant rats were randomly selected to be intraperitoneally injected sodium valproate solution(the dosage was 600 mg / kg),and the offspring were the model rats.In addition,four pregnant rats were intraperitoneally injected with the same amount of normal saline on the 12.5th day of pregnancy and its offspring was normal.The offspring were screened for developmental behavior(development,orientation,forced swimming,hot plate pain).In Experiment 1,the model rats were randomly divided into model group,hand acupuncture group,EA 5sA group,EA 10 sA group,EA 20 sA group and EA 60 sA group.The blank group and the model group only simulated the grasping process.The hand acupuncture group needled the GV1 and kept the needle for 30 min.The EA group needled the GV1 through the skin to the left and right sides respectively,connecting the electroacupuncture instrument.It stimulated 5s,10 s,20s,60 s respectively according to the group,intermittently for 2min,then stimulated 5s,10 s,20s,60 s respectively.It repeated for 15 times,and frequency 2Hz,continuous wave,continuous intervention for 7 days.The intensity was determined by the slight twitching of the local muscles of the rats without obvious struggle.In the second experiment,the offspring rats were randomly divided into EA 5sB group,EA 10 sB group,EA 20 sB group and EA 60 sB group.Each group had six rats.The way of electroacupuncture was the same as before.The total duration of fixed cumulative intervention was 240 s,and the single stimulation was 5 seconds,10 seconds,20 seconds and60 seconds,once every 2 minutes.The times of four groups were 48,24,12 and 4 times respectively.All groups of rats were tested for open field behavior before intervention(PN23)and after intervention(PN30).Then the hippocampus of rats was taken.The expression of BDNF,Bcl-2,CREB and p-CREB were analyzed by immunohistochemistry and Western blotting.Result:1.Body weight index: compared with the blank group,there was a significant difference in the body weight of PN7(postnatal day 7),PN 14,PN 21 and PN 30 in the model group(P< 0.05).2.Eye opening experiment: compared with the blank group,there was a significant difference in the time of opening eyes in PN 15,PN16(P < 0.05)and no significant difference in PN 12,PN 13,PN 14,which suggested that the time of opening eyes in the model group was later.3.Forced swimming: compared with the blank group,the swimming ability of the model group in PN9,PN11,PN13 and PN15 was lower than that of the blank group of the same agewith a significant difference(P < 0.05).4.Direction tendency: compared with the blank group,there were significant differences in PN7,PN8,PN9 and PN10 in the model group(P < 0.001).5.Hot board induced pain experiment: compared with the blank group,the model group was significantly insensitive to pain,and there were significant differences in PN9,PN11,PN13,PN15 days(P < 0.05).6.Open field behavior: 1)before intervention:there was no significant difference between the groups except the blank group(P > 0.05).Compared with the blank group,autism model rats in experiment 1 and experiment 2 had shorter total distance of activity,slower average speed,less standing exploration,and more repetitive and stereotypical movements(P < 0.05).2)After intervention:(1)Experiment 1: compared with the model group,the total distance and average speed of activity in the hand-acupuncture group,the EA5sA?10sA?20sA?60sA group and the blank group increased significantly(P<0.05);the number of standing and repetitive stereotype decreased significantly in the EA 20 sA group,the EA 60 sA group and the blank group(P<0.05).(2)Experiment 2: Compared with the model group,the total distance and average speed of rats in the blank group,EA 20 sB and60sB group increased,and the number of standing explorations increased.Reduction of repetitive stereotyped actions in each group(P < 0.05).Compared with EA 60 sB,EA 5sB and EA 10 sB had fewer standing times and increased stereotypical movements(P < 0.05).7.Western blot:Experiment 1: 1)Compared with the model group,BDNF protein expression was increased in the blank group,EA 20 sA and 60 sA group,and Bcl-2 protein expression was decreased(P < 0.05).(2)Compared with EA 60 sA group,the BDNF protein expression in hand acupuncture group,EA 5sA and 10 sA group decreased(P < 0.05).2)Compared with the model group,the expressions of CREB and p-CREB in groups EA 20 sA and 60 sA increased(P < 0.05).(2)Compared with EA 60 sA group,the expression of CREB protein in hand acupuncture group and EA 5sA group decreased(P < 0.05).Experiment 2: 1)(1)Compared with the model group,BDNF protein expression increased in the blank group(P < 0.01).Bcl-2 protein expression decreased in the blank group and the EA groups of 10 sB,20sB and 60sB(P < 0.05).Compared with EA 60 sB group,the expression of bcl-2 protein in EA 5sB group was relatively low(P < 0.05).2)(1)Compared with the model group,the expressions of CREB and p-CREB in the EA groups of 20 sB and60sB increased(P < 0.05).(2)Compared with the model group,the expressions of CREB and p-CREB in the EA groups of 20 sB and 60 sB increased(P < 0.05).(3)Compared with EA20 sB group,the expression of CREB in EA 5sB group decreased significantly(P < 0.05).8.Immunohistochemistry:Experiment 1:(1)Compared with the model group,the expression of CREB and p-CREB proteins increased in EA 20 sA and 60sA(P < 0.05).(2)Compared with the EA 20 sA group,CREB and p-CREB protein expression decreased in the EA 5sA group(P < 0.05).(3)Compared with the EA 60 sA group,CREB and p-CREB protein expression decreased in hand acupuncture group,the EA 5sA and 10 sA group(P < 0.05),and p-CREB protein expression decreased in the EA 20 sA group(P < 0.05).Experiment 2:(1)Compared with the model group,the expression of CREB and p-CREB proteins increased in EA 20 sB and 60 sB group(P < 0.05).(2)Compared with the EA 10 sB group,the average optical density of CREB protein in model group and EA 5sB group was decreased significantly(P < 0.05).(3)The average optical density of CREB and p-CREBprotein in EA 5sB group was lower than that in the EA 20 sB group(P < 0.05).(4)Compared with the EA 60 sB group,the average optical density of CREB and p-CREB protein in EA 5sB group and EA 10 sB group was decreased(P < 0.05),while the average optical density of p-CREB protein in EA 20 sB group was decreased(P < 0.05).Conclusion:1.Electroacupuncture at GV1 acupoint can improve the autonomic activity of autistic rats,and the stimulation time of electroacupuncture at 20s(cumulative stimulation for 15times)can improve the abnormal behavior patterns such as stereotyped repetition and reduced interest of autistic rats.2.Compared with normal rats,the expression of BDNF protein in the hippocampus of autistic rats is down-regulated and the expression of Bcl-2 protein is up-regulated.Taking electro-acupuncture 20 s as a unit of stimulation time(cumulative stimulation for 15 times),the expression of BDNF protein can be up-regulated and the expression of Bcl-2 protein can be down-regulated.3.Electroacupuncture,using 20 s as a unit of stimulation time(cumulative stimulation for15 times)can activate the expression of p-CREB and CREB protein in the hippocampus of autistic rats and cause acupuncture response effect.4.Whether the electroacupuncture 20s(cumulative intervention for 15 times)or the electroacupuncture 20s(cumulative intervention for 240s),the stimulation duration in 20 s is the minimum effective time. |
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