| Objective: To observe the changes of behavior and sleep in rats that were acupuncture on“Qiao” meridians,and investigate the effect of acupuncture on the Clock and Per2 gene expression in SCN,and to observe whether the efferent pathway has an effect on the Glu/ GABA-Gln metabolic loop by real-time fluorescent quantitative PCR(RT-PCR)and Western Blot(WB)technology,immunohistochemical staining(IHC),enzyme-linked immunosorbent(ELISA)technology.Then we can explore the mechanism of acupuncture “Qiao” meridians to treat insomnia.Methods: 1.Basic physical conditions changes of insomnia SD rats: 48 male SD rats were randomly divided into 4 groups with 12 rats each group: control group,model group,estazolam group and acupuncture group.The insomnia model was reproduced by intraperitoneal injection of PCPA.Rats of estazolam group will be treated by gavage with eszolam solution(0.08mg/ml,0.558mg/kg)once a day for five days.Rats in acupuncture group will be treated by acupuncture on “shenmai” and “zhaohai”.To observe the changes of body weigh,urine and stool between different stages.2.Behavioral change of insomnia SD rats: After Grouping,model building and treating(the same as method 1),the behavioral difference(Spatial memory ability,cognitive ability,spontaneous activity)between different groups and stages will be observed through water maze experiments and open field test.3.The change in sleep experiment of insomnia SD rats: After model building and treating,the effect of different treatment on sleep latency and sleep duration in rats induced by pentobarbital sodium(subthreshold dosage and above-threshold dose)were studied.4.The change in Per2,Clock genes and Glu/GABA-Gln metabolism loop of insomnia SD rats: At the end of the intervention treatment,Half of the rats in each group were removed from the hypothalamus and suprachiasmatic nucleus to observe the content of Per2,Clock,GABA,Glu,GAD and GS;The other half of the rats were used to observe the positive expression rate of GABAA after cerebral perfusion Results: 1.Basic physical conditions changes of insomnia SD rats(1)The baseline of the 4 groups showed no statistical differences on body weigh,urine and stool(p>0.05).(2)Body weight: compared with the control group,The decrease in weight gain of other three groups were statistically significant(p<0.05);Compared with the model group,acupuncture and estazolam could slow down the decrease and the difference was statistically significant(p<0.05).(3)Defecation: compared with thecontrol group,the amount of growth in defecation were statistically significant(p<0.05);After the intervention,estazolam group and acupuncture group were tend to be normal and showed a statistically significant compared with model group(p<0.05).2.Results of behavioral changes of insomnia SD rats:(1)The baseline of the 4 groups showed no statistical differences on behavioral behavior(p>0.05).(2)Water maze experiment: compared with the control group,making model can reduce the number of target quadrant entry,the percentage of target quadrant entry,the target quadrant retention time and the percentage of target quadrant retention time,with statistically significant differences(p<0.05).After treatment,all of the four index were tend to normal,but only the number of target quadrant entry show a significant difference(p<0.05).(3)Open-field test: compared with the control group,after making model,the increase of standing times were statistically significant(p<0.05),but the rest time increased,the exercise time decreased,and the distance decreased has no statistically significant(p>0.05);compared with the model group,the standing times of rats in the estazolam group and acupuncture group were decreased,but only the acupuncture group showed statistically significant(p<0.05).The rest time decreased,while the exercise time increased with a statistically significant(p<0.05),the exercise distance increased but difference has no statistically significant(p>0.05).3.The change in sleep experiment of insomnia SD rats(1)Effects of modeling on sleep in rats Synergistic sedation experiment with pentobarbital sodium: compared with control group,the rat in other three groups have a longer sleep latent period and a shorter sleep time,which the difference was statistically significant(p<0.05).Synergistic hypnosis experiment of pentobarbital sodium: compared with control group,the other three groups have a longer sleep latent period and a shorter sleep time,which the difference was statistically significant(p<0.05).(2)Effects of treating on sleep in rats Synergistic sedation experiment with pentobarbital sodium: compared with control group,model group has a longer sleep latent period,which the difference was statistically significant(p<0.05),and a shorter sleep time has no statistically significant(p>0.05).Compared with the model group,no matter estazolam or acupuncture all can reduce the sleep latent period(p<0.05),but only estazolam can improve the sleep time(p<0.05).Synergistic hypnosis experiment of pentobarbital sodium: compared with the control group,model group has a longer sleep latent period and shorter sleep time,the difference was statistically significant(p<0.05).Compared with the model group,the sleep latency was shortened in estazolam group and acupuncture group,but the difference was not statistically significant(p>0.05).However,estazolam and acupuncture can improve the sleep time significantly(p<0.05).4.Effects of acupuncture on Clock,Per2 gene and Glu/ GABA-Gln metabolic loop in SD rats with insomnia(1)The results of the ELISA showed :compared with the blank group,the concentration of GABA in the hypothalamus of the model group decreased,while the concentration of Glu increased,and the difference was statistically significant(p<0.05).The concentration of GABA increased and the concentration of Glu decreased in the hypothalamus of estazolam group and acupuncture group,and the difference was statistically significant(p<0.05).(2)Immunohistochemical results of GABAA in the hypothalamus showed that:compared with the blank group,there were more GABAA positive expressions in the hypothalamus of the rats in the model group,and the average optical density level increased,with statistically significant differences(p<0.05).There were less GABAA positive expression in the hypothalamus of estazolam group and acupuncture group,and the average optical density value was lower than that of the model group,and the difference was not statistically significant compared with the blank group(p>0.05).(3)WB test results showed that: compared with the blank group,the expressions of PER2 and CLOCK in SCN of the model group rats decreased,the differences were statistically significant(p<0.05).After treatment,the CLOCK and PER2 protein expressions in estazolam group and acupuncture group were higher than those in the model group,and the difference was not statistically significant(p>0.05)compared with the blank group.Compared with the blank group,the GAD protein expression level in the hypothalamus of the model group decreased and the GS protein expression level increased,the difference was statistically significant(p<0.05).The GAD protein expression level in the hypothalamus of estazolam group and acupuncture group increased,while the GS protein expression level decreased,has no statistically significant compared with control group(p>0.05).(4)RT-PCR results showed that compared with the blank group,the relative expressions of Clock mRNA and Per2 mRNA in SCN of rats in the model group decreased,and the difference was statistically significant(p<0.05).After treatment,the relative expressions of Clock mRNA and Per2 mRNA in SCN of rats in estazolam group and acupuncture group increased,the difference was not statistically significant compared with the blank group(p>0.05).Conclusion: 1.Acupuncture“Qiao”merdians to treat insomnia is safe and effective,which can shorten the latency of sleep and prolong the sleep time.2.Acupuncture“Qiao”merdians can effectively alleviate the metabolic disorders caused by insomnia,aslo have the effect of stabilize body weight,and maintain the normal amount of defecation.3.Acupuncture“Qiao”merdians can effectively alleviate the effects on spatial memory ability,cognitive ability and spontaneous activity caused by insomnia and make them back to normal.4.Acupuncture“Qiao”merdians can increase the mRNA and protein expression levels of Per2 and Clock genes in SCN,meanwhile,it can increase GABA concentration and GAD protein expression level In the hypothalamus,while Glu concentration,GS protein expression levels and GABAA positive expression were decreased.5.The mechanism of acupuncture“Qiao”merdians for insomnia treatment may be related to the core clock genes and the Glu/ GABA-Gln metabolic loop. |
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