Effects Of“Shugan Tiaoshen”Acupuncture Method On 5-HT_1AR、_2AR In Hippocampus And GABA_AR-α₁ In Hypothalamus Expression Of Insomnia Rats

Objective:To observe and analyze the effects of“Shugan Tiaoshen”acupuncture method on behavior,sleep time,5-HT_1AR,_2AR and hypothalamus of sleep deprived rats objective to reveal and explore the molecular biological mechanism of“Shugan Tiaoshen”acupuncture method intervention on sleep deprivation effect from the perspective of neurotransmitter receptor signal response in related brain regions,so as to provide the basis for the research and clinical application of acupuncture treatment of insomnia.Methods:70 SPF healthy Wistar rats（male）were randomly divided into blank group（n=10）,and the rest of the rats were clamped tail as stimulation combined with injected intraperitoneally p-chlorophenylalanine（PCPA）to establish sleep deprivation model.After 16days of modeling,50 rats were randomly divided into model group,grab group,western medicine group,acupuncture group and sham acupuncture group.The grab group was grabbed according to the fixed way of the acupuncture group every day;the western medicine group was given estazolam by gavage every day and grabbed the same way;the acupuncture group was given acupuncture at“Baihui”,“Shenmen（shuang）”,“Neiguan（shuang）”and“Taichong（shuang）”,and each point was given acupuncture for 1 minute,once a day,for 7days;the sham acupuncture group was given acupuncture at at the tail root,and 1/3 of upper,middle and lower parts of the tail,once a day one time,7 minutes each time,for 7 days.After treatment,the behavioral changes of rats in each group were compared by high plus maze and open field test;the changes of sleep latency and sleep wake cycle were analyzed by pentobarbital sodium sleep test;western blot was used to analyze the effects of“Shugan Tiaoshen”acupuncture method on the expression of 5-HT_1AR,5-HT_2AR in hippocampus and GABA_AR-α₁in hypothalamus.Result:1.Changes of general signs of rats in each group（1）Changes of physical signs:the rats in the blank group had normal hair color,good mental state and no abnormal performance;the rats in the model group showed increased excitability and aggressiveness after the first injection of PCPA,and these rats showed disordered and dim fur,spirit dispirited and other depressive states 72 hours later;the rats in grab group and sham acupuncture group were similar to the rats in model group;the hair color and general state of rats in western medicine group and acupuncture group were improved.（2）Changes of diet,drinking water and body weight:compared with the blank group,the daily diet,drinking water and body weight of rats in model group decreased significantly（P<0.01,P<0.01,P<0.05）;compared with the model group,there was no significant difference in diet,drinking water and weight gain of grab group（P>0.05）;compared with grab group,western medicine group and acupuncture group had more diet（P<0.05,P<0.01）,less drinking water（P<0.01）and more weight gain（P<0.05）;compared with the western medicine group,there was no difference in the above indexes in acupuncture group（P>0.05）;compared with sham acupuncture group,there were significant differences in diet,drinking water and weight gain indexes in the acupuncture group（P<0.01）.2.Behavioral changes of rats in each group（1）Comparison of high plus maze test in rats of each groupCompared with the blank group,the open arm entry ratio and the open arm time ratio of model group were significantly decreased（P<0.01）;compared with the model group,there was no difference in grab group（P>0.05）;compared with grab group,the open arm entry ratio of western medicine group was increased（P<0.05）,the acupuncture group was significantly increased（P<0.01）,and the open arm time ratio of the two groups was significantly increased（P<0.01）;compared with western medicine group,there was no difference in the frequency ratio of entering the open arm in the acupuncture group（P>0.05）,and the time ratio of entering the open arm was higher（P<0.05）;compared with the sham acupuncture group,the above indexes in the acupuncture group were significantly increased（P<0.01）.（2）Comparison of open field test of rats in each groupCompared with the blank group,the horizontal cross grid score and upright score of model group were significantly reduced,and the number of feces was significantly increased（P<0.01）;compared with the model group,there was no statistical difference in the grab group（P>0.05）;compared with the grab group,the horizontal cross grid score and upright score of western medicine group and acupuncture group were significantly increased,and the feces were significantly reduced（P<0.01,P<0.01,P<0.01）;compared with the western medicine group,acupuncture group had no statistical difference（P>0.05）;compared with the sham acupuncture group,acupuncture group had significant difference（P<0.01）.3.Changes of pentobarbital sodium sleep test in rats of each groupsCompared with the blank group,the sleep latency of the model group was significantly longer and the sleep time was significantly shorter（P<0.01,P<0.01）;compared with the model group,there was no difference in the grab group（P>0.05）;compared with the grab group,the sleep latency of western medicine group and acupuncture group was significantly shorter（P<0.05,P<0.01）and longer sleep time（P<0.01）;compared with the western medicine group,there was no difference in sleep latency（P>0.05）and longer sleep time（P<0.05）in acupuncture group;compared with the sham acupuncture group,there was a significant difference in acupuncture group（P<0.01）.4.Changes of neurotransmitter receptor protein expression in brain tissue of rats in each group（1）Comparison of expression of 5-HT_1AR and 5-HT_2AR in hippocampus of rats in each group Compared with the blank group,the expression of 5-HT_1AR in the hippocampus of the model group was significantly decreased,and the expression of 5-HT_2AR was significantly increased（P<0.01）;compared with the model group,there was no difference in grab group（P>0.05）;compared with the grab group,the expression of 5-HT_1AR protein in hippocampus of western medicine group and acupuncture group was significantly increased（P<0.01）,and the expression of 5-HT_2AR protein was decreased（P<0.05,P<0.01）;compared with the western medicine group,the expression level of 5-HT_1AR in acupuncture group was higher（P<0.05）,but there was no difference in the expression level of 5-HT_2AR（P>0.05）;compared with the sham acupuncture group,the above indexes in acupuncture group were significantly different（P<0.01）.（2）Comparison of expression of GABA_AR-α₁in hypothalamus of rats in each groupCompared with the blank group,the expression level of GABA_AR-α₁in the hypothalamus of model group was significantly lower（P<0.01）;compared with the model group,there was no difference in grab group（P>0.05）;compared with the grab group,the expression of GABA_AR-α₁in the hypothalamus of western medicine group and acupuncture group were significantly higher（P<0.01）;compared with the western medicine group,the increase of GABA_AR-α₁in the hypothalamus of acupuncture group had no difference（P>0.05）;compared with the sham acupuncture group,acupuncture group had significant difference（P<0.01）.Conclusion（1）The results showed that insomnia rats showed irritability,delayed reaction,abnormal drinking water and diet,slow weight growth,prolonged sleep latency and shortened sleep time.Both acupuncture and western medicine can improve the general condition and sleep of rats,and the effect of“Shugan Tiaoshen”acupuncture method is better in the change of behavior and prolonging sleep duration of rats.（2）The expression of 5-HT_1AR,5-HT_2AR in hippocampus and GABA_AR-α₁in hypothalamus of insomnia rats were abnormal.Acupuncture and western medicine could up regulate the expression of 5-HT_1AR in hippocampus and GABA_AR-α₁in hypothalamus,down regulate the expression of 5-HT_2AR in hippocampus,and play a role by enhancing inhibitory response and weakening excitatory response.The effect of acupuncture on inhibitory neurons was better than that of western medicine.This provides a neurobiological basis for the intervention of insomnia by“Shugan Tiaoshen”acupuncture method.