Study On The Effect Of Electroacupuncture On 5-HT Rhythm In Brain And Liver Tissues Of Mice Without Circadian Rhythm

Objective:To observe the effects of electroacupuncture in "Ganshu"(BL18)on the expression of 5-ht in the brain and liver of mice with no circadian rhythm sleep disorders,to investigate the regulation of electroacupuncture on the circadian rhythm of central and peripheral systems,and to preliminarily explain the mechanism of electroacupuncture in "Ganshu"(BL18)on the regulation of sleep-wake cycle disorder.Method:1.A total of 144 SPF grade male C57BL/6J mice at the age of 6 weeks were randomly divided into control group(normal group),model group,acupuncture group,and non-acupoint group by random number table method,with 36 in each group.In the room temperature of 20℃ ～ 25℃ SPF feeding room for 1 week,the control group was raised conventionally,and the model group,acupuncture group and non-acupoint group were built by 2h light/2h dark sleep deprivation method for 5 days.The success of modeling was judged according to the behavioral characteristics of the mice.The acupuncture group began to receive acupuncture on the 13 th day."Giangshu"(BL18)points on both sides were taken and connected to 6805-AII electroacupuncture apparatus for treatment.Both ends of the output wire were connected to bilateral acupuncture points.The density of period is 6 seconds,until the needle’s handle slightly shakes.Each treatment was retained for 20 minutes and continued for 5 days.The control points were fixed under both sides of the mouse without meridian or acupoint,and the method of electroacupuncture was the same as that of acupuncture group.The model group was only fixed in the same way every day without acupuncture intervention.2.After acupuncture treatment,the mice were sacrificed for neck fracture at 12 time points in one day,namely 10:00(CT10)、12:00(CT12)、14:00(CT14)、16:00(CT16)、18:00(CT16)、20:00(CT20)、22:00(CT22)、0:00(CT0)、2:00(CT2)、4:00(CT4)、6:00(CT6)and 8:00(CT8).Three were sacrificed at each time point.The brain tissue was removed and the brain stem tissue was placed in the fixative for later use.After that,the mice were fixed on a plate,and the abdominal skin and subcutaneous tissues were cut open with surgical scissors to exposethe abdominal cavity.The liver tissues were removed and placed in the refrigerator at-80℃for refrigeration.3.Immunohistochemistry was used to detect 5-ht in the mice brain and enzyme-linked immunoassay was used to detect 5-ht rhythm expression in the liver.And then analyzed the data.Results:1.Before the modeling,the mice in each group showed a stable lifestyle,and there was no statistical difference between daytime and night activity levels in each group(P>0.05).After modeling,the mice in the modeling group began to show mania,increased food intake,frequent daytime activities,and had no regular sleep time.There was a significant difference between the modeling group and the control group(P<0.01),indicating that the modeling was successful.2.After acupuncture treatment,the expression of 5-ht rhythm in the brain of the mice showed that the circadian rhythm existed in the blank control group(P < 0.05),the circadian rhythm disappeared in the model group(P > 0.05),the rhythm was restored in the acupuncture group(P < 0.05),and the rhythm did not return to the normal rhythm in the non-meridians &non-acupoints group(P > 0.05).Compared with the blank control group,the acupuncture group had a similar expression with the blank group(P < 0.01).3.After acupuncture treatment,the expression of 5-ht rhythm in the liver tissues of mice showed that the circadian rhythm was significant in the control group(P < 0.01),the circadian rhythm was disordered in the model group and the non-meridians & non-acupoints group(P >0.05),and the circadian rhythm returned to normal in the acupuncture group(P < 0.05).Compared with the control group,the acupuncture group had a similar expression with it(P <0.01).Conclusion:1.2h light/2h dark alternate light can cause the sleep-wake cycle disorder in mice,which can be used as a modeling method to replicate the no sleep rhythm disorders in mice.2.Acupuncture of "Gan shu"(BL18)can restore the normal expression of 5-HT in the brainand of mice with no circadian rhythm sleep disorders.3.Acupuncture of "Gan shu"(BL18)can restore the normal expression of 5-HT in the liver tissues of mice with no circadian rhythm sleep disorders.