Electroacupuncture On The Expression Of Clock And Bmal1 In The Liver Of C57BL/6J Mice With Circadian Rhythm Disorder

Aim: To investigate the effect of electroacupuncture intervention on circadian rhythm of C57BL/6J mice.The expression of mRNA and protein in Clock and Bmal1 was observed with liver as the main research organization.The possible mechanism of acupuncture on the circadian rhythm disorder in C57BL/6J mice was also explored.Methods: Using video recording method,the spontaneous activity and vertical movement(�standing up�)of the experimental animals were monitored and recorded.A total of 72 C57BL/6J mice were randomly divided into four groups including blank group,model group,acupuncture Gan Shu group,and acupuncture sham group,with 18 mice being allocated into each group.The mice were light synchronized(light: 08:00-20:00;dark: 00 PM-08:00)for 7 days;after that the mice were entrained for 5 days to a 2-hour light: 2-hour dark cycle;following that,the mice were kept under natural lighting conditions(light: dark = 12: 12).After the mice model was successfully built,the acupuncture was conducted at 15:00 everyday for 5 days.The liver extraction started 10 hours after the last acupuncture was conducted(02:00 of the sixth day),and 3 mice were sacrificed for liver extraction every four hours thereafter.For the acupuncture Gan Shu group,the acupuncture was conducted at a frequency of 4/20 hz and current of 0.2 mA for 15 minutes,once a day for five consecutive days;for the model group,the mice were only fixed without acupuncture being applied;and for the acupuncture sham group,the mice were fixed,and sham acupuncture was applied to non-acupoint.A quantitative real-time PCR method was used to quantify the mRNA expression levels of the Clock and Bmal1 genes in the liver.The circadian rhythm of each gene was analyzed using R software.The protein expression levels of Clock and Bmal1 in the liver were detected using Western-blot.Results: The mRNA expression levels of clock and bmal1 genes in the acupuncture Gan Shu group restored normal circadian rhythm,and the result was statistically significant(P < 0.05);there was no circadian rhythm established in the mRNA expression levels of Clock and Bmal1 in the model group,and this was also statistically significant(P > 0.05).A reduced vertical movement was observed in the acupuncture Gan Shu group as compared with that of the model group,and the difference reached statistical significance(P < 0.05).Based on the Western-blot results,significantly decreased protein expressions of Clock and Bmal1 were observed in the acupuncture Gan Shu group as compared with those in the model group(P < 0.05).Conclusion: 1 Acupuncture at BL18(Gan Shu)restored the liver clock of Clock and Bmal1 of C57BL/6J mice with circadian rhythm disorder;while the sham acupuncture group did not show re-establishment of the circadian rhythm,indicating that acupuncture at BL18 is able to restore the circadian rhythm of clock and bmal1 genes.2 Western-blot results demonstrated that the protein expressions of Clock and Bmal1 in the liver were decreased upon acupuncture at BL18 as compared with the levels in the model group,suggesting that acupuncture at BL18 is able to reduce the protein expressions of Clock and Bmal1,which may contribute to the re-establishment of the circadian rhythm.3 Following acupuncture treatment,the mice with circadian rhythm disorder showed similar vertical movement as compared to that of the blank group;and the spontaneous activity as well as the vertical movement were decreased after acupuncture treatment.4 These observations indicate that acupuncture could alleviate the upward trend of spontaneous activity and vertical movement of mice with circadian rhythm disorder,and the decreased expressions of Clock and Bmal1 might be one of the molecular mechanisms that contributes to such effect.