| Objective: Explore the temporal characteristics of Tuina regulating circadian rhythm and the effect of its intervention on VIP and VPAC2 receptors in hypothalamus,to provide a theoretical basis for the clinical treatment of circadian rhythm disorders.Methods: 24 male C57BL/6J mice were randomly divided into Control group(C),Model group(M),Tuina group(T)and Sham Tuina group(JT),with 6 mice in each group.Each mouse was reared in isolated cells,and the first one-week light/dark synchronous acclimation(Light: 8:00-20:00;Dark: 20:00-08:00),then adjust the light to constant darkness,modeling 14 days.The Clock Lab system was used to monitor and record the spontaneous activities of mice in real time,including the onset time point,phase transfer value,acrophase and total activity.Observe the temporal characteristics of the circadian rhythm spontaneous activity in different groups.After5 days of Tuina or sham Tuina intervention,the hypothalamus of mice was taken out.The content of VIP was detected by Elisa,the expression of Per2 mRNA was detected by PCR,and the expression of VPAC2 receptor protein was detected by Western Blot.SPSS 26.0 statistical software was used for data analysis,mainly used two-way repeated measures ANOVA,one-way ANOVA,and nonparametric tests,Graph Pad Prism 9.0 software was used for drawing,P<0.05 was statistically significant.Results: 1.General observation: After modeling,the body weight of mice increased slightly,and no hair loss or glossiness was observed.The mice showed mental hyperstimulation and more agile behavioral response.The mental state and behavioral response agility of mice in the T group and JT group gradually recovered,and T group was slightly better than the JT group.2.Characteristics of circadian rhythm of spontaneous activity in mice: the onset time point and acrophase of circadian rhythm were delayed,and the phase transfer value was decreased,the difference was statistically significant(P<0.05).There was no significant difference in the total activity of mice in each group(P>0.05).3.VIP content level: The M group VIP concentration decreased compared with the C group,the difference was statistically significant(P<0.05).The VIP concentration in the T group increased compared with the M group,and the difference was statistically significant(P<0.05).But there was no significant difference between the JT group and M group(P>0.05).4.VPAC2 receptors protein expression results: The expression of VPAC2 receptors protein in the M group increased compared with the C group,and the difference was statistically significant(P<0.05).The expression of VPAC2 receptor protein in the T group decreased compared with the M group,and the difference was statistically significant(P<0.05).But there was no significant difference between the JT group and M group(P>0.05).5.Expression of Per2 mRNA: The expression of Per2 mRNA in the M group decreased compared with the C group,and the difference was statistically significant(P<0.05).The expression of Per2 mRNA in the T group increased compared with the M group,and the difference was statistically significant(P<0.05).But there was no significant difference in the expression of Per2 mRNA in the JT group(P>0.05).Conclusion: 1.Under the condition of LD,the spontaneous activity of mice in each group showed obvious circadian rhythm,while under the condition of DD,the spontaneous activity showed free running state and showed advanced transfer.2.Tuina can adjust the circadian rhythm phase of spontaneous activity in mice,which shows that the onset time point and acrophase of circadian rhythm delayed,the phase shift value decreases,but there was no significant change in total activity.3.Tuina may increase the expression of Per2 mRNA by up-regulating VIP and downregulating the expression of VPAC2 receptor,thereby promoting the recovery of circadian rhythm in mice. |
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