| Objective To investigate the effects of circadian disturbances on endogenous circadian rhythms in body by examining the changes of central and peripheral circadian clock genes and the expression rhythms of serum melatonin and corticosterone after circadian disturbances in mice.To further explore the impact of circadian disturbances on the immune function in mice,elucidate the potential mechanism of peripheral neutrophils reduction caused by circadian disturbances,and provide a scientific basis for the health protection of body immune function damage caused by shift work.Methods1.Construction of circadian disturbances model: 72 C57bl/6j mice were randomly divided into normal circadian rhythm group(LD group)and circadian disturbances group(CD group).Mice in LD group were exposed to the normal light cycles(12 h light / 12 h dark cycle),and mice in CD group were exposed to the reversed light cycles(12 h light / 12 h dark cycle with reversed every three days for total 15 cycles).On the second day after the model,six mice of each group were sacrificed at every six time points,and serum,hypothalamus,cortex,liver,heart,spleen,and bone marrow tissue were harvested for analysis.2.Circadian activity of mice were assessed by measuring food consumption at day and night during modeling,and mice were weighed every two cycles.3.The 24 h expression profiles of the clock genes in central(hypothalamus and cortex)and peripheral(liver,heart,and spleen)tissues were determined by q-PCR in mice.4.The 24 h expression profiles of serum melatonin and corticosterone were determined by Elisa in mice.5.The absolute Neut counts in peripheral blood and bone marrow were monitored and analyzed by flow cytometry.6.The 24 h expression profiles of clock genes(bmal1、clock、npas2、per1、per2、per3、cry1、cry2、rev-erbα、rev-erbβ、dbp and ciart),transcription factors(G-CSFR、PU.1 and C/EBPα)and chemokines(CXCL12,CXCR4 and CXCR2)were determined by q-PCR in the bone marrow.7.Histological changes of bone marrow were detected by hematoxylin-eosin staining method(HE)in mice.8.The 24 h expression profiles of serum norepinephrine was determined by Elisa in mice,and the 24 h expression profiles of AR in bone marrow tissue were measured by q-PCR in mice.Results1.Compared with the LD group,the mice in the CD group changed the habits of sleeping at day and moving at night,with irregular feeding day and night,and the weight increase was significantly higher than the LD group(P<0.05).2.Under the LD light conditions,the expression of clock genes in the hypothalamic(bmal1,npas2,per1,cry2,rev-erbα,dbp and ciart),cortex(bmal1,clock,npas2,per1,per2,per3,cry1,rev-erbα,rev-erbβ,dbp and ciart),liver,heart and spleen(bmal1,clock,npas2,per1,per2,per3,cry1,cry2,rev-erbα,rev-erbβ,dbp and ciart)showed 24 h rhythmic oscillations.Under the CD light conditions,the expression of clock genes in the hypothalamic(bmal1,npas2,per2,per3,cry1,rev-erbα,dbp and ciart),cortex(bmal1,npas2,per2,cry1,rev-erbα,dbp and ciart),liver and heart(bmal1,clock,npas2,per1,per2,per3,cry1,cry2,rev-erbα,rev-erbβ,dbp and ciart),and spleen(bmal1,npas2,per2,per3,cry1,cry2,rev-erbα,rev-erbβ and dbp)showed 24 h rhythmic oscillations(P<0.05).Compared with the LD group,most of the core clock genes had a phase shift of close to 12 h in the CD group.Furthermore,circadian disturbances could affect endogenous rhythms of mice by enhancing or attenuating the amplitude of clock genes.3.Under LD light conditions,the expression of melatonin and corticosterone in serum had 24 h rhythmic oscillations,and daytime concentrations were higher than nighttime concentrations(P<0.05).Under CD light conditions,the expression of melatonin and corticosterone in serum lost 24 h rhythmic oscillations(P>0.05),and the overall concentrations of melatonin and corticosterone in serum were significantly lower than the LD group(P<0.0001).4.Compared with the LD group,The circadian disturbances caused reductions of maturation neutrophile counts in peripheral and bone marrow(P<0.05).5.Under LD and CD light conditions,the expression of clock genes all have 24 h rhythmic oscillations in the bone marrow tissue(P<0.05).Compared with the LD group,the period,amplitude,and phase of the core clock genes occurred different degrees of variation in the CD group.6.Compared with the LD group,adipocytes of the CD group increased in bone marrow tissue,which caused the decrease of hematopoietic cell density.7.Under LD light conditions,the expression levels of transcription factors(G-CSFR,PU.1 and C/EBPα)and chemokines(CXCL12,CXCR4 and CXCR2)had the 24 h rhythmic oscillations in bone marrow tissue,and the expression levels were higher in the dark phase than in the light phase(P<0.05).Compared with the LD group,the transcription factors and chemokines in bone marrow tissue showed different 24 h rhythmic expression profiles in the CD group(P <0.05).8.Under LD light conditions,serum NE and its bone marrow receptors(Adrβ1 and Adrβ2)had the 24 h rhythmic oscillations(P<0.05).Under CD light conditions,the expression of serum NE lost the 24 h rhythmic oscillation in the CD group(P>0.05),and the overall concentration of NE was significantly lower than the LD group(P<0.0001).Compared with the LD group,The expression of bone marrow receptors(Adrβ1 and Adrβ2)showed different 24 h rhythmic expression profiles in the CD group(P<0.05).Conclusions1.Chronic circadian disturbances interfered with the endogenous circadian rhythms by disturbing with the expression profiles of circadian clock genes in the central and peripheral as well as neuroendocrine hormones in serum.2.Chronic circadian disturbances reduced the number of neutrophils in the body.It could be speculated that neutrophils is a key effector cell to interfere with the body’s immune function.The circadian disturbances decreased peripheral neutrophils’ counts,and the possible inducements were that circadian disturbances interfered with the proliferation and differentiation of neutrophils in bone marrow and the migration of mature neutrophils from bone marrow to the periphery. |
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