| Many allergic diseases exhibit time-of-day-dependent variations associated with the endogenous circadian system.For example,in most allergic rhinitis(AR)patients,clinical symptoms often worsen during nighttime or the early morning,compromising nighttime sleep and resulting in poor performance during the daytime.Actually,the clock genes existed in the central and peripheral oscillators may affect the maintenance of the nasal cycle and play a critical role in the daily changes of nasal symptomology,however,the precise mechanisms involved in AR remain to be further investigated.The circadian clock drives ~24-h rhythms in many physiological and phathological processes,such as sleep-wake cycles,hormonal secretions,and food intake.In mammals,the master clock located in the hypothalamic suprachiasmatic nucleus(SCN)is activated to synchronize peripheral clocks distributed throughout the body via neural and endocrine pathways.The molecular circadian clock is generated by autoregulatory transcriptional–translational feedback loops comprising many clock genes,e.g.,Clock,Bmal1,Period(Per),cryptochrome(Cry),and their proteins.How these clock genes work in relation to human physiology and pathology has been described in detail elsewhere.Interestingly,this circadian oscillation lasts ~24 h for one cycle of the feedback loop.Some immune responses and allergic reactions are under the control of the circadian clock.The clock genes Per(Per1,Per2 and Per3)are the core negative regulators of the circadian timing system that mediate rhythmic variability in immune-inflammatory processes.In particular,endogenous glucocorticoids have been considered as Zeitgebers(time givers),which receive signals from SCN to synchronize peripheral clocks,such as nasal mucosa tissues 4.Glucocorticoid hormones are secreted by the adrenal gland that is regulated by the hypothalamus-pituitary gland-adrenal axis(HPA axis)in a circadian manner,which are potent phase-shifting agents for circadian gene expression in peripheral mouse tissues.In nocturnal animals,serum glucocorticoids reach peak levels around or just before the onset of the activity phase(between ZT12 and ZT16),with a nadir of a short duration between ZT0 and ZT4.A recent study confirmed a circadian rhythm in PER2 level in the mouse nasal mucosa,with peaks in the PER2 expression observed at the phase corresponding to the early dark period(ZT12)and as consistent with peak levels of serum glucocorticoids.The data suggest that endogenous glucocorticoids are important timing signals for peripheral oscillators and can reset the nasal cyclic activities via Per2 expression in the nasal mucosa.However,the regulatory mechanism of circadian clock on AR at the molecular level has yet to be determined.It’s well known that AR is an IgE-mediated allergic inflammation characterized by a predominant Th2 and Th17 type response,and involves eosinophils(EOS)infiltration,goblet cell hyperplasia,and mast cell(MC)degranulation in the local nasal mucosa.Nevertheless,the periodic changes in AR symptoms may be partly explained by the circadian rhythms in the neural-immune-humoral mechanism of AR.New evidence indicates that circadian clock regulates the adaptive immune system,mainly by influencing CD4+ T cell lineages immigration and differentiation.Some T cells-associated cytokines and transcription factors,such as nuclear factor/IL-3-regulated protein(E4BP4),and retinoic acid receptor-related orphan receptor(ROR)?t synthesis,display a circadian fashion.Furthermore,recent study show that E4BP4 is a basically negative transcription factor of Th2 and Th17 cells that is directly controlled by clock genes Rev-erbα and is linked to the clock regulatory network.Therefore,wehypothesized that the daily changes of nasal symptoms in the development of AR might be regulated by the circadian clock genes through alteration of different Th-specific transcription factors under the rhythmic secretion of endogenous glucocorticoids.Based on our preliminary study,we performed this study following three ways as:(1)According to literatures,adrenalectomized(ADX)mice and AR model were established.The study was aimed to study the effect of dysfunction of the HPA axis on the pathogenesis of AR,and further to explore the mechanism of neural-endocrine regulation.(2)Then,we chose ZT4 and ZT16 as suitable time points as reported previously in literature to observe circadian variability of AR symptoms and circadian expression of the core clock genes Per1,Per2 and Per3 in nasal mucosal tissues and peripheral blood mononuclear cells(PBMCs)of an OVA-sensitized mouse AR model,aiming to further determine whether endogenous glucocorticoids influence the diurnal severity of AR symptoms via regulating clock genes and explore the possible molecular mechanism and potential chronotherapeutic strategy in the pathophysiology of AR.(3)The aberrant light/dark cycles-induced CRD and OVA-sensitized AR models were successfully established.Here,we used an AR mouse model to explore how circadian rhythm disruption(CRD)affected nasal symptoms and anxiety-like behaviors.PartⅠ: Effects of endogenous glucocorticoid deprivation on immune deviation of allergic rhinitis in miceObjectives: To study the effect of dysfunction of the hypothalamic-pituitary-adrenal(HPA)axis on the pathogenesis of allergic rhinitis(AR)by the mouse model of decreased endogenous glucocorticoid(GC)after adrenalectomy,and further to explore the mechanism of neural-endocrine regulation.Methods: According to literatures,adrenalectomized(ADX)mice and AR model were established.Eighty mice were randomly divided into four groups(n=20 per group)including control group,AR group of normal mice(AR group),AR group of bilateral ADX(bilateral ADX/AR group)and AR group of unilateral ADX(unilateral ADX/AR group).In order to assess the model of ADX,adrenal gland tissue was assayed by HE staining and the serum adrenocorticotropic hormone(ACTH)and cortisol(CORT)concentrations were measured by enzyme-linked immunosorbent assay(ELISA).The behavioral observation with OVA-s Ig E assessments and count of eosinophils/mast cell by the HE/Toluidine Blue staining of nasal septum mucosa tissue were performed to evaluate the AR model.The expression of peripheral blood CD4+IL4+T cells(Th2 cells)and CD4+IFN-γ+T cells(Th1 cells),splenocytes of CD4+CD25+Treg cells(Treg cells)were measured by flow cytometry to study endogenous GC on the influence of immunological indexes in different groups of mice.SPSS 16.0 software was used to analyze the data.Results: The concentrations of OVA-s Ig E in control group,AR group,bilateral ADX/AR group and unilateral ADX/AR group mice was(28.86±3.62)ng/ml,(76.27±16.47)ng/ml,(48.37±8.89)ng/ml,(49.86±7.19)ng/ml,respectively.There was statistically significant difference between control group and AR group(t=7.09,P<0.05),AR group and bilateral ADX/AR group(t=4.81,P < 0.05),AR group and unilateral ADX/AR group(t=5.21,P<0.05).The level of Th2 cells in different four groups was(0.71±0.24)%,(7.03±1.95)%,(2.44±2.06)%,(3.20±1.21)%,respectively.There was statistically significant difference between control group and AR group(t=-2.93,P <0.05),AR group and bilateral ADX/AR group(t=-4.67,P < 0.05),AR group and unilateral ADX/AR group(t=-3.61,P<0.05).There was comparatively lower expression in bilateral ADX/AR group than that in unilateral ADX/AR group with no significant difference(t=4.39,P>0.05).Meanwhile,the level of Th1 cells in different four groups was(0.58±0.76)%,(0.57±0.59)%,(0.72±0.34)%,(1.03±0.32)%,respectively,with no significant difference among these groups.The proportion of CD4+CD25+Treg cells was(11.10±2.18)%,(4.10±1.07)%,(7.15±0.92)%,(4.58±1.05)%,respectively,with significant difference between control and other groups(t value was-7.171,-8.273,-8.360,respectively,all P < 0.05).Compared with AR group,CD4+CD25+Treg cells increased significantly in bilateral ADX/AR group(t=-2.607,P < 0.05).In addition,lower expression of eosinophil and mast cell were detected in the local nasal tissue of bilateral ADX/AR group,and mast cell degranulation wasn’t observed.Conclusions: Unilateral or bilateral ADX leads to HPA axis dysfunction and endogenous GC deprivation,possibly regulating the mechanism of AR through Th1/Th2 immune bias and Tregs cell activity.PartⅡ: Clock gene Period2 regulates diurnal changes of nasal symptoms in an allergic rhinitis mouse modelBackground: The allergic rhinitis(AR)symptoms exhibit prominent 24-h variations associated with the biological clock.Although endogenous glucocorticoids synchronize circadian oscillator in the nasal mucosa,the precise mechanism of AR remains unclear.Therefore,we investigated the association between circadian-clock genes and AR-symptom at various time points in the mouse model.Materials and Methods: Based on the rhythmic secretion of corticosterone levels,we chose two different time points at ZT4(10 A.M.)and at ZT16(10 P.M.)for observations,detections and comparisons.In both AR and control group,AR symptoms including nasal rubbing and nasal sneezing were observed,nasal mucosa tissues were stained by H&E and Toluidine Blue and the numbers of EOS and MC were statistically counted;the plasma levels of total Ig E,OVA-specific Ig E,IFN-γ,IL-4,IL-13,IL-17 A were measured by ELISA,the expressions of Th1,Th2 and Th17 cells in isolated peripheral blood mononuclear cells(PBMCs)were analyzed by flow cytometry,the diurnal changes of PBMCs-derived circadian clock genes Per1,Per2,Per3 m RNA and Th-related transcription factors T-bet,Gata3,E4bp4,Rorgt m RNA were detected by q RT-PCR.Results:(1)In AR group,the frequency of nasal rubbing was significantly higher at ZT4(resting phase)than at ZT16(active phase)(tnasal rubbing,AR=-3.391,p<0.01)in the AR group and accompanied by increased sneezing(tnasal sneezing,AR=-1.779,p<0.05),meanwhile,nasal symptom scores(8.63±0.52)at ZT4 were significantly higher than that (6.50±0.53)at ZT16(tscores,AR=-3.475,p<0.01).(2)Plasma corticosterone levels were increased significantly at ZT16 than ZT4 in both AR and control groups(tcorticosterone,AR=-3.477,tcorticosterone,control=-3.099,p<0.01).Interestingly,the fluctuation of plasma corticosterone levels was negatively correlated with nasal symptom scores.(3)In addition,EOS numbers(22.80±3.27)and MC numbers(9.67±2.02)at ZT4 were significantly higher than those(14.76±2.93)and(7.34±1.27)at ZT16(t EOS,AR=1.143,t MC,AR=0.999,p<0.05).And plasma levels of total Ig E(529.51±27.74)at ZT4 in the AR group were significantly higher than those(414.79±24.09)at ZT16(t total Ig E,AR=0.734,p<0.05).(4)Moveover,plasma IFN-γ levels and the proportion of Th1 cell in PBMCs at ZT4 were significantly lower than those at ZT16 in the AR group(t IFN-γ,AR=-7.449,p<0.01;t Th1,AR=-2.306,p<0.05),whereas plasma IL-4,IL-13,IL-17 A levels and the proportion of Th2 and Th17 cell in PBMCs at ZT4 were significantly higher than those at ZT16 in the AR groups(t IL-4,AR=6.446,t IL-13,AR=3.363,t IL-17 A,AR=5.630,p<0.01;t Th2,AR=3.201,p<0.01;t Th17,AR=3.756,p<0.01).(5)Furthermore,Per2 levels from nasal mucosa and PBMCs at ZT4 were significantly lower than those at ZT16 in the AR group(t Per2,AR=-2.970,p<0.01;t Per2,PBMCs,AR=-4.204,p<0.05).Compared with data at ZT16,Th2/Th17-positive transcription factor Gata3 and Rorgt levels at ZT4 increased in the AR group(t Gata3,AR=1.096,t Rorgt,AR=1.437,p<0.05),and Th2/Th17-negative transcription factor E4bp4 levels at ZT4 decreased in the AR group(t E4bp4,AR=0.733,p<0.05).(6)Importantly,plasma corticosterone levels and Per2 expressions between ZT4 and ZT16 in the AR group showed a similar pattern and rhythmicity,but the fluctuation of nasal symptom scores and Per2 expressions between ZT4 and ZT16 changed apparently in an opposite way.On the other hand,Per2 expressions and Th2-specific Gata3,Th17-specific Rorgt levels changed differently and showed negative interaction between ZT4 and ZT16 in the AR group.Due to the negative transcription factor of Th2-and Th17-related E4bp4,Per2 expressions showed similar fluctuation of E4bp4 levels at both time points in the AR group,with a nadir value at ZT4 and a top value at ZT16.Conclusions: Taken together,these results demonstrated that Per2 gene might regulate daily variations of nasal symptoms severity in an AR mouse model,partially through its possible anti-inflammatory function,as well as circadian regulation of Th2 and Th17 allergic responses.Our results suggests that circadian clock are involved in the pathogenesis of AR,thus laying the theoretical foundation of neural-immune-endocrinal mechanism in AR and prompting us to further explore the clinically appropriate chronotherapy for AR.Part Ⅲ: Chronic circadian rhythm disruption exacerbates inflammatory response via lowering tight junction expressions in an allergic rhinitis mouse modelObjectives: Epidemiologic data showed that an increased risk of allergic rhinitis(AR)was correlated with environmental disturbance of circadian clock.However,little is known about the precise mechanism.Here,we used an AR mouse model to explore how circadian rhythm disruption(CRD)affected nasal symptoms and anxiety-like behaviors.Methods: According to the previously described protocol,the aberrant 8:16h light/dark cycles for the fowolling 5 weeks induced CRD mice models by weekly 8 h phase advances,and OVA-sensitized AR mice models were prepared at the same time.The mice were randomly divided into negative control(NC)group,AR(AR)group,(CRD+NC)group and(CRD+AR)group(n=12).NC group and AR group were supplied with a 12:12h light/dark cycle when the light turned on at 06:00 AM and turned off at06:00 PM until the end of the experiment.After successfully establishing the animal models,nasal allergic symptoms were observed and anxiety-like behaviors in the open field test were evaluated.Some immunological parameters from nasal mucosa,plasama,splenocyte in the four groups were measured and determined by different experimental techniques.Results:(1)The behavioral observations showed that nocturnal activities in both CRD+NC group and CRD+AR group gradually disappeared and diurnal activitiesobviously increased from the beginning of the 5th week,accompanying the frequently restless act of moving up and down.With the delay of the modeling time,these behavioral characteristics became more obvious.Till the end of the 7th week,CRD mice models were finished successfully.Additionally,the expression of the core genes Clock,Bmal1,Per1 and Per2 were loss of rhythmicity in both CRD+NC group and CRD+AR group,further suggesting that aberrant light-dark cycles could successfully induce and establish CRD animal models.(2)After the final intranasal administration,the incidence of nasal rubbing and sneezing in each mouse significantly increased in both AR group and CRD+AR group,meanwhile,the total nasal symptom scores were calculated >5points,indicating that OVA-induced AR models were successfully established in mice.The scores(6.63±1.06)in AR group were significantly lower than those(8.38±0.74)in CRD+AR group,with significant difference between the two groups(P < 0.05).Meanwhile,compared to AR group,the time of CRD+AR group spent in center decreased significantly(P < 0.05),thus suggesting that CRD contributed to allergic symptoms and anxiety-like activities in AR mice.(3)The EOS numbers(21.38±2.73)and MC numbers(14.50±3.50)in the nasal mucosa in CRD+AR group significantly increased as compared to those(12.71±2.11)and(9.26±1.87)in AR(P<0.05),whereas goblet cells hyperplasia and mucus secretion in the nasal mucosa of CRD+AR group were greater increase than those of AR group,with significant difference(P <0.05).Moreover,the expression of Occludin and ZO-1 m RNA from nasal mucosa tissues in CRD+AR group was significantly lower than that in AR group(P < 0.05).(4)The plasma levels of Th2-associated cytokines IL-4,IL-6,IL-13 and IL-17 A in CRD+AR group significantly increased as compared to those in AR group(P<0.05),whereas the level of Th1-associated cytokine IFN-γ in CRD+AR group significantly decreased as compared to that in AR group(P < 0.05).Furthermore,the numbers of Th2 cells and Th17 cells were significantly higher in CRD+AR group than those in AR group(P<0.05),whereas the numbers of Th1 and Treg cells were significantly lower in CRD+AR group than those in AR group(P<0.05).Conclusions: The jet lag-induced CRD could exacerbate AR-like symptoms and psychological behaviors in mice,partially through regulating prominent Th2/Th17 response in the local(nasal mucosa)and systemic(spleen)tissues.These results implied that keeping regular lifestyle might help patients to better manage allergic episode in modern society. |
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