The Significance Of Bmal1 Dominanted "Active-rest Cycle Synchronizes To Nature" Behavivor In The Prevention Of Respiratory Viral Infection

Objective:Respiratory viral infection is characterized by high incidence,rapid spread,wide prevalence,and large variation in viral evolution.Different respiratory viral infections can cause various clinical symptoms ranging from mild upper respiratory tract infection to severe pneumonia.The common cold caused by rhinovirus of which the course of the disease is self-limiting while severe acute respiratory distress syndrome(ARDS)caused by SARS coronavirus(SARS-CoV)is a serious threat to human health.Respiratory virus infection not only endangers the public health,but also in recent years,another important harm of respiratory virus infection is to promote and aggravate the development of other diseases.For example,there are approximately 235 million asthmatic patients around the world,and about 85% of asthma attacks are related to respiratory virus infection.Therefore,the prevention and treatment of respiratory virus infection is of great significance.However,due to the high variability of respiratory virus groups,the retardation of vaccine development and the limitations of antiviral drugs,modern medicine is still unable to effectively control respiratory virus infection,therefore,the search for cost-effective and non-pharmacological interventions may provide new ideas for the prevention and control of respiratory virus infection.Respiratory virus infection belongs to the research category of exogenous febrile diseases in TCM(Traditional Chinese Medicine).TCM has accumulated rich experience in the prevention and treatment of exogenous diseases.In addition to the use of various internal and external Chinese herbal medicine for prevention and treatment of exogenous febrile diseases,TCM also attaches great importance to some non-drug control methods which was demonstrated by using a variety of external treatment,such as acupuncture,special eating and living habits,massage and so on to prevent epidemic disease in ancient times.Although these are important means of preventing and treating respiratory virus infection in TCM,and can best embody the ideological characteristics of preventive treatment of disease in TCM.However,the research on this area is far from enough.What's worse,the research quality is beyond satisfaction on the whole,most of them can only be found in the popular science readings rather than academic literature.This study is based on the idea of “preventive treatment of disease” and the holism concept of TCM.It is believed that the “ActiveRest Cycle Synchronizes to Nature” is of great significance in the prevention of respiratory viral infection.We plan to use arrhythmic mice to mimic abnormal active-rest cycle in human beings and observe whether or not it will affect the antiviral immunity to respiratory viral infection.Indeed,if our study can successfully demonstrate that the prevention of respiratory viral infection could be achieved simply by maintaining normal active-rest cycle that would be definitely intriguing,as it can not only enhance the confidence of nonpharmacological interventions in the prevention of respiratory viral infection,but also avoid over-medication or over-treatment in respiratory viral prevention which is the main cause of drug resistance or side effects.In addition,by revealing the key molecules involved in the mechanism of this antiviral immune response can also provide novel targets for antiviral drug developmentMethods:This study includes three parts: mouse experiment,in vitro cell culture and human sample detection.1.Mouse experiment : Three different kinds of arrhythmic mice were used to mimic abnormal active-rest cycle in human beings,including bmal1 knockout(bmal1-null),acquired inducible bmal1 gene knockout(bmal1-iKO)and chronic jet-lag mice(CJL).Wild type mice(wt)housed in standard lighting conditions were used as the control.Here,we used a Sendai Virus(SeV)infection model of acute and chronic airway disease to examine how abnormal active-rest cycle affects respiratory viral infection and asthmatic lung phenotypes.During the acute phase of SeV infection,we evaluate mouse's susceptibility to SeV infection by comparing the body weight,viral titer in lung tissues,SeV RNA expression and survival frequency after infection.Bronchio-alveolar lavage(BAL)were also collected to detect the protein contents,cytokines,cell counts and inflammatory cell classifications.Effects of the chronic SeV lung pathology were represented by airway resistance and muc5 ac expression 49 days after infection.To investigate whether our results were reflective of circadian gene deletion besides bmal1,we also inoculate per2-null mice with SeV in parallel;SeV inoculation was conducted on both wt mice and bmal1-null mice at various times(ZT0,ZT6,ZT12,ZT18)of day represented in Zeitgeber Time(ZT).Expression of ifnb1 was measured 24 h post infection while weight loss and SeV RNA expression were measured during the course of 5 days infection.2.In vitro cell experiment: Mouse trachea epithelial cells(mTECs)and bone marrow derived macrophages(BMDM)were isolated from bmal1-null mice and wt mice respectively.Cells were then infected with SeV-GFP(Sendai virus expressing green fluorescent protein)after grown to confluency.The changes of viral titer in the supernatant of the cell culture after virus infection were observed.The expression of?-tubulin(cilia-associated protein)and SeV-GFP in the airway epithelial cells were counted under fluorescence microscope using a double-blinded methodology;3.Human sample detection : 1)Clock gene(bmal1,npas2,per2,dbp,nr1d1,nr1d2)expression in nasal wash samples previously obtained from young children hospitalized for Respiratory Syncytial virus(RSV)bronchiolitis(n=10)and from healthy children(n=5)matched for age,sex,and collection time were detected.2)Expression of circadian clock genes(bmal1,npas2,per2,dbp,nr1d1,nr1d2,spon,clock)in bronchial brush samples obtained from adult patients with severe asthma(n=9),mild/moderate asthma(n=9),and healthy volunteers(n=6)were measured.4.Statistics: The log rank test was used for survival curve analysis.Data were pooled from at least two independent experiments and were expressed as mean±SE.We performed Student's two-tailed t-tests or one-way analysis of variances when comparing data between groups.All statistical analyses were performed in Microsoft Excel(Redmond,WA).P <0.05 was regarded as a statistically significant difference.Results:1.Mouse experiment: 1)All animals suffered from body weight loss after SeV infection during 7 days infection.However,the body weight for wt mice was gradually increasing from day 8 and returned back to normal by day 10 while that of most bmal1-null mice decreased all the way until death on day 8.The body weight and survival rate of wt mice is generally higher than that of bmal1-null mice during the course of infection(P < 0.05);SeV RNA expression and viral load in bmal1-null is higher than that of wt mice.However,there was no difference in the viral load between per2 knockout and wt mice on day 5(P > 0.05);It was estimated that bmal1-null mice are more than 62.5 folds more vulnerable to SeV infection.Further more,bmal1-null mice are also more vulnerable to influenza A virus as gauged by more severe weight loss than wt mice post H1N1 infection.The BAL protein concentration and leukocyte counts in bmal1-null mice is higher than that of wt mice(P < 0.05);For all mice,most cytokines(IFN-?,IL-28/IFN-?2,IL-6,RANTES/CCL5,ENA78/CXCL5)in BAL show an increasing trend and peak on day 5 but then drop gradually.Compared to wt mice,the cytokine levels in bmal1 mice are higher especially on day 5(P < 0.05).2)The airway resistance and muc5 ac of both CJL and bmal1-null mice are higher than that of wt mice(P < 0.05).3.Similar to bmal1-null mice,bmal1-iKO mice are more vulnerable to SeV infection than wt mice as indicated by greater weight loss and higher SeV virus expression on day 5 of infection(P < 0.05).4)We found that lung expression of ifnb1 varies significantly with the time of SeV inoculation in wt mice but not in bmal1-null mice.Time of SeV infection also affected the degree of weight loss in wt mice,but interestingly,viral RNA levels were not impacted by infection time,as observed at day 1 and day 5 post infection.2.In vitro cell experiment : We found that SeV infection was equally productive in BMDMs derived from bmal1-null and wt mice(P > 0.05);There was no difference in susceptibility to SeV-GFP between bmal1-null and wt ciliated mTECs(P > 0.05);bmal1-null un-ciliated mTECs seems to be more vulnerable to SeV infection than wt un-ciliated mTECs as indicated by higher SeV RNA in cell culture supernatant;Besides,by counting the co-location of SeV-GFP and ?-tublin in un-ciliated mTECs on day 7 of SeV-GFP infection,SeV-GFP can be found in both ?-tublin positive mTECs and ?-tublin negative mTECs.However,bmal1 deletion increases SeV-GFP infection in ?-tublin positive mTECs subset(P < 0.05)but have no effect on ?-tublin negative mTECs subset(P > 0.05).3.Human samples detection : bmal1 expression in nasal lavage fluid of all respiratory syncytial virus infected children was lower than that in healthy volunteers(P < 0.05);The expression of bmal1,npas2,per2,dbp,nr1d1,nr1d2 in the BAL of all asthmatic patients was lower than that of healthy volunteers(P < 0.05);In contrast,the expression of clock gene in BAL of asthmatic patients was higher than that of healthy volunteers(P < 0.05);The expression of spon2 gene was lower in mild asthma than that in healthy volunteers,but it was higher in severe asthma than that of healthy volunteers(P < 0.05).Conclusions:1.“Active-Rest Cycle Synchronizes to Nature” pattern protected mice from suffering severe weight loss,airway inflammation and overwhelming virus replication as mice with abnormal active-rest cycle did after SeV infection which demonstrated that synchronizing active-rest cycle to nature is of great importance as it helps the host to maintaining proper antiviral immunity against acute infection.2.The production of interferon and weight loss varies significantly with the time of SeV infection suggests that the antiviral immunity is characterized by daily fluctuation which encourage the host to synchronizing active-rest cycle to nature in order to maximize physiological fitness.3.Synchronizing active-rest cycle to nature can not only reduce acute respiratory viral infection,but also in the long run,it helps to alleviate asthmatic like chronic lung pathology.4.bmal1 is the key molecule and is required for “Active-Rest Cycle Synchronizes to Nature” pattern to exert antiviral response as deletion of bmal1 worsen respiratory infection and asthmatic phenotype.5.Airway epithelial cells is probably the critical place for bmal1 to take effect against respiratory infection.6.bmal1 may also play an important role in human airway remodeling as it is down-regulated both in RSV infected children and asthmatic adults.