Effects Of CLOCK Gene On Biorhythm Of Upper And Lower Respiratory Tract In Mice With Allergic Rhinitis

Objective:Recent studies have found that the symptoms of allergic rhinitis?nasal itching,stuffy nose,sneezing,and runny nose?show a day-of-time dependent variations changes,but the specific mechanism is still unclear.The aim of this study was to establish and evaluate the allergic rhinitis model in mice,to analyze the circadian rhythm of allergic rhinitis symptoms and the expression of CLOCK genes.Methods:1.Establishment of OVA-induced allergic rhinitis?AR?mouse modelMale Balb/c mice aged 8-10 weeks were purchased,about 18-25g.The mice were randomly divided into AR group and control group.OVA basal sensitization was performed in AR group on the 1st,7th and 14th day respectively,i.e.intraperitoneal injection of 1 ml suspension containing ovalbumin?OVA?50?g and Al?OH?₃4 mg.On the 21st day after basal sensitization,3%OVA nasal drip was used continuously for 12days,and 50?g OVA nasal drip was used on each side of nostril once a day.In blank control group,phosphate buffer?PBS?was used instead of OVA.2.Symptomatological score of allergic rhinitis?AR?modelThe nasal scratching,sneezing and runny nose were observed 10 minutes after the first nasal drip.At the end of the last nasal drip,two time points?04:00 and 16:00?were used to observe and record the number of times of nose scratching,sneezing and runny nose clearance in each group within 10 minutes.3.HE staining and toluidine blue staining of nasal mucosa and lung tissueThe mice were sacrificed at different time points?04:00,16:00?to extract nasal mucosa and lung tissue.The sections were stained with hematoxylin-eosin?HE?and toluidine blue,respectively.The number of eosinophils?EOS?and mast cells?MC?in nasal mucosa and lungs at different time points?04:00,16:00?was counted under highpower microscopy?x 400?.4.ELISA determination of serum OVA-sIgE in mice of each groupThe mice were anesthetized at different time points?04:00,16:00?the next day after the last nasal instillation for 24 hours.Blood was collected by removing the eyeball to EP tube,3000r/min,and centrifuged for 5 minutes.Serum was collected and stored at-20?for reserve.According to the instructions of ELISA reagent,the content of OVA specific IgE in serum of mice in each group was detected.5.Changes of Th2 cytokine?IL-4?in serum of mice in each group determined by ELISAAfter the mice were killed at different time points?04:00 and 16:00?,serum was collected by centrifugation and stored at-20?for reserve.According to the instructions of ELISA reagent,after adding sample,incubating,washing,colouring and terminating reaction,the concentration of IL-4 in serum of mice in experimental group and control group was detected.6.Detection of CLOCK expression in nasal mucosa and lung tissues by RT-qPCRThe nasal mucosa was obtained at different time points?04:00 and 16:00?under aseptic operation.The total RNA was extracted by Trizol method.The reverse transcription was used to amplify the DNA.The expression of CLOCK gene in nasal mucosa was detected by PCR.Similarly,lung tissues were obtained at different time points?04:00 and 16:00?under aseptic operation.Lung tissues were ground to fine powder with a bowl under liquid nitrogen freezing.Total RNA was extracted by Trizol method,which was consistent with the procedure of qPCR in nasal mucosa.Clock gene CLOCK expression in lung tissue was detected and analyzed after statistical induction.Results:1.Compared with the blank control group,mouse in AR group were significantly higher than those in control group in symptomatic score?the number of times of nose scratching and sneezing?,nasal mucosal staining?eosinophil and mast cell counts?,and immunological parameters?OVA-sIgE and IL-4?,suggesting that OVA-induced AR mice were successful in modeling.2.The results of symptom scoring showed that the nasal symptoms of mice in the 04:00AR group were significantly weaker than those in the 16:00 AR group,and there was a significant difference between the two groups.3.The results of nasal mucosa staining showed that the number of eosinophils and mast cells in AR group was significantly higher than that in control group.The number of eosinophils and mast cells in nasal mucosa was significantly different in AR group at different time points?04:00 and 16:00?,but there was no significant difference in the number of eosinophils and mast cells in lung tissue.4.The results of immunology showed that the levels of OVA-sIgE and IL-4 in serum of mice in the 04:00 AR group were significantly lower than those in the 16:00 AR group,and there was a significant difference between the two groups.5.The results of RT-qPCR showed that the expression of CLOCK in nasal mucosa and lung tissues changed periodically.The expression of CLOCK decreased at 04:00 and increased at 16:00.Conclusion:Respiratory symptoms induced by allergic rhinitis?AR?have time-dependent rhythmic changes,in which the expression of CLOCK gene in nasal mucosa?upper respiratory tract?and lung?lower respiratory tract?fluctuates in a circadian rhythm.This finding suggests that the CLOCK gene may participate in and regulate the occurrence and development of AR-related allergic diseases.