The Possible Mechanisms Of YKL40 Promoting Ptlcs Proliferation And IL-6,IL-8,IL10,TNF-α Secretion In Pediatric OSAS

Background:Obstructive Sleep Apnea Syndrome(OSAS)is a common disease in children with high incidence and it can cause near-term and long-term complications.So far the pathogenesis is still unclear.Recent studies have shown that OSAS is associated with systemic inflammation and inflammatory microenvironment.YKL40(human chondroglycoprotein 39),also known as chitinase-3-like protein 1(CHI3L1),is a proinflammatory factor involved in the inflammatory response and cell proliferation.YKL40 has been reported to be associated with OSAS in adults.At present,there is no related study between YKL40 and OSAS in children.In this experiment,we plan to clarify that YKL40 promotes lymphoid tissue proliferation and inflammatory factors secretion in pediatric OSAS,and explore the mechanism of YKL40 promoting cell proliferation and pro-inflammatory effects.Objective:1.To clarify the expression difference of YKL40 in pediatric PS and OSAS;2.To explore the mechanism of YKL40 promoting the proliferation of PTLCs;3.To explore the mechanism of YKL40 promoting IL-6,IL-8,IL10,TNF-α secretion in PTLCs;4.To explore the secretion of YKL40 under CIH and determine the cellular origin of YKL40.Methods:1.IHC was used to identify expression of YKL40 and PCNA and the correlation between YKL40 and PCNA in tonsils of children with PS and OSAS.QPCR and Western blot were used to identify expression of YKL40 in tonsils of children with PS and OSAS.The correlation between YKL40 and OAHI was analyzed.ELISA and qPCR were used to identify expression of YKL40,IL-6,IL8,IL-10,TNF-α in tonsils of children with PS and OSAS,then the correlation among them was analyzed.2.The primary tonsil lymphocytes(PTLCs)from children with OSAS were cultured and recombinant human YKL40 protein(rhYKL40)was added to culture media.After the stimulation with rhYKL40 of different concentration and time points,the optimal concentration and time of lymphocytes proliferation was assessed by CCK8 assay.The proliferation of CD4+T,CD8+T and CD19B cells was assessed by flow cytometry.The activation of ERK1/2 which is a key molecule of the MAPK pathway and the effects of ERK1/2 agonists and inhibitors on the proliferation of PTLCs were observed by Western blot.3.After the stimulation with rhYKL40 of different concentration and time points,the relative expression of IL-6,IL-8,IL-10 and TNF-α was assessed by qPCR.The activation of P65 which is a key molecule of the NF-κb pathway and the effects of NF-κb agonists and inhibitors on the secretion of PTLCs were observed by Western blot.4.The primary lymphocytes from mouse were cultured by intermittent hypoxia and normoxia and YKL40 in cell supernatants was determined by ELISA.Immunofluorescence(IF)colocalization was used to determine the cellular source of YKL40 in tonsil lymphoid tissue of children with OSAS.Results:1.The expression of YKL40 and PCNA in OSAS group was higher than PS group.Significant differences(P<0.001)were noted between the two groups.The expression levels of YKL40 are positively related to PCNA(P<0.001).The relative mRNA expression of YKL40 in OSAS group was higher than PS group.Significant differences(P<0.01)were noted between two groups.The relative mRNA expression of YKL40 in mild OSAS group was higher than PS group(P<0.05),MS group was higher than PS group(P<0.01)and M-S group was higher than mild OSAS group(P>0.05).The YKL40 mRNA was positively correlated with the OAHI(P<0.05).The relative mRNA expression of IL-6,IL-8,IL-10 and TNF-αin OSAS group was higher than PS group.Significant differences of IL-6,IL-8 and TNF-α mRNA were noted between the two groups.But the relative mRNA expression of IL-10 was no significant difference between two groups(P>0.05).The mRNA of YKL40,IL-6,IL-8 and TNF-α was positively correlated between every two pairs.The relative protein expression of YKL40 in OSAS group was higher than PS group.Significant differences(P<0.01)were noted between the two groups.The relative protein expression of YKL40 in mild and M-S OSAS groups were higher than PS group(P<0.05),but there was no significant difference between M-S group and mild OSAS group(P>0.05).2.100 ul volume were transferred into 96-well plates when the concentration of PTLCs reached 1×106 viable cells per 6-well plate,and proliferation of PTLCs could be stimulated with rhYKL40 in a concentration and time-dependent condition by CCK-8 assay.Proliferation of PTLCs reached a peak when a concentration was increased to 100 ng/ml and at a time point of 24h.The effects of rhYKL40 on the proliferation of CD4+T,CD8+T and B cells in PTLCs were assessed by flow cytometry after 100 ng/ml rhYKL40 for 24h.Percentage of CD4+T cells in YKL40 group was higher than control and significant difference was noted(P=0.042).Western blot showed that rhYKL40(100 ng/ml for 24 hours)significantly increased the phosphorylation of ERK1/2 which could be stimulated by ERK1/2 activator(PMA)and EKR1/2 phosphorylation was blocked by pretreatment with ERK1/2 inhibitor(AG126).The effects of rhYKL40,ERK1/2 activator(PMA)and ERK1/2 inhibitor(AG126)on the proliferation of CD4+T,CD8+T and B cells in PTLCs were assessed by flow cytometry.The proliferation of CD4+T cells could be stimulated by rhYKL40 and PMA,but AG126 could partly inhibit the effect.The proliferation of CD8+T and B cells was no significant difference in four groups.3.The relative mRNA expression of IL-6,IL-8,IL-10 and TNF-α of PTLCs could be stimulated with rhYKL40 in a concentration and time-dependent conditions after different concentrations(Control,10ng/ml,100ng/ml,1000ng/ml)for different time(6h,12h,24h,48h).The relative mRNA expression of IL-6 and IL-8 reached a peak with 100ng/ml rhYKL40 at a time point of 24h(P<0.05).The relative mRNA expression of TNF-α reached a peak with 1000ng/ml rhYKL40 at a time point of 24h(P<0.05).RhYKL40 significantly increased the phosphorylation of P65 which could be stimulated by NF-κB activator(3-oxo)and P65 phosphorylation was blocked by pretreatment with NF-κB inhibitor(Bay).4.The secretion of YKL40 was determined by ELISA after MLC culture in Nor and CIH for 8h,24h,48h and 72h.YKL40 secretion reached a peak at the time of 24h and YKL40 secretion in CIH group was higher than Nor group at the time of 48h and the difference was significant(P=0.026).Co-localization of YKL40 with CD4,CD8,CD20,CD68 and CK8 were operated by immunofluorescence.YKL40 was located in lymphatic epithelial area of tonsils,paracfollicular T cell zone and germinal center of the tonsil tissues.CD4 was located in paracfollicular T cell zone.CD8 was located in lymphatic epithelial area of tonsils and germinal center of the tonsil tissues.CD20 was mainly located in germinal center of the tonsil tissues and a small amount in paracfollicular T cell zone.CD68 was mainly located in germinal center of the tonsil tissues and a small amount in paracfollicular T cell zone and lymphatic epithelial area of tonsils.CK8 was mainly located in lymphatic epithelial area of tonsils.Colocalization were found in YKL40 and CD8,YKL40 and CD68.Conclusion:1.This is the first study to find that the expression of YKL40 and PCNA in OSAS group was higher than PS group.The expression levels of YKL40 are significantly related to PCNA and OAHI.This indicates that elevated YKL40 may promote the hyperplasia of tonsillar tissue in children with OSAS,elevated YKL40 may be a risk factor for airway stenosis on pediatric OSAS and YKL40 may be used as an indicator to determine the severity of pediatric OSAS.2.The expression of YKL40 in the tonsils of children with OSAS is positively correlated with IL-6,IL-8 and TNF-a.The positive correlation is also found between these pro-inflammatory factors.These findings indicate that there may be inflammatory microenvironment in children with OSAS and the inflammatory microenvironment may play an important role in the pathogenesis of pediatric OSAS.3.YKL40 activates ERK1/2 of the MAPK pathway in the process of PTLCs proliferation and activates P65 in the NF-κB pathway in the process of inflammatory factors secretion in PTLCs.Inhibitors of YKL40,ERK1/2 and P65 may serve as new targets for drug therapy in pediatric OSAS.4.This is the first study to find that intermittent hypoxia may induce YKL40 secretion in MLC.We first study colocalization of YKL40 and lymphocyte subtypes in OSAS by immunofluorescence.We find YKL40 colocalize with both CD8+T cells and CD68 cells.It is suggested that intermittent hypoxia induced YKL40 secretion by CD8+T cells and CD68 in tonsil tissue,YKL40 stimulates CD4+T cells proliferation and secretion of proinflammatory factors in tonsil tissue,causing or aggravating OSAS in children.These findings may provide new ideas for the study of the pathogenesis of pediatric OSAS.