Study On The Expression Of HMGB-1 And The Effect Of EMT On Human Bronchial Epithelium Induced By YKL-40

Background and Objectives:YKL-40 belongs to one of the highly conserved chitinase family members,which is deficient of chitinase activity,which is a newly discovered inflammatory factor.In recent years,a number of studies have confirmed that YKL-40 protein plays an important role in the pathology of inflammation and tissue remodeling.Bronchial asthma is a chronic airway inflammatory disease,with airway remodeling as one of the main pathological features.It has been confirmed that YKL-40 is closely related to airway remodeling in the pathogenesis of bronchial asthma.Epithelial-mesenchymal transition is one of the mechanisms of airway remodeling.Epithelial-mesenchymal transition is a process of dynamic changes in epithelial cells to obtain stromal cell phenotypes.It has been reported that this pathological condition of epithelial-mesenchymal transition can promote pulmonary fibrosis and the occurrence of airway remodeling in asthma.So,it need to be further studied that YKL-40 could induce bronchial epithelial cells to occur the interstitial changes and promote the occurrence and development of airway remodeling.HMGB-1 is a non-histone protein which belongs to a member of the "Damage-Associated Molecular pattern" family.HMGB-1 can be triggered to release actively or passively in the stress state.In recent years,the relationship between HMGB-1 and bronchial asthma have been gradually confirmed in several sudies.The specific pathogenesis of bronchial asthma is not yet fully elucidated.YKL-40 and HMGB-1 are both important regulatory factors in asthma.maybe there is a relationship between them for asthma.Therefore,the main purpose of this study is to understand the function of YKL-40 protein,and to clarify whether YKL-40 can induce the effect of EMT on bronchial epithelial cell in vitro,and to investigate whether it can induce the expression of HMGB-1.Methods:To investigate whether YKL-40 regulates the epithelial-mesenchymal transition in bronchial epithelial cells,we successfully recovered and cultured the human bronchial epithelial cells(Beas-2B cells).And then,these cells were stimulated with different concentrations of YKL-40 protein for 24 h.First,we observed the morphological changes of Beas-2B cells under the inverted microscope.Later,The fluorimetric changes of E-cadherin,N-cadherin,Vimentin and α-SMA in the cells were detected by theimmunofluorescence assay.Next,Western blot was used to detect the protein expression of E-cadherin,N-cadherin,Vimentin and Desmin.In addition.after Beas-2B cells were treated with different concentrations of YKL-40 protein solution for 12 h,the mRNA expression levels of E-cadherin,N-cadherin,Vimentin and Desmin were determined by quantitive real time RT-PCR.To test the relationship between YKL-40 and HMGB-1,we investigated the expression levels of HMGB-1 in human bronchial epithelial cells which were stimulated by YKL-40 via Elisa,quantitive real time RT-PCR and Western-Blot methods.According to the experimental design,multiple groups of different concentrations of YKL-40 protein solution were prepared and mixed well.The YKL-40 protein with a volume concentration of 1μg / m L was added to each of the six-well plate so that the final concentration of each hole was 0ng / mL,10 ng / mL,100 ng / mL and 400 ng / mL in order,and then put them into the incubator to stimulate and culture cells for 24 h.Then respectively collect the supernatant and cell lysate of the culture medium,later detailed and named the labels for retention.The protein levels of HMGB-1,E-cadherin,N-cadherin,Vimentin and Desmin were detected by Western Blot.The real-time quantitative PCR was used to determinate the expression of HMGB-1,E-cadherin,N--cadherin.The expression changes of E-cadherin,N-cadherin,Vimentin and α-SMA in the cells were observed by the immunofluorescence staining.At the same time,the morphological changes of bronchial epithelial cells(Beas-2B)under the control of various concentrations of YKL-40 were observed under the inverted microscopeResults:With the stimulation by different concentrations of YKL-40 protein,we observed that epithelial cells owned the interstitial cell-like shape under the microscope,The majority of cells from the original epithelial cell "paving stone" shape into the fusiform and spindle interstitial cells;the fluorescent staining result showed that the increased expression of N-cadherin,α-SMA and Vimentin and the decreased expression of E-cadherin.The above results are also confirmed by RT-PCR and Western Blot.It indicated that YKL-40 protein can promote the EMT effect in a concentration-dependent manner.In addition,we used Elisa,RT-PCR and Western-Blot experiments to show that YKL-40 could promote HMGB-1 expression in a concentration-dependent manner.Conclusions:YKL-40 can induce the EMT effect of bronchial epithelial cells and simultaneously promote the expression and secretion of HMGB-1 in bronchial epithelial cells.This study suggests that YKL-40 is associated with HMGB-1 and is expected to provide the new evidence for the study of the pathogenesis of bronchial asthma.