| BackgroundRespiratory epithelium is the first line of defense against inhaled pathogens and foreign particles.Mucociliary clearance is one of the important defense mechanisms.The mucociliary system consists of cilia and the surrounding mucus.The wobble frequency and effectiveness of cilia are the main determinants of mucociliary clearance,but the quantity and characteristics of mucin may also be important variables.The composition of mucus ensures the normal function of respiratory epithelium,and any change in the thickness and composition of the mucus layer will affect the wobble of cilia,which in turn leads to the decrease of the clearance function of mucus cilia.Nasal mucus is mainly secreted by nasal subepithelial glands,as well as goblet cells and plasma.Glands include serous glands and mucous glands.Serous gland cells produce aqueous fluids containing a variety of antibacterial defense factors.For example,lysozyme is one of the non-specific antibacterial proteins with broad-spectrum antibacterial activity.Mucous gland cells secrete highly viscous mucus and are rich in mucin.Mucins are a kind of macromolecules.The main mucins in the nose are mucin 5AC(MUC5AC)secreted by nasal epithelial goblet cells and mucin 5B(MUC5B)secreted by mucin glandular cells.Chronic rhinosinusitis(CRS)is an inflammatory disease that occurs in the nasal and sinus mucosa.Common symptoms include nasal congestion,increased nasal secretions,dysosmia and facial pain.According to the existence of nasal polyps,CRS can be divided into chronic rhinosinusitis with nasal polyps(CRSwNP)and chronic rhinosinusitis without nasal polyps(CRSsNP).Patients with CRS are often characterized by excessive secretion of nasal mucus and poor drainage.Mucus retention in the nasal cavity and paranasal sinuses will further aggravate inflammation,which will also lead to local hypoxia,resulting in the expression of hypoxia inducible factor(HIF),which is one of the main triggers of tissue remodeling and inflammatory cell aggregation.Chronic inflammation will cause changes in the proportion of secretory cells of goblet cells and glandular cells in the nasal cavity,resulting in changes in the physical and chemical properties of nasal mucus.The purpose of this study was to detect the changes of nasal mucus composition of CRS and to analyze the role of HIF in causing nasal mucus composition abnormality.MethodsA total of 24 cases of control group and 99 cases of CRS were included in this study,including 36 cases of CRSsNP group and 63 cases of CRSwNP group.Samples were taken from polyps of CRSwNP patients undergoing transnasal endoscopic surgery or sinus mucosa of CRSsNP patients,and inferior turbinate mucosa obtained from non-CRS patients during nasal septum deviation correction.(1)Immunohistochemical staining of lysozyme was used to evaluate the distribution of serous gland cells,Alcian blue staining was used to evaluate the distribution of epithelial goblet cells and mucous gland cells;(2)Western blotting was performed,MUC5AC was used to represent goblet cells and lysozyme represented serous gland cells,and the ratio of MUC5AC to lysozyme protein expression was analyzed to represent the ratio of goblet cells to serous gland cells;(3)Serous gland cell area and mucous gland cell area were counted,and the ratio of serous gland cell area to mucous gland cell area was analyzed;(4)The expression sites of HIF-la and HIF-2? in nasal mucosa were detected by immunofluorescence double staining;(5)The mRNA expression levels of all the above factors were analyzed by real-time quantitative PCR.Results(1)The distribution of nasal secretory cells in the nasal mucosa of healthy controls and CRS patients was analyzed.The results showed that compared with the control group,the lysozyme positive staining in CRSsNP patients increased,indicating that the serous gland cells increased,while the lysozyme positive staining in CRSwNP patients decreased,indicating that the serous gland cells decreased.Compared with the control group,the number of Alcian blue positive cells in the nasal epithelium of the two groups of CRS patients increased,suggesting that the number of goblet cells increased,and the increase of goblet cells was more obvious in the CRSwNP group.Compared with the control group,the area of mucous gland cells increased in CRSsNP group and decreased in CRSwNP group.(2)The expression levels of lysozyme,MUC5AC and MUC5B were compared.The results of immunofluorescence double staining showed that the epithelial goblet cells mainly secreted MUC5AC,and the mucous glandular cells mainly secreted MUC5B.Compared with the control group,the ratio of MUC5AC to lysozyme increased significantly in CRSwNP group,but there was no significant difference in CRSsNP group,indicating that the ratio of goblet cells to serous gland cells only increased significantly in CRSwNP group.Compared with the control group,the ratio of MUC5B to lysozyme increased significantly in both groups of CRS,suggesting that the ratio of serous gland cell area to mucous gland cell area increased significantly in both groups of CRS.The above results showed that the ratio of goblet cells to serous gland cells and the ratio of mucous gland cells to serous gland cells in CRSwNP group were significantly higher than those in control group,while those in CRSsNP group only showed an increase in the ratio of mucous gland cells to serous gland cells.(3)To detect the expression of HIF-1? and HIF-2? in nasal mucosa.Immunofluorescence double staining showed that both HIF-1? and HIF-2? were expressed in glandular epithelial cells and located in serous glandular cells.Compared with the normal control group,there was no significant difference in the mRNA level of HIF-1? in CRS between the two groups,but the mRNA level of HIF-2? was significantly higher in both groups.In terms of mRNA level,lysozyme was positively correlated with HIF-1? and HIF-2?,which further confirmed the co-localization of lysozyme with HIF-1? and HIF-2? in serous gland cells,indicating that HIF-1? and HIF-2? were involved in the secretory function of serous gland cells.ConclusionThere were mucus oversecretion and composition changes in nasal mucosa of CRS.In CRSsNP,serous gland cells and mucous gland cells increased significantly,but goblet cells had no significant change,which indicated that mucus oversecretion in CRSsNP was mainly related to glandular hyperplasia,while in CRSwNP,serous gland cells and mucus glandular cells decreased significantly,while goblet cells increased significantly,suggesting that mucus oversecretion in CRSwNP was mainly related to goblet cell proliferation.HIF is located in serous gland cells and may be involved in the secretory function of serous gland cells.The abnormal mucus composition results in the increase of mucus viscosity and the decrease of antibacterial components,thus reducing the innate immune function of nasal mucosa.BackgroundChronic rhinosinusitis(CRS)can be divided into primary and secondary.In primary CRS,the tissue inflammatory response of a very important nasal polyp(NP),NP belongs to diffuse type ? CRS,is characterized by eosinophilia and enhanced type 2 helper T cell(Th2)response.At present,the etiology of NP is still unclear,and the interaction between microorganisms and the host immune system may be related to the pathogenesis of NP.As the main gateway for outside air to enter the human body,the respiratory tract is vulnerable to pathogens,toxins and pollutants in the air.Respiratory epithelial cells and surface mucus are the first physical barriers against inhaled allergens and pathogens.The main components of the mucus layer are macromolecular polymers called mucins.In addition to secretory mucins,there is also a subfamily of mucins called cell surface mucins,which also play an important role in mucosal defense,such as mucin 4(MUC4).MUC4 is a heterodimer formed by post-translational processing of a gene,which consists of an O-glycosylated extracellular subunit(MUC4-a)and an N-glycosylated transmembrane subunit(MUC4-?).Recent studies have shown that MUC4 may play an important role in the normal polarization and differentiation of respiratory tract cells.Polarized ciliated epithelium acts as a barrier between nasal mucosa and the external environment.We speculate that MUC4 may play an important role in the host defense function of nasal epithelium.Bacteria can be detected in the nasal cavity of normal people and patients with NP.When the nasal mucosa is healthy,the defense mechanism of nasal epithelium can effectively remove bacteria and other pathogens.However,in the case of defective host defense,there will be the growth of bacteria and the spread of infection.Through pattern recognition receptors,immune system cells can recognize related proteins on pathogens and activate downstream pathways,thus effectively eliminating pathogens.One of the most representative pattern recognition receptors is the Toll-like receptor(TLR)family.Among them,TLR4 is the receptor of lipopolysaccharide(LPS)from Gram-negative bacteria.The combination of LPS and TLR4 can activate TLR4 pathway and induce the expression of Tumor necrosis factor-?(TNF-?)and Interleukin-1?(IL-1?)downstream.Specific nuclear factor-kappa B(NF-?B)is an important transcription factor in TLR4 signal pathway.Mucin and TLR are both important parts of mucosal defense and have synergistic effect,but their relationship in nasal mucosal epithelial cells is not clear.In this study,we detected the expression of MUC4 and TLR4 in healthy control group and NP tissue.In addition,we also studied the effect of interleukin-13(IL-13)on the expression of these factors in human nasal epithelial cells as a key Th2 cytokine mediating NP inflammatory response,and the role of TLR4 pathway in it.MethodsIn vivo study:(1)22 cases of control group and 38 cases of NP patients were included in this study.Polyp samples were taken from nasal polyps of NP patients during nasal endoscopic surgery,and those of the control group were non-CRS patients with deviation of nasal septum,and the inferior turbinate mucosa samples were taken as healthy control group during nasal septum surgery;(2)The mRNA expression of MUC4 and TLR4 in healthy control group and NP group was detected by real-time quantitative PCR;(3)Immunofluorescence staining and Western blotting were used to detect the protein expression of MUC4 and TLR4 in healthy control group and NP group.In vitro study:(1)To construct the model of human nasal mucosal epithelial stem/progenitor cells(hNESPCs)in vitro,and inoculate hNESPCs at the air-liquid interface for differentiation and culture until the epithelial cells were completely differentiated into human nasal epithelial cells(hNECs);(2)On the day of complete differentiation,the cells were treated with IL-13 for 48 days,and then treated with or without LPS for 48 hours to observe whether the effect of LPS was affected by IL-13;(3)In order to study whether the activation of TLR4 pathway is related to IL-13 stimulation,TLR4 specific inhibitor TAK242 or NF-?B specific inhibitor BAY11-7082 was added to the medium containing IL-13 for hNECs5 days;(4)The expressions of MUC4,TLR4,TNF-? and IL-1? in different treatment groups were detected by real-time quantitative PCR,immunofluorescence staining and Western blotting.Results(1)Immunofluorescence staining showed that the expression of MUC4 and TLR4 was low in normal nasal mucosa,while the expression of MUC4 and TLR4 was significantly increased in NP,which was located at the top of the epithelial cells of nasal mucosa.The mRNA expression levels of MUC4 and TLR4 in NP tissues were significantly higher than those in healthy controls,and there was a positive correlation between MUC4 and mRNA of TLR4(r=0.74,p<0.01).Western blotting showed that the protein expression levels of MUC4 and TLR4 in NP tissues were significantly higher than those in healthy controls.(2)the results of in vitro experiment showed that hNECs stimulated by IL-13 could significantly enhance the positive staining results of MUC4 and TLR4,and the protein expression levels of MUC4(2.16-fold,p<0.05)and TLR4(6.22-fold,p<0.05)were significantly higher than those of the untreated group.After IL-13 stimulation,the mRNA expression of MUC4 and TLR4 was significantly higher than that of the untreated group,which further confirmed this result.(3)The immunofluorescence results showed that the staining intensity of MUC4 and TLR4 in hNECs increased after IL-13 stimulation,and the staining intensity of MUC4 and TLR4 in IL-13 co-treatment with TAK242 or BAY11-7082 was lower than that in IL-13 stimulation alone,indicating that the inhibitor could inhibit the effect of IL-13.The results of Western blotting showed that the inhibitor could significantly reduce the protein expression of MUC4 and TLR4 after IL-13 stimulation.After IL-13 stimulation,the ratio of pNF-?B/NF-?B increased,which indicated that IL-13 could induce the phosphorylation of NF-?B.when IL-13 was co-treated with TAK242 or BAY11-7082,the ratio of pNF-?B/NF-?B was lower than that of IL-13 alone,indicating that the inhibitor could inhibit the phosphorylation of NF-?B induced by IL-13.The trend of transformation at the mRNA level was consistent with that of the protein,which further confirmed this result.(4)Although MUC4 and TLR4 increased significantly after hNECs was stimulated by IL-13,there was no significant change in mRNA and protein levels of MUC4 and TLR4 in LPS treatment group alone.In order to study the effect of IL-13 on the effect of LPS,we detected the expression of LPS downstream effector molecules TNF-? and IL-1?.The results of immunofluorescence showed that the percentage of TNF-? and IL-1? positive cells in hNECs decreased after IL-13 stimulation.After LPS stimulation alone,the percentage of positive cells of TNF-? and IL-1? increased significantly,while the percentage of positive cells of TNF-? and IL-1? in IL-13 and LPS co-stimulation group was significantly lower than that in LPS alone group.The results of PCR also showed that LPS stimulation could increase the mRNA expression of TNF-? and IL-1?,but this effect could be inhibited by IL-13.ConclusionOur study shows that MUC4 is increased in patients with NP,and IL-13 stimulation can up-regulate the expression of MUC4 in hNECs.IL-13 stimulation could activate NF-?B,TLR4 inhibitor TAK242 and NF-?B inhibitor BAY11-7082 could attenuate the up-regulation of MUC4 induced by IL-13.The increased MUC4 may participate in the imbalance of nasal mucosal defense function through inhibition of LPS response. |
PDF Full Text Request