| BackgroundChronic rhinosinusitis(CRS),the chronic inflammation of nasal cavity and sinus mucosa persistent for at least 12 weeks,is one of the most common ENT diseases.Multi-center statistical studies show that the incidence of CRS is increasing year by year,which severely affects human health and quality of life.Advanced mechicine knowledge and endoscopic surgical techniques have greatly improved the diagnosis and treatment of CRS.However,there are still high rates of recurrence associated with repeated infections and delayed recovery.This might be due to our inadequate understanding of the virulence factors and pathogenic mechanisms of chornic sinusitis.At present,the main pathogencity factors include bacteria,viruses,abnormal nasal anatomy,epithelial dysfunction,allergic reactions and immune system disorder,among which bacterial infection appears to be the most common and important one.Lipopolysaccharide(LPS),a key component of the outer membrane of Gram-negative bacteria,can activate immune cells,promote the release of various cytokines and chemokines,and participate in the inflammatory response of nasal mucosa.The epithelial cells of nasal mucosa functions as a physical barrier and are capable of cytokine secretion in response to external physical and chemical stimuli.The released cytokines act on other cells,which play a crucial role in the processes of chronic inflammation,injury repair and tissue remodeling.Autophagy is a cellular process featured by the formation of double-membrane vesicles(autophagsomes)engulfing damaged proteins,organelles or invaded pathogens destined for degradation.Autophagsomes are subsequently fused with lysosomes to generate autolysosomes,in which the autophagsome contents are digested by acidic lysosomal hydrolases to provide raw materials and energy for the biosynthesis of new macromolecules.Autophagy is evolutionarily conserved in eukaryotic cells;whereby cellular self-protection is achieved through digestion of its own components.However,under certain specific conditions,excessive consumption of organelles or biomacromolecules trigger programmed cell death(autophagic cell death).Therefore,the autophagy may have completely different impacts in different diseases,and even in distinct developmental stages of the same disease.Previous studies showed that autophagy is regulated by more than 30 molecules,collectively termed as the Atg(Autophagy-related)family.Microtubule-associated protein 1 light chain 3(LC3)is a mammalian homolog of yeast autophagy gene Atg8.When autophagy occurs,LC3 is first cleaved by Atg4 to produce water-soluble LC3-I which is then converted to its lipidated version LC3-II by conjugation to lipomidoethanol at the surface of autophagosomal membrane under the catalytic activities of Atg7 and Atg3.The amount of LC3-II is proportional to the number of autophagosomes,and it is used as a marker protein to study autophagy.LC3-?/LC3-?has been accepted as the gold standard for Western blot detection of autophagy.In recent years,many studies have indicated that autophagy has fundemental effects in the pathogenesis of respiratory infection,bronchial asthma,chronic obstructive pulmonary disease,etc.Tanaka A et al.found that cigarette smoking induced reactive oxygen species(ROS)in airway epithelial cells,phosphorylated JNK and activated LC3-II and the ROS inhibitor inhibited LC3-II expresion.Thus,blocking JNK activation and reducing LC3-? level could serve as an effective apporach to prevent and treat chronic obstructive pulmonary disease.In asthma,autophagy can affect the differentiation and balance of T helper(Th)1 and Th2 and mediate the immune inflammatory response of asthma-associated pathogens.Dickinson JD et al.suggested that IL-13 modulates the synthesis and secretion of airway cup-like cells by inducing autophagy.Martin et al.reported that children's asthma was associated with the polymorphism of Atg5 and Atg7,and the Atg5 level in nasal mucosa epithelial cells in patients with acute asthma was significantly higher than that in the control group.In recent years,scholars put forward the theory of "the same airway,the same disease".Therefore,we speculate that autophagy is related to the pathogenesis of CRS.So far only a Taiwan research group linked the expression of cyclooxygenase-2 with autophagy level in nasal mucosa fibroblasts.However,the role of autophagy in chronic sinusitis needs to be further studied and discussed.In the first part of this study,the expression of autophagy-associated gene LC3 in chronic nasosinusitis was detected at both mRNA and protein levels.Primary human nasal mucosa epithelial cells were isolated and cultured,and autophagy structure was observed by transmission electron microscope and fluorescence microscope.In the second part,the human nasal mucosa epithelial cells cultured in vitro were stimulated by LPS,and autophagy was examined by transmission electron microscope and immunofluorescent microscope.The expression of autophagy-related proteins was checked at different stimulation concentrations and different stimulation times.The molecular pathways of autophagy in human nasal mucosa epithelial cells were detected by Western blot,and the molecular pathways were further verified by specific inhibitors.Part? Study on the autophagy level of nasal mucosa epithelial cells in chronic rhinosinusitisObjectivesThis study first aims to compare the expression of autophagy-associated gene LC3-II in nasal mucosa from patients with CRS as well as those from healthy donors.This study further aims to determine the autophagy level in primary nasal mucoa epithelial cells isolated from CRS patients by electron microscope and immunofluorescence microscope.Methods1.The nasal mucosa were collected from 18 patients with chronic rhinosinusitis and 10 healthy donors.2.The expression and localization of LC3-? in nasal mucosa from each group by immunohistochemical staining.3.The total RNA from 28 samples was extracted by TRIzo1 and were reverse transcribed into cDNA.The expression of LC3-? in each group were determined by quantitative Real-time PCR(qRT-PCR).4.The primary nasal mucosa epithelial cells were isolated from chronic rhinosinusitis patients and healthy donors.The distribution and quantity of fluorescent spots were checked under the fluorescence microscope,and the number of self-hagy bodies was detected by transmission electron microscopy(TEM).The cells were further cultured in vitro and transfected with vector expressing LC3-B fused with green fluorescent protein(GFP-LC3).5.The SPSS 18.0 software was used for data analysis,and the difference was statistically significant when P<0.05.Results1.Immunohistochemical staining results showed that the autophagy protein LC3-II in the nasopharyngeal mucosa of chronic rhinosinusitis and normal controls were all expressed in the cytoplasm,which is mainly yellow or brown.When the tissue is strong positive,it is brown,and the cell membrane and interstitium can be stained.LC3-? was observed highly expressed in 88.9%(16/18)chronic rhinosinusitis patients while only in 30%(3/10)of healthy controls(P = 0.003).2.qRT-PCR showed that the expression levels of LC3-? mRNA in chronic nasosinusitis and normal controls were(3.188±0.3887)and(1.025±0.1543),respectively.The statistical analysis indicated a significantly higher expression of LC3-II mRNA in chronic sinusitis compared to the control group(P<0.001).3.Under the transmission electron microscope,autophagosomes were observed in the nasal mucosa primary cells from CRS patients.4.GFP-LC3 plasmid was transfected into primary nasal mucosa epithelial cells and expression of GFP-LC3 was checked under fluorescence microscope.In contrast to the normal nasal mucosa epithelial primary cells which showed low exprssion of GFP-LC3,the patient cells showed much higher expression of GFP-LC3,which were mainly located in cytoplasm.ConclusionsThe autophagy in nasal mucosa was significantly increased in CRS patients,suggesting a potential role of autophagy in the development of CRS.Part ? LPS regulates autophagy in human nasal epithelial cells through AMPK-mTOR signaling pathwayObjectivesTo investigate the role of AMPK-mTOR signaling pathway in LPS-induced autophagy of human nasal epithelial cells and whether TLR4/MyD88 signaling pathway is involved in autophagy.Methods1.The effect of LPS on the proliferation and apoptosis of human nasal mucosa epithelial cells was determined by CCK-8 assay and flow cytometry analysis respectively.2.The human nasal mucosa epithelial cells were treated with different dose of LPS(0,1,5,10?g/ml)for 12h.In another experiment setting,the cells were treated with LPS(10?g/ml)for different time(0,6,12,24h).The total protein was extracted,and the levels of autophagy-labeled protein LC3-II/LC3-I and SQSTM1(P62)protein were detected by Western blot.The final LPS treatment time and dose for the following experiments were decided according the Western blot data.3.Transmission electron microscopy(TEM)was used to detect the number of cellular double-membraned autophagosome in human nasal mucosa epithelial cells after treated with different dose of LPS.4.The distribution and number of fluorescent spots were determined by fluorescence microscope in human nasal mucosa epithelial cells transfected with GFP-LC3 plasmid.5.The human nasal mucosa epithelial cells were exposed to various doses of LPS(0,1,5,10?g/ml).The expression of autophagy-related proteins,including P-AKT(Ser473),AKT,P-AMPK(Tyr172),AMPK,P-mTOR(Ser2448)and mTOR were detected by Western blot.Further,the levels of these autophagy-related proteins were determined in LPS-stimulated nasal mucosa epithelial cells with or without the presence of AMPK inhibitor compound C.6.To furhter verify the role of AMPK-mTOR signaling pathway in mediating LPS-induced autophagy,the ratio of LC3?/LC3? and P62,P-AMPK,AMPK,P-mTOR and mTOR protein were detected by Western blot both in epithelial primary cells isolated from chronic nasosinusitis patients and from normal mucosa tissues.7.The protein levels of TLR4,MyD88 and LC3-?/LC3-? were detected in human nasal mucosa epithelial cells treated with different doses(0,1,5,10?g/ml)of LPS,and the expression of autophagy-related proteins were checked in LPS stimulated human nasal mucosa epithelial cells with or without the presence of TLR4 inhibitor Polymyxa B.Results1.In LPS-treated human nasal mucosa epithelial cells,the ratio of LC3-?/LC3-?increased in a LPS dose-dependent maner and reached the highest value at the dose of 10?g/ml LPS where stimulation group and the control group showed significant difference(P<0.01).In contrast,P62 protein decreased gradually upon the increase in LPS concentration.Additionally,the ratio of LC3-?/LC3-? increased in a time-dependent manner after LPS treatment and reached the peak value 12h after LPS treatment,where the difference was significant between treated and control group(P<0.01).The change in P62 protein level was opposite.2.12h after treatment with 10?g/ml LPS,the autophagosome,autolysosome and a large number of autophagic vacuoles were observed in human nasal mucosa epithelial cells under transmission electron microscope which is the gold standard for autophagy occurrence.3.The nasal mucosa epithelial cells transfected with GFP-LC3 plasmid were stimulated with different concentrations(0,1,5,10?g/ml)of LPS for 12h,and the distribution and expression of GFP-LC3 in cells were determiend under fluorescence microscope.The GFP-LC3 fluorescence distributed in the cytoplasm,indicating an increase in autophagosome.The percentage of GFP-LC3 and autophagosome-positve increased significantly with the elevated LPS concerntration,which was consistent with the Western blot data.4.After stimulating human nasal mucosa epithelial cells with LPS,there was a gradual increase in the expression of P-AMPK and a decrease in the expression of P-mTOR with the increasing LPS concentration.Instead no significant change was observed in p-AKT level.At the presence of AMPK inhibitor compound C,the LC3-II/LC3-I ratio induced by LPS significantly decreased whereas P-mTOR increased.5.Western blot showed that ratio of LC3-II/LC3-I decreased and p62 prtoein increased in primay nasal mucosa epithelial cells from chronic rhinosinusitis compared to the control counterparts.Meanwhile,there was an increased P-AMPK coupled with decreaed P-mTOR.All together,these data indicated a critical role of AMPK-mTOR signaling pathway in the pathogenesis of chronic rhinosinusitis.6.After LPS stimulation,TLR4,MyD88 expression and LC3-II/LC3-I ratio increased stepwisely with the increasing stimulation concentration.The administration of TLR4 inhibitor Polymyxa B supressed LPS-induced TLR4,MyD88 expression and reduced LC3-II/LC3-I ratio.Conclusions1.LPS induced autophagy in human nasal mucosa epithelial cells in a dose-and time-dependent manner.2.The P-AMPK level was increased and accordingly the P-mTOR was decreased in human nasal mucosa epithelial cells;LPS induced autophagy through the AMPK-mTOR signaling pathway.3.TLR4/MyD88-dependent signaling pathway also participated in LPS-induced autophagy in human nasal mucosa epithelial cells. |
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