| Background and objectiveChronic rhinosinusitis(CRS)is a common chronic inflammatory disease of the upper respiratory tract in otolaryngology head and neck surgery,with a high incidence,poor quality of life,and high cost of treatment.It is characterized by inflammation of the nasal cavity and paranasal sinus mucosa that lasts for more than 12 weeks.The main symptoms include nasal congestion,nasal drip,hyposmia or facial tenderness.CRS is clinically divided into two subtypes:chronic rhinosinusitis with nasal polyps(CRSwNP)and chronic rhinosinusitis without nasal polyps(CRSsNP)according to the presence or absence of polyps.The overall symptoms of CRSwNP and CRSsNP are significantly overlapping,and the symptom scores of qualities of life are similar.According to the number of eosinophils in nasal polyps,CRSwNP can be further divided into eosinophilic chronic rhinosinusitis with nasal polyps(eosCRSwNP)and non-eosinophilic chronic rhinosinusitis with nasal polyps(non-eosCRSwNP).CRS is a multifactorial disease,which includes environmental exposure,microbial infection,immune dysfunction,and genetic susceptibility.Bacterial infection plays an important role in the initiation and maintenance of inflammatory reaction in CRS.There are many kinds of microflora in the nasal cavity of CRS patients,but the exact composition varies with clinical condition(complication),age and anatomical location.At present,it is generally believed that Staphylococcus aureus plays a dominant role in CRS,and the positive rate of Staphylococcus aureus in CRSwNP group is higher than that in CRSsNP group and control group.Therefore,Staphylococcus aureus plays a specific role in the pathogenesis of CRS,and may play a more important role in the pathogenesis of CRSwNP.The colonization rate of Staphylococcus aureus is related to the percentage of eosinophils in peripheral blood and the degree of mucosal eosinophil infiltration in CRSwNP.In addition,Staphylococcus aureus is also abundant in the paranasal sinuses of healthy people,indicating that Staphylococcus aureus plays a dual role in the pathogenesis of CRS,that is,it is usually necessary for normal function and plays a pathogenic role in the pathogenesis of CRS,but the trigger factor of this role shift is not clear.Corynebacterium and Staphylococcus are present in the sinuses of most healthy volunteers and CRS patients,indicating that these two bacterial genera may have an important symbiotic relationship.There are several different kinds of Corynebacteria in the nasal cavity,of which Corynebacterium accolens is the most common one.These Corynebacteria help to maintain the normal function of the nasal cavity.However,the number of Corynebacterium decreased significantly in CRSwNP.Therefore,we speculate that the decrease of Corynebacterium in CRSwNP,especially C.accolens,affects the expression of virulence genes of Staphylococcus aureus,which leads to disease.CRSwNP is a more serious and resistant disease form in CRS.It has been associated with virulence factors of Staphylococcus aureus,especially superantigens.CRSwNP is characterized by type Ⅱ inflammatory reaction dominated by Th2 cell,80%of CRSwNP is eosCRSwNP,characterized by the increase of eosinophils,mast cells and basophils,and the increase of inflammatory cytokines such as IL-4,IL-5 and IL-13.Therefore,exploring the mechanism of Th2 cell activation is very important to understand the pathogenesis of CRSwNP.However,the molecular mechanism of Th2 cell activation is not very clear.Long non-coding RNAs(lncRNAs)is a type of non-coding RNA,discovered in recent years,which is more than 200bp in length.They play an important role in many diseases,such as immune diseases,cancer,cardiovascular diseases and so on.Long non-coding RNA nuclear paraspeckle assembly transcript 1(NEAT1)is a lncRNAs discovered in recent years,which plays an important role in the formation and maintenance of subnuclear paraspeckles.It’s up-regulated expression is an important general marker of cell differentiation.NEAT1 plays an important role in the occurrence and development of a variety of tumors,and may be used as a potential biomarker in human HIV infection,tuberculosis,sepsis,and cervical lesions.In the immune response,NEAT1 plays an important role in T cell-mediated immune inflammation by negatively regulating the expression of splicing factor proline/glutamine rich(SFPQ),mitogen-activated protein kinase fMAPK signal pathway and signal transduction and transcriptional activator protein 3(STAT3).However,it is rarely reported whether NEAT 1 can regulate the activation of Th2 cells.CRSwNP genome sequencing shows that the expression of NEAT 1 in eosCRSwNP is higher than that in non-eosCRSwNP and healthy controls.Therefore,we speculate that NEAT1-mediate Th2 cell activation plays an important role in the pathogenesis of eosCRSwNP.To sum up,this research is the first to study the virulence changes of Staphylococcus aureus caused by the change of C.accolens flora at home and abroad,and explain the mechanism of Staphylococcus aureus in the pathogenesis of CRS from the perspective of flora imbalance,which lays a foundation for the role of flora imbalance in the pathogenesis of CRSwNP.Detect the expression difference of NEAT1 in the serum of CRSwNP,explore its clinical application value as a serum marker,further study the mechanism of NEAT1 regulating Th2 cell activation,explore the role of NEAT 1 in the pathogenesis of eosCRSwNP,to provide theoretical basis for subsequent targeted therapy of eosCRSwNP.The main objectives and contents of the study are as follows:1.Isolate and identify C.accolens from the nasal cavity of healthy volunteers and CRSwNP patients,explore its co-growing conditions with Staphylococcus aureus,and further study the effects of different concentrations of C.accolens on Staphylococcus aureus planktonic bacteria and biofilm forms.2.Isolate human nasal epithelial cells(HNECs),culture HNECs at the air-liquid interface in a serum-free medium to differentiate into ciliated columnar epithelial cells and form a complete intercellular tight junction structure.Put the supernatant of Staphylococcus aureus cocultured with C accolens on HNECs cultured at the air-liquid interface to simulate the pathological infectious microenvironment of nasal mucosa,and detect the effect of Staphylococcus aureus infection on the tight junction,mucosal epithelial permeability,cell morphology and cell viability of HNECs after intervention,so as to explore the effect of C.accolens on the toxicity of Staphylococcus aureus.3.Detect the difference of NEAT1 expression in the serum of eosCRSwNP and healthy people,as well as in the serum of eosCRSwNP after operation,to explore its role in the diagnosis and postoperative monitoring of eosCRSwNP,and evaluate its clinical value as a serum marker.4.Isolate and culture CD4+T cells from the peripheral blood of healthy volunteers,construct NEAT1 and signal transduction and activator of transcription 6(STAT6)lentivirus and interference siRNA,and then transfect into CD4+T cells.Real-time PCR,Western Blot and ELISA determine the expression of genes,proteins and cytokines,and experimental techniques such as RNA-binding protein immunoprecipitation,RNA pull-down and immunoprecipitation to verify the relationship between NEAT1 and STAT6,and confirm the molecular mechanism of NEAT1 in regulating the activation of Th2 cells.Part one Isolation and identification of C.accolens from nasal cavity and its effect on Staphylococcus aureusMaterials and methods1.Dip the secretion of the lower nasal meatus gently with a disposable aseptic cotton swab to obtain clinical bacterial samples under nasal endoscope.Streak and spread the cotton swab on a tryptone agar(TSA)with 0.5%tween80 plate,37℃incubate for 48h.Identify as Corynebacterium according to the growth characteristics,colony characteristics and Gram staining characteristics of the isolated strains,and identify the strains by Corynebacterium biochemical reaction identification strip.2.Spread and inoculate Staphylococcus aureus and C.accolens on TSA,TSA+0.5%tween80 and TSA+1%tween80 plates.Then incubate plates for 48h at 37℃ to determine the optimal tween80 concentration for Staphylococcus aureus and C.accolens growing on TS A plates.3.Adjust the concentration of Staphylococcus aureus and C.accolens to a value of OD600 at 0.05,then transfer to a 50ml centrifuge tube containing tyrosinase soybean broth(TSB)+0.5%tween80,37℃ incubate at the speed of 200rpm.Measure the value of OD600 every hour to prepare a standard growth curve of bacteria.4.Spray C.accolens solution onto TSA+0.5%tween80 plate.Then,drop the Staphylococcus aureus on the plate sprayed with C.accolens,37℃ incubate for 48h,and quantify the inhibition or promotion of bacterial growth by measuring the distance between the colony edge inhibition or growth zone.5.Culture C.accolens solution in a centrifuge tube of TSB+0.5%tween80 at 37℃ 200 rpm,and collect the supernatant after 24h.Incubate bacterial suspension of C.accolens with a concentration of 1McF in a 6-well plate at 37℃ 70rpm for 48h to form a biofilm.Then,collect the supernatant.Determine the protein concentration in the sample by a BCA kit.6.Add Staphylococcus aureus solution and different concentrations supernatant(10%-90%)of C.accolens planktonic and biofilm into a 96-well black culture plate,and 37℃ 200rpm incubate for 24h.Then add alamarBlue,incubate at 37℃ for another 3h in the dark,and read the fluorescence intensity of the sample with a FLUOstar OPTIMA microplate reader.The effect of C.accolens supernatant on Staphylococcus aureus is also observed by laser scanning confocal microscope(CLSM).7.Add Staphylococcus aureus solution and different concentrations supernatant(10%-90%)of C.accolens planktonic and biofilm into a 96-well transparent microtiter plate,and incubate at 37℃ 70rpm for 48h to form a biofilm.Rinse the biofilm twice with saline to remove planktonic cells,add alamarBlue,incubate at 37℃ for 3h in the dark,and measure the fluorescence intensity of the sample with a microplate reader.8.Use statistical software SPSS 17.0 for statistical analysis.Data are expressed as mean ± standard deviation.According to the type of data,use Kruskal-Wallis test or One-way ANOVA for comparison between multiple groups,t test for pairwise comparison between groups,P<0.05 is considered statistically different.Results1.Collected 6 strains of C.accolens,1 strain of Staphylococcus aureus clinical strain,reference strain is Staphylococcus aureus ATCC51650.There was no significant difference in the growth of Staphylococcus aureus standard strains and clinical strains in TSA,TSA+0.5%tween80 and TSA+1%tween80 at 37℃ for 48h.C.accolens had no obvious growth after cultured in TSA medium at 37℃ for 48h,but grew well in TSA+0.5%tween80 and TSA+1%tween80 medium,and there was no significant difference in growth between the two groups.2.Staphylococcus aureus standard strains and clinical strains began to enter the logarithmic phase after 3h culture,and the logarithmic phase ended into the stable phase 10h later.C.accolens began to enter the logarithmic phase after 9h culture,and the logarithmic phase ended into the stable phase after 17h.3.Collected the supernatants of 6 strains of C.accolens planktonic and biofilm,and there was no significant difference in protein concentration between 6 strains of planktonic bacteria and biofilm.4.Co-culture of Staphylococcus aureus and C.accolens showed that Staphylococcus aureus standard strains,Staphylococcus aureus clinical strains and 6 C.accolens strains could grow normally without obvious inhibition or promotion.5.The supernatant of C.accolens planktonic strains had obvious inhibitory effect on the growth of standard strains and clinical strains of Staphylococcus aureus from the concentration of 60%.CLSM observation showed that a large number of red dead strains appeared after adding C.accolens supernatant,which significantly inhibited the growth of Staphylococcus aureus standard and clinical strains.The supernatant of C.accolens biofilm had obvious inhibitory effect on the growth of standard strains and clinical strains of Staphylococcus aureus from the concentration of 30%.6.The supernatant of C.accolens planktonic strain had obvious inhibitory effect on the biofilm growth of Staphylococcus aureus standard strains from the concentration of 80%,but had no obvious inhibitory effect on the biofilm growth of Staphylococcus aureus clinical strains.The supernatant of C.accolens biofilm had obvious inhibitory effect on the biofilm growth of Staphylococcus aureus standard strains from the concentration of 30%,and has a significant inhibitory effect on the growth of the biofilm of Staphylococcus aureus clinical strains from the concentration of 20%.Part two The changes of Staphylococcus aureus virulence mediated by C.accolens and its effects on human nasal mucosal epithelial cells Materials and methods1.Brush human nasal epithelial cells(HNECs)gently from the inferior turbinate,and culture in a collagen-coated flask at 37℃ with 5%CO2.Resuspend the cells when the HNECs are 80%confluence in the primary culture flask,and add the cells to a 12-well air-liquid interface(ALI)cell culture plate,70000 cells/well.Put the cells into a 37℃ 5%CO2 incubator and culture them until the cells cover the ALI culture wells after 3 weeks.2.Add different proportions(50%,70%,90%)of C.accolens to the Staphylococcus aureus solution,and place the mixed bacteria solution in a 50ml centrifuge tube in a 37℃ incubator at 200rpm for overnight culture.Collect the supernatant after 24h and determine the protein concentration in the supernatant by a BCA kit.3.Adjust the protein concentration of planktonic bacteria supernatant to 35-40mg/ml,and add it to the upper chamber of ALI cell culture plates after the HNECs cover the air-liquid interface.Measure the transepithelial cell impedance(TEER)values of HNECs at 0min,15min,30min,1h and 2h.4.Place FITC-dextran in the upper chamber of ALI cell culture plates after HNECs are intervened,and incubate at 37℃ for 2h.Transfer the cell culture solution from the lower chamber of ALI culture plates to 96-well plate in the dark,and measure the fluorescence intensity of cell culture medium with a microplate reader.5.Take the cell culture medium after different interventions,use LDH detection kit to determine the content of LDH released into the cell culture medium,and detect the cell viability after different interventions.6.Adjust the protein concentration of planktonic bacteria supernatant to 35-40mg/ml,add it to the upper chamber of ALI cell culture plates,and collect the culture solution in the lower chamber after culture for 24h.Measure the levels of IL-6 and IL-8 according to the IL-6 and IL-8 enzyme-linked immunosorbent assay(ELISA)kits.7.Use statistical software SPSS 17.0 to perform statistical analysis.Data are expressed as mean ± standard deviation.One-way ANOVA compares the differences between the negative control group and different Staphylococcus aureus strains intervention groups.T-test compares the statistical differences between Staphylococcus aureus intervention groups and the control group,P<0.05 is considered statistically different.Results1.The concentrations of protein in the supernatant were 35mg/ml-40mg/ml and did not have significant changes after the Staphylococcus aureus were cultured with different proportions of C.accolens(50%,70%,90%).2.The supernatant of C.accolens had no significant effect on the TEER value of HNECs.The supernatant of Staphylococcus aureus standard strains and clinical strains could significantly change the TEER value of HNECs after 15 min treatment.The supernatants of Staphylococcus aureus standard strains and clinical strains containing 50%C.accolens could significantly change the TEER value of HNECs after 30 min treatment.As the proportion of C.accolens increased,the time needed for Staphylococcus aureus to change the TEER value of HNECs was significantly prolonged.3.The supernatants of Staphylococcus aureus standard strains and clinical strains cultured with different proportions of C.accolens could significantly affect the permeability of HNECs.The supernatants effects of Staphylococcus aureus standard strains and clinical strains cultured with 70%and 90%C.accolens on HNECs permeability were significantly weaker than that of the corresponding Staphylococcus aureus group.4.The supernatants of Staphylococcus aureus standard strains and clinical strains cultured with different ratios of C.accolens had no statistically different effects on LDH in HNECs culture medium.5.The levels of IL-6 and IL-8 had no significant difference in Staphylococcus aureus standard strains and clinical strains cultured with 50%C.accolens compared with the corresponding Staphylococcus aureus standard strains and clinical strains groups.The levels of IL-6 and IL-8 were significantly lower in Staphylococcus aureus standard strains and clinical strains cultured with 70%and 90%C.accolens groups.Part three Clinical significance of serum NEAT1 in patients with chronic sinusitis with nasal polypsMaterials and methods1.Collect the nasal polyps of CRSwNP patients under functional endoscopic sinus surgery(FESS)treatment in our hospital.All patients meet the diagnostic criteria of CRSwNP in our country and Europe.Stain the pathological tissues with HE,and then collect images under a microscope to observe the eosinophils(Eos)infiltration in the tissues.Use tissue eosinophil ratio>15%as the criterion for eosCRSwNP,and divide CRSwNP into eosCRSwNP group and non-eosCRSwNP group.In addition,select healthy subjects as the control group.2.Collect venous peripheral blood from the patients in a fasting state in the second morning,and use an automatic hematology analyzer to detect the total number and proportion of eosinophils in the peripheral blood.3.Grade the main clinical symptoms such as nasal congestion,snot,head or face pain and hyposmia,evaluate the degree and scope of nasal lesions in CRSwNP according to Lund-Kennedy score and Lund-Mackay score.4.Extract total RNA from peripheral blood serum with RNA extraction kit,and use NanoDrop 2000 spectrophotometer to determine the purity and concentration of RNA.Then,use the Prime Script RTreagent kit from Takara,Japan for RNA reverse transcription reaction.5.Design NEAT1 and GAPDH primers according to the gene sequences in the GenBank database,and synthesized by Shanghai Bioengineering Technology Co.,Ltd.Perform fluorescent quantitative PCR reaction with cDNA produced above as the template and GAPDH as the internal reference.6.Use statistical software SPSS 17.0 to perform statistical analysis.Data are expressed as mean ± standard deviation.Use T-test for comparison between two groups,One-way ANOVA test for comparison between multiple groups,chi-square test for comparison of qualitative data,and ROC curve to judge the diagnosis sensitivity and specificity of the plasma NEAT1mRNA in CRSwNP patients.P<0.05 is considered statistically different.Results1.Collected 72 cases of CRSwNP patients,including 39 eosCRSwNP patients and 33 non-eosCRSwNP patients.30 cases in healthy control group.There was no statistical difference in demographic data such as the number of cases,age and gender among the groups(P>0.05).The percentage of eosinophils in the peripheral blood of the eosCRSwNP group was significantly higher than that of the non-eosCRSwNP group and the healthy control group(P<0.05).The Lund-Kennedy score and Lund-Mackay score in the eosCRSwNP group were not significantly different from the non-eosCRSwNP group(P>0.05).2.The results of real-time fluorescent quantitative PCR showed that the relative expression of NEAT1 in the serum of eosCRSwNP group was significantly higher than that of non-eosCRSwNP group and the control group(P<0.05).The relative expression of NEAT 1 in the serum of non-eosCRSwNP group was not significantly different from that of the control group(P>0.05).3.The ROC curve AUC of serum NEAT1 in the diagnosis of eosCRSwNP was 0.9248,95%CI was 0.8533-0.9963,P<0.01.The sensitivity was 97.4%,and specificity was 90%at the cutoff value(1.93).4.The relative expression of NEAT 1 in eosCRSwNP patients at 1 and 3 months after surgery was significantly lower than that before surgery(P<0.05),but the relative expression of NEAT1 was still significantly higher than that in the control group until 3 months after surgery(P<0.05).Part four Study on the mechanism of NEAT1 promotes Th2 cells activation via inhibiting STAT6 ubiquitinationMaterials and methods1.Collect peripheral blood from healthy volunteers in a fasting state in the morning,and separate CD4+T cells from peripheral blood mononuclear cells(PBMC)according to the instructions of the human CD4+T cell isolation kit.CD4+T cells were incubated in RPMI 1640 medium supplemented with 10%fetal bovine serum(FBS)and 1%penicillin/streptomycin at 37℃ with 5%CO2.2.Use calcium phosphate-mediated method to construct pcDNA3.1-NEAT1 plasmid.When CD4+T cells grow to 80%confluence in a six-well plate,resuspend the cells and use Lipofectamine 2000 cell transfection reagent to transfect the empty plasmid pcDNA3.1-NC,pcDNA3.1-NEAT1 plasmid,Si-NEAT1 or Si-STAT6 small interfering RNA into CD4+T cells at MOI=20.3.Extract the total RNA of CD4+T cells with TRIzol reagent,and use NanoDrop 2000 spectrophotometer to determine the purity and concentration of RNA.Use PrimeScriptTM RT kit from Takara,Japan for RNA reverse transcription to generate cDNA.Design NEAT1 and GAPDH primers according to the gene sequences in the GenBank database,and synthesized by Shanghai Bioengineering Technology Co.,Ltd.Perform fluorescent quantitative PCR reaction with cDNA produced above as the template and GAPDH as the internal reference to determine the level of NEAT1.4.Extract the total protein of CD4+T cells after intervention,determine the protein concentration with a BCA protein detection kit,and then perform Western Blot to analyze the relative expression of protein STAT6.5.Detect the levels of IL-4,IL-5 and IL-13 in CD4+T cells after intervention according to the instructions of human enzyme-linked immunosorbent assay(ELISA)kit.6.Lyse the intervened CD4+T cells,capture RNA in the cell lysate by magnetic bead method,separate and extract the RNA-bound protein,and then perform Western Blot experiment on the eluted protein.7.Lyse the CD4+T cells after intervention,separate RNA-binding protein by immunoprecipitation,extract RNA with TRIzol reagent,and detect RNA quality and concentration.Then perform qRT-PCR to quantify the immunoprecipitated RNA.8.Lyse the intervened CD4+T cells,collect total protein,immunoprecipitated with anti-STAT6(1:5000)and anti-ubiquitin(1:1000)for 12h,and incubate the protein with A/G agarose beads at 4℃ overnight.Then,boil the magnetic beads at 100℃ for 5 min.Finally,perform Western Blot to check the levels of Ub-STAT6 and STAT6 in the supernatant.9.Use statistical software SPSS 17.0 to perform statistical analysis.Data are expressed as mean ± standard deviation.Use T-test for comparison between two groups,One-way ANOVA test for comparison between multiple groups.P<0.05 is considered statistically different.Results1.The expression of NEAT1 in CD4+T cells transfected with pcDNA3.1-NEAT1 was significantly up-regulated,and the expression of NEAT 1 in CD4+T cells transfected with Si-NEAT1 was significantly down-regulated.ELISA test showed that over expression of NEAT1 enhanced the expression of IL-4,IL-5 and IL-13 in CD4+T cells.Silencing NEAT1 significantly reduced the expression of IL-4,IL-5 and IL-13 in CD4+T cells.2.Silencing STAT6 inhibited the expression of IL-4,IL-5 and IL-13 in CD4+T cells,and abolished the influence of NEAT1 over expression on the levels of IL-4,IL-5 and IL-13 in the CD4+T cells.3.Overexpression or down-regulation of NEAT1 had no effect on the expression of STAT6 mRNA in CD4+T cells.Western Blot showed that overexpression of NEAT1 could lead to up-regulation of STAT6 in CD4+T cells.Si-NEAT1 down-regulated the expression of NEAT 1 and STAT6 in CD4+T cells.4.CHX inhibited the expression of STAT6 in CD4+T cells and MG132 significantly enhanced the expression of STAT6 in CD4+T cells in the presence of pcDNA3.1-NEAT1 or pcDNA3.1-NC.Co-IP analysis showed that NEAT1 inhibited the ubiquitination of STAT6 in CD4+T cells.Conclusions1.C.accolens and Staphylococcus aureus can grow together in vitro without obvious inhibition or promotion.The supernatant of planktonic C.accolens can significantly inhibit the growth of Staphylococcus aureus at high concentration,but has no obvious inhibitory effect on Staphylococcus aureus biofilm.The supernatant of C.accolens biofilm can significantly inhibit Staphylococcus aureus in planktonic and biofilm forms from low concentration.2.The protein secreted in the supernatant of Staphylococcus aureus cultured with C.accolens can significantly reduce the damaging effect on HNECs cell barrier,that is,C.accolens significantly reduce the toxicity of Staphylococcus aureus.3.NEAT 1 in peripheral blood has good sensitivity and specificity in the diagnosis of eosCRSwNP,and can be used as a diagnostic marker of eosCRSwNP.The relative expression of NEAT1 in peripheral blood of eosCRSwNP patients are significantly decreased after surgery,which is expected to be used for monitoring the recovery and predicting recurrence of eosCRSwNP after surgery.4.NEAT 1 inhibits the ubiquitination of STAT6 by binding to STAT6 and promotes the expression of STAT6,thereby promoting the activation of Th2 cells. |
PDF Full Text Request