| Background:Chronic rhinosinusitis with nasal polyps(CRSw NP)is a disease associated with chronic inflammation of nasal mucosa epithelium with various etiologies.Its main symptoms are anosmia,thick nose,dizziness,headache,nasal obstruction,which seriously affects the quality of life and working state of the patients.Meanwhile,its recurring characteristics also cause huge social burden.Although with the advancement of medical methods,the curative effect of CRSw NP with antibiotics,nasal hormones and surgical treatments is now accepted by patients,satisfactory therapeutic effects are still not achieved.In recent years,many studies have found that CRSw NP can divided into eosinophilic chronic rhinosinusitis with nasal polyps(eos CRSw NP)and non-eosinophilic chronic rhinosinusitis with nasal polyps(Non-eosinophilic chronic rhinosinusitis with nasal polyps,non-eos CRSw NP).The eos CRSw NP is mainly characterized by the presence of a large number of eosinophils in the tissues,often accompanied by asthma and allergic rhinitis,mainly in European and American populations;the non-eos CRSw NP is mainly characterized by non-eosinophil infiltration such as neutrophils,mainly distributed in Asian people such as China.Due to the complex etiology and the different mechanisms,for eos CRSw NP and non-eos CRSw NP,explore their respective pathological processes and mechanisms,and achieve differentiated treatments for different types of CRSw NP,and make them individualized and precise treatment is more necessary to achieve better treatment effect.Hypoxia inducible factor-1α(HIF-1α),as a product of hypoxia,is decomposed under normoxia.Although recent studies have confirmed that the pathogenesis and development of CRSw NP are related to hypoxia-mediated inflammation,its mechanism of action in different types of CRSw NP,as well as the effect in NOD-like receptor family,pyrin domain containing 3(NLRP3)inflammasomes is still unclear.Therefore,our study mainly explored the expression of HIF-1α and NLRP3 inflammasome in CRSw NP,and further investigated the effect of HIF-1α on the differentiation of epithelial cells and macrophages in CRSw NP under hypoxia,as well as its effect on NLRP3 inflammasome,so as to explain the pathological mechanism of HIF-1α in CRSw NP.Part 1: HIF-1α promotes the activation of NLRP3 inflammasomes in noneosinophilic chronic sinusitis with nasal polyps by regulating M1 macrophagesObjective:To investigate the expression levels of HIF-1α and NLRP3 inflammasomes in different types of CRSw NP,confirm the cell types that mediate the effects of HIF-1α and NLRP3,and analyze the relationship between HIF-1α and the prognosis of CRSw NP.Methods:Patients with CRSw NP who underwent surgical treatment at the Otolaryngology-Head and Neck Surgery Department,West China Hospital,Sichuan University were enrolled in this study between October 2018 and October 2019.Patients with septal flexion and no inflammatory nasal disease served as control group.We collected the preoperative basic information,preoperative VAS score,preoperative Land-Mackay score,preoperative Lund-Kennedy score,and followed up the recurrence time of CRSw NP patients.Hematoxylin-eosin staining(HE)was used to detect CRSw NP,protein and m RNA quantification of HIF-1α and NLRP3 inflammasome were analyzed by Western blotting(WB)and reverse transcriptation-polymerase chain reaction(RT-PCR).The levels of HIF-1α and NLRP3 in different types of CRSw NP were analyzed by immunohistochemical staining.The distribution of HIF-1α and NLRP3 in different types of CRSw NP was analyzed by immunofluorescence costaining and the active cells were located.Phorbol-12-myristate-13-acetate(PMA)was used to induce the transformation of THP-1 monocytes into macrophages.Cobalt chloride(Co Cl2)was added to simulate hypoxia environment.WB and RT-PCR were used to detect protein and messenger ribonucleic acid(m RNA)to determine the type of macrophages.HIF-1α and NLRP3 were inhibited by small interfering ribonucleic Acid(si RNA),respectively.The protein and m RNA were detected by WB and RTPCR again,and the levels of HIF-1α and NLRP3 were analyzed and detected.Preoperative VAS score,preoperative Lan-Mackay score,preoperative Lund-Kennedy score and HIF-1α number in immunohistochemistry were collected to analyze the relationship between the above indexes and CRSw NP recurrence.Results:The healthy nasal mucosa of 25 patients and 75 patients with CRSw NP were collected and detected,which were divided into 32 cases of eos CRSw NP and 43 cases of noneos CRSw NP by HE staining.WB assay showed HIF-1α,NLRP3,cleaved caspase-1 in non-eos CRSw NP and eos CRSw NP,apoptosis associated speck-like protein containing a CARD(ASC),Interleukin(IL)-1β,and apoptosis associated speck-like protein containing a CARD(ASC),IL-18 and p38 mitogen-activated protein kinase(p38 MAPK)were higher than those in the control group,but the above indexes in non-eos CRSw NP were higher than those of eos CRSw NP.RT-PCR results also indicated that HIF-1α,NLRP3,caspase-1,ASC,IL-1β and IL-18 were higher in eos CRSw NP and non-eos CRSw NP than in the control group,while non-eos CRSw NP was higher than eos CRSw NP.In addition,the levels of IL-6 and IL-8 in noneos CRSw NP were also higher than those in eos CRSw NP and the control group.Similarly,immunohistochemical staining results indicated that HIF-1α and NLRP3 were higher in non-eos CRSw NP and eos CRSw NP than in the control group,while non-eos CRSw NP was higher than eos CRSw NP.The results of immunofluorescence co-localization indicated that HIF-1α mainly co-stained with INOS antibody of M1 macrophages,and the amount of HIF-1α in non-eos CRSw NP was higher than that in eos CRSw NP and the control group.Similarly,the number of NLRP3 and INOS coinfection in non-eos CRSw NP was also higher than that in eos CRSw NP and control group.Next,we used Cocl2 to stimulate macrophages to build a model of hypoxic environment.Protein and m RNA results suggested that hypoxia enhanced the expression of INOS,promoted the polarization of M1 macrophages,increased the expression of HIF-1α and NLRP3 inflammasome,and increased the protein and m RNA levels of cleaved caspase-1,ASC,IL-1β,IL-18,and phosphorylated p38 MAPK,as well as IL-6 and IL-8.In addition,Si RNA experiments showed that inhibition of HIF-1α decreased protein and m RNA levels of NLRP3,cleaved-caspase-1,IL-1β,IL-18,and phosphorylated p38 MAPK,as well as IL-6 and IL-8,while inhibition of NLRP3 also decreased protein and m RNA levels of cleaved-caspase-1,IL-1β,IL-18,and phosphorylated p38 MAPK,as well as IL-6 and IL-8.However,NLRP3 expression had no effect on HIF-1α.We also found that preoperative VAS score,preoperative Lan-Mackay score,preoperative Lund-Kennedy score,and the amount of immunohistochemical HIF-1α were associated with the recurrence of non-eos CRSw NP.The immunohistochemical HIF-1α was more accurate in predicting the recurrence of non-eos CRSw NP than the preoperative VAS score,the preoperative Lan-Mackay score and the preoperative Lund-Kennedy score.Conclusion:The present study suggested that HIF-1α and NLRP3 inflammasomes mainly existed in non-eos CRSw NP.HIF-1α may promote the activation of NLRP3 inflammasome in non-eos CRSw NP by promoting the polarization of M1 macrophages,leading to the occurrence of T helper cell(Th)type I inflammation.In addition,the amount of immunohistochemical HIF-1α can be used as a predictor of non-eos CRSw NP recurrence.Part II: HIF-1α is involved in the pathological process of chronic rhinosinusitis with nasal polyps by regulating the activation of NLRP3 inflammasome to promote epithelial cell differentiationObjective:To investigate the expression of HIF-1α and NLRP3 inflammasome in the epithelium of CRSw NP,confirm the cell types that HIF-1α and NLRP3 mainly existed in the epithelium,and analyzed the role and mechanism of HIF-1α and NLRP3 inflammasomes on nasal mucosa epithelial differentiation.Methods:Patients with CRSw NP who underwent surgical treatment at the Otolaryngology-Head and Neck Surgery Department,West China Hospital,Sichuan University were enrolled in this study between October 2018 and October 2019.Patients with septal flexion and no inflammatory nasal disease served as control group.WB and RT-PCR experiments were performed on the CRSw NP to detect the protein and m RNA levels of ciliary cells,goblet cells and basal cells in epithelial cells,and the levels of FOXJ1,YAP and Ki67 in CRSw NP were also detected.Immunofluorescence staining was used to detect the co-staining and expression of HIF-1α,NLRP3,FOXJ1,YAP and Ki67 in CRSw NP with ciliary cells,goblet cells and basal cells in the epithelium.Then,healthy nasal mucosa and CRSWNP tissues were collected for the culture of human nasal epithelial cells(h NECs).After the culture reached a certain concentration,the specimens were collected to detect the protein and m RNA expression of HIF-1α and NLRP3 inflammasome in the healthy nasal mucosa and the h NECs of CRSWNP.Subsequently,we added Cocl2 to stimulate healthy h NECs,and further detected the RNA and protein expressions of HIF-1α,NLRP3,caspase-1,ASC,IL-1β and IL-18 in h NECs under hypoxia at 0h,12 h,24h and 48 h.In order to further confirm the effect of hypoxia on epithelial differentiation,we established human nasal epithelial stem cells /progenitor cells(h NESPCs)in a gasliquid culture system.Different doses of Cocl2 were added to the hypoxic model,meanwhile,HIF-1α inhibitor KC7F2(KC),NLRP3 inhibitor CP-456773 sodium salt(CP)and YAP inhibitor Verteporfin(VP)were added,respectively.HIF-1α,NLRP3,Tubulin,FOXJ1,Mucin5 AC,P63,YAP and Ki67 were collected on days 7,14 and 21 for immunofluorescence,RT-PCR and WB detection.Cell number and mortality were calculated by trypan blue staining.Ciliary oscillations were observed using chargecoupled element microscope and visual video analysis software.Results:The results of WB and RT-PCR indicated that the protein and m RNA levels of Mucin5 AC in non-eos CRSw NP and eos CRSw NP were higher than those in the control group.Immunofluorescence results indicated that Mucin5 AC in non-eos CRSw NP and eos CRSw NP was higher than that in the control group,and increased with the increase of HIF-1α.In addition,both HIF-1α and NLRP3 were co-stained with Mucin5 ACpositive goblet cells.The protein and m RNA levels of Tubulin and FOXJ1 were lower in non-eoscrswnp and eos CRSw NP than in the control group.The results of immunofluorescence also suggested that the numbers of Tubulin and Fox J1 were lower in non-eos CRSw NP and eos CRSw NP than in the control group,and decreased with the increase of HIF-1α and NLRP3.The protein and m RNA levels of P63,YAP and Ki67 in non-eos CRSw NP and eos CRSw NP were higher than those in the control group.Immunofluorescence co-staining also indicated that the levels of P63,YAP and Ki67 were higher than those in the control group,and increased with the increase of HIF-1α and NLRP3.It is worth mentioning that YAP is mainly expressed in p63-positive basal cells.The results of h NEPCs indicated that Cocl2 promoted the expression of HIF-1α and NLRP3 in h NESPCs,and the expression of Tubulin and Fox J1 decreased as the expression of HIF-1α and NLRP3 increased.KC and CP inhibited the expression of HIF-1α and NLRP3,and increased the levels of Tubulin and Fox J1,but VP had no significant effect.Mucin5 AC was co-stained with HIF-1α and NLRP3,respectively,and increased with the increase of HIF-1α expression.In addition,the expression of YAP and Ki67 also increased with the increase of HIF-1α expression,and were costained with Mucin5 A respectively.KC,CP and VP all inhibited the expression of Mucin5 A YAP and Ki67,but the inhibitory effect of KC was stronger than that of CP and VP.The expression of P63 also increased with the stimulation of Cocl2 and increased with the dose.YAP,Ki67 and P63 were co-stained and increased with the expression of HIF-1α.Both KC and VP could inhibit the expression of P63,but CP had no obvious effect.m RNA and protein levels of HIF-1α and NLRP3 were increased after Cocl2 stimulation,while Tubulin and Fox J1 were decreased.KC and CP inhibited the m RNA and protein expressions of HIF-1α and NLRP3,and increased the levels of Tubulin and Fox J1.The same VP has no obvious effect.Cocl2 stimulation increased the m RNA expression of Mucin5 AC,YAP and Ki67,and increased with the increase of concentration,while KC,CP and VP all inhibited the expression of the above indexes,but KC and VP were stronger than CP.The expression of YAP and Ki67 protein levels also increased with the increase of Cocl2 stimulation concentration,and the expression of KC,CP and VP were also inhibited,but the inhibition intensity of KC and VP was higher than that of CP.Similarly,Cocl2 also increased the expression of P63 m RNA,while both KC and VP inhibited the expression of P63.But CP seemed to have no significant effect.Cell count and mortality tests showed that Cocl2 reduced cell count and increased mortality.KC and CP reversed this trend,and the effect of KC was stronger than that of CP.VP further reduced the number of cells and increased mortality.Through the detection of cilia swing,it was found that Cocl2 reduced the frequency of h NESPCs,while KC and CP increased the frequency of ciliary beat frequency,while VP had no significant effect.Conclusion:Our study suggested that the expression of HIF-1α and NLRP3 inflammasome produced by hypoxia mainly existed in CRSw NP,while non-eos CRSw NP was higher than eos CRSw NP.HIF-1α reduced ciliary cell production by reducing FOXJ1 expression,and activated the expression of YAP and Ki67 to promoted the proliferation of goblet cells and basal cells,thereby further affecting the differentiation of epithelial cells and participating in the pathological mechanism of CRSw NP.In addition,increased HIF-1α stimulated the production of NLRP3 inflammasome,which reduced ciliary cell production through apoptosis and affected goblet cell proliferation,but had no significant effect on basal cells. |
PDF Full Text Request