The Mechanism Of IL-33 And TSLP In The Pathogenesis Of Allergic Rhinitis And The Effect Of Hypoxia

Objective: Interleukin-33(IL-33)and thymic stromal lymphoietin(TSLP)are the main epithelial cell-derived factors,which can induce and promote Th2 immune response and play a role in the pathogenesis of allergic respiratory diseases.IL-33/ST2 and TSLP/OX40 L pathways also play an important role in the occurrence and development of allergic diseases,but whether there is interaction between them has not confirmed.In this study,the level of IL-33 protein in allergic rhinitis rat model was up-regulated and inhibited by animal experiments.The inflammatory changes of nasl mucosa,the expression of TSLP in nasal epithelium and serum of type 2immunoreactive cytokines(IL-4,IL-5,Tim-1)were observed.Methods:Wistar rats were sensitized by OVA and stimulated locally to prepare allergic rat medels.Set up four groups:1.normal saline group(negative control group,N=6);2.OVA group(N=6);3.OVA+IL-33 group(N=6);4.OVA+Anti-IL-33group(N=6).Hemotoxylin-eosin staining(HE)was used to observe the changes of airway inflammatory cells infiltration and eosinophil secretion.The expression of IL-33,ST2 and TSLP in nasal epithelium of rats were observed by immunohistochemistry(IHC).The level of Th2 cytokines(IL-4,IL-5,Tim-1),IL-33,TSLP and ovalbumin-specific Ig E(OVA-s Ig E)in blood supernatant were detected by enzyme-linked immunosorbent assay(ELISA).At the same time,rat nasal epithelial cells(RNEp Cs)were cultured by differential attachment method and conditioned medium for subsequent experiments.Results: 1.HE staining of nasal mucosa in allergic rhinitis rat model showed that the number of submucosal vessels and glands increased,mucosal cilia fell off,and eosinophils infiltrated significantly in nasal mucosal epithelium of OVA group and IL-33+OVA group allergic rhinitis rats.2.TSLP immunohistochemical products are located in the surface and propria of nasal epithelium;IL-33 is expressed in the cytoplasm and nucleus of nasal epithelial cells;ST2 is also expressed in the cytoplasm of nasal epithelial cells.Compared with the control group,the expression of IL-33,ST2 and TSLP are increased in the experimental groups.3.The expression of s Ig E,TSLP,IL-33,IL-4,IL-5,Tim-1 cytokines in serum increased significantly.Inhibiting IL-33 can inhibit the expression of ST2 and TSLP.Conclusion:1.Th2 cytokines are abundantly expressed in serum and nasal epithelium of rats with allergic rhinitis.IL-33,ST2 and TSLP are involved in the pathogenesis of allergic rhinitis.2.IL-33,ST2 and TSLP are expressed in the cytoplasm of RNEp Cs.Inhibition of IL-33 can down-regulate the expression of ST2 and TSLP.Positive synergistic effect may be between TSLP and IL-33/ST2 pathway.Objective: Based on normal rat nasal epithelial cells(RNEp Cs)and allergic rhinitis RNEp Cs,to construct normal and allergic rhinitis RNEp Cs’ model under normoxia and hypoxia.To construct TSLP gene knockdown cell model by lentvirus transfection.Aimed to study the interaction of IL-33/ST2 and TSLP in RNEp Cs,and to further explore the effects under hypoxia.Methods: Normal RNEp Cs and allergic rhinitis RNEp Cs were cultured to establish normoxic and hypoxic cell models.Normal RNEp Cs were cultured to construct anti-IL-33(IL-33 inhibition medel)and TSLP knock down cell models.Randomized groups: Normoxia group(N),TSLP knock down group(TSLP-KD),normoxia with anti-IL-33 group,normoxia allergic rhinitis group(ARS),hypoxia allergic rhinitis group(H-ARS).Normal oxygen condition:21%O2,5%CO2,74%N2,37℃;Hypoxia condition: 5%O2,5%CO2,90%N2,37℃.Immunohistochemical staining was used to identify cells;CCK-8 staining was used to detect cell proliferation;Use TUNEL fluorescence to detect apoptosis of RNEp Cs in each group.Tranwell method was used to detect the effect of different concentration of IL-33(0,5,10,20ug/ml)on cells’ migration.The secretion of IL-33 in the supernatant of RNEp Cs was detected by ELISA,and the expression of IL-33,TSLP and ST2 was detected by Western blot.Results:1.Cell growth: primary RNEp Cs were round or oval shape with abundant protrusions;AE1/AE3 was positive by immunochemical staining.2.Difference in cell proliferation(CCK-8 A value): Compared with normal oxygen control group(N),the proliferation of RNEp Cs in ARS group and H-ARS group increased,and the expression of H-ARS was more significant.After intervention with Anti-IL33 and TSLP-KD,the proliferation of RNEp Cs was inhibited.There was a significant difference between the two groups(p <0.05).With the increase of IL-33 concentration(0,5,10,20ug/ml),all groups showed a proliferation trend,especially in the hypoxic group(p <0.01).3.Apoptotic difference(TUNEL): Compared with the normoxic control group,the area of apoptotic cells in Anti-IL-33 group and TSLP-KD group increased,while the area of apoptotic cells in ARS and H-ARS group decreased significantly(p <0.01).4.Cell migration(Tranwell chamber): Compared with the normoxic control group,Anti-IL-33 group inhibited cell migration(p <0.01),while H-ARS group promoted cell migration(p <0.01).Under the action of IL-33 at different concentration gradients,cell migration in TSLP-KD and H-ARS was dose-effective.5.Elisa: Compared with the normoxic group,the secretion of IL-33 in ARS and H-ARS increased(p <0.01),the secretion of IL-33 increased with time in each group,but the secretion of IL-33 did not increase significantly in TSLP-KD group during the first 18 hours,whether under normoxic or hypoxic conditions.The secretion of IL-33 at 24 th hour was higher than that in 18 th hour(p <0.01).6.The expression of endogenous IL-33,ST2 and TSLP in each group(Western blot): The expression of the IL-33,ST2 and TSLP proteins in H-ARS and ARS group increased(p<0.05),while the expression in Anti-IL-33 and TSLP-KD group was inhibited(p<0.01).Hypoxia promoted the expression more than normoxia(H-ARS VS ARS,p<0.05).Conclusion:1.IL-33,ST2 and TSLP are abundant in RNEp Cs of allergic rhinitis;2.There is a positive synergistic effect between TSLP and IL-33/ST2 signaling pathway.3.Positive feedback between TSLP and IL-33 promotes the proliferation of RNEp Cs,inhibits cell apoptosis and enhances cell chemotaxis.4.Inflammatory response of RNEp Cs in allergic rhinitis is more significant under hypoxic conditions;it can effectively improve the local hypoxic environment of RNEp Cs,and inhibit the expression of hypoxia-related signaling pathway proteins in the treatment of allergic rhinitis,or to be a new idea for treatment of allergic rhinitis.