The Mechanism Of Intermittent Hypoxia-induced HIF-1α Regulation Of NLRP3-mediated Pyroptosis In The Inflammatory Response Of Asthma

Objective: To explore whether intermittent hypoxia（IH）can activate hypoxia inducible factor-1α（HIF-1α）and regulate NOD like receptor thermal protein domain associated protein 3（NLRP3）inflammasome-mediated pyroptosis to exacerbate the inflammatory response during asthma and to further clarify the potential molecular mechanism.Methods: Patients diagnosed with moderate and severe bronchial asthma who underwent polysomnography monitoring were enrolled.The general data of the patients,polysomnography monitoring results,induced sputum cell classification counts and interleukin-6（IL-6）,IL-8,HIF-1α and NLRP3 levels were collected,the impact of different degrees of OSAHS on asthma was analysed.The human bronchial epithelial cell line BEAS-2B was cultured in vitro,IL-13 and/or IH intervention was performed,cell damage and the expression of HIF-1α,pyroptosis-related signal molecules: NLRP3,GSDMD,IL-1β,cleaved caspase-1 and IL-18 and inflammatory indicators tumor necrosis factor-α（TNF-α）,IL-6,IL-8,matrix metalloprotein-9（MMP-9）were detected.HIF-1α and NLRP3 regulated the inflammatory response induced by IH and IL-13.OVA was used to establish a mouse asthma model that was subjected to CIH or normoxia.The treatment group was intraperitoneally injected with the HIF-1α inhibitor LW6.After the completion of modelling,bronchoalveolar lavage fluid（BALF）was collected for cell classification and quantification,cytokine levels: TNF-α,IL-1β,IL-6,IL-8 and NE were detected.The pathological changes in lung tissue were evaluated,HIF-1α levels in lung tissue homogenate and the expression of pyroptosis-related molecules: NLRP3,GSDMD,cleaved caspase-1 and IL-18 were detected.Immunofluorescence double staining was used to determine the expression of HIF-1α and NLRP3 in lung tissue.Results: The clinical study showed that asthma patients with OSAHS were older.With the exacerbation of OSAHS,the degree of obesity gradually increased,the proportion of asthma that was partially controlled and uncontrolled also gradually increased.The proportion of eosinophils of induced sputum was decreased with the exacerbation of OSAHS and the proportion of neutrophils of induced sputum was increased with the exacerbation of OSAHS.With increasing OSAHS severity,the expression of IL-6,IL-8,HIF-1α and NLRP3 in induced sputum increased.Correlation analysis showed that HIF-1α was positively correlated with AHI,BMI,the proportion of neutrophils,IL-6 and NLRP3.HIF-1α was negatively correlated with mean blood oxygen saturation and the proportion of eosinophils.NLRP3 was positively correlated with AHI and the proportion of neutrophils.NLRP3 was negatively correlated with mean blood oxygen saturation and the proportion of eosinophils.The results of in vitro experiments showed that IH exacerbated IL-13-induced injury of BEAS-2B cells and the expression of pyroptosis-related molecules,promoting the expression of neutrophil inflammatory indicators.After silencing HIF-1α expression,IH and IL-13 induced BEAS-2B cell damage,the expression of neutrophil inflammatory indicators decreased.After silencing HIF-1α,Nigerian bacteriocin upregulated the expression of NLRP3,increased the expression of neutrophil inflammatory indicators and increased the expression of pyroptosis-related pathway molecules but HIF-1α showed no changes.Animal experiments confirmed that the airway inflammation induced by CIH combined with OVA was regulated by the HIF-1α/NLRP3/GSDMD signaling pathway and mainly involved neutrophil inflammation.After HIF-1α suppression,the airway inflammation score was significantly reduced,the proportion of neutrophils in BALF was significantly reduced,the level of inflammatory factors was reduced and the expression of NLRP3 pathway-related molecules was inhibited.However,mice treated with OVA combined with normoxic intervention did not exhibit these changes.Conclusion: OSAHS is a risk factor for asthma exacerbation.OSAHS combined with asthma can upregulate HIF-1α expression,activation of NLRP3 inflammasome-mediated pyroptosis induces airway inflammation dominated by neutrophils,inhibition of HIF-1α can regulate inflammatory reactions by mediating the NLRP3 signaling pathway.Part Ⅰ: Effect of Obstructive Sleep Apnea on Airway Inflammatory Response in Asthmatic PatientsObjective: To explore the effects of mild,moderate and severe OSAHS on the control of asthma and to determine whether OSAHS exacerbates asthma airway inflammation,affects the expression of HIF-1α and NLRP3 and changes the type of asthma airway inflammation.Methods: According to inclusion and exclusion criteria,patients diagnosed with moderate and severe bronchial asthma at Tongji Hospital affiliated with Tongji Medical College of Huazhong University of Science and Technology from January 2021 to December 2022 were enrolled,the patients were diagnosed by polysomnography.General data,polysomnography monitoring results,induced sputum cell counts and induced sputum levels of IL-6,IL-8,HIF-1α and NLRP3 were collected from the enrolled patients,the impact of different degrees of OSAHS on asthma was analysed.Results: A total of 105 patients were included in this study,including 16 patients with simple asthma,37 patients with mild and moderate OSAHS and 52 patients with severe OSAHS.The age of asthma onset in each group was analysed,and it was found that the age of asthma onset in the simple asthma group was younger.With the exacerbation of OSAHS,the degree of obesity in patients gradually increased,and the proportion of patients with partially controlled and uncontrolled asthma also increased.With an increase in OSAHS,the sleep apnoea hypopnea index（AHI）,oxygen reduction index（ODI）,the time when blood oxygen saturation was Below 90%（T90%）,the percentage of time when blood oxygen saturation was below 90%（TS90%）and snoring index increased,the lowest oxygen saturation（LSpO₂%）and average blood oxygen saturation decreased.The induced sputum cells were classified and counted,the proportions of cell types among total cells were calculated.The results showed that the proportion of eosinophils decreased with the exacerbation of OSAHS and the proportion of neutrophils increased with the exacerbation of OSAHS.With increasing OSAHS severity,the expression of IL-6,IL-8,HIF-1α and NLRP3 in induced sputum increased.Correlation analysis showed that HIF-1α was positively correlated with AHI,BMI,neutrophils,IL-6 and NLRP3.HIF-1α was negatively correlated with mean blood oxygen saturation and eosinophils.NLRP3 was positively correlated with AHI and neutrophils.NLRP3 was negatively correlated with mean blood oxygen saturation and eosinophils.Conclusion: OSAHS is a risk factor for asthma deterioration.Age of asthma onset and BMI were related to the severity of OSAHS.With increasing OSAHS severity,the proportion of partially controlled and uncontrolled asthma patients also increased,the proportion of neutrophils in induced sputum increased,as well as the expression of IL-6,IL-8,HIF-1α and NLRP3.Part Ⅱ: IH Regulates NLRP3-Mediated Pyroptosis Through Activation of HIF-1α Aggravate IL-13 Induced Human Bronchial Epithelia Cell Injury and InflammationObjective: To explore the effect and mechanism of IH on IL-13-induced inflammatory reactions in BEAS-2B cells in vitro and to explore whether IH can promote HIF-1α-mediated regulation of NLRP3-mediated pyroptosis and affect the occurrence and development of IL-13-induced inflammatory reactions.Methods: BEAS-2B cells were grown to the appropriate fusion degree and treated with IL-13（4 ng/m L）and/or IH（21% O₂-25 minutes,5% O₂-35 minutes;1 cycle/1 hour）for 24 hours,apoptosis and the expression of HIF-1α,pyroptosis-related signaling molecules（NLRP3,GSDMD,IL-1β,cleaved caspase-1 and IL-18）and inflammatory factors（TNF-α,IL-6,IL-8 and MMP-9）were detected.HIF-1α expression was silenced with siRNA,and the effects of signaling molecules related to pyroptosis and inflammatory factors in BEAS-2B cells were detected.After silencing HIF-1α and administering nigericin（10 μmol/L）,the effect on signaling molecules related to pyroptosis and inflammatory indicators in BEAS-2B cells was detected.Results: IH or IH and IL-13 activated HIF-1α expression.IH could exacerbate BEAS-2B cell injury induced by IL-13 and the related molecules NLRP3,GSDMD,IL-1β,cleaved caspase-1 and IL-18 and could promote the expression of the inflammatory factors TNF-α,IL-6,IL-8 and MMP-9.After silencing HIF-1α with siRNA,IH and IL-13 induced BEAS-2B cell pyroptosis and damage,the inflammatory factors TNF-α,IL-6,IL-8 and MMP-9 were decreased.After silencing HIF-1α,the expression of NLRP3 was upregulated by nigerian bacteriocin,the inflammatory indicators TNF-α,IL-6,IL-8 and MMP-9 were increased,the pyroptosis-related pathway proteins NLRP3,GSDMD,IL-1β,cleaved caspase-1 and IL-18 were increased,but HIF-1α showed no significant changes,confirming that NLRP3 was a downstream molecule of HIF-1α.Conclusion: In BEAS-2B cells,IH promotes IL-13-induced pyroptosis,exacerbates cell damage and inflammatory reactions and induces the expression of neutrophil chemokines,including TNF-α,IL-6,IL-8 and MMP-9 via the HIF-1α/NLRP3/GSDMD signaling pathway.Part Ⅲ: CIH Exacerbates Pulmonary Neutrophilic Inflammatory Responses in Mouse Asthmatic Models by Promoting HIF-1α to Regulate NLRP3-Mediated PyroptosisObjective: To verify whether activation of the HIF-1α/NLRP3 signaling pathway by CIH exacerbates the airway inflammatory response induced by OVA in vivo.Methods: C57BL/6J mice were exposed to CIH or air for 1-28 days.The asthma model groups were sensitized by intraperitoneal injections of a mixture of OVA and Al（OH）₃ as an adjuvant on the 3rd,10 th and 17 th days.The mice were given OVA solution by nasal drops on the 24 th,25th,26 th,27th and 28 th days.In the treatment group,the HIF-1α inhibitor LW6（10 mg/kg）was injected intraperitoneally three times per week,the control group was treated with the same volume of normal saline.After the completion of modelling,bronchoalveolar lavage fluid（BALF）was collected for cell classification,the cytokines（TNF-α,IL-6,IL-8,IL-1β and NE）were detected,the pathological changes in lung tissue were evaluated,HIF-1α in lung tissue homogenate was detected,and the pyroptosis-related molecules（NLRP3,GSDMD,cleaved caspase-1 and IL-18）was detected.Immunofluorescence double staining was used to detect the expression of HIF-1α and NLRP3.Results: Airway inflammation was significantly increased in mice in the OVA group and CIH+OVA group.The airway inflammation induced by OVA alone was mainly eosinophilic inflammation and the increase in neutrophils was not significant.The airway inflammation induced by CIH plus OVA was mainly caused by an increase in neutrophils.Although eosinophils were increased,the increase in neutrophils was significant.In lung inflammation induced by CIH plus OVA,the levels of the inflammatory indicators TNF-α,IL-6,IL-8,IL-1β especially NE were significantly higher than those induced by OVA alone.In the lung tissue in the CIH+OVA group,the expression of HIF-1α and pyroptosis-related signaling molecules（NLRP3,GSDMD,cleaved caspase-1 and IL-18）increased significantly.After inhibiting HIF-1α,the decrease in the inflammation score in the OVA plus normoxia group was not significant,as shown by HE staining.The results of BALF cell classification showed that the proportions of various cell types did not change significantly,the level of inflammatory factors did not decrease and there were no significant changes in the expression of HIF-1α/NLRP3 pathway indicators.However,the inflammation score induced by OVA plus CIH was significantly reduced and the results of BALF cell classification showed that neutrophils were significantly reduced,the level of inflammatory factors was reduced and the expression of the HIF-1α/NLRP3 pathway was significantly reduced.These results indicate that HIF-1α/NLRP3 mediates neutrophil-based lung inflammation caused by OVA+CIH but does not participate in eosinophil-based lung inflammation caused by OVA alone.Conclusion: In this study,CIH+OVA promoted airway neutrophil inflammation in mice and HIF-1α was significantly increased.HIF-1α blockade could inhibit the NLRP3-mediated pyroptosis pathway,reduce neutrophil-related inflammatory factors such as IL-1β,TNF-α,IL-6,IL-8 and NE,alleviate airway inflammation mediated by neutrophils and further solidify the connection between HIF-1α/NLRP3 and neutrophil inflammation,showing that HIF-1α plays an important stimulatory role in CIH+OVA-driven neutrophil airway inflammation,the NLRP3 inflammasome and inflammatory factor synthesis and release.