| Chronic obstructive pulmonary disease(COPD)is a common,preventable and treatable chronic inflammatory disease of the airways.It has now become the third leading cause of death in the world.By 2060,more than5.4 million people will die of COPD and related diseases yearly.Globally,due to the increasing incidence and aging population,the social burden of COPD will continue to grow in the next few decades.Therefore,it is essential to elucidate the pathogenesis of COPD further and seek new treatment methods.Chronic inflammation of the lung parenchyma and peripheral airways is central to the inflammatory mechanisms of COPD and is vital to the induction of progressive airflow limitation.The inflammation,characterized by increased macrophages,neutrophils,T lymphocytes,and B lymphocytes in the lungs and airways,involves innate and adaptive immune responses.The imbalance of the two immune responses leads to damage to the regular repair and defense functions of the lung tissue,triggers a persistent chronic inflammatory response,and causes lung parenchyma damage,emphysema changes,and a decline in lung function.Repeated attacks of acute exacerbation of chronic obstructive pulmonary disease(AECOPD)lead to the continuous deterioration of lung function and aggravated inflammatory damage in the lungs,eventually leading to respiratory failure and even death.Lipopolysaccharide(LPS)is a component of the outer cell wall of Gramnegative bacilli.It is also the main pollution component in cigarette smoke or atmospheric particles,which can induce AECOPD.Previous studies have confirmed that many macrophages infiltrate the lungs after acute exposure to LPS,accompanied by increased cleavage of cysteine-containing aspartate specific proteinase-1(Caspase-1),interleukin-1β(Interleukin-1β,IL-1β)is actively secreted,but the exact mechanism is still unclear.Alveolar macrophages(AM)are essential in clearing and resolving lung inflammation.Pyroptosis is an inflammatory programmed death process.Once the pyroptosis pathway is activated,it can induce a robust inflammatory response to eliminate invading pathogens and maintain body homeostasis.But excessive pyroptosis can damage the body itself and lead to disease progression.Studies have shown that AM cell pyroptosis releases many proinflammatory mediators while eliminating "dissidents," leading to a "cascade amplification effect" of inflammation,which can cause excessive damage to lung tissue in a short period.NOD-like thermal receptor protein domain associated protein 3(NLRP3)is a critical element of the pyroptosis Caspase-1-dependent classic pathway,and its activation is considered to be the initiation signal of pyroptosis.HSP90 is a well-known molecular chaperone in the heat shock protein(HSP)family.NLRP3 is a client protein of HSP90.HSP90 affects the assembly and stability of its inflammasome.Without HSP90,NLRP3 will be degraded by proteases due to misfolding.The release of IL-1β is a downstream event after Caspase-1-dependent plasma membrane pore formation.Irisin is a muscle-derived or lipid-derived cytokine with antiinflammatory properties and can regulate various intracellular signaling pathways to exert its biological effects.Studies have found that when LPS stimulates macrophages to induce an inflammatory response,Irisin can reduce the release of IL-1β and reduce inflammation.In clinical case studies,the serum irisin level in COPD patients was lower than that of healthy controls.Its status was further reduced in AECOPD,indicating that Irisin was negatively correlated with inflammatory responses.Whether there is,some relationship between irisin and pyroptosis and inflammatory response and its specific mechanism deserves further exploration.This study explored the correlation between serum HSP90 levels and disease severity in patients with AECOPD.To analyze the protective effect and potential mechanism of Irisin in LPS-induced acute inflammatory injury of mouse lung tissue and MH-S cells and to provide a theoretical basis for finding potential targets in treating COPD inflammatory lung injury using HSP90 as an entry point.This study is divided into three parts,as follows:Part One Correlation between HSP90 level in serum and clinical parameters of patients with AECOPDObjective:Analyze the correlation between serum HSP90 level and inflammatory indicators,lung function,and other clinical indicators in AECOPD patients,explore the relationship between HSP90 and AECOPD inflammatory storm and seek new ideas for AECOPD treatment.Methods:We collected the serum,general clinical data,relevant laboratory,imaging,and pulmonary function test results of 60 AECOPD patients hospitalized in the Second Hospital of Hebei Medical University.The serum of 20 cases of healthy persons who underwent physical examination in the same period was collected in the control group.The expression level of serum HSP90 was detected by the ELISA method.We analyzed the correlation between HSP90 and various clinical indicators.Results:Compared with healthy controls,the expression level of serum HSP90 in AECOPD patients was significantly increased;high expression of HSP90 and low level of FEV1 may be high-risk factors for AECOPD patients with pneumonia;IL-6 and IL-1β,The levels of IL-17 and PCT were significantly higher than those of non-pneumonia patients(both P<0.05);HSP90 has higher sensitivity and specificity for the diagnosis of AECOPD complicated with pneumonia;high level of HSP90 is a poor prognostic factor for AECOPD patients.Conclusion:A high level of HSP90 is a high-risk factor for pneumonia in AECOPD and is associated with worsening lung function and disease progression.Part Two Irisin attenuates LPS-induced acute lung injury by inhibiting the activation of NLRP3 inflammasomeObjective:To explore the protective effect of Irisin on inflammatory injury and its specific mechanism in acute lung injury induced by LPS activation of NLRP3 in mice.Methods:Sixty healthy C57BL/6J mice(30 female and 30 male)aged 6-8weeks were randomly divided into control group,intervention by group: LPS-12 h group,LPS-24 h(LPS)and LPS-48 h group,high concentration LPS group(LPS-H),LPS+low concentration Irisin group(LPS+IR-L),LPS+high concentration Irisin group(LPS+IR),LPS+low concentration MCC950group(LPS+M-L),LPS+high concentration MCC950 group(LPS+M),LPS+Dexamethasone group(LPS+Dex),6 mice per group.We establish a mouse model of acute lung injury and drug intervention.The mice in the Con group were given intratracheal instillation of 50 ul normal saline,and the other nine groups were given an intratracheal infusion of 50 ul LPS.The concentration of LPS was 4 mg/kg in the LPS-H group,and LPS in the remaining eight groups was 2mg/kg.Irisin was administered by intraperitoneal injection 0.5 hours before LPS intervention.The concentration of Irisin was 0.5 mg/kg in the LPS+IR group,and Irisin in the LPS+IR-L group was 0.25 mg/kg.MCC950(NLRP3 Inhibitor)was administered by intraperitoneal injection 12 hours before LPS intervention.The concentration of MCC950 was 50 mg/kg in the LPS+M group,and MCC950 in the LPS+ML group was 25 mg/kg.Dexamethasone(0.5 mg/kg)was administered intraperitoneally,0.5 hours before LPS intervention.The mice in the LPS intervention 12 h group and the 48 h group were euthanized by 1%pentobarbital sodium(35 mg/kg)deep anesthesia combined with rapid bloodletting at 12 and 48 h of LPS intervention,respectively.The remaining eight groups were euthanized after 24 h of LPS intervention.We used hematoxylin-eosin staining to examine histopathological changes in mouse lung tissue.Detection of the expression of Irisin/FNDC5 in mouse lungs by immunohistochemical method.Immunofluorescence detection of the expression levels of macrophage-specific marker proteins CD68,CD80,i NOS,CD206,and GSDMD in mouse lung tissue.Use the cell counter to detect the total cell number and neutrophil count in the mouse BALF and the BCA protein concentration assay kit to detect the total protein concentration in the BALF.We used ELISA to detect the expression levels of IL-1β,IL-18,and TNF-α in mouse serum and BALF and the levels of Irisin in the serum of Con and LPS groups.We detected the Caspase-1 activation by the small animal in vivo imaging technology.The m RNA expression levels of IL-1β,IL-18,TNF-α,i NOS,IL-6,CD206,IL-10,NLRP3,HSP90,GSDMD,and Caspase-1 in mice lung tissue were detected by the q RT-PCR.We detected the protein expression level of NLRP3,HSP90,GSDMD,Pro-caspase-1,Cleavedcaspase-1,and IL-1β in mice lung tissue homogenate by western blotting.Results:1.Compared with the Con group,the protein expression of Irisin in the lung tissue of the mice in the LPS group was significantly increased(P<0.05),and the content of Irisin in the serum was significantly increased(P<0.05).2.After 24 hours of LPS intervention,the expression level of NLRP3 protein was significantly higher than that of the Con group,12 hours,and 48 hours group(P<0.05).3.The results of HE staining of mouse lung tissue showed that compared with the Con group,a large number of inflammatory cell infiltration and alveolar structure destruction appeared in the lung interstitium and around the trachea in the LPS and LPS-H groups,and the changes in the LPS-H group were more pronounced(P <0.05);the above-mentioned inflammatory damage in the other groups was alleviated(P<0.05),and there was no significant difference among the three groups of LPS+IR,LPS+M,LPS+Dex(P>0.05).4.The total number of cells,neutrophils,and complete proteins in the BALF of the mice in the LPS group were significantly more than those in the Con group(P<0.05);The total number of cells,neutrophils,and complete proteins in the mice BALF in the LPS+IR group were considerably lower than that of the LPS group(P<0.05).5.The secretion levels of IL-1β,IL-18,and TNF-α in BALF and serum of mice in the LPS group were significantly higher than those in the Con group(P<0.05);Compared with LPS group,the expressions of IL-1β and IL-18 was decreased considerably,the presentation of TNF-α decreased in LPS+IR group(P<0.05).However,There was no difference in the TNF-αproduction between the LPS+M and LPS groups(P>0.05).6.LPS significantly upregulated the m RNA expression level of IL-1β,IL-18,and TNF-α compared with the Con group(P<0.05).Compared with the LPS group,the m RNA expression level of IL-1β,IL-18,and TNF-α was significantly upregulated in the LPS+IR group(P<0.05).The m RNA expression level of IL-1β and IL-18 of LPS+M was lower than in the LPS group(P<0.05).However,there was no significant difference in the expression of TNF-α between the two groups(P > 0.05).The m RNA expression level of IL-1β and IL-18 showed no significant difference between the LPS+IR and the LPS+M(P>0.05).The m RNA expression of TNF-α in LPS+IR was lower than that in LPS+M(P<0.05).7.Compared with the Con group,the protein expressions of CD68,i NOS,and CD206 in the LPS group were significantly increased,and the m RNA expressions of i NOS,IL-6,CD206 and IL-10 were upregulation(P<0.05).CD68 and i NOS protein expression in the LPS+IR group was lower,and CD206 protein expression was higher than in the LPS group(P<0.05);The expression of i NOS and IL-6 m RNA was significantly reduced and CD206 and IL-10 m RNA expressions greatly increased in the LPS+IR group compared with the LPS group(P<0.05).There were no significant difference in the protein expressions of CD68,i NOS,and CD206 between the LPS+M and LPS groups,CD206 and IL-10 m RNA expressions were no significant difference(P>0.05);i NOS and IL-6 m RNA expressions in the LPS+M group were lower than in the LPS group(P<0.05).8.The activity of Caspase-1 in vivo in the LPS group was significantly higher than in the Con group(P<0.05).And the activation of Caspase-1 in the LPS+IR group was less than in the LPS group(P<0.05).9.The fluorescence intensity of GSDMD and Caspase-1 in the LPS group were significantly higher than in the Con group(P<0.05).Compared with the LPS group,the fluorescence intensity of GSDMD and Caspase-1 in LPS+IR and LPS+M groups decreased(P<0.05).10.The m RNA expression levels of NLRP3,HSP90,GSDMD,and Caspase-1 in the LPS group were significantly higher than those in the Con group(P<0.05);Compared with the LPS group,the m RNA expressions of NLRP3,HSP90,GSDMD,and Caspase-1 in the LPS+IR group decreased(P<0.05),while the m RNA expression of NLRP3,GSDMD,and Caspase-1 in the LPS+M group decreased(P<0.05),there was no significant difference in the expression of HSP90 m RNA(P>0.05).11.The protein expression levels of NLRP3,HSP90,GSDMD,Procaspase-1,Cleared-caspase-1,and IL-1β in the LPS group were significantly higher than in the Con group(P<0.05).Compared with the LPS group,the protein expression levels of NLRP3,HSP90,GSDMD,Pro-caspase-1,Cleared-caspase-1,and IL-1β in the LPS+IR group were decreased(P<0.05),the protein expression levels of NLRP3,GSDMD,Pro-caspase-1 and IL-1β in LPS+M group were reduced,but,there were no difference in the level of HSP90 and Cleared-caspase-1(P>0.05).Conclusion:LPS induces acute lung injury in mice.Irisin plays a role in repairing lung injury by regulating the polarization of macrophages in the lung.At the same time,it inhibits the activation of NLPR3 and reduces the expression of HSP90,GSDMD,Cleaved-caspase-1,and IL-1β.Irisin inhibits the activation of NLRP3 through a different signaling pathway from MCC950.Part Three Irisin protects against acute lung injury by inhibiting MH-S pyroptosis.Objective:To explore the interaction between Irisin,HSP90,NLRP3 inflammasome,and pyroptosis in the process of LPS-induced MH-S cell inflammatory injury and to elucidate the potential mechanism of Irisin inhibiting alveolar macrophage pyroptosis from playing a protective role.Methods:We added different concentrations of LPS to the culture medium to culture MH-S cells.We screened out the optimal concentration and intervention time of LPS-induced inflammatory injury of MH-S cells.According to the purpose of the experiment,MH-S cells were divided into a control group(Con),LPS group,LPS+Irisin(LPS+IR)group,LPS+17-AAG(LPS+17)group,and the CCK-8 test was used to screen out each group concentration of drug intervention.The ELISA method detected the secretion levels of IL-1β,IL-18,and TNF-α in the supernatant of cells in each group.We used the high-content imaging analysis system to sort macrophage types preliminarily.The expression levels of macrophage-specific marker proteins CD68,CD80,i NOS,CD206,and GSDMD in MH-S cells in each group were detected by immunofluorescence.MH-S cell membrane integrity was detected by the Live Cell/Membrane Damaged Cell Double Staining method.We used the FAMFLICA probe to detect intracellular Caspase-1 activity in MH-S cells.We deteced the m RNA expression levels of IL-1β,IL-18,TNF-α,i NOS,IL-6,CD206,IL-10,NLRP3,HSP90,GSDMD,and Caspase-1 in MH-S cells by q RT-PCR.At the same time,we used WB to detect the protein expression level of NLRP3,HSP90,GSDMD,Pro-caspase-1,Cleared-caspase-1,and IL-1β in MH-S cells.We also observed the effect of Irisin on MH-S cell pyroptosis by transmission electron microscopy.Results:1.The expression of NLRP3 protein in cells after LPS intervention for 4h was significantly higher than that of the Con group,LPS intervention 2 h,or12 h,or 24 h groups(P<0.05).We use the CCK-8 experiment to screen out the best intervention concentration of drugs: LPS 10ug/ml,Irisin 200ng/ml,17-AAG 100ng/ml.2.The expression levels of IL-1β,IL-18,and TNF-α in the cell supernatant of the LPS group were significantly higher than those of the Con group(P<0.05);Compared with the LPS group,the protein expression of IL-1β,IL-18 of LPS+IR,and LSP+17 was significantly reduced(P<0.05),and the level of TNF-α of LPS+IR was lower than LPS group(P<0.05),but there was no significant with the LPS+17 and LPS group(P>0.05).3.The m RNA expression level of IL-1β,IL-18,TNF-α in the LPS group was significantly higher than that in the Con group(P<0.05);The m RNA expression level of IL-1β,IL-18,TNF-α in LPS+IR group was significantly lower than that in LPS group(P<0.05);The m RNA expression level of IL-1 β,IL-18 in LPS+17 group decreased compared to LPS group(P<0.05),but there was no significant difference in TNF-α m RNA expression(P>0.05).4.Compared with the Con group,the protein expression of i NOS and the m RNA level of i NOS,IL-6 in the LPS group were significantly increased(P<0.05);The protein level of i NOS in LPS+IR group was considerably lower than that in the LPS group and LPS+17 group(P<0.05),and the m RNA expression of i NOS and IL-6 were significantly lower than those in LPS group and LPS+17 group(P<0.05);There was no significant difference in the protein expression level of i NOS and the m RNA expression level of i NOS,IL-6 between the LPS+17 group and LPS group(P>0.05).5.Compared with the Con group,LPS significantly reduced the number of cells stained green in the cell membrane integrity experiment and increased the number of red cells.Some cells were double-stained in green and red,indicating that the MH-S cell membrane was destroyed after LPS stimulated.The number of red-stained cells in the LPS+IR and LPS+17 groups was less than that in the LPS group,indicating that Irisin and 17-AAG can protect cells and maintain structural integrity.6.The fluorescence intensity of GSDMD and Caspase-1 in the LPS group was significantly higher than that in the Con group(P<0.05);Compared with the LPS group,the fluorescence intensity of GSDMD and Caspase-1 in the LPS+IR and LPS+17 groups significantly decreased(P<0.05).7.The m RNA expression levels of NLRP3,HSP90,GSDMD,and Caspase-1 in the LPS group were significantly higher than those in the Con group(P<0.05);Compared with the LPS group,the m RNA level of NLRP3,HSP90,GSDMD,and Caspase-1 in LPS+IR and LPS+17 groups decreased(P<0.05).8.The protein expression level of NLRP3,HSP90,GSDMD,Procaspase-1,Cleaved-caspase-1,and IL-1β in the LPS group was significantly higher than in the Con group(P<0.05).NLRP3,HSP90,GSDMD,Pro caspase-1,Cleared caspase-1,and IL-1 β protein expression in LPS+IR and LPS+17 groups were significantly lower than LPS group(P<0.05).9.The transmission electron microscopy results showed that before the plasma membrane ruptured after LPS stimulation,many vesicles,namely pyroptotic bodies,were formed in the MH-S cells.Then pores were included on the cell membrane,the cell membrane ruptured,and the contents flowed out,confirming that LPS induced MH-S cells desperate.Compared with the LPS group,the pyroptotic bodies in the LPS+IR group were reduced,and the lipid membrane was intact.Conclusion:LPS activates the NLRP3 inflammasome in MH-S cells and induces pyroptosis,prompting MH-S cells to express the M1 phenotype and release many cytokines to trigger inflammatory response.Irisin inhibits the Caspase-1-dependent pyroptosis pathway to reduce MH-S cell pyroptosis,regulates MH-S repolarization to minimize cytokine release,and plays a protective role in inflammatory injury.Conclusions:HSP90 is an independent risk factor for AECOPD with pneumonia;HSP90,IL-1β,IL-6,and IL-17 jointly affect lung function and disease progression in patients with AECOPD.Irisin reduces the pyroptosis of macrophages in the lungs of mice by inhibiting the activation of NLRP3 inflammasome induced by LPS,regulates the polarization of macrophages in the lungs of mice,and alleviates acute lung injury in mice.Irisin alleviates the inflammatory injury of LPS-induced MH-S cells by regulating the polarization of MH-S cells and inhibiting the pyroptosis signal transduction pathway of HSP90/NLRP3/ Caspase-1/GSDMD. |
