Mechanism Of Hederagenin Alleviating Lipopolysaccharide-induced Acute Lung Injury By Regulating NF-κB/NLRP3 Inflammasome

Objective:Sepsis is a systemic inflammation caused by bacterial infection which can lead to multiple organ failure.The lung is the most susceptible organ when sepsis occurs.Acute lung injury(ALI)is an inflammatory disease caused by sepsis.The excessive activation of M1 macrophages is an important pathological feature of ALI.Inhibiting the activation of M1 macrophages can reduce the release of inflammatory factors and alleviate pulmonary inflammation.In addition,many studies have shown that the NLRP3 inflammasomemediated inflammatory response is involved in the pathogenesis and progression of ALI.At present,there is no effective method to treat ALI caused by sepsis.How to prevent and treat ALI caused by sepsis is still the main problem in clinical treatment of sepsis.Therefore,it is necessary to explore the polarization law of macrophages and the activation mechanism of NLRP3 inflammasome during the occurrence of ALI,and find new antiinflammatory drugs to prevent and improve ALI,which will help to alleviate the pulmonary inflammatory response in patients with sepsis and improve the patient quality of life.Hedera sapogenin(HG)is mainly extracted from Hedera nepalensisvar.sinensis.It is a kind of triterpene saponins with anti-inflammatory,antibacterial,liver protection and other pharmacological effects.Studies have shown that HG has anti-inflammatory effects both in vivo and in vitro,and has broad application prospects in the treatment of inflammationrelated diseases.HG can inhibit the level of inflammatory factors in macrophages induced by lipopolysaccharide(LPS)and reduce the infiltration of inflammatory cells.In addition,HG reduces the release of pro-inflammatory factors in atherosclerotic rats by inhibiting the NF-κB pathway.Recent studies have shown that HG can alleviate pulmonary fibrosis and exert lung protection.However,the specific mechanism of HG in ALI caused by sepsis is still unclear.Therefore,this study aimed to explore the mechanism of HG in NLRP3 inflammasome activation and M1 macrophage polarization,and to evaluate the therapeutic effect of HG on sepsis-induced ALI in vitro and in vivo.To this end,the anti-inflammatory effect of HG was first evaluated at the cellular level,and its effect and mechanism on M1-type polarization of macrophages and NLRP3 inflammasome activation were explored.Secondly,a sepsis rat model was established,and HG intervention was given to further verify the protective effect of HG on the lung tissue of sepsis rats at the animal level,in order to provide a new therapeutic strategy and theoretical basis for the clinical treatment of ALI.Methods:Part I: THP-1 cells were treated with phorbol 12-myristate 13-acetate(PMA)combined with LPS to establish a sepsis cell model,and the effect of HG intervention on M1 polarization of macrophages was detected.Western blot was used to detect the expression of NLRP3 inflammasome and cleaved/pro-caspase-1 in cells.ELISA was used to detect the levels of IL-1β,IL-18,IL-6 and MCP-1 in the cell supernatant to evaluate the effect of HG on the production of inflammatory factors in M1 macrophages.The level of M1 macrophage marker CD8 was detected by flow cytometry,and the expression of i NOS and COX-2 was detected by Western blot to evaluate the effect of HG on macrophage polarization.The changes of NF-κB pathway-related molecules in macrophages were detected by immunofluorescence and Western blot to evaluate the effect of HG on NF-κB signaling pathway in macrophages.Part Ⅱ: The mechanism of HG inhibiting NLRP3 inflammasome activation and M1 macrophage polarization.After administering HG and NF-κB inhibitor BAY11-7082 to macrophages separately or jointly,LPS treatment was added,and the expression of p65 in the nucleus was detected by Western blot to evaluate the nucleus of p65.The expression of CD86 in cells was detected by flow cytometry,and the levels of i NOS and COX-2 were detected by Western blot to evaluate the effect of inhibiting the NF-κB signaling pathway on the polarization of macrophages.The expression of NLRP3 inflammasome and cleaved/pro-caspase-1 in the cells was detected by Western blot.ELISA detected the levels of IL-1β and IL-6 in the cell supernatant to analyze the effect of inhibiting the NF-κB signaling pathway on the activation of NLRP3 inflammasomes.Part Ⅲ:The sepsis rat ALI model was established by cecal ligation and puncture(CLP),and the therapeutic effect and mechanism of HG on sepsis-related ALI were explored.Eighty male rats were randomly divided into 5 groups: Sham group(n=16),CLP group(n=16),CLP+HG-12.5 mg/kg(n=16),CLP+HG-25 mg/kg(n=16),CLP+HG-50mg/kg(n=16).After modeling,10 rats in each group were selected to observe the survival rate,and the remaining 6 rats were given HG after intragastric administration of bronchoalveolar lavage fluid,blood and lung tissue for experiments.The effect of HG on ALI lung tissue injury was evaluated by HE staining,detecting the dry/wet ratio,MPO and SOD activity,MDA and GSH content of lung tissue.The expression of i NOS and COX-2in lung tissue was detected by Gimusa staining,immunofluorescence staining,Real-time PCR and Western blot.The levels of TNF-α,IL-6 and MCP-1 in bronchoalveolar lavage fluid were detected by ELISA to evaluate the effect of HG on inflammatory response and M1 macrophage activation in lung tissue.Expression of NLRP3 inflammasome and cleaved/pro-caspase-1 in lung tissue by Western blot and immunofluorescence staining.The levels of IL-1β and IL-18 in bronchoalveolar lavage fluid and lung tissue were detected by ELISA to evaluate the activation of NLRP3 inflammasome in lung tissue.The effect of HG on NF-κB signaling pathway in lung tissue was detected by immunohistochemistry and Western blot.Results:HG can reverse the protein expression levels of NLRP3 inflammasome and cleaved/pro-caspase-1 in M1 macrophages induced by LPS and reduce the expression level of pro-inflammatory cytokines such as IL-1β,IL-18,IL-6 and MCP-1 in the cell supernatant.The results of flow cytometry and Western blot showed that HG intervention reduced the level of M1 macrophage marker CD86 and inhibited the expression of i NOS and COX-2 in the cells.Further detection of the expression of NF-κB signaling pathway related molecules in cells found that HG intervention inhibits the activation of NF-κB signaling pathway.The mechanism of HG inhibiting NLRP3 inflammasome activation and M1 macrophage polarization: Both HG and BAY11-7082 can down-regulate the expression of p65 in the nucleus,and their joint intervention further reduces the expression level of p65 in the nucleus.The results of flow cytometry showed that the co-intervention of HG and BAY11-7082 can reduce the expression of CD86 in cells compared with individual intervention.Western blot results also showed that the combined intervention of HG and BAY11-7082 can synergistically inhibit the protein expression levels of i NOS and COX-2in cells.Western blot detection of the expression of NLRP3 in cells found that both HG and BAY11-7082 treatments significantly reversed the activation of NLRP3 inflammasomes induced by LPS and the expression level of cleaved/pro-caspase-1,and reduced IL-1β in the cell supernatant.The release of IL-6 inflammatory factors,and the combined treatment of the two further reduces the expression of the above-mentioned inflammatory factors.HG treatment can improve the survival rate of rats,improve the pathological damage of lung tissue in septic rats,and reduce the lung histopathological score and dry/wet ratio.The results of kit detection of MPO and SOD activity,MDA and GSH content in lung tissue show that HG can alleviate lung tissue oxidative damage.In addition,HG can reduce the number of neutrophils in the alveolar lavage fluid,reduce the levels of TNF-α,IL-6,MCP-1,IL-1β,and IL-18,and alleviate lung inflammation.Further detection of the polarization of M1 macrophages in lung tissue found that HG inhibited the polarization of M1 macrophages in lung tissue.Finally,the effects of HG on the NF-κB signaling pathway and NLRP3 inflammasomes in lung tissues were tested.The results showed that HG inhibited the activation of NF-κB signaling pathways in lung tissues and reduced NLRP3 inflammasomes and cleaved/pro-caspase-1 expression.Conclusion:(1)HG plays an anti-inflammatory role in sepsis-induced ALI.(2)HG plays a protective role in sepsis-induced ALI by blocking the NF-κB signaling pathway,inhibiting M1 macrophage polarization and NLRP3 inflammasome release.