The Mechanism Of Sodium Butyrate Regulating Neutrophil Migration And Extracellular Trapping Net Formation In Calculous Cholecystitis

Neutrophil Extracellular Traps(NETs)has been demonstrated to initiate gallstone formation.Increasing macrophages in plasma and gallbladder tissue is pathological characteristics of cholecystitis.CXCL16 is involved in inflammation mediated macrophages polarization and exosomal communication between cells.As a short-chain fatty acids(SCFAs),Butyrate acid have anti-inflammatory effects.However,the role of Butyrate acid in NETs accompanied with macrophages and molecular mechanism remains unclear.In this study,relevant indexes of plasma and gallbladder tissue of patients with calculous cholecystitis were detected and correlation analysis was conducted.Further,LPS induced macrophage polarization model was established.The mechanism of reducing NETs formation by butyric acid was explored from the perspective of inhibiting macrophage polarization,and the effect of CXCL16 on NETs formation was further studied from the aspect of CXCL16 carried by exosomes.Purpose:1.Explore the clinical characteristics and correlation analysis of NETs levels and intestinal flora and its metabolite butyric acid in acute and chronic gallstone cholecystitis.2.Investigate the effect of sodium butyrate on LPS-induced macrophage polarization in mice.3.Explore the effect of sodium butyrate-induced M2-type polarization on neutrophil chemotaxis and NETs formation.4.Investigate the effect of sodium butyrate on LPS-induced macrophage polarization in mice and the effect of sodium butyrate-induced M2-type polarization on neutrophil chemotaxis and NETs formation.Methods:1.Explore the clinical characteristics and correlation analysis of NETs levels and intestinal flora and its metabolite butyric acid in acute and chronic gallstone cholecystitis.Peripheral blood and stool specimens from patients with calculous cholecystitis and healthy volunteers of the same period in the Second Affiliated Hospital of Baotou Medical College from June 2019 to September 2020,as well as gallbladder tissues from gallstone patients after surgery,were collected and divided into:(1)healthy control group;(2)chronic calculous cholecystitis group;(3)acute calculous cholecystitis group;according to clinical manifestations and laboratory indices.The expression levels of neutrophil elastase(NE)were measured by immunohistochemical staining of gallbladder tissues;the expression of ds DNA,MPO-DNA and related inflammatory factors were measured by enzyme-linked immunosorbent assay in peripheral blood,and the concentration of short-chain fatty acids was measured by gas chromatography;the distribution of intestinal microorganisms was detected by 16 S rRNA gene sequencing in fecal specimens,and correlation analysis was performed.The distribution of intestinal microorganisms was determined by 16 S rRNA sequencing and correlation analysis.2.Investigate the effect of sodium butyrate on LPS-induced macrophage polarization in mice.The macrophages were treated with LPS RAW264.7 to obtain M1 phenotype,and then macrophages were incubated with different concentrations of sodium butyrate for 24 h.The expression levels of CD86 and CD206 were detected by cellular immunofluorescence staining,and the expression levels of inflammatory factors and chemokine CXCL16 in the culture medium were detected by ELISA.3.Explore the effect of sodium butyrate-induced M2-type polarization on neutrophil chemotaxis and NETs formation.The experimental study of sodium butyrate-regulated LPS-induced macrophages affecting neutrophil chemotaxis and NETs was performed by first obtaining and culturing neutrophils using a mouse bone marrow neutrophil isolation kit,co-culturing neutrophils with sodium butyrate-induced macrophage culture supernatant in Transwell chambers,and observing the migration of neutrophils;then adding CXCL16-containing antibody CXCL16 antibody was added to the culture medium to observe the migration of neutrophils;the normal culture medium of neutrophils was replaced with sodium butyrate-induced macrophage culture medium,and the expression of MPO was detected by cellular immunofluorescence,and the content of ds DNA and MPO-DNA in the culture medium was detected by ELISA.4.Investigate the effect of sodium butyrate on LPS-induced macrophage polarization in mice and the effect of sodium butyrate-induced M2-type polarization on neutrophil chemotaxis and NETs formation.Exosomes were isolated from peripheral blood of stone gallbladder infection and healthy controls and morphologically identified by transmission electron microscopy and NST;the markers CD63,TSG101 and HSP70,and the expression level of CXCL16 in exosomes were detected by Western blot.The exosomes obtained were co-cultured with normal human peripheral blood neutrophils,and the migration of neutrophils was detected by transwell method;the expression of MPO was detected by cellular immunofluorescence;the content of ds DNA and MPO-DNA in the culture medium was detected by ELISA;the expression level of CXCR6 in neutrophils was detected by Western blot method.The expression level of CXCR6 in neutrophils was detected by Western blot.The cell culture medium of M1-type macrophages treated with different concentrations of sodium butyrate was collected and exosomes were obtained by gradient centrifugation,the obtained exosomes were co-cultured with mouse neutrophils for the detection of NETs formation.Results:1.The clinical characteristics and correlation analysis of NETs levels and intestinal flora and its metabolite butyric acid in acute and chronic gallstone cholecystitis.(1)The levels of ds DNA and MPO-DNA,markers of neutrophil NETs formation in peripheral blood,were significantly increased in patients with cholecystitis compared with healthy controls,with statistically significant differences(P<0.05);the expression of neutrophil elastase in gallbladder tissue was significantly increased in the acute group compared with the chronic group(P<0.05).(2)Peripheral blood short-chain fatty acid content was reduced in patients with cholecystitis compared with healthy controls,with the expression of butyric acid significantly lower than in healthy controls(P<0.05).(3)The results of 16 s RNA gene sequencing of intestinal flora showed that,the ratio of Firmicutes/Bacteroidetes was decreased in the disease group compared with the normal group(P<0.01);Species abundance of Lachnospiracea and Roseburia were also significantly decreased in calculous cholecystitis group.(4)Pearson correlation analysis showed that ds DNA and MPO-DNA were negatively correlated with butyric acid content with correlation coefficients of(r =-0.579,P<0.001)and(r =-0.322,P<0.05),respectively.2.The effect of sodium butyrate on LPS-induced macrophage polarization in mice.(1)Sodium butyrate promoted LPS-induced macrophage M2 type conversion and reduced CXCL16 expression levels.3.The effect of sodium butyrate-induced M2-type polarization on neutrophil chemotaxis and NETs formation.(1)LPS-induced macrophages promoted neutrophil migration and NETs formation via CXCL16,and sodium butyrate reversed the effect of LPS.(2)The expression levels of CXCL16 and its ligands were increased in the gallbladder tissue of patients with acute calculous cholecystitis compared with the chronic group,and the ratio of M1/M2 was increased.4.The effect of sodium butyrate on LPS-induced macrophage polarization in mice and the effect of sodium butyrate-induced M2-type polarization on neutrophil chemotaxis and NETs formation.(1)The concentration of peripheral blood exosomes was significantly increased in patients with calculous cholecystitis compared with normal controls.Co-culture with normal human neutrophils resulted in an increased proportion of NETs formation and increased migration of neutrophils.(2)Decreased expression of sodium butyrate-induced exosomes from inflammatory macrophages and the CXCL16 they carry.(3)The exosomes secreted by M1-type macrophages promoted the migration of neutrophils and the formation of NETs in mice,and the exosomes obtained after sodium butyrate treatment had a reduced effect on the migration of neutrophils and the formation of NETs in mice.Conclusion:1.Butyric acid was negatively correlated with NETs,suggesting that butyric acid may have a regulatory effect on the formation of NETs.2.Sodium butyrate regulates the polarization of situation cells and decreases the expression of chemokine CXCL16.3.Sodium butyrate-induced macrophages reduce the migration of neutrophils and the formation of NETs by inhibiting the release of CXCL16.4.Sodium butyrate can affect the formation and migration of neutrophil NETs by regulating the exosomes secreted by macrophages,and the chemokine CXCL16 is involved in this regulatory process.