Effects Of HMGB1-TLR4/RAGE Signaling Pathway On The Inflammatory Process Of LPS-induced ALI In Mice

Objective: By observing the changes of inflammatory factors in lung tissues and serum at different time points,the influence of HMGB1-TLR4/RAGE signaling pathway on the inflammatory process of LPS-induced ALI in mice was explored.Methods:Seventy-two adult male mice with SPF grade C57BL/6 were divided into three groups.Control group(N group)was intranasally injected with 50 ul PBS buffer+30min,and then injected with 2ml PBS buffer intraperitoneally.The experimental group(LPS group)was intranasally injected with LPS solution(40mg/ml)for 50 ul+30min,and then injected intraperitoneally with 2ml PBS buffer solution;The intervention group(LPS+ anti-HMGB1 antibody group)was intranasally injected with LPS(40mg/ml)for 50 ul +30min,and then injected intraperitoneally with2.5mg/kg anti-HMGB1 antibody.Mice in each group were sacrificed and collected at8 h,16h,24 h and 48 h after administration.The general condition of mice and the morphology of lung tissue specimens were observed at each time point.The pathological injury of lung tissue was observed by HE staining.W/D value of lung tissue was calculated.The m RNA expression levels of NF-κBp65 and STAT3 in lung tissues were detected by q PCR.TLR4,RAGE and HMGB1 protein expression levels were determined by Western Blot.The expression levels of IL-1β,MIP-1 and HMGB1 in serum were detected by ELISA.Results:1.General situation of mice: at each time point in group N,mice were in good spirits,with smooth hair,smooth breathing,red lips and normal diet.When grasping mice,they were agile in evading.Mice in the LPS group showed listlessness,hair standing up,shortness of breath,cyanosis of the mouth,poor diet,slow escape action when grasping mice,and symptoms gradually worsened with time.Compared with the LPS group,the above conditions were improved in the LPS+ anti-HMGB1 antibody group.But it’s not back to normal.2.Morphology of gross lung tissue: The surface of lung tissue of N group was light pink,without obvious edema and hemorrhage.The lung tissue of mice in the LPS group was obviously red and swollen,with scattered ecchymosis and bleeding on the surface.The lung tissue of mice in the LPS+ anti-HMGB1 antibody group was more red and swollen,with scattered ecchymosis and hemorrhage on the surface,but it was less than that in the LPS group.3.Pathological injury of lung tissue: under the condition of HE staining,the lung tissue structure of mice in the N group was basically complete at each time point,with almost no red blood cells,inflammatory cells and fluid exudation.At each time point in the LPS group,the lung tissues of mice had different degrees of vascular congestion and hemorrhage,and a large number of red blood cells and inflammatory cells exudes into the lung interstitium and alveolar cavity,and the alveolar wall thickens,some alveolar walls break,and the alveolar cavity fusion expands.With the time prolongation,the lung tissue damage gradually aggravates.The degree of lung tissue injury in the LPS+ anti-HMGB1 antibody group was less than that in the LPS group at each time point.4.W/D value of lung tissue: W/D value of lung tissue in LPS group and LPS+anti-HMGB1 antibody group was significantly higher than that in N group at each time point(P<0.05),and this ratio had a tendency to increase with the prolonging of modeling time.The W/D value of lung tissue in LPS+anti-HMGB1 antibody group was lower than that in LPS group at all time points(P<0.05).5.The m RNA expression levels of NF-κBp65 and STAT3 in lung tissue were detected by q PCR.The m RNA expression levels of NF-κBp65 and STAT3 in lung tissue of LPS group and LPS+ anti-HMGB1 antibody group were higher than those of N group at 8h,16 h,24h and 48h(P<0.05).After anti-HMGB1 antibody intervention,the m RNA expression levels of NF-κBp65 and STAT3 in lung tissue were lower than those in LPS group at 8h,16 h,24h and 48h(P<0.05),but still higher than those in N group(P<0.05).6.Western Blot analysis of TLR4,RAGE and HMGB1 protein expression levels:compared with the N group,the expression of TLR4,RAGE and HMGB1 protein in the LPS group and the LPS+ anti-HMGB1 antibody group at 8h,16 h,24h and 48 h were increased to varying degrees(P<0.05).After anti-HMGB1 antibody intervention,the protein expression levels of TLR4,RAGE and HMGB1 in lung tissues of mice at8 h,16h,24 h and 48 h were lower than those in LPS group(P<0.05),but still higher than those in N group(P<0.05).7.Serum IL-1β,MIP-1 and HMGB1 expression levels: Serum IL-1β,MIP-1 and HMGB1 concentrations in LPS group and LPS+ anti-HMGB1 antibody group were higher than those in N group at each time point(P<0.05);After anti-HMGB1 antibody intervention,serum IL-1β,MIP-1 and HMGB1 concentrations at all time points were lower than those in LPS group(P<0.05),but still higher than those in N group(P<0.05).Conclusion:1.In the process of LPS-induced ALI inflammation in mice,the HMGB1-TLR4/RAGE signaling pathway is activated,and the expressions of NF-κB-p65 and STAT3 are up-regulated,promoting the release of IL-1β and MIP-1early inflammatory cytokines.2.Anti-HMGB1 antibody alleviates the severity of lung injury to a certain extent and controls the inflammatory response of ALI by affecting the expression of the above related proteins at different times and in different degrees.