The Reserch Of HMGB1-RAGE Signal Pathway On AMs Polarizations In The Process Of ALI Induced By LPS In Mice

Objective: to explore the effect of high-mobility group box 1(HMGB1)-advanced glycation end product receptor(RAGE)signal pathway on lipopolysaccharide(LPS)-in duced acute lung injury(ALI)in mice and its influence on the polarization of AMs to M1 type in the process of ALI in mice.Methods: 80 C57BL/6 mice(18-22g)were randomly divided into 4 groups: 20 mice were in the control group(Control group): PBS was nasally dripped into the mouse trachea,30 minutes later PBS was injected into the abdominal cavity;20 mice were in the model group(LPS group): LPS was instilled into the mouse trachea via the nose,and PBS was injected into the abdominal cavity 30 minutes later;20 mice were in the anti-HMGB1 antibody intervention group(LPS+anti-HMGB1 group): LPS was injected Nasal instillation into mouse trachea,30 minutes later anti-HMGB1 antibody was injected into the abdominal cavity;20 mice were in the RAGE inhibitor FPS-ZM1 intervention group(LPS+FPS-ZM1 group): LPS was nasally instilled into mouse trachea,After 30 minutes,the RAGE inhibitor FPS-ZM1 was injected into the abdominal cavity.After intraperitoneal injection of PBS,FPS-ZM1 or anti-HMGB1 antibody in each group,5 mice were sacrificed at each time point at 8h,16 h,24h,48 h.The Morphology of lung tissue specimen was observed by naked eyes,the wet/dry weight ratio(W/D)of lung tissue was calculated for statistical analysis,the pathological damage of lung tissue was observed by hematoxylin-eosin(HE)staining,the expression of HMGB1 and RAGE in lung tissue was detected by Western blot(WB),the expression of serum HMGB1 and tumor necrosis factor-α(TNF-α)was detected by enzyme-linked immunosorbent assay(ELISA),and the expression of M1 type alveolar macrophages(AMs)marker CD38 was detected by flow cytometry.Results:1.Morphology of lung tissue specimen: The Morphology of lung tissue specimen in the Control group were tender,without edema,petechiae,congestion,etc.at each time point;the lung tissue in the LPS group was swollen to varying degrees,and some showed local congestion,congestion or even hemorrhage,It was obvious at 24 and 48 hours,and the heaviest at 48 hours;the lung tissue swelling,congestion and ecchymosis in the LPS+anti-HMGB1 group and LPS+FPS-ZM1 group were slightly reduced compared with the LPS group.2.Lung tissue W/D ratio: Calculating mouse lung tissue W/D ratio for statistical analysis.W/D ratio increased at each time point in LPS group,LPS+anti-HMGB1 group and LPS+FPS-ZM1 group Compared with Control group,mostly significant in LPS group(P<0.05);while the ratio in LPS+anti-HMGB1 group and LPS+FPS-ZM1 group was less than LPS group,The difference was statistically significant(P<0.05).3.HE staining of lung tissue: the alveolar structure of the Control group is complete,there is no obvious inflammatory cells and fluid exudation in the alveolar cavity and interstitium;the alveolar septum in the LPS group is thickened and broken in different degrees,and the alveolar cavity and interstitial fluid in different degrees are exuded and inflammation Cells infiltrate,the above-mentioned changes aggravate with time,the mostly obvious at 48hours;the pathological changes in LPS+anti-HMGB1 group and LPS+FPS-ZM1 group at each time point were improved compared with LPS group at the corresponding time point,but were serious compared with the Control group.4.The Serum HMGB1 and TNF-α detection by ELISA method: The expression of HMGB1 protein in LPS group was the lowest at 8h,the highest at 16 h,and maintained at a high level at 24 h and 48 h,and the expression at each time point was higher than that at the corresponding time point in Control group,the difference was statistically significant(P<0.05);while the expression in LPS+anti-HMGB1 group and LPS+FPS-ZM1 group was lower than that in LPS group at each time point,but higher than that in Control group,the difference was statistically significant(P<0.05).The expression of TNF-α protein in the LPS group was highest at 8h,decreased slightly at 16 h,increased at 24 h compared with16 h,and decreased at 48 h,and at each time point the expression was higher than that at the corresponding time point in Control group,the difference was statistically significant(P<0.05);while the expression in LPS+anti-HMGB1 group and LPS+FPS-ZM1 group was lower than that in LPS group at each time point,but higher than that in Control group,the difference was statistically significant(P<0.05).5.The detection of HMGB1 and RAGE in lung tissue expression by WB: the expression of HMGB1 and RAGE in LPS group at each time point increased to different degrees than that in Control group,the difference was statistically significant(P<0.05).After the intervention of anti-HMGB1 antibody and FPS-ZM1,the expression intensity of HMGB1 and RAGE in the lung tissue of mice was lower than that in LPS group at each time point,the difference was statistically significant(P<0.05).6.The detection of AMs surface marker CD38 by Flow cytometry: The proportion of AMs expressing CD38 at each time point in LPS group,LPS+anti-HMGB1 group and LPS+FPS-ZM1 group was higher than that in Control group to varying degrees,the difference was statistically significant(P<0.05),after intervention of anti-HMGB1 antibody and RAGE inhibitor FPS-ZM1,The proportion of AMs expressing CD38 was lower than that in LPS group,the difference was statistically significant(P<0.05).Conclusion:1.HMGB1-RAGE signal pathway is involved in the systemic inflammatory response process of ALI and LPS-induced lung injury2.Blocking the HMGB1-RAGE signal pathway may partially inhibit the polarization of AMs to M1 macrophages,thereby reducing the LPS-induced ALI in mice.The polarization of AMs may be one of the important mechanisms of the HMGB1-RAGE signal pathway involved in the process of ALI.