Effects Of Ulinastatin On Macrophages Activity And TLR4 Signaling Pathway In Mice With Acute Lung Injury

Macrophages are important antigen presenting cells,which play an important roal in immune defense,immune self-stabilization and immune surveillance.Macrophages show unique phenotypes and functions in different tissues.Currently we think it has two kinds of macrophages,type M1 and type M2.Recently years,the roal of macrophages functional in the immunomodulatory of sepsis has attracted much attention.Sepsis is most caused by Gram-negative bacteria,the high mortality rate remains a challenge for us.Toll like receptor 4 is an important lipopolysaccharide receptor,which can initiate and activate endogenous signaling pathways.It is a bridge that links innate and adaptive immunity.TLR4 is mainly expressed on the surface of macrophages and dendritic cells.Ulinastatin(UTI)is a spectral serine protease inhibitor isolated.It was found that UTI has protective effect on LPS-induced acute lung injury in mice,but the mechanism of action was unclear.Aim:To observe the effect of UTI on the functional of macrophages and the effect of TLR4 signaling pathway on the surface of pulmonary macrophages of mice with acute lung injury induced by LPS.Further research the mechanism of UTI inhibiting inflammatory response,and to understand the pathophysiological mechanism and therapeutic targets of UTI.Provide a new theoretical basis for the treatment of septic acute lung injury.1.Effects of Ulinastatin on Macrophages Differentiation in Mice with Acute Lung Injury Methods:Total of 64 SPF male C57/BL mic were randomly divided into four groups: Sham,Sham+UTI,LPS,LPS+UTI,16 in each group.The mouse model of ALI were established by instilled the lipopolysaccharide(5mg/kg)into the trachea.In Sham+UTI group and LPS+UTI group,mice were treated with UTI(10,000U/kg)hypodermic injected before model establishment 30 minutes.Sham group and LPS group were treated with the equivalent normal saline.The general state,respiratory rate and 72 h survival rate of each group were recorded.Another 64 mice were divided into the above mentioned method.The bronchoalveolar lavage fluid(BALF)and lung tissue were obtained at 24 h after LPS treatment.The total cell count,neutrophil and macrophage count in BALF were determinated;the wet/dry weight ratio of the lung tissue were measured;hispathological changes and pathology scores of the lung tissue were observed;the nitric oxide synthase and the mRNA level of inflammatory cytokines such as TNF-α、IL-1β 、IL-6 and iNOS which related with M1 macrophages in BALF were determined via real-time quantitative PCR;the level of IL-10 、CD206、Arg-1 mRNA which related with M2 macrophages were determined.Results:The survival rate,lung W/D ratio,hispathological changes,pathological scores,mRNA level of TNF-α、IL-1β 、IL-6、iNOS、IL-10 were all lower in LPS+UTI group than those in LPS group,the difference is statistically significant(P<0.05);the level of CD206 and Arg-1 mRNA were no difference among groups.Conclusions:UTI has protective effect on acute lung injury in mice,the alveolar macrophages showed a tendency of M1 to M2 functional activation.UTI may play an immunomodulatory role by regulating the functional state of M1/M2.2.Effects of Ulinastatin on LPS-induced TLR4 Signaling Pathway in Mice with Acute Lung Injury Methods:Group of mice,pre-operative treatment and surgical procedures are the same as before.TLR4 mRNA on macrophages surface of mice in each group were detected via RT-PCR.TLR4/NF-Κb and p65 protein expression on macrophages surface were detected via Western-blot.Results:The level of TLR4 mRNA and TLR4/NF-κB、p65 protein expression in LPS group were significantly increased(P<0.05).UTI+LPS group was significantly lower than that in the LPS group(P<0.05).Conclusions:UTI can inhibits TLR4 signaling pathway induced by LPS in mice with acute lung injury.It can significantly reduced TLR4 expression.It indicate that this protective effects may be work by effecting TLR4/NF-κB signaling pathways.