Studies On The Expression Of Tribbles Homolog 3 On Lipopolysaccharide Induced Acute Lung Injury

Objective To examine the expression of tribbles homologous 3(TRB3)on lipopolysaccharide(LPS)induced acute lung injury(ALI)and its association with p38-mitogen-activated protein kinase(MAPK)pathway.Methods In vivo,rats received a intravenous injection by LPS(5 mg/kg)as models of ALI and a intravenous injection by NS(5ml/kg)as the control.In rat lung tissue the expression of TRB3 protein was examined using immunohistochemical staining,the expression of TRB3 m RNA was determined by reverse transcript polymerase chain reaction(RT-PCR).In vitro,cultured rat pulmonary microvascular cells(PMVEC)were randomly divided into dose-dependent,time-dependent and intervention groups.In dose-dependent group,PMVEC were stimulated by various concentrations of LPS(0,2,4,10 ?g/ml)for 4 h,and in time-dependent group PMVEC were challenged by 10?g/ml LPS for different time(0,4,8,12 h).In intervention group,PMVEC grown in normal medium or medium with 10?g/ml LPS for 4 h were pretreated using p38-MAPK inhibitor(10 ?mol/L SB203580)for 2 h.Protein expression of TRB3,p-p38 and p38-MAPK were examined using Western blot analysis.Results Immunohistochemical staining determined that TRB3 protein distributed in rat alveolar walls and glandular epithelium.Increased TRB3 m RNA expression using RT-PCR were found in lung tissue of rats injected by LPS when compared to those in NS group(3.675 ± 0.423 vs 1.056 ± 0.209,t =15.524,P < 0.01).Increased TRB3m RNA expression using RT-PCR had also been found in PMVEC stimulated by LPS when compared to those in the control group(2.098 ± 0.317 vs 0.612 ± 0.314,t =7.549,P < 0.01).In PMVEC,LPS significantly increased the expression of TRB3 protein in a dose-dependent manner(0,2,4,10 ?g/ml)after stimulation for 4 h(0.169 ± 0.089,0.198 ± 0.071,0.338 ± 0.089 and 0.494 ± 0.118,respectively,F = 12.619,P < 0.001).At indicated time-points after PMVEC were challenged by 10 ?g/ml LPS,the expression of TRB3 protein raised at 4 h(0.443 ± 0.087),then decreased gradually at 8 h(0.303 ±0.107),but still was higher than the control group(0.159 ± 0.073),there were significant difference(F = 11.273,P < 0.001).LPS significantly increased the expression of p-p38 protein after stimulation for 4 h when compared to the control group(0.660 ± 0.100 vs 0.227 ± 0.085,t = 49.121,P < 0.001).LPS also increased the expression of TRB3 protein after stimulation for 4 h when compared to the control group(0.461± 0.097 vs 0.178 ± 0.084,t = 15.113,P < 0.001).SB203580 decreased the protein levels of p-p38 in response to LPS(0.557 ± 0.125 vs 0.660 ± 0.100,t = 7.040,P< 0.05).SB203580 also decreased the protein levels of TRB3 in response to LPS(0.306± 0.077 vs 0.461 ± 0.097,t =11.900,P < 0.001).SB203580 alone has no effect on the expression of p-p38 and TRB3.Conclusion Levels of TRB3 protein increase in LPS-induced acute lung injury,and is regulated by p38-MAPK pathway.