| Objective:Acute lung injury(ALI)is a common clinical emergency with complex etiology,unknown mechanism without effective treatment.Its high morbidity and mortality seriously affect the life and health of people.It is necessary to explore new protective measures for ALI.In this study,we established a mouse model of ALI by intratracheal instillation of Lipopolysaccharide(LPS)to investigate whether e TAT-protein phosphatase 1B(ppm1b)can alleviate LPS-induced ALI and the mechanism,thereby providing a new direction for the treatment of clinical ALI.Methods:In vivo studies:1.SPF grade 8-week-old C57B/L6 mice were randomly divided into2 groups:Normal saline(NS)group,model group(LPS group).NS or LPS(2mg/kg)were used to construct the ALI model by intratracheal instillation.2.SPF grade 8-week-old C57B/L6 mice were randomly divided into 4 groups:placebo group(e TAT group),ppm1b protein group(e TAT-ppm1b group),solvent control group(e TAT+LPS group)and treatment group(e TAT-ppm1b+LPS group).Protein containing e TAT or e TAT-ppm1b(10mg/kg)was injected into mice tail vein in e TAT group or e TAT-ppm1b group.e TAT+LPS group and e TAT-ppm1b+LPS group were administrated with e TAT or e TAT-ppm1b protein(10mg/kg)by tail intravenous.Three hours later,the two groups received LPS(2mg/kg)by intratracheal instillation.Modeling after 24 hours,the mice were sacrificed and the whole lung was extracted for general observation,lung index and lung wet/dry ratio(W/D)detection.HE staining was applied to observe the pathological damage of lung tissues.The lung infiltration of macrophages with F4/80~+,neutrophils with MPO~+and myeloid cells with CD11b~+was detected and measured by immunohistochemistry.The expression of p-S6 and p-P44/42 protein was analyzed by immunohistochemistry.Real-time fluorescent quantitative PCR(q RT-PCR)was used to identify the expression of inflammatory cytokines(TNF-α,IL-1β,IL-6,CCL2,IL-8,IL-18,IL-10)and apoptosis-related genes(Bcl2,Bcl-xl,Puma,Bak and Bax)in m RNA levels.Western Blot was used to examine the expression changes of ppm1b,receptor-interacting protein 3(RIP3),phosphorylated RIP3,P38MAPK,phosphorylated P38MAPK.3.The LPS control group(e TAT+LPS group,n=23;)and the treatment gro up(e TAT-ppm1b+LPS group,n=19;)were modeled at the same time and then allowed to drink and eat freely without any treatment,and the number of dead individuals in each group was recorded every 24 h for 5 consecutive days.In vitro studies:human bronchial epithelial(HBE)cells were divided into 2 groups:PBS group and LPS group.The cells were given LPS(10ng/ml)or PBS and collected at 2 hours,4 hours and 6 hours after treatment.Expression of inflammatory cytokines was measured by q RT-PCR.The HBE cells were divided into 4 groups:e TAT group,e TAT-ppm1b group,e TAT+LPS group,e TAT-ppm1b+LPS group.The cells were preprocessed with e TAT-ppm1b(250n Mol)in three hours before LPS(10ng/ml)administration.Six hours after LPS treatment,expression of inflammation factors was examined by q RT-PCR.Results:1.At the end of day 5,13 mice in the LPS control group died(68.4%);7 mice in the e TAT-ppm1b treatment group died(30.4%).2.Gross lung view:Compared with the NS group,mice in the LPS group had enlarger lung tissue and significant bleeding;compared with the e TAT+LPS group,mice in the e TAT-ppm1b+LPS group had less bleeding in the lung tissues.3.Lung index and wet/dry weight(W/D)ratio:Compared with the NS group,lung index and W/D ratio were significantly increased in the LPS group;e TAT-ppm1b treatment significantly decreased lung index and W/D ratio after LPS stimulation when compared to the e TAT+LPS group(P<0.01).4.HE staining analysis:Compared with the NS group,lung in the LPS group had disorganized alveolar structure,dramatically hemorrhage,thickened alveolar septum and decreased mean alveolar area.Compared with the e TAT+LPS group,lungs in the e TAT-ppm1b+LPS group had clear alveolar structure with mitigating hemorrhage and notably increased mean alveolar area(P<0.01).5.Immunohistochemical analysis:Compared with the NS group,distribution of F4/80(P<0.05),MPO(P<0.01)and CD11b(P<0.05)positive cells were significantly increased in lung tissues in the LPS group.Compared with the e TAT+LPS group,the mean fluorescence intensity of F4/80(P<0.01),MPO(P<0.0001)and CD11b(P<0.05)positive cells were significantly reduced in e TAT-ppm1b+LPS group.6.q RT-PCR analysis:Compared with the NS group,the expression of inflammatory factors,such as TNF-a and IL-6,was elevated in the LPS group while the changes of apoptosis-related gene expression were not significant.The expression of inflammatory factors was blunted in the e TAT-ppm1b+LPS group when compared to the e TAT+LPS group.The expression of TNF-αand IL-6 was significant reduced in e TAT-ppm1b+LPS group when compared to those in the e TAT+LPS group(P<0.01).In in vitro experiments,the expression of inflammatory factors(TNF-α,IL-6,IL-1β)of HBE cells in the e TAT-ppm1b+LPS group was statistically significantly decreased when compared to those in the e TAT+LPS group(P<0.01).7.Western blotting:The RIP3,p-RIP3 protein expression was increased in the LPS group compared with the NS group.ppm1b protein was highly expressed in lung tissues in the e TAT-ppm1b group than that in the e TAT group at 3 hours and 24 hours after administration.In in vitro experiments,HBE cells treated with e TAT-ppm1b had higher levels of ppm1b protein than that in the e TAT group.8.The changes of signaling pathways in the ALI mouse model induced by LPS:There were significant positive staining for p-S6 and p-P44/42 in the lung after LPS treatment.e TAT-ppm1b have not affected changes of p-S6 and p-P44/42 caused by LPS treatment.LPS could remarkably elevate the expression of p-P38MAPK in the lung after LPS administration.e TAT-ppm1b administration effectively inhibited P38MAPK activation caused by LPS with increased expression of CDK4 and CDK6 genes.Conclusion:1.Tail vein injection of e TAT-ppm1b resulted in the stable presence of pp m1b protein in lung tissues,which prolonged the survival time of mice,reduc ed mortality,attenuated acute lung injury in model mice,reduced inflammatory response of lung and improved pulmonary edema;in vitro,it reduced inflam matory factor expression.2.The protective effect of e TAT-ppm1b on acute lung injury may occur b y inhibiting the P38MAPK pathway while promoting cell proliferation. |
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