Fucntional And Mechanistic Investigation Of Tumor Metastasis Associated Protein1 (MTA1) During The Pathogenesis Of Acute Lung Injury

Acute lung injury(ALI)is a severe disease of the respiratory system,its prevention and therapies remains a major problem in the medical field.The innate immune response plays a profound role in the pathophysiology of ARDS.Histologically,ALI/ARDS is characterized by a massive apoptosis in alveolar epithelial cells(AECs),especially type II alveolar epithelial cells(AECs II),and impairment of physiologic barrier result in pulmonary edema and abnormal activations of intravascular coagulation.However,the pathogenesis of ALI/ARDS remains controversial,the molecular mechanisms through which these responses are directed remain largely unknown.From a therapeutic standpoint,it is required for emerging aspects of ARDS pathophysiology that encompass modulators of the innate immune response,damage signal pathways that may serve as a foundation of future potential therapeutic targets.Metastasis-associated protein 1(MTA1),modifies the acetylation status of the target gene chromatin,functions in mediate transcriptional regulation.In order to analysize the molecular function of MTA1,we observed that it was upregulated in lung tissue of ALI mice induced by LPS,combined with references,we speculated that MTA1 may play an important role in ALI/ARDS induced by LPS.In this study,we have established a stable murine model of ALI/ARDS,and investited the relationships between the expression of MTA1 and pathological changes,inflammatory factors and apoptosis of alveolar epithelial cells.We further explored the regulatory mechanism(s)of MTA1,to further clarify the significance of its expression in ALI/ARDS.This experiment is divided into two parts.Part?: Variation of MTA1 levels in ALI mice's model induced by intratracheal instillation of LPSObjective To build the ARDS model in mices,analyze the expression of MTA1 in lungs of ALI mices 18 h after intratracheal instillation of LPS,and observed the expression of IL-1b,IL-6,IL-10 and TNF-?.Methods Twenty healthy C57BL/6 mices,weighed between 20 g and 30 g,were divided randomly into two groups according to the disposal of LPS.After 7 days of acclimatization,mice in experimental group were challenged with intratracheal instillation of 800 mg of LPS,Na?ve mice(without LPS challenge)were injected with saline only.Then,Mice were sacrificed 18 h following LPS instillation.Sections were then stained with hematoxylin and eosin,then estimated the lung injury and scored.The expression and location of MTA1 in the experimental group and control group were detected by Methods ABC immunostaining and quantitative determination of MTA1 by Western-Blot.The expression of IL-1b,IL-6,IL-10 and TNF-? in lung tissue were detected by RT-PCR..Results(1)HE staining showed that the pathological feature of mice,18 h following LPS in-stillation,up to diagnosis standard of ALI.(2)The expression of MTA1 was location in nuclear and significantly upregulated in the ALI group.The upregulation of MTA1 expression in lung mainly occurred in the AECs,as revealed by immunohistochemistry and Western blot.(3)Compared with na?ve mice,IL-1b,IL-6,IL-10 and TNF-? expression level obviously increased in the ALI group.Conclusion MTA1 is widely expressed in mice alveolar epithelial cells.The upregulation of MTA1 expression in lung occurred in ALI group induced by LPS.Part?: Affects of Mta1/MTA1 depletion on ALI Pathology and its relative molecular mechanism.Objective To observe Affects of Mta1/MTA1 depletion on Pathology,the expression of inflammatory cytokines,activity of NF-kB and apoptosis of alveolar cells in ALI model of mices and explore the mechanism of MTA1 on ALI.Methods Fifty healthy C57BL/6 mices were divided randomly into five groups according to their treatment.“Naive group”:10 mices;“Ctrl Si RNA Group”:10 mices were injected through the tail vein with Ctrl Si RNA;” Mta1 Si RNA Group”: 10 mices were injected through the tail vein with Mta1 Si RNA;” Mta1 Si RNA+LPS Group” 10 mices were injected through the tail vein with Mta1 Si RNA one day before LPS challenge.” Ctrl Si RNA+LPS Group” 10 mices were injected through the tail vein with Ctrl Si RNA one day before LPS challenge.Control group was injected Ctrl-Si RNA instead of Mta1 Si RNA.All mices were sacrificed 18 h after the disposal of LPS.HE staining showed that the pathological feature of mice.NF-k B filter assay was carried out using a commercial kit to evaluate the activity of NF-k B pathway as instructed by the manufacturer.Apoptosis of alveolar epithelial cell in each group was assay by TUNEL using a Situ Cell Death Detection Kit.The expression of inflammatory cytokines(IL-1??TNF-??IL-6?IL-10)and biomarkers of AECs were analyzed by RT-PCR.Results(1)Mta1-Si RNA designed in our experiment can effectively inhibit the expression level of MTA1 in mouse lung tissue.(2)Compared with LPS treatment only,injury of mices,injected Mta1 Si RNA through the tail vein before LPS treatment,was severe aggravated,showed that alveolar collapse,a large number of inflammatory cell infiltration,outline of alveolar disappeared,the significant increase in the NF-k B activity was substantially abolished by MTA1 ablation,apoptosis of alveolar epithelial cells was obviously increased in the group,injected Mta1-Si RNA through the tail vein before LPS challenge.(3)To compared with LPS treatment only,the expression of IL-1?,TNF-?,and IL-6 were upregulated in ” Mta1 Si RNA+LPS” Group(P?0.05),but there is not significant difference in the expression of IL-10(P?0.05).(4)The expression of Ttf-1 and ICAM-1 were significantly inhibited in ALI mice,and expression level of Ttf-1 was further downregulated once Ablation of MTA1,but this situation had not occurred in expression level of ICAM-1(P?0.05).(5)After inhibition of MTA1 gene expression,The number of alveolar epithelial cells apoptosis increased significantly.Conclusion(1)Injury was aggravated,NF-kB signaling pathway induced by LPS activation was inhibited and inflammatory mediators,IL-1b,TNF-?,IL-6 increased when expression level of MTA1 was reduced,but there was not influence in the expression of IL-10.(2)Apoptosis of alveolar epithelial cells was obviously increased.it infer ed that MTA1 played crucial role in apoptosis of AECs II,as expression level of Ttf-1(biomarker of AECs II)was further downregulated once ablation of MTA1.