The Role And Mechanism Of Activating Transcription Factor 3(ATF3)on The LPS Induced Acute Lung Injury In Mice

1.Background and objectiveThe acute lung injury(ALI)and the severe form of it called acute respira ory distress syndrome(ARDS)is caused by a variety of factors including inside and outside the lung caused by diffuse alveolar capillary membrane injury and severe hypoxemia of critical respiratory disease,because its pathogenesis is not clear,so there is still a high mortality rate.Among them,the imbalance of inflammatory response may plays an important role in the occurrence and development of ALI.Because its pathogenesis is not clear,so there is still a high mortality ratr.The sepsis caused by gram negative bacteria infection is an important cause of ALI/ARDS.The LPS which is one of the components of the cell wall of gram negative bacilli is the promoter of the innate immune response and inflammatory reaction.Therefore it is important to clarify the pathogenesis of LPS induced acute lung injury for the treatment.It has been proved that activating transcription factor 3(ATF3)is a fast response gene,which is closely related to the inflammatory stress,the maintenance of the body homeostasis,and the occurrence of tumor.In a variety of inflammatory models,ATF3 has a braking effect on the inflammatory response.However,the role of ATF3 in the acute lung injury induced by LPS is not completely understood.In view of this,this study will focus on the role of ATF3 on the LPS-induced ALI.The model of ALI was made through intraperitoneal injection of LPS in wild-type mice and ATF3 gene knockout mice,and screened the differential genes in the lungs of two groups mice,finally we construction the plasmid to down-regulation the expression of ATF3 in RAW264.7 cells,to explore the possible mechanism of LPS regulation of ATF3 leading to the inflammatory response,so as to provide a potential new goal of providing potential points for the Prevention of ALI.(1)Construction animal model of acute lung injury,defined the expre ssion of ATF3 in lung tissue.Intraperitoneal injection of LPS to mice,injection volume of 15mg/kg,so as to establish the animal model of acute lung injury of mice,and then detect the expression of ATF3 in lung tissue after stimulate of LPS by Western and RT-PCR,and identify its expression pattern.(2)ATF3 alleviates LPS-induced ALI in miceConstruction the ATF3 knockout mice,the expression of ATF3 in knockout mice was identified by Western blot.Subsequently,we compared with wild type mice and ATF3 gene knockout mice lung tissue biopsy,lung wet dry ratio,expression of inflammatory cytokines in lung tissues,protein concentration in bronchoalveolar lavage fluid and 7 day survival rate to evaluate the protective effect of ATF3 in LPS induced acute lung injury in mice.(3)Screening of ATF3 related differentially expressed genes in lung tissues of mice with acute lung injury by gene chipThe C57 mice and ATF3 knockout mice were injected intraperitoneally with LPS for 2 hours,then the total RNA of the lung tissues of the two groups were extracted,and the differentially expressed genes were screened by gene chip technology.Combined with literature reading,the final focus on TL1 A molecules,and then suggested that ATF3 may be protective the inflammation injury of ALI through down regulating the expression of TL1 A.(4)The underlying mechanisms for ATF3 regulation of LPS-induced ALI were investigated with the model of RAW264.7 cells in vitroThe expression pattern of ATF3 in RAW264.7 cells after LPS challenged was investigated.Subsequently,the effects of ATF3 on LPS-induced RAW264.7 cell inflammatory injury and TL1 A expression were determined after ATF3 expression was down-regulated by ATF3 si RNA plasmid.Furthermore,to determine the role of TL1 A in ATF3 regulation of LPS-induced ALI,we decreased both ATF3 and TL1 A expression in RAW264.7 cells by plasmid to observe the inflammatory cytokine IL-6 expression.3.Results(1)ATF3 expressed in normal lung tissues of mice,after intraperitoneal injection of LPS,the expression level of ATF3 in the lung tissues were significantly increased(P < 0.05),the results of RT-PCR and blot Western are consistent.(2)Wild type mice and ATF3 knock-out mice as compared to LPS after intraperitoneal injection,aggravate lung pathological injury of ATF3 gene knockout mice,lung wet / dry ratio 2.Methods increased,inflammatory cytokines IL-6,IL-1?,TNF-? expression increased,BALF protein concentration increased,the 7 day survival rate of mice decreased significantly(P < 0.05).(3)Screening of ATF3 related differentially expressed genes in lung tissues of mice with acute lung injury by gene chip.Combined reading literature,put forward that ATF3 might down regulate TL1 A expression and regulation of the scientific hypothesis of inflammation of LPS induced acute lung injury.(4)LPS significantly increased ATF3 expression in RAW264.7 cells,knockdown of ATF3 can significantly increased LPS-induced inflammatory cytokine TNF-? and IL-6 secretion.Compared with LPS and NCsi RNA+LPS group,the TL1 A expression was significantly increased in the ATF3 si RNA and ATF3 si RNA+LPS groups in RAW264.7 cells(P < 0.05).To further demonstrate the role of TL1 A signaling pathway in ATF3's modulation of LPS-induced ALI,we knockdown both ATF3 and TL1 A expression at the same time,the IL-6 expression in ATF3 si RNA+ TL1 Asi RNA group was significantly increased compared to ATF3 si RNA+LPS group(P < 0.05).4.Conclusion(1)ATF3 has a protective effect on LPS induced acute lung injury.(2)TL1A signaling pathway may be involved in the regulation of ATF3 on LPS induced acute lung injury.