| BackgroundAcute lung injury(ALI/ARDS)and its severe form acute respiratory distress syndrome(ARDS)is a fatal respiratory disease,which can be induced by a variety of injury factors.ALI/ARDS has the high level of morbidity and mort ALI/ARDSty,however,the molecular mechanism of ALI/ARDS is still unclear,which leads to the the limitation of diagnosis,prevention and treatment.Innate immune-mediated inflammatory response is the key link of the pathological process of ALI/ARDS.Inflammatory cascade amplification effect and anti-inflammatory imbalance can cause excessive inflammatory activation.Now,lots of studies focus on the inflammatory regulatory mechanisms of ALI/ARDS.Zinc finger CCCH domain-containing protein 12d(ZC3H12D)is a newly discovered zinc finger protein.It is high expression in human lung tissue.ZC3H12 D has a unique zinc finger domain and a Proline-rich region(PRR).CCCH-zinc finger may be a novel RNA or proteins binding motif.Studies found ZC3H12 D is mainly transcribed by NF-?B,and it has anti-tumor effect and anti-inflammatory effect,but the underlying mechanism is still unknown.We have found that in intestine ischemia reperfusion induced ALI,the expression level of ZC3H12 D is changed.The evidences portends ZC3H12 D may be involved in ALI.But Whether ZC3H12 D play a protective role in ALI/ARDS is still unclear.Thus,in this study,we explored the protective effect of ZC3H12 D on ALI and investigated the underlying molecule mechanism.Significance:Through in vitro and in vivo experiments to explore the protective effect of ZC3H12 D on LPS-induced ALI and the molecule mechanism of ZC3H12 D.Objective: 1.To Clarify whether ZC3H12 D is involved in LPS induced ALI;2.To observe the effects ZC3H12 D in LPS induced ALI and to explore the underlying mechanisms;3.To clarify whether ZC3H12 D protects alveolar epithelial cells through inhibiting inflammatory response,and to further investigate the molecular mechanism.Methods: Part I: Investigating the expression of ZC3H12 D in ALI induced by LPS 1.Establishing animal model.Male C57 BL / 6 mice(20 to 25 g)were given LPS(10 mg/kg)to induce ALI by intratracheal instillation.Mice were randomly divided into 5 groups: normal group(control group),2 hours group(2h group),4 hours group(4h group),8 hours group(8h group)and 12 hours group(12h group).At the indicated time points,the mice were sacrificed and the alveolar lavage fluid,plasma and lung tissue were collected to investigate alveolar lavage fluid content,lung wet dry weight ratio(W/D),pro-inflammatory factor TNF ??IL-1??IL-6,the pathological change(H&E staining)and the activation of NF-?B(immunohistochemical staining).2.Investigating ZC3H12 D expression in different time points.ZC3H12 D mRNA and protein expression level were measured respectively by qRT-PCR and Western blot.The tissue positioning of ZC3H12 D were investigated by immunehistochemical staining.Part II: Observing the protective effect of overexpression of ZC3H12 D on ALI mice 1.The adeno-associated virus(AAV)infect mice.C57 BL / 6 mice(15 to 18 g)were randomly divided into normal group,ZC3H12 D group and GFP group,each group of 20.ZC3H12 D group mice and GFP group mice respectively received ZC3H12 D adeno-associated virus or GFP adeno-associated virus 50 ul by intratracheal instillation.After 3 weeks,mice were sacrificed and collect lung tissue to make frozen section and analysis the expression of ZC3H12 D by Western Blot.2.To observe the effect of ZC3H12 D on ALI/ARDS.The 60 mice were randomly divided into 6 groups: LPS group,LPS + GFP group,and LPS + ZC3H12 D group,control group,GFP group and ZC3H12 D group.mice from LPS group,LPS + GFP group,and LPS + ZC3H12 D group were received LPS by intratracheal instillation to induce ALI/ARDS.After 12 hours,the mice were sacrificed and the alveolar lavage fluid,plasma and lung tissue were collected to investigate alveolar lavage fluid content and LDH activity,lung wet dry weight ratio(W/D),lung tissue MPO activity,lung tissue iNOS,Cox2 and ZC3H12 D expression(Western Blot),pro-inflammatory factor level(TNF ??IL-1??IL-6),the pathological change(H&E staining)and the activation of NF-?B and AP-1(Western Blot).Part III : exploring the protective effect of ZC3H12 D on LPS-insulted alveolar epithelial cell(A549 cell)and the underlying mechanism 1.To observe the expression of ZC3H12 D in A549 cells.1)The expression of ZC3H12 D in different time points after LPS stimulation: A549 cells were seeded in 6 well plates and were divided into six groups: control group(normal group),2h,4h,8h,16 h and 24 h group and the cells were stimulated with LPS(25 mu g/ml)for 2h,4h,8h,16 h and 24 h respectively.Then the cells were collected to measure the expression of ZC3H12 D.2)The expression of ZC3H12 D with various concentrations of LPS(25 ?g/ml,50 ?g/ml and 75 ?g/ml)stimulation: A549 cells were seeded in 6 well plates and were divided into control group(normal group),25ug/ml group,50ug/ml group and 50 ug/ml group.Cells were stimulated with 25ug/ml,50ug/ml and 50 ug/ml respectively for 2h or 16 h and collected to measure the expression of ZC3H12 D.2.To observe the protective effect of ZC3H12 D on LPS-insulted alveolar epithelial cell(A549 cell).A549 cells were seeded in 6 well plates and were divided into six groups: control group,ZC3H12 D group,LPS group and LPS + ZC3H12 D group.Cells were transfected with ZC3H12 D expression plasmid,then were stimulated with 50?g/ml LPS for 6h.Culture medium and cells were collected to measure the level of TNF??IL-1? and IL-6 and the activation of NF-?B and AP-1.A549 cells were transfected with GFP plasmid and ZC3H12 D expression plasmid respectively and were seeded in 96 well plates.The cells were divided into four groups: control group,ZC3H12 D group,LPS group and LPS + ZC3H12 D group,and were stimulated with LPS(100 ug/ml)for 24 h.After stimulation,cell survival rate were measured by CCK 8 method.3.exploring the mechanism of ZC3H12 D suppressing inflammation.THP 1 cells were seeded in 24 well plates and were divided into three groups: control group,sh ZC3H12 D group and ZC3H12 D group.The cells were transfected or infected with GFP plasmid,ZC3H12 D expression plasmid or shZC3H12 D lentivirus respectively and were treated with actinomycin D for 1 h,3 h and 6 h.Cells were harvested to measure c-fos,c-jun,TNF??IL-1??IL-6 and NF-?B p65 mRNA expression by qRT-PCR.Results: Part I: Investigating the expression of ZC3H12 D in ALI induced by LPS 1.The ALI/ARDS mice model were established successfully.After lung injury,the total cells in alveolar lavage fluid,lung wet/dry weight ratio(W/D)and pro-inflammatory factor level are increased significantly(p <0.05).The histopathology results showed typical ALI/ARDS pathological changes.The evidence show that the ALI/ARDS model were established successfully.The results showed that the most serious lung injury appeared on 8 hour and NF-?B pathway were activated.2.The expression of ZC3H12 D in ALI/ARDS mice model.The expression of ZC3H12 D mRNA and protein all decreased on 2 hours and began to increase from 4 hour,and reach the highest on 8 hour after lung injury(p <0.05).The results show that ZC3H12 D is involved in ALI/ARDS induced by LPS.Part II: Observing the protective effect of overexpression of ZC3H12 D on ALI 1.In vivo,ZC3H12 D adeno-associated virus can induce the overexpression of ZC3H12 D in lung tissue.After the virus infection,we first observed the lung tissue frozen section and measured the the expression of ZC3H12 D by immunohistochemical staining and Western blot.The results shows there are a large number of green fluorescence in the lung tissue and the expression of ZC3H12 D were significantly improved(p <0.05).2.In LPS-induced ALI/ARDS mice model,the overexpression of ZC3H12 D can effectively ameliorate the lung tissue injury.Then we established ALI/ARDS model to observe the effect of ZC3H12 D on ALI/ARDS.Overexpression of ZC3H12 D can significantly reduce lung tissue cell infiltration and pulmonary edema(p<0.05),alleviate lung tissue injury(p <0.05)and ameliorated histopathological alterations(p<0.05).The evidence showed overexpression of ZC3H12 D can effectively ameliorate the lung tissue injury.3.In vivo,overexpression of ZC3H12 D can reduce the level of pro-inflammatory cytokines.overexpression of ZC3H12 D can significantly reduce the level of pro-inflammatory cytokines(TNF?? IL-1? ? IL-6)(p <0.05)and mitigate lung tissue inflammation.Part III : exploring the protective effect of ZC3H12 D on LPS-insulted alveolar epithelial cell(A549 cell)and the underlying mechanism 1.the expression of ZC3H12 D in LPS-insult alveolar epithelial cells(A549 cells).The expression of ZC3H12 D decreased on 2 hours and began to increase from 4 hour,and reach the highest on 16 hour after LPS stimulation(p <0.05).Different dose of LPS(25 mu g/ml,50 mu g/ml and 75 mu g/ml)significantly reduced the expression of ZC3H12 D on 2 hours in a dose-dependent fashion(p <0.05)and significantly improve the expression of ZC3H12 D on 16 hours.2.In vitro,overexpression of ZC3H12 D can ameliorate LPS induced A549 cells injury and reduce the expression of inflammatory cytokinesOverexpression of ZC3H12 D can significantly reduce the level of inflammatory cytokines of LPS-insulted A549 cells and suppress the activation of NF-?B and AP-1(p <0.05).Overexpression of ZC3H12 D also can improve LPS-insulted A549 cells survival rates(p <0.05).3.ZC3H12 D can promote c-fos?TNF??IL-1??IL-6 and NF-?Bp65 mRNA degradation.we used THP-1 cells to explore the anti-inflammatory mechanism of ZC3H12 D.The results showed that ZC3H12 D can reduce c-fos?TNF??IL-1??IL-6 and NF-?B p65 mRNA stability,which suggests that ZC3H12 D can mitigate inflammatory reaction by targeting pro-inflammatory cytokines mRNAs.Conclusions: 1.ZC3H12 D is involved in LPS induced ALI.2.Overexpression of ZC3H12 D can effectively ameliorate lung injury by reducinginflammation factors and suppressing inflammatory signaling pathways.ZC3H12 D isa kind of protective molecular on ALI.3.ZC3H12 D can promote TNF ??IL-1??IL-6?c-fos and NF-?B p65 mRNA degradationand can protect alveolar epithelial cells through inhibiting inflammation response. |
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