| Part I DAPK1 is essential for the induction of apoptosis by Aza in d HL-60 cellsObjective:Investigate the effect of Aza on apoptosis and DAPK1 expression in d HL-60 cells in vitro,and determine whether Aza can be used as activator of DAPK1for subsequent experiments,as well as explore the role of DAPK1 in Aza induced d HL-60 cells apoptosis.Methods:All-trans retinoic acid(ATRA)was used to induce human promyelocytic leukemia cells(HL-60)to neutrophil-like cells(d HL-60)in vitro.The experiment was divided into A and B two parts.The part A was to study whether Aza could induce apoptosis and activate DAPK1 in d HL-60 cells.Firstly,different concentrations of Aza(0,1,2,and 4?M)were used to stimulate d HL-60 cells for 72 h respectively.Then,4?M Aza was used to stimulate d HL-60 cells for 0,24,48 and 72 h.The d HL-60 cells in each group were collected after the respective stimulation.TUNEL staining and flow cytometry were used to detect cell apoptosis,western blotting was used to detect the expression of DAPK1,p DAPK1 and p MLC.The part B was to investigate whether the regulation of DAPK1 expression affects Aza-induced d HL-60cells apoptosis.Firstly,the experimental groups:(1)control group;(2)control plasmid group:cells were transfected with control plasmid;(3)DAPK1 plasmid group:cells were transfected with DAPK1?Ca M-/-plasmid;(4)negative si RNA group:cells were transfected with control si RNA;(5)DAPK1 si RNA group:cells were transfected with DAPK1 si RNA.The expression of DAPK1 was detected by western blotting.After that,the experimental groups were divided into six groups:(1)control group;(2)Aza group:4?M Aza stimulated cells for 72 h;(3)DAPK1 plasma group:cells were transfected with DAPK1?Ca M-/-plasmid;(4)DAPK1 plasma+Aza group:DAPK1?Ca M-/-plasmid transfected cells were stimulated with Aza(4?M)for 72 h;(5)DAPK1 si RNA group:cells were transfected with DAPK1 si RNA;(6)DAPK1si RNA+Aza group:DAPK1 si RNA transfected cells were stimulated with Aza(4?M)for 72 h.The cells in each group were collected.The apoptosis was detected by TUNEL staining and flow cytometry.The expression of NF-?B p65,p NF-?B p65,Bcl-2 and Bax were detected by western blotting.Results:The results of part A showed that compared with 0?M Aza stimulation group,the number of apoptotic cells in 1,2 and 4?M Aza stimulation groups showed increased significantly(P<0.01),and the expression of DAPK1 and p MLC in cells in the above three groups also increased significantly(P<0.01),but the expression of p DAPK1 decreased(P<0.01).The above results changed gradually with the increase of Aza concentration and reached maximum or minumim at 4?M Aza.When the d HL-60 cells were stimulated by 4?M Aza for different durations(0,24,48 and 72 h),compared with 0 h group,the 24,48 and 72 h stimulation groups showed more apoptotic d HL-60 cells(P<0.01),higher levels of DAPK1 and p MLC(P<0.01)and lower level of p DAPK1(P<0.01).Aza-induced cell apoptosis and DAPK1 activation showed time-dependent manner.The results of part B showed that there were no significant difference in DAPK1 expression among control group,negative si RNA group and control plasmid group(P>0.05).Compared with the control group,the level of DAPK1 in cells showed apparently lower in DAPK si RNA group(P<0.01)and apparently higher in DAPK1 plasma group(P<0.01).When the cells overexpressed DAPK1 after transfected DAPK1?Ca M-/-plasmid,Aza further promoted cell apoptosis(P<0.01),decreased the expression of p NF-?B p65 and Bcl-2(P<0.01),and increased the expression of Bax(P<0.01).When DAPK1 was silenced in d HL-60 cells after DAPK1 si RNA transfection,Aza lost the capacity to increase apoptosis cells and affect NF-?B pathway.Compared with Aza group,the cells in DAPK1 si RNA+Aza group showed less apoptotic cells(P<0.01),higher levels of p NF-?B p65 and Bcl-2(P<0.01),and lower level of Bax(P<0.01).Conclusion:(1)Aza can up-regulate the expression of DAPK1 and induce cell apoptosis in d HL-60 cells.(2)DAPK1 is essential for Aza-induced cell apoptosis by affecting the expression of Bcl-2 family proteins which are the downstream of NF-?B.Part II Aza accelerates inflammatory resolution in lipopolysaccharide(LPS)-induced acute respiratory distress syndrome(ARDS)in miceObjective: To study the effect of Aza on the inflammatory resolution in lung tissues of mice after lipopolysaccharide(LPS)-induced acute respiratory distress syndrome(ARDS).Methods: Male C57BL/6 mice(aged 6-8 weeks old,weighting 23-25 g,80 mice in total)were fed in SPF grade environment.An ARDS model was evoked in C57BL/6 mice by intratracheal instillation of LPS at a dose of 3 mg/kg.After LPS instillation,the mice were randomly divided into two groups(40 mice in each group).(1)LPS group: one hour after LPS instillation,mice were injected with normal saline(NS;0.1 ml/animal,i.p.);(2)LPS+Aza group: one hour after LPS instillation,mice were injected with Aza(1 mg/kg,i.p.).After the above administration,8 mice in each group were sacrificed on days 0,1,2,4 and 7 after anesthesia.The right upper lung tissue was embedded in paraffin for hematoxylin and eosin(HE)staining.The sections were examined for pulmonary pathology under microscope and the lung injury scores were also calculated.The right middle lung tissue was used to examine for the wet / dry weight ratio(W/T).The left lung was lavaged with normal saline(NS)and bronchoalveolar lavage(BALF)was collected.The total protein in BALF was detected by BCA method,the total cell number was counted,and the neutrophils and macrophages in BALF were distinguished after Gemsa staining,and then counted under microscope respectively.One day after LPS instillation,the levels of pro-inflannatory cytokines TNF-?,IL-1?,IL-6 and MCP-1 and anti-inflammatory cytokine(IL-10)in BALF in these two groups were detected by ELISA.Results: After LPS instillation,pulmonary histopathological analysis showed important changes,including hemorrhage,increased thickness of the alveolar wall and infiltration of inflammatory cells in the LPS group and LPS+Aza group,and the inflammation of lung tissue were most apparent on day 1.With the prolongation of time,the inflammation of lung tissue in both groups decreased gradually,but the difference was that on days 1,2 and 4,the lung injury score in the LPS+Aza group was significantly lower than that in the LPS group at the same time point(P<0.01).Compared with LPS group at corresponding time point,the W/T in LPS+Aza group decreased on day 1(P<0.05),and on days 2 and 4,the W/T decreased dramatically(P<0.01).The total protein in BALF in the LPS+Aza group was significantly lower than that in the LPS group on days 1,2 and 4(P < 0.01).Moreover,on day 4,the total protein in BALF in the LPS+Aza group returned to the basic normal level,while that in the LPS group returned to normal until day 7.The results of cell counts in BALF showed that the number of total cells and neutrophils in BALF in both two groups decreased gradually with the time duration,but on days 1,2 and 4,the number of BALF total cells in the LPS+Aza group was significantly lower than that in the LPS group at the same time point(P<0.01).On days 1 and 2,the number of BALF neutrophils in the LPS+Aza group was also apparently lower than that in the LPS group at the same time point(P<0.01).Just like the change of total protein in BALF,the number of BALF neutrophils in the LPS+Aza group returned to normal on day 4,and was lower than that in the same time LPS group(P<0.05).And the number of BALF neutrophils in the LPS group returned to normal level until day 7.On the other hand,Aza also increased BALF macrophage numbers dramatically on day 2(P<0.01).On day 1,compared with LPS group,the levels of TNF-?,IL-1?,IL-6 and MCP-1 in BALF in LPS+Aza group were significantly lower(P<0.01),while the levels of anti-inflammatory cytokine IL-10 were significantly higher(P < 0.05).Conclusion: Aza can reduce inflammation and accelerate inflammatory resolution in LPS-induced ARDS in mice.Part III Effect of DAPK1 on neutrophil apoptosis affects the pulmonary inflammation in lipopolysaccharide(LPS)-induced acute respiratory distress syndrome(ARDS)in miceObjective: To investigate whether DAPK1 participates in Aza-induced neutrophil apoptosis in vivo and to determine whether regulating of DAPK1 could affect the protective role of Aza in LPS-induced ARDS in mice.Methods: The experiment was divided into two parts: A and B two parts.The part A mainly studied the effect of DAPK1 on Aza-induced neutrophil apoptosis in LPS-induced ARDS mouse model.Male C57BL/6 mice(aged 6-8 weeks old,weighting 23-25 g,80 mice in total)were fed in SPF grade environment,and randomly divided into 5 groups(16 mice in each group).(1)Sham group: After 14 days of continuous intraperitoneal injection of 10% DMSO(0.15 ml/day),PBS(50 ?l/mouse)was instillated into the trachea,and one hour after PBS instillation,normal saline(NS;0.1 ml/mouse)was intraperitoneally injected;(2)LPS group(Vehicle): After 14 days of continuous intraperitoneal injection of 10% DMSO(0.15 ml/day),LPS(3 mg/kg)was instillated into the trachea,and one hour after LPS instillation,NS(0.1 ml/mouse)was intraperitoneally injected;(3)LPS+Aza group(Aza): After 14 days of continuous intraperitoneal injection of 10% DMSO(0.15 ml/day),LPS(3 mg/kg)was instillated into the trachea,and one hour after LPS instillation,Aza(1 mg/kg)was intraperitoneally injected;(4)DAPK1 small molecule inhibitor+LPS group(DI): After 14 days of continuous intraperitoneal injection of DAPK1 small molecule inhibitor(DI;1 mg/kg/day),LPS(3 mg/kg)was instillated into the trachea,and one hour after LPS instillation,NS(0.1 ml/mouse)was intraperitoneally injected;(5)DAPK1 small molecule inhibitor+LPS+Aza group(Aza+DI): After 14 days of continuous intraperitoneal injection of DI(1 mg/kg/day),LPS(3 mg/kg)was instillated into the trachea,and one hour after LPS instillation,Aza(1mg/kg)was intraperitoneally injected.24 hours after administration,the mice in each group were anesthetized,and the peripheral blood was collected through picked the eyeballs,and neutrophils in peripheral blood were separated by magnetic beads.Western blotting was used to detect the expression of DAPK1 in neutrophils.After the right upper lung was embedded in paraffin,the sections were stained with immunohistochemistry and Ly6 G antibody was used to label accumulating neutrophils in the lung tissue.The left lung was lavaged with normal saline,and bronchoalveolar lavage fluid(BALF)was collected.The number of apoptotic neutrophils in BALF was detected by flow cytometry.The part B mainly investigated whether regulating DAPK1 affects the protective role of Aza on LPS-induced pulmonary inflammation in ARDS mice.The group was the same as part A.24 hours after LPS or PBS administration,the mice were anesthetized and sacrificed.The right upper lung was embedded in paraffin and the sections were stained with H&E to observe the pathological changes of lung tissue and make lung injury scores under microscope.The right middle lung was used for wet/dry weight ratio detection.The left lung was lavaged with normal saline to collect BALF.BCA was used to detect the total protein in BALF;the total number of BALF cells was calculated;and the number of BALF neutrophils and macrophages were also calculated respectively after Giemsa staining.ELISA was used to detect the levels of pro-inflammatory cytokines(TNF-?,IL-1?,IL-6 and MCP-1)and anti-inflammatory cytokine(IL-10)in BALF.Results: The results in part A showed that in LPS induced ARDS mice,compared with Vehicle group,both the expression of DAPK1 in neutrophils and the number of BALF apoptotic neutrophils in Aza group were significantly increased(P<0.01),and the number of accumulating neutrophils in lung tissue was dramatically reduced.When DAPK1 was inhibited by small molecule inhibitor,Aza could not promote DAPK1 expression and neutrophil apoptosis,which showed lower levels of DAPK1 in neutrophils and decreasing number of BALF apoptotic neutrophils in Aza+DI group compared with Aza group(P<0.01).And there was no significant difference in DAPK1 expression and cell apoptosis between DI group and Aza+DI group(P>0.05).The experiments in part B found that in LPS induced ARDS mice,the Aza+DI group,compared with Aza group,showed severe pathological changes,increasing ratio of lung wet/dry weight,total protein,number of total cells and neutrophils and levels of pro-inflammatory cytokines(P<0.01),and decreasing number of macrophages and IL-10 level(P<0.05).Conclusion: This study suggests that DAPK1 can mediate Aza-induced neutrophil apoptosis in vivo and affects the protective role of Aza in pulmonary inflammatory response in LPS-induced ARDS. |
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