| Part ? The influence of cyclic stretch for different time length on theapoptosis and expression of DAPK1 in mouse alveolar epithelial ? cells Objective To investigate the effect of cyclic stretch for different time length on the apoptosis and DAPK1 expression in mouse alveolar epithelial II cells, exploring the experimental condition for inducing cell apoptosis and the change pattern of DAPK1 expression.Methods Cyclic stretch was applied on mouse alveolar epithelial ? cells (MLE-12) by using Flex-5000 tension system. The stretch pattern was 30% stretch magnitude, 0.5Hz,sine wave for 6h, 12h and 24h respectively. In each time point, there were two groups:control group and cyclic stretch group. In order to investigate the different influence of cyclic stretch and static stretch on cell, we set a static .stretch group. The stretch condition was 30% stretch magnitude, 0.5Hz, 24h. The experiment was divided into two groups:control group and static group. Cell morphology was observed by using microscope. Cell viability was evaluated by MTT detection. The concentration of LDH in supernatant was determined in order to evaluate cell injury. Cell apoptosis was detected by flow cytometry.The expression of DAPK1, p-DAPK1 and p-MLC was determined by western bloting.Results 6h: Compared with control group, the cell morphology in cyclic stretch group was irregular. Cells partly grown branches and the viability increased significantly (P<0.05).LDH concentration in supernatant was elevated (P<0.05). Cell apoptosis was not changed(P>0.05). The expression of DAPK1, p-DAPK1 and p-MLC were not changed (P>0.05).12h: Compared with control group, a portion of cells in cyclic stretch group were round and clumped together. The number of cells which grown branches was significantly increased. Cell viability was not changed (P>0.05). LDH concentration in the supernatant was significantly up-regulated (P<0.05). Apoptotic cells were increased, but there was no statistical significance (P>0.05). The level of DAPK1, p-DAPK1 and p-MLC were not changed (P>0.05).24h: Compared with control group, cells in cyclic stretch group were round,flowing in the cultural medium. A small part of cells were adherent and clumped together.These cells possessed several slender branches. The cell viability was markedly decreased(P<0.05). LDH concentration in supernatant and apoptotic cell number was elevated significantly (P<0.05). The expression of DAPK1 and p-MLC was increased (P<0.05) and p-DAPK1 level was reduced (P<0.05).24h: Compared with control group, the morphology of cells in static stretch group was changed slightly. Cell viability, LDH concentration in supernatant and apoptosis rate were not changed (P>0.05). There were no significant changes in the expression of DAPK1,p-DAPK1 and p-MLC (P>0.05).Conclusion A high amplitude of mechanical stretch changed cell morphology as well as growth status. A short time of stretch did not affect cell apoptosis rate, but induced cell injury and promoted cell viability; As the stretching time prolonged, cell injury aggravated,cell viability decreased and the apoptotic cell increased. The expression of DAPK1 was significantly elevated as mechanical stretch induced cell apoptosis, along with a promotion of catalytic activity of DAPK1 (p-MLC); p-DAPK1, the inactive status of DAPK1, was reduced. In the same condition, static stretch could not induce cell injury or apoptosis.Meanwhile, there was no significant effect of static stretch on the expression of DAPK1,p-DAPK1 or p-MLC.Part ? DAPK1 regulated cyclic stretch induced mouse alveolar epithelial ? cell apoptosisObjective In order to investigate whether DAPK1 participated the high amplitude cyclic stretch induced mouse alveolar epithelial II cell apoptosis.Methods The experiment was divided into A and B two parts. The A part was to verify that inhibition of DAPK1 expression could reduce mouse alveolar epithelial II cell apoptosis. The experimental groups: control group (control); control siRNA+ cyclic stretch group (control-si+CS): cells received cyclic stretch after transfected by control siRNA;DAPK1-siRNA+cyclic stretch group (DAPK1-si+CS): cells received cyclic stretch after transfected by DAPK1. Stretch pattern: 30% amplitude, 0.5Hz, 24 h. Cell morphology was observed by microscope; MTT method was used to detect cell viability; Cell apoptosis was evaluated by flow cytometry. The B part was to investigate whether an increase of DAPK1 level could induce mouse alveolar epithelial ? cell apoptosis. The experimental groups:control group (control); control plasmid group (control plasmid): transfected by control plasmid; DAPK1-CaM plasmid group (DAPK1-CaM): transfected by DAPK1-CaM plasmid. The cells were cultured for 48 hours after transfection. Microscope was applied to observe cell morphology change. Cell viability and cultural LDH concentration were detected. The cell apoptosis rate was analyzed by flow cytometry.Results In the A part, compared with control-si+CS group, the morphology changes of cells in DAPK1-si+CS group was attenuated. The numbers of round cells and cells possessed branches were reduced markedly. Cell viability was elevated (P<0.05). LDH concentration in cultural supernatant and apoptotic cell number were decreased significantly (P<0.05). In the B part, compared with the control plasmid group, cell morphology in DAPK1-CaM group changed significantly. Most of the cells were round and shrinkage, flowing in the cultural medium. Cell viability decreased (P<0.05) and LDH concentration was up-regulated (P<0.05). The cell apoptosis rate was significantly increased (P<0.05).Conclusion DAPK1 participated in cyclic stretch induced mouse alveolar epithelial ?cell apoptosis. A reduction of DAPK1 expression inhibited cyclic stretch induced cell injury and apoptosis; Overexpression of DAPK1 induced apoptosis in mouse alveolar epithelial ? cell.Part ? DAPK1 promoted mechanical ventilation induced lung injury though inducing alveolar epithelial cell apoptosisObjective In order to investigate whether DAPK1 participated mechanical ventilation induced lung injury though inducing alveolar epithelial cell apoptosis and the inhibition of DAPK1 expression and activity could attenuate mechanical ventilation induced lung injury.Methods The experiment was divided into A and B two parts. The A part was to study the effect of different volume tidal on alveolar epithelial cell apoptosis and the expression and activity of DAPK1. Twenty four male C57 mice,6-8 weeks old,body weight 23-23g,were used in this part. Mice were randomly divided into three groups (n=8): (1) control group(control): no treatment; (2) low volume tidal mechanical ventilation group (LVt): mice were ventilated by a tidal volume 8ml/kg with 120 breaths/min; (3) high volume tidal mechanical ventilation group (HVt): mice were ventilated by a tidal volume 35ml/kg with 80 breaths/min.The ventilation time was 4 hour. After the experiment, mice were sacrificed and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. Pulmonary pathological changes were evaluated by HE staining; Protein concentration and cell count in BALF was detected; Lung tissues wet to dry (W/D) ratio was examined; The apoptotic alveolar epithelial cell number in lung tissues was evaluated by Tunel staining; DAPK1 and p-MLC level were determined by western bloting. The B part was to investigate whether DAPK1 inhibitor could attenuate mechanical ventilation induced alveolar epithelial apoptosis. Twenty four male C57 mice,6-8 weeks old, body weight 23-23 g, were used in this part. Mice were randomly divided into three groups (n=8): (1) control group (control): no treatment; (2) DMSO treatment group(DMSO): mice received mechanical ventilation after 10% DMSO injection (100?l per mouse,intraperitoneally, 4 weeks); (3) DAPK1 inhibitor treatment group (DI): mice received mechanical ventilation after DI injection (500?g/kg, intraperitoneally, 4 weeks). mice were ventilated by a tidal volume 35ml/kg with 80 breaths/min. The ventilation time was 4 hour.After ventilation, mice were sacrificed and collected samples. Pulmonary pathological changes were evaluated by HE staining; Protein concentration and cell count in BALF was determined; Lung tissues wet to dry (W/D) ratio was examined; The apoptotic alveolar epithelial cell number in lung tissues was evaluated by Tunel staining; DAPK1, p-MLC and P53 level were detected by western bloting.Results The A part: compared with control group, alveolar structures were damaged slightly and there was no inflammatory cells infiltration in LVt group; In HVt group,alveolar structures were damaged seriously, along with the thickening of alveolar septal,inflammatory cells infiltration and hemorrhage. Protein concentration in BALF, cell count and W/D ratio were not changed in LVt group (P>0.05); however, these indexes were elevated in HVt group (P<0.05). Compared with control group, the apoptosis cell number in lung tissue in LVt group was not change (P>0.05); the number increased in HVt group(P<0.005). There was no significant changes in the expression of DAPK1 and p-MLC in LVt group as compared with control group; but the levels of DAPK1 and p-MLC were markedly promoted in HVt group (P<0.05).The B part: The damages of lung structure in DI group were significantly attenuated as compared with DMSO group. Compared with DMSO group, protein concentration in BALF, cell count, W/D ratio and apoptosis cell number in lung tissue were decreased in HVt group (P<0.05); The expression of DAPK1, p-MLC and P53 were reduced markedly in HVt group (P<0.05).Conclusions Low tidal volume ventilation could not destroy the lung tissues, induce alveolar epithelial cell apoptosis and change the expression of DAPK1. High tidal volume ventilation induced lung injury and alveolar epithelial cell apoptosis. Meanwhile, the high tidal volume ventilation promoted DAPK1 expression and activity. Pretreatment of DAPK1 inhibitor reduced high tidal volume ventilation induced alveolar epithelial cell apoptosis,the expression and activity of DAPK1. The results indicated that DAPK1 inhibitor played a protective role in mechanical ventilation induced lung injury. |
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