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Objective To observe the effect of excess oxygen supply for different time periods on the mitochondrial energy metabolism in alveolar epithelial type Ⅱ cells. Methods Rat RLE-6TN cells were assigned into a control group (21% O2 for 4 h) and excess oxygen supply groups (95% O2 for 1,2,3,and 4 h,res-pectively).The content of adenosine triphosphate (ATP),the activity of mitochondrial respiratory chain complex V,and the mitochondrial membrane potential were determined by luciferase assay,micro-assay,and fluorescent probe JC-1,respectively.Real-time fluorescence quantitative PCR was employed to determine the mRNA levels of NADH dehydrogenase subunit 1 (ND1),cytochrome b (Cytb),cytochrome C oxidase subunit I (COXI),and adenosine triphosphatase 6 (ATPase6) in the core subunits of mitochondrial respiratory chain complexes Ⅰ,Ⅲ,Ⅳ,and Ⅴ,respectively. Results Compared with the control group,excess oxygen supply for 1,2,3,and 4 h down-regulated the mRNA levels of ND1 (q=24.800,P<0.001;q=13.650,P<0.001;q=9.869,P<0.001;q=20.700,P<0.001),COXI (q=16.750,P<0.001;q=10.120,P<0.001;q=8.476,P<0.001;q=14.060,P<0.001),and ATPase6 (q=22.770,P<0.001;q=15.540,P<0.001;q=12.870,P<0.001;q=18.160,P<0.001).Moreover,excess oxygen supply for 1 h and 4 h decreased the ATPase activity (q=9.435,P<0.001;q=11.230,P<0.001) and ATP content (q=5.615,P=0.007;q=5.029,P=0.005).The excess oxygen supply for 2 h and 3 h did not cause significant changes in ATPase activity (q=0.156,P=0.914;q=3.197,P=0.116) and ATP content (q=0.859,P=0.557;q=1.273,P=0.652).There was no significant difference in mitochondrial membrane potential among the groups (F=0.303,P=0.869). Conclusion Short-term excess oxygen supply down-regulates the expression of the core subunits of mitochondrial respiratory chain complexes and reduces the activity of ATPase,leading to the energy metabolism disorder of alveolar epithelial type Ⅱ cells.
Subject(s)
Animals , Rats , Energy Metabolism , Adenosine Triphosphate , Adenosine Triphosphatases , RNA, Messenger , OxygenABSTRACT
Objective To investigate the effects of heme oxygenase-1/carbon monoxide (HO-1/CO) pathway on mitochondrial fusion in rat alveolar epithelial type Ⅱ cells (AECⅡ) stimulated by lipopolysaccharide (LPS). Methods Once the cultured in vitro rat AECⅡcells line RLE-6TN reached confluency of 85%, they were subcultured and randomly divided into seven groups (n = 5 each). RLE-6TN cells were routinely cultured in control group. The cells in LPS group was stimulated with 10 mg/L LPS to reproduce the model of endotoxin challenge in AECⅡ cells. The cells in carbon monoxide-releasing molecule-2 (CORM-2, in vitro CO release agent) + LPS group (CL group) and Hemin (HO-1 inducer) + LPS group (HL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin for 1 hour, respectively, followed by 10 mg/L LPS stimulation. The cells in zinc protoporphyrin-Ⅸ (ZnPP-Ⅸ, HO-1 inhibitor) + LPS group (ZL group) was pretreated with 10 μmol/L ZnPP-Ⅸ for 0.5 hour followed by 10 mg/L LPS stimulation. The cells in CORM-2 + ZnPP-Ⅸ + LPS group (CZL group) and Hemin + ZnPP-Ⅸ + LPS group (HZL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin respectively for 1 hour, and other treatments were similar to those previously described in ZL group. At 24 hours after LPS stimulation, interleukin-6 (IL-6)and tumor necrosis factor-α (TNF-α) in the supernatant were determined by enzyme linked immunosorbent assay (ELISA), the protein expressions of HO-1, mitochondrial fusion related proteins 1 and 2 (Mfn1, Mfn2) and optic atrophy 1 (OPA1) were determined by Western Blot. Results Compared with control group, IL-6 and TNF-α contents in the supernatant were increased, HO-1 protein expression was up-regulated, Mfn1, Mfn2 and OPA1 protein expressions were down-regulated in all treatment groups. Compared with LPS group, IL-6 and TNF-α contents were significantly decreased after CORM-2 or Hemin pretreatment [IL-6 (ng/L): 48.6±3.7, 48.4±3.1 vs. 58.7±2.5; TNF-α (ng/L):40.7±5.3, 39.4±4.3 vs. 51.8±5.1], the protein expressions of HO-1, Mfn1, Mfn2 and OPA1 were significantly up-regulated (HO-1 protein: 0.873±0.051, 0.839±0.061 vs. 0.671±0.044; Mfn1 protein: 0.673±0.037, 0.654±0.025 vs. 0.568±0.021; Mfn2 protein: 0.676±0.044, 0.683±0.035 vs. 0.571±0.043; OPA1 protein: 0.648±0.031, 0.632±0.031 vs. 0.554±0.032; all P < 0.05); while opposite effects were found after ZnPP-Ⅸ preincubation, and there were significant differences in IL-6 and TNF-α contents and protein expressions of HO-1, Mfn1, Mfn2 and OPA1 as compared with those of LPS group [IL-6 (ng/L): 69.8±5.1 vs. 58.7±2.5, TNF-α (ng/L): 61.9±3.3 vs. 51.8±5.1, HO-1 protein: 0.545±0.023 vs. 0.671±0.044, Mfn1 protein: 0.406±0.051 vs. 0.568±0.021, Mfn2 protein:0.393±0.051 vs. 0.571±0.043, OPA1 protein: 0.372±0.050 vs. 0.554±0.032; all P < 0.05]. There were no significant differences in the parameters mentioned above between HL group and CL group, as well as among LPS, CZL and HZL groups. Conclusion HO-1/CO pathway promotes mitochondrial fusion and alleviates inflammatory response in LPS-induced rat AECⅡ cells.
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Objective To investigate the effect of all-trans retinoic acid (at-RA) on fetal alveolar epithelial type Ⅱ cells (fAEC Ⅱ s) proliferation and the expression of pulmonary surfactant C (SPC) as well as aquaporin 5 (AQP5).Methods fAEC Ⅱ s were isolated and purified from fetal lung of pregnant SD rats (19 days).After being cultured for 1 day,and the fAEC Ⅱ s were interfered by at-RA for 1,2 and 3 days.Cell proliferation,viability as well as growth state,expressions of SPC mRNA as well as AQP5 mRNA and expressions of protein SPC as well as AQP5 were respectively detected by using 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT),inverted microscope,real-time fluorescence quantitative PCR (RT-PCR) and Western blot.Results (1) When fAEC Ⅱ s were treated with at-RA for 1 day,and the cell proliferation and viability did not change (P ＞ 0.05),while the proliferation and viability were significantly improved in 2 days (P ＜ 0.05),and the difference was the most obvious (P ＜ 0.05) at 3 days.(2)Compared with the control group,the cell growth state was better,and the cell adherence was tighter and the refraction was higher in at-RA group.(3) Compared with the control group,at-RA up-regulated the expressions of AQP5 mRNA and AQP5 protein(t =-19.58,-10.44,-16.01,-46.25,-12.79,-27.96,all P ＜ 0.05),and the percentages of control group were 281.07％,766.67％,1 163.33％ and 792.65％,1 310.52％,1 561.56％ in 1,2 and 3 days respectively.(4) Compared with control group,the expressions of SP-C mRNA and SPC protein were up regulated when cells were exposed to at-RA for 1 and 3 d,but while they were down-regulated at 2 days(protein:the percentages of control group were 615.480％,369.450％ and 11.269％,respectively ; mRNA:728.33 ％,400.83 ％,66.57％,respectively)(t=-26.34,-25.26,13.80,-25.25,-31.71,9.12,all P＜0.05).Conclusions at-RA can promote the proliferation and differentiation of fAEC Ⅱs,enhance the fAEC Ⅱ s viability,and improve the expression of SPC and AQP5.
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Objective To investigate the dynamic expression of mRNA and protein of surfactant protein C (SPC),E-cadherin (E-cad),N-cadherin (N-cad) and α-smooth muscle actin (α-SMA) and elucidate the significance of epithelial-mesenchymal transition (EMT) in a newborn rat model of Bronchopulmonary dysplasia (BPD).Methods A newborn rat model of BPD in hyperoxia was established,and a control group exposed to air was established.Lung tissue was collected on days 3,7,14,and 21,respectively.Alveolar development was evaluated by radical alveolar counts(RAC),including thickness of alveolar septum,ratio of alveolar and septa.Real-time PCR and Western-blot were used to detect levels of markers of epithelial cells (SPC,E-cad) and interstitial cells (N-cad,α-SMA) in AT2 and protein expression.Results On day 7,14,and 21,compared with the control group,RAC (7.38 ± 0.92,9.25 ± 0.70,9.88 ± 0.99) and alveolar area/pulmonary septal area ratios (A/S) (2.53 ± 0.02,3.34 ± 0.09,3.96 ± 0.13) were all higher in BPD group [RAC (5.88 ± 0.83,5.14 ± 0.83,4.38 ± 0.52) and A/S (1.88 ± 0.03,1.95 ± 0.03,1.89 ± 0.02)] (all P ＜ 0.05) ; the alveolar septum (8.53 ± 0.04,10.75 ± 0.46,13.55 ± 0.84) in BPD group were thicker than those (5.77 ± 0.09,4.40 ± 0.12,3.67 ± 0.18) in the control group (all P ＜ 0.01).The expressions of SPC and α-SMA in BPD group were significantly higher than those in the control group on day 14 and 21 (all P ＜ 0.05).The level of E-cad mRNA (2.43 ± 0.60,2.59 ± 0.48,3.37 ± 0.53) and protein (18.39 ± 1.77,18.29 ± 1.52,11.48 ± 1.72) for E-cad were higher in the control group than those (mRNA:1.48 ± 0.55,1.57 ± 0.48,1.12 ± 0.45 ;protein:9.50 ± 1.38,8.57 ± 1.06,8.22 ± 1.31) in BPD group was lower(P ＜ 0.05),while the level of N-cad was significantly higher(P ＜ 0.05) on day 7,14,and 21.Conclusions In the development of BPD,the markers of lung epithelial cells were down regulation,while the markers of interstitial cells were up-regulation,and these findings suggest that EMT from lung epithelial cells contributes to the occurrence of BPD.
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Objective To study the temporal changes of alveolar epithelial type Ⅱ cells and surfactant pro-tein A in young rats with acute lung injury induced by lipopolysaecharide. Method Totally 110 SD young rats (male:53, female : 57) were randomly divided into ALI and normal control groups (six subgroups in each group).LPS(4 mg/kg) was given intraperitoneally in ALI group. The same amount of normal saline was given in the con-trol groups. Eight rats in each subgroup were sacrificed at 6, 12, 24, 36, 48 and 72 hours after the injection.Lung samples were taken for transmission electron microscope examination. RT-PCR was epmloyed for the mea-surement of SP-A mRNA. Western blot was used for the detection of SP-A in the lung tissue. ANOVA and homo-geneity of variance test were performed by SPSS 12.0. Results The microvilli disappeared at 24 hours after the injection of LPS. The number of lamellar body (LBs) was provisionality increased at 24 hours and 48 hours. The ring-like an'angement of LBs around nucleus and the giant LB with vacuole-like deformity were found as the main characteristics of AEC- Ⅱ in ALI at 48 hours. The number of LBs reduced and broken and residual LB remained at 72 hours. SP-A elevated greatly from 24 to 48 hours (P < 0.01), reached peak at 36 hours (6.94 ± 0.80, P <0.01),reached the lowest level(3.87 ±0.50, P <0.01)at 72 hours. Conclusions The pathological changes of AEC-Ⅱ and SP-A in lung tissue wiht ALI are time-dependent. The typical alterations of AEC- Ⅱ occurs at 48 hours accompanied by the compensatory increase of SP-A. AEC- Ⅱ is seriously injuried with the typical changes of LBs and the diminishing of SP-A in lung tissue.
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Objective To investigate the mechanism of growth hormone inhibiting IPS-induced apoptosis of alveolar type Ⅱ epithelial cells in rats. Method Isolated and purified AEC Ⅱ cells of SD rats were divided into 5 groups,8 duplicate wells in each group. Group I served as control group; group Ⅱ:LPS 10 ug/ml;group Ⅲ:LPS 10 ug/ml + GH 50 ng/ml;gronp IV :LPS 10 ug/ml + GH 100ng/ml; group V: LPS 10 ug/ml + GH 200 ng/ml. LPS was finally added into wells in group Ⅱ～V . After the cells were incubated for 24 hours, the apoptosis rate and necrosis rate of AEC Ⅱ cells stained with Annexin V/PI were detected by flow cytometry and Fas protein of AEC Ⅱ cells were measured by immunocytochemistry. Results (1) The apoptosis rate and necrosis rate of AECⅡ cells in group Ⅱ,Ⅲ, Ⅳ and V were significantly hitOer than those in group Ⅰ( qapoptosis rate Ⅰ, Ⅱ =12.26,qnecroeis Ⅰ,Ⅱ=18.34, qapoptosisⅠ.Ⅱ=9.63,qnecrosisⅠ,nⅡ=5.75,qapotosisⅠ,Ⅳ= 9.15,qnecrosisⅠ,Ⅳ= 5.39, qapotosisⅠ,Ⅴ = 10.87, qnecrosisⅠ,Ⅴ = 5.91, P 0.05), but lower in group Ⅲ,IV and V than those in group Ⅱ(qapoptosis Ⅱ,Ⅲ= 15.24, qpecrosisⅡ,Ⅲ=16.38, qapoptosisⅡ.Ⅳ = 15.95,qnecrosisⅡ.Ⅳ=16.95, qapoptosis rate Ⅱ,Ⅴ=14.57, qnecrosisⅡ.Ⅴ = 15.61,P<0.05). (2)The positive rate of Fas expression on AEC Ⅱ cells in group Ⅱ,Ⅲ, Ⅳ and V was obviously higher than that in group Ⅰ. ( q Ⅰ.Ⅱ=35.67, qⅠ ,Ⅲ=14.32, qⅠ,Ⅳ = 13.87, qⅠ.Ⅴ=26.16, P<0.05), but lower in gronpⅢ ,Ⅳ and Ⅴ than that in gronp Ⅱ(qⅡ,Ⅲ=12.54, qⅡ,Ⅳ = 13.02, qⅡ,Ⅴ =6.96, P<0.05). Conclusions GH can probably de-crease the apoptosis of AEC Ⅱ cells by inhibiting Fas expression.
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Objective To observe the effects of hyperoxia on Notch 1 receptor of alveolar epithelial type Ⅱ cells (AEC Ⅱ), in a hetcrocellular culture of alveolar epithelial type Ⅱ cells and lung flbroblasts(LF), in order to explore Notch signaling in hyperoxic induced lung injury and thus make theoretical basis for prevention and treatment of a acute/chronie neonatal lung injury. Method Twelve Spragne Dawkey female rats with 200～220 g and 3 Spragne Dawkey male rats with 220～250 g were offered from experimental animal centre of Tongji Medical Colleege, Huazhong University of Science and Technology. The AEC Ⅱ/ LF co-culture system was established successfully. AEC H s from premature rats were randomly assigned to 2 groups: air control group and hyperoxia group. Air control group was kept in room air 50% ml/L CO2 enviromnent at 37°C, while hyperoxia group was exposed to 950 ml/L O2 + 250 ml/L CO2. Immuno-histochemistry was taken to detect Notch 1. Fluorescent quantitafive PCR was used to quantify the Notch 1 mRNA. MTT method was taken to assess cen proliferation viability.Flow eytometry double label method was used to detect cell percentages. Results In hyperoxia group:Notch 1 activation was inhibited, and Notch 1 mRNA decreased to 0.43,0.29,0.11,0.03 fold of control (95% confidence limit). AEC Ⅱ percentage descended predominantly[ 24 h hyperoxia group vs. control group: (68.92±6.88)%vs. (90.35±4.01)%, P =0.006;48 h hyperoxia group vs. control group: (38.03±3.27) vs. (61.47±4.81)%, P =0.000;72 h hyperoxia group vs. control group:(20.13±4.45)% vs. (52.05±3.35)%, P =0.000;96 h hyperoxia group vs. control group:(8.17±1.99)% vs. (52.59±2.93)%, P =0.0001 while that d AECI rised[24 h hyperoxia group vs. contrd group:(0.11±0.03)% vs. (0.01±0.01)%, P=0.006;48h hyperoxia group vs. control gnmp:(49.73±3.45)% vs. (16.13±2.13)%, P =0.000;72 h hyperoxia group vs. control group: (52.43±3.14) % vs. (5.98±0.95) %, P = 0.000;96h hyperorxia group vs. control group:(19.85±3.26)% vs. (29.03±3.16)%, P =0.007]. Comclusions Hyperoxia may inhibit Notch signaling pathway, which can weaken proliferation and disdifferentiation of AEC Ⅱ s. Investigations on how to control Notch signaling will provide fresh thoughts for alveolar epithelium repairing.
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Objective:To investigate survival and apoptotic responses of alveolar epithelial typeⅡcells(ATⅡ cell)under oxidative stress and to discuss the regulation mechanism mediated by c-jun NH2-terminal kinase(JNK).Methods:Primary rat alveolar epithelial typeⅡcells were attacked by Hydrogen peroxide(H2O2)and cells were pretreated with SP600125 in some groups.Cell viability,apoptotic rate and the expression of phosphorylated JNK(p-JNK)were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,flow cytometry and western blot analysis,respectively.Results:Compared with control group,decreased cell viability and increased apoptotic rate in ATⅡ cells occurred in time-dependent manner when treated with 500?mol/L H2O2 in different time.H2O2 induces phosphorylation of JNK.SP600125 enhanced cell viability and decreased apoptotic rate after H2O2-exposure.Conclusion:Apoptosis could be induced by H2O2 in ATⅡ cells in time-dependent manner.JNK signaling pathway may play a proapoptotic role in the regulation of apoptosis induced by H2O2.