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Objective:To explore the dynamic phenotype of type Ⅱ alveolar epithelial cells(AEC Ⅱ)in radiation-induced lung fibrosisand its role in the formation of fibrosis.Methods:Totally 90 C57BL/6J female mice were divided into 2 groups: irradiation group (50, thoracic irradiation with a single dose of 20 Gy X-rays), control group (40, sham irradiation). At 24 h, 4 and 12 weeks after irradiation, 5 mice were euthanized and the lungs were collected for pathological observation. The other lungtissues were collected for the isolation of primary AEC Ⅱ cells with microbeadssorting.The mRNA expressions of proSP-C, HOPX, vimentin, β-catenin and TGF-β1 in AEC II cells were detected by RT-PCR.Results:Acute pneumonitis was observed in the lungs at 24 h after irradiation and alleviated in accompany with partial alveolar septal thickening and a small amount of collagen deposition at 4 weeks after irradiation. The collagen deposition became more pronounced at 12 weeks after irradiation, together with collapsed and fused alveolar cavities, alveolar septal hyperplasia, and pulmonary fibrosis formation.The mRNAexpression levels of proSP-C and HOPX in primary AEC Ⅱ cells increased at 24 hours after irradiation and then approached to a peak value at 4 weeks after irradiation ( F=8.441, 3.586, P=0.036). The mRNA expression levels of vimentin, a biomarker of EMT, was increased significantly at 4 weeks and continued up to 12 weeks after irradiation( F=8.358, P=0.001). The mRNA expression levels of profibrotic factors β-catenin and TGF-β1 were both significant increased at 12 weeks after irradiation( F=4.62, 3.279, P=0.044). Conclusions:The phenotypeof AECⅡ cells could not only be transformed from proSP-C+ to HOPX+ /proSP-C+ , HOPX+ /proSP-C+ /vimentin+ , and vimentin+ /proSP-C, but also differentiated into mesenchymal cells with highly expressed profibrotic factors, thereby inducing EMT process, which either played a role in the repair of radiation-induced lung injury or triggered radiation-induced fibrosis.
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Objective To investigate the effects of agmatine (AGM) on the apoptosis of type Ⅱ alveolar epithelial cells (AECⅡ) in rats with hyperoxia-induced acute lung injury (HALI) and provide a theoretical basis for the treatment of HALI. Methods A total of 24 Sprague-Dawley (SD) rats were randomly divided into three groups:normal control group (fed in air), HALI model group and AGM pretreatment group (400 mg/kg AGM was given before the hyperoxia treatment or HALI model establishment), each group with 8 rats. The rats were placed in a self-made high oxygen model box with oxygen concentration of > 90%, temperature of 25-27 ℃, humidity of 50%-70% and carbon dioxide concentration < 0.5% to replicate the HALI rat model; no any treatment was given to the normal control group. After the hyperoxia was treated for 48 hours, the arterial blood was taken from the rat carotid artery for blood gas analysis;under light microscope, the pathological changes of lung tissues were observed and the pathological evaluation scores were carried out; the contents of tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1) in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA); the apoptosis of AECⅡ of lung tissues was determined by flow cytometry, and the apoptotic rate was calculated; the expressions of the apoptosis related protein Bcl-2 and Bax were detected by Western Blot. Results Compared with the normal control group, the oxygenation index (OI) and Bcl-2 of HALI model group and AGM pretreatment group were significantly decreased [OI (mmHg, 1mmHg = 0.133 kPa): 135.04±16.82 vs. 463.74±22.04, Bcl-2 protein expression (A value): 0.35±0.18 vs. 0.89±0.08], while the respiratory index (RI), pathological scores of lung injury, TNF-α, IL-6, IL-1, the apoptosis rate of AECⅡ, Bax protein expression were all significantly increased [RI: 1.29±0.15 vs. 0.24±0.03, pathological score of lung tissue: 4.72±1.32 vs. 0, TNF-α (μg/L): 44.48±1.42 vs. 14.12±0.88, IL-6 (μg/L): 51.46±1.62 vs. 23.20±0.89, IL-1 (μg/L): 44.03±2.45 vs. 11.64±1.34, apoptosis rate of AECⅡ: (56.24±1.14)% vs. (22.64±0.58)%, Bax protein expression (A value): 2.37±0.34 vs. 1.41±0.48, all P < 0.05]. Compared with HALI model group, the OI and Bcl-2 of AGM pretreatment group were significantly increased [OI (mmHg): 364.72±14.56 vs. 135.04±16.82, Bcl-2 protein expression (A value): 0.68±0.10 vs. 0.35±0.18, all P < 0.05], while the RI, pathological scores of lung injury, TNF-α, IL-6, IL-1, apoptosis rate of AECⅡ, and Bax protein expression were significantly decreased [RI: 0.45±0.09 vs. 1.29±0.15, pathological score of lung tissue: 2.30±0.96 vs. 4.72±1.32, TNF-α (μg/L):22.98±0.72 vs. 44.48±1.42, IL-6 (μg/L): 35.79±0.86 vs. 51.46±1.62, IL-1 (μg/L): 24.06±0.86 vs. 44.03±2.45, apoptosis rate of AECⅡ: (28.58±1.21)% vs. (56.24±1.14)%, Bax protein expression (A value): 1.98±0.42 vs. 2.37±0.34, all P < 0.05]. Conclusion The apoptotic rate of AECⅡ in HALI rats is reduced by AGM, and the regulatory mechanism needs to be further studied.
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Objective To analyze the PM2.5 components collected during winter in Xinxiang city and their inflammatory effects on human type lⅡ alveolar epithelial cells.Methods Fine particulate matter (PM2.5) was collected during winter in Xinxiang using a high-volume air sampler.The composition and mass concentration of soluble anions and metal elements in PM2.5 were determined with ion chromatography and inductive coupled plasma emission spectrometer,respectively.3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was used to examine the effect of PM2.5 on viability of human type Ⅱ alveolar epithelial cell line A549;enzyme-linked immunosorbent assay was used to examine the expression of interleukin-1β(IL-1β) and interleukin-8 (IL-8) in A549 cells.Results Airborne PM2.5 contained higher levels of NOx and SO42-during winter in Xinxiang city.The amount of PM2.5-derived metal elements in PM2.5 varied significantly,with higher levels of Ca,Mg,Zn and Al.The inhibition ratio of 12.5,25.0,50.0,100.0,200.0,400.0 mg · L-1 pM2.5 group on A549 of human type Ⅱ alveolar epithelial cell was higher than that of 0.0 mg · L-1 PM2.5 group (P ＜ 0.05).The inhibition ratio of 200.0,400.0 mg · L-1 PM2.5 group on A549 of human type Ⅱ alveolar epithelial cell was higher than that of 12.5,25.0,50.0,100.0 mg · L-1 PM2.5 group(P ＜ 0.05).There was no significant difference in the inhibition ratio in the 12.5,25.0,50.0,100.0 mg · L-1 PM2.5 group (P ＞ 0.05),and there was no significant difference in the inhibition ratio between 200.0 mg · L-1 PM2.5 group and 400.0 mg · L-1 PM2.5 group(P ＞ 0.05).Compared with 0.0 mg · L-1 pM2.5 group,the IL-1βand IL-8 protein expression of the human type Ⅱ alveolar epithelial cells culture supernatants in the 25.0,50.0,100.0 mg · L-1PM2.5 group was higher(P ＜0.05).But there was no significant difference in the IL-1β and IL-8 protein expression of human type Ⅱ alveolar epithelial cell culture supernatants among 25.0,50.0,100.0 mg · L-1 pM2.5 group (P ＞ 0.05).Conclusion Analysis of source apportionment suggests that construction and automobiles are main sources of ambient PM2.5 air pollution.Moreover,exposure to PM2.5 can cause damage and pro-inflammatory response of human type Ⅱalveolar epithelial cells.
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Objective Idiopathic pulmonary fibrosis ( IPF) is a chronic inflammatory disease with unknown etiology and is lack of effective therapy. The aim of this study is to explore the function of Ca2+ and PI3K/AKT/mTOR pathway in the pathogenesis of IPF, and the impact of 1,25?( OH) 2 D3 on Ca2+ and PI3K/AKT/mTOR pathway in type Ⅱalveolar epithelial cells of rat with IPF. Methods 150 male SD rats were randomly divided into 2 groups: prevention group ( control groupⅠ, model groupⅠ, medication groupⅠ) and treatment group ( control groupⅡ, model groupⅡ, medication groupⅡ) . The tracheal exposure surgery was operated in control groupⅠ/Ⅱ, and then 200μL sterile physiological saline was administered by intraperitoneal injection of each rats 2 days and 14 days after surgery, separately. Bleomycin(BLM)(5 mg/kg) was in?jected into the trachea of model groupⅠ/Ⅱ, and then vitamin D3 solvent(0.1%ethanol and 99.9%glycol propylene, 1μL/g) was ad?ministered by intraperitoneal injection 2 days and 14 days after surger?y, separately. Bleomycin( BLM) ( 5 mg/kg) was injected into the tra?chea of medication groupⅠ/Ⅱ, and then 1,25?( OH) 2 D3( 2μg/kg) was administered by intraperitoneal injection 2 days and 14 days after surgery, separately. IPF model was built by injecting Bleomycin into the trachea of rats, 1,25?(OH)2D3(2μg/kg) was used to prevent and treat IPF by intraperitoneal injection in medication group. The hydroxyproline content of lung tissue in each group was measured, type Ⅱalveolar epithelial cells were separated from lung tissue and labeled with Fluo?3AM, then concentration of Ca2+ was detected by Laser scanning confocal microscope. The mRNA levels of PI3K, AKT and mTOR in the typeⅡalveolar epithelial cells were tested by RT?PCR. Results Compared with control groupⅠ/Ⅱ at each time point, hydroxyproline content of lung tissue, Ca2+ concentration and expression of PI3K, AKT and mTOR in typeⅡalveolar epithelial cells in model groupⅠ/Ⅱand medication groupⅠ/Ⅱwere sig?nificantly raised( P<0.05 or P<0.01) , but these were significantly reduced in medication groupⅠ/Ⅱcompared with model groupⅠ/Ⅱ( P<0.05 or P<0.01) . Correlation analysis showed that there is significant positive correlation between Ca2+ concentration and mRNA expression levels of PI3K, AKT and mTOR in model groupⅠ/Ⅱ(r=0.5988, r=0.6230, r=0.6603,P<0.01)and medication groupⅠ/Ⅱ( r=0.701 2, r=0.632 3,r=0.740 3,P<0.01) . Conclusion The PI3K/AKT/mTOR pathway plays an important role in devel?opment of IPF. 1,25?( OH) 2 D3 is able to reduce Ca2+concentration in typeⅡalveolar epithelial cells and inhibit the PI3K/AKT/mTOR pathway, and then inhibit the development of IPF.
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AIM: To detect whether N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits epithelial-mes-enchymal transition in A549 cells induced by TGF-β1 through suppressing the expression of heat shock protein 27 (HSP27) and zinc finger proteins Snail (including SNAI1and SNAI2) which ultimately inhibited the deposition of type I and type III collagens.METHODS:The colocalizations of HSP27 and SNAI1/SNAI2 respectively on A549 alveolar epi-thelial cells induced by TGF-β1 were measured by confocal microscopy .The expression of HSP27, SNAI1 and SNAI2 at mRNA level was detected by real-time PCR.Western blotting analysis was used to detect the expression of HSP 27, SNAI1 and SNAI2 on epithelial-mesenchymal transition in A549 cells induced by TGF-β1 and also the deposition of type I and type III collagens in A549 cells transfected with HSP27shRNA prior to TGF-β1 stimulation.RESULTS: Compared with control group, TGF-β1 increased the expression of HSP27, SNAI1, SNAI2, type I and type III collagen, which decreased significantly followed by Ac-SDKP intervention.The expression of SNAI1, type I and type III collagen decreased signifi-cantly after transfected with HSP27shRNA in A549 cells, which had the similar effect on Ac-SDKP intervention.CON-CLUSION:Ac-SDKP inhibits the transition of cultured A 549 cells to myofibroblasts and attenuates collagen synthesis by suppressing the expression of HSP 27 and zinc finger proteins SNAI 1 and SNAI2.
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Objective To study the effects of the Notch ligands Dlk1 and recombinant human nucleu factorκB (Jagged1) on the proliferation and transdifferentiation of the type Ⅱ alveolar epithelial cells when the Notch signaling pathway activated.Methods The primary type Ⅱ alveolar epithelial cells (AEC Ⅱ) cultured with recombinant protein Dlk1 and recombinant human nucleu factor-κB (rhNF-κB) (activator of Jagged1),respectively,and then cultured with DMEM (containing 120 mL/L FBS) as controls.Proliferation and differentiation conditions of the AEC Ⅱ were observed at 48 h,72 h,96 h time point by the light microscope and electron microscopes separately.Cell number was counted with hemacytometer; the proliferation rate was measured by methyl thiazolyl tetrazolium (MTT) ; Immunofluorescence double standard method was used to detect the AEC Ⅱ specific surfactant protein C (SP-C) and AEC Ⅰ specific protein aquaporin5 (AQPS) ;the expression of SP-C,AQPS,Dlk1,Jagged1,Notch1 and Hes1 mRNA were detected by real time-PCR.Results The cell population and proliferation:compared with control group,AEC Ⅱ proliferation was promoted in the Dlk1 group [cell numbers (× 109/L) 9.05 ± 0.45 vs 7.95 ± 0.65,11.68 ± 0.43 vs 8.68 ± 0.52,11.55 ± 0.17 vs 8.73 ± 0.48,all P ＜ 0.05 ; MTT results (value A) 0.699 ± 0.050 vs 0.462 ± 0.080,0.912 ± 0.080 vs 0.535 ±0.040,0.726 ±0.050 vs 0.540 ±0.020,all P ＜0.05] and decelerated AEC Ⅱ transdifferentiation into AEC Ⅰ ; while AEC Ⅱ proliferation was inhibited in rhNF-κB group [cell numbers (× 109/L) 4.95 ± 0.33 vs 7.95 ± 0.65,4.73 ±0.71 vs 8.68 ± 0.52,4.04 ± 0.11 vs 8.73 ± 0.48,all P ＜ 0.05; MTT results (value A) 0.398 ± 0.030 vs 0.462 ± 0.080,0.402 ± 0.070 vs 0.535 ± 0.040,0.380 ± 0.110 vs 0.540 ± 0.020,all P ＜ 0.05] and accelerated AEC Ⅱ transdifferentiation into AEC Ⅰ.One-Way ANOVA showed that the difference among the 3 groups had statistical significance (cell numbers:F =486.73,P =0.02; cell proliferation:F =37.16,P =0.02).The mRNA expression:compared with control group,the expression of SP-C mRNA of Dlk1 group was significantly higher (P ＜ 0.05) while the expression of AQP5 mRNA was remarkably lower and delayed (P ＜ 0.05),the expression of Jagged1 mRNA was weak or little,Dlk1 and Notch1 mRNA were up-regulated (P ＜ 0.05),and the Hes1 mRNA was reduced (P ＜ 0.05) ; the expression of SP-C mRNA of rhNF-κB group was significantly reduced (P ＜ 0.05),while the AQP5 mRNA expressed ahead of time and increased (P ＜ 0.05),Jagged1,Hes1 and Notch1 mRNA were higher (P ＜ 0.05),and the Dlk1 mRNA was weak.One-Way ANOVA showed that the difference in the expressions of SP-C,AQP5,D1k1,Jagged1,Hes1 and Notch1 mRNA among the 3 groups had staistical significance (F =96.80,P =0.01 ; F =82.55,P =0.01 ; F =269.80,P=0.00;F =312.34,P =0.00;F =169.17,P =0.01;F =19.85,P =0.02).Conclusions There are varied effects on proliferation and differentiation of the AEC Ⅱ when the Notch signaling is activated by different ligands:Dlk1 promoted proliferation and inhibited differentiation,while Jagged1 inhibited proliferation and promoted transdifferentiation.