Objective To explore the migration and differentiation of bone marrow mesenchymal stem cells（MSCs） in lung . Methods MSCs were harvested from a male Wister rat. Sixty female Wister rats were randomly divided into four groups. The pulmonary fibrosis model was established by intratracheal instillation of bleomycin in group A-D. Immediately and 7 days after bleomycin administration respectively，the rats in group B and C received infusion with 5-bromodeoxynridine （BrdU） labeled MSCs via tail vein. And the rats in group D were infused MSCs without BrdU labeling serving as a negative control. The sry gene of Y chromosome was detected by polymerase chain reaction （PCR）. Double immunofluorescence staining was used to detected BrdU and surfactant associated protein-C （SP-C） expression in lung tissue，fresh bone marrow，and the 5th generation MSCs. Reverse transcriptipon-PCR was used to detect the expressions of SP-C mRNA and AQP-5 mRNA. Results The sry gene was detected in bleomycin induced lung injury tissues of the rats after MSCs infusion immediately and on the 7th day The MSCs in lung tissue could transformed into cells with ACEⅡ morphological features and molecular phenotype. The transformation rate was higher in the rats received MSCs infusion immediately than the rats received on 7th day. The 5th generation MSCs and fresh bone marrow expressed SP-C mRNA，without AQP-5 mRNA and SP-C expression. Conclusions Exogenous MSCs can be transplanted into injured lung tissues and transform into AECⅡ，especially in early stage of lung injury. The differentiation potential of MSCs can be activated in injury micro-environment.
Objective To explore whether microRNA-203 (miR-203) targets and regulates the Toll-like receptor 4 (TLR4)/nuclear transcription factor kappa B (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) to protect alveolar epithelial cells from lipopolysaccharide (LPS)-induced apoptosis and inflammation injury. Methods The alveolar epithelial A549 cells were used as the research objects and divided into: Control group (normal culture), LPS group (LPS treatment), LPS+miR-NC mimics group (LPS treatment after transfection of miR-NC mimics), LPS+ miR-203 mimics group (LPS treatment after transfection of miR-203 mimics), LPS+miR-203 mimics+pcDNA group (LPS treatment after transfection of miR-203 mimics and pcDNA), LPS+miR-203 mimics+pcDNA-TLR4 group (LPS treatment after transfection of miR-203 mimics and pcDNA-TLR4). Dual luciferase reporter gene was used to detect the targeting relationship between miR-203 and TLR4; Real-time quantitative reverse transcription-polymerase chain reaction was used to detect the relative expression levels of miR-203 and TLR4 mRNA; enzyme-linked immunosorbent assay was used to measure the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6; flow cytometry was used to detect the apoptosis rate of A549 cells; Western blot was used to detect the expression of B-cell lymphoma/leukemia-2 gene (Bcl-2) and Bcl-2 associated X protein (Bax), TLR4, NF-κB and NLRP3 proteins in A549 cells. Results There was a targeted regulation relationship between miR-203 and TLR4. Compared with the Control group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the level of Bcl-2 protein in cells decreased (P<0.05). Compared with the LPS+miR-NC mimics group, the expression of TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics group decreased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant decreased, the apoptosis rate decreased, the expression level of miR-203 and the level of Bcl-2 protein in cells increased (P<0.05). Compared with the LPS+miR-203 mimics+pcDNA group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics+pcDNA-TLR4 group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the expression level of miR-203 and the level of Bcl-2 protein in cells decreased (P<0.05). Conclusion MiR-203 can target TLR4/NF-κB/NLRP3 to protect alveolar epithelial cells from apoptosis and inflammation induced by LPS.
Objective To investigate the preventive therapeutic effects of CPD1, a novel phosphodiesterase 5 inhibitor, on lung pathological phenotype and interstitial fibrosis of lung in pulmonary fibrosis model rats caused by bleomycin (BLM). Methods Rats were randomly divided into a sham surgery group (n=10), a model group (n=14), a CPD1 treatment group (n=13), and a nintedanib positive control drug treatment group (n=13). Pulmonary fibrosis model was constructed by slowly instilling BLM (3 mg/kg) into the left bronchus in the model group and two treatment groups. Two hours after BLM infusion, the rats were treated with CPD1 (20 mg·kg–1·d–1), or positive control drug nintedanib (50 mg·kg–1·d–1) by intragastric administration, respectively, for 2 weeks. To observe the effect of CPD1 treatment on pathological structural damage, collagen deposition, and the expression of fibronectin (Fn), α-smooth muscle actin (α-SMA), Collagen Ⅰ, and E-cadherin (E-Cad) in the affected lung tissues of unilateral pulmonary fibrosis rats. Moreover, to further observed the effects of CPD1 intervention on the expression of transforming growth factor β1 (TGF-β1) and Smad3 in the cell model of human alveolar basal epithelial A549 cells. Results Compared with the control group, the lung tissue structure was seriously damaged in the BLM group, and with expansion of the alveolar space, collapse of the alveolar lumen. Significant widening of the alveolar septum and thickening of the alveolar wall were observed in the BLM group. There was a marked increase in collagen deposition in the thickened walls of the BLM group. Moreover, the expressions of Fn, α-SMA, Collagen Ⅰ, TGF-β and Smad3 were increased, while the expression of E-Cad significantly decreased in the BLM group (all P<0.05). Compared with the BLM group, the lung tissue damage was significantly improved in the CPD1 group rats. Furthermore, CPD1 inhibit the expression of Fn, α-SMA, Collagen Ⅰ, TGF-β and Smad3, and upregulate the expression of E-Cad (all P<0.05). Conclusions Prophylactic treatment with phosphodiesterase 5 inhibitor CPD1 strongly attenuates BLM-induced pulmonary fibrosis by inhibiting the lung injury and inflammation response via targeting TGF-β/Smad pathway, reducing the deposition of extracellular matrix.
Objective To investigate the effects of wedelolactone (WEL) on lipopolysaccharide (LPS)-induced pyroptosis of alveolar epithelial cells and AMP-activated protein kinase/nucleotide binding oligomeric domain like receptor 3 (NLRP3)/cysteinyl aspartate specific proteinase-1 (Caspase-1) signaling pathway. Methods Human lung epithelial cells BEAS-2B were treated with 5 - 200 μmol/L wedelolactone, and cell activity was detected using MTT assay. The alveolar epithelial cells were divided into control group, lipopolysaccharide group (LPS group), 10 μmol/L wedelolactone group (WEL-L group), 20 μmol/L wedelolactone group (WEL-M group), 40 μmol/L wedelolactone group (WEL-H group), 40 μmol/L wedelolactone+10 μmol/L AMPK inhibitor Compound C group (WEL-H+Compound C group), and 20 μmol/L Caspase-1 inhibitor Z-YVAD-FMK group (Z-YVAD-FMK group). Transmission electron microscopy was applied to observe the microstructure of cells. ELISA was applied to detect levels of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-8 (IL-8). Immunofluorescence was applied to detect Caspase-1 and gasdermin family proteins (DGSDMD). Western blot was applied to detect protein expression levels of AMPK, NLRP3, and Caspase-1. Results Wedelolactone concentrations of 10, 20 and 40 μmol/L were selected for follow-up experiments. Compared with Control group, LPS group showed decreased cell activity, severe damage, cell contraction, mitochondrial ridge breakage and decreased number, increased levels of TNF-α, IL-1β, IL-8 and GSDMD, NLRP3, Caspase-1 expression, and decreased p-AMPK/AMPK expression (P<0.05). Wedelolactone treatment could significantly improve LPS-induced pyrosis of alveolar epithelial cells (P<0.05). Compound C could partially reverse the effect of wedelactone on LPS-induced pyrodeath of alveolar epithelial cells (P<0.05). Z-YVAD-FMK treatment also significantly improved LPS-induced pyroptosis of alveolar epithelial cells (P<0.05). Conclusion Wedelolactone can inhibit LPS-induced pyroptosis of pulmonary alveolar epithelial cells by inhibiting AMPK/NLRP3/Caspase-1 signaling pathway.