As Fig.?2 shows, no significant differences were found in the retention of MSC with GFP in the lung tissue between the MSC-GFP and MSC-ShHGF groups (green fluorescent protein, hepatocyte growth factor gene knockdown HGF levels in the lung after MSC treatment To examine the effect of MSC infusion on HGF levels in ALI rats, we measured the HGF concentration in the lung. permeability. The VE-cadherin was recognized with inmmunofluorescence, and the lung endothelial cell apoptosis was assessed by TUNEL assay. The severity of lung injury was evaluated using histopathology. The cytokines and HGF levels in the lung were measured by ELISA. Results MSC-ShHGF with markedly lower HGF manifestation were successfully constructed. Treatment with MSC or MSC transporting green fluorescent protein (MSC-GFP) managed HGF manifestation at relatively high levels in the lung at 24?h. MSC or MSC-GFP decreased the LWW/BW and the Evans Blue Dye extravasation, safeguarded adherens junction VE-cadherin, and reduced the lung endothelial cell apoptosis. Furthermore, MSC or MSC-GFP reduced the swelling and alleviated lung injury based on histopathology. However, HGF gene knockdown significantly decreased the HGF levels without any changes in the MSC retention in the lung, and diminished the protective effects of MSC within the hurt lung, indicating the restorative effects of MSC on ARDS were partly associated with the NPS-2143 (SB-262470) HGF-expressing NPS-2143 (SB-262470) character of MSC. Conclusions MSC restores lung permeability and lung injury in part by keeping HGF levels in the lung and the HGF-expressing character is required for MSC to protect the hurt lung. Electronic supplementary material The online version of this article (doi:10.1186/s13287-016-0320-5) contains supplementary material, which is available to authorized users. Top10 (GenePharma, Shanghai, China). The recombinant plasmid DNAs were extracted from Top10 and purified using the Plasmid Preparation Kit (GenePharma, Shanghai, China). The purity of the DNA was assessed having a spectrophotometer (Tecan, Switzerland). A260/A280 nm absorbance ratios of 1 1.8C2.2 suggested a pure DNA sample. Theses plasmids were then separately co-transfected with three packaging plasmids (pGag/Pol, pRev, pVSV-G) into 293?T cells using RNAi-mate (Genepharma, Shanghai, China) according to the manufacturers instruction. The lentiviral particles were collected and stored at C80?C for future use. Titer was acquired by GFP manifestation assay [13]. MSC were seeded and cultured in six-well plates for 24?h. The lentiviral vectors (transporting LV3-GFP or LV3-GFP ShRNA HGF) were then added to the wells at a multiplicity of illness (MOI) value of 100:1 and cultured with MSC for 24?h. After 24?h, the tradition medium was changed, and puromycin was added in the minimal lethal concentration (1.5?g/ml) for transfected MSC. The puromycin-resistant cells NPS-2143 (SB-262470) were then collected. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) MSCs treated with LV3-GFP (MSC-GFP) or LV3-GFP-ShRNA HGF (MSC-ShHGF) were collected, respectively. Total RNA was isolated from MSC, MSC-GFP or MSC-ShHGF using TRIzol reagent (Takara Bio, Inc., Kyoto, Japan) according to the manufacturers protocol. The quality of the RNA was assessed having a spectrophotometer (Tecan, Switzerland). 260/280?nm absorbance ratios of 1 1.8C2.2 suggested a pure RNA sample. The RT-PCR primers for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rat HGF (Table?1) were provided by GenePharma (Shanghai, China). RT-PCR assays were performed following a One-Step RT-PCR protocol explained by Funglyn Biotech Inc. (Shanghai, China). Table 1 The primer sequence of genes NPS-2143 (SB-262470) foundation pair, glyceraldehyde-3-phosphate dehydrogenase, hepatocyte growth factor, polymerase chain reaction European blotting analysis MSC, MSC-GFP, and MSC-ShHGF were collected GHR after transduction with lentiviral vector. Total cellular protein from either MSC, MSC-GFP, or MSC-ShHGF was extracted and separated using SDS-PAGE gels (10?%), as previously described [14]. Protein was then incubated with main antibodies to HGF (1:600 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or -actin (1:10,000 dilution; Abcam Ltd., Cambridge, UK). The blots were washed three times and then incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP; Zhongshan Golden Bridge Biotechnology Co., Ltd, China). Immunoreactive complexes were visualized using chemiluminescence reagents (Thermo Scientific). Evaluation of HGF levels by ELISA MSC, MSC-GFP, and MSC-ShHGF were seeded inside a 12-well plate at a denseness of 1 1??105 cells per well. After 12?h the tradition medium was changed, and MSC were cultured in an incubator at 37?C, 5?% CO2 for 24?h. The tradition medium was then collected and HGF protein.