Non-muscle myosin II knockdown improves survival and therapeutic effects of implanted bone marrow-derived mesenchymal stem cells in lipopolysaccharide-induced acute lung injury

Abstract

Background: Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to have some beneficial effects in acute lung injury (ALI), but the therapeutic effects are limited due to apoptosis or necrosis after transplantation into injured lungs. Here, we aim to explore whether Non-muscle myosin II (NM-II) knockdown could enhance BMSCs survival and improve therapeutic effects in ALI.

Methods: MSCs, isolated from rat bone marrow, were transfected with the small interfering RNA (siRNA) targeted to NM-II mRNA by a lentivirus vector. Rats were equally randomized to four groups: the control group was given normal saline via tail vein; the other three groups underwent intratracheal lipopolysaccharide (LPS) instillation followed by administration with either normal saline, BMSCs transduced with lentivirus-enhanced green fluorescent protein (eGFP) empty vector, or BMSCs transduced with lentivirus-eGFP NM-II siRNA. Hematoxylin and eosin staining was used to evaluate lung histopathologic changes and Masson trichrome staining was used to assess lung fibrosis. The myeloperoxidase activity was also tested in lung tissues. The mRNA expression of inflammatory cytokines in lung tissues was determined via quantitative reverse transcription PCR. Sex-determining region of the Y chromosome gene expression was measured by fluorescence in situ hybridization (FISH) assay. The expression of self-renewal activity and apoptosis-associated proteins were measured by Western blot.

Results: Transplantation of NM-II siRNA-modified BMSCs could improve histopathological morphology, decrease inflammatory infiltrates, down-regulate the expression levels of inflammatory cytokines, and reduce pulmonary interstitial edema. NM-II siRNA-modified BMSCs showed antifibrotic properties and alleviated the degrees of pulmonary fibrosis induced by endotoxin. In addition, NM-II knockdown BMSCs showed slightly better therapeutic effect on lung inflammation when compared with control BMSCs. The beneficial effects of NM-II siRNA-modified BMSCs may be attributed to enhanced self-renewal activity and decreased apoptosis.

Conclusions: NM-II knockdown could inhibit the apoptosis of implanted BMSCs in lung tissues and improve its self-renewal activity. NM-II siRNA-modified BMSCs have a slightly enhanced ability to attenuate lung injury after LPS challenge.

Keywords: Bone marrow-derived mesenchymal stem cells (BMSCs); acute lung injury (ALI); gene therapy; non-muscle myosin II (NM-II).

Figure 1

Phenotype verification of primary BMSCs by flow cytometry for expression of multiple CD antigens. BMSC, bone marrow-derived mesenchymal stem cell.

Figure 2

Assessment of transduction efficiency by detecting NM-II expression with western blotting. Representative of three independent experiments (*, P<0.05, vs. BMSCs^ctrl). BMSC, bone marrow-derived mesenchymal stem cell.

Figure 3

LPS treatment produced alveolar wall thickening, neutrophil infiltration, and occasional alveolar hemorrhage. The inflammatory histologic changes produced by LPS in lung parenchyma were significantly attenuated after treatment with either BMSCs^ctrl or BMSCs^{NM-II KD}. A significant amelioration in histopathologic changes was achieved by treatment with BMSCs^{NM-II KD} relative to those seen after BMSCs^ctrl treatment. Histopathological features were tested by hematoxylin and eosin staining on day 1 after treatment, ×200 (A). Pathological score was measured (B). Data are presented as the mean ± SE. n=4 in each group. **, P<0.01 vs. control; ^#, P<0.05, ^##, P<0.01 vs. ALI; ^&, P<0.05 vs. BMSCs^ctrl. ALI, acute lung injury; BMSC, bone marrow-derived mesenchymal stem cell.

Figure 4

MPO activity (A), mRNA expression of inflammatory cytokines (B), concentrations of inflammatory cytokines in BAL fluids (C) and total cells counts in BAL fluids (D) were measured on day 1 after treatment. Data are presented as the mean ± SE. n=4 in each group. **, P<0.01 vs. control; ^#, P<0.05, ^##, P<0.01 vs. ALI; ^&, P<0.05 vs. BMSCs^ctrl. MPO, myeloperoxidase; BAL, bronchoalveolar lavage; BMSC, bone marrow-derived mesenchymal stem cell.

Figure 5

Both BMSCs^ctrl and BMSCs^{NM-II KD} treatments markedly reduced the collagen deposition observed in LPS exposed rats. Lung fibrosis was measured at 21 days after treatment by Masson trichrome after staining, ×200 (A). The degree of lung fibrosis was scored (B). Data are presented as the mean ± SE. n=4 in each group. **, P<0.01 vs. control; ^#, P<0.05, ^##, P<0.01 vs. ALI.

Figure 6

SRY (sex-determining region of the Y chromosome) gene expression was measured by FISH at 24 hours, 7 days and 14 days after treatment.

Figure 7

NM-II knockdown inhibited the decreased of BMSCs viability. Nanog and Ang-1 were significantly upregulated in BMSCs upon NM-II knockdown, while the activity of Caspase-3 was decreased in BMSCs upon NM-II knockdown. Cell viability was measured by CCK8 assay (A). Expression of self-renewal and apoptosis-associated proteins were evaluated by western blotting (B). Representative of three independent experiments. Relative protein levels were quantified by densitometry and expressed as optical density ratio with GAPDH serving as internal standards (C). *, P<0.05, **, P<0.01 vs. control.