Therapeutic effects of conditioned medium from bone marrow-derived mesenchymal stem cells on epithelial-mesenchymal transition in A549 cells.

Pulmonary fibrosis (PF) is a chronic lung disease. The transforming growth factor-ß1 (TGF-ß1)/Smad3 signaling pathway plays an important role in the pathogenesis of pulmonary fibrosis. Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to be a modulator of the molecular aspects of the fibrosis pathway. However, it is still unknown as to whether the conditioned medium from BMSCs (BMSCs-CM) inhibits the epithelial-mesenchymal transition (EMT) process. This study confirmed the hypothesis that BMSCs-CM exerts an anti-fibrotic effect on human type II alveolar epithelial cells (A549) by suppressing the phosphorylation of Smad3. We used the A549 cells in vitro to detect morphological evidence of EMT by phase-contrast microscopy. These cells were randomly divided into 4 groups as follows: the control group, the TGF-ß1 group, the SIS3 (specific inhibitor of Smad3) group and the BMSCs-CM group. The immunofluorescence method was used to determined the location of E-cadherin (E-calcium mucins; E-cad), α-smooth muscle actin (α-SMA) and p-Smad3. The expression levels of E-cad, CK8, α-SMA, vimentin, p-Smad3, Snail1, collagen I (COLI) and collagen III (COLIII) were detected by western blot analysis. Following exposure to TGF-ß1, the A549 cells displayed a spindle-shaped fibroblast-like morphology. In accordance with these morphological changes, the expression levels of E-cad and CK8 were downregulated, while the expression levels of α-SMA and vimentin were upregulated. Along with this process, the expression levels of p-Smad3, Snail1, COLI and COLIII were increased. However, the cells in the BMSCs-CM group and SIS3 group exhibited a decrease in the levels of α-SMA and vimentin (which had been upregulated by TGF-ß1), and an increase in the levels of E-cad and CK8 expression (which had been downregulated by TGF-ß1). On the whole, these results indicated that BMSCs-CM suppressed the EMT which might be associated with TGF-ß1/Smad3. This study provides the theoretical basis for the research of the mechanisms responsible for pulmonary disease.

Effects of bone marrow-derived mesenchymal stem cells on the autophagic activity of alveolar macrophages in a rat model of silicosis.

The aim of the present study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) on the expression of the autophagy-associated proteins, microtubule-associated protein light chain 3 (LC-3) and autophagy-related gene Beclin-1 (Beclin-1), in alveolar macrophages (AMs) in a rat model of silicosis. Furthermore, the study investigated the molecular mechanisms underlying the effects of BMSC treatment. A population of 60 adult female Sprague-Dawley (SD) rats were allocated at random into three groups, namely the control, model and BMSC treatment groups (n=20 per group). BMSCs were isolated from five male SD rats (age, 6-8 weeks) and cultured in vitro. The silicosis model was established using a single 1.0-ml infusion of silicon dioxide suspension administered via non-exposed tracheal intubation. Rats in the BMSC treatment group received a 1.0-ml transplantation of BMSCs (1×106/ml). The rats were sacrificed on days 1, 7, 14 and 28 after modeling, and AMs were extracted from the rats using bronchoalveolar lavage. Third-generation BMSCs were identified using flow cytometry with fluorescein isothiocyanate staining, and the morphological characteristics of the AMs were observed using hematoxylin and eosin staining. The expression levels of LC-3 and Beclin-1 were determined using immunocytochemistry sand western blot analysis. The expression levels of LC-3 and Beclin-1 were found to be increased at all the time points in the model group. LC-3 and Beclin-1 levels began to increase at day 1, peaked at day 14 and decreased after day 28; however, the levels remained elevated compared with the basal expression levels. The AMs of the BMSC treatment group exhibited significantly alleviated pathological symptoms compared with the model group AMs, as indicated by significantly decreased expression levels of LC-3 and Beclin-1 at each time point. Therefore, the results indicated that autophagy was promoted in the AMs of the silicosis model rats. Furthermore, treatment with BMSCs was demonstrated to reduce the expression levels of LC-3 and Beclin-1, subsequently inhibiting autophagic activity and mitigating the damage associated with silicosis.

Neuroprotective effects of brilliant blue G on the brain following traumatic brain injury in rats.

The P2X7 inhibitor, brilliant blue G (BBG), has been reported as a neuroprotective drug against a variety of disorders, including neuropathic pain and brain ischemia. Currently, no studies have examined the potential for BBG to provide neuroprotection in animal models of TBI. The aim of the present study was to investigate the neuroprotective effect of BBG on TBI and to determine the underlying mechanisms. The rats were subjected to a diffuse cortical impact injury caused by a modified weight-drop device, and then divided randomly into three groups: the sham-operated, BBG treatment and vehicle groups. In the BBG treatment group, 50 mg/kg brilliant blue G (BBG; 100% pure), a highly specific and clinically useful P2X7 antagonist, was administered via the tail vein 15 min prior to or up to 8 h following TBI. The co-localization of NeuN and protein kinase Cγ (PKCγ) was followed with immunofluorescent staining. The expression of P2X7, PKCγ and inflammatory cytokines was identified by western blot analysis. Wet-dry weight method was used to evaluate brain edema, and motor function outcome was examined using the neurological severity score. The present study demonstrated that the administration of BBG attenuated TBI-induced cerebral edema and the associated motor deficits. Following trauma, BBG treatment significantly reduced the levels of PKCγ and interleukin-1ß in the cortex. The results provide in vivo evidence that BBG exerted neuroprotective effects by attenuating brain edema and improving neurological functions via reducing PKCγ and interleukin-1ß levels following TBI.