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.

[Regulation of organic anion transporting polypeptides expression and activity].

Organic anion transporting polypeptides (OATP), a member of solute carrier (SLC) superfamily, is considered as an important transmembrane uptake transporters. OATP is involved in the transport of a variety of endo- and xenobiotics (bile acids, bilirubin, prostaglandin, thyroid hormones, steroid hormone conjugates), drugs and toxins in a Na+ and ATP independent manner. Multiple factors (eg. hormones, proinflammatory cytokines, drugs) can affect the distribution, expression and activity of OATPs, leading to an altered accumulation of OATP substrates and related food-drug and drug-drug interactions. Changes in the distribution and expression of OATPs in malignant tissues may be related to the pathological process of cancer, while the modulation epigenetic mechanism also contributes to its distribution patterns. This review describes the factors that can affect the expression or function of OATPs, which may provide a valuable reference for drug development and the clarification of pathogenesis.

Hydrogen sulfide targets EGFR Cys797/Cys798 residues to induce Na(+)/K(+)-ATPase endocytosis and inhibition in renal tubular epithelial cells and increase sodium excretion in chronic salt-loaded rats.

AIMS: The role of hydrogen sulfide (H2S) in renal sodium and water homeostasis is unknown. We investigated whether H2S promoted Na(+)/K(+)-ATPase endocytosis via the H2S/EGFR/gab1/PI3K/Akt pathway in renal tubular epithelial cells. RESULTS: H2S decreased Na(+)/K(+)-ATPase activity and induced its endocytosis in renal tubular epithelial cells, which was abrogated by small interfering RNA (siRNA) knockdown of epidermal growth factor receptor (EGFR) and gab1, a dominant-negative mutant of Akt and PI3K inhibitors. H2S increased EGFR, gab1, PI3K, and Akt phosphorylation in both renal tubular epithelial cells and kidneys of chronic salt-loaded rats. These increases were abrogated by siRNA knockdown of EGFR, but not of c-Src. Radiolabeled H2S exhibited transient, direct binding to EGFR and directly activated EGFR. Some disulfide bonds in EGFR intracellular kinase domain were susceptible to H2S-induced cleavage. Mutations of EGFR Cys797 (human) or Cys798 (rat) residues increased EGFR activity and prevented H2S-induced Na(+)/K(+)-ATPase endocytosis. H2S also inhibited sodium hydrogen exchanger-3 (NHE3) activity in renal tubular epithelial cells. H2S treatment increased sodium excretion in chronic and acute salt-loaded rats and decreased blood pressure in chronic salt-loaded rats. INNOVATION AND CONCLUSION: H2S directly targets some disulfide bonds in EGFR, which activates the EGFR/gab1/PI3K/Akt pathway and subsequent Na(+)/K(+)-ATPase endocytosis and inhibition in renal tubular epithelial cells. EGFR Cys797/Cys798 residues are essential for an intrinsic inhibitory mechanism and for H2S actions in renal tubular epithelial cells. Other pathways, including NHE3, may be involved in mediating the renal effects of H2S. Our results reveal a new renal sodium homeostasis mechanism, which may provide for novel treatment approaches for diseases related to renal sodium homeostasis dysfunction.

Therapeutic effect of exogenous bone marrow­derived mesenchymal stem cell transplantation on silicosis via paracrine mechanisms in rats.

Silicosis is a well-known occupational disease, characterized by epithelial injury, fibroblast proliferation, expansion of the lung matrix and dyspnea. At present, no effective treatment methods for silicosis have been identified. The present study aimed to investigate the protective potential of exogenous bone marrow-derived mesenchymal stem cell (BMSC) transplantation on experimental silica-induced pulmonary fibrosis in rats and analyze the underlying paracrine mechanisms associated with its therapeutic effects. BMSCs were isolated, cultured and passaged from male Sprague-Dawley (SD) rat bone marrow. Third-generation BMSCs were identified by flow cytometry using FITC staining. Following the successful establishment of the silicosis model, exogenous BMSCs were infused into female adult SD rats via the tail vein. Lungs were evaluated using hematoxylin and eosin (H&E) staining. The expression of interleukin-1 receptor antagonist (IL­1RA), interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) protein was detected by immunohistochemistry and western blot analysis. Co-localization of sex determining region Y (SRY) and IL-1RA expression was determined by double-label immunofluorescence. The distribution of transplanted BMSCs was tracked by monitoring the expression of SRY in rats. Treatment with BMSCs was found to protect the lungs against injury and fibrosis by the suppression of upregulated IL-1 and TNF-α protein, via triggering IL-1RA secretion. This mechanism was hypothesized to be mediated by paracrine signaling. These results indicate that the release of IL­1RA from BMSCs via paracrine mechanisms significantly blocks the production and/or activity of IL-1 and TNF-α. The present study provides an experimental basis for cellular therapy in silicosis.

[Impact of H2S on oxidized-low density lipoprotein-stimulated nuclear factor-κB in human monocytes/macrophage and its mechanisms].

OBJECTIVE: To investigate the role of hydrogen sulfide (H2S) on oxidized-low density lipoprotein (ox-LDL)-stimulated NF-κB pathway in human monocytes/macrophages and its mechanisms. METHODS: THP-1 cells were induced to differentiate into macrophages by incubation with phorbol myristate acetate (PMA) . The human monocytes/macrophages were divided into 4 groups: control group, ox-LDL group, ox-LDL + H2S 100 µmol/L group and ox-LDL + H2S 500 µmol/L group. The expression of IκBα and phosphorylation of NF-κB p65 in the cells were detected by Western blotting. The expression of IκBα and nuclear translocation of NF-κB p65 in the cells were observed by laser confocal method. The interaction between NF-κB p65 and IκBα in the nuclear extracts was detected by coimmunoprecipitation method. RESULTS: Compared with the control group, the phosphorylation of NF-κB p65 in the human monocytes/macrophages of ox-LDL group was increased significantly (0.855 ± 0.116 vs. 0.502 ± 0.218, P=0.046), while the expression of IκBα in the cells of the ox-LDL group was decreased (0.612 ± 0.216 vs. 0.997 ± 0.167, P=0.029). Compared with the ox-LDL group, the phosphorylation of NF-κB p65 in the cells of the ox-LDL + H2S 100 µmol/L group and the ox-LDL + H2S 500 µmol/L group was decreased significantly (0.424 ± 0.225 vs. 0.855 ± 0.116, P=0.020; 0.378 ± 0.071 vs. 0.855 ± 0.116, P=0.011, respectively), while the expressions of IκBα in the cells of the ox-LDL + H2S 100 µmol/L group and the ox-LDL + H2S 500 µmol/L group were increased (1.037 ± 0.111 vs. 0.612 ± 0.216, P=0.015; 1.046 ± 0.084 vs. 0.612 ± 0.216, P=0.013, respectively). The results from laser confocal method demonstrated that the IκBα expression in the cytoplasma of cells in the ox-LDL group was lower than that in the control group, and the nuclear translocation of NF-κB p65 in the cells of the ox-LDL group was higher than that in the control group. The IκBα expression in the cytoplasma of cells in the ox-LDL+ H2S 100 µmol/L group and ox-LDL + H2S 500 µmol/L group was higher than that in the ox-LDL group, and the nuclear translocation of NF-κB p65 in the cells of ox-LDL group was lower than that in the ox-LDL group. The coimmunoprecipitation experiment showed that no IκBα integrated with NF-κB p65 was detected in the nuclear extracts of cells in the control group, a small amount of IκBα integrated with NF-κB p65 was detected in the nuclear extracts of cells in the ox-LDL group, but a large amount of IκBα integrated with NF-κB p65 was detected in the nuclear extracts of cells in the ox-LDL+H2S 100 µmol/L group and the ox-LDL + H2S 500 µmol/L group. CONCLUSION: H2S inhibited the activation of NF-κB p65 pathway in the ox-LDL-induced human monocytes/macrophages. The mechanisms might involve the prevention of the degradation of IκBα, then the inhibition of the phosphorylation and nuclear translocation of NF-κB p65, thus promoting the IκBα integrated with NF-κB p65 in the nuclei, and then inhibiting the activity of NF-κB.

The PI3K/Akt pathway mediates the protection of SO(2) preconditioning against myocardial ischemia/reperfusion injury in rats.

AIM: To explore the mechanisms underlying the protection by SO2 preconditioning against rat myocardial ischemia/reperfusion (I/R) injury. METHODS: Male Wistar rats underwent 30-min left coronary artery ligation followed by 120-min reperfusion. An SO2 donor (1 µmol/kg) was intravenously injected 10 min before the ischemia, while LY294002 (0.3 mg/kg) was intravenously injected 30 min before the ischemia. Plasma activities of LDH and CK were measured with an automatic enzyme analyzer. Myocardial infarct size was detected using Evans-TTC method. The activities of caspase-3 and -9 in myocardium were assayed using a commercial kit, and the levels of p-Akt, Akt, PI3K and p-PI3K were examined with Western blotting. RESULTS: Pretreatment with SO2 significantly reduced the myocardial infarct size and plasma LDH and CK activities, as well as myocardial caspase-3 and -9 activities in the rats. Furthermore, the pretreatment significantly increased the expression levels of myocardial p-Akt and p-PI3K p85. Administration of the PI3K inhibitor LY294002 blocked all the effects induced by SO2 pretreatment. CONCLUSION: The results suggest that the PI3K/Akt pathway mediates the protective effects of SO2 preconditioning against myocardial I/R injury in rats.

Hydrogen sulfide treatment promotes glucose uptake by increasing insulin receptor sensitivity and ameliorates kidney lesions in type 2 diabetes.

AIMS: To examine if hydrogen sulfide (H2S) can promote glucose uptake and provide amelioration in type 2 diabetes. RESULTS: Treatment with sodium hydrosulfide (NaHS, an H2S donor) increased glucose uptake in both myotubes and adipocytes. The H2S gas solution showed similar effects. The NaHS effects were blocked by an siRNA-mediated knockdown of the insulin receptor (IR). NaHS also increased phosphorylation of the IR, PI3K, and Akt. In Goto-Kakizaki (GK) diabetic rats, chronic NaHS treatment (30 µmol·kg(-1)·day(-1)) decreased fasting blood glucose, increased insulin sensitivity, and increased glucose tolerance with increased phosphorylation of PI3K and Akt in muscles. Similar insulin-sensitizing effects of NaHS treatment were also observed in Wistar rats. Moreover, glucose uptake was reduced in the cells with siRNA-mediated knockdown of the H2S-generating enzyme cystathionine γ-lyase in the presence or absence of exogenous H2S. Moreover, chronic NaHS treatment reduced oxygen species and the number of crescentic glomeruli in the kidney of GK rats. INNOVATION AND CONCLUSION: This study provides the first piece of evidence for the insulin-sensitizing effect of NaHS/H2S in the both in vitro and in vivo models of insulin resistance. REBOUND TRACK: This work was rejected during a standard peer review and rescued by the Rebound Peer Review (Antoxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Jin-Song Bian, Samuel Dudley, Hideo Kimura, and Xian Wang.