RESUMEN
Background: In our studies, it was shown that the effectiveness of the conditioned medium obtained by cultivating mesenchymal stem cells depends on the microenvironment conditions used to cultivate the cells. It was demonstrated that the conditioned medium obtained by culturing cells with low oxygen content (10%) has a much more pronounced protective effect. Methods: Protein compositions obtained from MSCs cultured under hypoxic (10% O2 hc-MSC) and normal (21% O2 nc-MSC) conditions were used to treat acute liver failure (ALF) induced in mice by acetaminophen injection. Thus, we obtained fractions normalized by volume, which predominantly contained proteins with masses > 50, 50-30, 30-10, and 10-3 kDa. Results: The data from biochemical studies have shown that only fractions from 10 to 30 kDa (hcMSC and ncMSC) significantly reduced the level of liver enzymes in the beginning of the acute period after acetaminophen administration. Mass spectrometry analysis of the proteins contained in the isolated fractions showed a sharp increase in the protein levels in the 10-30 kDa hcMSC fraction as compared with that in 10-30 kDa ncMSCs. The composition obtained from MSCs cultured at lower O2 level (fraction 10-30 kDa hcMSC) was shown to be more potent than the composition prepared from normoxic cells. Conclusion: The results have shown that a composition obtained by culturing the cells under a reduced content of O2 (10%), significantly improves the biochemical parameters, and histological arrester reduces the degree of inflammation and stimulates regenerative processes in liver, compared to both the control group and group treated with the composition that was obtained by culturing the cells under normal oxygen content.
RESUMEN
Chronic overuse of common pharmaceuticals, e.g. acetaminophen (paracetamol), often leads to the development of acute liver failure (ALF). This study aimed to elucidate the effect of cultured mesenchymal stem cells (MSCs) proteome on the onset of liver damage and regeneration dynamics in animals with ALF induced by acetaminophen, to test the liver protective efficacy of MSCs proteome depending on the oxygen tension in cell culture, and to blueprint protein components responsible for the effect. Protein compositions prepared from MSCs cultured in mild hypoxic (5% and 10% O2) and normal (21% O2) conditions were used to treat ALF induced in mice by injection of acetaminophen. To test the effect of reduced oxygen tension in cell culture on resulting MSCs proteome content we applied a combination of high performance liquid chromatography and mass-spectrometry (LC-MS/MS) for the identification of proteins in lysates of MSCs cultured at different O2 levels. The treatment of acetaminophen-administered animals with proteins released from cultured MSCs resulted in the inhibition of inflammatory reactions in damaged liver; the area of hepatocyte necrosis being reduced in the first 24 h. Compositions obtained from MSCs cultured at lower O2 level were shown to be more potent than a composition prepared from normoxic cells. A comparative characterization of protein pattern and identification of individual components done by a cytokine assay and proteomics analysis of protein compositions revealed that even moderate hypoxia produces discrete changes in the expression of various subsets of proteins responsible for intracellular respiration and cell signaling. The application of proteins prepared from MSCs grown in vitro at reduced oxygen tension significantly accelerates healing process in damaged liver tissue. The proteomics data obtained for different preparations offer new information about the potential candidates in the MSCs protein repertoire sensitive to oxygen tension in culture medium, which can be involved in the generalized mechanisms the cells use to respond to acute liver failure.
Asunto(s)
Acetaminofén/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/terapia , Medios de Cultivo Condicionados/metabolismo , Fallo Hepático Agudo/etiología , Fallo Hepático Agudo/terapia , Células Madre Mesenquimatosas/metabolismo , Proteoma , Animales , Biomarcadores , Biopsia , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Hipoxia/metabolismo , Fallo Hepático Agudo/patología , Masculino , Espectrometría de Masas , Células Madre Mesenquimatosas/citología , Ratones , Consumo de Oxígeno , Sustancias Protectoras/metabolismo , Sustancias Protectoras/farmacología , Proteómica/métodosRESUMEN
BACKGROUND: Mesenchymal stem cells based paracrine bioactive factors that deploy their task as an essential mechanism, but their efficiency for skin regeneration still requires clarification. METHODS: The mesenchymal stem cell-based paracrine factors were administered by subcutaneous injection of 0.5 mL peptides (general protein 8 mg/mL). These were performed after radiation on different days like the first, third, sixth, eighth, and 10th. To determine the consequences, we performed photography, planimetry, and preclinical test each week after 15 days of radiation. MSC-based peptides were injected into a rat that had radiation burns, and its observation encouraged cell-free therapeutic remedies to regenerate skin. Both control and experimental groups were exposed to 110 Gy of X-rays, which resulted in the formation of localized radiation burns on the skin (S=6 cm2) 15 days later. Thirty days after radiation, the wound stabilized (surface of the wound was S=2.2±0.2 cm2) and fluctuated throughout the course of the pathological process. RESULTS: The wounded area on the skin from the 15th to the 29th day after radiation was practically the same in both groups. The wounded area gradually reduced by 6.1±0.4 cm2 (experimental group) and 5.9±0.6 cm2 (control group) 15 days after radiation up to 2.2±0.3 cm2 (in both control and experimental groups) on the 29th day after radiation. However, starting from the 36th day, there was a constant reduction in the burn area in the experimental group up to 0.2±0.1 cm2 till the 71st day after radiation. CONCLUSION: In the control group, the area of the lesion ranged from 1.4±0.6 cm2 on the 50th day to 1.9±0.8 cm2 on the 71st day. During the 57th to the 71st day, the difference between the affected area in the experimental and control groups was 1:8. The experimental group has a significantly higher level of skin regeneration and significant decrease in the level of leukocyte infiltration, thereby reducing necrosis.
RESUMEN
Ischemia/reperfusion (I/R) injury of the small intestine caused by occlusion of the superior mesenteric artery affects the intestinal tissue as well as components of the blood circulatory system from the microvasculature to mesenteric vessels. The aim of this work was to study the correlation between the dynamics of destruction development in the intestinal tissue, microvasculature, and mesenteric vessels in I/R of the small intestine. The microvasculature was analyzed by whole-organ continuous monitoring of the intestinal mucosal blood perfusion by laser Doppler flowmetry during the entire I/R. Real-time RT-PCR was used to assess gene expression of NF-κB, caspase-3, Ki67, and TNF-α in blood vessels. At the start of reperfusion, the first targets to be disrupted are microvessels in the apical villi. Injury of the apical part of the microcirculatory bloodstream correlates with the reduction in intestinal mucosal blood perfusion, which occurred simultaneously with apical villous destruction. By the end of the reperfusion period, the low intestinal mucosal blood perfusion is mirrored by the destruction of the microvasculature and mucosal structures in the entire organ. The development of mesenteric vessel injury is characterized by a change in NO metabolism and damaged endothelial cells concomitant with an alteration in the expression of genes encoding NF-κB, caspase-3, and Ki67 by the end of the reperfusion period. In I/R injury, detrimental effects on the intestinal tissue, microvasculature, and mesenteric vessels develop and exhibit common mechanisms of function, which show strong correlations.
Asunto(s)
Intestino Delgado/irrigación sanguínea , Intestino Delgado/patología , Daño por Reperfusión/patología , Animales , Apoptosis/genética , Vasos Sanguíneos/patología , Diferenciación Celular/genética , Proliferación Celular/genética , Intestino Delgado/fisiopatología , Masculino , Microcirculación , Microscopía Fluorescente , Óxido Nítrico/metabolismo , Ratas Wistar , Flujo Sanguíneo Regional , Daño por Reperfusión/fisiopatologíaRESUMEN
The purpose of this review is to systematize data from many studies and observations of proliferative, anti-apoptotic and anti-inflammatory effects of mesenchymal stromal cell (MSC) paracrine factors and their biologic effects in models of acute organ injury.