RESUMEN
While insulin is an anabolic hormone, AMP-activated protein kinase (AMPK) is not only a key energy regulator, but it can also control substrate metabolism directly by inducing skeletal muscle protein degradation. The hypothesis of the present study was that insulin inhibits AMPK and thus down-regulates the expression of the ubiquitin E3 ligases, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in skeletal muscle cells. Differentiated L6 myotubes were treated with 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside (AICAR) and/or compound C to stimulate and/or block AMPK respectively. These treatments were also conducted in the presence or absence of insulin and the cells were analysed by western blot and quantitative real-time PCR. In addition, nucleotide levels were determined using HPLC. The activation of AMPK with AICAR enhanced the mRNA levels of MAFbx and MuRF1. Insulin reduced the phosphorylation and activity AMPK, which was accompanied by reduced MAFbx and MuRF1 mRNA levels. Using a protein kinase B (PKB/Akt) inhibitor, we found that insulin regulates AMPK through the activation of Akt. Furthermore, insulin down-regulated AMPK α2 mRNA. We conclude that insulin inhibits AMPK through Akt phosphorylation in L6 myotubes, which may serve as a possible signalling pathway for the down-regulation of protein degradation. In addition, decreased expression of AMPK α2 may partially participate in inhibiting the activity of AMPK.
Asunto(s)
Proteínas Quinasas Activadas por AMP/biosíntesis , Regulación hacia Abajo/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Fibras Musculares Esqueléticas/enzimología , Ubiquitina-Proteína Ligasas/biosíntesis , Aminoimidazol Carboxamida/análogos & derivados , Animales , Línea Celular , Fibras Musculares Esqueléticas/citología , Proteínas Musculares/metabolismo , Ratas , Ribonucleótidos/biosíntesis , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Burn-blast combined injury has a complex pathological process that may cause adverse complications and difficulties in treatment. This study aims to establish a standard animal model of severe burn-blast combined injury in rats and also to investigate early phasic changes of blood coagulation. By using 54 Wistar rats, distance from explosion source (Hexogen) and size of burned body surface area were determined to induce severe burn-blast combined injury. Thereafter, 256 rats were randomly divided into four groups (n = 64): blast injury group, burn injury group, burn-blast combined injury group, and sham injury group. Gross anatomy and pathological changes in lungs were investigated at 3, 24, 72, and 168 h, respectively. Blood was also collected for analyzing coagulation parameters as prothrombin time, activated partial thromboplastin time, and plasma levels of fibrinogen, D-dimer, antithrombin III, and α2-antiplasmin from 0 to 168 h after injury. Severe burn-blast combined injury was induced by inflicting rats with a moderate blast injury when placing rats 75 cm away from explosion source and a full-thickness burn injury of 25% total body surface area. The rats with burn-blast combined injury had more severe lung injuries when compared with the other three groups. Pathological examination in the BBL group showed diffused alveolar hemorrhage, fluid filling, alveolar atelectasis, rupture and hyperplasia of partial alveolar septum, emphysema-like change, reduced capillary bed, and infiltration of extensive polymorphonuclear cells after injury. The blood of combined injured rats was in a hypercoagulable state within 24 h, shortly restored from 24 to 48 h, and rehypercoagulated from 48 to 72 h after injury. A secondary excessively fibrinolytic function was also found thereafter. The rat model of burn-blast combined injury was successfully established by simulating real explosion characteristics. Rats with burn-blast combined injuries suffered from more severe lung injuries and abnormal coagulation and fibrinolytic function than those induced by a burn injury or a blast injury component. Hence, a time-dependent treatment strategy on coagulation function should be emphasized in clinical therapy of burn-blast combined injury.
Asunto(s)
Traumatismos por Explosión/sangre , Traumatismos por Explosión/complicaciones , Coagulación Sanguínea , Quemaduras/sangre , Quemaduras/complicaciones , Animales , Traumatismos por Explosión/patología , Quemaduras/patología , Modelos Animales de Enfermedad , Fibrinólisis , Pulmón/patología , Lesión Pulmonar/sangre , Lesión Pulmonar/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Neutrophil elastase (NE) takes part in the pathogenesis of acute lung injury. However, its role in lung injury of burn-blast combined injury is unclear. Our objective was to assess the role of NE, and effect of sivelestat, a specific NE inhibitor, in lung injury induced by burn-blast combined injury in rats. METHODS: One hundred and sixty male Sprague-Dawley rats were randomly subjected to burn-blast combined injury (BB) group, burn-blast combined injury plus sivelestat treatment (S) group or control (C) group. Blood gas, protein concentration and NE activity in bronchoalveolar lavage fluid (BALF), pulmonary myeloperoxidase (MPO) activity, serum concentrations of TNF-α and IL-8, etc. were investigated from 0 h to 7 d post-injury. RESULTS: In BB group, PaO2 decreased, while NE activity in BALF, total protein concentration in BALF, pulmonary MPO activity and W/D ratio, serum concentrations of TNF-α and IL-8 increased with neutrophil infiltration, progressive bleeding and pulmonary oedema. Compared with BB group, sivelestat treatment decreased the NE activity and ameliorated the above indexes. CONCLUSION: Sivelestat, exerts a protective effect in lung injury after burn-blast combined injury through inhibiting NE activity to decrease pulmonary vascular permeability, neutrophil sequestration, and production of TNF-α and IL-8.
Asunto(s)
Traumatismos por Explosión/complicaciones , Quemaduras/complicaciones , Elastasa de Leucocito/fisiología , Lesión Pulmonar/enzimología , Animales , Traumatismos por Explosión/tratamiento farmacológico , Traumatismos por Explosión/enzimología , Líquido del Lavado Bronquioalveolar/química , Quemaduras/tratamiento farmacológico , Quemaduras/enzimología , Dióxido de Carbono/metabolismo , Modelos Animales de Enfermedad , Glicina/análogos & derivados , Glicina/uso terapéutico , Interleucina-8/metabolismo , Lesión Pulmonar/tratamiento farmacológico , Lesión Pulmonar/etiología , Masculino , Oxígeno/metabolismo , Presión Parcial , Proteínas Inhibidoras de Proteinasas Secretoras/uso terapéutico , Ratas , Ratas Sprague-Dawley , Inhibidores de Serina Proteinasa/uso terapéutico , Sulfonamidas/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To observe the growth and migration of human umbilical cord mesenchymal stem cells (hUCMSCs) on polycarbonate membrane with different pore sizes and explore the criteria of selecting optimal Transwell insert for indirect co-culture to induce the differentiation of hUCMSCs. METHODS: hUCMSCs were isolated in vitro and then expanded in culture medium. After the treatment of mitomycin C, the cells were seeded on porous membranes of 6-well-dish Transwell inserts with different pore sizes of 0.4, 3.0 and 8.0 µm respectively. After culturing for 7 days, the cells were observed and counted on the bottom of each porous membrane. Then the calculation of migration ratio was performed. The growth and migration of hUCMSCs on porous membranes were also examined under scanning electron microscope (SEM). RESULTS: The migration ratios of hUCMSCs on membranes of 0.4, 3.0 and 8.0 µm pore sizes were 0, 1.8% and 8.0% respectively. The migration ratio of cells on 0.4 µm pore size membrane was statistically different from that of the other two pore size groups (P < 0.01). Under SEM, a small portion of cells were growing on the bottoms of membranes and moving through the pores. But there was no cell movement through 0.4 µm pore size membrane. CONCLUSIONS: hUCMSCs can migrate through the polycarbonate membranes of 3.0 µm and 8.0 µm pore sizes but not through the 0.4 µm one. Thus both sides of polycarbonate membrane of 0.4 µm pore size may be used for close indirect co-culture to induce the differentiation of hUCMSCs.
Asunto(s)
Diferenciación Celular , Células Madre Mesenquimatosas/citología , Cemento de Policarboxilato , Técnicas de Cultivo de Célula , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Humanos , Cordón Umbilical/citologíaRESUMEN
OBJECTIVE: To study the modulatory effect of insulin on apoptosis of skeletal myoblast (L6 cells) by serum of scalded rat and its mechanism. METHODS: L6 cells cultured with DMEM medium containing 10% FBS were divided into control (C, added with 20% normal rat serum), serum from rat with scald injury (S, added with 20% serum from scalded rat), insulin (I, added with 20% normal rat serum and 100 nmol/L insulin), and serum of scalded rat + insulin (SI, added with 20% serum of scalded rat + 100 nmol/L insulin) groups according to the random number table. After being cultured for 48 hours, apoptosis was observed with Hoechst 33258 staining and its number counted, annexin V -FITC/PI double-labeling method was used to assess apoptosis rate, the protein levels of phosphorylated (p-) Akt, p-PI3K, Bax, Bcl-2, and active caspase-3 were determined by Western blotting. Data were processed with grouped or paired t test. RESULTS: (1) The amount of apoptosis with typical morphological change in S group [(59.6 +/- 3.9) per visual field] was more than that in C, I, and SI groups [(4.9 +/- 2.6), (5.5 +/- 2.1), (19.7 +/- 2.3) per visual field, with t value respectively 28.53, 29.86, 21.53, P values all below 0.01]. (2) Apoptotic rate in S group was (18.5 +/- 1.8)%, which was markedly higher than that in C, I, and SI groups [(1.1 +/- 0.6)%, (1.5 +/- 0.3)%, (7.8 +/- 0.6)%, with t value respectively 22.41, 22.83, 13.92, P values all below 0.01]. (3) Compared with those in C group, the protein levels of Bax and active caspase-3 in S group were up-regulated (1.12 +/- 0.63 vs. 0.16 +/- 0.03, 2.15 +/- 0.51 vs. 0.21 +/- 0.03, with t value respectively 3.80, 10.69, P values all below 0.01), the protein level of p-Akt was lowered (0.20 +/- 0.03 vs. 0.42 +/- 0.07, t = -8.46, P < 0.01), and the protein levels of p-PI3K and Bcl-2 showed no statistical difference (0.19 +/- 0.03 vs. 0.26 +/- 0.09, 0.17 +/- 0.03 vs. 0.28 +/- 0.07, with t value respectively -2.73, - 1.14, P values all above 0.05). The protein levels of Bax (0.40 +/- 0.14) and active caspase-3 (0.83 +/- 0.18) in SI group were lowered (t = -3.23, P < 0.05; t = 6.66, P < 0.01) and the protein levels of p-Akt, Bcl-2, and p-PI3K in SI group were elevated (0.39 +/- 0.10, 0.78 +/- 0.03, 0.47 +/- 0.12, with t value respectively 4.07, 18.71, 5.05, P < 0.05 or P < 0.01) as compared with those in S group. CONCLUSIONS: Serum from scalded rat can induce apoptosis in skeletal myoblast, and the effect can be inhibited by insulin through PI3K/Akt signal pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Quemaduras/metabolismo , Insulina/farmacología , Mioblastos Esqueléticos/efectos de los fármacos , Animales , Quemaduras/sangre , Quemaduras/patología , Caspasa 3/metabolismo , Línea Celular , Masculino , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/patología , Ratas , Ratas Wistar , Suero/inmunología , Transducción de Señal , Proteína X Asociada a bcl-2/metabolismoRESUMEN
OBJECTIVE: To explore the early changes in serum neutrophil elastase (NE) in rats with burn, blast injury or combined burn-blast injury and its significance. METHODS: A total of 176 male Sprague Dawley rats were randomly divided into four groups: control (C), burn (BU), blast injury (BL) and burn-blast combined injury (BB). Rats in C group were not injured. Animals in BU group were subjected to 25% TBSA full-thickness burn on back with 94 degrees C water for 12 seconds; Animals in BL group were inflicted with moderate blast injury with 5 g 8701 compressed dynamite stick as the explosion source 75 cm away while left chest facing the explosive source; Rats in BB group were burned immediately after the blast injury similarly as in BL group. During the first 24 h post-injury, animals in BU and BB groups received intraperitoneal injection of sodium lactate Ringer's solution at a dose of 50 ml x kg(-1) x 12 h(-1). Protein concentration in bronchoalveolar lavage fluid (BALF), water content of lung tissue and NE content in serum were determined at 0 h (C group), 3 h, 6 h, 12 h, 1 d, 2 d, 3 d, 7 d post-injury. RESULTS: Protein concentration in BALF, water content of lung tissue and NE content in serum in SD rats of the injured groups were significantly higher than those in C group (P < 0.01 or P < 0.05), peaked within 2 d post-injury, especially at 2 d post-injury (NE content in serum: BU group, 319. 85 +/- 19.50 ng/ml; BL group, 467.43 +/- 31.64 ng/ml; BB group, 626.00 +/- 26.38 ng/ml vs. C group, 78.53 +/- 25.10 ng/ml). Overall, protein concentration in BALF, water content of lung tissue and NE content in serum in BB group were significantly higher than BU and BL groups (P < 0.01 or P < 0.05). Correlation analysis showed that within 3 d postinjury, a significant positive correlation was found between the protein concentration in BALF, water content of lung tissue and NE content in serum (r = 0.7910, 0.8078, P < 0.05) in BU group. NE content in serum and protein concentration in BALF were significantly positively correlated in BB group (r = 0.8672, P < 0.05). CONCLUSION: NE may play an important role in early lung injury of burn or blast injury, especially in combined burn-blast injury.
Asunto(s)
Quemaduras/sangre , Elastasa de Leucocito/sangre , Lesión Pulmonar/sangre , Heridas y Lesiones/sangre , Animales , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Insulin resistance, hyperglycemia, inflammatory disorders and immune dysfunction cause high morbidity and mortality in patients with severe trauma, burn injuries, or sepsis. Many studies have shown that intensive insulin therapy can combat insulin resistance, decrease blood glucose levels, and induce anabolic processes, thus, decreasing morbidity and mortality. Moreover, in recent years, it has been proven that insulin can attenuate systemic inflammatory responses and modulate the proliferation, apoptosis, differentiation and immune functions of certain immune cells, especially monocytes/macrophages, neutrophils, and T cells associated with severe trauma, burn injury, or sepsis. This effect of insulin may expand our understanding of intensive insulin therapy in critically ill patients. This review attempts to summarize studies on the modulatory effects and mechanisms of insulin therapy on systemic inflammation and immune cells in severe trauma, burn injury and sepsis, and further propose some questions for future studies.
Asunto(s)
Quemaduras/inmunología , Inflamación/tratamiento farmacológico , Insulina/uso terapéutico , Linfocitos/efectos de los fármacos , Fagocitos/efectos de los fármacos , Sepsis/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Heridas y Lesiones/inmunología , Quemaduras/tratamiento farmacológico , Quemaduras/metabolismo , Humanos , Hiperglucemia/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Modelos Inmunológicos , Sepsis/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Heridas y Lesiones/tratamiento farmacológico , Heridas y Lesiones/metabolismoRESUMEN
AIM: To express and purify fusion protein of human CD112 extracellular region, prepare monoclonal antibodies (mAb) to CD112 and investigate the distribution of CD112 molecule. METHODS: The CD112-Fc fusion protein was expressed with gene recombinant technique in eukaryotic system and purified with affinity chromatography column. The BALB/c mice were immunized with purified CD112-Fc for preparation of mAb by hybridoma technique. The prepared mAbs were identified by indirect ELISA, Western blot and flow cytometry (FCM). Subsequently, the distribution of CD112 on different human cell lines was investigated by FCM. RESULTS: The effective expression plasmid pIg3C-CD112 was constructed, and human CD112-Fc was successfully expressed and purified with a purity of more than 90%. Nine clones of hybridoma were obtained, which were designated as FMU-CD112.1-FMU-CD112.9. FMU-CD112.1, 3, 6 and 8 specifically reacted with recombinant CD112-Fc protein in Western blot and FMU-CD112.1, 4, 6 and 8 could be used in FCM. The investigation of CD112 distribution showed high expression on the cell lines differentiated from epithelial cells. CONCLUSION: The hybridomas secreting mAbs to human CD112 are established successfully, which may lay the foundation for further research on the biological function of CD112.