Objective To elucidate whether glucose transporters-4 （GLUT-4） takes part in glucose uptake of mesenchymal stem cells （MSCs） and whether Akt gene improves translocation and expression of GLUT-4 in MSCs under hypoxic environment ex vivo. Methods MSCs, transfected by Akt gene and no, were cultured with normoxia (5% CO2) or hypoxia (94%N2, 1%O2 and 5% CO2) at 37 ℃ for 8 h. Glucose uptake was assayed by using radiation isotope 2-［3H］-deoxy-Dglucose (3H-G) and the expression of GLUT-4 protein and mRNA was assayed by immunocytochemistry, Western blot and RT-PCR, respectively. Results ①3 H-G intake of MSCs was significantly increased in hypoxiatransfection group than that in hypoxia-non-transfection 〔（1.39±0.13） fold, P＜0.05〕, but which was lower than that in normoxia-non-transfection group, P＜0.05. ②GLUT-4 was expressed by MSCs under any conditions. Compared with normoxia-non-transfection group, hypoxia decreased the expressions of GLUT-4 mRNA and protein significantly (P＜0.05). ③Compared with hypoxianontransfection group, the expression of GLUT-4 〔mRNA（1.756±0.152） fold, total protein in cell （1.653±0.312） fold, protein in plasma membrane （2.041±0.258） fold〕 was increased in hypoxia-transfection group significantly (P＜0.05), but which was lower than that in normoxianontransfection group (P＜0.05). ④There was significantly positive relation between 3H-G intake and GLUT-4 protein expression in plasma membrane (r=0.415, P=0.001).Conclusion GLUT-4 may take part in glucose uptake of MSCs, and the capability of Akt gene to improve MSCs anti-hypoxia may be finished by its role in increasing the expression and translocation of GLUT-4.
Objective To explore the impact of ischemic postconditioning on ischemia-reperfusion injury in isolatedelderly rat hearts and their relation with P-Akt. Methods A total of 30 healthy elderly SD rats (21-23 months old， male or female) with their body weight of 450-500 g were divided into 3 groups: control group， ischemia-reperfusion group， and postconditioning group， with 10 rats in each group. Coronary artery blood flow，myocardial infarction size， phosphorylatedAkt （p-Akt） expression， and changes in myocardium and mitochondria were detected. Results Coronary artery blood flow of the postconditioning group was significantly higher than that of the ischemia-reperfusion group （6.4±1.2 ml/min vs.3.1±1.2 ml/min， P＜0. 01）， and myocardial infarction size of the postconditioning group was significantly smaller thanthat of the ischemia-reperfusion group （35.0%±2.0% vs. 55.7%±3.6%， Plt;0. 05）. The expression of P-Akt was significantlyhigher， and myocardial fibers and mitochondria were preserved better in the postconditioning group than the ischemia-reperfusion group. Conclusion Ischemic postconditioning can protect isolated elderly rat hearts against ischemia-reperfusion injury， which may be related to P-Akt activation.
ObjectiveTo investigate the effect and mechanism of caveolion-1 on the growth and proliferation of human pancreatic carcinoma cell Panc1, in vitro. MethodsThe plasmid pCI-neo-cav-1 and its corresponding empty vector (pCI-neo) were transfected into Panc1 cell line (study group and control group, respectively). Expressions of caveolin-1, Akt, and Aktphosphate (p-Akt) were determined in transfectants by Western blot analysis. The cell growth curve was drawn and the double time was calculated in each group, and the cell cycle was analyzed by flow cytometry. The colony formation ability of tumor cells was detected by anchorageindependent growth assay. ResultsCaveolin-1 expression was up-regulated (Plt;0.01) and the growth of Panc1 cell was inhibited significantly (Plt;0.01) in the study group comparing with the control group. Caveolin-1 overexpression inhibited proliferation of Panc1 cell by arresting the cell cycle in the G0/G1 phase (Plt;0.05), the rate of S phase in the study group was lower than that of the control group (Plt;0.01). Proliferation index of the study group was also lower than that of the control group (Plt;0.01). Caveolin-1 overexpression reduced the capacity of the cells to form colonies in soft agar (Plt;0.01). p-Akt protein was reduced in the study group as compared with the control group (Plt;0.05). ConclusionCaveolin-1 can function as a cancer suppressor through inhibiting the activation of PI3K/Akt signaling pathway in Panc1 cell.
Objective To construct green fluorescent protein (GFP)/Akt fusion gene vector for observing the expression and localization of GFP/Akt in rats bone marrow-derived mesenchymal stem cells （MSCs）. Stem cell factor （SCF） effected expression of c-kit, Akt and VEGF mRNA and protein in MSCs transfected by pEGFP-C1/Akt through PI3-Akt pathway.Methods Akt recombined GFP vector by restriction enzymes, MSCs was transfeced by GFP/Akt and GFP through cationic liposomes, and then veritied by restriction endonuclease assay and sequence analysis. Transfection and localization of GFP were evaluated by fluorescene microscopy. The expressions of c-kit, Akt and VEGF mRNA and protein were examined by RT-PCR and Western blot after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. SCF effected the expression of c-kit, Akt and VEGF mRNA and protein after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. Results Restriction endonuclease assay and sequence analysis verified that thesuccessfulconstructionoftherecombinantvectorpEGFP-C1/AktandefficienthighexpressionofpEGFP-C1/Akt fusion protein in the MSCs of rats. Under fluorescent microscence, green flurescence was seen homogeneously distributed in the entire cell of the cells transfected by the recombinant vector pEGFP-C1, and diffusely in the cytoplasm of the cells transfected by the recombinant vector pEGFP-C1/Akt. The expression of Akt and VEGF mRNA and protein were significantly higher in MSCs transfected by pEGFP-C1/Akt (plt;0.05). The expression of c-kit, Akt and VEGF mRNA and protein were significantly higher in experiment group (SCF+pEGFP-C1/Akt) and control group (SCF+pEGFP-C1), plt;0.05. In experiment group， SCF stimulation enhanced expression of Akt and VEGF mRNA and protein (plt;0.01). Conclusion GFP/Akt fusion gene vector is successfully construted and the fusion protein expressed in the MSCs of rats induces the expression of Akt and VEGF mRNA and protein. SCF stimulation enhanced expression of c-kit, Akt and VEGF mRNA and protein through PI3/Akt pathway.
Objective To investigate the effect of bone mesenchymal stem cells (MSCs) transfected with pEGFP-C1/Akt after intramuscular injection on angiogenesis in ischemic hindlimb of mice. Methods A total of 30 Wistar mice used in the present experiments were divided into gene therapy group (injected MSCs transfected with pEGFP-C1/Akt), non-gene therapy group （injected MSCs） and control group (injected PBS). Mice skin temperature in left hindlimbs was measured by a infrared ray temperature meter before and right after ischemia and 1-7 days after transfection, respectively. Digital subtraction angiography was carried out to observe angiogenesis in hindlimb of mice after 28 days. The capillary density was determined with immunohistochemical analysis in the resected specimen. The expressions of Akt mRNA and protein, VEGF mRNA and protein were measured via RT-PCR and Western blot. Results Skin temperature increased significantly on 3 d after transfection in gene therapy group. On 28 d after transfection， angiography showed that the number of collateral vessels markedly increased in gene therapy group. Green fluorescence cells were observed in ischemic tissue from the adductor and semimembranous muscles of gene therapy group under fluorescent microscope. The result of immunohistochemical analysis of capillary density showed that the capillary density was significantly higher in gene therapy group 〔(7.1±0.3)/high power〕 than that in non-gene therapy group 〔(4.2±0.4)/high power〕 and control group 〔(1.3±0.2)/high power〕, with significantly statistical differences among three groups (P＜0.01). Akt mRNA (2.44±0.14) and protein (1.1±0.13), VEGF mRNA (1.1±0.11) and protein (0.97±0.13) of gene therapy group markedly increased compared with Akt mRNA (1.58±0.13) and protein (0.78±0.12), VEGF mRNA (0.78±0.14) and protein (0.67±0.11) of non-gene therapy group as well as Akt mRNA (0.64±0.11) and protein (0.36±0.12), VEGF mRNA (0.56±0.11) and protein (0.33±0.13) of control group (P＜0.01). Conclusion The effect of MSCs transfected with pEGFP-C1/Akt on angiogenesis is significantly better than only MSCs therapy, which may offer a new way for the ischemic diseases.
ObjectiveTo investigate the possible mechanism of the improvement of type 2 diabetes mellitus with insulin resistance of skeletal muscles after Roux-en-Y gastric bypass surgery (RYGB). MethodsThirty GK rats were randomly divided into GK-RYGB group, sham operation group (GK-SO group), and control group (GK-control group); in addition, 10 Wistar rats served as normal control group.On day 28, the animals were sacrificed.The ghrelin concen-tration and PI3Kp85α, Akt/PKB, and GLUT4 levels were measured by ELISA, Western blot, and real-time PCR me-thods, respectively. Results①Compared with the GK-SO group and GK-control group, the plasma ghrelin levels were significantly increased in the normal control group (P < 0.01) and GK-RYGB group (P < 0.01).②Compared with the GK-SO group and GK-control group, p-/t-PI3Kp85α, p-/t-Akt/PKB, and m-/t-GLUT4 proteins were significantly incre-ased in the normal control group (P < 0.01, P < 0.05, and P < 0.01, respectively) and GK-RYGB group (P < 0.01, P < 0.05, and P < 0.01, respectively).③Compared with the GK-SO group and GK-control group, PI3Kp85α, Akt, and GLUT4 mRNA were significantly increased in the normal control group (P < 0.01, P < 0.05, and P < 0.05, respectively) and GK-RYGB group (P < 0.01, P < 0.05, and P < 0.05, respectively). ConclusionRYGB could elevate the ghrelin level obviously and upregulate PI3Kp85α, Akt/PKB, and GLUT4 levels and thus improve the insulin resistance of skeletal muscles of rats with T2DM.
ObjectiveTo investigate the effect of PI3K/Akt/mTOR signaling pathway on liver injury induced by severe acute pancreatitis (SAP). MethodsForty healthy adult male Sprague-Dawley (SD) rats were randomly divided into 4 groups: Sham operation group (SO group), SAP group, PI3K inhibitor LY294002 group (LY294002 group), and mTOR kinase inhibitor rapamycin group (rapamycin group). The rat model with SAP was made by injection with 5% sodium deoxycholate through retrogradely bilio pancreatic duct. Serum levels of amylase (AMY), alanine aminotransferase (ALT), and aspartate transaminase (AST) were detected through the inferior vena at 6 h after modeling. Pathologic change of the liver was observed under the light microscope. TUNEL analysis was used to detect apoptotic index (AI) of the heptocyte. Expressions of Akt, phosphated-Akt (p-Akt), mTOR, phosphated-mTOR (p-mTOR) protein were evaluated by Western blot. Results①Compared with the SO group, the serum levels of AMY, ALT, AST, and the hepatocyte AI were significantly increased among the other three groups (P < 0.05). Compared with the SAP group, the serum levels of AMY, ALT, AST, and the hepatocyte AI were significantly decreased in the LY294002 group and rapamycin group (P < 0.05).②Compared with the SO group, the damages of the liver tissues were aggravated among the other three groups. The pathologies of the liver tissues were ameliorated in the LY294002 group and rapamycin group as compared with the SAP group.③Compared with the SO group, the levels of p-Akt/Akt, p-mTOR/mTOR were significantly increased among the other three groups (P < 0.05). Compared with the SAP group, the levels of p-Akt/Akt, p-mTOR/mTOR were significantly decreased in the LY294002 group (P < 0.05), but in the rapamycin group, only the p-mTOR/mTOR level was significantly decreased (P < 0.05). ConclusionThe activation of PI3K/Akt/mTOR signaling pathway might be one of the reasons for the liver injury induced by SAP and blocking this signaling pathway might be a potential target of preventing progress of SAP and alleviating liver injury induced by SAP.
ObjectiveTo investigate whether Akt1 gene transfection mediated by recombinant lentivirus (LVs) in the bone marrow mesenchymal stem cells (BMSCs) could enhance the ability of hypoxia tolerance so as to provide a theoretical basis for improving the effectiveness of stem cells transplantation. MethodLVs was used as transfection vector, enhanced green fluorescent protein (EGFP) was used as markers to construct the pLVX-EGFP-3FLAG virus vector carrying the Akt1 gene. The 3rd generation BMSCs from 3-5 weeks old Sprague Dawley rats were transfected with pLVX-EGFP virus solution as group B and with pLVX-EGFP-3FLAG virus solution as group C; and untransfected BMSCs served as control group (group A). At 2-3 days after transfection, the expression of green fluorescent was observed by fluorescence microscope; and at 48 hours after transfection, Western blot method was used to detect the expression of Akt1 protein in groups B and C. BMSCs of groups B and C were given hypoxia intervention with 94%N2, 1%O2, and 5%CO2 for 0, 3, 6, 9, and 12 hours (group B1 and group C1) . The flow cytometry was used to analyze the cell apoptosis rate and cell death rate, and the MTT method to analyze the cell proliferation, and Western blot to detect the expression of apoptosis related gene Caspase-3. ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in groups B and C, the transfection efficiency was about 60%. Akt1 expression of group C was significantly higher than that of group B (t=17.525, P=0.013) . The apoptosis rate and cell death rate of group B1 increased gradually with time, and difference was significant (P<0.05) . In group C1, the apoptosis rate and cell death rate decreased temporarily at 3 hours after hypoxia intervention, then increased gradually, and difference was significant (P<0.05) . The apoptosis rate and cell death rate of group C1 were significantly lower than those of group B1 at each time point (P<0.05) except at 0 hour. MTT assay showed that absorbance (A) values of groups B and C were significantly higher than those of groups B1 and C1 at each time point (P<0.05) ; the A value of group B was significantly lower than that of group C at each time point (P<0.05) . The A value of group B1 was significantly lower than that of group C1 at 6, 9, and 12 hours after hypoxia intervention (P<0.05) . Western blot results showed that the Caspase-3 expression of group C1 significantly reduced when compared with group B1 at each time point (P<0.05) . ConclusionsAkt1 gene transfection mediated by recombinant LVs could significantly improve hypoxia tolerance of BMSCs by inhibiting the apoptosis, which could provide new ideas for improving the effectiveness of stem cells transplantation.
This study aimed to explore the role of miR-130a-3p in cardiomyocyte hypertrophy and its underlying mechanisms. Pressure-overload induced myocardial hypertrophy mice model was constructed by thoracic aortic constriction (TAC). In vitro, norepinephrine (NE) was used to stimulate neonatal rat cardiomyocytes (NRCMs) and H9c2 rat cardiomyocytes to induce hypertrophic phenotypes. The expression of miR-130a-3p was detected in mice hypertrophic myocardium, hypertrophic NRCMs and H9c2 cells. The mimics and inhibitors of miR-130a-3p were transfected into H9c2 cells to observe the role of miR-130a-3p on the hypertrophic phenotype change of cardiomyocytes separately. Furthermore, whether miR-130a-3p regulated hypertrophic related signaling pathways was explored. The results showed that the expression of miR-130a-3p was significantly decreased in hypertrophic myocardium, hypertrophic NRCMs and H9c2 cells. After transfection of miR-130a-3p mimics, the expression of hypertrophic marker genes, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), and the cell surface area were notably down-regulated compared with the control group (mimics N.C. + NE group). But after transfection of miR-130a-3p inhibitor, the expression of ANP, BNP and β-MHC in H9c2 cells increased significantly, and the cell area increased further. By Western blot, it was found that the protein phosphorylation level of Akt and mTOR were down-regulated after over-expression of miR-130a-3p. These results suggest that miR-130a-3p mimics may alleviate the degree of cardiomyocyte hypertrophy, meanwhile its inhibitor can further aggravate cardiomyocyte hypertrophy. Over-expression of miR-130a-3p may attenuate cardiomyocytes hypertrophy by affecting the Akt pathway.
Objective To investigate the role and regulatory mechanism of ring finger protein 11 (RNF11) on Akt signaling pathway in the process of osteogenesis of bone marrow mesenchymal stem cells (BMSCs) to provide ideas for further clarifying its osteogenesis mechanism and its use in clinical treatment in the future. Methods BMSCs were isolated and cultured from fresh bone marrow of healthy donors and subcultured. The 4th generation cells were used in experiments after identification by flow cytometry, and osteogenic, chondrogenic, and adipogenic induction. BMSCs were cultured in osteogenic differentiation medium for 0-14 days. The degree of osteogenic differentiation was detected by Alizarin red staining and alkaline phosphatase (ALP) staining, and the protein expression of RNF11 was detected by Western blot. The 4th generation BMSCs were divided into blank control group (group A), empty lentivirus (Lv-NC) group (group B), and knockdown RNF11 (Lv-ShRNF11) group (group C). Osteogenesis was induced and cultured for 0-14 days. The expression of RNF11 protein was detected by Western blot, the degree of osteogenic differentiation was detected by Alizarin red staining and ALP staining, and the relative mRNA expressions of Runx2, osteocalcin (OCN), and osteopontin (OPN) were detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein relative expressions of Akt, Smad1/5/8, and β-catenin signaling pathway were detected by Western blot, expressed as the ratio before and after phosphorylation. In order to study the effect mechanism of RNF11 on Akt signaling pathway, the 4th generation BMSCs were divided into Lv-NC transfection group (group A1), Lv-ShRNF11 transfection group (group B1), and Lv-ShRNF11 transfection supplemented with Akt signaling pathway activator SC79 group (group C1). The protein relative expressions of RNF11 and Akt signaling pathway were detected by Western blot, the related osteogenesis indexes were detected by Alizarin red staining, ALP staining, and qRT-PCR. ResultsThe flow cytometry, and osteogenic, chondrogenic, adipogenic induction culture identification showed that the isolated and cultured cells were BMSCs. The protein relative expression of RNF11 increased gradually with the extension of osteogenic differentiation time (P<0.05); after knockdown RNF11, Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C were significantly lower than those in groups A and B, and qRT-PCR detection showed that the relative expression of Runx2, OCN, and OPN mRNA significantly decreased (P<0.05). The protein relative expressions of RNF11 and Akt signaling pathway significantly increased with the extensions of osteogenic differentiation time (P<0.05). After knockdown RNF11, the protein relative expression of Akt signaling pathway in group C was significantly lower than that in groups A and B (P<0.05), while Smad1/5/8 and β-catenin signaling pathway had no significant effect (P>0.05). Compared with group A1, the protein relative expression of RNF11 in groups B1 and C1 significantly decreased (P<0.05). Compared with groups A1 and C1, the protein relative expression of Akt signaling pathway in group B1 was significantly lower (P<0.05); Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C1 were slightly lower than that of group A1 (P>0.05), but significantly higher than that of group B1 (P<0.05); qRT-PCR detection showed that the relative expressions of Runx2, OCN, and OPN mRNA in group C1 were slightly lower than those of group A1 (P>0.05), but were significantly higher than those of group B1 (P<0.05). ConclusionRNF11 promotes the differentiation of BMSCs into osteoblasts by positively regulating the activation level of Akt signaling pathway. RNF11 can be used as a potential target to improve the bone repair efficacy of BMSCs and treat bone metabolic diseases.