CTRP3 increased insulin sensitivity of insulin resistant 3T3-L1 adipocytes via decreasing expression of inflammatory factors

AIM:To investigate the effects of C1q/TNF related protein 3 (CTRP3) on the insulin sensitivity of insulin resistant 3T3-L1 adipocytes. METHODS:The insulin resistance model of 3T3-L1 adipocytes was induced by palmic acid cultivation. The adipocytes were treated with different concentrations of recombinant CTRP3 protein (10, 50, 250,1 250 μg/L) for 12 h, and for different times (2, 6, 12, 24 h) at the concentration of 250 μg/L. The glucose consumption was detected by the glucose oxidase method. The glucose transport ratio was measured by 2-deoxidation-［3H］-glucose intake method. The contents of TNF-α and IL-6 in the supernatant were detected by ELISA. The mRNA expression of TNF-α, IL-6 and glucose transporter-4 (GLUT-4) was measured by real-time PCR. The protein expression of GLUT-4 was detected by Western blotting. RESULTS:Compared with normal control (NC) group, the glucose consumption and glucose intake ratio of insulin resistance (IR) group was decreased by 50.6% and 57.9%, respectively. Compared with IR group, with the increase in CTRP3 (10, 50, 250,1 250 μg/L) in intervention groups, the glucose consumptions were increased by 22.1%, 42.9%, 76.6% and 80.5%, respectively, and the glucose intake ratios were increased by 39.0%, 68.0%, 108.0% and 111.0%, respectively. With the increased duration (2, 6, 12 and 24 h) of CTRP3 treatment at the concentration of 250 μg/L, the glucose intake ratio was increased by 23.0%, 79.0%, 109.0% and 114.0%, respectively. The contents of TNF-α and IL-6 in the supernatant were decreased by 17.4% and 17.1% respectively as treated with CTRP3 at the concentration of 250 μg/L for 12 h, and the mRNA expression of TNF-α and IL-6 was decreased by 26.0% and 18.9% respectively, while the mRNA and protein expression of GLUT-4 was increased by 61.5% and 55.6% respectively. CONCLUSION: CTRP3 may increase the insulin sensitivity of insulin resistant 3T3-L1 adipocytes by down-regulating the expression of inflammatory factors, improving the insulin signal transduction and increasing the expression of GLUT-4.     

Effect of glucocorticoid on PI-3K and p38 MAPK signaling pathways in 3T3-L1 adipocytes

AIM: To study the effects of dexamethasone (DEX) on the glucose transport system and the PI-3K/Akt and p38 MAPK insulin signaling pathways in 3T3-L1 adipocytes,and to investigate the possible mechanism in glucocorticoid induced insulin resistance. METHODS: The 3T3-L1 adipocytes were exposed to DEX for 48 h and incubated with 100 nmol/L insulin for additional 30 min. The glucose uptake was measured by detecting the glucose content in cell culture supernatants. Then expression and distribution of Glut4 was measured. The insulin signaling proteins Akt,phospho-Akt,p38MAPK and phospho-p38MAPK were also measured with Western blotting. RESULTS: DEX inhibited insulin stimulated glucose transport capacity in 3T3-L1 adipocytes. DEX did not alter the amount of Glut4 protein in total cell lysates but attenuated the insulin-stimulated Glut4 translocation to the plasma membrane. DEX significantly inhibited insulin stimulated phosphorylation of Akt and p38 MAPK. CONCLUSION: These results suggest that DEX alters insulin stimulated glucose transport capacity in 3T3-L1 adipocytes,which is mediated by attenuating insulin stimulated activation of PI3K-Akt and p38 MAPK pathways,and reducing insulin stimulated Glut4 translocation and transport activity. These may lead to insulin resistance in 3T3-L1 adipocytes.     

Total triterpenoids from Psidium Guajava leaf improves insulin resistance in 3T3-L1 adipocytes

AIM: To investigate the effects of total triterpenoids from Psidium guajava leaf (TTPGL) on 3T3-L1 adipocyte insulin resistance (IR) and to explore the possible mechanism. METHODS: 3T3-L1 pre-adipocytes were cultured and induced to differentiate into 3T3-L1 adipocytes, then treated with TTPGL (0.3, 1, 3, 10 μg/L) for 48 h. The cells were divided into 0.1% DMSO group, positive drug sodium orthovanadate (Van, 10 μmol/L) group, model group and control group. The effect of TTPGL on the cell activity of pre-adipocytes was detected by MTT assay and its influence on the cellular differentiation was observed by oil red O staining. The IR model of the 3T3-L1 adipocytes was established successfully and then treated with different drugs for 48 h. The glucose consumption in the supernatant of IR adipocyte's culture medium was assayed by glucose oxidase-peroxidase (GOD-POD), free fatty acid (FFA) levels were measured by colorimetric method, and adipocytokines levels were assayed by ELISA. The mRNA expression of protein tyrosine phosphatase-1B (PTP1B) of IR adipocyte was detected by real-time PCR. The protein levels of phosphorylated insulin receptor substrate 1/insulin receptor substrate 1 (p-IRS-1/IRS-1) and phosphorylated protein kinase B/protein kinase B (p-Akt/Akt) were determined by Western blot. RESULTS: Compared with DMSO group, TTPGL treatment significantly promoted the cell activity of 3T3-L1 pre-adipocytes and inhibited its differentiation (P < 0.01). TTPGL (1~10 μg/L) improved glucose consumption of IR adipocytes significantly (P < 0.01), with or without insulin stimulation, and TTPGL (0.3~3 μg/L) restrained FFA production remarkably(P < 0.01). Compared with model group, TTPGL (0.3 and 3 μg/L) significantly increased the secretion of adiponectin in IR adipocytes (P < 0.05), and inhibited the secretion of tumor necrosis factor-α (TNF-α) (P < 0.01). TTPGL (3 μg/L) restrained the secretion of resistin significantly (P < 0.05), and showed no significant effect on secretion of leptin. It also down-regulated the mRNA expression of protein tyrosine phosphates 1B (PTP1B) in IR adipocytes significantly (P < 0.01), and increased the protein levels of p-IRS-1/IRS-1. TTPGL (0.3 and 3 μg/L) up-regulated the protein level of p-Akt/Akt in IR adipocytes significantly (P < 0.05).CONCLUSION: TTPGL reduces IR in 3T3-L1 adipocytes. The mechanism may be that TTPGL significantly down-regulated mRNA expression of PTP1B and increased the protein levels of p-IRS-1/IRS-1 and p-Akt/Akt in IR adipocytes.     

Oxymatrine ameliorates high fat-induced insulin resistance in ApoE-/- mice

ATM: To investigate the effect of oxymatrine (OXY) on high fat-induced insulin resistance in mice, and to investigate the mechanism. METHODS: ApoE^-/-mice with high-fat diet for 16 weeks were divided into insulin resistance group, and OXY groups at concentrations of 25, 50 and 100 mg/kg. C57BL/6J mice served as normal control group. The mice in OXY groups were gavaged with OXY for 8 weeks. Glucose tolerance test in the mice was performed. Fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), fatty acid (FFA) and fasting insulin (FINS) in the plasma were measured. The mRNA expression of insulin receptor (INSR), insulin receptor substrate-2 (IRS-2), glucose transporter 2 (GLUT2) in the liver tissues was examined by RT-qPCR. The protein levels of GLUT2, INSR, IRS-2, p-INSR, p-IRS-2, PI3K, p-PI3K, serine/threonine protein kinase (AKT) and p-AKT were examined by Western blot.RESULTS: OXY reduced the levels of FBG, TC, TG, FFA and FINS, and attenuated insulin resistance. Compared with insulin resistance group, the mRNA expression of INSR, IRS-2 and GLUT2 significantly increased in OXY groups (P<0.05). The protein levels of p-INSR/INSR, p-IRS-2/IRS-2, p-PI3K/PI3K, p-AKT/AKT and GLUT2 also increased in OXY groups (P<0.05).CONCLUSION: OXY ameliorates high fat-induced insulin resistance in mice via PI3K/AKT pathway.     

Mechanism of interleukin-6 induced insulin resistance in 3T3-L1 adipocytes

AIM: To investigate the molecular mechanism of interleukin-6 induced insulin resistance in 3T3-L1 adipocytes.METHODS: 3T3-L1 adipocytes were treated with IL-6 at concentration of 20 μg/L within 48 hours. Insulin stimulated glucose uptake was measured by 2-deoxy ［3H］ glucose. Western blotting was used to measure insulin receptor substrate-1(IRS-1), protein kinase B(PKB) expression, tyrosine phosphorylation on IRS-1, and PKB phosphorylation. RESULTS: On basal status, glucose uptake in 3T3-L1 cells, PKB phosphorylation and tyrosine phosphorylation of IRS-1 were all at low level. Insulin stimulation induced a rapid increase in glucose uptake, PKB phosphorylation and IRS-1 tyrosine phosphorylation. IL-6 inhibited insulin-induced glucose uptake and PKB phosphorylation level about 50%. After IL-6 treatment, IRS-1 protein expression and tyrosine phosphorylation of IRS-1 were decreased 35% and 40%, respectively. The inhibitor of mammalian target of rapamycin(mTOR), rapamycin, reversed above effects of IL-6. CONCLUSION: IL-6 induced insulin resistance in 3T3-L1 adipocytes is related to decrease IRS-1 expression and impairs IRS-1 tyrosine phosphorylation. IL-6 induced insulin resistance in adipocytes may be related to the activity of mTOR.     

Effects of adiponectin siRNA on the glucose transport in 3T3-L1 adipocytes

AIM: To construct the adenovirus vector with adiponectin (Acrp30) siRNA, and to observe its effect on the Acrp30 expression and glucose transport in 3T3-L1 adipocytes. METHODS: Mouse Acrp30 siRNA fragment was designed, synthesized and cloned into the adenovirus vector. 3T3-L1 cells were infected with the two recombinant adenoviruses, respectively. The mRNA expression and protein levels of Acrp30 in these cells were evaluated by RT-PCR and ELISA. Glucose transport was measured by 2-Deoxy-［3H］-D-glucose incorporation method. RESULTS: The recombinant adenoviruses were successfully constructed. They remarkably downregulated the expression of Acrp30 at both mRNA and protein levels in 3T3-L1 cells, and decreased the glucose transport in 3T3-L1 adipocytes (P<0.05). CONCLUSION: The siRNA expression vectors effectively inhibit the expression of Acrp30 in 3T3-L1 adipocytes, and decrease the glucose transport.     

Role of asiatic acid in treatment of insulin resistance in mouse adipocytes

AIM：To investigate the effects of asiatic acid, one of triterpenoids from Psidium guajava leaves, on the proliferation and differentiation of 3T3-L1 preadipocytes, and glucose and lipid metabolism of insulin-resistant adipocytes. METHODS：The proliferation of 3T3-L1 preadipocytes was tested by MTT assay, and the accumulation of lipid droplets in differentiated preadipocytes was measured by oil red O staining. The insulin-resistant cell model was established by exposure of the cells to dexamethasone. The cellular glucose uptake was determined by glucose oxidase-peroxidase assay. The free fat acid (FFA) concentration was detected by colorimetric method. Secreted adiponectin were measured by ELISA. The protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and protein tyrosine phosphatase 1B (PTP1B) in insulin-resistant adipocytes were analyzed by Western blotting. RESULTS：Compared with medium group, asiatic acid increased the proliferation of 3T3-L1 preadipocytes and inhibited their differentiation at a concentration range of 10~100 μmol/L (P<0.05 or P<0.01). At concentrations of 30 μmol/L and 100 μmol/L, asiatic acid enhanced cellular glucose uptake in the insulin-resistant adipocytes both in basic and insulin-stimulation states. Asiatic acid decreased FFA production (P<0.05), and down-regulated the protein expression of PTP1B (P<0.05, or P<0.01). However, no effect on the secretion of adiponectin and the protein expression of PPARγ was observed (P>0.05). CONCLUSION：Asiatic acid enhances glucose uptake and inhibits FFA production in insulin-resistant adipocytes via down-regulating the protein expression of PTP1B, all of which play the roles of increasing insulin signaling sensitivity to improve insulin resistance.     

Effect of protein kinase C signal pathway on resistin expression in 3T3-L1 adipocytes

AIM：To investigate the effect of protein kinase C on resistin expression in 3T3-L1 adipocytes.METHODS：The differentiated 3T3-L1 adipocytes were incubated with 50 nmol/L phorbol 12-myristate 13-acetate (PMA) or 5 μmol/L Ro-31-8220 for 24 h.Expression of resistin mRNA was detected by RT-PCR and expression of resistin protein was detected by Western blotting.RESULTS：Compared with control,PMA increased the expression of resistin mRNA and protein in 3T3-L1 adipocytes significantly (P<0.01),while Ro-31-8220 decreased the expression of resistin mRNA and protein in 3T3-L1 adipocytes obviously (P<0.01).CONCLUSION：Protein kinase C signal pathway may regulate resistin expression in 3T3-L1 adipocytes.     

DHA induced cytotoxicity in NSCLC cells via inhibiting PI3K pathway activation and GLUT2 expression

AIM: To investigate the molecular mechanisms of cytotoxicity induced by dihydroartemisinin (DHA) in non-small cell lung cancer (NSCLC) cells. METHODS: NSCLC cell lines A549 and NCI-H1650 were treated with various concentrations of DHA for indicated time. Subsequently, the effects of DHA on the cell activity, colony formation ability and apoptosis were determined by MTT assay, colony formation assay, Annexin V-FITC/PI staining and flow cytometry, respectively. At the same time, the effects of DHA on glucose, ATP and lactate levels were assessed, and the PI3K pathway activation and glucose transporter 2(GLUT2) expression were detected by Western blot in the A549 cells and NCI-H1650 cells. Overexpression of GLUT2 and Rheb was established in A549 and NCI-H1650 cells by transfection with GST-GLUT2 and GST-Rheb plasmids, respectively, and the effects of DHA on cell activity, apoptosis, glucose level, ATP content and PI3K pathway activation were analyzed in A549 cells and NCI-H1650 cells. The effect of glucose deprivation on the cytotoxicity triggered by DHA in NSCLC cells was also determined. RESULTS: Compared with control group, DHA significantly inhibited cell activity and colony formation ability, and induced remarkable cell apoptosis in the A549 cells and NCI-H1650 cells. At the same time, DHA reduced ATP and lactate contents, and hindered glucose uptake in a time-and dose-dependent manner in A549 cells and NCI-H1650 cells. The activity of PI3K pathway and GLUT2 expression were downregulated, while upregulated GLUT2 expression and activated PI3K pathway reduced the cytotoxicity induced by DHA in NSCLC cells. Glucose deprivation increased DHA-mediated cytotoxicity in NSCLC cells. On the contrary, high levels of glucose inhibited DHA-mediated cytotoxicity in NSCLC cells. CONCLUSION: DHA restrains cell activity and colony formation, and induces apoptosis. DHA induces cytotoxicity via inhibiting PI3K pathway activation and GLUT2 expression, leading to inhibit glycolytic metabolism in NSCLC cells.     

Visfatin gene expression and secretion are regulated by aldosterone in 3T3-L1 preadipocytes and adipocytes

AIM: To explore the effect of aldosterone on visfatin gene expression and secretion in 3T3-L1 preadipocytes or adipocytes. METHODS: Aldosterone at concentration of 10-8 or 10-6 mol/L with or without 10-6 mol/L spironolactone was added to cultured 3T3-L1 preadipocytes or adipocytes for 24 h or 48 h. The mRNA levels of visfatin and mineralocorticoid receptor were measured using real time PCR. The concentration of visfatin in the culture medium was determined by ELISA. RESULTS: In 3T3-L1 preadipocytes treated with aldosterone, the mRNA expression of visfatin reduced and the mRNA expression of mineralocorticoid receptor(MR) increased, but the concentration of visfatin in culture medium was not regulated significantly by aldosterone. In adipocytes with aldosterone treatment, the mRNA expression of visfatin and visfatin concentration in culture medium reduced, and mRNA expression of MR increased. The effect of aldosterone was blocked by spironolactone to some extent. CONCLUSION: Aldosterone inhibits the gene expression and protein secretion of visfatin in 3T3-L1 adipocytes.     

Effects of propofol on lung injury and PI3K/Akt pathway in rats after liver ischemia and reperfusion

AIM：To study the effect of propofol on phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway in the model of rat lung injury after hepatic ischemia and reperfusion (IR). METHODS：Sixty-six SD rats were randomly divided into 4 groups：sham operation group (n=6), IR group (n=24), propofol group (n=24) and propofol+wortmannin group (n=12). The rats in IR group and propofol group were further divided into 4 subgroups according to the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The rats in propofol+wortmannin group were also divided into 2 subgroups according to the time points of 3 h and 6 h after reperfusion. The rats in sham group were only dissected porta without ligation. The ligation of hepatic pedicle in the rats in IR group was performed to induce liver ischemia for 30 min and then reperfusion was conduced. The rats in propofol group were given slow injection of propofol (20 mg/kg) from the caudal vein 10 min before ischemia, and then propofol was continuously pumped at dose of 20 mg·kg-1·h-1 until rats' death. Other procedures were the same as the rats in IR group. The rats in propofol+wortmannin group were injected with wortmannin, a blocker of PI3K, at dose of 15 μg/kg before ischemia, and also given the injection of propofol as the rats in propofol group. The lung tissues of the rats were collected at the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The protein levels of total Akt (t-Akt), phosphorylated Akt (p-Akt) and Bcl-2 in the lung tissues were detected by Western blotting. The apoptotic rate was analyzed by flow cytometry with annexin V-FITC/PI staining. RESULTS：Compared with sham group, the protein levels of p-Akt and Bcl-2, and the cell apoptotic rate in the lung tissues in IR group, propofol group and propofol+wortmannin group increased. Compared with IR group, the protein levels of p-Akt and Bcl-2 increased, and cell apoptotic rate of the lung tissues decreased in propofol group. Compared with propofol group, the protein levels of p-Akt and Bcl-2 decreased, and cell apoptotic rate in the lung tissues increased in propofol+wortmannin group. CONCLUSION：Propofol reduces the lung injury in rats induced by liver ischemia and reperfusion, and its mechanism may be involved in PI3K/Akt signaling pathway.     

Knockdown of growth hormone receptor prevents growth hormone induced inflammatory cytokine production in 3T3-L1 adipocytes

AIM: To investigate the effect of growth hormone receptor (GHR) knockdown on nuclear factor-κB (NF-κB) activity and inflammatory cytokine production stimulated by growth hormone (GH) in 3T3-L1 adipocytes. METHODS: The specific siRNA for GHR was transfected into 3T3-L1 adipocytes to silence GHR expressions. The effects of GH on NF-κB activation and inflammatory cytokine production in 3T3-L1 adipocytes transfected with siRNA-GHR or siRNA-control were measured by dual-luciferase system analysis, real-time RT-PCR and ELISA. RESULTS: The protein expression of GHR was diminished after transfection with GHR specific siRNA. Dual-luciferase reporter system analysis revealed that GHR knockdown resulted in attenuation of GH-stimulated NF-κB activation in the 3T3-L1 adipocytes. GHR knockdown ameliorated the GH-induced production of inflammatory cytokines TNF-α, IL-1β, IL-6, MCP-1 and MIP-1α in the 3T3-L1 adipocytes. CONCLUSION: Knockdown of GHR might be efficacious to prevent GH-induced inflammatory responses in the 3T3-L1 adipocytes.     

Mechanism of acylation stimulating protein resistance induced by fatty acid in 3T3-L1 adipocytes and preadipocytes

AIM: To evaluate the potential acylation stimulating protein (ASP) resistance in both adipocytes and preadipocytes under the conditions by which insulin resistance is produced by the stimulation of free fatty acids (FFA), and to explore the mechanism of ASP resistance on post-receptor level. METHODS: 3T3-L1 preadipocytes were induced to differentiate. Then the cells were treated with oleate or palmitate at concentration of 0 mmol/L (FFA-free DMEM/F12), 0.125 mmol/L, 0.5 mmol/L or 1.0 mmol/L overnight. Glucose transport was assessed by ［3H］ 2-deoxyglucose uptake to evaluate insulin resistance and ASP resistance. Both non-FFA treated and FFA treated 3T3-L1 cells were cultured with ASP at concentration of 5.0 μmol/L for 4 h, then the cell proteins were extracted, and the expressions of guanine nucleotide binding protein beta (Gβ), guanine nucleotide-binding protein alpha-q/11(Gαq/11), phosphorylated-protein kinase Cα (p-PKCα) and phosphorylated-protein kinase Cζ (p-PKCζ) were measured by Western blotting. RESULTS: Both adipocytes and preadipocytes were responsive to ASP. ASP stimulation increased glucose transport by 198% in adipocytes and by 287% in preadipocytes (P<0.01 vs PBS). FFA at concentration of 0.125 mmol/L did not change ASP-stimulated glucose transport significantly, but high dose of oleate or palmitate effectively reduced the ASP response with a significant reduction by 47% (P<0.05 for oleate) and 34% (P<0.05 for palmitate) at 1 mmol/L FFA in adipocytes. Similarly in preadipocytes, glucose uptake rates were decreased by 43% (P<0.05 for oleate) and 62% (P<0.01 for palmitate) at 1 mmol/L FFA. Effects were comparable to those obtained with insulin. After overnight incubation with oleate or palmitate in adipocytes and preadipocytes, Gβ, Gαq/11, p-PKCα and p-PKCζ were downregulated both in the absence of ASP treatment and in the presence of ASP treatment in adipocytes. At concentration of 1.0 mmol/L, oleate inhibited the expressions of ASP-induced Gβ, Gαq/11, p-PKCα and p-PKCζ in adipocytes by 47％, 44%, 39% (P<0.05, P<0.01) and 20% (P>0.05), respectively. Palmitate also effectively blocked the expressions of ASP (at concentration of 1.0 mmol/L)-induced Gβ, Gαq/11, p-PKCα and p-PKCζ by 50%, 43%, 44% and 43% (P<0.05, P<0.01) in adipocytes. In preadipocytes, oleate only inhibited ASP-induced p-PKCα and p-PKCζ significantly by 39% and 19%, respectively (P<0.05). However, overnight exposure of 3T3-L1 preadipocytes to 1 mmol/L palmitate leaded to 45%, 50%, 52% and 21% (P<0.05, P<0.01) inhibition of ASP-induced expressions of Gβ, Gαq/11, p-PKCα and p-PKCζ, respectively. CONCLUSION: Oleate and palmitate inhibit ASP-mediated stimulation of glucose transport both in adipocytes and preadipocytes. The study provides direct evidence of ASP resistance under the condition of insulin resistance induced by FFA in a cellular model. The mechanism of action involves both changes in expression of C5L2 as well as signaling parameters. Fatty acid-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance and obesity phenotype.     

Regulatory effects of reactive oxygen species on the production of PAI-1 in 3T3-L1 adipocytes and its related possible mechanisms

AIM: To investigate the regulatory effects of reactive oxygen species (ROS) on the production of plasminogen activator inhibitor 1 (PAI-1), and try to determine the signaling cascades involved in it. METHODS: 3T3-L1 cells were cultured and differentiated into mature adipocytes. Cell viability was measured by MTT. The PAI-1 mRNA expression levels were evaluated by quantitative real-time PCR. Quantification of the PAI-1 protein levels secreted into conditioned medium was performed by multiplex immunoassay and sandwich ELISA. The phosphorylation status of protein kinases was determined by Bio-Plex phosphoprotein assays. RESULTS: In 3T3-L1 adipocytes, H2O2 significantly augmented the expression of PAI-1. Also, H2O2 activated several signaling pathways including ERK1/2, JNK, Akt, p70 S6K and JAK/STAT. Verified by protein kinase inhibitors, Akt, JAK/STAT and ERK1/2 may participate in the H2O2-induced increase in PAI-1. CONCLUSION: H2O2 markedly up-regulates the production of PAI-1 in 3T3-L1 adipocytes via some intracellular signaling pathways such as Akt, JAK/STAT and ERK1/2.     

Expression of programmed cell death factor 4 in pulmonary fibrosis model

AIM:To detect the expression of programmed cell death protein 4 (PDCD4) in pulmonary fibrosis model and its effect on cell viability and pulmonary fibrosis indicators, and to explore its mechanism. METHODS:The expression level of PDCD4, α-smooth muscle actin (α-SMA) and collagen type I (COL-I) in human embryonic lung fibroblasts cell line HFL-1 group and HFL-1+TGF-β1 group were detected by Western blot and RT-qPCR. The plasmid pEZ-M03-PDCD4 and empty vector pEZ-M03 were transfected into myofibroblasts (MB), and the protein expression level of PDCD4 was detected by Western blot. The protein levels of p-AKT and AKT in blank control group, pEZ-M03-PDCD4 group, pEZ-M03 group and LY294002 group and the expression of cell cycle-related proteins c-Myc and cyclin D1 were determined by Western blot. The effect of PDCD4 on the viability of MB was measured by CCK-8 assay. The hydroxyproline content in the culture supernatant of HFL-1 cells and transfected MB was detected by hydroxyproline digestion method. The expression of PDCD4 in the lung tissues of the mice in model group and control group was detected by Western blot. RESULTS:Compared with HFL-1 group, the expression of α-SMA and COL-I at mRNA and protein levels in HFL-1+TGF-β1 group was significantly increased (P<0.01), the mRNA expression of PDCD4 was not significantly changed, while PDCD4 protein was significantly down-regulated (P<0.01). Compared with blank control group and pEZ-M03 group, the protein expression of PDCD4 in pEZ-M03-PDCD4 group was significantly increased (P<0.05), the protein expression of c-Myc and cyclin D1 was significantly decreased (P<0.05), the cell viability was also significantly inhibited (P<0.01), and the content of hydroxyproline in the culture supernatant was significantly reduced (P<0.05). Compared with blank control group, the protein levels of p-AKT were significantly decreased in pEZ-M03-PDCD4 group and LY294002 group, and no significant difference between blank control group and pEZ-M03 control group was observed. Compared with control group, PDCD4 expression was decreased in model group (P<0.01).CONCLUSION:PDCD4 is low expressed in the process of pulmonary fibrosis. Over-expression of PDCD4 inhibits the viability of MB, decreases the content of hydroxyproline, and inhibits the PI3K/AKT signaling pathway.     

Effect of lentinan on apoptosis and PI3K/AKT signaling pathway in leukemic HL-60 cells in vitro

AIM:To study of the regulatory effect of lentinan on human leukemic HL-60 cell apoptosis and its effect on PI3K/AKT signaling pathway in HL-60 cells in vitro.METHODS:Lentinan at concentrations of 0 mg/L, 15 mg/L, 30 mg/L and 45 mg/L was applied to HL-60 cells cultured to the logarithmic phase in vitro, and the inhibitory effect of lentinan on the viability of HL-60 cells was measured by MTT assay after 24 h, 48 h and 72 h. The apoptosis induced by lentinan was analyzed by flow cytometry. The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3, cleaved caspase-8, cytochrome C, PI3K, AKT and p-AKT were determined by Western blot. After treatment with PI3K inhibitor LY294002 at 5 mg/L for 72 h, the apoptosis of HL-60 cells was analyzed by flow cytometry. RESULTS:The viability of HL-60 cells was inhibited after treatment with lentinan at concentrations of 15 mg/L, 30 mg/L and 45 mg/L for 24 h, 48 h and 72 h in concentration-dependent and time-dependent manners (P<0.05). The apoptosis of HL-60 cells was promoted after treatment with lentinan (15 mg/L, 30 mg/L and 45 mg/L) for 72 h in a concentration-dependent manner (P<0.05). The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3 and cytoplasmic cytochrome C in the HL-60 cells induced by 30 mg/L lentinan were increased significantly with the increase in the treatment time (P<0.05), but caspase-8 did not show any change. The protein levels of PI3K, AKT and p-AKT were decreased obviously with the increase in the lentinan concentration (P<0.05). Treatment of HL-60 cells with LY294002, a PI3K pathway inhibitor, produced apoptosis-inducing effect similar to lentinan (P<0.05). CONCLUSION:Lentinan induces HL-60 cell apoptosis by inhibiting PI3K/AKT signaling pathway.     

Effect of FGFR1 expression knock-down on biological characteristics of infantile hemangioma endothelial cells

AIM: To study the effect of fibroblast growth factor receptor 1 (FGFR1) expression knock-down on the viability, apoptosis, invasion and migration of infantile hemangioma endothelial cells (HemECs). METHODS: FGFR1 was down-regulated by FGFR1 small interfering RNA (si-FGFR1) transfection. The viability of the cells was measured by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry and the invasion and migration abilities were determined by Transwell assay. The protein levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and phosphorylated AKT (p-AKT) were examined by Western blot. RESULTS: Transfection of si-FGFR1 into HemECs had significant effects on inhibiting cell viability (P<0.05), promoting apoptosis (P<0.05), and decreasing cell invasion and migration abilities (P<0.05). The results of Western blot showed that knockdown of FGFR1 gene expression in the cells reduced the protein levels of PI3K and p-AKT (P<0.05), and had no significant effect on AKT protein level. CONCLUSION: Knock-down of FGFR1 expression changes the biological characteristics of endothelial cells in infantile hemangiomas by regulating PI3K/AKT signaling pathway.     

Effects of apelin-13 on oxidative stress induced by high uric acid in adipocytes

AIM: To study the effects of apelin-13 on oxidative stress induced by high uric acid in 3T3-L1 adipocytes and its underlying mechanisms. METHODS: 3T3-L1 adipocytes were stimulated with uric acid at 10 mg/dL for 48 h. Some of the adipocytes were administered with 1 μmol/L apelin-13 in the presence of uric acid at 10 mg/dL. The adipocytes stimulated with 100 μmol/L H₂O₂ were served as positive controls. The intracellular reactive oxygen species (ROS) concentrations were detected by flow cytometry. The biochemical kits were used to measure the activities of superotide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and NADPH oxidase (NOX) activity, and the content of malondialdehyde (MDA) in the cell lysate and the supernatant. The mRNA levels of renin-angiotensin system (RAS) components, including angiotensinogen (AGT), angiotensin-converting enzyrne1 (ACE1), angiotensin II type 1 receptor (AT1R) and AT2R, as well as angiotensin II receptor -like 1 (APJ) were measured by real-time PCR. The concentrations of angiotensin II (AngⅡ) in the cell lysate and the supernatant were measured by ELISA. RESULTS: Adipocytes stimulated with uric acid at 10 mg/dL had lower activities of antioxidant enzymes (SOD, GSH-PX and CAT) and higher levels of NOX activity and MDA content (P < 0.05). Accordingly, the intracellular ROS levels were found to be dramatically increased. However, apelin-13 administration attenuated uric acid-induced oxidative stress in the 3T3-L1 adipocytes. Uric acid at 10 mg/dL upregulated the mRNA expression of local RAS, enhanced AngⅡ concentrations both in the cell lysate and the supernatant, and down-regulated the mRNA level of APJ in the adipocytes (P < 0.05). Conversely, apelin-13 partially reversed these parameters. CONCLUSION: Apelin-13 attenuates oxidative stress induced by uric acid, may be via down-regulation of local RAS expression in the 3T3-L1 adipocytes.