Quercitrin promotes apoptosis of gastric cancer cell line SGC7901 by inhibiting PI3K/AKT signaling pathway

AIM: To investigate whether quercitrin induces apoptosis of gastric cancer cell line SGC7901 by inhibition of PI3K/AKT signaling pathway. METHODS: The human gastric cancer SGC7901 cells were selected as the research object. The cytotoxicity of quercitrin was detected by MTT assay, and IC₅₀ value of quercitrin was calculated. The SGC7901 cells were divided into control group, quercitrin group (incubated with 200 μmol/L quercitrin), insulin-like growth factor-1 (IGF-1) group (incubated with 100 μg/L IGF-1) and quercitrin+IGF-1 group (incubated with 200 μmol/L quercitrin and 100 μg/L IGF-1). After 48 h, the apoptosis of SGC7901 cells was analyzed by flow cytometry, and the protein levels of cleaved caspase-3, p-AKT (Ser473), AKT, p-PI3K (Tyr508) and PI3K were determined by Western blot. RESULTS: The viability of SGC7901 cells was significantly decreased as the concentration of quercitrin increased, starting at 100 μmol/L (P<0.05). The IC₅₀ value of quercitrin for 48 h was 275.40 μmol/L. After treatment with 200 μmol/L quercitrin for 48 h, the apoptosis rate and the protein level of cleaved caspase-3 in quercitrin group were significantly increased (P<0.05), and the phosphorylated levels of AKT and PI3K were significantly decreased compared with control group (P<0.05). Treatment with quercitrin and IGF-1 inhibited the effect of quercitrin on SGC7901 cells compared with quercitrin group. CONCLUSION: Quercitrin may induce apoptosis of gastric cancer cell line SGC7901 by inhibiting the activation of PI3K/AKT signaling pathway.     

Effects of leptin on hypoxia-induced apoptosis in cultured alveolar typeⅡ cells of fetal rat and its mechanism

AIM: To investigate the effects of leptin (LEP) on the alveolar type Ⅱ cells(AECⅡ) apoptosis induced by Na₂S₂O₄ and explore the molecular mechanisms. METHODS: Primary AECⅡ culture was prepared according to a specific immunosorption procedure with slight modification and the cells were identified by transmission electron microscope and immunocytochemistry. AECⅡ damage was induced by 5 mmol/L Na₂S₂O₄. LEP group cells were treated with LEP at concentrations from 100 μg/L to 1 600 μg/L. The cell survival rate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell cycle and apoptosis were analyzed by flow cytometry and the level of caspase-3 was measured by Western blotting. RESULTS: Highly purified AECⅡ, obtained by the method of modified immunosorption, were identified with the positive expression of SP-A and intracellular lamellarbodies were found under electron micrography. The cells, exposed to 5 mmol/L Na₂S₂O₄, showed characteristic changes of apoptosis and activation of caspase 3. These damages were relieved by the treatment of LEP (100-1 600 μg/L), with survival increasing, apoptosis peak decreasing, cell morphology restoring and caspase 3 activation inhibiting.CONCLUSION: Leptin prevents AECⅡ from apoptosis induced by Na₂S₂O₄ or hypoxia. The potential mechanism of its action may be related to promoting cell cycle from G₁ phase to S phase and inhibiting the activating of caspase 3.     

Inhibitors of CaMKⅡ suppress the expression of TNF-α, TGF-β1 and collagen I,III in cardiac fibroblasts treated with angiotensin II or electrical field stimulation

AIM: To observe the role of calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) in the proliferation, released cytokines and expression of collagen Ⅰ and Ⅲ in rat cardiac fibroblasts induced by angiotensin Ⅱ (AngⅡ) or electrical field stimulation (EFS).METHODS: The cultured cardiac fibroblasts were isolated from the neonatal rats of 1-3 days and used in the 3rd passage. The cells were divided into 10 groups: control group, 0.1 μmol/L AngⅡ group, 0.1 μmol/L AngⅡ+0.5 μmol/L KN92 group, 0.1 μmol/L AngⅡ+0.5 μmol/L KN93 group, 0.1 μmol/L AngⅡ+0.5 μmol/L AIP group; 10V 1.0 Hz EFS group, 10 V 1.0 Hz EFS+0.5 μmol/L KN92 group, 10 V 1.0 Hz EFS+0.5 μmol/L KN93 group, 10 V 1.0 Hz EFS+0.5 μmol/L AIP group, 10 V 1.0 Hz EFS+0.1 μmol/L AngⅡ group.MTT was used to detect the proliferation of cardiac fibroblasts. The release of cytokines was measured by ELISA. The mRNA expression of TNF-α, TGF-β₁ and collagen Ⅰ, Ⅲ was determined by RT-PCR.RESULTS: CaMKⅡ inhibitors (0.5 μmol/L KN93 or 0.5 μmol/L AIP) prevented the proliferation and the increase in the expression of TGF-β₁ and TNF-α in cardiac fibroblasts induced by AngⅡ (0.1 μmol/L) or EFS (10 V 1.0 Hz). CaMKⅡ inhibitors (0.5 μmol/L AIP or 1.0 μmol/L AIP) also prevented the increase in mRNA expression of collagen Ⅰ and Ⅲ induced by 0.1 μmol/L AngⅡ. CONCLUSION: Inhibition of CaMKⅡ prevents the proliferation of cardiac fibroblasts induced by AngⅡ or EFS. The possible mechanism of CaMKⅡ inhibitors may be involved in preventing the mRNA expression and release of cytokines (TGF-β₁ and TNF-α), and regulating collagen I and III expression.     

Effects of TNF-α-mediated insulin resistance on INSIG2 and SCAP expressions in vivo

AIM: To investigate the effects of TNF-α induced insulin resistance (IR) on INSIG1, INSIG2, SCAP and SREBP expressions in mice. METHODS: Male C57BL/6J mice were randomly divided into 4 groups. The mice were given an intraperitoneal injection of TNF-α (6 μg·kg-1·d-1; 3 μg·kg-1·d-1 and 1 μg·kg-1·d-1) and saline (NC group) twice daily for 7 d. The insulin sensitivity and glucose metabolism in awaken mice were evaluated by intravenous glucose tolerance test (IVGTT). The mRNA expression and protein levels of gene were measured by RT-PCR and Western blotting. RESULTS: After TNF-α treatment, fasting blood glucose (FBG), plasma insulin and free fatty acids (FFA) were significantly elevated in TNF-α (6 μg·kg-1·d-1) group compared to NC, TNF-α (1 μg·kg-1·d-1) and TNF-α (3 μg·kg-1·d-1) groups (P<0.01 and P<0.05, respectively). There was a lower glucose tolerance in TNF-α (6 μg·kg-1·d-1) group than that in other three groups during IVGTT. In TNF-α (6 μg·kg-1·d-1) group, the insulin release of glucose-stimulation was higher than that in NC and TNF-α (1 μg·kg-1·d-1) groups (P<0.01 and P<0.05). The INSIG2 mRNA expression of adipose tissues in TNF-α (6 μg·kg-1·d-1) group was significantly increased compared with NC group (P<0.01), and INSIG2 protein levels were also increased (P<0.05). In TNF-α treatment mice, SCAP mRNA level in adipose tissues was significantly up-regulated than that in the controls (P<0.05). The mRNA expressions of INSIG1 and SREBP1 in two groups were not significantly changed (P>0.05). CONCLUSION: In TNF-α induced insulin resistance, INSIG2 and SCAP may be involved in the pathways of lipid metabolism.     

Effects of leptin on hypoxia-reoxygenation induced apoptosis in human L02 cells and expression of Fas and FasL

AIM: To observe the effect of leptin (LEP) on hypoxia-reoxygenation induced apoptosis in L02 cells.METHODS: In the experiment, L02 cell injury was induced by hypoxic air (95%N2 and 5% CO2). The cultured L02 cells were divided into hypoxic 12 h group (IR group) alone, normal control group and the hypoxic plus leptin (100 μg/L, 200μg/L, 400 μg/L, 800 μg/L and 1 600 μg/L) treatment groups in vitro. Flow cytometry, terminal dUTP nick-end labeling (TUNEL) assay and fluorescent quantitative PCR were used to measure the changes of apoptosis in L02 cells and expression of Fas/FasL mRNA.RESULTS: (1) The percentage of L02 cells apoptosis and positive TUNEL cells significantly increased in IR group compared to control group (P<0.01). When L02 cells were treated with LEP, the percentage of cell apoptosis and positive TUNEL cells were decreased compared to IR group. (2) Compared to control group, the Fas/FasL mRNA expression significantly increased in IR group (P<0.01). When L02 cells were treated with LEP, the Fas/FasL mRNA expression decreased compared to IR group, the effect of LEP at concentration of 400 μg/L was obviously (P<0.05). CONCLUSION: LEP decreases the apoptosis of hypoxic-reoxygenation L02 cells by down-regulation of Fas/FasL mRNA expression in L02 cells.     

Effect of recombinant MIF on lung fibroblast proliferation and collagen synthesis in vitro

AIM: To investigate the influence and mechanism of recombinant macrophage migration inhibitory factor (rMIF) on fibroblasts. METHODS: MRC-5 fibroblasts were divided into two groups: the treated group was treated with rMIF (25-100 μg/L, 12 h, 24 h or 48 h) and the control was non-rMIF treatment. The activity of proliferation in both groups was investigated and compared by CCK-8 means. Synthesis of collagen in the culture supernatants was detected by the hydroxyproline. The expression of collagen type I mRNA was examined using RT-PCR analysis. The level of collagen type I protein induced by rMIF was quantified by Western blotting. RESULTS: The production of proliferation ratio of fibroblasts treated with 50 μg/L and 100 μg/L rMIF at 24 h or 48 h were increased obviously (P<0.05 or P<0.01). The collagen synthesis significantly increased after stimulation with 100 μg/L rMIF for 48 h (P<0.01). rMIF significantly increased the expression of collagen type I mRNA and protein in a dose dependent manner compared with control (P<0.05 or P<0.01). CONCLUSION: rMIF upregulates the proliferation of fibroblasts and has an effect on the production of collagen. These results suggest that MIF may play important roles in the pathogenesis of airway remodeling.     

Improving effects of ginsenoside Rb1 on glucose metabolism in cardiomyocytes under hypoxia by hypoxia-inducible factor 1α

AIM: To elucidate the effect of ginsenoside Rb1 (Gs-Rb1) on the glucose metabolism to improve the viability of the cardiomyocytes under hypoxia, and whether hypoxia-inducible factor 1α (HIF-1α) and/or AMPKα are involved in the process.METHODS: The neonatal rat cardiomyocytes were cultured, and randomly divided into control group, hypoxia (1% O₂, 94% N₂ and 5% CO₂) group, Gs-Rb1 (200 μmol/L) group, Ara-A (500 μmol/L) group, Gs-Rb1+Ara-A group, YC-1 (5 μmol/L) group, Gs-Rb1+YC-1 group, Ara-A+YC-1 group and Gs-Rb1+YC-1+Ara-A group. After the intervention for 8 h, the cell viability was analyzed by MTT assay. The protein levels of AMPK, HIF-1α and glucose transporter-4 (GLUT-4) were determined by Western blot. The activities of heterophosphatase (HK), phosphofructokinase (PFK) and lactic dehydrogenase (LDH) were measured by ELISA.RESULTS: Gs-Rb1 significantly improved the viability of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 and Ara-A. In addition, YC-1 and Ara-A had a synergistic effect. Gs-Rb1 increased the protein levels of AMPK and HIF-1α in the hypoxic cardiomyocytes, which was significantly inhibited by Ara-A and YC-1. Gs-Rb1 significantly increased the expression of GLUT-4 on the cytomembrane of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 or Ara-A, especially Ara-A+YC-1. Gs-Rb1 significantly increased the activities of HK, PFK and LDH, all those were significantly inhibited by YC-1 or Ara-A. Besides, YC-1 and Ara-A had a synergistic effect.CONCLUSION: Gs-Rb1 improves the viability of hypoxic cardiomyocytes, which may be related to the regulation of glucose uptake and enhancement of glycolysis by synergy of both HIF-1α and AMPK.     

Relationship between zinc and zinc transporter expression in human lung cancer cells

AIM: To study the changes of zinc transporter gene expression in A-549 cell line exposed to ZnCl2 and N,N,N’,N’-tetrakis 2-pyridylmethyl ethylenediamine (TPEN).METHODS: Human lung cancer cell line A-549 was exposed to different concentrations of ZnCl2 (0, 50, 100, 150, 200 μmol/L) and TPEN (0, 5, 10, 15, 20 μmol/L), respectively. Twelve hours later, the cell viability was measured by MTT (methyl thiazolyl tetrazolium) and levels of zinc transporter mRNA was detected by RT-PCR. Zinquin was used to estimate the intracellular zinc concentrations.RESULTS: A-549 cell viability rate was significantly decreased when exposed to ZnCl2 at concentrations of 150 and 200 μmol/L, and to TPEN. The intracellular zinc concentration was significantly increased when exposed to ZnCl2 and decreased when exposed to TPEN. Zinc transporter (ZnT-1) mRNA level was increased along with the increase in the concentration of ZnCl2 but decreased when exposed to TPEN. The expressions of ZIP-1 and ZIP-10 (Zrt-and Irt-like protein) were increased along with the increase in the concentration of TPEN but decreased when exposed to ZnCl2.CONCLUSION: ZnT-1 expression is induced by zinc supplement. ZIP-1 and ZIP-10 expressions are induced by zinc deficiency and repressed by zinc supplement.     

The relationship between serum IGF-1, IGFBP-3 levels,bone mineral density and bone metabolic markers in postmenopausal patients with osteoporosis

AIM:To study the relationship between serum insulin-like growth factor- 1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3) levels, bone mineral density(BMD) and bone metabolic markers in postmenopausal women. METHODS:Serum IGF-1, IGFBP-3, osteocalcin(BGP), isomeric C-telopeptide of type I collagen (β-CTX), estradiol(E₂), calcitonin(CT), parathormone(PTH), calcium (Ca), and phosphorus(P) were measured in 90 postmenopausal osteoporosis patients and 70 healthy postmenopausal women. BMD of lumbar vertebra and left femoral neck were determined by dual energy X-ray absorptiometry.RESULTS:BMD of lumbar vertebra and left femoral neck decreased significantly (P<0.01), serum IGF-1, IGFBP-3, E₂, CT and BGP decreased significantly (P<0.01), serum β-CTX and PTH increased significantly in postmenopausal osteoporosis group (P<0.01). There were no significantly differences in serum Ca, P between two groups (P>0.05). BMD of lumbar vertebra and left femoral neck were positively correlated with serum IGF-1, IGFBP-3, E₂, CT and BGP, but negatively correlated with β CTX and PTH. There were no correlation with serum Ca, P and BMD.CONCLUSION:Serum IGF-1, IGFBP-3, E₂, CT, BGP, β-CTX and PTH level were correlated with BMD of lumbar vertebra and left femoral neck. These markers can be one of the valuable evidences for screening osteoporosis in postmenopausal women.     

Effect of extracts of ginkgo biloba leaves on activation of RBL-2H3 cells

AIM: To investigate the mechanism of extracts of ginkgo biloba leaves (EGB) on degranulation from mast cells. METHODS: Wistar rats were randomly divided into 6 groups, including normal group, control group, high dose of EGB group (30 mg/kg), moderate dose of EGB group (10 mg/kg), low dose of EGB group (3 mg/kg) and positive control medicine group (azelastine, 5 mg/kg), each group had 10 rats (half female and half male). The experiment of passive cutaneous anaphylaxis reaction (PCA) and colorimetry were used to determine the effect of EGB on degranulation of mast cells in vivo. For in vitro study, various doses of EGB (200 mg/L, 100 mg/L and 50 mg/L) were added to the culture medium of RBL-2H3 cells cultured with 200 μg/L of dinitrophenyl (DNP) specific IgE overnight. The azelastine (30 mg/L) was selected as the positive control. The antigen (DNP-human serum albumin, DNP-HSA, 20 μg/L)-induced release of degranulation was measured by enzymatic assay, histamine by EIA, leucotriene C4 (LTC4), interleukin-4(IL-4) and tumor necrosis factor-α (TNF-α) by ELISA, respectively. In addition, the effect of EGB on phosphorylation of Akt and p38 was observed by Western blotting. RESULTS: The results showed that treatments with high dose of EGB (30 mg/kg) and moderate dose of EGB (10 mg/kg) were followed by a decrease in PCA of rats. All doses of EGB (200 mg/L, 100 mg/L and 50 mg/L) obviously suppressed the degranulation from RBL-2H3 cells, whereas results in high dose group (200 mg/L) and moderate dose of EGB group (100 mg/L) indicated significantly inhibitory effect on β-hexosaminidase, histamine, LTC4, IL-4,TNF-α, phosphorylation of Akt and p38 of RBL-2H3 cells induced by antigen. CONCLUSION: EGB may suppress the anaphylactic reaction by inhibiting the action of mast cells. Akt and p38 at least in part contribute to this event.     

Effect of siRNA on myocarditis induced by MCMV in mice

AIM: To investigate the effect of siRNA on myocarditis induced by MCMV in BALB/c mice. METHODS: One handred and twenty male BALB/c mice (4 weeks old) were divided randomly into 6 groups with 20 mice each. Among these groups, one normal control group, one viral control group and 4 experimental groups were assigned. Mice in viral control and 4 experimental groups were inoculated intraperitoneally injection (ip) with 100 μL of DMEM medium containing TCID5010-4 MCMV, and followed by 200 μL of different doses of liposomally endocapsulated siRNA in 4 experimental groups and by 200 μL of vehicle solution of siRNA in viral control group 30 min later. Mice in normal control group were first inoculated ip 100 μL of DMEM medium and followed by 200 μL of vehicle solution of siRNA 30 min later. At day 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 of postinfection, all mice were killed and blood samples were collected for detecting serum zymogram (CK, CK-MB, GOT, LDH and LDH₁/LDH₂) and the anti-cardiac β₁ receptor autoantibody titer level. Hearts were removed aseptically for detecting cardiac histological lesions and ultrastructure changes. RESULTS: Decreased morbidity of myocarditis, lightened cardiac pathologic lesions and ultrastructural changes in 4 experimental groups were observed. Serum zymogram (CK, CK-MB, GOT, LDH and LDH₁/LDH₂) and β₁ autoantibody titer level in viral control group were significantly higher than those in normal control group, but zymogram and β₁ autoantibody titer levels in 4 experimental groups were between those in viral control group and normal control group. The activities of CK, CK-MB, GOT, LDH and LDH₁/LDH₂ and β₁ autoantibody titer level decreased accompanied with siRNA dose increasing. These effects were in a dose dependent manner. CONCLUSION: siRNA protects myocardial tissue against MCMV infection.     

Effect of HMGA2 gene on high glucose-induced apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway

AIM:To investigate the effect of HMGA2 down-regulation on apoptosis and Notch signaling pathway in renal tubular epithelial cells exposed to high glucose (HG). METHODS:D-glucose at 5, 10, 20 and 30 mmol/L was used to stimulate human renal tubular epithelial HK-2 cells for 2 h, and D-glucose at 30 mmol/L was used to stimulate the HK-2 cells for 10 min, 60 min and 120 min. The protein expression of HMGA2 was determined by Western blot. The HK-2 cells were divided into normal glucose (NG) group, HG group, HG+si-HMGA2 group and HG+NC group, in which siRNA was transfected by Lipofectamine^TM 2000 for 48 h. Flow cytometry was used to analyze the apoptotic rate, reactive oxygen species (ROS) assay kit was used to detect ROS content, and Western blot was used to detect the protein levels of Notch1, Hes1 and Bcl-2. The HK-2 cells were treated with the Notch signaling pathway inhibitor DAPT, and then the cells were divided into HG group, HG+DAPT group and HG+si-HMGA2+DAPT group. The apoptotic rate was analyzed by flow cytometry. RESULTS:Exposure of the HK-2 cells to D-glucose at different concentrations for different time significantly increased the expression of HMGA2 (P<0.05). Compared with NG group, the protein expression of HMGA2, Notch1 and Hes1 in HG group was increased, the expression of Bcl-2/Bax was decreased, the apoptotic rate was increased, and the content of ROS was increased obviously (P<0.05). Compared with HG group, the protein expression of HMGA2, Notch1 and Hes1 of HG+si-HMGA2 group was decreased, the expression of Bcl-2/Bax was increased, the apoptotic rate was decreased, and the content of ROS was decreased significantly (P<0.05). The apoptotic rate in HG+DAPT group was significantly lower than that in HG group, while the apoptotic rate in HG+si-HMGA2+DAPT group was significantly lower than that in HG+DAPT group (P<0.05). CONCLUSION:Down-regulation of HMGA2 expression inhibits the apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway and decreasing ROS production.     

Properties of ACh-sensitive BK channels in mice type Ⅱ vestibular hair cells and its modulation by calcium

AIM: To explore the properties of the acetylcholine (ACh)-sensitive potassium channel in type Ⅱ vestibular hair cells (VHCs Ⅱ) in mice saccular macula and the modulation effect of calcium ions.METHODS: Under the whole-cell patch mode,the pharmacology properties of ACh-sensitive potassium channel and the modulation of calcium ions on ACh-sensitive potassium channel were investigated.RESULTS: Following extracellular perfusion of ACh,VHCs Ⅱ displayed a slow and sustained outward current,which was sensitive to tetraethylammonium (TEA,5 mmol/L) and charybdotoxin (CTX,100 nmol/L),but not sensitive to 4-aminopyride (4-AP,15 μmol/L).ACh-sensitive potassium current was inhibited by intracellular application of ethylene glycol-bis (B-aminoethylether)-N,N,N,N-tetraacetic- acid (EGTA,5 mmol/L) and extracellular perfusion of Cd2+ and Ni2+,respectively.Intracellular application of heparin (8 g/L) failed to inhibit ACh-sensitive potassium current.CONCLUSION: Extracellular application of ACh activates the big conductance,calcium-dependent potassium current (BK) in VHCs Ⅱ of mice,which is potently modulated by extracellular Ca2+ ions.However,intracellular IP3-dependent Ca2+ ions release mechanism is not involved in the activation of the ACh-sensitive BK channel.     

皇帝蕉薄片外植体愈伤组织的诱导及植株再生

将皇帝蕉试管苗茎段徒手切成厚约1mm的薄片,经无菌的0.5%柠檬酸溶液处理片刻后,接入各种培养基中。结果表明:(1)适度的暗培养预处理有利于愈伤组织诱导,合适暗培养天数为4d;(2)诱导愈伤组织最佳培养基为:B5+2,4-D13.572μmol/L+IBA4.921μmol/L+NAA5.371μmol/L+KT13.94μmol/L+椰乳5%+PP3330.0001mg/L,愈伤组织诱导率为86.0%;(3)最佳愈伤组织分化芽培养基为:改良MS培养基+BA13.6831μmol/L+NAA0.537μmol/L,芽分化率达到87.0%;(4)诱导芽生根的最佳培养基是:1/2MS+IBA0.492μmol/L(或+NAA0.537μmol/L),生根率达到100%。     

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.     

Effects of hyaluronan on secretion of MMPs from murine alveolar macrophage and lung fibroblast

AIM:To investigate the effects of different molecular weights of hyaluronan (HA) on secretion of matrix metalloproteinases (MMPs) from macrophages and fibroblasts as well as contraction and degradation of collagen gels.METHODS:(1) Alveolar macrophages were obtained from lungs of normal Wistar rats by bronchoalveolar lavage.Lung fibroblasts were obtained by performing primary culture of lung tissue which was acquired from rat born first day.(2) HA was divided into 4 different molecular weights (MW):100 kD, 200 kD, 500 kD and 1 000 kD.(3) Mixing collagen, HA and cell suspension so that the final mixture resulted in 0.75 g/L collagen, 5×108 cells/L macrophages or fibroblasts.The mixture was cast into 24-well plates.Gelation occurred within 30 min, after which gels were released from the wells and floated in culture medium.(4) The areas of collagen gels were measured every 24 h, and the media were changed everyday.(5) The supernatants of culture media were collected after first 24 h and the concentration of MMPs was measured using an enzymed-linked immunosorbent assay (ELISA).(6) Collection of collagen gel after 72 h, spectrophotometry was used to measure hydroxyproline.RESULTS:(1) HA with different MW induced MMP-9, 12, 13 releases from alveolar macrophages, among which 200 kD had the most significant effect.(2) HA with different MW induced MMP-1, 9, 13 releases from lung fibroblasts, among which HA of 100 kD and 200 kD had the most significant effect.(3) HA with MW 100 kD, 200 kD and 500 kD augmented macrophage-mediated collagen gel contraction at 24 h, and the effect continued for 72 h.(4) HA with MW 100 kD, 200 kD and 500 kD augmented fibroblast-mediated collagen gel contraction at all time points, but only HA of 100 kD and 200 kD had significant effect compared with that of 500 kD and 1 000 kD after 72 h.(5) Over three-day culture period, addition of HA resulted in decrease of hydroxyproline in macrophage collagen gel, HA with 100 kD and 200 kD MW had significant effect than that of 500 kD and 1 000 kD.(6) Over three-day culture period, HA added resulted in decrease of hydroxyproline in fibroblast collagen gel, 200 kD HA had significant effect than that of others.CONCLUSION:(1) HA induces MMPs secretion from macrophages and fibroblasts when casted into collagen gel, among which HA with MW 200 kD has the strongest effect.(2) HA augmentes the contraction of collagen gels in both cells, and HA of 100 kD, 200 kD and 500 kD has the most significant effect.(3) Hyaluronan results in the degradation of the collagen within the gel; HA of 100 kD and 200 kD decreases the hydroxyproline to the lowest level.     

Rapamycin inhibits HMGB1 expression and releases in RAW264.7 cells induced by lipopolysaccharides in vitro

AIM: To observe the mechanism that rapamycin (RPM) affects HMGB1 expression and release in RAW264.7 cells induced by lipopolysaccharides (LPS).METHODS: RAW264.7 cells were cultured in six wells plate and divided into five groups: control group, 250 μg/L LPS treatment group, 100 μg/L RPM treatment group, 50 μg/L rTNF-α treatment group and 100 μg/L TNF-α antibody treatment group. After 4 h treatment, the TNF-α level in the culture media was evaluated by ELISA assay. After 24 h, the expression of HMGB1 mRNA was measured by RT-PCR, and HMGB1 protein level in the culture media was determined by Western blotting analysis.RESULTS: TNF-α level in the culture media of RAW264.7 cells has no significant difference between RPM treatment group and control group (P>0.05). Both HMGB1 mRNA expression and HMGB1 protein level were remarkably higher in LPS treatment group than that in control group (P<0.05). RPM attenuated LPS-induced HMGB1 mRNA and HMGB1 accumulation. Compared with that in RPM treatment group, HMGB1 accumulation was increased in rTNF-α treatment group, and had no significant difference in TNF-α antibody treatment group (P>0.05).CONCLUSION: RPM inhibits HMGB1 expression not only by directly suppressing STAT3 activation, but also by indirectly reducing TNF-α level.     

Effect of exogenous hydrogen sulfide on expression of NLRP3 inflammasome in hepatocytes

AIM: To evaluate the effect of exogenous hydrogen sulfide (H₂S) on the expression of NLRP3 inflammasome in hepatocytes.METHODS: The hepatocytes L02 and SMMC-7721 were used to establish the model of inflammation by stimulating with lipopolysaccharide (LPS) at different concentrations in vitro. The expression of NLRP3 inflammasome in the hepatocytes was detected by Western blot and the cell viability was measured by MTT assay for determining appropriate concentration of LPS. The hepatocytes were divided into 4 groups:the cells in control group were incubated with normal medium for 18.5 h; the cells in LPS group were incubated with normal medium for 0.5 h followed by 100 μg/L LPS for 18 h; the cells in LPS+H₂S group and H₂S group were incubated with 200 μmol/L sodium hydrosulfide hydrate (NaHS) for 0.5 h followed by 100 μg/L LPS or normal medium for 18 h, respectively. The protein expression of NLRP3 and caspase-1 in the cells of every group was determined by Western blot. RESULTS: Compared with control group, the protein expression of NLRP3 and caspase-1 increased significantly in LPS group (P<0.05) and had no significant change in H₂S group. Compared with LPS group, the protein expression of NLRP3 and caspase-1 in LPS+H₂S group decreased significantly (P<0.05). CONCLUSION: In hepatocytes, exogenous H₂S suppresses the expression of NLRP3 inflammasome.     

Contributions of cardiotrophin-1 to cardiac fibroblast proliferation

AIM: To investigated the role of CT-1 in the hypertensive ventricular remodeling. METHODS: The cardiac fibroblasts in 3-4 passages were cultured in vitro, and divided into eight groups according to the different intervention factors: group C (control); group DM: dimethyl sulphoxide (DMSO); group P: cultured under high hydrostatic pressure (160 mmHg); group ASODN: interfered with CT-1 antisense oligodeoxynucleotides (10 μmol/L); group SODN: incubated with CT-1 sense oligodeoxynucleotides (10 μmol/L); group AG: interfered with AG490 (25 μmol/L); group PD: interfered with PD98059 (20 μmol/L); group LY: interfered with LY 294002 (10 μmol/L). Western blotting was employed to assess the expression of STAT3, ERK1/2 and PI3-K respectively at protein level. Cell proliferation was quantified by MTT. RESULTS: High hydrostatic pressure stimulated the proliferation of cardiac fibroblasts, upregulated the expression of CT-1. CT-1ASODN inhibited the proliferation of cardiac fibroblasts (0.132±0.013 vs 0.154±0.011, P<0.05). ASODN extensively inhibited the expression of STAT3, ERK1/2 and PI3-k respectively at protein level (2.09±0.25 vs 2.47±0.28, P<0.05), (1.13±0.19 vs 1.61±0.22, P<0.05), (1.25±0.23 vs1.71±0.25, P<0.05). AG490, a JAK-STAT3 inhibitor, reversed the increase in the proliferation of cardiac fibroblasts stimulated by high hydrostatic pressure and the expression of ERK1 phosphorylation (0.118±0.018 vs 0.155±0.010, P<0.05). PD98059, a MAPK-ERK1/2 inhibitor, increased the proliferation of cardiac fibroblasts stimulated by high hydrostatic pressure (0.185±0.011 vs 0.155±0.010, P<0.05) and the expression of STAT3 phosphorylation (1.83±0.23 vs 1.58±0.22, P<0.05). LY294002, a PI3-K inhibitor, had no effect on the proliferation of cardiac fibroblasts stimulated by high hydrostatic pressure (0.157±0.015 vs 0.155±0.010, P>0.05). No difference of the expression of the above factors was observed in SOND (0.151±0.010 vs 0.154±0.011, P>0.05) and DMSO (0.141±0.017 vs 0.155±0.010, P>0.05) groups as compared with the control group. CONCLUSION: Under high hydrostatic pressure, the proliferation of cardiac fibroblasts is essentially mediated by STAT3, independent of PI3-K and the action is negatively regulated by ERK1/2 via inhibiting STAT3.The interaction between STAT3 and ERK1/2 may assist CT-1 in developing adequate cardiac fibroblast proliferation.