Effects of aldosterone on expression of angiotensin Ⅱ receptors in cultured rat mesangial cells

AIM: To investigate the effect of aldosterone (ALD) on the mRNA expression of angiotensin Ⅱ (Ang II) type 1 (AT-1a R and AT-1bR) and 2 (AT-2R) receptors in cultured rat mesangial cells (RMCs) treated with high glucose. METHODS: Rat mesangial cells were cultured in high glucose medium containing different concentrations of ALD (10-8-10-6 mol/L). The antagonists of ALD and Ang II receptors including pironolactone (10-7 mol/L, aldosterone receptor antagonist, SPI), losartan (10-7 mol/L, Ang II type 1 receptor blocker, Los) or PD123319 (10-9 mol/L, Ang II type 2 receptor antagonist, PD) were added in the cell culture for 12 h. The control cells were only treated with high (30 mmol/L) or normal (5.6 mmol/L) glucose medium. The viability and proliferation of the RMCs were evaluated by MTT assay. The mRNA expression of AT-1aR, AT-1b R and AT-2R was detected by semi-quantitative RT- PCR. The expression of MCP-1 in cultured RMCs was detected by ELISA. RESULTS: The mRNA expression of AT-1aR, AT-1b R and AT-2R was increased significantly by treatment with ALD in a dose-dependent manner (1.62-1.77, 9.61-9.89 and 7.26-7.35 folds of high glucose control, respectively, P<0.01). SPI significantly reduced the mRNA expression of AT-1aR and AT-1b R (P<0.01) but not affected the mRNA expression of AT-2R. The ratio of AT-1aR/AT-1b R in cultured RMCs treated with high glucose decreased significantly after stimulated with ALD (P<0.01). However, the effect of ALD was inhibited by SPI (P<0.01). Aldosterone treatment induced a significant upregulation of MCP-1 expression in a dose-dependent manner, and previous treatment with spironolactone, losartan or PD123319 abolished this aldosterone-induced MCP-1 expression. CONCLUSION: The results suggest that aldosterone is involved in the inflammatory response by up-regulating the expression of AT-1aR, AT-1bR and AT-2R, changing the proportion of AT-1R subtype, and inducing MCP-1 overproduction in cultured RMCs treated with high glucose.     

Effect of insulin on the SGK1 expression and extracellular matrix synthesis in human mesangial cells cultured in high glucose

AIM:To study the effect of insulin on the serum and glucocorticoid-inducible kinase 1 (SGK1) expression and extracellular matrix synthesis in human glomerular mesangial cells (HMC) cultured in high glucose. METHODS:The HMCs were cultured in the presence of 5.5 or 25 mmol/L glucose with or without 100 nmol/L insulin (i.e. NG, HG, NI and HI groups). 4 h latter, expressions of SGK1, insulin receptor substrate-1 (IRS1) and IRS2 in corresponding groups were detected by immunofluorescence or examined by Western blotting. The phosphorylation of IRS1 and IRS2 was measured by immunoprecipitation. 24 h latter, connective tissue growth factor(CTGF) and fibronectin (FN) were also examined by RT-PCR and ELISA, respectively. RESULTS:Compared with NG, the SGK1 protein expression in HG, NI and HI groups was significantly higher (P<0.01). High glucose mainly caused IRS2 protein and its phosphorylation level increase (P<0.01). When treated with 100 nmol/L insulin, IRS1 protein and its phosphorylation in HI group apparently elevated while slight inhibition of IRS2 protein expression and its phosphorylation were observed (HI vs HG, P<0.05). High glucose enhanced the expression of CTGF and FN, and insulin strengthened this effect.CONCLUSION:Insulin and high glucose up-regulate the expression of SGK1 in mesangial cells through different target molecular pathways and ultimately enhance ECM synthesis. The effect of insulin is highly associated with IRS1 signaling cascades.     

Ubiquitin-dependent degradation of SnoN/Ski protein in glomerular mesangial cells induced by high glucose

AIM: To observe the protein expression of SnoN/Ski and ubiqutin ligase Arkadia in rat glomerular mesangial cells (GMCs) exposed to the high glucose. METHODS: Cultured rat glomerular mesangial HBZY-1 cells were divided into control group, 20 mmol/L glucose group, 30 mmol/L glucose group, 30 mmol/L glucose+MG132 group (culture medium containing 30 mmol/L glucose and 0.5 μmol/L specific proteasome inhibitor MG132), and mannitol group. The expression levels of SnoN, Ski and Arkadia were measured by Western blotting analysis, immunofluorescence and laser scanning confocal microscopy. RESULTS: In control GMCs, the expression of SnoN/Ski was rich and Arkadia was weak. After stimulated with high glucose, the expression of SnoN/Ski was decreased and Arkadia was gradually increased (P<0.05). Compared with high glucose group, the levels of SnoN/Ski and Arkadia were mostly reverted by adding the proteasome inhibitor MG132 at concentration of 0.5 μmol/L (P<0.01). The expression levels of SnoN/Ski and Arkadia were not significantly changed in mannitol group in comparison with control group (P>0.05). CONCLUSION: High glucose decreases the expression of SnoN/Ski through ubiquitin-dependent degradation of SnoN/Ski. The degradation of SnoN/Ski mediated by Arkadia may play an important role in the pathogenesis of diabetic nephropathy.     

Angiotensin II-induced matrix metalloproteinase-9 expression mediated by NF-κB pathway in human THP-1 cells

AIM: The present study was undertaken to investigate the effect of angiotensin II (AngⅡ) on expression of MMP-9 in THP-1 macrophages. METHODS: Macrophages converted from THP-1 monocytes by incubating with PMA (0.1 μmol/L) for 48 h were divided into PMA group; PMA+AngⅡ group (10-7mol/L, 1 h); PMA+AngⅡ+PDTC group (10 μmol/L, 30 min) and PDTC group. Western blotting was used to detect the MMP-9 and phosphorylation of NF-κB p65, and the expression of MMP-9 mRNA in THP-1 macrophages was measured by RT-PCR.RESULTS: Compared to control group, the expression of MMP-9 (1.06±0.11, P<0.05) and phosphorylation of NF-κB p65 (1.02±0.10, P<0.05) in THP-1 macrophages were expressed when treated with AngⅡ (10-7mol/L); and the expression of MMP-9 mRNA were upregulated (1.22±0.08, P<0.05). However, NF-κB inhibitor PDTC reduced the NF-κB p65 (0.99±0.12, P<0.01) and MMP-9 (1.04±0.14, P<0.01) expressions and decreased the expression of MMP-9 mRNA (0.90±0.06,P<0.01). CONCLUSION: NF-κB signaling pathway contributes to the expression of MMP-9 in THP-1 macrophage induced by AngⅡ.     

Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.     

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.     

Minocycline suppresses sodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling

AIM：To explore the role of PI3K/Akt signaling in the anti-apoptotic effect of minocyline (MC) on the apoptosis of PC12 cells induced by sodium nitroprusside (SNP). METHODS：PC12 cells were divided into 4 groups: blank control group, SNP (500 μmol/L) group, MC (10 μmol/L)+SNP group and LY294002+MC+SNP group. The cell viability was observed by MTT assay. The expression of Akt and p-Akt was determined by Western blotting. RESULTS： The viability of the PC12 cells decreased after exposed to 500 μmol/L SNP for 24 h. Meanwhile, MC at concentration of 10 μmol/L significantly blocked the effect of SNP, such as decreasing the cell viability. Pretreatment with LY294002 for 60 min prior to exposure of the PC12 cells to MC and SNP down-regulated the expression of p-Akt induced by SNP. CONCLUSION：Minocycline regulates PI3K/Akt signaling pathway to restrain the apoptosis of PC12 cells induced by SNP.     

Effect of intermedin1-53 on aldosterone-induced cardiac fibroblast proliferation

AIM: To investigate the roles of intermedin_1-53 (IMD_1-53) and exteracellular signal-regulated kinase (ERK) signal pathway in the proliferation of rat cardiac fibroblasts (CFBs).METHODS: Isolated and cultured CFBs from new born SD rats were randomly divided into control group, aldosterone (ALD) groups (at different concentrations) and ALD+IMD groups (IMD at different concentrations). The viability of CFBs was determined by MTT assay. Western blotting was used to observe the effect of IMD on ALD-induced ERK phosphorylation.RESULTS: IMD_1-53 had no significant effect on the proliferation of CFBs in basal state, but inhibited the CFBs growth stimulated by ALD in a concentration (10^-9~10^-7 mol/L)-dependent manner. IMD_1-53 also inhibited ERK phosphorylation stimulated by ALD in a concentration (10^-9~10^-7 mol/L)-dependent manner.CONCLUSION: IMD_1-53 inhibits the proliferation of CFBs by ERK signal pathway.     

Minocycline inhibits BV-2 cell activation by regulating P2X7 receptor

AIM：To explore the role of P2X7 receptor in inhibition of lipopolysaccharide (LPS)-stimulated BV-2 cell activation by minocycline. METHODS：BV-2 cells were divided into 5 groups: control group, LPS group, LPS+0.1 μmol/L Mino group, LPS+1 μmol/L Mino group and LPS+10 μmol/L Mino group. The expression of P2X7 receptor was determined by real-time PCR and Western blotting. The levels of TNF-α and IL-1β in the microglia culture supernatants were measured by ELISA. The morphological changes of the cells were also observed. RESULTS：After exposed to LPS, the expression of P2X7 receptor increased in BV-2 cells at mRNA and protein levels. The concentrations of TNF-α and IL-1β in the microglia culture supernatants also increased. Meanwhile, 0.1~10 μmol/L minocycline inhibited those changes in a dose-dependent manner. CONCLUSION：Minocycline inhibits the activation of microglia. The mechanism may be related to the P2X7 receptor.     

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 interleukin-10 on the calcineurin activity in cultured cardiac fibroblasts induced by arginine vasopressin

AIM: The purpose of the present study was to investigate the effect of interleukin-10 (IL-10) on the proliferation and calcineurin (CaN) activity in cultured cardiac fibroblasts (CFs) induced by arginine vasopressin (AVP).METHODS: The CFs of left ventricle in neonatal Sprague-Dawley rats were isolated and cultured by trypsin digestion and selective plating technique. Then the proliferation rates of cells were determined by using the MTT ［3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide］ assay (A490 value). Cell cycle distribution was determined with flowcytometry technique. The CaN activity was measured by ultra-violet spectrophotography.RESULTS: (1) MTT colorimetry showed that 10-7 mol/L AVP significantly increased A490 value of CFs in comparison with control group (P<0.01). IL-10 attenuated the A490 value of AVP group in a concentration dependent manner. The A490 value of the 10-8, 10-7, 10-6, 10-5 g/L IL-10+10-7 mol/L AVP groups was 0.201±0.007, 0.187±0.006, 0.173±0.010 and 0.157±0.029 respectively, all data significantly lower than those in the presence of AVP alone (P<0.05 or P<0.01). (2) The percentage of the cells in S stage and proliferation index were markedly increased in 10-7mol/L AVP group compared with the control (P<0.01, respectively). In the 10-6 g/L IL-10+10-7 mol/L AVP group, the percentage cells in S stage and proliferation index were significantly lower than those in AVP group (P<0.01, respectively). IL-10 itself had no effect on fibroblast proliferation, but reduced AVP-induced fibroblast proliferation. (3) There was a significantly increase in CaN activity in AVP group compared with control (P<0.01). In the 10-8, 10-7, 10-6 and 10-5 g/L IL-10＋10-7 mol/L AVP groups, the CaN activity was 3.22±0.04, 3.06±0.06, 2.53±0.04 and 2.22±0.04, respectively. IL-10 dose-dependently down-regulated the CaN activity induced by AVP (P<0.01, respectively). However, the CaN activity was still higher in IL-10+AVP group than that in control group (P<0.05 or P<0.01).CONCLUSION: Our data indicate that IL-10 regulates the CaN activity of CFs in the cell proliferation induced by AVP, suggesting that IL-10 plays a role in the regression of cardiac remodeling.     

朱顶红无菌苗叶片高效再生体系

以朱顶红（Hippeastrum vittatum）叶片为外植体进行离体培养，具有取材方便、试材充足、成本低等优势，但叶片诱导再生率极低，是朱顶红离体培养的一大难题。本试验中分别以‘花孔雀’和‘黑天鹅’朱顶红无菌苗叶片为外植体，探究了不同植物生长调节剂和不同取材部位对不定芽诱导和继代增殖的影响。结果表明：最佳外植体为 MS 培养基中培养 10 d 形成的幼嫩叶片基部（0.5 cm），在光照 16 h · d-1（光照强度 36 μmol · m-2 · s-1）下，不定芽诱导的最适培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 2 mg · L-1 TDZ，两个品种的不定芽均以间接途径发生，其中‘花孔雀’在培养 40 d 后形成愈伤组织，55 d形成不定芽，诱导率可达 69.44%；‘黑天鹅’在培养 45 d 后形成愈伤组织，65 d 形成不定芽，诱导率达到 66.67%；最适体细胞胚诱导培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 PIC，‘花孔雀’和‘黑天鹅’的诱导率分别达到 66.67%和 63.89%；最佳不定芽增殖培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 1 mg · L-1 TDZ，‘花孔雀’和‘黑天鹅’的增殖系数分别达到 4.67 和 3.46；在不添加植物生长调节剂的 MS培养基中进行生根培养，30 d 后两个品种的生根率均达到 100%；将生根培养 30 d 的小植株转移至室温条件下放置 3 d，摘去封口膜再驯化 3 d 后，移栽至经高温消毒的草炭︰蛭石（体积比）为 1︰1 的基质中，成活率达到 100%     

Breast cancer stem cells cultured without insulin and effect of estrogen induction

AIM:To determine whether suspension culture medium without insulin can be used to feed breast cancer tumorsphere, or not. METHODS:MCF7 cells were used to build tumorsphere. The morphological changes, CD44+ CD24- expression, aldehyde dehydrogenase 1 (ALDH1) expression and multiple division ability were measured to identify the breast cancer stem cells and to detect the function of 17β-estradiol (E2β) in tumorsphere of MCF7 cells. RESULTS:The tumorshere, each containing 30 to 60 cells, was obtained by the method of insulin-removal suspension culture. These cells were cytokeratin 18 and CD10 proteins positive, and the number of CD44+ CD24- cells and ALDH1 protein expression were significantly higher than the adherent cultured cells (P<0.05). Using 10-10 mol/L E2β to treat the tumorshere for 7 d, the tumor cell number and volume were significantly increased. Using 10-10 mol/L E2β to treat the tumorshere for 24 h, the CD44+ CD24-cells and ALDH1 protein expression were significantly higher than those in non-treatment group (P<0.05). CONCLUSION: Suspension culture medium without insulin can be used to feed breast cancer tumorsphere. These tumorsphere could be used as a model to determine the function of E2β in breast cancer stem cell research.     

Expression of SGK isoforms in human proximal tubular epithelial cells under the condition of high glucose concentration

AIM: To investigate the effect of high glucose concentration on serum and glucocorticoid induced protein kinase (SGK) mRNA and protein expressions in human proximal tubular epithelial cells (HKC) and the possible role of SGK in the production of extracellular matrix (ECM) of HKC under the condition of high glucose. METHODS: HKC was divided into 3 groups: control glucose group (CG group, 5.5 mmol/L D-glucose); high glucose group (HG group, 25 mmol/L D-glucose) and osmotic control group (MG group, 19.5 mmol/L mannitol and 5.5 mmol/L D-glucose). The expressions of SGK mRNA and protein were assessed by semi-quantitative RT-PCR and Western blotting respectively. The level of secretary and cytoplasmic fibronectin (FN) were detected by enzyme-linked immunoabsordent assay (ELISA) and indirect-immunofluorescence. RESULTS: HKC expressed SGK1, SGK2 and SGK3 at mRNA and protein levels. Their mRNA level were up-regulated since 2 hours after cells exposed to D-glucose and this up-regulation persisted to the end of 8th hour, and SGK1 protein level elevated simultaneously. On the other hand, the increased FN secretion by high glucose was in a time-dependent manner and its improved secretion threshold was just followed by the high expression of SGK1. CONCLUSIONS: In response to high glucose, the expression of SGK1, SGK2 and SGK3 in human proximal tubular epithelial cells were up-regulated which was accompanied with FN accumulation. The high expression of SGK may mediate overproduction of ECM in proximal tubular epithelial cells and contribute to the diabetic nephropathy.     

Inhibitory effects of Lobelia Chinensis Lour alkaloid on the proliferation of human umbilical artery vascular smooth muscle induced by endothelin-1

AIM: To investigate the effects of Lobelia Chinensis Lour Alkaloid (LCLA) on the proliferation of cultured vascular smooth muscle cells (VSMC) induced by ET-1. METHODS: Human umbilical artery VSMC was cultured and divided into five groups: ET group, ET+LCLA group, ET+BQ-123 group，ET+ staurosporine （ST） group and control group. The cell proliferation activity was subsequently quantified by cell counting kit-8 (CCK-8) and ［3H］-TdR incorporation. Flow cytometry was used to examine cell cycle. Quantitative immunohistochemical technique was used to investigate the expression of proliferating cell nuclear antigen (PCNA) and confocal microscope was used to measure the fluorescent intensity of Ca2+. Cytotoxicity was measured by Trypan blue exclusion and LDH colorimetry tests. RESULTS: BQ-123 (10-6mol/L), ST (10-7mol/L) and LCLA (100, 200 and 400 mg/L) inhibited the increase in cell number, ［3H］-TdR incorporation, the percentage of the S phase and markedly decreased the expression of PCNA and fluorescent intensity of Ca2+ in response to ET-1 of VSMC (P<0.05). CONCLUSION: LCLA (100-400 mg/L) inhibits ET-1-induced proliferation of VSMC in a dose-dependent manner and the anti-proliferative effect is realized by reducing the Ca2+ concentration in VSMC.     

AngⅡ/AT1R pathway leads to down-regulation of endothelial nitric oxide synthase phosphorylation

AIM:To investigate the mechanism of angiotensinⅡ (AngⅡ)/angiotensinⅡ type 1 receptor (AT₁R) pathway activating protein phosphatase 2A (PP2A) which leads to down-regulation endothelial nitric oxide synthase (eNOS) phosphorylation level in mesenteric arteries of rats. METHODS:The mesenteric arteries of adult male SD rats (weighing 160~180 g; n=90) were isolated under aseptic conditions. Firstly, to determine the effect of angiotensinⅡ down-regulated eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into normal control (control) group and AngⅡ group. The mesenteric arteries in AngⅡ group were incubated with AngⅡ at 1×10^-7 mol/L, 1×10^-6 mol/L and 1×10^-5 mol/L for 6 h, 12 h and 24 h, respectively. Secondly, to investigate the molecular mechanism by which angiotensinⅡ activated PP2A leading to down-regulation eNOS (Ser1177) phosphorylation level, the mesenteric arteries were randomly divided into control group, AngⅡ group and candesartan (CAN; a specific AT₁R blocker)+AngⅡ group. The mesenteric arteries were pretreated with 1×10^-5 mol/L CAN for 1 h, then incubated with 1×10^-7 mol/L AngⅡ for 12 h in CAN+AngⅡ group. The protein levels of eNOS, p-eNOS (Ser1177), PP2Ac, p-PP2Ac (Tyr307) and protein phosphatase 2A inhibitor 2 (I₂^PP2A) in the arteries were determined by Western blot. The activity of PP2A in the arteries was detected by PP2A activity kit. RESULTS:Compared with the control group, the protein level of p-eNOS (Ser1177) in the mesenteric arteries was decreased after incubated with AngⅡ for 6 h, 12 h and 24 h (P<0.05). The decreasing tendency of p-eNOS (Ser1177) showed concentration-dependently, especially in 12 h and 24 h groups. The expression of eNOS protein showed no significant difference in each group. Compared with the control group, the mesenteric arteries of the rats were incubated with AngⅡ at 1×10^-7 mol/L for 12 h in vitro, the protein levels of p-eNOS (Ser1177) were down-regulated (P<0.05); pretreatment with CAN significantly increased the protein level of p-eNOS (Ser1177) (P<0.05); the protein levels of eNOS showed no significant difference in each group. Compared with the control group, the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A were decreased after the mesenteric arteries were treated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesartan pretreatment restored the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A (P<0.05), however the expression of PP2Ac protein showed no significant difference in each group. Compared with the control group, the activity of PP2A was increased in the mesenteric arteries incubated with AngⅡ at 1×10^-7 mol/L for 12 h (P<0.05). Candesarten pretreatment inhibited the activity of PP2A significantly (P<0.05). CONCLUSION:AngⅡ increases PP2A activity via AT₁R pathway, thus leading to down-regulation eNOS (Ser1177) phosphorylation level in mesenteric arteries. The molecular mechanism of PP2A activation may be associated with decreasing the protein levels of p-PP2Ac (Tyr307) and I₂^PP2A.     

Effects of SUMOylation on IKKγ/NF-κB signaling in cultured rat glomerular mesangial cells treated with high glucose

AIM：To investigate the effects of SUMOylation on IκB kinase γ (IKKγ)/NF-κB signaling in cultured rat glomerular mesangial cells (GMCs) treated with high glucose. METHODS：Cultured HBZY-1 rat GMCs were divided into normal glucose group and high glucose groups, and mannitol was used for osmotic control. The expression of SUMO1, SUMO2/3, IKKγ and NF-κB p65 was measured by Western blotting and RT-PCR. Interaction between SUMO and IKKγ was detected by co-immunoprecipitation. RESULTS：Compared with normal glucose group, the expression of SUMO and NF-κB p65 was increased in high glucose groups in a dose- and time-dependent manner. The expression of IKKγ was not changed by high glucose. The SUMOylation of IKKγ in high glucose groups was significantly decreased as compared with normal glucose group. CONCLUSION:High glucose obviously changes the interaction between SUMO and IKKγ in cultured rat mesangial cells, which may be involved in the activation of NF-κB by taking a special influence on the SUMOylation of IKKγ/NF-κB signaling molecules.     

Protective effect of atrial natriuretic peptide on alveolar type II cells

AIM: To study the protective effect of atrial natriuretic peptide (ANP) on alveolar type II cells (AT-Ⅱ) damaged by lipopolysaccharide (LPS).METHODS: AT-Ⅱ were placed in a 6 well cell culture cluster (0.5×106 cells/cm2) and divided into 3 groups: (1) control group (n=6), the medium consisted of RPMI-1640 without FBS. (2) LPS group (n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS (1 mg/L). (3) ANP group (n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS (1 mg/L) and ANP (10-8, 10-7, 10-6 mol/L). After 4, 12 and 24 h, the cell culture mediums of control group, LPS group and ANP (10-7 mol/L) group were collected, and those of the ANP (10-6, 10-8 mol/L) group were collected after 12 h. Alkaline phosphatase(AKP), lactate dehydrogenase(LDH), malondialdehyde(MDA), total phospholipids (TPL) and surface tension (ST) in the medium of every group were examined. RESULTS: AT-Ⅱ were characterized by AKP staining. The contents of LDH, AKP and MDA in the medium of every ANP group were lower than those in the corresponding LPS group. The TPL content in the medium of every ANP group was higher than that in the corresponding LPS group, and the change of ST of the medium was opposite to that of TPL. The effect at 12 h was the most significant, for example, at 12 h, the activities of AKP in the mediums were: control (43.5±10.4) U/L, LPS (98.1±16.4) U/L, LPS+ANP (10-6) (46.4±10.5) U/L, LPS+ANP(10-7) (60.7±9.5) U/L, LPS+ANP(10-8) (91.3±13.9) U/L.CONCLUSION: ANP protects the AT-Ⅱ from being damaged by LPS and promotes the secretion of pulmonary surfactants.     

Effects of leptin on BSEP protein expression and signaling pathway in HepG2 cells

AIM: To investigate the effects of leptin on the expression of bile salt export pump (BSEP) and signaling pathway in human hepatocellular carcinoma cell line HepG2. METHODS: HepG2 cells were cultured in vitro. Leptin at concentrations of 10^-8, 10^-7 and 10^-6 mol/L was used as a stimulating factor. The protein levels of adenosine monophosphate-activated protein kinase alpha subunit (AMPKa), phosphorylated AMPKa (p-AMPKa) and BSEP in the HepG2 cells at 24 h, 48 h and 72 h were detected by Western blotting. The optimal culture time and leptin concentration were selected, and compound C at concentration of 10 μmol/L was added to this group. The protein expression of BSEP was detected by Western blotting. RESULTS: Intervention of HepG2 cells with leptin for 72 h increased the protein expression of AMPKa gradually in a concentration-dependent manner, and leptin at concentration of 10^-6 mol/L induced the strongest AMPKa expression (P<0.01). Intervention of HepG2 cells with leptin for 24 h increased the phosphorylation level of AMPKa gradually in a dose-dependent manner (P<0.01). The effect of leptin on the increase in the protein expression of p-AMPKa was also in a time-dependent manner (P<0.01). After intervention with different concentrations of leptin for 24 h, the protein expression of BSEP in the HepG2 cells was gradually increased by the stimulation of leptin in a concentration- and time-dependent manner (P<0.01). Compared with NC group, the protein expression of BSEP in 10^-6 mol/L leptin group and 10^-6 mol/L leptin+10 μmol/L compound C group was increased at 72 h (P<0.01), and that in 10^-6 mol/L leptin+10 μmol/L compound C group was lower than that in 10^-6 mol/L leptin group at 72 h (P<0.01). CONCLUSION: Leptin promotes the protein expression of BSEP in HepG2 cells by leptin-AMPK-BSEP signaling pathway. Leptin promotes the increases in AMPKa protein and the level of phosphorylation of AMPKa in HepG2 cells.     

Effect of fluvastatin on migration of vascular smooth muscle cells from rats

AIM: To investigate the effect and mechanism of fluvastatin on the migration induced by platelet derived growth factor-BB (PDGF-BB) and endothelin-1 (ET-1) in cultured vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs derived from spontaneously hypertensive rats (SHR) were used. Cell migration was determined by modified Boyden chamber assays. Intracellular free calcium (［Ca2+］i) was measured with fluorescent Ca2+ indicator Fura-2/AM. RESULTS: PDGF-BB and ET-1 significantly induced VSMCs migration, which was inhibited by pretreatment of VSMCs with fluvastatin (10-9-10-5 mol/L) in a dose-dependent manner, and the peak inhibition rate of migration induced by PDGF-BB and ET-1 was over 86.67%. Fluvastatin also attenuated the increase in ［Ca2+］i induced by PDGF-BB and ET-1, with a peak inhibition rate of 86.76% and 65.32%, respectively. CONCLUSION: PDGF-BB and ET-1 promote migration of VSMCs from SHR．Fluvastatin may have direct inhibitory effects on cell migration induced by PDGF-BB and ET-1. The increase in ［Ca2+］i may acts as intracellular signaling in the migration in response to PDGF-BB and ET-1 in VSMCs.