Role of caveolae in high glucose-induced extracellular matrix production in rat mesangial cells

AIM：To investigate the role of caveolae in high glucose (HG)-induced extracellular matrix (ECM) production in rat mesangial cells (MCs). METHODS：Synchronized rat MCs were divided into normal glucose group, HG group, HG+methyl-β-cyclodextrin (β-MCD) group and HG+β-MCD+cholesterol (Chol) group. Western blotting was used to detect the protein expression of caveolin-1 (Cav-1), phosphorylated caveolin-1 (p-Cav-1-Y14) and collagen type 1 (Col I). The mRNA expression of Cav-1 was determined by real-time PCR. ELISA was used to measure the level of fibronectin (FN) in the supernatant. RESULTS：High glucose significantly increased the expression of FN and Col I. In HG 12, 24 and 48 h groups, the mRNA and protein levels of Cav-1 were not significantly different from those in HG 0 h group, whereas the level of p-Cav-1-Y14 was significantly increased. β-MCD significantly attenuated HG-induced elevation of p-Cav-1-Y14 and FN production, but had no effect on HG-induced Col I expression. All these responses to β-MCD were abolished by Chol. CONCLUSION：High glucose significantly increases the production of Col I and FN in rat MCs. FN production induced by high glucose is mediated by p-Cav-1-Y14.     

Effects of lipopolysaccharide and interleukin 1 receptor antagonist on proliferation and nitric oxide synthesis of mesangial cells

AIM: To investigate the effects of lipopolysaccharide (LPS) and interleukin-1 receptor antagonist (IL-1ra) on mesangial cells proliferation and nitric oxide synthesis. METHODS: Glomerular mesangial cells from SD rats were cultured. The first and second passages of cultured cells were used for the experiment. LPS and LPS plus IL-1ra were added in cell cultures, respectively. By using chemical method the nitrite in supernatants was measured, [³H]-TdR incorporation was determined to evaluate the GMC proliferation. Northern and slot hybridizations were performed to detect the expression of iNOS mRNA. RESULTS: There were expression of iNOS mRNA, more production of nitrite (0.64±0.25 vs 0.12±0.06 nmol/10⁴ cell) in supernatants and GMC proliferation (3735±1177.9 vs 1785±280.6) in LPS group compared to the control. While compared with LPS group, in LPS+IL-1ra GMC group, expression of iNOS mRNA decreased by 40%, nitrite increased (3.28±0.33nmol/10⁴ cell), proliferation of GMC decreased (818±77.27). CONCLUSION: LPS could activate the GMC to express iNOS mRNA and produce more nitrite. IL-1ra could partially inhibit the effects of LPS on the expression of iNOS mRNA in GMC, but not nitrite. There is no synchronous correlation between NO production and GMC proliferation.     

Effect of salvianolic acid B on high glucose-induced phenotypic transition and extracellular matrix secretion in human glomerular mesangial cells

AIM:To investigate the effect of salvianolic acid B (Sal B) on high glucose-induced phenotypic transition and extracellular matrix (ECM) secretion in human glomerular mesangial cells (HGMCs) and the underlying mechanisms. METHODS:HGMCs were randomly divided into control group, high glucose group and high glucose plus high dose, medium dose and low dose of Sal B groups. The HGMCs except those in control group were exposed to high glucose (33.3 mmol/L) for 72 h, while those in Sal B groups were co-incubated with indicated concentrations of Sal B. The protein levels of α-smooth muscle actin (α-SMA), transforming growth factor-β₁ (TGF-β₁) and phosphorylated Smad2 and p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. The secretion levels of collagen type I (Col I), collagen type Ⅲ (Col Ⅲ), fibronectin (FN) and laminin (LN) were measured by ELISA. RESULTS:Exposure to high glucose markedly increased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs (P<0.01). The phosphorylation levels of Smad2 and p38 MAPK were also significantly increased (P<0.01). Co-incubation with Sal B evidently decreased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs induced by high glucose (P<0.05 or P<0.01). The phosphorylated levels of Smad2 and p38 MAPK were also reduced noticeably (P<0.05 or P<0.01). CONCLUSION:Sal B significantly suppresses high glucose-induced phenotypic transition and ECM secretion in the HGMCs, which might be attributed, at least partly, to inhibition of TGF-β₁/Smad signaling pathway and p38 MAPK activation.     

Mechanisms of nitric oxide synthesis induced by human complement C5b-9complex in glomerular mesangial cells of rats

AIM: To explore the mechanism of the nitric oxide (NO) synthesis induced by human C5b-9 complex in glomerular mesangial cells(MC) of rats. METHODS: The MC of rats were cultured and stimulated with human complement C5b-9 complex to induce TNFα and IL-1β. At the same time, several parameters related to NO synthesis were measured at 3 h, 6 h and 24 h after C5b-9 stimulation. The effects of monoclonal antibodies against TNFα and IL-1β on NO synthesis were examined in this system. RESULTS: TNFα concentration in supernatant from MC in C5b-9 group was higher than that of control group at 6, 24 h after stimulation with C5b-9 complex and reversed by adding anti-TNFα McAb. C5b-9 complex didn't stimulate the release of IL-1β in same system. In addition, the expression of iNOS mRNA in MC was observed at 3 h after stimulation with C5b-9. Levels of iNOS mRNA expression and cGMP in MC and NO₃^-/NO₂^- in supernatant from MC in C5b-9 group were higher than those in control group at 6, 24 h after C5b-9 stimulation, these changes were also reversed by adding monoclonal antibody against TNFα. CONCLUSION: C5b-9 complex could induce iNOS mRNA expression at 3 h after C5b-9 stimulation, and the synthesis of NO at 6, 24 h was related to TNFα released from cultured MC of rats by C5b-9 complex to a certain extent.     

Changes in L-arginine/NO pathway of erythrocytes in patients with essential hypertension

AIM:To investigate the changes of L-arginine/nitric oxide(L-Arg/NO) pathway and the characteristics of L-Arg transport in erythrocytes (RBC) in patients with essential hypertension(EH). METHODS: 12 EH patients were studied with 10 normotensive people as control. The nitric oxide (NO) production was assayed by measuring the nitrite in medium using Greiss reaction. NO synthase activity was measured according to the method described by Ghigo. In order to achieve zero-trans condition, erythrocytes were incubated at 37℃ for 3 h and then L- Arg transport of RBC were determined by measuring the influx of radiolabelled L-arginine by Deliconstantins described method. RESULTS: In EH patients, NO production was lower than that in control (P＜0.01) and the cGMP quantity was only 72.8% of control (P＜0.01). NOS activity of EH patients decreased to 47.8% of control. Saturable influx of L-Arg was mediated by the classical cationic amino acid transport system Y^＋ and Y^＋L system with high affinity. Under zero-trans condition, the ability of L-Arg transport by RBC in EH patient was lower than control. The V_max for total L-Arg transport and via system Y^＋ respectively decreased 36.8% and 42.5% compared with control. But V_max or L-Arg transport via system Y^＋L was not affected by EH. In EH patients K_m value for total, system Y^＋and system Y^＋L had no difference with control. CONCLUSION: The present study suggested that there is disturbance of L-Arg/NO pathway in human erythrocytes from patient with EH.     

Effect of L-arginine on intimal proliferation and expression of related cell cycle regulatory factors after vascular injury in rats

AIM:To investigate the effect of L-arginine (L-Arg) on intimal proliferation and expression of related cell cycle regulatory factors after vascular injury in rats. METHODS:Rats were divided into three groups :sham operation group, balloon injury group(this group included balloon 48 h,7 d and 14 d subgroup) and balloon+L-Arg group. Neointima area were calculated morphologiocally. The expression of cyclin dependent kinase-2(CDK2),cyclin E and proliferation cell nuclear antigen (PCNA) were measured by means of immunohistochemical technique and computer image analyzer. RESULTS:After vascular balloon injury, the level of plasma NO decreased, CDK2、cyclin E and PCNA expressed in the media at 48 h and in the neointima at 7 d and 14 d but with low and undetected expression in the media, the expression of CDK2, cyclin E and PCNA increased with the intima thickening. Compared with balloon 14 d group, the plasma NO level increased (P＜0.01), the neointima area reduced by 59.1%(P＜0.01) and the positive expression indexes of CDK2, cyclin E and PCNA decreased by 36.1%, 46.3% and 76.2% respectively in balloon+L-Arg group (P all＜0.01). CONCLUSION:L-Arg can effectively repress intima proliferation after vascular injury, which may be associated with its inhibiting the proliferation of vascular smooth muscle cell through downregulating the excessive expression of CDK2, cyclin E and PCNA.     

Effect of L-arginine on nitric oxide and brain edema after subarachnoid hemorrhage in rats

AIM:To investigate the role of nitric oxide in the development of brain edema after subarachnoid hemorrhage(SAH), and the influence of L-arginine on them. METHODS:Noncraniotomy models of SAH in Wistar rats were used and animals were divided into sham-operated group, SAH group and SAH plus L-arginine group. Dynamic changes of regional cerebral blood flow within 24 hours were measured. Serum nitric oxide level, brain water and sodium content at different time points within 24 hours were also detected. RESULTS:Regional cerebral blood flow and serum nitric oxide level at every time point after operation in SAH group were lower than those in sham-operated group, while brain water content and sodium content in the former group were higher than those in the latter group. Above pathological alterations in SAH plus L-arginine group were not so obvious as in SAH group.CONCLUSION:Decrease in serum nitric oxide plays a role in the development of brain edema after SAH, which may be partly reversed by administration of L-arginine.     

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.     

Effects of endothelial nitric oxide synthase gene transfection on cytokines and cAMP production in human phagocytic cells

AIM and METHOD: Human endothelial nitric oxide synthase (eNOS) gene was transfected into human phagocytic cell U937 and the effects of gene transfer on cytokines and cAMP production were observed. RESULTS: A functional eNOS was stably expressed in transfected U937 cells, but NO release was undetectable in intact transfectants. However, eNOS gene expression upregulated tumor necrosis factor-α release and downregulated interleukin-10 and cAMP production in either presence or absence of NOS inhibitor N^ω-monomethyl-L-arginine. CONCLUSION: The function of tranfected eNOS gene product showed cellular speciality. The effector molecule that changed the produced pattern of cytokines and cAMP in phagocytic cells seems not likely the nitric oxide.     

Effects of basic fibroblast growth factor on proliferation and collagen production in human umbilical cord mesenchymal stem cells

AIM：To observe the growth pattern and surface markers of human umbilical cord mesenchymal stem cells(hUCMSCs) and to explore the influence of basic fibroblast growth factor (bFGF) on the proliferation and collagen production in hUCMSCs cultured in vitro. METHODS：hUCMSCs were isolated by enzyme digestion method and adherent culture. The surface markers CD45, CD34, CD105, CD29 and HLA-DR of the cells were analyzed by flow cytometry. The osteogenic ability and adipogenic differentiation were confirmed with oil red O and alizarin red staining. The optimal concentration of bFGF to promote the proliferation of hUCMSCs was 20 μg/L. The cells in control group were cultured in the growth medium consisting of DMEM/F12 and 10% volume fraction of fetal bovine serum. The cells in experiment group were cultured under the same condition of control group but plus 20 μg/L bFGF. The proliferation of hUCMSCs was analyzed by MTT assay. The expression of type I and III collagens at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS：The growth curves indicated that bFGF promoted the proliferation of hUCMSCs. The hUCMSCs expressed CD29, but did not express CD34, CD45 or HLA-DR in the presence or absence of bFGF unanimously. The cells were alizarin red staining-positive and oil red O staining-positive. Compared with control group, the expression of type I and III collagens significantly decreased at mRNA and protein levels in experiment group. CONCLUSION：bFGF promotes the proliferation of hUCMSCs and does not change the expression of the surface markers. bFGF inhibits the expression of type I and III collagens at mRNA and protein levels, indicating that bFGF enhances the healing of wound without inducing scar hyperplasia.     

Recombinant human defensin α1 induces proliferation of rat glomerular mesangial cells in vitro

AIM: To study the effects and mechanism of recombinant human defensin α₁ on cell proliferation in cultured rat glomerular mesangial cells.METHODS: The influences of defensin α₁ at various concentrations on rat 1097 mesangial cell line cultured in vitro were evaluated with MTT assay.The different concentrations of U0126,signal-regulated protein kinase (MEK) inhibitor,were added into the culture mediums of mesangial cells to do blocking test.Incubated with a final concentration of 3 mg/L defensin α₁,the phosphorylation of extracellular signal regulated kinase (ERK)1/2 and type IV collagen of mesangial cells in different times were evaluated by Western blotting.RESULTS: Defensin α₁ at 3-20 mg/L enhanced proliferation of rat glomerular mesangial cells.The incubation times for the maximum effect on proliferation was 12 h (P<0.01),whereas defensin α₁ concentration >20 mg/L decreased cell proliferation.The cell proliferation induced by defensin α₁ was inhibited by U0126.Stimulation of the cells with defensin α₁ at concentration of 3 mg/L for 5 minutes induced a maximum effect on a ratio of phosphorylation of ERK1/2 to total ERK.After 12 h incubation with defensin α₁,an increase in type IV collagen was observed by Western blotting and continued to increase at 24 h and 48 h (P<0.01).CONCLUSION: Defensin α₁ enhances rat glomerular mesangial cell proliferation and induces type IV collagen production by MAPK signaling pathway.     

Effect of cyclophosphamide on proliferation and apoptosis of rat mesangial cells in vitro

AIM:To investigate the effect of cyclophosphamide(CTX) on proliferation and apoptosis of mesangial cells(GMC) of rat in vitro. METHODS:GMC proliferat ion was detected by MTT method,GMC apoptosis was examined by inverted microscopy for phase-contract and fluoroscopy and flow cytometry analysis.The levels of Fas and Bcl-2 were also detected by immunohistology. RESULTS:The proliferation of GMC were inhibited by CTX, methylprednisolone(MP), low molecular weight heparin(LMWH). Apoptosis of GMC was induced by CTX, the apoptosis rate of GMC was 8.2%, and the Fas level was increased. CONCLUSION:CTX could inhibit proliferation and induce apoptosis of GMC possibly by enhancing the Fas level.     

Effect of L-arginine on function and structure of mitochondria in ischemia-reperfusion myocardial cells in rabbits

AIM: To study the effect of L-arginine (L-Arg) on function and structure of mitochondria in ischemia-reperfusion (MRI) myocardial cells. METHODS: Thirty rabbits were randomly divided into three groups (n=10 in each), control group, MIR group and MIR+L-Arg group. The mitochondrial respiratory function, Ca²⁺ concentration ([Ca²⁺]m), malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. Meanwhile, the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), total adenylic acid number (TAN) and energy charge (EC) in the myocardial tissue were respectively measured. Moreover, the ultrastructure changes in myocardial mitochondria were observed during MIR. RESULTS: The mitochondrial respiratory control rate (RCR), velocity 3 (V₃), SOD, surface density (Sv) and specific surface (δ) in MIR+L-Arg group were higher than those in MIR group, velocity 4 (V₄), [Ca²⁺]m, MDA, volume density (Vv), horizental diameter (Hd) were lower than those in MIR group. ATP, ADP, TAN and EC levels of myocardial tissue were higher than those in MIR group. There was no significant difference between MIR+L-Arg and control group in V₃, V₄, SOD, MDA, Vv, Sv, δ, Nv, Vd, AMP and TNA. CONCLUSION: It is suggested that L-Arg improves the function and structure of mitochondria in myocardial cells in the reperfusion injury after myocardial ischemia by decreasing oxygen free radical level and Ca²⁺ overload in the mitochondria.     

Effect of L-arginine liposome on L-arginine transport of pulmonary artery in rats chronically exposed to hypoxia-hypercapnia

AIM: To investigate the effect of chronic hypoxia-hypercapnia and L-arginine (L-Arg) liposome on L-Arg transport in rats pulmonary artery. METHODS: Forty Sprague-Dawley rats were randomly divided into four groups, normal control group (NC), chronic hypoxia-hypercapnia group (HH), chronic hypoxia- hypercapnia group+L-Arg (HL) and chronic hypoxia-hypercapnia group+L-Arg liposome (HP). Changes in pulmonary artery L-Arg transport and pulmonary arterial microscopy were observed. RESULTS: (1) The mean pulmonary artery pressure (mPAP) and weight ratio of right ventricle to left ventricle and septum (RV/LV+S) in HH group were higher than those in NC group, and in HP group was lower than that in HH group and HL group, but there was no significant difference between HL group and HH group; (2) At 0.005 mmol/L, 0.01mmol/L, 0.02mmol/L, 0.05 mmol/L, 0.1 mmol/L and 0.2mmol/L concentration of L-Arg, the velocity of L-Arg transport in HH group was lower than that in NC group, and in HL group higher than in HH group, and in HP group was much higher than that in HH group and in HL group. (3) Light microscopy showed that vessel well area/total area (WA/TA) and media thickness of pulmonary arterioles (PAMT) were much higher in rats of HH group than those in NC group, WA/TA and PAMT in HP group were obviously improved. CONCLUSION: The above results indicated that there existed a functional disturbance in L-Arg transport of pulmonary artery in rats chronically exposed to hypoxia-hypercapnia, and it was obviously enhanced when liposome was used as L-Arg carrier. Thus, it appears that liposome-L-Arg may have clinical perspective in the treatment of chronic hypoxic pulmonary hypertension.     

Effects of aspirin on production of nitric oxide and inducible nitric oxide synthase mRNA expression under inflammatory conditions in human vascular endothelial cells

AIM: To explore the effect of aspirin on inducible nitric oxide synthesis and gene expression under inflammation in endothelial cells. METHODS:Using NADPH, Griess methods and RT-PCR, the activity of isozymes of NO synthase (NOS), nitric oxide (NO) level, and iNOS mRNA expression were examined respectively. Also, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content and cell viability were measured. RESULTS: Aspirin (3 mmol/L) reduced inducible NO production and NOS activity(P＜0.05), caused a significant decrease in LDH release rate and MDA content with a further increase in cell viability. Aspirin inhibited inducible NO excretion and alleviated the damage caused by NO in a concentration-dependent manner. However,aspirin had no effect on basal NO levels in the absence of stimulation by inflammatory factor. On the other hand, under middle concentration (＜10 mmol/L), aspirin was able to reduce enzymatic activity of NOS and protein expression by increasing the stability of iNOS mRNA. In contrast, at high concentration (20 mol/L), aspirin could decrease the stability of iNOSmRNA. Sodium salicylate and indomethacin did not inhibit inducible NO production. CONCLUSION:Aspirin could significantly inhibit inducible NO production in vascular endothelial cells during inflammation.     

Effect of IL-10 on IL-1β-induced cyclooxygenase-2 expression and prostaglandin E2 release in human mesangial cells

AIM: To investigate the effect of IL-10 on IL-1β-induced prostaglandin E₂(PGE₂) release and cyclooxygenase-2(COX-2) expression in human mesangial cells and to examine whether IL-10 has effect on the biological function of IL-1β.METHODS: The PGE₂ concentration in supernatants of HMC was measured by radioimmunoassay. The COX-2 mRNA and protein were measured by RT-PCR and Western blot, respectively. RESULTS: PGE₂ and COX-2 were significantly increased after treatment with IL-1β(P＜0.01 for both) in cultured human mesangial cells. IL-10 had no effects on basical production of COX-2 and PGE₂(P＞0.05, respectively), while it inhibited IL-1β-elicited PGE₂ production, as well as COX-2 mRNA and protein expression in a concentration-dependent fashion. CONCLUSIONS: These results indicated that IL-10 depressed the IL-1β-induced release of PGE₂ and expression of COX-2. These data suggested that IL-10 could exert anti-inflammatory actions at several levels, not only by inhibiting the production of pro-inflammatory cytokines but also by suppressing their biological function.     

Effects of interleukin -13 on interleukin-12 production in mesangial cells induced by LPS

AIM: To investigate the effect of interleukin-13 (IL-13) on interleukin-12 (IL-12) production in mesangial cells.METHODS: The protein synthesis of IL-12 in mesangial cells was measured by ELISA.The expression of IL-12 mRNA in mesangial cells was evaluated by RT-PCR.RESULTS: The production of IL-12 in mesangial cells stimulated by lipopolysaccharide(LPS) was significantly increased (P<0.01).IL-13 (1-100 μg/L) inhibited the protein and mRNA expression of IL-12 in a dose-dependent manner (P<0.05 or P<0.01).CONCLUSION: IL-13 inhibits IL-12 expression induced by LPS in mesangial cells.IL-13 may regulate immune responses by balancing Th1/Th2 in glomerulonephritis.     

Effect of some valid components of chinese medicinal herbs on IL-6 mRNA expression in rat mesangial cells

AIM and METHODS: Cultured mesangial cells were stimulated by lipopolysaccharide(LPS), then were treated with some valid components in BUSHENHUOXUEXIEZHUOTANG: emodin, ligustrazine and ganoderma polysaccharide. The effects of these components on interleukin-6(IL-6) mRNA expression in mesangial cells were identified by Northern hybridization. RESULTS: Emodin, ligustrazine and ganoderma polysaccharide inhibited LPS-induced IL-6 gene expression in mesangial cells.CONCLUSION: BUSHENHUOXUEXIEZHUOTANG may relieve mesangial cells proliferation by inhibiting IL-6 mRNA expression.