Effect of γ radioactive stent on proliferation and apoptosis of smooth muscle cells

AIM: To observe effect of γ radioactive ［103Pd］ stent on the proliferation and apoptosis of smooth muscle cells, the mechanism of radioactive stent preventing in-stent restenosis was explored. METHODS: Fifty male New Zealand rabbits were randomized into stent group and ［103Pd］ stent group. Control group was set up. The materials were harvested on 3, 7, 14, 28, 56 days after operation and the following investigation were carried out, including pathomorphology, immunohistochemistry, apoptosis (TUNEL) and in situ hybridization studies.RESULTS: ① The severity of the stenosis in ［103Pd］ stent group was less severe than that in stent group. It was most obvious on 56 th day (P<0.01). ② The expression of proliferating cell nuclear antigen(PCNA) of ［103Pd］ stent group was lower than that in stent group on 3 to 28 days. It was most obvious on 7th day, 16.35%±0.79% vs 24.36±0.55% (P<0.01). ③ TUNEL method showed that the ［103Pd］ stent group had much more apoptosis of VSMC than that in stent group. The highest rate of apoptosis appeared on day 7, 14.72%±0.53% vs 12.42%±1.13% (P<0.01). ④ By calculating the ratio of PCNA/apoptosis (P〖KG*6〗∶〖KG-*2〗A), a much lower ratio was seen in ［103Pd］-stent groups than that in stent group at 3 to 28 days. There was significant statistic difference (P<0.05). ⑤ For bcl-2/bax ratio, the result in ［103Pd］-stent group was lower than that in stent group at 3 to 28 days. There was significant statistic difference (P<0.05).CONCLUSION: γ radioactive stent inhibits the proliferation and accelerates apoptosis of injured media vascular smooth muscle cells. It decreases the ratio of proliferation to apoptosis and relieves the severity of restenosis.     

Selenium-enriched Spirulina platensis promotes proliferation of hepatocytes in rat partial hepatectomy

AIM: To explore the effects of selenium-enriched Spirulina platensis (Se-SP) on proliferation of hepatocytes in rat hepatectomy. METHODS: Rat hepaectomy model was conducted using male Wistar rats. The rats were randomized into five groups: operation groups with 150 (H), 50 (M) and 15 (L) mg·kg^-1·d^-1 of Se-SP, placebo-control (P) and sham operation group (F). Activities of glutathione peroxidase (GPx) and thioredoxin reductases (TR) in hepatocytes were determined by chemical colorimetry. The expression index of proliferating cell nuclear antigen (PCNA) in hepatocytes was detected by immunohistochemistry, and the level of ［³H］-TDR incorporation in regenerative hepatocytes was analyzed by radio-immunity. RESULTS: Activity of GPx and TR, PCNA expression index as well as ［3H］-TDR insertion in hepatocytes (in vitro) were obviously higher (P<0.05) in L groups than those in P and F groups. All parameters were significant changed (P<0.05) after operation in H, M, L and P groups whereas some slightly change in F group. Furthermore, correlation analysis showed that the levels of GPx and TR in hepatocytes all showed positive correlation with PCNA expression (r²=0.77 and 0.87, respectively) and with ［³H］-TDR incorporation level (r² = 0.73 and 0.84, respectively) in hepatocytes. CONCLUSION: Se-SP enhances hepatocyte proliferation in rat hepatectomy, up-regulation of selenoenzymes might be responsible for this effect.     

Agmatine inhibits proliferation of rat PASMCs induced by hypoxia

AIM: To determine the effect of agmatine on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by hypoxia. METHODS: Primary culture of rat PASMCs was prepared from adult male Wistar rat pulmonary artery by the method of tissue block anchorage. PASMCs were divided into two groups: control group and agmatine-treated group. The activity of LDH in the medium was measured by chromatometry. ［3H］-TdR incorporation was measured by liquid scintillation counting. The cell cycle was measured by flow cytometry, and the PCNA content was measured by image analysis. RESULTS: Agmatine did not exert significant effect on the activity of LDH in the medium, but significantly decreased the ［3H］-TdR incorporation and PCNA content of PASMCs. Agmatine significantly decreased the cell ratio of G2/M phase and increased the cell ratio of G0/G1 phase. With the increment of the concentration of agmatine, ［3H］-TdR incorporation was significantly decreased. CONCLUSION: Agmatine has no significant cytotoxic effect on PASMCs and agmatine dose-dependently inhibits the proliferation of rat PASMCs induced by hypoxia.     

Expression of PI3K in airway smooth muscle cell of asthmatic rats

AIM: To investigate the expression of PI3K in airway smooth muscle cell (ASMC) of asthmatic rats.METHODS: 16 Wistar rats were divided into two groups, asthma and normal control at random. After establishment of asthmatic model, flow cytometry, immunofluorescence and Western blotting were applied to detect the growth fraction of ASMC and the expression of PI3K in cultured ASMC from each rat.RESULTS: It was revealed from flow cytometry that the ratio of S + G2/M to total number of cells in asthma group ［ (27.90±3.44) % ］ was higher than that in normal control group ［ (13.00±1.56) %, P<0.05］. The expression of PI3K was observed in both asthma and normal control group. However, it was much higher in asthma group than that in normal control group. There was a positive correlation between the expression of PI3K and the growth fraction in ASMC. CONCLUSION: The increased expression of PI3K might play an important role in regulating the proliferation of ASMC in asthma.     

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.     

Decreased fibroblast proliferation and collagen content in the wound of diabetic rats caused by streptozotocin

AIM: To study fibroblast proliferation and collagen synthesis during wound healing in diabetic rats induced by streptozotocin. METHODS: 30 Wistar male rats were randomly divided into control group and model group. 55 mg/kg STZ were given intraperitoneally to model rats. After 3 weeks, a round skin of 2.04 cm2 was excised on all dorsal back of rats. The healing time and healing rate were observed according to re-epithelization. The numbers of fibroblasts and the expression of proliferating cell nuclear antigen (PCNA) were observed by Hematoxylin-Eosin (HE) staining and immuno-histochemistry assay. Collagen Ⅰ and Ⅲ stained by Picric acid-Sirius red were calculated by image analysis. RESULTS: The healing time in model group was (27.13±1.81) days，significantly longer than that in control group ［(15.25±1.67） days， P<0.01］. The healing rates in model group were significantly less than that in control group at day 3, day 7 and day 15 (P<0.01). The amount of fibroblasts and the expression of PCNA in model group were significantly less than those in control group on day 3, day 5, day 7 and day 9, respectively (P<0.05, P<0.01). Even the content of collagen I in the wound of both groups increased with time, the values were much higher than that in model group at different times (P<0.05), respectively. For model group, the ratio of collagen Ⅰ/Ⅲ was less than that in control group 3, 7 and 11 days after wound (P<0.01). CONCLUSION: STZ impaires wound healing in rats, which is possible caused by the disturbance of fibroblast proliferation and collagen synthesis in the wound.     

Inhibitory effect of ferulate on attachment and migration induced by fibronectin and fibrinogen in cultured vascular smooth muscle cells from SHR

AIM: To investigate the effect of sodium ferulate (SF), one of the principal components of rhizoma ligustici wallichii, on the attachment and migration induced by fibronectin (FN) and fibrinogen (Fg) in vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs derived from spontaneously hypertensive rats (SHR) were used. Cell attachment was conducted using a flat-bottomed 96 well polystyrene plate. Cell migration was determined by modified Boyden chamber assays. Intracellular free calcium (［Ca2+］i) was measured with fluorescent Ca2+ indicator Fura-2/AM. RESULTS: FN and Fg significantly induced the attachment and migration of VSMCs in a dose-and time-dependent manner, which was inhibited by pre-treatment of VSMCs with SF (10-7-10-3mol/L) a dose-dependentl fashion. The peak inhibition rate of attachment induced by FN and Fg was 67.12% and 70.23%, respectively, and the peak inhibition rate of SF on migration induced by FN and Fg was 69.79% and 87.06% (P<0.01). The rise of ［Ca2+］i in VSMCs provoked by FN and Fg was significantly suppressed by 10-3mol/L SF (P<0.01). CONCLUSION: The attachment, migration and increase in ［Ca2+］i induced by FN and Fg in VSMCs from SHR are suppressed by SF.     

Role of intracellular free Ca2+ concentration in the regulation of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells

AIM: To investigate the role of intracellular free Ca²⁺ concentration (［Ca²⁺］_i) in the regulation of calcium-activated chloride (Cl_Ca) channels in pulmonary artery smooth muscle cells (PASMCs) of rats under normoxic, acute and chronic hypoxic conditions. METHODS: Acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe ［Ca²⁺］_i of rat PASMCs in normal and chronic hypoxic condition. The influences of Cl_Ca channels on PASMCs proliferation were assessed by MTT assay. RESULTS: (1) The Cl_Ca channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) produced inhibitory effects on acute hypoxia-evoked contractions in pulmonary artery. (2) Under chronic hypoxic condition, ［Ca²⁺］_i was increased. In normoxic condition, ［Ca²⁺］_i was (123.63±18.98) nmol/L, and in hypoxic condition, ［Ca²⁺］_i was (281.75±16.48)nmol/L (P<0.01). (3) In normoxic condition, ［Ca²⁺］_i had no significant change and no effect on Cl_Ca channels was observed (P>0.05). (4) Chronic hypoxic increased ［Ca²⁺］_i which opened Cl_Ca channels. The NFA and IAA-94 blocked them and decreased ［Ca²⁺］_i from (281.75±16.48)nmol/L to (117.66±15.36)nmol/L (P<0.01). (5) MTT assay showed that in chronic hypoxic condition NFA and IAA-94 decreased the value of absorbing light degree (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). CONCLUSION: Hypoxia increased ［Ca²⁺］_i which opened Cl_Ca channels and had a positive-feedback to ［Ca²⁺］_i. This may play an important role in hypoxic pulmonary hypertension. In chronic hypoxic condition, Cl_Ca channel may play a role in the regulation of PASMCs proliferation.     

Deoxyribozyme inhibits the proliferation of human umbilical artery smooth muscle cells

AIM: To observe the inhibition effects of 10-23 deoxyribozyme (DNAzyme) specific to human proliferating cell nuclear antigen (PCNA) gene on the proliferation of human umbilical artery smooth muscle cells (HUASMC) in vitro. METHODS: Using lipofectamine (Lip)-mediated method, the DNAzyme specific to PCNA was introduced into HUASMC. ［3H］-TdR incorporation was determined. The cell proliferation was examined by MTT assay and cell cycle was detected by flow cytometry. RESULTS: ［3H］-TdR incorporation in 1.0 μmol/L DNAzyme group was lower than that in control group (P<0.05) at 2 days after transfection. Compared with control group, the A value of MTT cholorimetric analysis decreased in 1.0 μmol/L DNAzyme group and antisense oligodeoxynucleotide (ASODN) group, at 2, 3 and 5 days after transfection significantly (P<0.01). The decrease in A value showed a dose-dependent manner within the experimental range at 5 days. After 2 days, the percentage of quiescence cells (G0/G1) was 73.8%, 54.7% and 41.1% in DNAzyme, ASODN and control groups, respectively. CONCLUSION: The DNAzyme specific to PCNA suppresses the proliferation of HUASMC effectively in vitro.     

Effect of angiotension-Ⅱ on rat cardia fibroblasts

AIM: To investigate the effects of angiotensin-Ⅱ (Ang-Ⅱ) on the proliferation and collagen synthesis in rat cardiac fibroblasts and the expression of Ang-Ⅱ receptor on fibroblasts. METHODS: ［3H］-TdR and ［3H］-prolin at different concentrations were incubated with the confluent fibroblasts of newborn rats stimulated by Ang-Ⅱ. Receptor of Ang-Ⅱ on fibroblasts and intracellular free calcium ions were examined by autoradiograms and fluorescence technique. RESULTS: In the presence of Ang-Ⅱstimulation, the increase in incorporation of ［3H］-TdR and ［3H］-prolin was much higher than that of control in a dose-dependent manner. Autoradiograms showed that Ang-Ⅱ was uniformly distributed over the membrane of the fibroblasts. The silver grains on fibroblasts were obviously decreased after adding unlabeled Ang-Ⅱ by the autoradiograms. The concentration of free calcium ions was increased in fibroblast. CONCLUSION: These findings suggest that Ang-Ⅱ promotes fibroblast proliferation and the syntheses of collagen protein, in which Ang-Ⅱ binds to the membrane receptor of fibroblasts to activate the signal system in cells, such as intracellular free calcium ions.     

Heme oxygenase decreases the generation of ROS in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells

AIM:To investigate the role of heme oxygenase (HO) in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells.METHODS:(1) Western blotting analysis was carried out to detect protein level of HO-1 in the tissues.(2) ［3H］-TdR, ［3H］-leucine incorporation was measured in cultured vascular smooth muscle cells.(3) 2,7-dichlorofluorescin diacetate (DCFH-DA) as an index was used to determine the cellular reactive oxygen species (ROS) level.RESULTS:(1) No significant difference in HO-1 protein expression level between AngⅡ-stimulated and control groups was observed, but HO-1 protein level in Hemin-induced group was higher than that in other two groups (P<0.01).No significant increase in HO-1 protein expression was found in zinc-protoporphyrin IX (ZnPPIX) group.(2) After AngⅡ stimulation, ［3H］-TdR and ［3H］-leucine incorporations of vascular smooth muscle cells (VSMCs) were increased.Hemin inhibited this increase.The higher concentration of Hemin, the more significant was the inhibitory effect.On the contrary, ZnPPIX promoted the increase in the effect of AngⅡ by inhibiting HO.(3) Fluorescence intensity in AngⅡ group was obviously higher than that in control groups (P<0.01).Compared with AngⅡ group, Hemin group decreased 62.7%, but ZnPPIX group increased 39.5%.CONCLUSION:Hemin induces HO-1 expression and inhibits the effect of AngⅡ to stimulate proliferation and hypertrophy of VSMCs.The mechanism may be related to its inhibition of ROS production.     

Expression and function of calcium-sensing receptor in mouse embryonic stem cells

AIM: To observe the functional expression of calcium-sensing receptor (CaSR) in the mouse embryonic stem cells (mESCs). METHODS: The expression and distribution of CaSR were detected by Western blotting and immunofluorescence observation in 129 mouse ES-D3 cells. The intracellular concentration of free calcium (［Ca2+］i) was determined by confocal laser scanning microscopy. The cell viability was analyzed by MTT assay and flow cytometry. RESULTS: CaSR protein was expressed in mESCs. Extracellular calcium or neomycin significantly increased the expression of CaSR and ［Ca2+］i. Neomycin increased the cell viability, up-regulated the protein expression of p-ERK2. These effects of neomycin were inhibited by NPS2390. CONCLUSION: CaSR is expressed in mESCs. The activation of CaSR is involved in the proliferation of mESCs.     

Effect of granulocyte-colony stimulating factor on the reendothelialization and intima hyperplasia of ballon-injured rat carotid artery

AIM:To study the effect of mobilization of stem cells by exogenous recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the repairing process of reendothelialization and neointima hyperplasy on ballon injured rat carotid arteries.METHODS:Male Wistar rats were randomly divided into rhG-CSF group and NS+injury group.The animals were injected daily with 30 μg/kg rhG-CSF or 0.9% NaCl for 7 days,then underwent balloon angioplasty of the common carotid arteries which were harvested and processed for scanning electron microscopy (SEM),Evans blue staining,morphometric analysis of endothelialization and neointimal formation at 1 h,3 d,5 d,7 d,14 d after injury.Immunohistochemistry for proliferation cell nuclear antigen (PCNA) and RT-PCR for eNOS mRNA were also conducted for evaluating the proliferation of cells of the vessel wall and the possible mechanism of the repairing.RESULTS:SEM and Evan’s blue staining showed increased reendothelialization of the denuded vessels in rhG-CSF-treated animals compared with that NS+injury animals ［(60.6±7.3)% vs (41.6±3.3)%，P<0.01］.Neointima thickness was reduced by 37.3% in rhG-CSF group compared with NS+injury group 2 weeks after injury.Immunohistochemical staining for PCNA positive cells was less in rhG-CSF group compared with that in NS+injury group (42.6% vs 72.8%,P<0.01).CONCLUSION:rhG-CSF has beneficial effects on the reendothelialization and neointima thickness of the ballon-injured arteries.Mobilization of EPCs by exogenous granulocyte colony stimulating factor may be a potential therapeutic strategy for prevention of restenosis after percutenous coronary artery intervention.     

Inducing vascular smooth muscle cell proliferation by insulin involves in phosphatidylinositol 3-kinase and ERK1/2

AIM: To investigate the effect of insulin on vascular smooth muscle cells (VSMCs) proliferation and to evaluate the intracellular signaling pathways involved.METHODS: VSMCs separated from Sprague-Dawley rats were used in this study. The proliferation of VSMCs induced by insulin was assayed by ［3H］-thymidin incorporation. The protein expression and activity of p-ERK1/2 were determined by immunblot and ［γ-32P］ATP incorporation.RESULTS: Insulin induced cell proliferation in a concentration-dependent manner. The proliferative effect of insulin on VSMCs was inhibited partly by LY294002 (48.8%), an inhibitor of PI-3 kinase, and the ERK1/2 inhibitor PD98059 (43.6%), respectively. Moreover, phosphorylation of ERK1/2 and activity of ERK1/2 induced by insulin were also inhibited partly by LY294002.CONCLUSION: PI-3 kinase and ERK1/2 are involved in insulin induced VSMCs proliferation.     

Endothelin receptor antagonist BQ123 inhibits calcification in vascular smooth muscle cells induced by β-glycerophosphate

AIM:To observe the effect of endothelin receptor antagonist (BQ123) on calcification in rat vascular smooth muscle cells (VSMCs) in vitro. METHODS:Calcification of cultured rat VSMCs was produced by incubation with β-glycerophosphate. Calcium content, Ca2+ deposition and alkaline phosphatase activity were analyzed to estimate the extent of calcification. The DNA synthesis was detected by ［3H］ -TdR and ［3H］-Leu incorporation. Osteopontin (OPN) mRNA was measured by competitive quantitative RT-PCR. Content of ET was measured by radioimmunoassay (RIA). RESULTS:The results showed that compared with the control, the content of calcium, ［45Ca2+］ uptake and alkaline phosphatases activities in calcified VSMCs increased by 118%, 174% and 7-fold (all P<0.01), respectively. The expression of OPN mRNA in calcified VSMCs was up-regulated by 86% (P<0.01). The calcified VSMCs grew rapidly, in which ［3H］-TdR and ［3H］-Leu were elevated by 71% and 35%. The content of ET in calcified VSMCs medium was increased by 35% as compared with control. Furthermore, calcified VSMCs plus BQ123 groups obviously relieved degree of calcification, of which calcium content, Ca2+ deposition and alkaline phosphatase activities were 33%, 37%, 40% lower than those in calcified VSMCs (P<0.01), respectively. The expression of OPN mRNA was down-regulated by 25% (P<0.01) and significantly inhibited VSMCs proliferation. CONCLUSION:BQ123 reduces VSMCs calcification, suggesting that ET promotes calcification in VSMCs mainly by ET/ ETA receptor pathway.     

Pancreatic kininogenase protects against renal fibrosis in rat model of unilateral ureteral obstruction

AIM To assess the beneficial effects of pancreatic kininogenase （PK） on renal fibrosis in rat model of unilateral ureteral obstruction （UUO）. METHODS Male Sprague-Dawley rats were randomly assigned to 5 groups and treated daily with PK for 7 d and 14 d. Masson trichrome and HE staining were used to assess the degree of tubulointerstitial fibrosis, and immunohistochemistry and Western blot were employed to evaluate the expression of profibrotic and proinflammatory cytokines, and apoptosis- and autophagy-related proteins. RESULTS PK treatment significantly decreased expression of profibrotic and proinflammatory cytokines, and these were paralleled with attenuation of tubulointerstitial inflammation ［monocyte chemoattractant protein-1 （MCP-1） and Toll-like receptor 2 （TLR-2）］ and fibrosis ［transforming growth factor β1 （TGF-β1） and connective tissue growth factor （CTGF）］ in a time-dependent manner. Oxidative stress induced by UUO manifested by augment of oxidant enzymes ［NADPH oxidase-2 （NOX-2） and NOX-4］ and decrease in antioxidant enzymes ［superoxide dismutase 1 （SOD1） and SOD2］, which was closely associated with dysregulation of apoptosis- and autophagy-related proteins subsequent excessive apoptotic （Bcl-2/Bax and cleaved caspase-3） and autophagy （LC3B, beclin-1 and P62）, and all of these were eliminated by administration of PK. CONCLUSION PK treatment protects against the progression of renal fibrosis in obstructed kidneys probably by interfering oxidative stress and programmed cell death.     

Effect of inhibiting nuclear factor-kappa B on TNF-α-induced expression of angiotensinogen and production of angiotensinⅡ in glomerular mesangial cells

AIM: To study the effect of inhibiting nuclear factor-kappa B (NF-κB) activity on the expression of angiotensinogen (AGT) and the production of angiotensinⅡ (AngⅡ) induced by tumor necrosis factor-α (TNF-α) in glomerular mesangial cells (MCs) of SD rats. METHODS: The MCs of SD rats were isolated and divided into three groups as follows: control; MCs treated with TNF-α, and the MCs treated with TNF-α + pyrrolidinedithiocarbamate (PDTC). The activity of nuclear factor-kappa B was measured by electrophoretic mobility shift assay. The expression of AGT was determined by RT-PCR for mRNA and Western blotting for protein. The concentration of angiotensinⅡ in supernatant was measured by RIA. RESULTS: The NF-κB activity in the MCs treated with TNF-α (20.67±9.14)×102 μg/cell was significantly higher than that in control cells ［(8.25±4.35)×102 μg/cell, P<0.01］ and the MCs treated with TNF-α+PDTC ［(7.20±4.57)×102 μg/cell, P<0.01］, and no significant difference was found between control and the MCs treated with TNF-α＋PDTC (P>0.05). The AGT mRNA level in the MCs treated with TNF-α (0.27±0.05) was higher than that in the control cells (0.20±0.05, P<0.05), and no significant difference was observed when compared with that in the MCs treated with TNF-α＋PDTC (0.22±0.06, P>0.05). The expression of AGT protein in the MCs treated with TNF-α (0.60±0.19) μg/cell was higher than that in the control ［(0.37±0.15)μg/cell, P＜0.05］ and the MCs treated with TNF-α＋PDTC ［(0.37±0.17)μg/cell, P<0.05］, and no significance was found between the MCs treated TNF-α＋PDTC and the control (P>0.05). The AngⅡ level in supernatant of cultured MCs treated with TNF-α ［(9.73±2.38)×10-5 ng·L-1/cell］ was significantly higher than that in the control ［(7.50±1.51)×10-5 ng·L-1/cell, P<0.05］ and in the MCs treated with TNF-α＋PDTC ［(6.94±1.46)×10-5 ng·L-1/cell, P<0.05］, however, the difference between the MCs treated with TNF-α＋PDTC and the control was of no significance (P>0.05). CONCLUSION: TNF-α activates the NF-κB in glomerular MCs, induces the AGT expression and the production of AngⅡ. Inhibition of NF-κB decreases the AGT expression and the production of AngⅡ. Therefore, the effects of TNF-α on AGT and AngⅡ may be mediated by NF-κB.     

Effects of adrenomedullin 2 on proliferation of microvascular endothelial cells from the rat cerebral cortex

AIM: To investigate the effects of adrenomedullin (ADM) 2 (AM2) on proliferation of microvascular endothelial cells from the rat cerebral cortex. METHODS: Microvascular endothelial cells (MEC) were isolated from the cerebral cortex of SD rats and cultured. The cultured cells were identified using immunocytochemistry assay with antibody for factor VIII-related antigen and randomly distributed into eight experimental groups as follows: control, AM2 10-7 mol/L, 10-8 mol/L, 10-9 mol/L, ADM, ADM+AM2, 10% fetal bovine serum (FBS) stimulated, and 10% FBS＋AM2 10^-7 mol/L groups. The proliferation of MEC was detected using ［3H］-TdR incorporation assay. RESULTS: Compared with control, AM2 (10^-7-10^-9 mol/L), ADM (10^-7 mol/L), and AM2 (10^-7mol/L) co-incubated with ADM (10^-7 mol/L) had no effects on ［3H］-TdR incorporation into the MEC (P>0.05). 10% FBS induced ［3H］-TdR incorporation increased by 87.5% (vs control, P<0.05), which was abolished by co-incubated the MEC with 10^-7 mol/L AM2 (P<0.05). CONCLUSION: AM2 inhibits FBS-stimulated proliferation of MEC from the rat cerebral cortex.     

Effects of PAR-2 agonist peptide on proliferation and cytosolic calcium level in hepatoma cells

AIM: To investigate the effects of PAR-2 agonist peptide on the proliferation and cytosolic calcium concentration (［Ca2+］c) in human hepatoma cells HepG2. METHODS: Human hepatoma cell line HepG2 was cultured. The cells were treated with PAR-2 agonist peptide SLIGKV-NH2 and the reverse PAR-2 agonist peptide VKGILS-NH2, respectively. The ［Ca2+］c of hepatoma cells were measured by microfluorimetric techniques based on calcium indicator fura-2/AM. The influences on proliferation of hepatoma cells were determined by MTT method. The changes of cell cycle were evaluated by flow cytometry, and the changes of cyclin D1 mRNA expression were detected by RT-PCR. RESULTS: After treated with 50 μmol/L SLIGKV-NH2, a rapid rise of ［Ca2+］c in HepG2 cells was induced (P<0.01), percent S phase, G2/M phase and proliferation index (PI) of HepG2 cells were elevated (P<0.01), and cyclin D1 mRNA expression was significantly upregulated (P<0.01). The proliferation rates of HepG2 cells treated with 1-50 μmol/L SLIGKV-NH2 were significantly increased, and the effect was in a dose-dependent manner (P<0.01 or P<0.05). No statistical significance of the difference between VKGILS-NH2 and control group was observed (P>0.05). CONCLUSION: PAR-2 agonist peptide induces the rise of ［Ca2+］c in HepG2 cells, upregulates the expression of cyclin D1 mRNA, accelerates the progress of cell cycle, promotes the synthesis of DNA and the proliferation of hepatoma cells via activating PAR-2 in vitro.