Effects of SOCS3 gene on the expression of c-myc mRNA and proliferation of rat pulmonary arterial smooth muscle cells under hypoxia conditions

AIM: To explore the effect of SOCS3 gene on the expression of c-myc mRNA and proliferation of rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxia conditions. METHODS: PASMCs was cotransfected with pEFSOCS3 and pSV2neo by lipofectamine, and positive cell clones were obtained after being screened with G418. Expressions of SOCS3 protein in PASMCs before and after transfection were detected by immunocytochemistry, respectively. Before and after transfection, PASMCs were exposed to normoxic and hypoxia conditions at various time points, respectively, and the expressions of c-myc mRNA were assessed by semi-quantitive RT-PCR. ［3H］-TdR incorporation method was used to detect the cell proliferation. RESULTS: The expression of SOCS3 protein was confirmed by immunocytochemistry in PASMCs transfected with SOCS3 gene. c-myc mRNA level in the SOCS3 gene-transfected cells exposed to hypoxia were remarkablely lower than that in the control cells, respectively (P<0.01). Compared with the control groups at the same time points, ［3H］-TdR incorporation in SOCS3 gene-transfected cells was significantly low. CONCLUSION: SOCS3 protein may inhibit the proliferation of PASMCs by downregulating the c-myc gene expression under hypoxia conditions.     

Effects of Gax gene transfection on proto-oncogene expression and proliferation of PASMCs in rats under hypoxia conditions

AIM: To explore the effects of Gax gene transfection on expressions of c-fos and c-jun mRNA and proliferation of pulmonary arterial smooth muscle cells (PASMCs) in rat under hypoxia.METHODS: PASMCs were transfected with Gax gene by Ad-Gax.Under normal oxygentention (21% O₂) or hypoxia (2.5% O₂) for 12 h condition,expressions of Gax mRNA and protein in PASMCs were detected by RT-PCR and immunocytochemistry.The expressions of c-fos and c-jun mRNA were evaluated by RT-PCR.［³H］-TdR incorporation was used to measure the PASMCs proliferation.RESULTS: The Gax overexpression in transfection group was confirmed by RT-PCR and immunocytochemistry.Under normal oxygentention or hypoxia,the c-fos and c-jun mRNA levels in transfection group were lower than those in the non-transfection group,respectively (P<0.05).［³H］-TdR incorporation in the transfection group was lower than that in non-transfection group (P<0.05,P<0.01).CONCLUSION: Gax overexpression might inhibit the PASMCs proliferation induced by hypoxia through downregulating the expressions of c-fos and c-jun.     

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.     

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.     

Effects of chronic hypoxia on intracellular calcium concentration in pulmonary arterial smooth muscle cells

AIM:To study the effect of chronic hypoxia (CH) on the intracellular calcium (［²⁺］_i) in pulmonary artery smooth muscle cells (PASMCs) and the role of L-type calcium channel and calcium store. METHODS:The rat chronic hypoxia model was set up and intervene the PASMCs with normal PSS, calcium-free PSS, nifedipine, and heparine respectively. The resting ［Ca²⁺］_i was determined with the Fura-2/AM calcium imaging technique. RESULTS:(1) The ［Ca²⁺］_i in CH group in normal PSS was higher than that in control group in normal PSS (P<0.05). The ［Ca²⁺］_i in CH group in normal PSS was higher than that in calcium-free PSS (P<0.05). (2) No obvious change of ［Ca²⁺］_i before and after application of nifedipine in PASMCs of CH groups was observed. (3) No difference of ［Ca²⁺］_i before and after application of heparine in PASMCs of CH groups was detected. CONCLUSION:Chronic hypoxia increased the ［Ca²⁺］_i in PASMCs. Chronic hypoxia induced increase in ［Ca²⁺］_i may relate to the influx of extracellular calcium, but not due to the L-type calcium channel or the IP3R modulation only.     

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.     

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.     

类番茄茄与番茄属间有性杂交的研究

用类番茄茄与番茄属的 9个种进行有性杂交 ,通过人工离体胚培养获得栽培番茄‘粤农二号’×类番茄茄的F1植株。杂种近于不育。用栽培番茄、类番茄茄、野生番茄及(栽培番茄×野生番茄 )与之回交及杂交 ,获得与栽培番茄的回交子一代及与秘鲁番茄、多腺番茄、潘那利番茄、粤农二号×秘鲁番茄F1的杂种 ,但它们的能育性很低。用栽培番茄、野生番茄与它们再回交及杂交 ,未能得到与栽培番茄的二次回交子一代 ,只获得 (粤农二号×类番茄茄 )× (粤农二号×秘鲁番茄 )的F1与秘鲁番茄、多腺番茄的复合杂种。人工苗期接种黄瓜花叶病毒 (CMV)结果表明 ,类番茄茄及其与番茄属的远缘杂种对CMV有较强的抗性。     

Effects of acute hypoxia on calcium signal of sarcoplasmic reticulum in pulmonary artery smooth muscle in rats

AIM: To investigate the effects of acute hypoxia on calcium of sarcoplasmic reticulum in pulmonary artery smooth muscle in rats. METHODS: The fluorescence Ca2+ indicator Fura-2/AM was used to observe intracellular free Ca2+ concentration (［Ca2+］i) in rat pulmonary artery smooth muscle cells (PASMCs) in the presence of ryanodine (RD) and cyclopiazonic acid (CPA) in normal (37 ℃, 5%CO2, 21%O2, 74%N2), acute hypoxic (37 ℃, 5%CO2, 2%O2, 93%N2) under Ca2+ and Ca2+ free conditions. Pulmonary artery ring was used to determine the pulmonary artery tension by using routine blood vascular perfusion in vitro under the same conditions. RESULTS: (1) Under acute hypoxic conditions, ［Ca2+］i was increased ［(96.99±7.16) nmol/L in normoxic condition and (257.06±32.48) nmol/L in hypoxic condition, P<0.01］. (2) Ryanodine or procain, an agent that blocks ryanodine receptor-seneitive (RyR) Ca2+ stores, inhibited hypoxia-induced increases in ［Ca2+］i { ［Ca2+］i decreased to (100.91±11.21) nmol/L, P<0.01}. CPA or thapsigargin (TG), the agent that inhibits sarcoplasmic reticulum (SR) Ca2+ -ATPase and inhibits SR uptake Ca2+, increased ［Ca2+］i. Under acute hypoxic and Ca2+ conditions, CPA or thapsigargin (TG) increased ［Ca2+］i more than that in Ca2+ free conditions. (3) Acute hypoxia evoked pulmonary artery contractions. Pulmonary artery tension had no effects under normoxic and increased under acute hypoxia condition. (4) Ryanodine or procain inhibited hypoxia-evoked contractions in the pulmonary artery. CPA or TG increased artery tension. Under acute hypoxic and Ca2+ conditions, CPA or TG increased tension more than that in Ca2+ free condition. CONCLUSION: The results indicate that release of Ca2+ from the SR, at least, RyR Ca2+ store, contributes to the mechanism of hypoxic pulmonary vasoconstriction in rat. This is a mechanism intrinsic to pulmonary artery without the need for Ca2+ influx across the plasmalemma or an endothelial factor.     

Role of calcium-sensing receptor in hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells

AIM: To explore the role of calcium-sensing receptor (CaSR) in rat pulmonary artery smooth muscle cells (PASMCs) and its effect on hypoxia-induced proliferation of PASMCs. METHODS: The expression and distribution of CaSRs were detected by Western blotting and immunofluorescence observation. The intracellular concentration of free calcium ([Ca]_i) was determined by confocal laser scanning microscopy. The cell viability was analyzed by MTT assay. The expression of PCNA and CaSRs was determined by Western blotting. RESULTS: CaSR protein was expressed in rat PASMCs. Hypoxia significantly increased the expression of CaSR and PCNA,[Ca]_i and the cell viability. GdCl₃ (an agonist of CaSR) or NPS2390 (an antagonist of CaSR) amplified or weakened the effect of hypoxia, respectively.CONCLUSION: CaSR is expressed in rat PASMCs. The activation of CaSR is involved in the proliferation of PASMCs induced by hypoxia.     

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.     

Calcium-sensing receptor mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells through PI3K pathways

AIM:To explore the signal transduction pathways of calcium-sensing receptor(CaSR) that mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells(PASMCs). METHODS:The expression of cyclin D1 and phosphorylated protein kinase B(p-Akt) was analyzed by Western blotting. Cell proliferation was tested using a BrdU incorporation assay, and cell cycle analysis was carried out using a flow cytometric assay. RESULTS:Hypoxia significantly increased the expression of cyclin D1 and p-Akt, the BrdU incorporation and the cell proliferation index. GdCl₃, an agonist of CaSR, amplified the effect of hypoxia. LY294002,a PI3K inhibitor, decreased the up-regulation of cyclin D1 expression and the BrdU incorporation, and also inhibited the increase in the cell proliferation index induced by hypoxia and GdCl₃ in PASMCs. CONCLUSION: The CaSR mediates hypoxia-induced proliferation of rat PASMCs through PI3K pathways.     

Chronic metabolic acidosis markedly induces proliferation of mesangial cells in rats

AIM: To study the effects of chronic metabolic acidosis on glomerulus, mesangial cells and the production of extracellular matrix. METHODS: Chronic metabolic acidosis was induced by addition of 0.28 mol/L NH4Cl to drinking water for 3, 7, or 14 days in male Wistar rats (n=10). Light microscope combined with computer software (Motic Images Advanced 3.2) was used to determine the effect of chronic acid loading on renal morphologic changes. The expressions of proliferation cell nuclear antigen (PCNA) and p27 in glomeruli were detected by Western blotting or immunohistochemistry. Fibronectin (FN) mRNA was detected by real-time PCR. The proliferation of mesangial cells in vitro was determined by ［3H］-TdR incorporation. The concentration of FN in cultured supernatant was detected by ELISA. RESULTS: On day 1, 3, 7 and 14, the arterial pH and plasma ［HCO3-］ in experimental rats were significantly decreased. There was a significantly increased in the kidney weight and the ratio of kidney to body weigh in experimental rats on day 3, 7 and 14. The glomerular area and cell numbers also increased significantly. Immunoblotting demonstrated decreased p27 expression and increased PCNA expression in isolated glomeruli, and the expression of PCNA increased in a time-dependent manner following the time of chronic metabolic acidosis. Immunohistochemistry showed increased positive PCNA expression mainly localized to mesangial cells. The expression of FN mRNA was significantly elevated in experimental rats on day 7 and 14. In vitro, acid loading induced mesangial cell proliferation and synthesis of FN. CONCLUSION: These results suggest that chronic metabolic acidosis induces mesangial cell proliferation, and its mechanism may be associated with the downregulation of cell cycle kinase inhibitor p27.     

Jagged1 knocking down in endothelial cells accelerates PDGF induced proliferation and migration of vascular smooth muscle cells in rats

AIM: To investigate the effect of Jagged1 expression in endothelial cells (EC) on platelet derived growth factor (PDGF) induced proliferation and migration of vascular smooth muscle cells (VSMC) in rat.METHODS: Rat aorta EC was inoculated in the lower chamber and VSMC were in the upper chamber of the cell coculture system. Three groups were divided: control, sicontrol and siJagged1. The EC Jagged1 protein expression was assayed by Western blotting to evaluate small RNA interfering (RNAi) efficiency. After the cells were cocultured with PDGF for 24 h, the proliferation and migration of VSMC were respectively evaluated by ［3H］-TdR incorporation and migrating cells counting. Protein expression of α-SM-actin in VSMC was assayed by Western blotting. RESULTS: The Jagged1 protein expression in EC was significantly lower in siJagged1 group than that in control group (0.26±0.02 vs 0.67±0.02, P<0.05), and no statistic significance was observed between control and sicontrol groups. The VSMC ［3H］-TdR incorporation and migration were higher in PDGF +siJagged1 group than those in PDGF group {［3H］-TdR incorporation (23 074±2 702) counts·min-1·well-1 vs (16 442±1 803)counts·min-1·well-1, n=5, P<0.05; migration (27±4) cells/field vs (15±3)cells/field, n=5, P<0.05}. The α-SM-actin protein in VSMC was lower in PDGF + siJagged1 group than that in PDGF group (0.25±0.06 vs 0.49±0.04, n=3, P<0.05).CONCLUSION: Jagged1 knock down in rat EC accelerates PDGF induced proliferation and migration of VSMC. These results suggest that Jagged1 expression in EC plays an important role in maintaining VSMC contract phenotype and inhibiting VSMC overgrowth after arterial injury.     

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 inhibiting myosin light chain kinase on endothelin-1 induced proliferation and apoptosis of pulmonary arterial smooth muscle cells in rats

AIM: To investigate the effect of inhibiting myosin light chain kinase(MLCK) on endothelin-1(ET-1) induced proliferation and apoptosis of rat pulmonary artery smooth muscle cells(PASMCs). METHODS: Rat PASMCs were cultured and stimulated with ET-1. The cells were randomly divided into control group, ET-1 group and ET-1+MLCK inhibitor group(ET-1+M). Western blotting, MTT assay, [³H]-TdR incorporation and flow cytometry were employed to test the expression of myosin light chain(MLC) and MLCK, cell proliferation, cell cycle and apoptotic rate of PASMCs,respectively. The phosphorylation of MLC was determined by glycerol-PAGE coupled with Western blotting. RESULTS: Compared with control group, the protein expression of MLCK and MLC phosphorylation significantly enhanced after ET-1 stimulation. ET-1 markedly induced the proliferation and decreased the percentage of apoptotic rate in the PASMCs. However, pretreatment with ML-7, a MLCK inhibitor, significantly reversed the above effects induced by ET-1. CONCLUSION: MLCK inhibitor effectively inhibits the ET-1-induced proliferation and the cell cycle progression.     

Effects of Kv1.5 on proliferation and apoptosis of rat PASMCs under hypoxia+hypercapnia condition and relationship with MAPK pathway

AIM：To investigate the effects of voltage-dependent K＋ channel 1.5 (Kv1.5) on the proliferation and apoptosis of rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia+hypercapnia condition and the relationship with mitogen-activated protein kinase(MAPK) signal pathway. METHODS：The PASMCs isolated from the male SD rat were cultured under hypoxia+hypercapnia condition, and randomly divided into normal group (N group), hypoxia+hypercapnia group (HH group), hypoxia+hypercapnia+DMSO incubation group (HD group), hypoxia+hypercapnia+U0126 (an extracellular signal-regulated kinase 1/2 inhibitor) incubation group (HU group), hypoxia+hypercapnia+SB203580 (a p38 mitogen-activated protein kinase inhibitor) incubation group (HS group), and hypoxia+hypercapnia+anisomycin (an agonist of MAPK) incubation group (HA group). Cell Counting Kit-8 was used to detect the cell viability. The protein expression of Kv1.5, PCNA and Bax was detected by Western blotting. RESULTS：Compared with N group, the cell viability and PCNA protein expression in HH group and HD group were significantly raised (P<001), but Kv1.5 and Bax proteins were significantly decreased (P<0.01). No difference between HH group and HD group was observed (P>005). Compared with HD group, the cell viability and PCNA protein expression in HU group, HS group and HA group were decreased (P<0.05 or P<0.01), but Kv1.5 protein and Bax protein were raised (P<0.01), with the most significant changes in HA group. ^{CONCLUSION：}The regulation of Kv1.5 to the proliferation and apoptosis of PASMCs under hypoxia+hypercapnia condition might have a relationship with the activation of MAPK signal pathway.     

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.     

Inhibitory effect of Ophiopogon japonicus on rat cardiac fibroblasts

AIM：To investigate the inhibitory effect of Ophiopogon japonicus on rat cardiac fibroblast (CFs) and the underlying mechanism. METHODS：Cultured CFs from Sprague-Dawley (SD) rats were randomized into 4 groups: control group (normal rat cardiac fibroblasts), Ophiopogon japonicus of 10 μg/L group, Ophiopogon japonicus of 20 μg/L group and Ophiopogon japonicus of 30 μg/L group. Cell vitality, ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 in CFs were determined. RESULTS：Compared with control, the cell vitality, ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 were significantly decreased in Ophiopogon japonicus of 10 μg/L group. Compared with Ophiopogon japonicus of 10 μg/L group, the cell vitality, ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 were significantly decreased in Ophiopogon japonicus of 20 μg/L group. Compared with Ophiopogon japonicus of 20 μg/L group, the cell vitality,［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 were significantly decreased in Ophiopogon japonicus of 30 μg/L group. CONCLUSION：Ophiopogon japonicus may inhibit CFs. These actions are related to the changes of ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3.     

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.