Signaling mechanism of dendritic cell maturation stimulated by uric acid

AIM: To investigate the effect of uric acid on the signal molecule expression involved in MAPKs and NF-κB pathways during the maturation of dendritic cells (DCs). METHODS: DCs were obtained from murine bone-marrow and cultured in vitro. After the immature DCs were stimulated with uric acid (200 mg/L) and NF-κB inhibitor PDTC, or MAPKs inhibitors SB203580, PD98059 or SP600125 for 15 min, 30 min or 45 min, the cytoplasmic and nuclear extracts of the cells were collected and were subject to immunoblot analysis with the antibodies specific for NF-κB p65 or phosphorylated forms of p38, ERK1/2 and JNK. The cell lysates from DCs treated with LPS or DMSO served as controls. After treated with uric acid and PDTC, SB203580, PD98059 or SP600125 for 48 h, DCs were collected. The cell surface markers were analyzed by flow cytometry. The production of IL-12 p70 in the culture supernatants was detected by ELISA. RESULTS: Within 15 min of uric acid conditioning in the immature DCs, increased expression of NF-κB p65 and the phosphorylation of p38, ERK1/2 and JNK in the nuclear or cytoplasmic extracts of DCs were observed. The expression of these proteins reached their peak at 30 min after stimulation. Pretreatment of DCs with PDTC, SB203580, SP600125 or PD98059 blocked the expression of NF-κB p65 and phosphorylation of p38, ERK1/2 and JNK in response to uric acid stimulation. Treatment of DCs with SB203580, SP600125 or PDTC reduced the uric acid-induced up-regulation of CD83, CD86 and IA/IE, and inhibited the effect of uric acid on the secretion of IL-12 p70 (P<0.05 or P<0.01). SB203580 and PDTC possessed a significant inhibitory effect on uric acid. Nevertheless, PD98059 increased the up-regulation of CD83, CD86, IA/IE and IL-12 p70 induced by uric acid (P<0.05). CONCLUSION: Uric acid controls the balance of signal molecule phosphorylation of p38 MAPK, ERK1/2 and JNK, and NF-κB pathways. A possible mechanism of the DCs maturation stimulated by uric acid may be the modulation of the threshold and duration of MAPKs and NF-κB signaling.     

Neuropeptide Y induced redistribution of intracellular free calcium in rat cardiomyocytes

AIM: To investigate the effect of neuropeptide Y (NPY) on intracellular free calcium(［Ca²⁺］_i) and Ca²⁺ sarcoplasmic reticulum of cardiomyocytes in rats.METHODS: Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY at concentration of 100 nmol/L for 24 h. Fluorescent indicator Fluo-4 AM was used to detect ［Ca²⁺］_i and Fluo-5N AM was used to detect Ca²⁺ in sarcoplasmic reticulum (SR). Calcium image was recorded by laser scanning confocal microscope. The SR Ca²⁺ load was estimated by caffeine-induced Ca2+ transient (CCT). RESULTS: 24 h after incubation with NPY, compared with control group, the concentration of ［Ca²⁺］_i was significantly elevated (P<0.05), and the concentration of free Ca2+ in SR (［Ca²⁺］_SR) was significantly decreased (P<0.05), and the peak of CCT was attenuated.CONCLUSION: Stimulation with NPY for 24 h causes redistribution of free calcium in rat cardiomyocytes, namely the elevation in ［Ca²⁺］_i and decline in ［Ca²⁺］_SR.     

Involvement of caspase-3 and p38MAPK in allogeneic CD8+T cell-induced apoptosis of vascular endothelial cells

AIM: To investigate the function of caspase-3 and mitogen-activated protein kinases (MAPKs) in allogeneic CD8+T cell-induced apoptosis of vascular endothelial cells. METHODS: Allogeneic CD8+T cells were isolated from PBMC by positive selection using magnetic beads coated with anti-CD8 antibody. After cocultured with allogeneic CD8+T cells, apoptosis of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs) were detected by AnnexinV-FITC labeling. Western blotting was used to examine the change of MAPK and caspase-3 expression in the vascular endothelial cells. The influence of SB203580 (inhibitor of p38MAPK), SP600125 (inhibitor of JNK), PD98059 (inhibitor of ERK), Z-DEVD-FMK (a caspase-3-specific peptide inhibitor) on apoptosis was also examined. RESULTS: At 24 h and 48 h time-point, the apoptosis rates of HUVECs were 41.7%±10.1% and 29.4%±8.3%, respectively (P<0.01, vs untreated HUVECs); the apoptosis rates of HDMECs were 28.9%±7.2% and 15.2%±4.8%, respectively (P<0.01, vs untreated HDMECs). These effects were largely prevented by Z-DEVD-FMK and SB203580 (P<0.05). Allogeneic CD8+T cells enhanced cleavage of caspase-3 and led to p38MAPK phospholation. CONCLUSION: Caspase-3 and p38MAPK mediate allogeneic CD8+T cells-induced apoptosis of vascular endothelial cells.     

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.     

Role of p38 MAPK signaling pathway in inflammatory effects on cerulein-induced pancreatic cells

AIM: To investigate the effect of p38 MAPK signal pathway on cerulein-treated pancreatic acinar AR42J cells.METHODS: AR42J cells were divided into control group, cerulein group (treated with 10^-8 mol/L of cerulein), and SB203580 group (treated with 10 μmol/L of SB203580 and 10^-8mol/L of cerulein).The cells were harvested 3 h after treatment.Secretion rate of amylase was measured.The translocation of p-p38 MAPK to nuclei was imaged by immunofluorescence.The protein expression levels of p-p38 MAPK and TNF-α were detected by Western blotting.The activation of NF-κB was measured by electrophoretic mobility assay.RESULTS: Compared with control group, cerulein resulted in increases in the secretion rate of amylase and protein level of TNF-α (P<0.01), as well as the expression levels of p-p38 MAPK and NF-κB (P<0.01).Cerulein induced nuclear translocation of p-p38 MAPK.Compared with cerulein group, the secretion rate of amylase and protein level of TNF-α in SB203580 group decreased significantly (P<0.01).The expression of p-p38 MAPK and NF-κB also decreased greatly (P<0.05).Nuclear translocation of p-p38 MAPK was inhibited by SB203580.CONCLUSION: The p38 MAPK pathway involves in cerulein-induced pancreatic inflammatory response via regulating NF-κB.     

Trichosanthin-induced apoptosis in melanoma B-16 cell line

AIM: To investigate the antitumor effect and mechanism of trichosanthin (TCS) on melanoma B-16 cells.METHODS: (1) The injury of B-16 cells by trichosanthin was observed with SCGE and hoechst33258 staining method.(2) LCSM and specificity fluorescent probe Fluo-3/AM,H₂DCF-DA,DAF-FM diacetate were applied to analyze the dynamic changes of Ca²⁺,ROS and NO in single cell cultured with TCS.Simultaneously,the relationship between ROS,NO and increase of Ca²⁺ was also revealed.RESULTS: (1)When treated with TCS (50 mg/L) for 3 h and 6 h,neither cytotoxicity assay nor SCGE showed the differences compared with control group.After 12 h incubation,specificity phenomena of DNA injury-comet tail appeared in SCGE and chromatin condensation even apoptotic body formation were seen by Hoechst33258 staining.（2） TCS (50 mg/L) evoked rapid enhancement of the production of Ca²⁺,ROS and NO in the cell and the differences between TCS and control group had statistical significance (P<0.01).Augment of ROS and NO was involved in the increase of Ca²⁺.CONCLUSION: Trichosanthin has relatively strong cytotoxicity on melanoma B-16 cells and contributes to apoptosis as well.Trichosanthin-induced apoptosis is related to the increase of Ca²⁺,NO and ROS in the cells.     

Effects of naringin on MAPK signal pathway in rat bone marrow mesenchymal stem cells during osteogenic differentiation

AIM: To study the effects of Chinese herbal monomer naringin (NG) on the MAPK signal pathway in bone marrow mesenchymal stem cells (MSCs) derived from SD rats during the differentiation into osteoblasts in vitro . METHODS: The changes of evaluating indicators alkaline phosphatase (ALP), bone gla protein (BGP) and type I collagen (Col I) in MSCs were observed under the conditions of normal, adding p38 pathway inhibitor SB203580, adding extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, adding c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125, and adding SB203580, PD98059 and SP600125 together. The protein phosphorylation of p38, ERK1/2 and JNK was measured by Western blotting. The mRNA expression levels of transforming growth factor beta 1 (TGF-β₁), bone morphogenetic protein 2 (BMP-2) and core binding factor α1 (Cbfα1) were measured by fluorescence quantitative PCR. RESULTS: The most effective concentration of NG to promote the differentiation of MSCs into osteoblasts was 10^-7 mol/L. The highest expression levels of both ALP and BGP were observed in NG group (P<0.05), while the expression of Col I did not reveal significant difference (P>0.05). Compared with NG group, the expression levels of ALP, BGP and Col I decreased differently after adding different inhibitors. Compared with control group, the protein phosphorylation of JNK was increased (P<0.05), and the phosphorylation of p38 was decreased (P<0.05), while the phosphorylation of ERK1/2 did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the protein phosphorylation of p38, ERK1/2 and JNK showed fluctuation with some increasing and others decreasing. Compared with control group, the expression of BMP-2 was increased (P<0.05), and the expression of Cbfα1 was decreased(P<0.05), while the expression of TGF-β₁ did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the mRNA expression levels of TGF-β₁, BMP-2 and Cbfα1 decreased differently after adding different inhibitors. CONCLUSION: Activation of ERK/JNK signaling and up-regulation of BMP-2 expression may be the main mechanism of NG to promote the differentiation of MSCs into osteoblasts. NG has strong impact on p38 pathway to improve the expression of BMP-2 in MSCs.     

p38 MAPK signaling pathway is involved in the regulation of receptor-mediated endocytosis

AIM: To investigate the role of p38 MAPK signaling pathway in the receptor-mediated endocytosis. METHODS: The effects of p38 MAPK on the receptor-mediated endocytosis were observed by using Alexa 594-conjugated Transferrin, in the presence of p38 specific inhibitor SB203580 or ERK pathway specific inhibitor PD98059, or using p38 knockout techniques. RESULTS: In the process of receptor-mediated endocytosis, p38 was activated by phosphorylation. Furthermore, the receptor-mediated endocytosis was inhibited by pretreatment with SB203580 or p38 knockout, while pretreatment with PD98059 had no effect on this process.CONCLUSION: p38 MAPK signaling pathway plays a role in the regulation of receptor-mediated endocytosis.     

Changes of RyR-mediated Ca2+ release in VSMCs is involved in the development of vascular hyporeactivity in hemorrhagic shock rats

AIM: In order to investigate the mechanisms involved in the vascular hyporeactivity after hemorrhagic shock, the changes of Ca²⁺ release from calcium store in vascular smooth muscle cells (VSMCs) with hypoxia were observed and the role of Ca²⁺ release from calcium store in the occurrence of vascular hyporeactivity to norepinephrine (NE) after hemorrhagic shock in rats was further explored.METHODS: A hemorrhagic shock model (40 mmHg for 2 h) in rats and a VSMCs hypoxic model were established. The changes of intracellular Ca²⁺ concentration in VSMCs were evaluated by fura3-AM and the role of IP₃R and RyR mediated Ca²⁺ release from calcium store was further observed. The role of IP₃R and RyR mediated Ca²⁺ release from Ca²⁺ store in the development of vascular hyporeactivity was measured with an isolated organ perfusion system. RESULTS: In the absence of extracellular Ca²⁺, NE upregulated by mobilizing Ca²⁺ release through calcium store. Compared to the normal control, the VSMCs had a slight increase when treated with hypoxia and NE-induced intracellular down-regulated, both without significant difference. Compared to the normal control cells, there was a significant change of Ca²⁺ release from calcium store in hypoxia-treated VSMCs, characterized by the significant increase in triggered by RyR-sensitive Ca²⁺ releasing activator caffeine. However, the increase in triggered by IP₃R-mediated Ca²⁺ release agonist adenophostin A (10^-5 mol/L) and ATP-Na₂ (10^-4 mol/L) had no significant difference in hypoxic VSMCs. Furthermore, the vascular reactivity to NE decreased in abdominal aorta in hemorrhagic shock (40 mmHg, 2 h) rats. The activation of IP₃R mediated Ca²⁺ release with ATP-Na₂ (10^-4 mol/L) did not improve the vascular reactivity to NE, while inhibition of IP₃R mediated Ca²⁺ release with heparin (10⁴ U/L) significantly antagonized the vascular reactivity to NE in hemorrhagic shock rats. In addition, in normal K-H solution (with about 2.2 mmol/L) and Ca²⁺-free K-H solution, RyR antagonist ryanodine (10^-5 mol/L) partly restored the vascular reactivity to NE in hemorrhagic shock rats, while RyR agonist caffeine(10^-3 mol/L) further decreased the vascular reactivity. CONCLUSION: The over-activation of RyR-mediated Ca²⁺ release from calcium store is partly involved in the development of vascular hyporeactivity after hemorrhagic shock in rats.     

Role of Toll-like receptor 4/MAPKs pathway on monocyte chemoattractant protein-1 secretion induced by oxidized low density lipoprotein in vascular smooth muscle cells

AIM: To investigate the role of Toll-like receptor 4/MAPKs pathway on the secretion of monocyte chemoattractant protein-1 (MCP-1) induced by oxidized low density lipoprotein (ox-LDL) in the vascular smooth muscle cells (VSMCs). METHODS: mRNA and protein expressions of MCP-1 in VSMCs stimulated with oxidized low density lipoprotein were determined by RT-PCR and ELISA, respectively. The phosphorylated forms of ERK1/2 and p38MAPK were determined by Western blotting. TLR4 neutralizing antibodies (a specific TLR4 inhibitor), PD98059 (ERK1/2 specific inhibitor), SB23015 (p38MAPK specific inhibitor) and SP600125 (JNK specific inhibitor) were used to investigate the underlying mechanisms. RESULTS: The mRNA and protein expressions of MCP-1 in VSMCs were up-regulated by ox-LDL (P<0.05), while those were inhibited by TLR4 neutralizing antibodies, PD98059 or SB23015 (P<0.05), but not by SP600125 (P>0.05). TLR4 had regulatory effect on the phosphorylation of ERK1/2 and p38MAPK. CONCLUSION: ox-LDL is an endogenous ligand of TLR4. The secretion of MCP-1 induced by ox-LDL in VSMCs is at least in part via TLR4/ERK1/2 and TLR4/p38MAPKs pathways.     

Changes of ［Ca2+］i and the activity of ACE in alveolar macrophages of rabbits with high-fat diet

AIM: To investigate the effects of high-fat diet on the level of intracellular free calcium (［Ca²⁺］_i) and the activity of angiotensinⅠconverting enzyme (ACE) in alveolar macrophages (AMs) of rabbits.The association between asthma and high-fat diet was also observed.METHODS: Twelve male New Zealand rabbits were medially divided into normal diet group and 1.2% high-cholesterol diet group randomly.8 weeks later,bronchial alveolar lavage was performed in vitro.［Ca²⁺］_i was determined by Fluo-2/am.The activity of ACE was detected with ultraviolet method.RESULTS: The levels of ［Ca²⁺］_i in AMs greatly increased (P＜0.01).The activity of ACE both in BALF and in culture supernatants of AMs was all greatly increased compared with normal diet group (P＜0.01).In hypercholesterolemic group the number of macrophages in BALF showed a positive correlation with the content of cholesterol in serum,such as the level of ［Ca²⁺］_i in AMs and the activity of ACE in the culture supernatants of AMs (all P＜0.05).CONCLUSION: The results suggest that AMs of rabbits may be activated by hyperlipoidemia and become ease to be stimulated.     

Akebia saponin D promotes differentiation of bone marrow mesenchymal stem cells into osteoblasts through MAPK signaling pathways

AIM:To explore the role of Akebia saponin D(ASD) in the differentiation of rat bone marrow-derived mesenchymal stem cells(BMSCs) into osteoblasts. METHODS:The rat BMSCs were cultured using routine methods. The effects of ASD on the differentiation of MSCs into osteoblasts were observed. The p38 mitogen-activated protein kinase(p38 MAPK) inhibitor SB203580 and extracellular signal-regulated kinase(ERK) inhibitor PD098059 were used to evaluate the mechanisms. The activity of alkaline phosphate(ALP) and content of osteocalcin(OC) were assayed during differentiation. The mRNA expression of osteoprotegerin(OPG) and receptor activator of nuclear factor-κB ligand(RANKL) was determined by real-time fluorescence quantitative PCR. The activity of p38 MAPK and ERK was measured by Western blotting. RESULTS:Six days after treatment with ASD, the mRNA expression of OPG significantly increased, while the mRNA level of RANKL significantly decreased in induced cells. ASD increased the activity of ALP and the content of OC. Moreover, ASD enhanced the activity of both p38 MAPK and ERK, which was inhibited by SB203580 and PD098059. SB203580 and PD098059 also inhibited the positive role of ASD in the differentiation of MSCs into osteoblasts. CONCLUSION:Akebia saponin D significantly enhances differentiation of rat BMSCs into osteoblasts in vitro, which may be mediated by the p38 MAPK and ERK signaling pathways.     

Angiopoietin-1 increases intracellular free Mg2+ by tyrosine kinase/PI3K in HUVECs

AIM:The mechanism of angiopoietin-1 (Ang-1) in mediating increase in intracellular free magnesium (［Mg²⁺］_i) in human umbilical vein endothelial cells (HUVECs) was investigated in this study. METHODS:The change of ［Mg²⁺］_i in HUVECs was quantitatively detected in intracellular cation measurement system via loaded with the fluorescent magnesium indicator mag-fura-2. RESULTS:Ang-1 increased ［Mg²⁺］_i, and there was not any significant difference among the groups of 0 mmol/L and 1 mmol/L of extracellular Mg2+. Similar results were obtained in groups done with Ca2+. Pretreatment with tyrosine kinase inhibitors (tyrphostin A23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) blocked the increase in ［Mg²⁺］_i induced by Ang-1. However, mitogen-activated protein kinase inhibitors (SB202190 and PD98059) had no effect on the Ang-1-induced ［Mg²⁺］_iincrease. CONCLUSION:These results suggest that the increase in ［Mg²⁺］_i by Ang-1 come from intracellular Mg2+ pools mediated by tyrosine kinase/PI3K -dependent signaling pathways.     

Calreticulin is involved in the protection of hypoxic preconditioning against cardiomyoblast H9c2 cell oxidative injury

AIM: To investigate whether hypoxic preconditioning (HPC) protects cardiomyoblast H9c2 cells against oxidative injury, and to discuss whether calreticulin (CRT) contribute to this protection through p38 MAPK signaling pathway. METHODS: Cardiomyoblast H9c2 cells were randomly divided into eight groups as follows: hydrogen peroxide stress (H₂O₂); brief hypoxic exposure of 20 min to simulate hypoxic preconditioning (HPC); 20 min of hypoxic exposure followed by 24 h of normoxic reoxygenation before hydrogen peroxide stress (HPC+H₂O₂), SB203580 (the specific inhibitors of p38 MAPK)+HPC+H₂O₂, antisense oligonucleotides transfection of calreticulin (AS), AS+H₂O₂, AS+HPC+H₂O₂ and control. Morphological studies, estimation of lactate dehydrogenase (LDH) leakage and flow cytometry were employed to assess the cell apoptosis and necrosis. RT-PCR and Western blotting analysis was used to detect calreticulin expression and phosphorylation of p38 MAPK. RESULTS: The results obtained are as follows: (1) HPC relieved cell injury caused by H₂O₂. Compared with those in H₂O₂ group, apoptosis rate and LDH leakage in culture medium in HPC + H₂O₂ group decreased 13.4% and 44.0%, respectively (P<0.05), and cell survive rate increased 12.7% (P<0.05). SB203580, a selective p38 MAPK inhibitor presented before HPC, eliminated the cytoprotection of HPC. Compared with HPC+H₂O₂ group, apoptosis rate and LDH leakage increased 5.4% and 45.0%, respectively (P<0.05), and cell survive rate decreased 5.0%(P<0.05). (2) Brief hypoxia intimating HPC resulted in mild CRT up-regulation (1.4-fold increased vs control group, P<0.05), but this up-regulation was lower than that of 3.6-fold increase induced by oxidative stress. HPC relieved the over-expression of CRT induced by H₂O₂ (26% decreased vs H₂O₂ group, P<0.05). (3) Transfection of antisense oligonucleotides of CRT before HPC reduced cytoprotection against oxidative stress. Correlative analysis indicated that mild up-regulation of CRT induced by HPC was positively correlated with survive rate (r=0.8573, P<0.05). (4) SB203580 suppressed CRT up-regulation (the expression of CRT decreased 38% or 23%, vs HPC+H₂O₂ group or HPC group, respectively). CONCLUSION: These results suggest that hypoxic preconditioning up-regulates calreticulin expression through p38 MAPK signaling pathway and protects cardiomyoblast H9c2 cells against oxidative injury.     

Effects of FKBP12.6 gene on intracellular Ca2+ concentration in mouse H9c2 (2-1) myocardial cells transfected by ultrasound-mediated destruction of microbubbles

AIM:To investigate the changes of intracellular calcium ion (Ca²⁺) concentration in mouse H9c2 (2-1) cells transfected with or without FK506 binding protein 12.6(FKBP12.6) gene by ultrasound mediated destruction of microbubbles. METHODS:The pcDNA3.1-FKBP12.6 plasmid, mingled with albumin-coated microbubbles agents, was transfected into H9c2 (2-1) cells by ultrasound-mediated destruction of microbubbles. The H9c2 (2-1) cell growth state was investigated by inverted microscope. The changes of intracellular Ca²⁺ concentration was determined by laser scanning confocal microscope. The FKBP12.6 protein expression was checked by immunohistochemistry. RESULTS:As compared with control cells, the H9c2 (2-1) cells, transfected with FKBP12.6 gene, grew better, had higher gross intracellular Ca²⁺ concentration. CONCLUSION:FKBP12.6 gene augments Ca²⁺ concentration in mouse H9c2 (2-1) cells, enhances the contractibility of the myocardial cell, which may be helpful to improve the myocardial dysfunction.     

p38 MAPK involves in cepharanthine-induced apoptosis in cardiomyocytes

AIM: To investigate the apoptotic effect of cepharanthine (CEP) on neonatal rat cardiomyocytes(NRCMs) and the underlying mechanisms. METHODS: MTT assay was used to detect the viability of the cells. CEP-induced apoptosis in NRCMs was evaluated by Hoechst 33342 staining and the expression of activated caspase-3. The phosphorylation levels of mitogen-activated protein kinases (MAPKs),such as extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38 MAPK,were examined by Western blotting. The specific inhibitors of ERK and p38 MAPK were applied for identifying the roles of the corresponding signal pathways in CEP-induced apoptosis of cardiomyocytes. RESULTS: CEP inhibited the viability of NRCMs in a dose-and time-dependent manners. Positive nuclear fragmentation and activated caspase-3 were found in CEP-treated NRCMs. The phosphorylation levels of ERK and p38 MAPK were significantly elevated in CEP-treated NRCMs, but the change of JNK was not obvious. SB203580, an inhibitor of p38 MAPK, significantly alleviated the apoptotic effect induced by CEP. However, PD98059, an inhibitor of ERK1/2, did not significantly reduce the apoptotic effect.CONCLUSION: p38 MAPK is involved in CEP-induced apoptosis in NRCMs.     

Calcineurin signal transduction pathway involves in cardiomyocyte hypertrophy induced by prostaglandin F2α

AIM: To study the role of prostaglandin F₂α (PGF₂α) in cardiac hypertrophy and its relation with calcineurin (CaN) signal transduction pathway in vitro. METHODS: The cultured neonatal rat cardiomyocyte was used to observe the hypertrophic effect of PGF2α, and the hypertrophic response was assayed by measuring the cell diameter, protein content and atrial natriuretic factor (ANF) mRNA expression. For mechanism studies, the intracellular free calcium concentration (［Ca²⁺］_i) in cultured cardiomyocytes was measured by using Fura-2/AM as a fluorescent indicator. ANF and CaN mRNA expressions, and the expressions of CaN and its downstream effectors, NFAT₃ and GATA₄ proteins were assayed by RT-PCR and Western blotting, respectively.RESULTS: In cultured cardiomyocytes, PGF₂α induced profound hypertrophic morphology change, the significant increase in cell diameter and protein content in a concentration-dependent manner compared with those in vehicle control (P<0.01). The same result was found in measuring the ［Ca²⁺］_i in cardiomyocytes (P<0.01). PGF₂α at concentration of 10-7 mol/L significantly promoted ANF and CaN mRNA expressions and the protein expressions of CaN/NFAT₃/GATA₄ compared with those in the vehicle control (P<0.05). Cyclosporin A, a CaN inhibitor, markedly inhibited the myocyte hypertrophy (P<0.01), reduced the increased ［Ca²⁺］_i(P<0.01) and decreased the expressions of CaN mRNA and CaN/NFAT₃/GATA₄ proteins (P<0.05) compared with those of only PGF₂α　10^-7 mol/L treatment. CONCLUSION: Cardiomyocyte hypertrophy induced by PGF2α may be, at least in part, mediated by CaN signal transduction pathway activated by increasing ［Ca²⁺］_i.