Signal mechanism of endothelin-1-mediated activation of airway fibroblasts induced by injured airway epithelial cells

AIM: To explore the effects of p38 mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinases (PI3K)/Akt on interleukin (IL)-6, the endothelin (ET)-1-mediated process of airway fibroblast activation induced by injured human bronchial epithelial cells (HBE). METHODS: Human primary cultured airway fibroblasts were co-cultured with HBE pre-treated with or without poly-L-arginine (PLA). The procedure was also performed in the presence or absence of p38 MAPK selective inhibitor SB203580, PI3K selective inhibitor LY294002 or ETA receptor blocker BQ123, respectively. Immunostaining, Western blotting or ELISA were used for detecting α-smooth muscle actin (α-SMA) expression, the activities of p38 MAPK and Akt in fibroblasts or IL-6 levels in supernatants of fibroblasts. In addition, fibroblasts were mixed with soluble collagen and cultured with HBE treated as the same mentioned above, the gel contraction was measured by serial area measurements. RESULTS: ET-1 and IL-6 levels ［(13.69±1.36) ng/L, (56.7±10.7) ng/L］ in the supernatants of fibroblasts cultured with injured HBE were significantly higher than those in the supernatants of fibroblasts cultured with HBE ［(3.79±0.64) ng/L, (15.5±3.2) ng/L］. BQ123, SB203580 or LY294002 decreased IL-6 levels ［(27.2±3.1) ng/L, (31.5±3.6) ng/L, (41.3±3.2) ng/L］ differently in the supernatants of fibroblasts induced by injured HBE. Activation of p38 MAPK preceded Akt in fibroblasts cultured with injured HBE. BQ123 reduced the phosphorylation levels of p38 MAPK and Akt. SB203580 concentration-dependently attenuated Akt phosphorylation, while LY294002 had little effect on p38 MAPK phosphorylation. Fibroblasts expressed more α-SMA after cultured with injured HBE and showed significant increase in the gel contraction compared to fibroblasts cultured with HBE ［percentage of gel contraction: (61.2±2.7)% vs (15.4±7.3)%］, all these effects were diminished or inhibited by BQ123, SB203580 or LY294002. Furthermore, the effects of BQ123 and SB203580 on decreased gel contraction were stronger than the effect of LY294002. CONCLUSION: ET-1 exerts a key role in the airway fibroblasts activation induced by injured HBE through activating p38 MAPK, PI3K/Akt signaling and promoting IL-6 expression.     

Effects of epithelial cells treated with poly (I:C) on activation of fibroblasts and roles of EMMPRIN

AIM: To investigate the effects of epithelial cells treated with polyinosinic-polycytidylic acid [poly(I:C)] on the proliferation, transdifferentiation and signaling mechanisms of airway fibroblasts.METHODS:Human alveolar epithelial cells were treated with poly(I:C). The cell culture supernatants were used to stimulate the airway fibroblasts or the fibroblasts growing in collagen gels. The proliferation of the fibroblasts, the expression of a-smooth muscle actin (α-SMA) in the fibroblasts and the contractility of the collagen gels containing fibroblasts, as well as the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) were observed. The proliferation of the fibroblasts and the expression levels of α-SMA, MMP-2 and MMP-9 in the fibroblasts stimulated with EMMPRIN were detected. The inhibitors specific for p38 MAPK or ERK1/2 were used to explore the effects on α-SMA expression and EMMPRIN secretion. RESULTS:The culture supernatants of the epithelial cells treated with poly(I:C) induced the proliferation, α-SMA expression and gel contraction as well as EMMPRIN secretion in the fibroblasts. EMMPRIN dose-dependently enhanced fibroblast proliferation, α-SMA expression and activity of MMP-2 and MMP-9. The supernatants of epithelial cells treated with poly(I:C) activated p38 MAPK and ERK1/2 signaling in the fibroblasts, as the specific inhibitors of MAPK or ERK1/2 signaling attenuated the expression of α-SMA and EMMPRIN in the fibroblasts. CONCLUSION: Poly(I:C) induces fibroblast proliferation, α-SMA expression and gel contraction by affecting the process mediated by p38 MAPK and ERK1/2 signaling pathways in epithelial cells. EMMPRIN may be the important media involved in this event.     

Mesenchymal stem cells contribute to the formation of liver fibrosis

AIM: Myofibroblasts play key roles in the formation of liver fibrosis. It has been reported that bone marrow mesenchymal stem cells can differentiate into myofibroblasts, and migrate to the damaged organs to participate the fibrotic process. Therefore, this study was designed to investigate the possible function and mechanism of bone marrow mesenchymal stem cells in developing liver fibrosis. METHODS: A mesenchymal stem cell line Ap8c3 was tagged with enhanced green fluorescent protein(eGFP)(Ap8c3-eGFP). The rat model of liver fibrosis was established by bile duct ligation(BDL) or injection of pig serum(IPS). Ap8c3-eGFP was intravenously injected into BDL or IPS-induced liver fibrotic rats. The eGFP positive(eGFP⁺) cells and expression of α-smooth muscle actin(α-SMA) in these eGFP⁺ cells in rat livers and bone marrow(BM) were detected. RESULTS: Intravenous engraftment of Ap8c3- eGFP resulted in homing to fibrotic livers as well as BM. Co-expression of α-SMA by eGFP⁺ cells was found in liver sections, and eGFP⁺ cells were found mainly in fibrotic septum in fibrotic livers. Ap8c3-eGFP was observed to differentiate into myofibroblasts, which specifically expressed α-SMA after homing to bone marrow.CONCLUSION: Differentiation of mesenchymal stem cells into myofibroblasts plays important roles in the formation of liver fibrosis. BM-derived myofibroblasts can migrate to damaged livers and participate in the formation of liver fibrosis.     

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.     

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.     

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.     

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.     

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.     

Hypoxia induces myofibroblast formation and stimulates production of collagen I in myofibroblasts through ERK1/2 pathway

AIM: To investigate the effect of hypoxia on the myofibroblast transdifferentiation from fibroblasts,and associated signaling of hypoxia on the production of collagen I in cultured rat renal cortical myofibroblasts.METHODS: The study is composed of two relevant parts.In the first part,a normal rat renal interstitial fibroblast cell line NRK-49F was treated with hypoxia (1% O2) or normoxia (21% O2) for 6 h,12 h and 24 h.The expression of hypoxia inducible factor-1α (HIF-1α) was examined by Western blotting in order to make sure the hypoxic condition is reliable.The myofibroblast transformation from fibroblasts induced by hypoxia was assayed by detecting the protein levels of α-smooth muscle actin (α-SMA).In the second part,the object was done on the primary cultured rat renal cortical myofibroblasts.Myofibroblasts were subjected to hypoxic or normoxic conditions for variety of times.The levels of HIF-1α in cell lysates and collagen I protein in supernatant culture medium and the activation of extracellular signal-regulated kinase (ERK)1/2 MAPK pathway were analyzed by Western blotting.RT-PCR was carried out to measure the levels of collagen I mRNA at different time points (2 h,4 h and 6 h).The distribution of HIF-1α in myofibroblasts was demonstrated by immunocytochemistry.The changes of collagen I production were detected after PD98059,a specific inhibitor of ERK1/2 activation pretreatment and during the hypoxia incubation.The activity of gelatinase matrix metalloproteinase-2(MMP-2) and MMP-9 in the supernatant medium from the cultured cells were assayed by gelatin zymography.RESULTS: Significant increased levels of HIF-1α protein appeared in cell lysates under hypoxia for 6 h.Furthermore,HIF-1α was translocated into nuclei of myofibroblasts after 6 h exposure of myofibroblasts to hypoxia.The levels of α-SMA protein increased in NRK-49F under hypoxia for 12 h (187%±32%,P<0.05).The level of collagen I protein in culture medium was increased in hypoxia treated myofibroblasts at 6 h (171%±27%,P<0.05) and 12 h (256%±61%,P<0.05).Collagen I mRNA expression was increased in cells under hypoxia condition for 4 h (189%±28%,P<0.05) and 6 h (221%±44%,P<0.05).The activities of MMP-2 and MMP-9 in the supernatant medium were not significantly changed at different experimental time points between the normoxic and hypoxic conditions.Activation of ERK1 /2 occurred as early as 15 min,sustained the high level at 30 min and 60 min and was back to the baseline level at 2 h.Blockade of ERK activation with PD98059 abolished hypoxia-induced expressions of collagen I protein.CONCLUSION: Hypoxia contributes to the renal interstitial fibrosis through inducing formation of myofibroblasts and stimulating the production of collagen I in myofibroblasts.     

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.     

Vasodilatatory effect and mechanism of CH2Cl2 extract of flos magnoliae on isolated rat thoracic aorta

AIM: To characterize the vasodilatatory effect of CH₂Cl₂ extract of flos magnoliae (CEF) on isolated rat thoracic aorta and to elucidate its possible mechanism. METHODS: The thoracic aorta was isolated from male Sprague-Dawley (SD) rats and the isometric tension of aortic rings with or without endothelium was measured. RESULTS: CEF (0.1-1 000 mg/L) produced concentration-dependent, endothelium-independent relaxations in phenylephrine (PE)-contracted aortic rings. The maximum relaxation induced by CEF was 78.68%±6.03% in endothelium intact rings and 64.98%±13.90% in endothelium removed rings while the forskolin (1 μmol/L)-induced vasodilation was obtained as 100%. The vasodilatatory effect of CEF was not statistically inhibited by 10 μmol/L glibenclamide (Glib), 3 mmol/L tetraethylammonium (TEA), 100 μmol/L BaCl₂ and 10 μmol/L 1H- -oxadiazole- -quinoxalin-1-one (ODQ) in the preparations without endothelium. The CEF pre-treatment significantly inhibited vasoconstrictions to angiotensin Ⅱ (AngⅡ), prostaglandin F_2α, (PGF_2α), dopamine (Dopa), vasopressin (Vaso), 5-hydroxytryptamine (5-HT) and PE by 91.31%, 82.11%, 95.32%, 90.53%, 72.22% and 83.63%, respectively (P<0.01). In Ca²⁺-free medium treated endothelium removed aortic ring, incubation with CEF at concentration of 82 mg/L significantly attenuated intracellular Ca²⁺ release by PE. In Ca²⁺-free + high potassium medium incubated aortic rings without endothelium, CEF (82 mg/L) markedly inhibited potassium-stimulated Ca²⁺-dependent contraction which was mainly due to Ca²⁺ influx (P<0.01).CONCLUSION: CEF induced vasorelaxation is mainly related to interfering intracellular calcium homeostasis by blocking Ca²⁺ influx and intracellular Ca²⁺ release.     

Role of TGF-β1-activated p38 MAPK in up-regulation of PAI-1 expression by TGF-β1 in human ovarian cancer cells

AIM: To investigate the relationship between up-regulation of plasminogen activator inhibitor-1(PAI-1) expression and activation of p38 mitogen-activated protein kinase(p38 MAPK) and extracellular signal-regulated kinase(ERK) pathways by TGF-β1 in human ovarian cancer cells. METHODS: PAI-1 expression in human ovarian cancer cells treated with TGF-β1(10 μg/L)was assayed by real-time PCR and Western blotting. The activation of p38 MAPK and ERK was determined by Western blotting using phosphorylated p38 MAPK and phosphorylated ERK antibodies. Specific p38 MAPK inhibitor(SB203580) or ERK inhibitor(PD98059) was used to inhibit their activation. RESULTS: TGF-β1 up-regulated the expression of PAI-1, and activated p38 MAPK and ERK pathways in the ovarian cancer cells. Inhibition of p38 MAPK activation by SB203580 resulted in significant inhibition of the mRNA expression of PAI-1 induced by TGF-β1. However, inhibition of ERK activation did not significantly alter TGF-β1-induced increase in PAI-1 mRNA level. CONCLUSION: TGF-β1-activated p38 MAPK pathway contributes to the up-regulation of PAI-1 expression by TGF-β1 in ovarian cancer cells.     

p38 MAPK-Pim-3 signal pathway may be involved in cardiomyocyte anoxia preconditioning against anoxia/reoxygenation injury

AIM: To investigate the role of mitogen-activated protein kinases (MAPKs) pathways and the molecular mechanism by which the proto-oncogene Pim-3 protects cardiomyocyte against anoxia/reoxygenation (A/R) injury. METHODS: The primarily cultured neonatal rat ventricular cardiomyocytes were randomly divided into 4 groups: control group; A/R group; APC+A/R group; SB203850, U0126 or SP600125+APC+A/R group. The cells were pre-incubated with U0126 (ERK1/2 inhibitor), SP600125 (SAPK/JNK inhibitor), or SB203850 (p38 MAPK inhibitor) at concentration of 10 μmol/L for 30 min before the APC. The activities of p38 MAPK, JNK and ERK1/2 were detected by Western blotting. The viability of cardiomyocytes was assayed by MTT and the apoptosis of cardiomyocyte was detected by TUNEL. RESULTS: U0126, SB203850, and SP600125 abolished the increased expression of ERK1/2, p38-MAPK, and JNK proteins induced by APC+A/R or A/R, respectively. The expression level of Pim-3 protein significantly decreased when the p38 MAPK signal pathway was inhibited. Meanwhile, the activity of LDH and the apoptosis index increased, and the viability of cardiomyocytes decreased. CONCLUSION: Pim-3 expression through a p38 MAPK signaling pathway may protect cardiomyocytes from A/R injury.     

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.     

Involvement of extracellular signal regulated kinase in the regulation of amyloid precursor protein processing in PC12 cells by TPPB

AIM: To explore the signal transduction pathways involved in the regulation of amyloid precursor protein (APP) processing by protein kinase C (PKC) activator TPPB.METHODS: PC12 cells were treated with TPPB (PKC activator) for 3 h and various signal transduction inhibitors were added to the conditioned medium to investigate their effects on α-secretase form of soluble amyloid precursor protein (sAPPα) secretion after TPPB treatment via Western blotting. Extracellular signal regulated kinase (ERK, p42/44MAPK) and phospho-p42/44MAPK were also measured after TPPB treatment.RESULTS: TPPB (1 μmol/L) significantly increased sAPPα secretion as compared with control group. The increase in sAPPα secretion by TPPB was partially blocked by ERK inhibitor U0126, c-Jun N-terminal kinase (JNK) inhibitor SP600125 and protein tyrosine kinase (PTK) inhibitor genistein, but not by p38MAPK inhibitor SB203580. TPPB (1 μmol/L) increased the expression of phospho-p42/44MAPK without altering total p42/44MAPK levels.CONCLUSION: ERK, JNK and PTK may be involved in the regulation of APP processing by TPPB.     

Influences of Vit-E on the mtDNA damage and Ca2+ homostasis of hippocampus and antioxidative ability in brain of aging mice induced by D-galactose

AIM: To study the influences of vitamin E (Vit-E) on the mtDNA damage and Ca²⁺ homeostasis in hippocampus and antioxidative ability in aging brain induced by D-galactose.METHODS: D-galactose (1 000 mg·k^-1·d^-1 ) was injected into mice hypodermically for 8 weeks to induce aging animal model, and Vit-E (100 mg·kg^-1; 250 mg·kg^-1) was administered for 6 weeks by ig at the 3rd week of making model. After Vit-E treatment for 8 weeks, water maze test was used to determine the ability of mice’s learning and memory. The activities of glutathione peroxidase (GSH-Px) and succinate dehydrogenase (SDH), the content of nitric oxide (NO) and activity of nitric oxide synthase (NOS) in the brain tissue were detected separately. Fura-2/AM, double-wave-length fluorospectrophotometer and PCR method were used to measure the concentration of calcium ion and mtDNA mutation in the hippocampus cells.RESULTS: Administration of Vit-E improved significantly the ability of learning and memory in model mice, inhibited the activity of NOS and decreased the amount of NO, and increased the activities of GSH-Px and SDH respectively in brain tissues, decreased the concentration of calcium ion (P<0.01, P<0.05), and prevented the damage of mtDNA in hippocampus.CONCLUSION: Vit-E can enhance the antioxidative ability, regulate the homeostasis of Ca²⁺ and inhibit the damage of mtDNA caused by oxidative stress in aging brain, and improve the ability of learning and memory in aging mice.     

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.     

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.