Effects of ginkgolide B on ［Ca2+］i and mitochondrial function of cultured rat retinal neurons in vitro

AIM: To observe the effect of ginkgolide B (GB) on the intracellular calcium ion concentration (［Ca2+］i) and mitochondrial function of cultured rat retinal neurons in vitro.METHODS: in vitro primary culture of rat retinal neurons was used in the experiment. The apoptosis model of glutamate-induced retinal neurons was established and co-cultured with ginkgolide B. The ［Ca2+］i and mitochondrial membrane potential of the retinal neurons were detected by laser scanning confocal microscope.RESULTS: Glutamate decreased the survival rate of retinal neurons, increased the apoptosis and the ［Ca2+］i, lowered the mitochondrial membrane potential. The ［Ca2+］i was clearly diminished and the mitochondrial membrane potential was significantly increased with the GB intervention, and the apoptosis decreased significantly.CONCLUSION: GB protects retinal neurons from glutamate induced neurotoxicity. The effect of GB on retinal neurons might be due to its ability to decrease the ［Ca2+］i and increase mitochondrial membrane potential.     

L-carnitine inhibits H2O2-induced rat cardiomyocyte apoptosis through suppressing Ca2+/CaMKII signaling pathway

AIM：To examine the inhibitory effect of L-carnitine on hydrogen peroxide (H2O2)-induced apoptosis of rat cardiomyocytes and to further explore the underlying mechanisms. METHODS：Primarily cultured neonatal rat myocardial cells were prepared and challenged by 200 μmol/L H2O2 to induce cell apoptosis. In order to evaluate the effects of Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid (BAPTA), calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 and L-carnitine on cell viability, apoptosis, resting intracellular free Ca2+ concentration (［Ca2+］i) and phospho-CaMKII (p-CaMKII) expression, these three agents were added 30 min or 1 h prior to H2O2 stimulation. Cell viability was measured by MTT assay and apoptosis was determined by flow cytomertry. The ［Ca2+］i was measured by laser confocal scanning. Cleaved caspase-3 and p-CaMKII expression was detected using Western blotting. RESULTS：Upon 200 μmol/L H2O2 stimulation for 12 h, cell viability decreased and apoptotic rate increased significantly compared with control．Pretreament with L-carnitine, BAPTA and KN93 significantly increased cell viability and decreased apoptosis．Furthermore, intracellular Ca2+ overload triggered by H2O2 could be greatly relieved by L-carnitine and BAPTA pretreatment, but not affected by KN93. H2O2-stimulated cleaved caspase-3 and p-CaMKII expression was also significantly inhibited by all these three agents. CONCLUSION：L-carnitine inhibits H2O2-induced rat cardiomyocyte apoptosis possibly via suppressing Ca2+/CaMKII signaling pathway.     

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.     

Protease-activated receptors in dorsal motor nucleus of vagus in rats with inflammatory bowel disease

AIM: To demonstrate the presence of protease-activated receptor-1 (PAR-1) and PAR-2 in the dorsal motor nucleus of the vagus (DMNV), and to elucidate the cellular mechanisms that are triggered upon receptor activation in the rat with inflammatory bowel disease. METHODS: Twenty rats were used to produce the animal model of inflammatory bowel disease. The tissues of DMNV were collected from these animals for measuring PAR-1 and PAR-2. By using the Fura-2-AM, the concentration of intracellular calcium in primary cultured DMNV cells from newborn rats was observed in the presence of PAR-1 or PAR-2. RESULTS: Thrombin and PAR-1 agonist peptide (PARP-1) activated PAR-1 with a maximum change in intracellular calcium concentration Δ［Ca2+］i. Trypsin and PAR-2 agonist peptide (PARP-2) activated PAR-2 with a maximum Δ［Ca2+］i. Inhibition of phospholipase C (PLC) by 1 μmol/L U73312 decreased Δ［Ca2+］i induced by PAR-1 activation. The PAR-2-mediated Δ［Ca2+］i decreased when PLC was inhibited. Blockade of IP3 receptor by 2APB decreased the Δ［Ca2+］i due to PAR-1 and PAR-2 activation. CONCLUSION: Our results indicate that PAR-1 and PAR-2 are present in the DMNV neurons, and their activation leads to increases in intracellular calcium via signal transduction mechanism that involves activation of phospholipase C and the production of inositol 1,4,5-trisphosphate.     

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.     

Effects of bortezomib-based therapy on hemostasis system and circulating microparticles in multiple myeloma patients

AIM：To investigate the changes of hemostasis, thrombosis and total microparticles (TMPs) in multiple myeloma (MM) patients receiving bortezomib-based induction therapy (bortezomib+adriamycin+dexamethasone, PAD). METHODS：The levels of TMPs were detected by flow cytometry in 38 newly diagnosed MM patients and 30 healthy people. The changes of the platelet, coagulation, anticoagulation, fibrinolytic activation and TMPs in the MM patients before and after PAD teatment were also studied.RESULTS：Before treatment, the values of FVIII:C and vWF:Rco in MM patient were elevated ［(152.89±31.14)% and (165.69±38.43)%］, the activation of platelet aggregation was inhibited ［(63.76±21.36)%］, and the PAI level increased ［3.98(1.63)U/mL］. Compared with the healthy people, higher concentration of TMPs was observed in MM patients ［(640.65±214.22)/μL vs (134.29±63.09)/μL, P<0.01］, and the level of TMPs was positively correlated with serum β2-MG (r=0.672, P<0.01).After PAD therapy, platelet aggregation activation was restored ［(77.83±15.62)%, P=0.01］, and PAI level decreased ［0.88(1.38)U/mL, P<0.01］. The level of TMPs also decreased, after 3 cycles of PAD, to the value of (184.25±93.35)/μL. CONCLUSION：MM patients were characterized by impaired platelet, coagulation and fibrinolysis functions, and increased level of TMPs. PAD restored platelet function, decreased the levels of PAI and TMPs, which might partially explain the low incidence of thrombosis in the MM patients receiving bortezomib-based treatment.     

Integrative therapy using coenzyme Q10 and minocycline in 1-methyl-4-phenylpyridinium-induced hemiparkinson rats

AIM：To investigate the integrative treatment of both coenzyme Q10 (CoQ10) and minocycline in the rats intranigrally intoxicated with 1-methyl-4-phenylpyridinium (MPP+). METHODS：The rat model of Parkinson disease (PD) was established by intranigral microinjection of MPP+. The degree of microglial activation was measured by immunofluorescent density of OX-42 (a microglia marker) in the substantia nigra (SN). The number of viable dopaminergic neurons was determined by counting the tyrosine hydroxylase (TH) positive neurons in the SN. The behavioral performances were revealed with the number of apomorphine-induced rotations, score of forelimb akinesia and vibrissae-elicited forelimb placing asymmetry. RESULTS：Pretreatment with CoQ10 or intracerebroventricular (icv) posttreatment with minocycline alone provided partial attenuation against MPP+-induced locomotor defects. Integrative therapy provided enhanced beneficial effects, and resulted in a significant attenuation of locomotor disability than any single therapy (all P<0.01). The results of immunohistological analysis showed that the TH positive neurons were maximally protected by integrative therapy compared with minocycline group and CoQ10 group (P<0.01) . CONCLUSION：The integrative therapy of CoQ10 combined with minocycline may offer additional therapeutic benefit to MPP+ -induced hemiparkinson rat model. Such neuroprotective strategy of targeting different aspect of the neurodegenerative phenotypes may highlight a new therapeutic strategy for future management of PD.     

Influences of coculture with bone marrow mesenchymal stem cells on dopaminergic neurons in brain slice treated with 1-methyl-4-phenylpytidinium

AIM: To study the protective effects of bone marrow-derived mesenchymal stem cells on damaged dopaminergic neurons induced by 1-methyl-4-phenylpytidinium(MPP+).METHODS: The parkinson disease(PD) models were established in newborn rats. Bone marrow mesenchymal stem cells(MSCs) obtained from adult bone marrow were cultured, isolated and purified. MSCs were co-cultured with brain slice and the immunohistochemical technique, electron microscopy, propidium iodide staining were used to observe the changes of neurons. RESULTS: In the MPP+ treatment group, the neurites grew slowly and sparsely, dead cells were found in all regions. In the co-culture group, the neuritis grew densely, only a few cells were dead, the number of tyrosine hydroxylase(TH)-stained neurons increased and the structure of organellae was normal. CONCLUSION: MSCs may protect dopaminergic neurons against damage induced by MPP+. These results provide some data for cell transplantation therapy to Parkinsons disease.     

Effects of phospholamban antisense RNA on SR Ca2+-ATPase and ［Ca2+］i in rat cardiomyocytes by adeno-associated virus vector

AIM: To investigate the effect of phospholamban antisense RNA (asPLB) on the activity of sarco-endoplasmic reticulum (SR) Ca2+-ATPase, and the change of intracellular free Ca2+ concentration (［Ca2+］i) in rat cardiomyocytes by adeno-associated virus(AAV) vector. METHODS: rAAV-asPLB and rAAV-LacZ were constructed by AAV Helper-Free System. RT-PCR and Western blotting were used to determine the mRNA and protein expression of PLB. The activity of SR Ca2+-ATPase and the ［Ca2+］i were measured. RESULTS: Compared to controls, the PLB mRNA and protein expression reduced in rat cardiomyocytes transfected with rAAV-asPLB. The activity of Ca2+-ATPase was increased. In rest state, the level of ［Ca2+］i in rAAV-asPLB transfected group was decreased. The level of ［Ca2+］i was increased when induced by isoproterenol. CONCLUSION: rAAV-asPLB vector disrupts the expression of PLB, enhances the activity of Ca2+-ATPase, reduces the resting ［Ca2+］i and enhances the isoproterenol-induced ［Ca2+］i.     

Inhibitory effects of Co-SZ eye drops on apoptosis of lens epithelial cells and their signal transduction mechanisms

AIM: To investigate the inhibitory effect of Co-SZ eye drops on apoptosis of lens epithelial cells (LEC) induced by H₂O₂ and to study the cellular and molecular mechanisms.METHODS: (1) All lenses of Sprague-Dawley rats were incubated with H₂O₂ and Co-SZ eye drops.The apoptosis rates of LEC were determined by TUNEL method.The changes of LEC ultrastructure and the formation of apoptotic body were observed by electron microscopy.(2) Bovine LEC were incubated with H₂O₂ and Co-SZ eye drops.The inhibitory of LEC apoptosis was detected by MTT after incubation.The changes of fractional DNA content in LEC were detected by flow cytometry (FCM).［Ca2+］i , cAMP and cGMP of LEC were determined by spectrofluoremeter and radioimmunoassay, respectively.RESULTS: The LEC apoptosis rates in Co-SZ eye drops group were decreased significantly compared with H₂O₂ group by TUNEL.The ultrastructure changes in LEC of Co-SZ eye drops group were lighter than that in H₂O₂ group.The LEC apoptosis rates of Co-SZ eye drops group were dose-dependently decreased significantly compared with H₂O₂ groups via MTT assay.LEC apoptosis induced by H₂O₂ was inhibited by Co-SZ eye drops, and showing dose-dependent.The DNA contents in LEC of Co-SZ group were increased.The ［Ca2+］i and cAMP in Co-SZ group were decreased obviously.The cGMP was increased.CONCLUSION: The LEC apoptosis induced by H₂O₂ was inhibited by Co-SZ eye drops.The mechanism of apoptosis inhibition by Co-SZ eye drops maybe contribute to the increase in DNA content.The signal transduction mechanisms are related to the decrease in ［Ca2+］i and cAMP and the increase in cGMP.     

Effects of ghrelin on firing activity of gastric distension-sensitive neurons in hypothalamic arcuatus nucleus and gastric motility in diabetic rats

AIM： To study the roles of ghrelin in the regulation of gastric distension (GD)-sensitive neurons in the hypothalamic arcuate nucleus (Arc) and gastric motility in diabetes mellitus (DM) rats. METHODS：DM rat model was made by intraperitoneal injection of streptozotocin. The effects of ghrelin and ［D-Lys3］-GHRP-6 on GD-sensitive neurons in the Arc of DM rats were observed by recording the extracellular potentials of single neurons, and the gastric motility was also monitored in vivo. The expression of ghrelin receptor,growth hormone secretagogue receptor (GHS-R), in Arc was studied by real-time PCR and immunofluorescene method. RESULTS：(1) Ninety-eight GD-sensitivity neurons were recorded in the Arc of normal rats, in which 64.3% were classified as GD-excitatory (GD-E) neurons and 35.7% were GD-inhibitory (GD-I) neurons. Microinjection of ghrelin excited 73.0% of the 63 GD-E neurons and the discharge frequency significantly increased as compared with the neurons treated with saline. Ghrelin inhibited 60.0% of the 35 GD-I neurons and the discharge frequency was significantly reduced (P<0.01). The effect of ghrelin was blocked by the antagonist of ghrelin ［D-Lys3］-GHRP-6. (2) Sixty-six GD-sensitive neurons were recorded in the Arc of diabetes rats, in which 64.3% were GD-E neurons, and 35.7% were GD-I neurons. Microinjection of ghrelin excited 35.1% of the GD-E neurons and the discharge frequency significantly increased as compared with the neurons treated with saline. Ghrelin inhibited 21 of the 29 GD-I neurons (72.4%) and the discharge frequency was significantly reduced. Compared with normal control group, the ratio of GD-E and GD-I neurons in diabetic rat Arc GD-sensitive neurons was not significantly changed. However, the ratio of GD-E neurons treated with ghrelin was significantly decreased, and the average increase rate of discharge frequency was significantly decreased. Ghrelin did not change GD-I neurons rejection ratio and discharge frequency average reduction rate. (3) Microinjection of ghrelin into the Arc significantly promoted gastric motility in normal and DM rats, and a significant dose-dependent manner was observed. However, the promotion effect of ghrelin on gastric motility in normal rats was stronger than that in DM rats, and the effect was completely blocked by ［D-Lys3］-GHRP-6. (4) The mRNA expression of GHS-R1a in the hypothalamic Arc of diabetic rats significantly reduced as compared with the normal control rats. The protein level of GHS-R1a in the hypothalamic Arc was also significantly reduced in DM rats. CONCLUSION：Ghrelin regulates the activity of GD-sensitive neurons in hippocampus Arc of diabetic rats by the action on ghrelin receptor.     

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.     

Protective effect of ethyl pyruvate on brain tissues in neonatal rats with hypoxic-ischemic brain damage

AIM：To study the effects of ethyl pyruvate (EP) on brain tissues in neonatal rats with hypoxic-ischemic brain damage (HIBD) and its underlying mechanisms. METHODS：A total of 165 seven-day-old Sprague-Dawley (SD) rats were randomly divided into 3 groups：sham operation group (n=43), HIBD group (n=61) and HIBD+EP group (n=61). The rats in HIBD+EP group were intraperitoneally injected with EP (50 mg/kg) 30 min before operation, and once a day after surgery. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in brain homogenate, water content of brain and apoptotic cells in cortex were detected 3 days later. Ischemic and non-ischemic brain tissues were weighed to assess the extent of brain atrophy 14 days later. RESULTS：Higher level of SOD ［(125.78±18.35)×103 U/(g protein)］ and lower level of MDA ［(4.42±1.04) μmol/(g protein)］ in HIBD+EP group than that in HIBD group ［(97.84±15.50)×103 U/(g protein) and (6.02±0.89) μmol/(g protein), respectively］ was observed (P<0.05)．In addition, the water content of ischemic hemisphere was significantly higher than that of non-ischemic one in HIBD group (P<0.05), and was indistinguishable from that of non-ischemic one after EP treatment (P>0.05), indicating the protective effect of EP against brain edema. The apoptotic cells in cortex and hippocampus in HIBD+EP group ［(96.63±10.08)/field and (41.91±9.96)/field, respectively］ were obviously decreased compared with HIBD group ［(111.54±1.64)/field and (51.73±1.77)/field, respectively］, but still higher than those in sham operation group (P<0.05). The atrophy ratio of ischemic hemisphere in HIBD+EP group was (13.25±5.19)%, significantly lower than that in HIBD group ［(20.32±5.10)%, P<0.05］. CONCLUSION：Ethyl pyruvate is neuroprotective against HIBD in neonatal rats via increasing SOD level, decreasing MDA level, attenuating brain edema, decreasing apoptotic cells in cortex and alleviating atrophy of hypoxic-ischemic hemisphere.     

Modulation of interleukin-2 on the positive effect of isoproterenol in the isolated cardiomyocytes

AIM: To explore the effects and mechanism of interleukin-2 (IL-2) on the positive effect of isoproterenol (ISO) in the isolated rat cardiomyocytes. METHODS: Enzymatically isolated cardiomyocytes were used. Peak twitch amplitude and maximal velocity of shortening/relaxation (±dL/dtmax) in the isolated cardiomyocytes were recorded with a microscope coupled to a charge-coupled device camera and ［Ca2+］i transients were determined with a fluorometric ratio method by using Fura-2/AM as Ca2+ indicators. RESULTS: ① ISO increased the peak twitch amplitude and ±dL/dtmax of the isolated cardiomyocytes. Perfusion for 15 min with IL-2 at 2×103 U/L, which had no effect at all, attenuated the enhancing effect of ISO on the peak twitch amplitude and ±dL/dtmax. ② ISO increased the ［Ca2+］i transients of the single ventricular myocytes in a dose dependent manner and the corresponding EC50 values of ISO was (0.12±0.01) μmol/L. Perfusion for 15 min with IL-2 at 2×103 U/L, which had no effect on the ［Ca2+］i transient at all, attenuated the enhancing effect of ISO and the corresponding EC50 was (0.44±0.06) μmol/L. ③ The electrically induced ［Ca2+］i transient was significantly increased by pretreatment with 20 mg/L cholera toxin for 12 h. The elevation of the ［Ca2+］i transient induced by cholera toxin was significantly attenuated by 2×103 U/L IL-2. ④ Forskolin (1 μmol/L), the activator of adenyl cyclase, significantly increased the electrically induced ［Ca2+］i transient, which was attenuated by IL-2 at 2×103 U/L. CONCLUSION: IL-2 inhibits the positive effect of isoproterenol in the isolated single ventricular myocytes, in which Gs protein and adenyl cyclase are involved.     

Effects of sodium deoxycholate on functional state of isolated pancreatic acinar cells of rat

AIM: To investigate the alteration of functional state of pancreatic acinar cells stimulated by sodium deoxycholate (SDOC), and to explore the possible signal pathway involved in the effects of SDOC. METHODS: Rat pancreatic acinar cells were isolated by collagenase digestion and were treated with varying concentration of SDOC or culture media respectively. At different time points (30 min, 1 h, 4 h, 10 h), cell viability was determined by MTT and supernatant of cells was collected to measurer the content of maloidialdehyde (MDA) and the activity of superoxide dismutase (SOD). Some cells were loaded with Fluo-3/AM, then exposed to varying doses of SDOC. ［Ca2+］i change of single pancreatic acinar cell in extracellular fluid with the absence or presence of Ca2+ was determined by laser scanning confocal microscopy. RESULTS: SDOC initiated cell damage in a time-and concentration-dependent manner (P<0.05). Egtazic acid (EGTA) at the concentration of 1 mmol/L decreased the cell mortality (P<0.05). SDOC did not induce a rise of ［Ca2+］i in the calcium-free extracellular fluid. Addition of extracellular calcium in the presence of SDOC resulted in a rapid and remarkable rise of ［Ca2+］i. The increase in ［Ca2+］i preceded the pathological and biological alteration of pancreatic acinar cells. The supernatant content of MDA increased (P<0.05) and the supernatant activity of SOD decreased in SDOC group(P<0.05). CONCLUSION: SDOC initiates cell damage in a time, concentration and calcium dependent manner. SDOC only induces the influx of Ca2+ from extracellular fluid. Calcium overload as an early pathogenetic event takes part in the damage of pancreatic acinar cells by the response of superoxidation. Calcium homeostasis disorder may be one of the causes or at least an important mediator of SDOC- induced pancreatic acinar cell damage.     

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.     

miR-155-specific siRNA enhances chemosensitivity of Burkitt lymphoma Raji cells to cytosine arabinoside by inducing apoptosis

AIM：To investigate the effect of miR-155-specific siRNA alone or in combination with cytosine arabinoside (Ara-C) on the growth and apoptosis of Burkitt lymphoma Raji cells. METHODS：miR-155-specific siRNA and/or Ara-C were used to treat the cells. Quantitative real-time polymerase chain reaction was used to detect the expression of miR-155. The growth of the cells was analyzed by CKK-8 assay. The cell apoptosis was determined by flow cytometry. RESULTS：The miR-155 expression level of the cells transfected with miR-155 siRNA was significantly lower than that in the 2 control groups. Ara-C or miR-155 siRNA alone inhibited the growth of Raji cells in a dose-depend manner. miR-155 siRNA combined with Ara-C produced more inhibition of cell proliferation (P<0.05). After treatment for 48 h, the apoptotic rate of Raji cells in miR-155 siRNA+Ara-C group ［(38.4±1.4)%］ was higher than that in Ara-C group ［(16.5±0.3)%］ and miR-155 siRNA group ［(14.6±0.3)%］, with statistically significant difference (P<0.05). The expression of caspase-3 in Ara-C+miR-155 siRNA group was increased significantly as compared with Ara-C group and miR-155 siRNA group. CONCLUSION：miR-155-specific siRNA enhances the chemosensitivity of Raji cells to Ara-C by inducing apoptosis through the caspase-3 pathway.     

Effects of Tribulus terrestris L. saponin on apoptosis and changes in cytosolic calcium induced by hypoxia/reoxygenation in rat cortical neurons

AIM: To study the effect of Tribulus terrestris L. saponin (TTLS) on apoptosis and changes in cytosolic calcium concentration induced by hypoxia/re-oxygenation in rat cortical neurons. METHODS: Rat cortical neurons in primary culture were used, and a apoptosis model was induced by hypoxia/reoxygenation. LDH releasing rate was detected by spectrophotometry. The apoptosis rate of cortical neurons was analyzed quantitatively by flow cytometry with Annexin V-FITC and PI staining. Intracellular free Ca2+(［Ca2+］i) was observed with a confocal laser-scanning microscope and determined by mean fluorescent value with Fluo-3 fluometry. RESULTS: Compared to control group, three hours of hypoxia and twelve hours of reoxygenation group induced cortical neuronal apoptosis and significantly increased the intracellular free Ca2+ concentration（P<0.01). Compared with model group, TTLS decreased the percentage of neuronal apoptosis and reduced neuronal ［Ca2+］i（P<0.01).CONCLUSION: TTLS could obviously reduce hypoxia/reoxygenation-induced apoptosis and alleviate the damage degree of rat cortical neurons.The mechanism might be related to inhibiting the calcium overload induced by hypoxia/reoxygenation in rat cortical neurons.