Phosphodiesterase 3 inhibitor attenuates cough response in a sensitized guinea pig model

AIM: To investigate the effects of selective phosphodiesterase 3 inhibitor olprinone on cough response in guinea pigs sensitized and challenged with ovalbumin.METHODS: Forty sensitized guinea pigs were randomly divided into control (n=10), challenged (n=10), olprinone (n=10) and aminophylline group (n=10). Two hours after challenged with the aerosol of 1% ovalbumin or saline, animals were intraperitoneally injected either with saline, 25 mg/kg of olprinone or 25 mg/kg aminophylline. At 24 h, the injection was repeated with 2.5 mg/kg and 25 mg/kg olprinone or 2.5 mg/kg and 25 mg/kg aminophylline respectively in olprinone and aminophylline group, cough response to inhaled capsaicin and airway responsiveness to methacholine (PC150) were measured. Then, total cell number and differential counts were analyzed in bronchoalveolar lavage fluid. RESULTS: The cough frequency was (5±2) times/3 min in control group and (24±3) times/3 min in challenged group (P<0.05), while PC150 was (659±57) mg/L in control group and (238±67) mg/L in challenged group (P<0.05). 25 mg/kg olprinone significantly inhibited the augmented cough response and airway hyperresponsiveness, the cough frequency and PC150 were (15±2) times/3 min and (580±45) mg/L (P<0.05), which differed significantly from (18±2) times/3 min and (438±52) mg/L in aminophilline group (P<0.05). However, olprinone failed to reverse the elevated total cell number and percentage of eosinophils in bronchoalveolar lavage fluid from guinea pigs challenged with ovalbumin (P>0.05).CONCLUSION: Phosphodiesterase 3 inhibitor attenuates cough response associated with eosinophilic airway inflammation by bronchodilatory effect.     

The relationship between cough response and airway inflammation in guinea pigs sensitized and challenged with ovalbumin

AIM: To investigate the effects of intensity of eosinophilic airway inflammation on cough response in guinea pigs sensitized and challenged with ovalbumin. METHODS: 34 sensitized guinea pigs were challenged with the aerosol of either saline (group A, n= 7) or 0.04% (group B, n= 7), 0.2% (group C, n= 8) and 1% (group D, n= 12) of ovalbumin. 24 hours later, cough response to inhaled capsaicin and airway responsiveness to inhaled methacholine (PC₁₅₀) were measured. Total cell number and differentials in bronchoalveolar lavage were analyzed. RESULTS: After challenge, one animal in group C and five animals in group D died from severe wheezing. With ascending concentration of ovalbumin, cough frequency induced by inhaled capsaicin increased from (6±2) times/3 min in group A to (22±4) times/3 min in group D( P< 0.05). PC₁₅₀, which did not change in group B, increased in both group C and group D significantly in addition to an increase in total cell number and eosinophils in bronchoalveolar lavage. Cough response to inhaled capsaicin was positively correlated with total cell number ( r= 0.84, P< 0.01) and eosinophils ( r= 0.78, P< 0.01) in bronchoalveolar lavage, and negatively correlated with PC₁₅₀ ( r= -0.78, P< 0.01). There was a negative correlation between PC₁₅₀ and total cell number ( r= -0.80, P< 0.01) or eosinophils ( r= -0.85, P< 0.01). CONCLUSION: Cough response in sensitized guinea pigs is enhanced and finally develops into wheezing with a progress in eosinophilic airway inflammation.     

Effect of BCG on experimental asthma in a guinea pigmodel of allergen sensitization

AIM: To examine the effect of bacillus calmette-guerin (BCG) on experimental asthma in guinea pigs. METHODS: Guinea pigs were sensitized with BCG and then with ovalbumin (ip). Two weeks later, guinea pigs were challenged with ovalbumin (OVA) aerosol inhalation. Thirty one Guinea pigs were divided into three groups at random control group,OVA-treated group, BCG and OVA-treated group.RESULTS: Ovalbumin inhalation caused a marked airway infiltration of eosinophils and all the animals exhibit asthmatic symptoms. Pretreatment with BCG induced typical increase in lymphocytes and monocytes in peripheral blood and in bronchoalveolar lavage fluid (BALF). BCG markedly inhibited eosinophil infiltration and attenuated the asthmatic symptoms. CONCLUSION: These data suggest that BCG exerts an inhibitory effect on asthmatic inflammation.     

Effects of M. vaccae on eosinophil apoptosis and Bcl-2 protein expression in lung tissues of asthmatic guinea pigs

AIM: To study the effects of M. vaccae on eosinophil apoptosis and Bcl-2 protein expression in lung tissues of asthmatic guinea pigs. METHODS: 30 guinea pigs were divided into normal saline (NS) group, asthma group and M. vaccae treatment group at random, every group included 10 guinea pigs. Guinea pigs in M. vaccae treatment group were injected intramuscularly with 22.5 μg M. vaccae 10 days before OVA immunization. TdT-mediated dUTP nick end labeling (TUNEL) technique was used to investigate the apoptosis of eosinophils and immunohistochemistry method was used to study the expression of Bcl-2 protein in lung tissues. RESULTS: The apoptosis index (AI) of eosinophils in lung tissues in M. vaccae treatment group was significant higher than that in asthma group ［(23.78±5.42)% vs (4.56±0.68)%, P<0.01］. The mean optical density value of Bcl-2 protein in lung tissues of M. vaccae treatment group was significant lower than that of asthma group ［(1 556.3±492.4) vs (2 321.9±751.2), P<0.05］. CONCLUSION: The apoptosis of eosinophils induced by M. vaccae in lung tissues of asthmatic guinea pigs may be due to the inhibition of Bcl-2 protein expression.     

Effect of rosiglitazone on myocardial energy substrate utilization in type 2 diabetic rats

AIM: To study the effect of rosiglitazone (RSG) to improve insulin sensitivity on myocardial energy substrate utilization as well as the cardiac function in a rat model of type 2 diabetes mellitus. METHODS: Sprague-Dawley rats were conducted into three groups: chow-fed rats were fed with normal chow (12% of calories as fat); fat-fed/STZ rats were fed with high-fat diet (40% of calories as fat) for 4 weeks and then injected with streptozotocin 35 mg/kg intraperitoneal; fat-fed/STZ/RSG rats were fat-fed/STZ rats treated with rosiglitazone (3 mg·kg-1·d-1) for 2 weeks. A cannula connected to a passive transducer was inserted the heart for the measurement of the cardiac function including heart rate (HR), left ventricular end-diastolic pressure (EDP) and ±dp/dtmax. Then the isolated hearts were mounted onto a Langendorff perfusion apparatus to perfuse with Krebs-Henseleit buffer in the presence of 5 mmol/L glucose and 0.4 mmol/L ［3H］ labelled palmitate. Glucose uptake and ［3H2O］ collection were measured to evaluate the rate of carbohydrate and fatty acid oxidation. RESULTS: Compared with the chow-fed rats, fat-fed/STZ rats had a significantly depression of glucose uptake in the hearts ［(54.7±6.2 vs 69.0±5.7) μmol·g-1 dry weight, P<0.01］ after 30 min perfusion. The oxidation of glucose and palmitate were 18% and 82%, respectively. Paralleling the reduction was a change of EDP ［(14.3±1.8 vs 10.5±1.1) mmHg, P<0.05］ and -dp/dt ［(550±57 vs 650±42) mmHg/s, P<0.01］, indicating a impaired left ventricular diastolic function. In the hearts subjected to fat-fed/STZ group, rosiglitazone treated for 2 weeks resulted in a elevated level of glucose uptake ［(63.5±6.4 vs 54.7±6.2) μmol·g-1 dry weight, P<0.05］. A protective role of the ventricular function ［EDP decreased from （14.8±1.9） to （11.0±0.8） mmHg/s and -dp/dtmax increased from （558±60） to （629±51） mmHg/s, P<0.05］ were observed. CONCLUSIONS: Our study indicates that there is a depression of glucose oxidation and at increase in fatty acid oxidation in type 2 diabetic hearts. Elevation of insulin sensitivity using rosiglitazone increases the myocardial glucose metabolism and shows a benefitial result to heart functions.     

Estrogen treatment enhances mesenteric lymphatic contractility in rats after hemorrhagic shock

AIM To investigate the effects of 17β-estradiol （E2） treatment on the mesenteric lymphatic microcirculation and isolated lymphatic contractility in rats after hemorrhagic shock, and to explore the relationship between contractility and the difference between intra- and extracellular calcium ion concentrations （［Ca²⁺］） of lymphatic smooth muscle cells （LSMCs）. METHODS Male Wistar rats were divided into sham group, shock group and shock+E2 group. The rats were subjected to hemorrhage ［（40±2） mmHg for 90 min］ and resuscitation with or without subcutaneous injection of E2 （2 mg/kg）. After resuscitation for 3 h, the mesenteric lymphatic microcirculation in vivo was observed. Moreover, the isolated mesenteric microlymphatic rings were prepared for the observations of lymphatic contractility evaluated by the indexes including end-systolic diameter, end-diastolic diameter, contraction frequency （CF） and passive diameter. Meanwhile, the difference between intra- and extracellular ［Ca²⁺］ of LSMCs was recorded during lymphatic contraction. RESULTS Treatment with E2 significantly enhanced the CF, total contractile fraction and lymphatic dynamics index in vivo in the rats after hemorrhagic shock, and increased the CF, the fractional pump flow and the difference between intra- and extracellular ［Ca²⁺］ of LSMCs in isolated lymphatics from the shocked rats （P<0.05）. CONCLUSION Estrogen treatment enhances lymphatic contractility in rats after hemorrhagic shock, which is related to enhancement of difference between intra- and extracellular ［Ca²⁺］ of LSMCs.     

COX-2 inhibitor protects rat heart against oxidative stress through a pathway independent of cyclooxygenase

AIM: To investigate whether nimesulide ［a selective cyclooxygenase 2 (COX-2) inhibitor］ and piroxicam (an inhibitor of COX-1) protect the rat hearts against oxidative stress induced by hydrogen peroxide,superoxide anion or hydroxyl free radical.METHODS: Cardiac contractility,lactate dehydrogenase (LDH) and malondialdehyde (MDA) were analyzed by the Langendorff method in isolated rat hearts.Production of 6-Keto-PGF1α,a marker of COX activity,was measured in isolated rat hearts.RESULTS: Rat hearts were exposed to hydrogen peroxide (H₂O₂),pyrogallol (which produced superoxide anion) or Vit C+Fe2+ (which produced hydroxyl free radical) for 10 min followed by reperfusion for 30 min.H₂O₂ decreased cardiac contractility and increased LDH release,which was inhibited by nimesulide (3 mg/kg) ［LVDP 72%±10% vs 61%±11%,LDH （5.5±2.5）U/L vs （8.0±2.1）U/L,P<0.05］.Piroxicam (3 mg/kg) increased systolic function (LVDP 73%±10% vs 61%±11%,P<0.05),but exacerbated diastolic function ［LVEDP （29.00±5.61）mmHg vs （23.16±3.57） mmHg,P<0.01］ in H₂O₂ treated rat hearts.Nimesulide also protected rat hearts against superoxide anion and hydroxyl free radical injury.Nimesulide and piroxicam had no effect on the content of 6-Keto-PGF_1α in rat hearts.Mitochondrial ATP sensitive potassium channel (mitoK_ATP) inhibitor 5-HD blocked the improvement of contractility (LVDP and ±dp/dt_max) induced by nimesulide in H₂O₂ treated rat hearts (53%±12% vs 69%±3%,58%±11% vs 72%±7% and 37%±8% vs 51%±4% respectively,P<0.01).CONCLUSION: The results suggests that COX-2 inhibitor nimesulide can protect rat hearts against oxidative injury.The protection is independent of COX activity.Activation of mitoK_ATP may be involved in nimsulide-induced cardioprotection in rat hearts.     

早熟大粒无核葡萄新品种‘超级无核’

 ‘超级无核’葡萄系从美国引进葡萄新品种‘Superior Seedless’优选单株培育出的优良品种。无核、大粒、早熟、优质、早实、丰产、生长势强健、耐病、耐不利栽培条件, 是适合高温、高湿、少日照地区栽培的无核葡萄新品种。     

Effects of salvianolic acid B on the energy metabolism and hydrocephalus in mice with cerebral ischemia

AIM: To explore the effects of salvianolic acid B (SalB) on the energy metabolism and hydrocephalus in mice with cerebral ischemia.METHODS: NIH mice were randomly divided into four groups: sham-operated group,cerebral ischemia group,SalB-treated group and nimodipine-treated group.The brain tissue energy charge (EC),phosphocreatine (PCr),the activity of ATPase,excitability amino acid (EAA) content and water content of brain were measured when cerebral ischemia for 30 min.RESULTS: EC (0.520±0.034),PCr content ［(98.344±13.249) μmol/g］,the activity of Na⁺-K⁺-ATPase ［(0.593±0.013)×10³ U/g］ and Ca²⁺-ATPase ［(0.484±0.053)×10³ U/g］ in SalB-treated group were significantly higher than those in cerebral ischemia group {EC (0.465±0.037),PCr content ［(81.614±9.919) μmol/g］ ,the activity of Na⁺-K⁺-ATPase ［(0.244±0.065)×10³ U/g］,the activity of Ca²⁺-ATPase ［(0.321±0.086)×10³ U/g］} (P<0.01).The glutamate (Glu) content ［(0.405±0.110) μmol/g］,aspartate (Asp) content ［(0.141±0.020) μmol/g］ and water content of brain ［(38.1±0.1)%］ in SalB-treated group were markedly lower than those in cerebral ischemia group ［ Glu content (0.550±0.140) μmol/g,Asp content (0.287±0.050) μmol/g,water content of brain (44.1±0.1)%］ (P<0.05,P<0.01).CONCLUSION: The increase in cerebral energy metabolism and the activity of ATPase,and decrease in EAA content in brain tissue are the mechanism of SalB alleviating hydrocephalus at the early stage of cerebral ischemia in mice.     

Meconium induces formation of nitrotyrosine and expression of inducible nitric oxide synthase in the rat lung

AIM: To evaluate the contribution of inducible nitric oxide synthase (iNOS) and nitrotyrosine to acute lung injury (ALI) in rats with meconium aspiration. METHODS: 16 health male Sprage-Dawley rats were randomized to control group and meconium group, followed by intratracheally administration of 1 mL/kg saline or 1 mL/kg 20% human newborn meconium suspension. The animals were killed after 24 h of treatment. The measurements included bronchoalveolar lavage fluid (BALF) cell count, pulmonary myoloperoxidase (MPO) activity and nitric oxide (NO) level. Western bloting was used to determine the expression of pulmonary nitrotyrosine-a specific “footprint” of peroxynitrite and iNOS. RESULTS: Compared to control group, the rats in the meconium group had increased BALF cell counts ［(4.04±1.01)×109cells/L vs （0.53±0.19)×109cells/L］, pulmonary MPO activity ［(1.49±0.22）U/g wet lung tissue vs (0.62±0.16) U/g wet lung tissue］, NO level ［(12.77±5.00） mmol/g protein vs （4.89±1.32) mmol/g protein］, increased expression of nitrotyrosine and iNOS (0.46±0.19 and 1.49±0.60 vs 0.15±0.04 and 0.09±0.04, respectively), all P<0.01. CONCLUSIONS: Meconium results in an increase in expression of pulmonary iNOS, leading to over production of NO and nitrotyrosine, which may be of pathogenic importance in the ALI with meconium aspiration.     

Effects of endogenous nitric oxide on potassium channels of bronchial smooth muscle cells from asthmatic rats

AIM: To investigate the effects of in vivo application of L-arginine on potassium channels in bronchial smooth muscle cells (BSMC) isolated from asthmatic model rats. METHODS: Male SD rats were randomly divided into control group, asthmatic group and asthmatic rats treated with L-arginine (L-Arg group). Single BSMCs were obtained by acute enzyme separation method. The resting membrane potential (Em), Ca2+-activated K+ channels (BKCa) currents and voltage-dependent K+ channel (Kv) currents in BSMCs were recorded under whole-cell patch clamp technique. RESULTS: (1) The Em of asthmatic group was significantly lower than that in control group (P<0.05). In vivo application of L-Arg significantly hyperpolarized BSMCs near to control group (P>0.05). (2) The peak current density at +50 mV of KCa: IKca in asthmatic group ［(43.8±16.5) pA/pF］ was significantly lower than that in normal group ［(72.5±19.9) pA/pF］ (P<0.01). Treatment with 300 mg/kg L-Arg significantly increased IKca in asthmatic group to (58.7±12.4) pA/pF (P<0.05). (3) The peak currents density at +50 mV of Kv: IKv in asthmatic group ［(32.4±8.7) pA/pF］ was significantly lower than that in control group ［(57.7±9.8) pA/pF］ (P<0.01). Treatment with L-Arg also significantly increased IKv in asthmatic group to (43.6±7.9) pA/pF, (P<0.05). CONCLUSION: Endogenous NO improves Em in asthmatic BSMCs, increases the activities of BKCa channels and Kv channel. These findings implicate that NO may have a potential therapeutically role in airway hypersensitivity.     

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.     

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.     

Genistein downregulates survivin gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To probe into the role of 1, 4, 5-trisphosphate inositol (IP3) and survivin protein in apoptosis of HepG2 cells induced by genistein. METHODS: HepG2 cells were treated with 60 μmol/L genistein for 12 h, 24 h, 48 h and 72 h. IP3, survivin and apoptosis rate were assayed by IP3-［3H］ Birtrak assay, Western blotting and flow cytometry, respectively. RESULTS: IP3 in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein were significantly lower than that in control (P<0.01) ［(12.0±1.4) pmol/106cells, (7.5±0.8) pmol/106 cells, (5.6±0.5) pmol/106cells, (3.3±0.6) pmol/106 cells， vs (29.2±0.6) pmol/106 cells］. V-survivin/ V-β-actin, which was the gray degree multiply area of survivin/the gray degree multiply area of β-actin in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein, were significantly lower than that in control (P<0.01) ［（0.36±0.13, 0.33±0.03, 0.23±0.04, 0.18±0.04）， vs 0.63±0.06］. The apoptosis rate in groups incubated with 60 μmol/L genistein for 24 h, 48 h and 72 h was significantly higher than that in control （P<0.01） ［(7.4%±0.5%, 20.5%±2.0%, 30.7%±1.6%) vs 2.6%±0.1%］. CONCLUSION: Genistein induces apoptosis in HepG2 cells by reducing IP3 production and survivin protein expression.     

Pathological features of airway inflammation in eosinophilic bronchitis

AIM: To explore the pathological features of airway inflammation in patients with eosinophilic bronchitis (EB) and compared to those with cough variant asthma (CVA). METHODS: Flexible fibre optic bronchoscopy was performed in 11 patients with EB, 10 with CVA, 14 with bronchial asthma and 10 normal controls. The mean thickness of the basement membrane was measured by light microscopy. Using immunohistochemical and special staining, the localization and density of inflammatory cells (eosinophils, mast cells, T lymphocytes) were detected in bronchial submucosa in EB and CVA patients. RESULTS: The mean thickness of the basement membrane was significantly increased in the subjects with EB ［2.92 μm (2.10-6.50 μm)］, CVA ［5.64 μm (3.23-8.48 μm)］ and bronchial asthma ［9.08 μm (6.61-11.99 μm)］ rather than that in the normal controls ［2.08 μm (1.62-3.40 μm)］. There were also significant differences among the three groups. The number of mast cells and eosinophils in the bronchial submucosal from subjects with EB ［75 cells/mm2 (35-112 cells/mm2), 7 cells/mm2 (0-31 cells/mm2)］ was substantially decreased than those in subjects with CVA ［148 cells/mm2 (34-200 cells/mm2), 114 cells/mm2 (1-768 cells/mm2); P<0.05］. There was no significantly difference in T lymphocyte counts between the EB and CVA. CONCLUSIONS: EB is an inflammatory disorder of the airways with the characteristics of various inflammatory cell (eosinophils, mast cells and T lymphocytes) infiltration. The mean thickness of the basement membrane is less severe than that in CVA and bronchial asthma and the level of infiltration of inflammatory cells is less than that in CVA, which may be one of the reasons that airway hyperresponsiveness is rarely seen in EB.     

Evaluation of the inflammatory response in two-hit acute lung injury model of rats using ［8F］ FDG microPET

AIM: To investigate whether two-hit acute lung injury (ALI) model is better than one-hit, and to evaluate the inflammatory response in the lungs during these models by using ［8F］FDG microPET. METHODS: Thirty three, adult, male Sprague-Dawley rats weighing 180-210 g were used and divided into 4 groups. Rats in LPS group (n=10) and LPS-HCl group (n=10) were challenged with intraperitoneal administration of LPS at the dose of 5 mg/kg, while rats in NS group (n=3) and HCl group (n=10) received normal saline solution intraperitoneally at the dose of 1 mL/kg, after 16 h, all animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg) and placed in a 60°inclined position, the femoral artery was cannulated and connected to a pressure transducer to record the arterial pressure on a polygraph recorder, the trachea was surgically exposed. Rats in HCl group and LPS-HCl group received direct intratracheal injection of HCl (pH=1.2) at the dose of 0.5 mL/kg while rats in NS group and LPS group received the same volume of normal saline solution. Blood gas samples (each 0.3 mL) were obtained at 30, 90 and 240 min after the instillation and replaced by the same volume of saline solution, the samples were analyzed using a blood gas analyzer. 240 min after HCl or NS administration, the rats underwent a microPET scanning, then, all the rats were sacrificed and the lungs were obtained for histological analysis. RESULTS: Blood gas analysis showed that rats in LPS-HCl group had higher PaO2 and lower PaCO2 than the other groups. MAP decreased markedly in LPS-HCl group, while MAP in other groups remained stable. The results of microPET showed that the ratio of ROI between the right lung and the muscle tissue of the right arm in LPS-HCl group was 9.00±1.41, and was significantly higher than that in LPS group (4.01±0.60) and HCl group (3.33±0.55). Histological examination showed that the mean lung injury score in LPS-HCl group was 12.70±0.95, while that was 8.40±1.26 in HCl group and 7.00±0.82 in LPS group, and there were significant differences (both P<0.01). CONCLUSION: LPS pretreatment significantly magnifies and prolongs the inflammatory response to the subsequent acid instillation in both lungs. When compared with “one-hit”, “two-hit” is easier to induce the ALI, and ［8F］FDG microPET is a useful tool to evaluate the inflammatory reaction during ALI.     

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.     

Restoration of cardiac function in failing beagle hearts by recombinant adeno-associated viral gene transfer of sarcoplasmic reticulum Ca2+-ATPase

AIM: Abnormal Ca2+ homeostasis is one basic cause of heart failure. Studies have recently shown that overexpression of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) by adenoviral/adeno-associated viral gene transfer restores contractile function ex vivo and in murine or rabbit models. We therefore hypothesized that an increase in SERC A2a protein will improve cardiac function in a pacing-induced big animal model of heart failure.METHODS: 17 beagles were randomized into control group (CG, n=4) and chronic heart failure group (n=11). Four weeks after right ventricular rapid pacing (230 beats/min), 11 beagles all got heart failure (documented by >29.3% decrease in ejection fraction). 4 of 11 were used as heart failure group (HF, n=4). 9 HF beagles were randomized to receive either a recombinant adeno-associated viral carrying the SERCA2a gene (HF+SERC A2a, n=5) or the reporter gene enhanced green fluorescent protein (HF+EGFP, n=4) by thoracotomy. All HF beagles paced by 180 beats/min in order to maintain failing state. Thirty days after infection, parameters of systolic and diastolic function were measured by doppler echocardiography and hemodynamic monitor in all beagles.RESULTS: At 30 days after gene transfer, symptoms of HF+SERCA2a dogs improved. Echocardiogram parameters were superior to those in HF+EGFP group (P<0.05). Cardiac hemodynamic parameters of HF+SERCA2a dogs strikingly improved: LVSP, +dp/dtmax and -dp/dtmax increased, mean value increased respectively 54.12％［(214.72±31.74) mmHg vs (139.32±36.79) mmHg］, 146.81％［(6 779.43±217.58) mmHg/s vs (2 746.85±931.2) mmHg/s］ and 71.52％［(-4 341.42±322.02) mmHg/s vs (-2 531.14±616.15) mmHg/s］; LVEDP lowered 63.43％［(21.86±6.95) mmHg vs (59.78±6.92) mmHg］ compared with the dogs in HF+EGFP group. No significant difference in all parameters compared with those of control group was observed. Under laser confocal microscopy, widespread green fluorescence was observed in the myocardial frozen section of dogs in HF+EGFP group. CONCLUSION: These results support the hypothesis that overexpression of SERCA2a improves cardiac function in big animal model of chronic heart failure. The study demonstrates that gene transfer of SERCA2a into cardiac with recombinant adeno-associated viral vector is a prospective therapy methods.