Construction of T vectors based on Xcm I recognition site and optimization of PCR fragments for ligation / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.</p><p><b>METHODS</b>We firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.</p><p><b>RESULTS</b>Our results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.</p><p><b>CONCLUSION</b>Our protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.</p>

Synergisms of cardiovascular effects between iptakalim and amlodipine, hydrochlorothiazide or propranolol in anesthetized rats / 中国应用生理学杂志

The primary object of this fundamental research was to survey the synergistic cardiovascular effects of iptakalim, a novel ATP-sensitive potassium channel (K(ATP)) opener, and clinical first-line antihypertensive drugs, such as calcium antagonists, thiazide diuretics and β receptor blockers by a 2 x 2 factorial-design experiment. It would provide a theoretical basis for the development of new combined antihypertensive therapy program after iptakalim is applied to the clinic. Amlodipine besylate, hydrochlorothiazide and propranolol were chosen as clinical first-line antihypertensive drugs. Blood pressure, heart rate (HR) and cardiac functions were observed in anesthetized normal rats by an eight-channel physiological recorder. The results showed that iptakalim monotherapy in a low dose could produce significant antihypertensive effect. There was no interaction between iptakalim and amlodipine on the maximal changes of systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MABP), the left ventricular systolic pressure (LVSP), and the left ventricular end-diastolic pressure (LVEDP) (P > 0.05). However, the effects of combination iptakalim/amlodipine on the maximal changes of SBP, DBP, MABP, LVSP and LVEDP were more obvious than those of iptakalim or amlodipine monotherapy. And there was strong positive interaction between iptakalim and amlodipine on the maximal changes of HR (P>0.05). According to the maximal changes of DBP, MABP, LVSP and LVEDP (P < 0.05) of combination iptakalim with hydrochlorothiazide, there was strong positive interaction between them. But there was no interaction between iptakalim and hydrochlorothiazide on the maximal drop of SBP and HR (P > 0.05). According to the maximal drops of DBP, MABP of combination iptakalim with propranolol, there was strong positive interaction between them (P < 0.05). But there was no interaction between iptakalim and propranolol on the maximal changes of SBP, LVSP, LVEDP and HR (P > 0.05). In conclusion, it was the first time to study the effects of amlodipine, hydrochlorothiazide or propranolol, which had different mechanisms of action from iptakalim, on cardiovascular effects of iptakalim in anesthetized normal rats. This study proved that the combination of iptakalim with hydrochlorothiazide or propranolol respectively had significant synergism on lowering blood pressure, while the combination of iptakalim/amlodipine had additive action on lowering blood pressure. Meanwhile the antihypertensive effect was explicit, stable and long-lasting. Iptakalim thus appears suitable for the clinical treatment of hypertensive people who need two or more kinds of antihypertensive agents.

Genome-wide association study of high altitude pulmonary edema / 中国应用生理学杂志

<p><b>OBJECTIVE</b>High altitue pulmonary edema (HAPE) impacts seriously people's health at high altitude. Screening of susceptibility genes for HAPE will be used for the evaluation and protection of susceptible people.</p><p><b>METHODS</b>We performed a genome-wide association study (GWAS) using Affymetrix SNP array 6.0 in 23 HAPE patients and 17 healthy controls. GO and Pathway analysis softwares were used to analyze and draw gene network.</p><p><b>RESULTS</b>Thirty-nine SNPs were found to be significantly different between case and control groups (P < 10(-4)). GO and Pathway analysis of 27 genes around the 39 SNPs indicated that these genes mainly participate in the regulating of cell proliferation, regulation of nitrogen compound metabolic process and G-protein coupled receptor protein signaling pathway and so on.</p><p><b>CONCLUSION</b>It suggests that these SNPs and genes found in this study may be associated with the susceptibility of HAPE.</p>

The construction and application of a novel apparatus for detecting oxygen consumption of mice under normobaric hypoxia / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To establish a method for real-time recording the oxygen consumption of mice under normobaric hypoxia.</p><p><b>METHODS</b>The experimental apparatus was made up of animal container, filling water control system, electronic balance, hose, a computer with weight recording software, etc. The working principle was that the oxygen consumed by animal was replaced by water filling which was controlled by the pneumatic and hydraulic actuator. The water was weighted by an electronic balance and the weight signal was recorded into excel file at the same time. The accuracy and precision of the apparatus were detected by a 10 ml syringe. The oxygen consumption characteristics of 6 acute repetitive hypoxia mice and 6 normal mice were observed.</p><p><b>RESULTS</b>The P value for the paired t test was 1 and the CV value was 4%. The survival time and total oxygen consumption of acute repetitive hypoxia mice were both significantly increased compared to normal mice (P < 0.05), which were (58.8 +/- 6.8) min and (46.0 +/- 8.7) min respectively for the survival time and (85.1 +/- 8.5) ml and (73.6 +/- 5.4) ml respectively for total oxygen consumption.</p><p><b>CONCLUSION</b>The hypoxia tolerance of the acute repetitive hypoxia mice can significantly improved by taking more oxygen in the animal cabin. The accuracy and precision of the apparatus are high and it can be used for the determination of oxygen consumption in hypoxia research.</p>

Stimulation of endothelial non-neuronal muscarinic receptor attenuates the progression of atherosclerosis via inhibiting endothelial cells activation / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of non-neuronal muscarinic receptors (NNMR) stimulation on atherosclerosis and endothelial cells activation.</p><p><b>METHODS</b>Atherosclerosis model was established in ApoE-/- mice by a high fat diet for 7 weeks. During the experimental periods, animals were received a low (7 mg/kg/d) or a high (21 mg/kg/d) dose of arecoline by gavage. At the termination of the treatments, serum total cholesterol and NO levels were measured, and the aorta morphology was analyzed by hematoxylin and eosin staining. The gene expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in the thoracic aortas was determined by RT-PCR, and the MCP-1 protein expression and NF-κB activity were detected by Western blot analysis. NO production, MCP-1 secretion in cultured rat aortic endothelial cells (RAECs), and monocyte-endothelium adhesion assay were also performed after arecoline treatments.</p><p><b>RESULTS</b>Arecoline efficiently decreased atherosclerotic plaque areas, increased serum nitric oxide (NO) content, suppressed the mRNA and protein expression of MCP-1, and modulated the IκB-α degradation and P65 phosphorylation in the aortae of ApoE-/- mice. Furthermore, arecoline promoted NO production and suppressed MCP-1 secretion in cultured RAECs after ox-LDL exposure, and either atropine or NG-nitro-L-arginine methylester could abrogate these effects. Arecoline also significantly inhibited the adherence of U937 monocytes to the ox-LDL injured human umbilical vein endothelial cells, which could be abolished by atropine.</p><p><b>CONCLUSION</b>Our results indicate that arecoline attenuates the progression of atherosclerosis and inhibits endothelial cells activation and adherence by stimulating endothelial NNMR. These effects, at least in part, are due to its modulation on NF-κB activity.</p>

Effects of ATP-sensitive potassium channel opener iptakalim against ventricular remodeling and its mechanisms of endothelial protection / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the effects of iptakalim (Ipt), an ATP-sensitive potassium channel opener, on cardiac remodeling induced by isoproterenol (ISO) in Wistar rats.</p><p><b>METHODS</b>ISO was given subcutaneously (85 mg/(kg x d), sc, 7 days) to induce cardiac remodeling in rats. The rats in Ipt treated group were administrated with Ipt 3 mg/kg (po) after ISO injection. After treated with Ipt for 6 weeks, the hemodynamic parameters were tested by an eight channel physiological recorder (RM-6000). Then the heart weight was weighed and the cardiac remodeling index was calculated. HE stain and Masson's stain were employed to perform histological analysis, the hydroxyproline(Hyp) content in cardiac tissue was detected by colorimetric method, radioimmunoassay was used to measure the plasma levels of endothelin-1 (ET-1) and prostacyclin (PGI2).</p><p><b>RESULTS</b>Six weeks after ISO injection, the cardiac functions of model group were damaged markedly compared with those of normal group. The characteristics of ventricular remodeling in model group included that the heart weight index, myocyte cross-sectional area, myocardial fibrosis, and the hydroxyproline content in cardiac tissue were all increased significantly. The plasma level of ET-1 was increased, while the plasma level of PGI2 was decreased significantly. These changes could be reversed by Ipt treatment (3 mg/(kg x d) for 6 weeks).</p><p><b>CONCLUSION</b>Ipt can reverse cardiac remodeling induced by isoproterenol in rats. The endothelial protective effect regulating effects of Ipt on the balance between the ET-1 and PGI2 system may be involved in its mechanisms.</p>

The characteristics of vascular endothelial injuries induced by extreme environmental factors / 中国应用生理学杂志

Vascular endothelium plays an important role in regulating vascular homeostasis. Over the past years, it has become clear that endothelial dysfunction is a key event of pathophysiological changes in the initiation and progression of injuries induced by extreme environmental factors. The present review summarizes current understanding of vascular endothelial dysfunction induced by hypoxia, cold and heat, and provides the information for prevention and treatment of environmental exposure injuries.

Proteomic identification of human serum biomarkers associated with high altitude pulmonary edema / 中国应用生理学杂志

<p><b>OBJECTIVE</b>High altitude pulmonary edema (HAPE), a life-threatening disease, has no biological markers used for the routine prevention, diagnosis and treatment. The aim of this study was to identify serum proteins differentially expressed in patients with HAPE for discovering essential biomarkers.</p><p><b>METHODS</b>A complete serum proteomic analysis was performed on 10 HAPE patients and on 10 high altitude and 11 sea level healthy people as control using two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting. Finally, two most significantly changed proteins were validated by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Eight protein spots stained with differential intensity, respresenting 5 distinct proteins were identified in patients compared with healthy controls through analysis of these composite gels. Among them, four proteins, namely alpha 1-antitrypsin(alpha1-AT), Haptoglobin(Hp), apolipoprotein A-1 (apoA-1) and Complement C3 increased remarkably, while one protein, apolipoprotein A-IV (apoA-IV) decreased significantly. The variation of alpha1-AT and Haptoglobin, as detected by ELISA, was consistent with the results from proteomic analysis.</p><p><b>CONCLUSIONS</b>It is well known that Hp, alpha1-AT and complement C3 are associated with inflammation and apoA-1 and apoA-IV play important roles in lipid absorption, transport and metabolism. Therefore, the significant expression changes of Hp, alpha1-AT and complement C3 and apoA-1 and apoA-IV between HAPE patients and their corresponding healthy controls highlight the role of inflammatory response system and lipid metabolism system in the pathophysiology of HAPE.</p>

Therapeutic intervention against deacclimatization to high altitude / 中国应用生理学杂志

The incidence of deacclimatization to high altitude syndrome (DAHAS) prevailed up to 80% in highland troops, and 100% in manual workers, and severe DAHAS could significantly affects patients' health, work and life. So it is imperative to develop effective prevention and treatment measures for DAHAS. The present review analyzes effective prophylactic and therapeutic measures against DAHAS, implemented at our hospital.

Non-neuronal muscarinic receptor activation prevents apoptosis of endothelial cells induced by homocysteine / 中国应用生理学杂志

<p><b>OBJECTIVE</b>Endothelial apoptosis plays an important role in the initiation of atherosclerosis. It would be useful to clarify whether activation of non-neuronal muscarinic receptor (NNMR) could prevent endothelial apoptosis and atherosclerosis. We investigated the effects of NNMR activation on regulating rat aortic endothelial cells (RAECs) apoptosis induced by homocysteine, an independent risk factor of atherosclerosis, and further studied its molecular mechanism.</p><p><b>METHODS</b>RAECs were incubated using homocysteine at the concentration of 2.7 mmol/L for 36 h. RAECs were also pre-treated with carbachol or arecoline to examine their effects. RT-PCR was used to assess changes in the gene expression related to cell apoptosis.</p><p><b>RESULTS</b>Incubation of RAECs with homocysteine at the concentration of 2.7 mmol/L resulted in morphologic changes, such as cellular shrinkage, membrane blebbing, chromatin condensation and margination. These could be attenuated by pretreatment with carbachol and arecoline at the concentration of 10 micromol/L for 12 h. Homocysteine induced apoptosis in RAECs and the molecular mechanisms were associated with the regulation of fas, fas-L and caspase-8 in the death receptor pathway, bcl-2, bcl-xL and bax in the mitochondrial pathway, caspase-12 in the endoplasmic reticulum pathway and caspase-3, caspase-6 and p53 as downstream effectors. Carbachol and arecoline attenuated the effects of homocysteine on genes in the death receptor pathway, in the mitochondrial pathway and in the downstream pathway. Atropine could reverse all of the effects of arecoline.</p><p><b>CONCLUSION</b>Activation of NNMR by carbacol and arecoline inhibits homocysteine-induced endothelial cell apoptosis mainly through regulation of death receptor pathway, mitochondrial pathway and downstream effectors.</p>

The acclimatization to extreme environments and its physiological mechanisms / 中国应用生理学杂志

Acclimatization is a process of biological adaptation when exposed to environmental factors such as hypoxia, cold and heat for prolonged periods of time, where non-genetical variations play a role in allowing subjects to tolerate hypoxic, cold or hot environments. This review focuses on the characteristics and mechanisms of acclimatization found through major research advances by our institute. First, the mechanisms underlying the acclimatization to extreme environments are complex. In our investigations, the physiological changes of multiple systems including the nervous, circulatory, respiratory, and hemopoietic system were demonstrated when the acclimatization to hypoxia was developed, and the underlying significance of hypoxia-inducible factor-1 (HIF-1) was investigated. Second, it is suggested that the development of acclimatization to extreme environments is complicated. Hypoxia and cold coexist at high altitude. Our investigations revealed the characteristics of negative cross-relationship in the acclimatization to hypoxia and cold. And third, it is interesting for us to understand that acclimatization to extreme environments is transferable among individuals, and the characteristics of heat acclimatization-inducing factor (HAlF) were presented. The above findings will provide a theoretical guidance for protective operations and help to establish a solid foundation for future research related to acclimatization.

Effects of iptakalim on pressure-overload induced cardiac remodeling and plasma PGI2 content in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the effects of iptakalim (IPT) on pressure-overload induced cardiac remodeling in rats, and investigate correlation between this protection effects and plasma PGI2 content.</p><p><b>METHOD</b>The pressure-overload induced cardiac remodeling model was induced by abdominal aorta constriction for 6 weeks, and the rats were divided into 5 groups repectively: (1) sham group, (2) control group, (3) IPT 3 mg/kg group (IPT 3), (4) indomethacin 2 mg/kg group (Indo 2), (5) indomethacin 2 mg/kg + IPT 3 mg/kg group (Indo 2 + IPT 3). RM6000 eight channel physiological recorder was used to record haemodynamics index, heart weight was weighed and the cardiac remodeling index was calculated, HE stain and Masson's stain were employed to perform histological analysis, colorimetric method was used to detect the hydroxyproline content in cardiac tissue, radioimmunological method was used to measure the plasma PGI2 content.</p><p><b>RESULTS</b>After 42 days of aortic banding, the hyperdynamic circulation state, cardiac remodeling and decreased plasma PGI2 content were observed in the model group compared with those in the sham group, which were effectively reserved by treatment with IPT 3 mg/kg. Single-use indomethacin led to further deterioration of this pathophysiological changes, however, combination administration of IPT 3 mg/kg prevented these from worsening characteristic by ameliorating hyperdynamic circulation state and cardiac remodeling, augmnent plasma PGI2 content.</p><p><b>CONCLUSION</b>IPT can significantly reverse abdominal aorta binding/pressure-overload induced cardiac remodeling, its mechanism may contribute to binding K(ATP) channel in endothelial cells, ameliorating endothelium cells function, augmenting PGI2 synthesis and secretion.</p>

The effect of pioglitazone on apoptotic cardiomyocytes for ischemia reperfusion / 中华心血管病杂志

<p><b>OBJECTIVE</b>This study was to investigate the effect of pioglitazone on apoptotic cardiomyocytes with the model of ischemia-reperfusion at rat heart in vivo.</p><p><b>METHODS</b>Sprague-Dawley rats were randomly divided into two groups. One was 30 min reperfusion group, which was subdivided into sham (n = 5), model (vehicle, n = 6) and pioglitazone 3 mg/kg (n = 7) with 30 min ischemia followed by 30 min reperfusion to detect the area of myocardial infarction (MI). Another was 2 h reperfusion group, which was further subdivided into sham (n = 5), model (vehicle, n = 6), and pioglitazone 0.3 mg/kg (n = 6), 1 mg/kg (n = 7) and 3 mg/kg (n = 6). Apart from the sham, pioglitazone and vehicle were administered intravenously 30 min before occlusion. Then hearts were excised, paraffined and cut into 4 microm thick. Immunohistochemistry, in situ hybridization, TUNEL and DNA agarose gel electrophoresis were performed to detect the expression of Bax, Bcl-2, Caspase-3 and PPARgamma protein and PPARgamma mRNA.</p><p><b>RESULTS</b>(1) Compared with model, nec/aar of pioglitazone decreased by 28% (P < 0.01). The nec/lv ratio reduced by 32% (P < 0.01). (2) In a dose-dependent manner, the expressions of Bax and Caspase-3 were depressed, while the expression of Bcl-2, PPARgamma protein and PPARgamma mRNA were enhanced by pioglitazone. (3) The apoptotic index of subgroups injected pioglitazone reduced significantly by TUNEL compared with model (P < 0.05). Agarose gel electrophoresis demonstrated that DNA ladder existed in model, pioglitazone 0.3 mg/kg and pioglitazone 1 mg/kg, but not pioglitazone 3 mg/kg.</p><p><b>CONCLUSIONS</b>Pioglitazone could protect the heart from I/R injury evidenced by the improvement in the expression of PPARgamma at the levels of protein and mRNA after pioglitazone administrated, and by the decrease in the apoptotic cardiomyocytes.</p>

Regulatory effects of nucleotides on ATP-sensitive potassium channel opener pinacidil-induced vascular relaxation / 中国应用生理学杂志

<p><b>AIM</b>In order to evaluate the regulatory effects of nucleotides and adenosine on ATP-sensitive potassium channel (K(ATP)) in artery smooth muscles, the effects of them on vascular relaxation induced by K(ATP) opener pinacidil(Pin) were investigated.</p><p><b>METHODS</b>The isolated endothelium- denuded aorta rings were preincubated with nucleotides or nucleotides and glibenclamide (Gli) for 10 min, the vascular relaxation induced by Pin in aorta precontracted with 20 mmol x L(-1) KCl was observed.</p><p><b>RESULTS</b>After the isolated endothelium-denuded aorta rings were preincubated with ATP, ADP, UDP, GTP and adenosine (Ade) 100 micromol x L(-1) respectively, the vascular relaxation induced by Pin was changed as following: (1) ATP could inhibit the K(ATP) activation by Pin and enhance the blockade of K(ATP) by Gli. (2) ADP could inhibit the K(ATP) activation by Pin and attenuate the blockade of K(ATP) by Gli. (3) The regulatory effect of Ade on K(ATP) was similar with that of ADP. (4) UDP could enhance the K(ATP) activation by Pin and attenuate the blockade of K(ATP) by Gli. (5) GTP could enhance the K(ATP) activation by Pin, but had no effects on the blockade of K(ATP) by Gli.</p><p><b>CONCLUSION</b>Nucleotides and adenosine, related to energy metabolism, could modulate the functions of K(ATP) in vascular smooth muscle. But their pharmacological characteristics were different.</p>

Effects of chronic intermittent hypoxic exposure on myocardial mitochondria ATPase and enzyme complexes of respiratory chain in rats / 中国应用生理学杂志

<p><b>AIM</b>To study the effects of acute and chronic intermittent hypoxic exposure on the activities of Na+ , K+ -ATPase, Ca2 + , Mg2 + -ATPase of myocardial mitochondria and enzyme complexes of respiratory chain in rats.</p><p><b>METHODS</b>The activities of Na , K+ -ATPase, Ca2+ , Mg2+ -ATPase of myocardial mitochondria and enzyme complexes of respiratory chain were investigated after chronic intermittent hypoxic exposure (3000 m and 5000 m, 4 h/d, 2 w respectively) and normoxic rats were exposed to hypoxia (8000 m) for 4h.</p><p><b>RESULTS</b>(1) Hypoxia had no effects on the activity of Na+, K+ -ATPase in myocardial mitochondria of rats. (2) Compared with normoxic control rats, the activity of Ca2+, Mg2+ -ATPase in myocardial mitochondria of acute hypoxic rats was reduced significantly. After chronic intermittent hypoxic exposure, its activity was increased significantly compared with that of acute hypoxic rats. (3) Compared with normoxic control rats, the activities of enzyme complex I, II and IV of respiratory chain in acute hypoxic rats were reduced significantly. After chronic intermittent hypoxic exposure, their activities were increased significantly compared with those of acute hypoxic rats. Under the same experimental conditions, hypoxia had no effects on the activity of enzyme complex III.</p><p><b>CONCLUSION</b>After chronic intermittent hypoxic exposure, the activities of Na+, K+ -ATPase, Ca2+, Mg2+ -ATPase of myocardial mitochondria and enzyme complexes of respiratory chain were increased significantly. These suggested that chronic intermittent hypoxic exposure could improve the functions of respiratory chain in myocardial mitochondria and keep the normal energy metabolism.</p>

Comparison of pharmacological characteristics of the endothelial target for acetylcholine between big artery and small artery / 中国应用生理学杂志

<p><b>AIM</b>To compare the differences of pharmacological characteristics of the endothelial target for acetylcholine (ETA) between rat aorta and tail artery.</p><p><b>METHODS</b>Differences in the endothelium-dependent relaxation induced by acetylcholine (ACh: 10(-8) - 10(-4) mol/L) were studied using isolated rat tail artery helical strips and aortic rings, so that the pharmacological characteristics of ETA in small artery can be observed.</p><p><b>RESULTS</b>ACh-induced endothelium-dependent relaxation was observed both in rat tail artery strips and in aortic rings precontracted with potassium chloride (60 mmol/L) in a concentration-dependent manner. In tail artery this effect was partially blocked by L-N(omega)-Nitro-arginine methyl ester (L-NAME: 10(-4) mol/L) or methylene blue (MB: 10(-5) mol/L), together with indomethacin (Indo: 10(-4) mol/L), but in aorta it was completely blocked by L-NAME or MB.</p><p><b>CONCLUSION</b>It is different of the pharmacological characteristics of ETA between big artery and small artery. A non-NO and non-PGI2 relaxing factor, together with nitric oxide (NO) and prostacyclin (PGI2), mediates endothelium-dependent vasorelaxation induced by ACh in small artery, but NO may be the principal endothelial vasodilator substance in big artery.</p>