Effect of endogenous carbon monoxide on intracellular calcium concentration in focal ischemic cerebral tissue in rats

AIM: To study effect of endogenous carbon monoxide on intracellular calcium concentration and explore the mechanism in brain protection of endogenous CO in focal cerebral ischemia in rats. METHODS: SD rats were divided into three groups randomly, which including hemin, ZnPP group and saline group as control. Respectively saline, hemin, ZnPP were injected intra-peritoneally twelve hours before middle cerebral artery was occluded. Twenty four hours after MCAO model was set up, the concentration of carbon monoxide in blood and intracellular calcium in neural cells was examined. RESULTS: Contrast to saline group, the concentration of CO in blood rose up while intracellular calcium in occluded side decreased in hemin group; the concentration of CO in blood went down while intracellular calcium in occluded side rose up in ZnPP group, there was significant difference among them (P<0.05). Hemin and ZnPP had no effect on intracellular calcium in non-occluded sides (P>0.05). CONCLUSIONS: It may be one of mechanisms on brain protection in ischemic cerebral tissue that carbon monoxide affected intracellular calcium concentration of neural cells by regulating Ca²⁺-K⁺ channel on cell membrane as a messenger gaseous molecular and neurotransmitter.     

The regulation of nitric-oxide synthase/nitric-oxide system by endogenous carbon monoxide in rats with pulmonary hypertension

AIM:To study the role and the mechanism of heme oxygenas/endogenous carbon monoxide on nitric oxide synthase/nitric oxide system in rats with pulmonary hypertension induced by hypoxic hypercapnia.METHODS:Sprague-Dawley rats were randomly divided into three groups: control group (A group), hypoxic hypercapnic group (B group), hypoxic hypercapnia+hemin group (C group). Blood CO concentration (COHb%), NO concentration, HO-1 activity, iNOS, cNOS in blood serum and lung homogenate were measured, respectively. RESULTS:① mPAP and RV/(LV+S) of B group were significantly higher than those of A and C group(P＜0.01).② Blood CO concentration, activity of HO-1in blood serum and lung homogenate in rats of B group were significantly higher than those of A group, but were significantly lower than those of C group (P＜0.01). ③ NO concentration in blood serum and lung homogenate in rats of B group were significantly lower than those of A group, those of C group were significantly higher than those of B group (P＜0.01).④The activity of iNOS in blood serum and lung homogenate in rats of B group were significantly higher than those of A group, but were significantly lower than those of C group (P＜0.01). Activity of cNOS in blood serum and lung homogenate of B group were significantly lower than those of A group (P＜0.01), and there was no significant difference between cNOS in B and C group.CONCLUSION:Endogenous carbon monoxide upregulated iNOS/NO system in rats with chronic pulmonary hypertension induced by hypoxic hypercapnia.     

Role of heme oxygenase-1 in cardioprotection against anoxia/re oxygenation induced injury and itsmechanisms

AIM:To investigate the role of HO-1 in pro tection of rat hearts against anoxia/reoxygenation-induced injury and its under lying mechanism.METHODS:Cardiac contractility,lactate dehydrogenase (LDH) and infarct area were analyzed by the Langendorff method in isolated rat hearts.RESULTS:After intraperitoneal injection of HO-1 inducer hemin,CO concentration in rat blood enhanced (P<0.01 vs control group).Pretreatm ent with hemin prevented the increase in LVEDP and decrease in LVDP,±dp/d tmax during the anoxia and reoxygenation period in hearts.Hemin had n o effect on changes of coronary flow,but it really inhibited the release of LDH from anoxia/reoxygenation hearts.Hemin also reduced the infarct area in anoxia heart after 2 h reoxygenation (P<0.01).CO concentration in rat blood redu ced after intraperitoneal injection of HO-1 inhibitor ZnPP (P<0.01 vs contr ol group).ZnPP aggravated the decrease in LVDP and ±dp/dtmax.Co mpared with anoxia/reoxygenation heart,pretreatment of ZnPP enhanced the LDH re lease and enlarged the infarct area (P<0.05).GC inhibitor methylene blue a nd cyclooxygenase-2 (COX-2) inhibitor celecoxib both partly abolished the protec tion effect of hemin on LVEDP,LVDP and ±dp/dtmax.Pretreatment o f methylene blue or celecoxib also cancelled the inhibition of LDH release and r eduction of infarct area caused by hemin (P<0.05).CONCLUSION:HO-1 inducer hemin protects heart from anoxia/reoxy genation-induced injury.The cardiac protection of HO/CO might be through GC pathway,and the activation of COX-2 might be also involved in this process.     

Inhibitory effect of carbon monoxide on proliferation of rat pulmonary artery smooth muscle cells under anoxia

AIM: To investigate the effect of endogenous and exogenous carbon monoxide on the proliferation of pulmonary artery smooth muscle cells under anoxic condition. METHODS: Primary culture of rat PASMCs were passed every 3 days, the 3-5 passages were used. PASMCs were divided into 5 groups, cultured under normoxia and hypoxia and treated with HO inducer hemin, CO scavenger bovine hemoglobin (Hb) and exogenous carbon monoxide (CO), respectively. After 48 hours incubation under the conditions mentioned above, the following assay were carried out: 1) the MTT colorimetric assay and immunocytochemical staining were used to study the energy metabolism and the expression of proliferating cell nuclear antigen (PCNA) in PASMCs. 2) flow cytometry was used to analyze the cell cycle of PASMCs. RESULTS: In comparison with the control group, the value of MTT colorimetric assay was higher, the immunocytochemical staining of PCNA was stronger and the percentages of PASMCs in S and G₂M phases in the anoxia group were higher (P＜0.01). After treatment with hemin and CO, the above indexes were decreased (P＜0.01 or P＜0.05). But treatment with Hb made the above indexes increased (P＜0.01 or P＜0.05). CONCLUSION: The endogenous CO suppress the proliferation of PASMC in an autocrine way. Both the induction of endogenous CO by hemin and the treatment with exogenous CO could suppress the rat PASMCs' proliferation under anoxic condition.     

Effects of ghrelin on brain edema and aquaporin 4 expression after cerebral ischemia/reperfusion in rats

AIM: To explore the effects of ghrelin on the brain edema, the permeability of blood-brain barrier (BBB) and the expression of aquaporin 4 (AQP4) after cerebral ischemia/reperfusion in rats. METHODS: Adult male Sprague-Dawley rats were randomly divided into sham operation group, middle cerebral artery occlusion (MCAO) group and ghrelin treatment group. The MCAO model was made with nylon thread for 2 h of occlusion following 22 h of reperfusion. Ghrelin at a dose of 10 nmol/kg was injected via femoral vein at the beginning of reperfusion. The cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Brain functional deficits were evaluated by determining the neurological scores. The changes of brain swelling and water content were analyzed through volume calculation and dry/wet weight measurement. The permeability of BBB was detected by collecting extravascular Evans blue (EB) in the brain cortex. The changes of AQP4 expression were assessed by the methods of immunohistochemistry and Western blotting. RESULTS: Compared with MCAO group, the rats in ghrelin treatment group had smaller brain infarct volume, lower EB exudation content and neurological scores. The percentage of brain swelling, water content and AQP4 expression were lower in ghrelin treatment group than those in MCAO group. CONCLUSION: Ghrelin reduces the injury of cerebral ischemia/reperfusion, and lightens the brain edema and BBB damage in rats. Ghrelin also inhibits the expression of AQP4 in brain tissue.     

Expression of tissue factor in cerebral microvascular thrombosis in rats

AIM: To study the expression of tissue factor (TF) in cerebral microvascular thrombosis and its dynamic changes in rats. METHODS: 50 female SD rats were randomized to control group, 2, 4, 6, and 24 hours after thrombosis groups, 10 rats in each group. The model of cerebral microvascular thrombosis was induced by photo-chemical method. ELISA and immunohistochemistry methods were used to observe the changes of TF contents in blood plasma and the expression of TF in cerebral microvascular in each group. RESULTS: Cerebral thrombosis was induced by photo-chemical method successfully. The TF content in plasma was obviously higher in 4 h and 6 h groups than that in control group (P＜0.01). TF intensely expressed on vascular endothelial cells in thrombosis groups and negatively expressed in control group. The average A value of TF in 6 h group and 24 h group was significantly higher than that in 2 h and 4 h groups (P＜0.01). It was also found that vWF content in plasma was obviously higher in 2 h group (P＜0.01) and 4 h group (P＜0.05) group than that in control group. AT activity in plasma showed obviously increased in 24 h group, compared with that in the others (P＜0.05). CONCLUSION: These findings suggest the photochemical infarct model is convenient to investigate cerebral thrombosis. TF plays an important role in thrombosis in this model.     

Effect of 3-n-butylphthalide pretreatment on structure and function chan-ges of blood brain barrier in rat model of focal cerebral ischemia-reperfusion injury

AIM: To investigate whether pretreatment with 3-n-butylphthalide (NBP) ameliorates blood brain barrier (BBB) dysfunction in a rat model of focal cerebral ischemia-reperfusion injury (CIRI). METHODS: Male SD rats (n=120, 24 rats in each group) were randomly divided into sham operation group (sham group), model group (IR group), low dose group of NBP pretreatment (NBP I group), medium dose group of NBP pretreatment (NBP II group) and high dose group of NBP pretreatment (NBP III group). The model of CIRI was established by a suture method. After ischemia for 2 h and reperfusion for 24 h, the contents of water and Evans blue (EB) were detected. The pathological changes of the BBB ultrastructure were observed under transmission electron microscope. The protein level of matrix metalloproteinases 9 (MMP-9) was measured by immunohistochemical technique. The mRNA expression of MMP-9 was determined by real-time PCR. RESULTS: After CIRI, the content of water and EB was progressively increased, the BBB was damaged seriously, and the expression of MMP-9 was significantly up-regulated compared with sham group (all P<0.01). Pretreatment with NBP significantly decreased the contents of water and EB, relieved morphological damage of the BBB, and reduced the expression of MMP-9 obviously (all P<0.01). Compared with NBP I group, the changes in NBP II and III group were remarkable (P<0.05), but the difference between NBP II group and NBP III group was not obvious (P＞0.05). CONCLUSION: Pretreatment of 3-n-butylphthalide has preventive effect against cerebral ischemia reperfusion injury in the rats, which may be related to decrease the expression of MMP-9 and reduce the permeability of blood brain barrier.     

Effects of progesterone on water,sodium, potassium and calcium contents in striatum of rats with focal cerebral ischemia/reperfusion

AIM: To explore the neuroprotective action of progesterone(PROG), which has been proved to be a "neuroactive steroid". METHODS: The model of focal cerebral ischemia was established in rats by reverserble inserting a nylon thread with a diameter of 0.2 mm into the anterior cerebral artery through the internal carotid artery. The effect of PROG was assessed by determining water,sodium, potassium, and calcium contents in striatum of rats subjected to 2 h ischemia followed by 22 h reperfusion. RESULTS: The water,sodium,and calcium contents of middle cerebral artery occlusion(MCAO) striatum were obviously higher,the potassium content was obviously lower than those of non-MCAO striatum in I/R and DMSO groups,but there was no significant difference between these two groups.Compared with the result in I/R and DMSO groups , water, sodium and calcium contents significantly decreased, but potassium(P＜0.01) obviously elevated in MCAO striatum in the PROG-pretreated group and PROG-pre or posttreated group.There was also significant reduction in water and sodium, but not significantly changed in calcium and potassium in PROG-posttreated group. The water,sodium,potassium,and calcium contents of MCAO striatum in each PROG-treated group were not remarkably different in comparision with those in dexamethasone treatment group(P＞0.05). CONCLUSION: Treatment with PROG can significantly reduce the striatal injury of rats with cerebral ischmia-reperfusion.     

Stability of cerebral focal ischemia/reperfusion model induced by monofilament in Kunming mice

AIM: To study the stability of mouse cerebral ischemia/reperfusion model induced by the method of monofilament. METHODS: Sixty male Kunming mice were divided into 3 groups according to the body weight: group A (18-21 g), group B (22-28 g) and group C (30-35 g). Ischemia/reperfusion (I/R) model was made by middle cerebral artery occlusion (MCAO) with nylon monofilament. To evaluate the mouse MCAO model, the method of PRM2 laser Doppler was used to detect the cerebral blood flow, the neurological deficit scores were determined by Longa standard and infarction volume was detected with TTC staining. The content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were also measured by ELISA. RESULTS: The successful rates of model establishment in both group A and group B were higher than that in group C (P＜0.05), especially the highest in group B . The mortality in group A was significantly higher than that in group B and group C (P＜0.05). The behavior scores and cerebral infarct volume in group A and group B were significantly higher than those in group C (P＜0.05). Obvious brain injury and neurological deficits were also observed in group A and group B with the higher content of MDA and the lower activity of SOD in the cerebral cortex of the injury side. CONCLUSION: There are three important factors to ensure the success and stability of MCAO mouse model induced by monofilament, i.e. the diameter of monofilament matching the body weight of the mice, the suitable length of monofilament within the blood vessel, as well as the maintaining of proper room temperature during experiment. The MDA content and SOD activity are also effective indexes for evaluating the cerebral I/R injury.     

Protective effects of basic fibroblast growth factor (bFGF) against to myocardial ischemia in rats

AIM: To study the protective effects of basic fibroblast growth factor (bFGF) on myocardial ischemia in rats and their underlying mechanism. METHODS: A rat myocardial ischemic injury model was established by left coronary artery ligation. The rats were killed at 2 h, 4 h, 8 h after coronary artery occlusion. The samples of blood and myocardium were collected for observing the expression of Bcl-2 and Bax in myocardial cells and the changes of superoxide dismutase (SOD) or myocardial enzymes. RESULTS: The amount of Bcl-2 protein expression of myocardial cells in ischemia + bFGF group was significantly higher than that in ischemia+saline group (P＜0.01) at 2 h, 4 h after coronary artery occlusion. However, the change of Bax protein expression was reversed (P＜0.05). The activity of SOD in ischemia+bFGF group was higher than that in ischemia+saline group, and the changes of LDH, CK-MB and α-HBDH in ischemia+bFGF group were reversed (P＜0.05) in serum. CONCLUSION: bFGF has protective roles against myocardial ischemia in rats.     

Effect of naoluo xintong on expression of Fas,Fas L in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats

AIM: To investigate the effets of naoluo xintong on the expression of Fas, FasL protein in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats. METHODS: The local cerebral ischemia /reperfusion model was established by intraluminal thread occlusion of the middle cerebral arteries (MCAO), the middle cerebral arteries of rats were occluded for 2 hours and reperfused for 1, 3 and 7 days. The animals were divided into pseudo surgery group(sham group), model group, Yiqi group, Huoxue group and naoluo xintong group. Using the techniques of immuno-histochemical staining and in situ hybridization, the expression of Fas and FasL was observed in hippocampus CA1 area, the expression of Fas mRNA was also observed in the cortex of frontal and parietal lobe. RESULTS: A value of Fas and FasL protein expression or A value and positive unit of Fas mRNA expression in control group were higher than those in sham in hippocampus CA1 area, the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats (P<0.01). A value and/or positive unit of their expression in naoluo xintong group were lower than those in control group (P<0.05 or P<0.01). A value and/or positive unit of their expression in Yiqi and Huoxue groups were higher than those in naoluo xintong group for 3 and/or 7 days (P<0.05 or P<0.01). CONCLUSION: naoluo xintong could resist neuron apoptosis, alleviate pathologic injury after local cerebral ischemia/reperfusion in MCAO rats by inhibiting the expression of Fas, FasL protein and Fas mRNA.     

Changes of heme oxygenase-carbon monoxide system-in vascular calcification in rats

AIM:To investigate the change of heme oxygenase (HO)-carbon monoxide (CO)-cyclic guanosine monophosphate (cGMP) pathway in vascular calcification, to clarify the cellular and molecular mechanimsm in vascular calcification.METHODS:Vascular calcification model was established in rats by using vitamin D₃ and nicotine. The relative content of HO-1 mRNA, immunochemistry (IH) for HO-1, HO activity, HbCO formation and content of cGMP in aorta were measured.RESULTS:Compared to those of control rats, the HO-1 mRNA level in vessels of rats in VDN group(vascular calcification group) were decreased by 34.9% (P＜0.05);expression of HO-1 protein were decreased too, there were trace positive staining of HO-1 in the endothelium, and no obvious immunoreactivity in the medial layer;HO-1 activity was decreased by 60.6% (P＜0.01), CO concentration was decreased by 53.9% (P＜0.01) and cGMP content was decreased by 77.1% (P＜0.01) in vessels of rats in VDN group.CONCLUSION:There were obvious down regulation in HO-CO-cGMP pathway in calcified vessels.     

Ischemic postconditioning ameliorates pia mater microcirculation in rats subjected to cerebral ischemia reperfusion

AIM:To investigate the ameliorative effect of ischemic postconditioning (I-postC) on pia mater microcirculation in rats subjected to cerebral ischemia reperfusion (I/R) and its mechanisms.METHODS:Thirty-two male Wistar rats were randomly divided into sham, I/R, I-postC, and ischemic preconditioning (IPC) groups.The global cerebral I/R injury was induced by shunting carotid artery in rats.Pia mater microcirculation and cerebral microcirculatory perfusion were measured after reperfusion.The content of soluble intercellular adhesion molecule-1 (sICAM-1) in plasma was detected using enzyme linked-immunosorbent assay (ELISA).Myeloperoxidase (MPO), malondialdehyde (MDA), and superoxide dismutase (SOD) in cerebral tissue were detected.The expressions of vascular endothelial cell cadherin (VE-cadherin) and NF-κB p65 in cerebral tissue were assayed by Western blotting.RESULTS:(1) The disturbance of the blood flow in microvessel induced by I/R was improved significantly by I-postC.In addition, I-postC alleviated significantly the decrease in diameters of microvesseles, cerebral microcirculatory perfusion and cerebral VE-cadherin content induced by I/R (P＜0.05).(2) sICAM-1 in plasma, MPO and MDA in cerebral tissue decreased, but SOD activity in cerebral tissue increased in I-postC group, compared with those in I/R group (P＜0.05 or P＜0.01).The over-expression of NF-κB p65 induced by I/R was relieved by I-postC (P＜0.05).CONCLUSION:I-postC ameliorates pia mater microcirculation in rats subjected to cerebral I/R through suppressing the activation of polymorphonuclear neutrophils mediated by ICAM-1.     

Relationship between expression of heme oxygenase-1 mRNA and pulmonary hypertention induced by hypoxic hypercapnia

AIM: To study the effect of chronic hypoxic hypercapnia on expression of heme oxygenase-1 (HO-1). METHODS: Sprague-Dawley rats were randomly divided into three groups: control group(A),hypoxic hypercapnic group(B), hypoxic hypercapnia+hemin group(C). HO-1 and HO-1 mRNA were observed in pulmonary arterioles by the technique of immunohistochemistry and in situ hybridization. RESULTS: ① mPAP and weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) were significantly higher in rats of B group than those of A and C group (P<0.01). Differences of mCAP were not significant in three groups(P>0.05). ② Blood CO concentration was significantly higher in rats of B group than that of A group (P<0.01), it was much higher in C group than that of B group(P<0.01). ③ Light microscopy showed that vessel well area/total area (WA/TA), density of medial smooth muscle cell (SMC) and media thickness of pulmonary arterioles were much higher in rats of B group than those of A and C group (P<0.01). ④ The observation by electron microscopy showed proliferation of medial smooth muscle cells and collageous fibers of pulmonary arterioles in rats of B group, hemin could reverse the changes mentioned above. ⑤ HO-1 and HO-1 mRNA in pulmonary arterioles was significantly higher in rats of B group than those of A group(P<0.01), and they were significantly higher in rats of C group than those of B group (P<0.01). CONCLUSION: Expression of HO-1 mRNA and HO-1 in pulmonary arterioles was enhanced by hypoxic hypercapnia. Hemin partly inhibited pulmonary hypertension and pulmonary vessel remodeling by enhancing the expression of HO-1 mRNA and HO-1.     

Effects of carbon monoxide on hypoxia inducible factor-1 expression in hypoxia preconditioned mice

AIM:To study the effect of carbon monoxide (CO) on hypoxia inducible factor-1α(HIF-1α) expression and on hypoxic tolerance duration in hypoxia preconditioned mice. METHODS:Mice were injected with normal saline (NS) or hemin intraperitoneally. Western blot was used to detect HIF-1α in hippocampus. The tolerant duration of each hypoxic run was recorded. RESULTS:After 1 and 2 runs of hypoxia exposure, there were no significant differences in HIF-1α expression and hypoxic tolerance duration between N and hemin injection groups. After 3 and 4 runs of hypoxia exposure, the content of HIF-1α was lower and hypoxic tolerance duration was shorter (P＜0.05) in hemin injection groups than that in NS injection groups. CONCLUSION:CO could decrease the expression of HIF-1α, and decrease hypoxic tolerance duration in hypoxia preconditioned mice. The mechanism may be that CO binds to hemoprotein and inhibits hemoprotein oxygen sensor.     

Role of the molar ratio of kaliuretic peptide/atrial natriuretic peptide in elderly essential hypertension

AIM: To investigate the role of the molar ratio of kaliuretic peptide/atrial natriuretic peptide in elderly essential hypertension(EH). METHODS: The levels of plasma kaliuretic peptide (KP) and atrial natriuretic peptide (ANP) in 46 elderly patients with essential hypertension (EH) and 11 spontaneuos hypertension rats (SHR) were determined by radioimmunoassay. Meanwhile, the concentation of KP and ANP in myocardium of rats were observed as well. RESULTS: Compared with control group, the plasma levels of KP and ANP in EH group were no significantly different, but the molar ratio of KP/ANP were lowered significantly (P＜0.05). The levels of plasma KP and ANP in SHR were significantly higher than those in Wistar-Kyoto rats (WKY)(P＜0.01 and P＜0.05 respectively). However, the molar ratio of KP/ANP were markedly lower(P＜0.01). The concentration of KP in atrium was significantly higher than that in ventricle in SHR(P＜0.01). Moreover, there are no significantly different between SHR and SKY in atrium concentration of KP. CONCLUSION: KP and the molar ratio of KP/ANP maybe play an important role in pathogenesis of elderly essential hypertension.     

Change of cPKC-α level in the ascitic white blood cells of AP rats and effects of tetrandrine and aspirin

AIM: To investigate the alteration of classical protein kinase C-α(cPKC-α)in the ascitic white blood cells of rats with acute pancreatitis (AP), and explore the effects of aspirin (ASP) or tetrandrine (Tet) on them. METHODS: The total of 56 health SD rats were divided into four groups, AP+ASP group, AP+Tet group, AP+normal saline (NS) group, and sham operation control (SO) group. AP model was induced by a retrograde injection of 3% sodium deoxycholate into the pancreatic duct. The AP+Tet group received a intraperitoneal injection of Tet (80 mg/kg), respectively. The AP+ASP group received an infusion of ASP (12.5 mg/100 g) by use of a nose-gastric catheter. At 1 h, 5 h after the treatment, cPKC-α in the ascitic white blood cells of AP rats was measured by Western blot and chemiluminescence, and the semi-quantitative values was obtained by Gel-pro analyzer. RESULTS: The values of cPKC-α decreased in AP+NS group, but increased significantly in the groups treated with ASP and Tet (P＜0.05, or P＜0.01). The levels of AMY increased in AP+NS group and decreased in the treated groups significantly (P＜0.05, or P＜0.01). The morphological lesions in the pancreas were attenuated by these medicines. CONCLUSIONS: 1. ASP and Tet increase cPKC-α levels in the ascitic white blood cells of rats with acute pancreatitis. 2. ASP and Tet have protective effects on the morphology and function of the pancreas in the course of AP.