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Objective:To evaluate the role of heme oxygenase-1 (HO-1) in hemopexin (HPX)-induced reduction of cerebral ischemia-reperfusion (I/R) injury in rats.Methods:One hundred and eighty male Sprague-Dawley rats, aged 7-8 weeks, weighing 250-280 g, were divided into 5 groups ( n=36 each) using a random number table method: sham operation group (group SH), I/R group, vehicle group (group V), HPX group, and HPX plus HO-1 specific inhibitor ZnPPIX(HZ group). Local cerebral I/R was produced by middle cerebral artery occlusion for 120 min followed by reperfusion in anesthetized rats.In I/R, V, HPX and HZ groups, 0.9% normal saline 10 μl, 0.1% NaN 3 10 μl, 1.86 g/L HPX (diluted to 10 μl in 0.1% NaN 3 solution), and 1.86 g/L+ ZnPPIX 20 μmol/L (diluted to 10 μl in 0.1% NaN 3 solution) were injected through the lateral ventricle, respectively, immediately after onset of reperfusion.Morris water maze test was used to detect the cognitive function on day 1 before ischemia and day 2 of reperfusion.Rats were sacrificed, brains were removed and brain tissues were obtained for determination of the permeability ratio of Evans blue (EB), brain water content, expression of VE-cadherin in ischemic penumbra (by Western blot), and expression of angiopoietin-1 (Ang1) mRNA and Ang2 mRNA (by real-time polymerase chain reaction). Ang1 mRNA/Ang2 mRNA ratio was calculated.CD31/vWF double labeling immunofluorescence was used to detect the density of neovascularization in hippocampal tissues in the ischemic penumbra. Results:Compared with SH group, the escape latency was significantly prolonged at 2-7 days of reperfusion, the percentage of time of staying at the target quadrant was decreased, the frequency of crossing the platform was decreased, the permeability ratio of EB in brain tissues was increased at day 7 of reperfusion, and the brain water content, Ang1 mRNA/Ang2 mRNA ratio, expression of VE-cadherin and density of neovascularization were decreased in I/R, V, HPX and HZ groups ( P<0.05). Compared with I/R group, the escape latency was significantly shortened at 2-7 days of reperfusion, the percentage of time of staying at the target quadrant was increased, the frequency of crossing the platform was increased, the permeability ratio of EB in brain tissues was decreased at day 7 of reperfusion, and the brain water content, Ang1 mRNA/Ang2 mRNA ratio, expression of VE-cadherin and density of neovascularization were increased in HPX group ( P<0.05), and no significant change was found in the parameters mentioned above in V and HZ groups ( P>0.05). Compared with HPX group, the escape latency was significantly prolonged at 2-7 days of reperfusion, the percentage of time of staying at the target quadrant was decreased, the frequency of crossing the platform was decreased, the permeability ratio of EB in brain tissues was increased at day 7 of reperfusion, and the brain water content, Ang1 mRNA/Ang2 mRNA ratio, expression of VE-cadherin and density of neovascularization were decreased in HZ group ( P<0.05). Conclusion:HO-1 is involved in HPX-induced reduction of cerebral I/R injury in rats.
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Objective To explore the potential effects of endothelial progenitor cells (EPCs)-angiogenesis on mechanism of alleviating cognitive dysfunction in rats subjected to cerebral ischemia-reperfusion (I/R) injury. Methods A total of 121 male Sprague–Dawley (SD) rats were randomly divided into four groups:Sham group (n=31), focal I/R(MCAO, 0.9%saline 10μL, n=30) group, MCAO+Vehicle (sodium azide, 0.1%Vehicle 10μL, n=30) group and MCAO+HPX (1.86 g/L HPX 10μL, n=30) group. The modified neurological severity scores (mNSS) was carried out to determine neurological function deficit after I/R. Morris water maze (MWM) was carried out to assess learning and memory abilities after I/R. The circulating EPCs after I/R were counted by flowcytometry (FCM) combined with double-immunofluorescence staining of CD34 and CD133. Angiogenesis in rat penumbra cortex after I/R was assessed by immunohistochemical technique combined with immunofluorescent chromogenic detection of CD31 and vWF. Results Compared with sham group, the mNSS scores, the escape latency and the circulating EPCs count were increased after I/R, the time percentage spent in target quadrant was reduced, and the new vessel density in penumbra cortex was increased after I/R in MCAO group (P 0.05). The mNSS score and the escape latency were significantly decreased, the circulating EPCs count and new vessel density in penumbra cortex were significantly increased after I/R in MCAO+HPX group compared with those of MCAO+Vehicle and MCAO group (P<0.05). Conclusion EPCs-angiogenesis signaling plays positive effects on HPX alleviating cognitive dysfunction in rats subjected to focal cerebral ischemia reperfusion injury.
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Objective To evaluate the changes in the expression of protein interacting with Cα kinase 1 (PICK1) in the spinal cord and dorsal root ganglion (DRG) neurons during remifentanil-induced hyperalgesia in rats with incisional pain.Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 42-49 days, were randomly divided into 4 groups (n =8 each) using a random number table: control group (group C) , incisional pain group (group Ⅰ) , remifentanil group (group R), and remifentanil + incisional pain group (group R + Ⅰ).In R and R+Ⅰ groups, remifentanil was infused intravenously for 60 min at the rate of 1.2 p,g · kg-1 · min-1.In C and Ⅰ groups, normal saline was infused intravenously for 60 min at the rate of 0.12 ml · kg-1 · min-1.In Ⅰ and R+Ⅰ groups, the model of incisional pain was established, and remifentanil and normal saline were infused intravenously, respectively, at the same time.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before normal saline or remifentanil infusion, and at 2, 6, 24, and 48 h after the end of normal saline or remifentanil infusion (T1-4).The rats were sacrificed after the last measurement of pain threshold.The lumbar segment (L4-6) of the spinal cord and left DRGs were removed for determination of the expression of PICKl mRNA (by quantitative real-time reverse transcriptase-polymerase chain reaction) and PICK1 protein (by Western blot).Results Compared with group C, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in R and R+Ⅰ groups, and the MWT was significantly decreased, the TWL was shortened (P＜0.05) , and no significant change was found in the expression of PICK1 protein and mRNA in group Ⅰ (P＞0.05).Compared with group Ⅰ, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in group R+Ⅰ (P＜0.05).Compared with group R, the MWT was significantly decreased, the TWL was shortened (P＜0.05) , and no significant change was found in the expression of PICK1 protein and mRNA in group R+ Ⅰ (P＞0.05).Conclusion The mechanism by which remifentanil induces hyperalgesia may be related to up-regulation of PICK1 expression in the spinal cord and DRG neurons of rats with incisional pain.
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Objective To study the expression of mammalian target of rapamycin(mTOR)in ischemic postconditioning(I-postC)-induced attenuation of ischemia/reperfusion(I/R)injury in rat skeletal muscle.Methods A total of 48 healthy male Wistar rats were randomly divided into 3 groups(n=16 each group):I/R group(4-hour ischemia followed by 12-or 24-hour reperfusion),ischemic preconditioning (IPC)group(3 cycles of 5-minute ischemia followed by 5-minute reperfusion),and I-postC group(3 cycles of 1-minute reperfusion followed by 1-minute ischemia).The rat model of I/R injury in right hind limb model was established by clamping the right femoral artery.The changes in the morphology,wet-to-dry weight ratio(W/D),malondialdehyde(MDA),and myeloperoxidase(MPO)in skeletal muscle were compared.The expression of mTOR was detected by Western blot and immunohistochemistry.Results In I-postC and IPC groups,the skeletal muscle edema was less severe,the levels of MDA and MPO significantly decreased,and the expression of mTOR significantly in creased,compared with I/R group(all P＜0.03).There was no significant difference between I-postC and IPC groups.Conclusion Ipostc may attenuate I/R injury in rat hind limbs by activating mTOR signal pathway,which is similar to the mechanism of IPC.
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Objective To study the expression of mammalian target of rapamycin (mTOR) in autogenous vein graft. Methods Autogenous vein graft model was established in 64 Wistar rats. Graft vein was harvested 6 hours, 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks after transplantation. mTOR mRNA was measured by RT-PCR and in situ hybridization. Western blotting and immunohistochemistry methods were used to detect the expression of mTOR. PCNA expression was detected. Results The vein graft mRNA and protein expression of mTOR increased soon after transplantation. mTOR mRNA reached the peak 1 to 2 weeks after surgery [(48?18)% and (33?11)% respectively, P
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Objective To study the effect of antisense RNA retroviral vector of mTOR on the proliferation of vascular smooth muscle cells(VSMC).Methods The conservative region of mTOR gene was inserted into pLXIN reversedly,then the vector was packaged in PT67 cells by transfection with lipofectamine and(transfected) to VSMC.The efficiency of antisense inhibition was verified,and the changes of p70S6k and(4E-BP1) were also determined at the same time.The proliferation activity was determined by flowcytometry and MTT.Results After the vector was successfully transfected to the cells.pLXIN-A-mTOR vector could(efficiently) decrease mRNA and protein expression of mTOR and p70S6k of the cell,while the expression of 4E-BP1 increased significantly.The cell cycle and proliferation activity of VSMC was also stunned in phase G_1.Conclusions The mTOR antisense retroviral vector,pLXIN-A-mTOR,has been constructed(successfully),which can significantly inhibit the proliferation of VSMC.
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Objective To investigate the expression and relationship of early growth response gene-1((Egr-1)),platelet-derived growth factor-B(PDGF-B) and tranforming growth factor(TGF-?_1) in autogenous vein graft in rats,and the role in vein graft intimal hyperplasia(IH).Methods Autogenous vein graft model was(established) in 90 wistar rats.The vein graft samples were harvested at 1,2,6,24 hours,and 3,7,14,28,42 days after surgery.Normal vein was used as control group.Egr-1、PDGF-B,TGF-?_1 mRNA was measured by reverse transcription-PCR and in situ hybridization.Western blotting and immunohistochemistry were used to detect the protein expression of Egr-1,PDGF-B and TGF-?_1. Results Expression of Egr-1,PDGF-B,TGF-?_1 mRNA and protein was not detected in normal vein.In grafting vein,expression level of Egr1mRNA reached a peak at 28days,and the positive rate of Egr-1mRNA was 45%?6%;(PDGF-BmRNA) reached a peak at 14days(48%?6%);a peak of TGF-?_1mRNA was 46%?9% reached at 7days;Egr-1 protein expression reached a peak at 28days, and the positive rate of Egr-1 protein was 40%?9%.PDGF-B protein reached a peak at 28days(45%?4%),TGF-?_1 protein reached a peak at 14days(41%?7%).Conclusions Intimal hyperplasia of vein graft is closely associated withexpression of Egr-1、PDGF-B and TGF-?_1;the activation and expression of PDGF-B and TGF-?_1 may be(modulated) by Egr-1,and they may contribute to increase expression of Egr-1 by feedback.
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Objective To study the effect of antisense mTOR gene transfection mediated by nanoparticles(NP) on intimal perliferation after vein grafting.Methods Nanoparticle antisense mTOR gene complex was prepared with PLGA and PVA.Autogenous vein graft model was established in 72 rats by transplanting internal branch of jugular vein to carotid artery.Three groups were studied:(1) antisense mTOR group,antisense mTOR gene mediated by NP was transfected into the veins before anastomosis.(2) Empty vector group,the vein was transfected by empty vector mediated by NP.(3) Control group,no transfection.The grafted veins were harvested 3 days,1 week,2 weeks and 4 weeks after operation,respectively.The exogenous mTOR mRNA and protein expression were determined and intimal hyperplasia(IH) was observed.The presence of apoptotic VSMC was also detected.Results Antisense mTOR gene transfection mediated by nanoparticle complex inhibited the mRNA and protein expression of mTOR gene(P
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Objective To study the effect of expression of RNA interference targeting protein kinase B(PKB) gene transfection mediated by nanoparticles(NP) on intimal hyperplasia(IH) in vein grafts of rats.Methods Nanoparticle PKB short hairpin RNA(shRNA) gene complex was prepared with PLGA and PVA.Autogenous vein graft model was established in 72 rats by transplanting internal jugular vein to carotid artery.The models were randomly divided into 3 groups:(1) PKB shRNA group: PKB-shRNA gene mediated by NP were transfected into the veins before anastomosis.(2) Empty vector group: the veins were transfected by empty vector mediated by NP.(3) Control group(no transfection).The grafted veins were harvested 3,7,14 and 28 days after the operation respectively.The mRNA and protein expression of PKB were determined by Northern blot and Western blot.IH was observed by HE and Verhoeff stain.The presence of apoptotic VSMC was detected by TUNEL stain.Results Compared to empty vector group and control group,PKB shRNA group had less expression of mRNA and protein of PKB gene(P
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Objective To study the expression of hypoxia-inducible factor(HIF)-1? and related genes in ruptured abdominal aorta aneurysm(RAAA) and explore the underlying pathogenesis. Methods 18 RAAA specimens were collected and 20 AAA tissues were used as control. Total RNA was extracted and Northern blot was used to evaluate the expression of HIF-1? mRNA. Western blot and immunohistochemistry method were used to determine the expression of HIF-1? and vascular endothelial growth factor(VEGF). Microvessel density(MVD) was measured for CD34. Results The expression of HIF-la mRNA was significantly higher in RAAA than that in AAA (P
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Objective To investigate the effect of survivin antisense oligodeoxynucleotides(ASODN) on(intimal) hyperplasia(IH) in vein graft in rats.Methods Autogenous vein graft model was established in 60 Wistar rats by transplanting the interior jugular vein to common jugular artery using microsurgical technique.The rats were divided into 5 groups according to different processing methods,including survivin ASODN 50?g and 200?g,scramble ODN 200?g,control group and lipoectin+pluronic group.Vein graft samples were harvested at 1 or 2 weeks after surgery and histomorphologic methods were used to compare the degree of IH.Survivin mRNA was measured by RT-PCR.Western blotting and immunohistochemistry methods also were used to detect the expression of survivin and PCNA.Apoptosis of VSMC was dectected by TUNEL.Results IH was evident within 1 to 2 weeks after vein grafting,but was markedly inhibited by 50?g of survivin ASODN(P
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In order to determine the pathophysiologic significance of electrocardiographic ventricular hypertrophy in athletes (LVH ath), echocardiographic measurements of left ventricular mass (LVM) were performed on 50 LVH ath. and 50 non-LVH matched ath. They were members of National Teams of track and field, swimming, football and cycling, with an average age of 20.74?3.3 years. Anatomic validation of the method was used for calculation of LVM in this study. Comparisons of LVM were made between LVH ath. and non-LVH ath., and between non-LVH ath. and the untrained:LVM=1.04?[(LVID+PWT+IVST)~3-(LVID)~3]-14Results showed no significant difference of LVM between LVH ath. and non-LVH ath. while LVM of non-LVH ath. were considerably greater than that of the untrained (P
