降钙素基因相关肽预适应心肌保护作用的实验研究

目的观察降钙素基因相关肽(CGRP)预适应在整体大鼠心肌缺血/再灌注损伤中的作用并探讨CGRP能否诱导延迟性预适应效应。方法50只SD大鼠随机平均分为5组。所有动物均接受1h缺血/2h再灌注处理,A组于术前24h及术前分别静脉注射CGRP10μg/kg;B组于术前静脉注射CGRP10μg/kg;C组于术前24h静脉注射CGRP10μg/kg;D组于术前24h及术前分别静脉注射相同体积的生理盐水;E组无预处理。用硝基四唑氮篮(TTC)染色判定梗死大小,并以坏死区占危险区的重量百分比表示;取血清测心肌酶学改变,用免疫组化方法检测心肌热休克蛋白70(HSP70)的表达。结果A组、B组、C组较D组、E组心梗面积明显缩小(P<0.01),心肌酶CK、LDH明显降低(P<0.01),A组心肌HSP70表达升高,C组心肌HSP70表达显著升高,B组、D组和E组心肌HSP70基本无表达,A组与B组、C组,D组与E组在心梗面积、心肌酶学的改变方面无明显差异(P>0.05)。结论CGRP预适应在整体大鼠能诱导早期及延迟性效应,缩小心梗面积,减少心肌酶漏出,对大鼠心肌缺血/再灌注损伤起保护作用;CGRP预适应在第一窗口期与第二窗口期的心肌保护作用无明显叠加。     

Limb ischemic preconditioning protects human umbilical vein endothelial cells from oxidative stress induced by H2O2

AIM: To investigate the effects of the sera from the rats after limb ischemic preconditioning (LIPC) on human umbilical vein endothelial cells (HUVECs) injured by hydrogen peroxide (H₂O₂). METHODS: The HUVECs were divided into 5 groups: the cells in control group were cultured without any intervention; the cells in model group (M) were damaged by 1 mmol/L H₂O₂ for 2 h; the cells in early preconditioning serum (EPS) group, delayed preconditioning serum (DPS) group or sham limb ischemic preconditioning serum (SPS) group were treated with the corresponding serum at 5% for 12 h, respectively, and then treaed with H₂O₂ for 2 h. The viability of the HUVECs was analyzed by flow cytometry. The lactate dehydrogenase (LDH) in the culture media was detected. The cell adhesion molecules in the HUVECs were detected by real-time PCR. The mRNA and protein expression of heme oxygenase-1 (HO-1) was also determined. RESULTS: The viability of HUVECs incubated with 1 mmol/L H₂O₂ for 2 h significantly decreased compared with the control cells, which was accompanied with the augmentations of LDH in the medium and the cell adhesion molecules in cells, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Preincubation with EPS and DPS derived from the rats subjected LIPC attenuated these injuries. Furthermore, pretreatment with EPS and DPS increased the expression of HO-1 at mRNA and protein levels. CONCLUSION: LIPC protects the HUVECs from H₂O₂-induced injury.     

Protective effect of delayed ischemic preconditioning on renal ischemia/reperfusion injury via induction of hypoxia inducible factor

AIM: To investigate the effects of delayed ischemic preconditioning on renal ischemia-reperfusion injury in mice and to study the role of hypoxia inducible factor 1α(HIF-1α). METHODS: Male C57/BL6N mice were randomly divided into 3 groups: sham operation group(sham), ischemia/reperfusion group(IR) and ischemic preconditioning group(IPC). Thirty-minute ischemia was induced by clamping renal bilateral pedicles followed by reperfusion in IR group. Fifteen-minute pre-ischemia was performed 4 days before IR in IPC group. Serum creatinine(Scr), blood urea nitrogen(BUN), kidney morphology and apoptosis were observed at different time points following reperfusion. The expression of HIF-1α in the renal tissues was evaluated by the methods of immunohistochemistry and Western blotting analysis. The mRNA expression of vascular endothelial growth factor(VEGF) and glucose transporter-1(Glut-1) was detected by real-time quantitative RT-PCR.RESULTS: Compared with IR group at 24 h following reperfusion, acute tubulointerstitial injury was significantly relieved in IPC group. The levels of Scr and BUN, and apoptosis of tubular epithelial cells were also decreased in IPC group. Nuclear expression of HIF-1α was higher in IPC group than that in IR group. The mRNA expression of VEGF and Glut-1, the target genes of HIF-1, was also increased significantly in IPC group. CONCLUSION: Delayed ischemic preconditioning attenuates both morphologic and functional injuries induced by renal ischemia/reperfusion. This protective effect may be related to the increased expression of hypoxia inducible factor.     

Effects of brain tissue extract of hypoxia-preconditioned mice at different concentrations on tolerance of PC12 cells to hypoxia

AIM: To investigate the effect of brain tissue extract of hypoxia-preconditioned mice (HP extract) on tolerance of PC12 cells to hypoxia. METHODS: The mice model of acute repetitive hypoxia was reproduced and brain tissue extracts were prepared. HP extract was added into the cultures of PC12 cells and the final concentrations of HP extracts were 0.2, 0.8, 3.2, 6.4 or 12.8 g/L (HP group), respectively. Brain tissue extract of normal mice (N extract) at the same five concentrations were used as controls (N group). The PC12 cells were cultured in hypoxia (2% O2). After hypoxia for 24 h, 48 h or 72 h, colorimetric method (A570) of tetrazolium salt MTT (3-［4,5-dimethylthiazol-2-yl］-2,5-diphenyl tetrazolium bromid) staining was adopted to determine the cell viability and lactate dehydrogenase (LDH) release percentage assay was also conducted after 24 h, 48 h or 72 h hypoxia. Besides, apoptotic percentages at early stage (24 h hypoxia) and late stage (72 h hypoxia) were detected respectively by means of annexin V-FITC/PI double-stained flow cytometry and Hoechst 33258 stained fluorescence microscopy. RESULTS: HP extract at the concentrations lower than 6.4 g/L (including 6.4 g/L) showed protective effect on PC12 cells in early stage of hypoxia (24 h). A570 values in HP group were significantly higher than those in N group, but LDH release percentages were significantly lower than those in N group after 24 h hypoxia. With hypoxia prolonging, HP extract at high concentrations gradually lost the protective effect. At the time point of 72 h hypoxia, HP extract at concentrations higher than 6.4 g/L (including 6.4 g/L) had pro-apoptotic effect. At this time point, A570 values of HP groups at these concentrations were significantly lower than those in the corresponding N group, both LDH release percentages and apoptotic percentages were significantly higher than those in the N group. CONCLUSION: The effects of HP extract on tolerance of PC12 cells to hypoxia depend on its concentrations and on the time of treatment.     

Phenotypic changes in progenies of northern clones of Picea abies (L.) Karst. grown in a southern seed orchard

Results are presented from three different field trials comparing controlled crossed materials of Norway spruce from the southern seed orchard at Lyngdal (58° N) with open‐pollinated progenies from the same mother trees standing in the northern natural forest (63–66° N). The seed orchard progenies flushed consistently later in the spring, terminated leader shoot growth later in the summer, had higher frequencies of lammas shoots, were delayed in lignification during autumn, and were 15% taller at age seven years from seed. The difference between the orchard‐ and the natural stand material appeared to be permanent from age four to age seven. It is suspected that the non‐native environment in the seed orchard could affect the genotypic performance of the seed orchard progenies.     

Hypoxic preconditioning protects rat myocardial microvascular endothelial cells against endoplasmic reticulum stress-induced injury

AIM：To investigate the effect of hypoxic preconditioning (HPC) on endoplasmic reticulum stress (ERS)-induced injury in cultured microvascular endothelial cells (MVECs) from rat hearts. METHODS：MVECs injury was induced by an ERS inductor thapsigargin (TG). Lactate dehydrogenase (LDH) leakage and apoptotic rate were detected to evaluate the injury of MVECs. Cytoskeleton and endoplasmic reticulum (ER) in MVECs were observed by phalloidin-FITC fluorescence staining and ER staining, respectively. Two-dimensional electrophoresis and mass spectrometry (MS) were used to identify proteomic profile in MVECs treated with TG. Western blotting was used to detect the expression of ERS markers, calreticulin (CRT) and glucose-regulated protein 78 (GRP78). RESULTS：TG induced the increase in LDH activity in medium and the apoptosis of MVECs in a dose-dependent manner. TG treatment up-regulated the expression of CRT and GRP78, while HPC attenuated the ERS-induced injury and the up-regulation of ERS markers in MVECs. CONCLUSION：HPC protects MVECs from ERS-induced injury.     

Myocardial sarcolemmal and mitochondrial KATP channels in ischemic preconditioning

The potential cardioprotection can be involved during ischemic preconditioning in heart which mechanisms remain unknown yet. It is reported that sarcolemmal and mitochondrial K_ATP channels mediate cardioprotection, and they play distinct myoprotective roles during ischemic preconditioning. This paper reviews the present progress in myocardial sarcolemmal and mitochondrial K_ATP Channels in ischemic preconditioning.     

Protective effect of ischemic preconditioning on rat brain with ischemia/reperfusion injury

AIM:To study whether ischemic preconditioning(IPC) has a protective effect against ischemia/reperfusion(I/R) injury in brain, and the possible relationship between IPC and the regulating function of microcirculation.METHODS:The I/R models were established both in I/R and IPC groups of Sprague-Dawley rats. Additional procedure was performed of short term cerebral ischemic preconditioning in IPC group 24 hours before I/R. Skull windows were performed through which microcirculation features were measured before ischemia, during ischemia, and reperfusion. Finally, brains were cut into slices and stained with red tetrazoline(TTC).RESULTS:Most TTC stained brains in I/R group presented irregular palely red areas which were few in IPC group. Compared with I/R group, IPC group presented relatively increase in accumulated length of capillaries, mean cerebral microcirculatory perfusion, and microcirculatory velocity in ischemic and reperfusion phase. There was no-reflow phenomenon in I/R group in reperfusion phase, which was substituted by the course of increasing reperfusion in IPC group.CONCLUSIONS:IPC could relieve the reduction of tissue perfusion during ischemia and the no-reflow phenomenon during reperfusion by improving the regulating function of microcirculation, which relatively promote the opening of capillaries and accelerating of microvascular flow, therefore protect brain from I/R injury.     

Association among COX2, iNOS and p38MAPK in late phase of preconditioning in rat cardiomyocytes

AIM:To explore the association among COX₂ , iNOS and p38MAPK in late phase of preconditioning. METHODS: The expression of COX₂ , iNOS and p38MAPK activity were determined by western blotting and the injury of cardiomyocyte was assessed by LDH release and trypan blue exclusion in four groups: control group, group IPC (ischemic preconditioning), group SMT (iNOS inhibitor), group NS₃₉₈ (COX₂ inhibitor) and group SB (p38MAPK inhibitor).RESULTS:The expression of COX₂ in group IPC increased markedly in comparision with group SMT and group SB.The expression of iNOS in group SB was lower than that in group IPC and group NS₃₉₈.The difference of the amount of iNOS was not significant between group IPC and group NS₃₉₈.The difference of the amount of phospho-p38MAPK was not significant among group IPC, group SMT and group NS₃₉₈(P>0.05).The LDH was lower, and cell viability was higher in group IPC than those in control group.The LDH was higher, and cell viability was lower in group SMT, group NS₃₉₈ and group SB than those in group IPC. CONCLUSIONS:In late phase of preconditioning , the p38MAPK activity and expression of iNOS and COX₂ increase significantly in rat cardiomyocytes, activited p38MAPK mediates iNOS, then promotes COX₂ expression.     

列当(Orobanche spp. and Phelipanche spp.)种子的采集与预处理方法

列当(Orobanche spp.and Phelipanche spp.)是一种在世界范围内危害严重的根寄生杂草,防除列当的研究也越来越多。正确地采集和预培养列当种子是获得正确防除列当研究结果的前提。列出了大田正确采集列当种子的方法,以及对采集到的种子进行过筛提纯、加洗洁精和吐温20清洗后,再表面消毒和预培养,并通过发芽试验检验此方法的可行性。结果表明,经过处理的向日葵列当、瓜列当种子的发芽率最高分别为54.4%、59.1%。此方法可以应用于列当的采集和预处理过程中,可为列当研究和防除提供参考。