肾缺血对缺氧诱导microRNAs及VEGF-NOTCH信号分子的影响

目的 观察肾缺血性损伤后,缺氧诱导microRNAs及VEGF-NOTCH信号分子的表达变化,探讨肾缺血损伤后血管新生的可能调控途径.方法 选择雄性Balb/c小鼠20只,采用夹闭双侧肾蒂方法制备急性缺血肾损伤模型,假手术组设为对照组.通过观察缺血恢复后24 h小鼠肾功能及肾组织病理学改变确定模型成功.实时定量RT-PCR检测缺血恢复后4 h,24 h时缺氧诱导miRNA-210,miRNA-92a,VEGF,Flk-1及Notch1 mRNA表达水平;Western-blot检测缺血恢复后24 h,72 h时Flk-1蛋白的变化;免疫组织化学染色检测CD31表达,判断缺血组织微血管内皮细胞增生状况.结果 肾缺血恢复24 h小鼠肌酐、尿素氮明显升高(P＜0.05),光镜下观察大量小管上皮细胞肿胀、空泡变性坏死,肾小管管腔扩张,见上皮细胞碎片和管型,表明成功建立肾缺血损伤模型.肾缺血恢复4 h,24 h后,与正常组比较,肾组织miRNA-210上调倍数分别为(2.02±0.29),(5.58±0.16);miRNA-92a的表达上调倍数分别为(3.23±0.74),(1.53±0.33),P＜0.05;VEGF,Flk-1及Notch1 mRNA表达水平均升高(P＜0.05).缺血恢复后24 h,72 h时,Flk-1蛋白水平显著升高(P＜0.05),肾组织微血管密度明显增加.结论 肾缺血性损伤后可出现代偿性血管新生,且缺氧诱导miRNA如miRNA-92a,miRNA-210表达上调,与血管新生相关的信号分子VEGF及Notch1表达均升高,提示miRNA/VEGF-Notch1可能是肾缺血性损伤后血管新生的主要调控通路,从而为探讨缺血性损伤后血管新生的机制提供了依据.

Abstract：

Objective To investigate the expression changes of microRNAs and VEGF-NOTCH in renal ischemic injury in mice, and to explore the potential mechanism associated with renal angiogenesis.Method Male Balb/c mice were subjected to a standard renal ischemia to induce acute kidney injury (AKI) after 45 min of bilateral renal artery clamping. Following 4 h, 24 h of reperfusion or sham operation, kindey tissues were collected and subjected to detect the expression changes of microRNAs which relatived with angiogenesis and VEGF, Flk-1, Notch1 mRNA by Quantitative Real-time RT-PCR. Flk-1 protein was detected by Western blotting analysis at 24 h and 72 h following Ischemia/Reperfusion(I/R) injury. The expression of CD31 was examined in tissue sections by immunohistochemistry staining, and the microvessels in ischemic region of each group were counted. Results miRNA-210 and miRNA-92a expression increased significantly, with prominent changes at 4 h and 24 h after reperfusion( P ＜ 0.05 ). VEGF and Flk-1 mRNA expression and Flk-1 protein were increased in renal I/R compared with control group respectively (P＜0.05 ).Immunohistochemistry staining results of CD31 showed a significant increase of microvessels in renal ischemic region. Conclusion This study first reported the changes in miRNAs expression in response to kidney I/R in mouse. our results implied that miRNAs may be involved in targeting VEGF-Notch pathway signaling to regulate angiogenesis after renal I/R injury. It provided novel insights into the angiogenesis mechanism of renal ischemic injury.     

Changes of expression of Fas and FasL protein in rats after renal ischemia/reperfusion injury

AIM：To study the relationship between the expression of Fas/FasL protein and apoptosis in rats after renal ischemia/reperfusion.METHODS:Establish the models of renal ischemia/reperfusion injury in rat and SABC immunohistochemical methods were used to detect the changes of expression of Fas/FasL protein.Pathomorphological changes in renal ischemia/reparfusion injury were observed.RESULTS:The expression of Fas/FasL proteins was negative in the sham operated group.Fas/FasL proteins were increased in renal in ischemia/reperfusion group,and gradually upregulated with the duration of ischemia or reperfusion and peaked at 72h of reperfusion.The expression of Fas/FasL proteins was stronger in 60min ischemia group than 30min ischemia group and they were mainly expressed in renal tubule.We observed local necrosis and inflammatory cells infiltration around infracted areain ischemia/reperfusion group by HE dyeing methods.And the necrosis area was mainly occurred around proximal convoluted tubule.CONCLUSIONS:These findings suggested that Fas/FasL proteins were over expressed after ischemia/reperfusion injury in rats renal.And Fas/FasL system was involved in the process of renal ischemia/reperfusion injury.     

大鼠肾缺血/再灌注损伤不同时相Fas、FasL蛋白表达及意义

AIM: To study the relationship between the expression of Fas/FasL protein and apoptosis in rats after renal ischemia/repernision. METHODS: Establish the models of renal ischemia/reperfusion injury in rat and SABC im-munohistochemical methods were used to detect the changes of expression of Fas/FasL protein. Pathomorphological changes in renal ischemia/reperfusion injury were observed. RESULTS: The expression of Fas/FasL proteins was negative in the sham operated group. Fas/FasL proteins were increased in renal in ischemia/reperfusion group, and gradually upregulated with the duration of ischemia or reperfusion and peaked at 72 h of reperfusion. The expression of Fas/FasL proteins was atronger in 60 min ischemia group than 30 min ischemia group and they were mainly expressed in renal tubule. We observed local necrosis and inflammatory cells infiltration around infracted area in ischemia/repernision group by HE dyeing methods. And the necrosis area was mainly occurred around proximal convoluted tubule. CO     

心肌缺血/再灌注损伤后瘦素的改变及机制初探

Objective To explore the effect of rat myocardial ischemia/reperfusion (I/R) injury on serum Leptin, endothelin (ET), C-reactive protein (CRP) and myocardial Leptin expression, and discuss the role of Leptin in myocardial I/R injury.Methods Fifty Sprague-Dawley (SD) rats were randomly divided into sham-operation, ischemia and I/R 1, 2, 3 hours groups, with 10 rats in each group.Anterior descending artery of the left coronary artery was ligated for 45 minutes and released for 1, 2 and 3 hours to establish myocardial I/R model, and the said artery of the rats in sham-operation group was not ligated.Blood from left femoral artery was collected at different time points, and serum Leptin, ET and CRP contents were detected.Myocardial tissue was harvested, and stained with hematoxylin-eosin (HE)and immunohistochemistry for its observation of the myocardial pathological changes and Leptin protein expression.Results Serum Leptin content (μg/L) of ischemia group was significantly lower than that of sham-operation group (4.69 ± 1.67 vs.6.48 ± 2.02, P＜ 0.05); as the reperfusion time was prolonged,serum Leptin level increased gradually, and the level of I/R 3-hour group recovered to that before injury [(6.59±2.58) μg/L].ET content (ng/L) of ischemia group was significantly higher than that of sham-operation group (110.58 ± 37.86 vs.80.74 ± 34.43, P＜0.05), the levels of ET in I/R 1, 2 and 3 hours groups were significantly lower than those of ischemia group (35.87 ± 13.56, 31.98 ±10.88,34.56±14.37 vs.110.58±37.86, all P＜0.05).CRP content (mg/L) of ischemia group was significantly higher than that of sham-operation group (13.12±4.82 vs.3.24±1.72,P＜0.01); as the reperfusion time was prolonged, serum CRP level increased gradually,and the levels of I/R 1, 2 and 3 hours groups were significantly higher than those of ischemia group (18.37 ± 6.48, 24.30 ± 9.51, 27.08 ± 8.32 vs.13.12 ±4.82, all P＜0.05).Pathological examination showed that there was necrosis of ischemic myocardial cells in ischemia group, with mild congestion and edema in interstitial spaces.After I/R injury, the myocardial cells showed coagulative necrosis, and there was severe congestion of myocardial interstitia.Immunohistochemistry results showed that there was a tendency of decrease in Leptin protein expression in the early phase but increase in the late phase after the injury.Conclusion Leptin content in the serum and myocardial tissue decreases significantly in the early phase after myocardial I/R but increases gradually in the rehabilitative phase, suggesting that Leptin maybe a stress protective factor against I/R-induced myocardial injury.There is a possible association between Leptin and the early increase followed by a delayed decrease of ET as well as the increase of CRP.     

磷酸肌酸钠对大鼠脑缺血-再灌注损伤的保护效应及机制

Objective To observe the effects of exogenous sodium phosphocreatine (PCr) on cerebral repeffusion injury of rats after ischemia in order to explore the potential mechanism. Method Thirty-six healthy adult male Wistar rats with body weight 200- 220 g were randomly (random number) divided into sham operation group, ischemic reperfusion (I/R) group and PCr treatment group. The I/R model was established by using electro-cauterizing bilateral vertebral arteries and occluding bilateral common carotid arteries with atraumatic carotid clasps for 10 min, and then the clasps were released for 48 hours reperfusion. In sham operation group, bilateral common carotid arteries were exposed without occlusion. In PCr treatnent group, PCr in dose of 150 mg/kg was administered intravenously 60 min before the occlusion of bilateral common carotid arteries. Normal saline was administered intravenously instead of PCr into rats of I/R group. After reporfusion for 48 hours, the rats were sacrificed and brains removed for detections of neuron apoptosis by using TUNEL, malondialdebyde (MDA) level by using chromtometry and calmodulin (CaM) activity by using ELISA. Results Compared with sham operation group, TUNEL-positive cells, MDA level and CaM activity increased in I/R group and PGr treatment group ( P ＜0.01). Compared with I/R group, TUNEL-positive cells, MDA level and CaM activity were lower significantly in PCr treatment group ( P ＜ 0.01). Conclusions PCr can lessen cerebral ischemic reperfusion injury and neuron apoptosis, the mechanism maybe relates to the attenuation of abnormalities in calcium balance and reduction of oxygen free radicals by PCr.     

Electro-acupuncture up-regulates the expression of stromal cell-derived factor-1α mRNA and its protein and promotes revascularization in the brain after focal cerebral ischemia and reperfusion

Objective To investigate the mechanism by which electro-acupuncture (EA) promotes revascularization in the brain after focal cerebral ischemia and reperfusion.Methods The Sprague-Dawley rat model of focal cerebral ischemia was made by filament occlusion. The rats were randomly divided into a control group, a model group, and an EA group. The model and EA groups were each divided into 5 subgroups receiving reperfusion 1, 3,7, 14 or 21 days after ischemia. EA was given at the bilateral Hegn point (LI 4) in the EA group. The expression of stromal cell-derived factor-1α(SDF-1α) mRNA was detected using a RT-PCR in the 3, 7 and 14 day subgroups.The immunohistochemical method was employed to detect the expression of SDF-1α protein. Results Compared with the control group, expression of SDF-1α protein increased significantly in the model and EA groups. Compared with the model group, the expression of SDF-1α mRNA increased significantly in the 3, 7 and 14 day subgroups.SDF-1α protein expression and microvessel count increased slightly but not significantly in the 1d subgroup, but the increases were significant in the 3, 7, 14 and 21 day subgroups.Conclusions EA may promote angiogenesis in an ischemic area of the cortex by increasing the expression of SDF-1αmRNA and its protein after focal cerebral ischemia and reperfusion.     

Electro-acupuncture up-regulates the expression of stromal cell-derived factor-1α mRNA and its protein and promotes revascularization in the brain after focal cerebral ischemia and reperfusion

Objective To investigate the mechanism by which electro-acupuncture (EA) promotes revascularization in the brain after focal cerebral ischemia and reperfusion.Methods The Sprague-Dawley rat model of focal cerebral ischemia was made by filament occlusion. The rats were randomly divided into a control group, a model group, and an EA group. The model and EA groups were each divided into 5 subgroups receiving reperfusion 1, 3,7, 14 or 21 days after ischemia. EA was given at the bilateral Hegn point (LI 4) in the EA group. The expression of stromal cell-derived factor-1α(SDF-1α) mRNA was detected using a RT-PCR in the 3, 7 and 14 day subgroups.The immunohistochemical method was employed to detect the expression of SDF-1α protein. Results Compared with the control group, expression of SDF-1α protein increased significantly in the model and EA groups. Compared with the model group, the expression of SDF-1α mRNA increased significantly in the 3, 7 and 14 day subgroups.SDF-1α protein expression and microvessel count increased slightly but not significantly in the 1d subgroup, but the increases were significant in the 3, 7, 14 and 21 day subgroups.Conclusions EA may promote angiogenesis in an ischemic area of the cortex by increasing the expression of SDF-1αmRNA and its protein after focal cerebral ischemia and reperfusion.     

脊髓损伤大鼠核因子kappa B与细胞凋亡及肢体功能间的相关性分析

Objective To investigate the relationship of nuclear factor kappa B(NF-κB),Bcl-2 and Bax with limb function after acute spinal cord injury in rats. Methods Forty-eight rats were divided at random into a control group and an experimental group with 24 rats in each.The spinal cords of the rats in the experimental group were injured at the T8,9,10 level through moderate compression.Four hours,8 h,and 1,3,7 and 14 days after the injury,4 rats were selected randomly from each group and graded with a BBB score.They were then sacrificed and their spinal cords were collected.Immunohistochemical measurements were used to observe the expression of NF-κB, Bcl-2 and Bax. Results NF-κB,Bcl-2 and Bax were observed in the injured spinal nerve cells of rats in the exper imental group but were absent in the control group.After injury,the expression of these factors increased at first and then decreased.BBB scores for limb function increased gradually.No correlation was found between the changes in NF-κB and Bcl-2,but the expression of NF-κB was positively correlated with that of Bax.There was negative correla tion between NF-kB levels and BBB scores,and between NF-kB levels and the ratio of Bcl-2 to Bax. Conclusion In rats,there is a close negative correlation between NF-kappa B levels,the ratio of Bcl-2/Bax and limb function after acute spinaI cord iujury.     

Pretreament with repeated electroacupuncture induced neuroprotection against spinal cord ischemiareperfusion injury in rabbits

AIM:To investigate whether pretreatment with repeated electroacupuncture (EA) could induce ischemic tolerance against transient spinal cord ischemia in rabbits.METHODS:24male New Zealand white rabbits were randomly assigned to 3 groups(n=8 each),animals in the control group received no treatment;animals in the SP and EA group received sodium pentobarbitone 30mg/kg each day for 5 days;animals in EA group were also received electroacupuncture at the Zusanli acupoint 30min a day for 5days.24hours after the last treatment,spinal cord ischemia was induced by an infrarenal aortic occlusion for 20min.Hind-limb motor function was determined with the Tarlov criteria at 4,8,12,24 and 48h after reperfusion.All animals were sacrificed at 48h after reperfusion and the spinal cords(I5) were remoed immediately for histopathologic study.RESULTS:The neurologic outcome and histopathology(48h) in the EA group were significantly better than the control group(P=0.006).CONCLUSION:Pre-ischemic treatment with electroacupuncture significantly reduces spinal cord ischemia-reperfusion injury in rabbits.     

多器官功能障碍综合征大鼠肺组织血栓调节蛋白和基质金属蛋白酶-9的表达

Objective To determine the expressions of thrombomodulin (TM) and matrix metalloproteinase-9(MMP-9) in the lung of rats with multiple organ dysfunction syndrome (MODS) and to investigate the mechanism of lung injury in MODS. Method Forty adult mule Sprague-Dawley (SD) rats were randomly divided into two groups,namely the normal control group and the MODS model group. The rats of model group were further divided into four subgroups as per different intervals (6 h, 12 h, 24 h and 48 h) ,and there were 8 rats in each groups. The animal models of MODS were estabhshed by two hits,the left eyeball of each model rat was removed to bleed to 2 mL/100g,and four hours later, lipopolysaccharide (LPS 5 mg/kg) was injected into intraperitoneal cavity of model rats. The same volume of saline was injected intraperitoneally into rats of control group instead of LPS. All rats were sacrificed at various intervals. The histological changes in lung tissue were observed by naked eye and light microscope. The expressions of TM and MMP-9 proteins were deteceted by using immunohistechemistry. One-way ANOVA was used for comparison among multiple group. Results (1)There were no histopathological changes in lung of rats of control group, and the lung injury was serious in MODS rots. (2) Compared with the rots of con-trol group, the expression of TM in lung tissue of MODS rats increased 6 hours after LPS, reached peak 12 hours later(P <0.01),and then decreased during 24～48 period.There was no significant difference in expression of TM between two groups 48 hours later. Compared with control group, the expressions of MMP-9 in lung tissue of MODS rats didn't significantly increase 6 ～ 48 hours after LPS (P < 0.01). Conclusions There are endothelium damage and extracellular matrix damage found in the lung tissue of rats at the early phase of MODS. TM and MMP-9 are good biomarkers of endothelium and extracellular matrix damage, and they can be used for diagnosing and es-timating the severity of injury lungs at the early phase of MODS.