RISK信号通路在β_2-肾上腺素受体激动剂Clenbuterol减轻心肌细胞缺氧/复氧损伤中的作用

目的研究β2-肾上腺素受体激动剂clenbuterol对原代培养的心肌细胞缺氧/复氧损伤的作用及其是否与激活再灌注损伤挽救激酶(reperfusion injury salvage kinase,RISK)信号通路有关。方法将原代培养的新生Wistar大鼠乳鼠心肌细胞分为8组,1正常培养组;2缺氧/复氧(A/R)组;3clenbuterol(1μmol·L-1)+A/R;4ICI118,551(10μmol·L-1)+clenbuterol(1μmol·L-1)+A/R组;5美托洛尔metoprolol(10μmol·L-1)+clenbuterol(1μmol·L-1)+A/R组;6 metoprolol(10μmol·L-1)+A/R组;7 PD98059(20μmol·L-1)+clenbuterol(1μmol·L-1)+A/R组;8LY294002(10μmol·L-1)+clenbuterol(1μmol·L-1)+A/R组。采用MTT法测定各组细胞存活率;比色法检测心肌细胞培养液的乳酸脱氢酶(LDH)含量;Hoechst 33342荧光染色法检测细胞凋亡率;分子探针DCFH-DA检测细胞内活性氧的水平;Western blot检测心肌细胞缺氧/复氧后ERK及p-ERK1/2蛋白的表达水平。结果与A/R组比较,clenbuterol+A/R组明显增高细胞存活率,降低LDH含量,降低细胞凋亡率,ROS产生减少,p-ERK1/2蛋白表达水平增高,而选择性β2受体阻断剂ICI 118,551可取消clenbuterol的上述作用,β1受体阻断剂Metoprolol对clenbuterol的作用无影响,PI3K抑制剂LY294002和ERK1/2抑制剂PD98059可阻断clenbuterol对心肌细胞缺氧/复氧损伤的保护作用。结论clenbuterol能够减轻心肌细胞缺氧/复氧损伤,加入选择性β2受体阻断剂ICI 118,551,PI3K抑制剂LY294002和ERK抑制剂PD98059均使clenbuterol的保护作用取消,表明clenbuterol可通过激动β2肾上腺素受体,激活RISK信号通路发挥抗心肌细胞缺氧/复氧损伤的作用。

Clenbuterol诱导载酯蛋白D在猪脂肪组织中表达加强

Clenbuterol(CLB,盐酸克伦特罗),俗称瘦肉精。由于其在动物体内广泛残留,可能导致食用此类产品的人中毒而被禁止使用,但已有研究表明该制剂可显著减少猪脂肪沉积,因此,对其分子机制进行深入研究将具有重要的理论与实践价值。本研究通过对家猪饲喂高剂量clenbuterol(≥1.5mg/kg体重),提高瘦肉率10%左右。通过实时荧光定量PCR技术对脂肪组织中与脂类代谢密切相关的4个基因研究发现,饲喂clenbuterol的猪脂肪组织内载酯蛋白D(apolipoprotein D,apoD)mRNA丰度增加超过2倍,而脂蛋白酯酶(lipoprotein lipase,lpl)、硬酯酰辅酶A去饱和酶(stearoyl-CoA desaturase,scd)和激素敏感脂酶(hormone sensitive lipase,hsl)mRNA丰度变化不明显。因此,推断clenbuterol通过改变部分脂代谢关键基因的表达来实现减少脂肪积累。载酯蛋白D可能是CLB应答基因之一。

饲料中克仑特罗(Clenbuterol)的气相色谱-质谱联用法测定

本研究建立了饲料中气相色谱 -质谱测定克仑特罗(Clenbuterol)的分析方法 ,试样中Clenbuterol经提取、净化、衍生化 ,采用GC/MSSCAN方式进行测定 ,外标法定量。研究表明 ,Clenbuterol的线性范围为0.5～10mg/kg,方法回收率为92 %～99 % ,检测底限为0.2mg/kg。

Clenbuterol对大鼠脊髓损伤后运动功能恢复作用研究

目的探讨肾上腺素能β2受体激动剂Clenbuterol对脊髓损伤后运动功能恢复的作用。方法重力冲击方法建立大鼠胸髓(T10)损伤动物模型,以不同高度确立试验组。根据BassoBeattieBresnahan(BBB)评分标准评测运动功能情况。试验动物存活6周,进行挫伤脊髓的组织形态学观察。结果Clenbuterol治疗组的平均BBB分值为(10.0±2.1),而对照组分值为(6.3±1.5),P<0.05。Clenbuterol治疗组周边组织残存区面积明显大于对照组。结论在脊髓损伤后应用肾上腺素能β2受体激动剂Clenbuterol可保护脊髓残存的神经组织,帮助改善运动功能的恢复。

Clenbuterol对肥育猪作用效果的研究

７０头“社长大”肥育猪始重（５８±ｌ．０）ｋｇ，按饲养试验要求分成５组，饲喂Ｃｌｅｎｂｕｔｅｒｏｌ含量（ｍｇ／ｋｇ）分别为５，１０，１５，２０的饲粮至猪体重９０ｋｇ左右时结束。结果表明：①饲粮中添加Ｃｌｅｎｂｕｔｅｒｏｌ能提高日增重、降低料重比。其中以１５ｍｇ／ｋｇ组效果最佳，日增重提高１５％（Ｐ＜０．０２），料重比下降１０％；②不同添加量的Ｃｌｅｎｂｕｔｅｒｏｌ均提高了猪胴体瘦肉率。其中１５ｍｇ／ｋｇ组瘦肉率升高５％（Ｐ＜０．０５），胴体脂肪比率则降低１５％（Ｐ＜０．０５），而且Ｃｌｅｎｂｕｔｅｒｏｌ对猪胴体组成的。影响存在部位间的差异，对前躯作用最明显；③提高瘦肉率的同时，Ｃｌｅｎｂｕｔｅｒｏｌ还使眼肌中的营养组成发生变化，蛋白质含量升高４．５％（Ｐ＜０．０５），脂肪含量降低３０％（Ｐ＜０．０５）。

Clenbuterol对海人藻酸所致大鼠海马组织培养损伤的保护作用研究

目的 探讨 β-肾上腺素能受体协同剂 Clenbuterol是否可保护海马神经组织对抗兴奋性氨基酸—海人藻酸 (KA)所致的神经损伤。方法 应用培养的海马组织切片模型 ,将 14d的海马脑片在高钙离子浓度的培养液中加用 5 0 μm ol/ L 的 KA3h,在应用 KA前 2 h、应用的同时及之后的 18h内 ,加用 10 0 μm ol/ L 的 Clenbuterol。KA所致的神经损伤由 Propidium Iodide(PI)的吸收量、乳酸脱氢酶 (L DH)的释放量来评价。结果 Clenbuterol可以保护海马神经组织由 KA所致的损伤 ,表现在 Clenbuterol和 KA同时用药组比 KA单独用药组 L DH释放量及 PI吸收量均降低 2 5 %～ 30 %左右。并证明 Clenbuterol的神经保护作用 ,是通过 β2 -肾上腺素能受体的激活来完成。结论 Clenbuterol可保护海马神经组织对抗 KA所致的兴奋性损伤 ,这种神经保护作用可以使它成为一种治疗缺血性卒中很有潜力的药物。

Clenbuterol肉品残留检测方法

本文对Clerbuterol(一种β—激动剂)的化学结构及其在肉品中残留检测方法作了较详尽的综述。

Clenbuterol，β_2-肾上腺素能受体激动剂促进成年大鼠脊髓半横切后红核脊髓束轴突再生和运动功能的恢复

目的研究Clenbuterol对于促进脊髓半横切(SCI)后轴突再生和运动功能恢复的功效。方法成年Wistar大鼠(n=10-12)接受硬脊膜内的第7颈段脊髓单侧半横切显微手术。Clenbuterol以9 mg/L的剂量加入饮用水中,而对照组未给予任何治疗。采用Cylinder试验和患肢抓力试验观察运动功能的恢复状况。行为学观察期(6周)结束后,大鼠接受第二胸段脊髓处椎板切开术,4%Fluorogold微量注射进入红核脊髓束,一周后被处死。结果运动功能逐渐恢复,从SCI后第3周开始,Clenbuterol治疗组显著优于对照组(最终患肢抓力:403.45±19.82g vs 335.13±13.48g,p<0.05);在Cylinder试验中,双侧前肢共同使用触壁的百分比,Clenbuterol组显著高于对照组(43.39%±5.85%vs 19.42%±6.61%,p<0.05)。Clenbuterol治疗显著地降低了SCI后继发性组织损伤,提高了pCREB阳性细胞核在整个SCI点的表达水平。与对照组相比,Clenbuterol显著地促进了红核脊髓束轴突的再生。结论脊髓损伤后,Clenbuterol可促进离断的轴突再生以及运动功能的恢复。

Clenbuterol 对鸭心电图及血糖皮质激素水平的影响

随日粮中添加 clenbuterol 剂量增加,经汽车长途运输后,肉鸭行为发生异常,死亡率明显提高;心率明显加快,心律不齐,T 波高耸;血清中皮质醇、皮质酮水平显著升高.剂量、心率、血清皮质酮三者呈高度正相关,提示高剂量 clenbuterol 增加了肉鸭对应激的敏感性.

Effect of Clenbuterol Hydrochloride on the in vitro Development of Mouse Embryo

To investigate the effect of clenbuterol hydrochloride on the in vitro development of both 1 cell and 2 cell mouse embryos. Methods The cultural systems of both 1 cell and 2 cell mouse embryo were used to determine the effect of clenbuterol hydrochloride at doses of 1 ng/mL, 3 ng/mL, and 10 ng/mL on developmental rates of mouse embryos. Results When 1 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, developmental rates from the 4 cell stage to blastocyst stage were significantly lower than those in the control group (P<0.05), but on dosages of 3 ng/mL and 10 ng/mL, the inhibiting effects on embryo development were significantly increased (P<0.01). When 2 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, obvious differences in developmental rates were not found between the 2 cell embryo group and the control (P>0.05). However, at levels of 3 ng/mL and 10 ng/mL, significant decrease of developmental rates in 2 cell embryos was observed from the 4 cell and from the 8 cell stage, respectively (P<0.05). Embryos cultured with clenbuterol hydrochloride appeared to have more granules, fragments and degeneration than those in the control. Conclusion Clenbuterol hydrochloride has a toxic effect on the mouse embryos, and the effect is in a dose dependent. 1 cell mouse embryos cultured with clenbuterol hydrochloride could be easily inhibited at 2 cell stage, but the effect of clenbuterol hydrochloride on development of the late 2 cell embryos would be reduced.

Development of a Porcine cDNA Microarray:Analysis of Clenbuterol Responding Genes in Pig(Sus scrofa) Internal Organs

Pig （Sus scrofa） fat accumulation can be reduced by feeding with high dosages of clenbuterol, but the molecular mechanism has not yet been explained. In our study, a porcine cDNA microarray representing 3 358 pig genes was successfully developed. This microarray is the first porcine DNA microarray in China and its false positive rate is 0.98%, which means the microarray platform is reliable. The microarray can be used to study gene expression profiles in multiple pig tissues because the present genes percentage of adipose, skeletal muscle, heart, liver, lung, kidney, and spleen were all more than 60%. This microarray was used to identify the genes responding to clenbuterol stimulation in pig internal organs, including heart, liver, lung, spleen, and kidney. Many genes were identified including enzymes involved in lipids metabolism （lipoprotein lipase up-regulated in liver, heart and lung, ATP-citrate lyase and carnitine palmitoyltransferase II precursor up-regulated in liver, succinyl-CoA up-regulated in lung, mitochondrial malate dehydrogenase down-regulated in spleen）, and signaling pathway genes （cAMP-protein kinase A signaling pathway was found up-regulated in liver, heart, lung, and kidney as reported previously, while transforming growth factor was found down-regulated in heart and lung）. However, no common gene responding to clenbuterol administration was found in all tissues. The expression levels of 14 genes were analyzed using real-time PCR with 82.1% of them induced to express similar magnitudes as in the microarray analyses. This work offers some understanding of how clenbuterol so effectively reduces pig adipose accumulation on the molecular level.

Studies on the Voltammetric Determination of Clenbuterol at One-touch Immunochips

A novel immunosensor for determination of clenbuterol was described in this paper. Horseradish peroxidase (HRP) and Monoclonal antibody against clenbuterol was covalently immobilized onto the cellulose acetate membrane which was used as one-touch immunochips. After 20 min of competitive reaction of clenbuterol in samples against glucose oxidase-conjugated clenbuterol (CLB-GOD) with anti-clenbuteol antibody immobilized on the membrane, the one-touch transducer was fixed on the Nafion - ferrocene modified glass-carbon electrode (GCE, working electrode) by “O” ring. The increased current changes (ΔI) were recorded for the quantitative determination of clenbuterol by derivative cyclic voltammetry. Experimental results showed that a very effective system for electron transmission had been established using dual-enzyme system and Nafion-ferrocene modified GCE. Quantitative analysis of clenbuterol could be easily performed on this kind of immunosensor, which showed the advantage of time-saving and high sensitivity (The detecting limit of this kind of immunosensor can be down to 100 ng/L). In addition, other conditions affecting the electrochemical response were also discussed in detail.

Electrochemical Detection of Clenbuterol in Pig Liver at Pyrrole DNA Modified Boron-doped Diamond Electrode

The direct detection of clenbuterol（CL） in pig liver without any extraction separation at a pyrrole-DNA modified boron-doped diamond（BDD） electrode is reported. The pyrrole-DNA modified BDD electrode has a strong electrocatalytic effect on the redox reaction of CL. One oxidization and two reduction peaks of CL appear at 340. 2, 299. 8 and 166. 6 mV（ versus SCE）, respectively. The pyrrole polymer alone cannot electrocatalyze the above reaction at a BDD electrode ; the electrocatalytic effect of a BDD electrode modified with DNA membrane is unsufficient for the analytical detection of CL; the replacement of boron-doped diamond by glass carbon makes the electrocatalytic reaction impossible ; the redox process is pH dependent. The influences of various experimental parameters on the pyrrole-DNA modified BDD electrode were investigated. A sensitive cyclic vohammetric response for CL was obtained in a linear range from 3.4 × 10^-6 to 5 × 10^ -4 mol/L with a detection limit of 8.5 × 10^-7 mol/L. A mean recovery of 102. 7% of CL in the pig liver sample solution and a reproducibility of 3.2% were obtained.

饲料中添加Clenbuterol对杂交肉猪的生产性能和胴体品质的影响

日龄与体重相近的长白×赣州白杂交肉猪１６头，随机分为两组：Ⅰ（对照组），Ⅱ（试验组），公母各半。猪体重接近４５ｋｇ时，Ⅱ组猪饲料添加０．８ｍｇ／Ｌβ－肾上腺素能受体激动剂Ｃｌｅｎｂｕｔｅｒｏｌ（ＣＬ），直到屠宰前８ｄ停止添加。在猪接近８５ｋｇ体重时，两组猪同时屠宰测定。结果表明，ＣＬ使猪的生长速度提高９．６％，平均日增重提高２０％，饲料转化率提高１７．４５％，眼肌面积增加１５．１％，瘦肉率增加１８．３％，背膘厚度减少１５．３％，脂肪率减少２３．２％（均Ｐ＜０．０５）。胴体在水中的比重（胴体在水中重／胴体重），试验猪比对照猪增加５０％以上（Ｐ＜０．０１），说明饲料中添加ＣＬ使猪胴体的瘦肉成分增多，脂肪成分减少。

Effects of Clenbuterol,Genotype,and Feeding Method on Bodyweight Gain of Meat Rabbits

Four-factor and three-level orthogonal experimental design(L9(34))was used in the experiment.The effects of three genotypes:Germany SAB three-bred-cross rabbits(S2),New Zealand purebred(N),and crossbred rabbits(ON);three levels of Clenbuterol treatment: 1 ng.g-1,2 ng.g-1and 3 ng.g-1,and three feeding methods:5-day,7-day and 10-day withdrawal period after two weeks of feeding Clenbuterol(CL)on bodyweight gain were evaluated.CL apparently improved average daily gain(ADG)of rabbits.70-day ADG of 5-day and 10-day withdrawal were higher than that of 7-day withdrawal,70-day ADG of S2 genotype was significantly higher than that of the control and N genotype was significant.Genotype,additive dose and feeding method had significant effects on overall-stage ADG.ON genotype,2 ng.g-1 dose and 5-day withdrawal feeding method was the best.Responses of different genotype to Clenbuterol appeared different at experimental prophase or whole experimental period.

Illegal Use of Clenbuterol in Cattle Production in Mexico

Slaughtered cattle (n = 582) from México State were bled for a serological screening of clenbuterol residues, using a commercial enzyme-linked immunosorbent assay (ELISA). Clenbuterol residues were found in a total of 153/582 (26.2%) sera analysed. These results reinforced the assumption of the illegal use of clenbuterol in cattle production in México;therefore, routine screening examinations in slaughtered cattle were strongly advised considering the toxic potential for humans.

Evaluation of the clenbuterol imprinted monolithic column prepared by reversible addition-fragmentation chain transfer polymerization

做更多的同质的器官的整体的结构，可逆增加破碎链转移(木排) 过程在 clenbuterol 的合成被采用印的聚合物。在合成，聚合物结构和分离效率上的合成条件的影响被学习。结果证明与椽子过程准备的印的柱比在礼品与常规聚合准备的柱让更高的柱效率和选择学习，它可以源于更高的表面区域，更小的毛孔尺寸和更狭窄的水珠在他们的结构的尺寸分发。如果为 clenbuterol 的更好的条件印了整体的聚合物准备，结果显示了那木排聚合。

Preparation of Hydroxypropyl-β-cyclodextrin Cross-linked Multi-walled Carbon Nanotubes and Their Application in Enantioseparation of Clenbuterol

A method of cross-linking multi-walled carbon nanotubes by a nucleophilic substitution of brominated multi-walled carbon nanotubes using hydroxypropyl-β-cyclodextrin anions was studied. The modified multi-walled carbon nanotube samples were characterized using thermogravimetric analysis, energy-dispersive X-ray spectroscopy, transmission electron microscopy, scanning electron microscopy, Raman spectroscopy and Fourier transform infrared spectroscopy. The hydroxypropyl-β-cyclodextrin modified multi-walled carbon nanotubes were used as a chiral stationary phase additive for thin-layer chromatography to separate clenbuterol enantiomers, and the chiral separation factor was increased.

Fast Determination of Clenbuterol and Salbutamol in Feed and Meat Products Based on Miniaturized Capillary Electrophoresis with Amperometric Detection

The fast separation capability of a novel miniaturized capillary electrophoresis with an amperometric detection （μCE-AD） system was demonstrated by determining clenbuterol and salbutamol in real samples. The effects of several factors such as the acidity and concentration of the running buffer, the separation voltage, the applied potential and the injection time on CE-AD were examined and optimized. Under the optimum conditions, the two β-agonists could be baseline separated within 60 s at a separation voltage of 2 kV in a 90 mmol/L H3BO3-Na2B4O7 running buffer （pH 7.4）, which was not interfered by ascorbic acid and uric acid. Highly linear response was obtained for above compounds over three orders of magnitude with detection limits ranging from 1.20×10^-7 to 6.50×10^-8 mol/L （S/N=3）. This method was successfully used in the analysis of feed and meat products with relatively simple extraction procedures.

Changes of testicular ultrastructure of rat after clenbuterol exposure

Objective To investigate effects of clenbuterol （CLB） on testicular ultrastructure of rat. Methods Twenty adult male Sprague-Dawley rats were randomly divided into four groups （5 rats per group）. CLB solved in normal saline solution was given at the dose of O mg/kg body weight （bw） （group A, as control）, 0.4 mg/kg bw （group B）, 2.0 mg/kg bw （group C）, and 18.5 mg/kg bw （group D）for 14 d by garage consecutively, respectively. Transmission electron microscopy was used to observe changes on testicular ultrastructure. Results In group B, some small vacuoles were found in Sertoli cells. In groups C and D, vacuoles were common in Sertoli cells and spermatogonia. The phenomenon of vacuolation in group D was more severe than that in group C. In group D, basal membrane showed some irregular and wrinkled changes, Leydig cells had more vacuoles and increased lipid droplets. Conclusion Testicular ultrastructure of rat had pathological changes after CLB exposure, and the alterations became more severe with the increasing doses.