Influence of Fluid Therapy on Gentamicin Pharmacokinetics in Colic Horses

The aminoglycoside antibiotic gentamicin is commonly used in equine medicine for the prevention and treatment of Gram-negative and staphylococcal bacteria in surgically treated colic patients. The pharmacokinetics of gentamicin in these patients might be altered by the disease status, and/or under the influence of fluid therapy. The purpose of this study was to investigate the effect of intravenous fluid treatment on gentamicin kinetics in colic patients. Colic patients subjected to laparotomy were given fluid infusions according to clinical status. Following gentamicin administration, blood samples were taken for gentamicin analysis at different time points, and the main pharmacokinetic parameters including Vc, Vss, t(1/2) and MRT were calculated. Horses undergoing fluid therapy showed a significantly different t(1/2), clearance and MRT as compared to non-infused patients. However, taking into account the clinical status of the patients receiving fluid support, the data suggest that endotoxaemia, rather than fluid therapy, influence gentamicin pharmacokinetics following laparotomy.     

刺糖对庆大霉素致小鼠肾损伤保护作用的初步研究

本试验旨在应用庆大霉素(GM)诱导建立小鼠亚急性肾损伤的模型,初探维吾尔药刺糖对庆大霉素肾损伤的保护作用。将健康昆明系小鼠28只随机分为7组,分别为A组(125 mg/kg GM)、B组(100 mg/kg GM)、C组(80 mg/kg GM)、D组(125 mg/kg GM+刺糖)、E组(100 mg/kg GM+刺糖)、F组(80 mg/kg GM+刺糖)及G组(对照组),连续用药7 d,于第8天采血后处死,分别测定血液中非蛋白氮(NPN)含量及肾脏、肝脏、脾脏的重量。试验结果显示,D组和F组血液中非蛋白氮(NPN)含量和肾脏系数分别与A组及C组相比差异显著(P<0.05);E组血液中NPN含量与B组相比差异不显著(P>0.05),而E组与B组肾脏系数相比差异显著(P<0.05)。A、B、C 3组对小鼠的肾脏都有不同程度的损伤。试验结果表明,刺糖对125和80 mg/kg GM所致的肾损伤起到了明显的保护作用,但对100 mg/kg GM所致的肾损伤未起到明显的保护作用。     

Effect of gentamicin on CD3+ T-lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study

Objective

The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection.

Animals Studied

Three adult horses with no evidence of ophthalmic or systemic disease.

Procedure

Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4′,6-diamidino-2-phenylindole (DAPI) and carboxyfluoroscein succinimidyl ester (CSFE) were used as markers of cell viability and cell proliferation, respectively. Following 5-day culture, live cell counts and CSFE fluorescent intensity data were collected via automated cell count and flow cytometry. The experimental design was duplicated using preservative-free gentamicin and a proprietary brand formulation. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test.

Results

No statistically significant comparisons in CD3+ T-lymphocyte live cell counts and geometric mean fluorescent intensity of CSFE were identified between gentamicin concentrations or formulations.

Conclusions

Gentamicin had no effect on equine peripheral blood CD3+ T-lymphocyte cell viability and proliferation in concentrations ranging from “safe” to “retinotoxic” in relation to intravitreal injection volumes. Low-dose intravitreal gentamicin may not suppress the Th1- and Th17-mediated immune response.     

青蒿琥酯通过Keap1/Nrf2通路抑制氧化应激改善犬急性肾损伤的体内外分析

本研究旨在观察青蒿琥酯对庆大霉素诱导犬急性肾损伤的抗氧化调节作用及其机制。将20只犬随机等分成4组：对照组(Control)、庆大霉素模型组(GM)、青蒿琥酯治疗组(GM+ART)、青蒿琥酯+ML385干预组(GM+ART+ML385)。除对照组,其他组犬采用注射GM建立AKI模型。成功造模后,GM+ART组给与青蒿琥酯,ART+ML385组给与ART和ML385,对照组和GM组给予生理盐水,试验期12 d。用不同浓度GM与MDCK细胞共培养,确定最佳浓度为4.0 mmol·L^-1,用相同方法确定ART最佳浓度为50.0 μmol·L^-1。将体外培养MDCK细胞、过表达Kelch样ECH相关蛋白1(Keap1)的MDCK细胞(M-K)和敲减核因子E2相关因子2(Nrf2)表达的MDCK细胞(M-SiNrf2)分别分成3组：健康对照组、GM对照组和GM+ART干预组,共培养24 h后用于检测。结果显示：1)在动物试验中,GM+ART组肌酐(Cr)、尿素氮(UN)及肾损伤因子1(KIM-1)水平显著低于GM组,GM+ART+ML385组Cr、UN及KIM-1水平显著高于GM+ART组。2)在动物试验中,与GM组比较,GM+ART组Nrf2和谷胱甘肽半胱氨酸连接酶催化亚基(GCLC)蛋白表达上调,Keap1蛋白表达下调,肾组织匀浆中总超氧化物歧化酶(T-SOD)和谷胱甘肽(GSH)水平升高,丙二醛(MDA)水平降低。与GM+ART组比较,给与ML385后Nrf2和GCLC蛋白表达下调,T-SOD和GSH水平降低。3)在细胞试验中,与4.0 mmol·L^-1 GM共培养的MDCK细胞比较,加入50.0 μmol·L^-1ART能显著提高细胞增殖率,降低ROS水平,下调Keap1表达,上调Nrf2、血红素氧合酶1(HO1)及GCLC表达。4)在细胞试验中,与MDCK细胞比较,在GM+ART相同处理下,M-K细胞和M-SiNrf2细胞Keap1蛋白表达上调,Nrf2、HO1及GCLC蛋白表达下调,细胞增殖率降低,ROS含量升高(P＜0.05或P＜0.01)。综上所述,青蒿琥酯对庆大霉素诱导犬急性肾损伤具有保护作用,其作用机制与青蒿琥酯通过Keap1/Nrf2信号通路抑制氧化应激反应有关。     

Gentamicin Nephrotoxicity – A Comparison of In Vitro Findings with In VivoExperiments in Equines

The aminoglycoside gentamicin is often used in equine practice. Despite its clinical use, concerns remain regarding the potential toxic side-effects, such as nephrotoxicity, in equine patients, particularly after repeated dosing. The aim of the study was to investigate first in vitro the mechanisms contributing to the renal toxicity of gentamicin and to identify sensitive biomarkers indicating proximal tubule damage. To this end, the kidney-derived cell lines LLC-PKI and MDCK were treated with gentamicin at different concentrations. Toxicity was assessed by measuring the release of gamma-glutamyl transferase (GGT), and the production of reactive oxygen species (ROS). Cell viability was measured using Alamar blue (AB) and Neutral red (NR) cytotoxicity assays. Gentamicin exerted a dose-dependent toxicity. Primarily, loss of brush border membrane integrity, indicated by GGT leakage, and an increased ROS production were observed. As GGT was found to be a sensitive marker for gentamicin-induced renal cell injury, in the subsequent in vivo experiments, in which ponies were given gentamicin (3.0 mg/kg bw three times daily and 4.5 mg/kg bw twice daily) for five consecutive days, plasma levels and the urinary excretion of GGT and creatinine were measured and the GGT:creatinine ratio was calculated. Elevated GGT levels in urine following gentamicin therapy were observed, but this enzyme leakage was transient and returned to baseline values after cessation of therapy. It could thus be concluded that even a conservative dose regimen of gentamicin did not result in significant renal toxicity in healthy ponies.     

同时检测牛奶中喹诺酮类和庆大霉素残留的胶体金免疫层析方法研究

【目的】喹诺酮类药物和庆大霉素均为高效、广谱抗菌药物,对大多数革兰氏阳性菌和革兰氏阴性菌都具有显著的抗菌效果,是中国畜牧业和水产业中常用的两类兽药。由于这两类药物在动物源性食品中的残留可能导致对人类健康的危害,因此,为了保护消费者的健康,研究和制定动物源性食品中同时检测这两类药物的残留检测方法对完善中国的食品安全监测体系具有重要意义。【方法】建立了同时检测牛奶中13种喹诺酮类和庆大霉素残留的胶体金免疫层析方法。采用柠檬酸三钠还原法制备胶体金颗粒,并对喹诺酮类和庆大霉素单克隆抗体按比例进行混合标记。同时,采用方阵法系统研究了胶体金标记这两类抗体时的pH值和抗体用量对灵敏度的影响,并对这两类药物抗原的包被条件进行选择确定。在此基础上研发出可同时检测牛奶中13种喹诺酮类药物和庆大霉素的胶体金快速检测试纸条,试纸条采用直接竞争法原理。【结果】该方法可同时检测恩诺沙星、环丙沙星、诺氟沙星、氟甲喹、培氟沙星、氧氟沙星、依诺沙星、噁喹酸、麻保沙星、氟罗沙星、奥比沙星、达氟沙星和洛美沙星这13种喹诺酮类药物和庆大霉素,对其他喹诺酮类药物如：沙拉沙星、二氟沙星、司帕沙星、帕珠沙星等无交叉反应,同时对其他氨基糖苷类药物如：链霉素、新霉素、卡那霉素等也无交叉反应。该试纸条对牛奶中这13种喹诺酮类药物和庆大霉素的检测限均为20 ng·mL^-1,完全满足国家对这两类药物的残留限量要求。牛奶样本直接检测,无需处理,整个检测过程5 min内完成。【结论】采用该方法和HPLC-MS/MS对60份牛奶盲样进行比对试验,阳性样品全部检出,同时筛选方法未出现假阳性和假阴性现象,二者的测定结果基本相符,表明该方法准确可靠,适用于现场大批量样本的快速检测和筛选。实际操作过程中,可以采用胶体金免疫层析对样品进行现场快速初筛;筛选的疑似阳性样品,可以采用HPLC-MS/MS方法对样品中QNS和GEN的含量进一步确认。     

猫脂肪间充质干细胞及其条件培养基对庆大霉素致损猫肾细胞增殖和凋亡的影响

试验旨在探究猫脂肪间充质干细胞（adipose mesenchymal stem cells,AD-MSCs）及其条件培养基（adipose mesenchymal stem cells conditioned medium,AD-MSCs-CM）对庆大霉素致损的猫肾细胞（crandell rees feline kidney,CRFK）增殖和凋亡的影响及机制。试验分为空白组、庆大霉素（gentamicin,GM）组、AD-MSCs组和AD-MSCs-CM组;用Ⅰ型胶原酶消化法分离猫AD-MSCs,并用流式细胞术鉴定分离的细胞;以0、2、4、8 mmol/L GM完全培养基处理CRFK,并于12、24和48 h用CCK-8法检测细胞活性,筛选最适造模浓度;致损的CRFK分别与猫AD-MSCs及AD-MSCs-CM共培养,于24、48和72 h后用CCK-8法检测细胞增殖活性,24 h后通过Annexin V流式细胞术检测细胞凋亡率,最后通过实时荧光定量PCR检测细胞周期蛋白D1（cyclin d1,CCND1）、细胞增殖核抗原（proliferative nuclear antigen,PCNA）、Bax和Bcl-2基因的表达水平。结果显示,分离培养的猫AD-MSCs在体外培养条件下呈典型的梭型,高表达MSCs表面标记物CD44、CD90和CD105,不表达白细胞表面标记物CD45;AD-MSCs和AD-MSCs-CM均能促进GM致损CRFK的增殖并抑制其凋亡,其中AD-MSCs和AD-MSCs-CM可通过上调增殖相关基因CCND1、PCNA和抗凋亡基因Bcl-2的表达并降低促凋亡基因Bax的表达而发挥促增殖、抑凋亡作用。试验成功分离得到猫AD-MSCs,并证实AD-MSCs及其条件培养基在体外能促进GM致损CRFK的增殖和迁移,为猫AD-MSCs治疗猫急性肾损伤提供依据。