车前子提取物抑制扑热息痛诱导小鼠肝损伤的作用研究

【目的】研究车前子提取物干预扑热息痛(Acetaminophen,APAP)引起小鼠肝损伤的作用,并探究其潜在作用机制。【方法】运用半仿生酶法提取车前子活性成分,采用液相色谱-质谱联用(LC-MS)检测其主要成分。体内研究车前子提取物对APAP肝损伤的保护作用,将60只雌性昆明小白鼠随机分为正常对照组(NC)、APAP肝损伤模型组(LD)、车前子对照组(PA)以及APAP干预的车前子提取物高、中、低剂量组(HPA+LC、MPA+LD、LPA+LD)。其中NC组和LD组小鼠灌胃生理盐水、PA组小鼠灌胃200μg/mL车前子提取物,HPA+LD,MPA+LD和LPA+LD组小鼠分别灌胃车前子提取物200,100,50μg/mL,每天2次,连续6 d;末次给药12 h后,采血并分离肝脏,检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)活性,评价肝脏损伤程度;检测肝脏中超氧化物歧化酶(SOD)活性及谷胱甘肽(GSH)、过氧化氢(H_2O_2)和丙二醛(MDA)含量,评价肝脏的氧化应激程度;检测CYP2E1 mRNA和蛋白表达,并通过细胞色素P450酶2E1(Cytochrome 2E1,CYP2E1)法体外筛选车前子抗APAP肝损伤可能的有效成分。【结果】LC-MS检测发现,车前子提取物有37种主要成分;血清生化指标检测发现,不同剂量的车前子提取物明显减少APAP诱导的氧化产物,增强抗氧化防御能力,减轻肝脏损伤且呈剂量依赖性;车前子提取物显著降低CYP2E1 mRNA和蛋白表达,说明车前子抑制CYP2E1的活性;车前子提取物中抗APAP肝损伤的有效成分为车前草苷D、车前草苷E、高车前素和高车前苷。【结论】车前子提取物的有效成分为车前草苷D、车前草苷E、高车前素和高车前苷。车前子提取物对APAP诱导的肝损伤具有保护作用,其潜在作用机制可能与抑制肝脏CYP2E1的表达和活性有关。     

对乙酰氨基酚对大鼠原代肝细胞的损伤机制

胶原酶两步灌流法获取SD大鼠的原代肝细胞,并用不同剂量的对乙酰氨基酚处理.MTT法测定肝细胞的存活率,分光光度法测定培养上清液中乳酸脱氢酶(LDH)的含量.Western blotting法测定大鼠肝细胞中细胞色素P450酶2E1(CYP2E1)的表达水平.结果表明,对乙酰氨基酚可导致肝细胞存活率降低,LDH含量显著升高,CYP2E1表达增加.说明达到一定的剂量时.对乙酰氨基酚可引起肝细胞的损伤.     

HPLC-PDA方法同时检测黄连解毒散中非法添加的对乙酰氨基酚和盐酸溴己新

为同时检测黄连解毒散中非法添加的对乙酰氨基酚和盐酸溴己新,以十八烷基键合硅胶为填充剂,1%冰醋酸溶液(用三乙胺调剂p H值至4.0)为流动相A,甲醇为流动相B,进行梯度洗脱,流速为1.0 m L/min,波长扫描范围为210~400 nm,柱温25℃,建立了HPLC-PAD检测方法,并采用峰纯度检查和光谱相似度检查辅助对照品比对方法,对非法添加药物进行确证。结果显示,对乙酰氨基酚和盐酸溴己新的回收率分别为98.6%和99.4%,RSD分别为0.6%和0.2%;线性方程分别为y=43408x-401.96,R2=1和y=14331x-1682.4,R2=1;检测限分别为1和5 mg/g;定量限分别为2和10 mg/g。本方法简洁、灵敏、可靠,可同时对黄连解毒散中非法添加的对乙酰氨基酚和盐酸溴己新违禁药物进行定性和定量检测。     

Comparative pharmacokinetics and a clinical laboratory evaluation of intravenous acetaminophen in Beagle and Galgo Español dogs

Objective

To assess the pharmacokinetics (PK) and conduct a clinical laboratory evaluation of acetaminophen in Beagle and Galgo Español (GE) dogs.

不同剂量对乙酰氨基酚诱导大鼠急性肝损伤的研究

通过测定SD大鼠血浆及肝组织中六种酶的活性或含量,探索对乙酰氨基酚短期内对大鼠肝脏的损伤作用。SD大鼠实验组分别皮下注射75、150、250 mg·kg-1剂量的对乙酰氨基酚,对照组注射生理盐水且于给药后1、6、24 h分别采取血浆和肝组织,采用比色法检测各项指标。结果显示:高剂量组大鼠血浆中AST(356.72±43.87 U·L-1)和ALT(325±40.56 U·L-1)以及肝匀浆中GSH(4.55±0.53 mmol·prot-1)、SOD(90.1±12.23 U·g-1)和MDA(230.24±40.43 nmol·g-1)与对照组相比,差异极显著(P0.01),说明250 mg·kg-1剂量的对乙酰氨基酚可对大鼠肝脏产生急性损伤作用。     

Pharmacokinetics of acetaminophen after intravenous and oral administration in fasted and fed Labrador Retriever dogs

Acetaminophen (paracetamol) is used in dogs to manage fever and mild pain. The aim of this study was to assess the pharmacokinetics of acetaminophen in both fed and fasted Labrador Retrievers after a single intravenous and oral administration (20 mg/kg). Six healthy dogs underwent three treatments in a randomized block study (a, n = 2; b, n = 2; c, n = 2). In phase one, group a received acetaminophen intravenously, group b and c orally after being fasted and fed, respectively. In phase two and three, groups were swapped, and the experiment was repeated. At the end of the trial, each dog received the same treatment. Acetaminophen plasma concentrations were detected using a validated HPLC‐UV method. The pharmacokinetic analysis was performed using a noncompartmental model. Clearance, volume at steady state and half‐life of acetaminophen in Labrador Retrievers were 0.42 L/kg hr, 0.87 L/kg and 1.35 hr, respectively. No significant statistical differences were found between fasted and fed dogs regarding maximum plasma concentration, time at maximum concentration and bioavailability as measured by the AUC. Feeding does not significantly affect the acetaminophen oral pharmacokinetics.     

Oral pharmacokinetics of acetaminophen to evaluate gastric emptying profiles of Shiba goats

The pharmacokinetics of acetaminophen was investigated following oral dosing to Shiba goats in order to evaluate the properties of gastric emptying. Acetaminophen was intravenously and orally administered at 30 mg/kg body weight to goats using a crossover design with a 3-week washout period. The stability of acetaminophen in rumen juice was also assessed. Acetaminophen concentrations were measured by HPLC. Since acetaminophen was stable in rumen juice for 24 hr, the extremely low bioavailability (16%) was attributed to its hepatic extensive first-pass effect. The mean absorption time and absorption half-life were unexpectedly short (4.93 and 3.35 hr, respectively), indicating its marked absorption from the forestomach, which may have been due to its smaller molecular weight. Therefore, acetaminophen was considered to be unsuitable for evaluating gastric emptying in Shiba goats.     

Time-course changes in the expression levels of miR-122, -155, and -21 as markers of liver cell damage,inflammation, and regeneration in acetaminophen-induced liver injury in rats

Drug-induced liver injury (DILI) is a significant threat to patient health and a major concern during drug development. Recently, multiple circulating microRNAs (miRNAs) have been reported to be potential biomarkers for DILI. To adapt and validate miRNAs for clinical use, we investigated the time-course changes in miR-122 expression levels in an acetaminophen-induced liver injury model in rats. In addition, miR-155 and miR-21 were evaluated as makers of inflammation and regeneration, respectively, to characterize liver status. Our results revealed that miR-122 is an early and sensitive biomarker of hepatocellular injury at a stage when alanine transaminase, aspartate transaminase, and total bilirubin were not detectable. However, no significant differences in the expression levels of other miRNAs (miR-155 and -21) were observed between treatment and vehicle groups. Collectively, these time-course changes in the expression levels of miRNAs may be useful as markers for clinical decision-making, in the diagnosis and treatment of DILI.