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蒲公英叶水提取物对 APAP 诱导的大鼠肝损伤的保护作用及其机制。

Protective Effect of Dandelion Leaf Water Extracts on APAP-Induced Liver Injury in Rats and Its Mechanism.

机构信息

Department of liver disease, Lanzhou University Second Hospital, Lanzhou 730030, China.

Department of Neonatology, Lanzhou University Second Hospital, Lanzhou 730030, China.

出版信息

Cell Mol Biol (Noisy-le-grand). 2022 May 31;68(5):24-33. doi: 10.14715/cmb/2022.68.5.4.

Abstract

with the advent of a large number of drugs in recent years and the aggravation of human aging, drug-induced liver injury is increasing year by year. The protective effect of dandelion extract on acetaminophen (APAP) - induced drug-induced liver injury in rats and its specific mechanism was studied by in vitro cell culture. For this aim,twenty healthy SD rats with the same physiological status were divided into model group and normal group, with 10 rats in each group. The drug-induced liver injury model was made by intragastric administration of 1 g/kg APAP for 14 days. The liver function lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected to verify the success of the model. After that, the liver tissues were aseptically isolated from the normal group and APAP model group, and the primary hepatocytes were cultured. They were divided into control group (control), liver injury model group (model), medium-dose dandelion extract group (1mm dlwe) and high dose dandelion extract group (2mm dlwe). The cell proliferation activity was detected by CCK-8 cell proliferation activity kit. Cell samples were collected at 72 hours to detect the contents of AST and ALT in cell supernatant. The contents of oxidative stress-activated oxygen (ROS), reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) were detected by colorimetry, and the apoptosis was detected by flow cytometry. Inflammatory factors, key genes of liver injury, drug metabolic enzymes cytochrome P450 2E1 (CYP2E1), mitogen-activated protein kinase (MAPK) and nuclear transcription factors were detected by RT-PCR- κ B (NF- κ B) P65 signaling pathway-related gene expression level. Finally, the expression of CYP2E1, MAPK and NF-kB signaling pathwayswere analyzed by Western blot. Results showed thatSerum ast, ALT and LDH increased (P<0.05), suggesting that the liver injury model was successful. The hepatocytes in the normal group were oval, flat, evenly distributed and well adhered to the wall. The liver injury model group had more suspended cells, pseudopodia, polygonal and poor growth state. The cells in the medium-dose dandelion extract group (1mm dlwe) and high dose dandelion extract group (2mm dlwe) adhered well, mostly oval, similar to the normal group and grew well. CCK8 found that the cells in the model group decreased significantly, the proliferation activity decreased significantly, the ast, alt, LDH and ROS in the cell supernatant of the model group increased compared with other groups (P<0.05), and the contents of GSH and GSH PX decreased (P < 0.05). Apoptotic cells in the model group increased, and TNF in the model group-α, COX-2, CYP2E1, MAPK, JNK and NF KB p65 increased (P < 0.05). CYP2E1, MAPK and NF KB p65 increased in the model group (P < 0.05).

摘要

近年来,随着大量药物的出现和人类老龄化的加剧,药物性肝损伤的发病率逐年增加。本研究通过体外细胞培养,探讨蒲公英提取物对乙酰氨基酚(APAP)诱导的大鼠药物性肝损伤的保护作用及其具体机制。为此,将 20 只具有相同生理状态的健康 SD 大鼠随机分为模型组和正常组,每组 10 只。采用灌胃 1g/kg APAP 14 天建立药物性肝损伤模型。检测肝功能乳酸脱氢酶(LDH)、天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT),以验证模型的成功。然后,从正常组和 APAP 模型组无菌分离肝组织,培养原代肝细胞。它们被分为对照组(control)、肝损伤模型组(model)、中剂量蒲公英提取物组(1mm dlwe)和高剂量蒲公英提取物组(2mm dlwe)。通过 CCK-8 细胞增殖活性试剂盒检测细胞增殖活性。在 72 小时收集细胞样本,检测细胞上清液中 AST 和 ALT 的含量。比色法检测氧化应激激活氧(ROS)、还原型谷胱甘肽(GSH)和谷胱甘肽过氧化物酶(GSH-Px)的含量,流式细胞术检测细胞凋亡。通过 RT-PCR 检测炎症因子、肝损伤关键基因细胞色素 P450 2E1(CYP2E1)、丝裂原活化蛋白激酶(MAPK)和核转录因子κB(NF-κB)P65 信号通路相关基因表达水平。最后,通过 Western blot 分析 CYP2E1、MAPK 和 NF-κB 信号通路的表达。结果表明,血清 ast、ALT 和 LDH 升高(P<0.05),表明肝损伤模型成功。正常组的肝细胞呈椭圆形、扁平状、均匀分布、贴壁良好。肝损伤模型组的悬浮细胞较多,伪足多,呈多边形,生长状态较差。中剂量蒲公英提取物组(1mm dlwe)和高剂量蒲公英提取物组(2mm dlwe)的细胞贴壁良好,多呈椭圆形,与正常组相似,生长良好。CCK8 发现模型组细胞明显减少,增殖活性明显下降,与其他组相比,模型组细胞上清液中 AST、ALT、LDH 和 ROS 含量增加(P<0.05),GSH 和 GSH PX 含量减少(P<0.05)。模型组凋亡细胞增多,TNF-α、COX-2、CYP2E1、MAPK、JNK 和 NF-κB p65 表达增加(P<0.05)。模型组 CYP2E1、MAPK 和 NF-κB p65 表达增加(P<0.05)。

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