CDK抑制剂(AT7519)对小鼠急性肝损伤的保护作用

肝脏 ›› 2021, Vol. 26 ›› Issue (4): 375-378.

CDK抑制剂(AT7519)对小鼠急性肝损伤的保护作用

刘彦君, 黄鹤鸣, 付荣, 石翠翠, 范建高, 张元元, 罗成, 李光明

200092 上海交通大学医学院附属新华医院消化内科(刘彦君, 黄鹤鸣, 付荣, 石翠翠, 范建高, 李光明);
中国科学院上海药物研究所药物研发与设计中心(张元元, 罗成)

收稿日期:2020-10-05 发布日期:2021-05-22
通讯作者: 李光明, Email:liguangming@xinhuamed.com.cn
基金资助:
国家自然科学基金(81070344, 81803554, 91853205, 81625022, 81821005)

Protective effect of cyclin-dependent kinase inhibitor AT7519 on carbon tetrachloride induced acute liver injury in mice

LIU Yan-jun¹, HUANG He-ming¹, FU Rong¹, SHI Cui-cui¹, FAN Jian-gao¹, ZHANG Yuan-yuan², LUO Cheng², LI Guang-ming¹

1. Department of Gastroenterology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, China;
2. Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China

Received:2020-10-05 Published:2021-05-22
Contact: LI Guang-ming, Email: liguangming@xinhuamed.com.cn

摘要/Abstract

摘要： 目的探索细胞周期蛋白依赖性激酶抑制剂AT7519对四氯化碳所致小鼠急性肝损伤的保护作用。方法采用LPS诱导的RAW264.7细胞炎症模型, 不同浓度(20 μmol、10 μmol、5 μmol)AT7519预处理1 h再以LPS刺激4 h, RT-qPCR检测AT7519对促炎细胞因子表达水平的影响。此外, 建立小鼠四氯化碳急性肝损伤模型, 将30只C57BL/6小鼠随机分成3组:正常对照组、模型组、药物干预组, 检测小鼠血清转氨酶水平, 并行HE、免疫组化染色评估肝组织病理学变化, 以及RT-qPCR检测肝组织中促炎细胞因子表达水平。结果 LPS诱导的RAW264.7细胞炎症模型中, LPS刺激模型组IL-6、IL-1β和TNF-α的mRNA分别为(299.8±6.1)、(8198±360.1)、(172.0±5.9), 较正常对照组(1.1±0.1)、(1.1±0.0)、(1.0±0.0)显著升高(P<0.05);而不同浓度AT7519干预组均可显著下调上述促炎细胞因子, 20 μmol AT7519处理组IL-6、IL-1β和TNF-α的mRNA相对表达量为(6.5±0.4)、(275.2±10.4)、(41.5±1.0), 10 μmol AT7519处理组为(36.7±5.4)、(1585±26.0)、(106.2±12.2), 5 μmol AT7519处理组为(71.7±7.1)、(1615±77.9)、(147.8±11.5), 均显著低于模型组(P<0.05)且效果呈剂量依赖性。小鼠四氯化碳急性肝损伤模型中, 模型组小鼠血清ALT、AST水平分别为(1116±197.0)U/L、(966±157.3)U/L, AT7519干预组ALT、AST水平分别为(230.6±103.1)U/L、(247.8±110.7)U/L, 差异有统计学意义(P<0.05)。组织病理分析结果显示, 模型组肝组织可见肝细胞大片坏死及炎性细胞浸润, 干预组小鼠上述病理变化均明显减轻。此外, 干预组小鼠肝组织促炎细胞因子IL-6、IL-1β和TNF-α的mRNA水平为(1.6±0.7)、(1.6±0.5)、(2.1±0.9)较模型组的(10.7±5.3)、(4.6±1.5)、(16.4±3.0)均显著下调(P<0.05)。结论 AT7519对四氯化碳诱导的小鼠急性肝损伤有显著保护作用, 其机制可能与减少炎性细胞浸润、抑制促炎细胞因子表达有关。

关键词: 四氯化碳, 急性肝损伤, 细胞周期蛋白依赖性激酶抑制剂, 炎性细胞, 细胞因子

Abstract: Objective To study on the hepatoprotective effect AT7519, a cyclin-dependent kinase (CDK) inhibitor, on carbon tetrachloride (CCl4) induced acute liver injury in mice.Methods Lipopolysaccharide (LPS)-induced RAW264.7 cell inflammation model was used for the in vitro study. RAW264.7 cells were pretreated with different concentrations of AT7519 (20uM, 10uM, and 5uM) for 1h followed by LPS stimulation for 4 h. The effect of AT7519 on the expression of proinflammatory cytokines were then evaluated with quantitative reverse transcription PCR (RT-qPCR) analysis. For the in vivo study, CCl₄^- induced acute liver injury model in mice was employed. Thirty C57BL/6 mice were randomly divided into three groups including a normal control group, a model group, and an AT7519 treated group. The levels of serum transaminases were investigated, along with hepatic histopathological evaluation with H&E and immunohistochemistry staining. The levels of proinflammatory cytokine expression were tested by RT-qPCR. Results RT-qPCR results of the in vitro experiment revealed that the mRNA expression levels of IL-6, IL-1β and TNF-? were markedly increased after LPS stimulation (299.8±6.1, 8198±360.1, 172.0±5.9, respectively) when compared with the normal control group (1.1±0.1, 1.1±0.0, 1.0±0.0, respectively), of which the changes could be reversed by AT7519 treatment. The relative expression of IL-6, IL-1β and TNF-α mRNA in 20uM AT7519 treated RAW264.7 cells were 6.5±0.4, 275.2±10.4, 41.5±1.0, in 10uM AT7519 treated RAW264.7 cells were 36.7±5.4, 1585±26.0, 106.2±12.2, and in 5uM AT7519 treated RAW264.7 cells were 71.7±7.1, 1615±77.9, 147.8±11.5, respectively, which were dose-dependently lower than those in the model group (P<0.05). In the murine model of CCl4-induced acute liver injury, the serum levels of ALT and AST presented a sharp increase in the model group [ (1116±197.0) U/L and (966±157.3) U/L], while AT7519 administration [ (230.6±103.1) U/L and (247.8±110.7) U/L] brought about a significant reduction in serum transaminase levels (P<0.05). As for liver histopathology, AT7519 treatment alleviated the pathologic changes in the livers of mice in model group, including massive hepatocytes necrosis and inflammatory cells infiltration. Moreover, the mRNA expression levels of IL-6, IL-1β and TNF-α in the livers were dramatically reduced after AT7519 treatment (1.6±0.7, 1.6±0.5, and 2.1±0.9, respectively) when compared with those of the model group (10.7±5.3, 4.6±1.5 and 16.4±3.0, respectively) (P<0.05).Conclusion AT7519 demonstrated a promising hepatoprotective effect against CCl4-induced acute liver injury in mice. The underlying mechanisms may correlate with a reduction of inflammatory cells infiltration and an inhibition of inflammatory cytokines expression.

Key words: Carbon tetrachloride, Acute liver injury, Cyclin-dependent kinase inhibitor, Inflammatory cells, Cytokine

刘彦君, 黄鹤鸣, 付荣, 石翠翠, 范建高, 张元元, 罗成, 李光明. CDK抑制剂(AT7519)对小鼠急性肝损伤的保护作用[J]. 肝脏, 2021, 26(4): 375-378.

LIU Yan-jun, HUANG He-ming, FU Rong, SHI Cui-cui, FAN Jian-gao, ZHANG Yuan-yuan, LUO Cheng, LI Guang-ming. Protective effect of cyclin-dependent kinase inhibitor AT7519 on carbon tetrachloride induced acute liver injury in mice[J]. Chinese Hepatolgy, 2021, 26(4): 375-378.

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