ADAM8的表达对急性酒精性肝损伤小鼠的影响

doi:10.11958/20231741

天津医药 ›› 2024, Vol. 52 ›› Issue (4): 346-349.doi: 10.11958/20231741

ADAM8的表达对急性酒精性肝损伤小鼠的影响

杨孟利(), 李三强^△(), 张凯杰, 崔钦奕, 冯家阳, 李依林, 李昊远, 齐婧含

河南科技大学基础医学与法医学院（邮编471000）

收稿日期:2023-11-11 修回日期:2023-12-07 出版日期:2024-04-15 发布日期:2024-04-19
通讯作者: ^△E-mail：sanqiangli2001@163.com
作者简介:杨孟利（1999），女，硕士在读，主要从事肝损伤与修复分子机制研究。E-mail：2186374936@qq.com
基金资助:
国家自然科学基金资助项目(82170606);河南省高等学校重点科研项目计划基础研究专项(23ZX006)

The effect of ADAM8 expression on acute alcoholic liver injury in mice

YANG Mengli(), LI Sanqiang^△(), ZHANG Kaijie, CUI Qinyi, FENG Jiayang, LI Yilin, LI Haoyuan, QI Jinghan

College of Basic Medicine and Forensic Medicine, Henan University of Science and Technology, Luoyang 471000, China

Received:2023-11-11 Revised:2023-12-07 Published:2024-04-15 Online:2024-04-19
Contact: ^△E-mail：sanqiangli2001@163.com

杨孟利, 李三强, 张凯杰, 崔钦奕, 冯家阳, 李依林, 李昊远, 齐婧含. ADAM8的表达对急性酒精性肝损伤小鼠的影响[J]. 天津医药, 2024, 52(4): 346-349.

YANG Mengli, LI Sanqiang, ZHANG Kaijie, CUI Qinyi, FENG Jiayang, LI Yilin, LI Haoyuan, QI Jinghan. The effect of ADAM8 expression on acute alcoholic liver injury in mice[J]. Tianjin Medical Journal, 2024, 52(4): 346-349.

摘要/Abstract

摘要：

目的探究CRISPR/Cas9技术靶向抑制去整合素-金属蛋白酶8（ADAM8）在小鼠急性酒精性肝损伤中的作用及分子机制。方法 18只6~8周龄健康雌性昆明小鼠根据随机数字表法分为正常组、酒精组和质粒组，每组6只。正常组不做处理，质粒组采用水流动力学注射法尾静脉注射利用CRISPR/Cas9技术抑制ADAM8基因的三合一重组质粒ADAM8-sgRNA3（3 g/kg），酒精组尾静脉注射等量生理盐水。酒精组和质粒组采用50%（V/V）分析纯乙醇一次性灌胃（14 mL/kg）诱导急性肝损伤。禁食16 h后进行眼球取血，检测丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）活性；苏木素-伊红（HE）和过碘酸-雪夫（PAS）染色检测肝脏损伤和糖原变化情况；蛋白免疫印迹法检测肝组织中ADAM8、细胞色素CYP450 2E1（CYP2E1）、热休克蛋白70（HSP70）及丝裂原活化蛋白激酶（MAPK）通路相关因子的表达。结果与正常组相比，酒精组ALT和AST升高，肝损伤评分增加，糖原含量减少，ADAM8、CYP2E1、磷酸化的细胞外调节蛋白激酶1/2（p-ERK1/2）、磷酸化的p38（p-p38）MAPK以及磷酸化的c-Jun氨基未端激酶（p-c-Jun）的表达升高，而HSP70的表达降低（P<0.05）。与酒精组相比，质粒组ALT和AST降低，肝损伤评分降低（P<0.05），糖原含量增多；ADAM8、CYP2E1、p-ERK1/2、p-p38 MAPK以及p-c-Jun的表达降低，HSP70的表达增加（P<0.05）。结论利用CRISPR/Cas9技术靶向抑制ADAM8的表达，可以通过MAPK信号通路改善小鼠急性酒精性肝损伤。

关键词: ADAM蛋白质类, 急性酒精性肝损伤, 丝裂原激活蛋白激酶类, CRISPR/Cas9

Abstract:

Objective To investigate the role and molecular mechanism of CRISPR/Cas9 technology targeting the inhibition of disintegrin and metalloprotease 8 (ADAM8) in acute alcoholic liver injury in mice. Methods Eighteen healthy Kunming female mice aged 6-8 weeks were randomly divided into the normal group, the alcohol group and the plasmid group, with 6 mice in each group. The normal group was given no treatment, and the plasmid group was injected with three-in-one recombinant plasmid ADAM8-sgRNA3 (3 g/kg) by CRISPR/Cas9 technology to inhibit the ADAM8 gene using hydrodynamic injection tail vein method. The alcohol group was injected with an equal amount of physiological saline through tail vein. After 3 days of regular feeding, the alcohol group and the plasmid group were subjected with 50% (V/V) alcohol analytical grade ethanol by a one-time gavage (14 mL/kg) to induce acute liver injury. After fasting for 16 hours, eyeball blood sample was taken to detect activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Mice were euthanized and liver tissue was separated and extracted. Hematoxylin-Eosin staining (HE staining) and Periodic Acid-Schiff staining (PAS staining) were used to detect liver injury and glycogen changes in each group of mice. The expression levels of ADAM8, cytochrome CYP450 2E1 (CYP2E1), heat shock protein 70 (HSP70) and mitogen-activated protein kinase (MAPK) pathway related factors were detected by Western blot assay. Results Compared with the normal group, ALT and AST were increased, liver injury score was increased, glycogen content was decreased, ADAM8, CYP2E1, phosphorylated extracellular regulated kinase 1/2 (p-ERK1/2), phosphorylated p38 (p-p38) MAPK and phosphorylated c-Jun aminoterminal kinase (p-c-Jun) were increased in the alcohol group. The expression of HSP70 was decreased (P<0.05). Compared with alcohol group, ALT and AST were decreased, liver injury score was decreased in the plasmid group (P<0.05). Glycogen content was increased. ADAM8, CYP2E1, p-ERK1/2, p-P38 MAPK and p-c-Jun expression levels were decreased, while HSP70 expression was increased (P<0.05). Conclusion Targeted inhibition of ADAM8 expression by CRISPR/Cas9 technology can improve acute alcoholic liver injury in mice through MAPK signaling pathway.

Key words: ADAM proteins, acute alcoholic liver injury, mitogen-activated protein kinases, CRISPR/Cas9

中图分类号:

R364.5

图/表 5

表1 各组小鼠肝损伤评分和糖原光密度比值比较（$\bar{x}±s$）

Tab.1 Comparison of liver injury scores and glycogen optical density ratio in liver tissue of mice between three groups

组别	n	肝损伤评分/分	糖原光密度比值
正常组	6	0.00±0.00	1.00±0.60
酒精组	6	5.00±2.00^a	0.00±0.00^a
质粒组	6	1.67±0.58^b	0.04±0.00^a
F		13.460^＊＊	7.911^＊

图1 各组小鼠肝脏组织病理学变化（×200）

Fig.1 Pathological changes in liver tissue of mice in each groups (×200)

表2 3组小鼠血清ALT和AST的活性比较（$\bar{x}±s$）

Tab.2 Comparison of the activity of serum ALT and AST between three groups of mice

组别	n	ALT/（U/L）	AST/（U/L）
正常组	6	13.09±2.05	16.78±1.97
酒精组	6	24.48±5.88^a	38.54±13.36^a
质粒组	6	15.40±1.63^b	18.71±6.14^b
F		15.750^＊＊	11.870^＊＊

表3 3组小鼠肝组织ADAM8、CYP2E1、HSP70、p-ERK1/2、p-p38以及p-c-Jun的蛋白表达比较（n=6，$\bar{x}±s$）

Tab.3 Comparison of expression levels of ADAM8, CYP2E1, HSP70, p-ERK1/2, p-p38 and p-c-Jun in liver tissue of mice between three groups

组别	ADAM8	CYP2E1	HSP70
正常组	1.00±0.00	1.00±0.00	1.00±0.00
酒精组	1.74±0.32^a	2.30±0.58^a	0.50±0.08^a
质粒组	0.69±0.30^b	0.77±0.55^b	0.71±0.11^b
F	13.540^＊＊	9.690^＊	30.240^＊＊
组别	p-ERK1/2	p-p38	p-c-Jun
正常组	1.00±0.00	1.00±0.00	1.00±0.00
酒精组	1.45±0.12^a	2.06±0.50^a	1.71±0.25^a
质粒组	0.84±0.16^b	1.10±0.42^b	1.19±0.11^b
F	22.170^＊＊	7.350^＊	16.330^＊＊

图2 3组小鼠肝组织中ADAM8、CYP2E1、HSP70、p-ERK1/2、p-p38以及p-c-Jun的表达情况

Fig.2 Expression of ADAM8, CYP2E1, HSP70, p-ERK1/2, p-p38 and p-c-Jun proteins in liver tissue of mice in three groups of mice

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ADAM8的表达对急性酒精性肝损伤小鼠的影响

The effect of ADAM8 expression on acute alcoholic liver injury in mice

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