[1]王换换,刘颖,李帅,等.肾素-血管紧张素系统2条通路在油酸致大鼠BRL-3A细胞非酒精性脂肪肝中的作用[J].南京农业大学学报,2020,43(5):910-918.[doi:10.7685/jnau.201906050]
 WANG Huanhuan,LIU Ying,LI Shuai,et al.Role of two pathways of renin-angiotensin system in oleic acid-induced non-alcoholic fatty liver disease in mouse BRL-3A cells[J].Journal of Nanjing Agricultural University,2020,43(5):910-918.[doi:10.7685/jnau.201906050]
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肾素-血管紧张素系统2条通路在油酸致大鼠BRL-3A细胞非酒精性脂肪肝中的作用()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
43卷
期数:
2020年5期
页码:
910-918
栏目:
动物科学
出版日期:
2020-09-15

文章信息/Info

Title:
Role of two pathways of renin-angiotensin system in oleic acid-induced non-alcoholic fatty liver disease in mouse BRL-3A cells
作者:
王换换 刘颖 李帅 何晟 朱斌 张源淑
南京农业大学农业农村部动物生理生化重点开放实验室, 江苏 南京 210095
Author(s):
WANG Huanhuan LIU Ying LI Shuai HE Sheng ZHU Bin ZHANG Yuanshu
Key Laboratory of Animal Physiology and Biochemistry and Rural Affairs, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
关键词:
血管紧张素系统(RAS)BRL-3A细胞非酒精性脂肪肝病(NAFLD)细胞模型
Keywords:
angiotensin system(RAS)BRL-3A cellsnon-alcoholic fatty liver disease(NAFLD)cell model
分类号:
S852.2
DOI:
10.7685/jnau.201906050
摘要:
[目的] 通过建立油酸诱导的大鼠肝脏间质BRL-3A细胞非酒精性脂肪肝(NAFLD)损伤模型,探讨BRL-3A细胞中RAS的ACE/AngⅡ/AT1R和ACE2/Ang-1-7/Mas这2条通路相互负向调节与NAFLD细胞损伤的关系。[方法] 采用不同浓度油酸(0.025、0.05、0.1和0.2 mmol·L-1)处理BRL-3A细胞24 h,通过检测细胞活力、细胞内甘油三酯(TG)含量、丙氨酸氨基转移酶(ALT)活性、天门冬氨酸氨基转移酶(AST)活性以及油红染色确立NAFLD细胞模型条件,然后选取低、中、高3个浓度(0.025、0.1和0.2 mmol·L-1)油酸作用于细胞24 h,用ELISA法检测细胞上清液中血管紧张素Ⅱ(AngⅡ)、血管紧张素1-7(Ang1-7)水平;Western blot分析细胞内血管紧张素转化酶(ACE)、血管紧张素转化酶2(ACE2)、血管紧张素Ⅱ受体1(AT1R)、Mas受体(MasR)蛋白水平;斯皮尔曼相关分析法分析油酸浓度与细胞内ACE、ACE2表达水平的相关性。[结果] 1)用0.1 mmol·L-1油酸处理BRL-3A细胞24 h,成功建立BRL-3A细胞NAFLD模型。2)油酸处理24 h后,BRL-3A细胞出现脂滴沉积,在0.025 mmol·L-1油酸(低浓度)组,ACE2、MasR表达水平和Ang1-7水平有升高趋势,AngⅡ水平与AT1R表达水平下降,ACE/ACE2值下降;0.1 mmol·L-1油酸(中浓度)组ACE、AT1R表达水平显著升高,其他RAS成员变化不明显;0.2 mmol·L-1油酸(高浓度)组,ACE2、MasR表达水平和Ang1-7水平下降,AngⅡ水平和ACE、AT1R表达水平及ACE/ACE2值升高。3)斯皮尔曼相关分析显示,油酸浓度、血液相关生化指标(TG含量、AST和ALT活性)和脂滴数及脂滴面积与ACE2表达水平和Ang1-7水平负相关,而与ACE表达水平和AngⅡ水平正相关。[结论] BRL-3A细胞中的2条轴共同参与了油酸致BRL-3A细胞NAFLD损伤的发生和发展过程。低浓度油酸时,ACE2介导的Ang1-7/MasR通路占优势;高浓度油酸处理细胞损伤加重,ACE介导的AngⅡ/AT1R通路占主导地位。ACE2与油酸致细胞炎性损伤的程度呈显著负相关关系。
Abstract:
[Objectives] The relationship between the negative regulation of ACE/AngⅡ/AT1R and ACE2/Ang-1-7/Mas pathways of RAS and the damage of non-alcoholic fatty liver disease (NAFLD) in mouse liver stromal cells (BRL-3A) was investigated by establi-shing injury model of NAFLD in mouse BRL-3A cells induced by oleic acid.[Methods] BRL-3A cells were treated with different concentrations (0.025,0.05,0.1 and 0.2 mmol·L-1) of oleic acid for 24 h. The cell viability assay,intracellular TG content,ALT and AST activities and oil red staining were used to confirm the NAFLD cell model. Then,three oleic acid concentrations (0.025,0.1 and 0.2 mmol·L-1) were applied to the cells for 24 h for subsequent experiments. The content of AngⅡ and Ang1-7 in the supernatant was detected by ELISA. The expression levels of angiotensin converting enzyme (ACE),angiotensin converting enzyme (ACE2),angiotensin type1 receptor (AT1R) and Mas receptor (MasR) in the cells were analyzed by Western blot. Meanwhile,Spearman correlation analysis was used to analyze the correlation between different concentrations of oleic acid treated for 24 h and the levels of intracellular ACE and ACE2.[Results] 1) Using 0.1 mmol·L-1 oleic acid to treat cells for 24,the NAFLD model of BRL-3A cells was successfully established. 2) Using oleic acid to treat cells for 24,lipid droplets were deposited in BRL-3A cells. The expression levels of ACE2,MasR and Ang1-7 content increased with 0.025 mmol·L-1 oleic acid,however,the expression level of AT1R,AngⅡ content and the ratio of ACE/ACE2 decreased. Besides,the expression levels of ACE and AT1R in the concentration of 0.1 mmol·L-1 group significantly increased,but the changes of other members in RAS were not significantly. On the contrary,the expression levels of ACE2,MasR and Ang1-7 content in 0.2 mmol·L-1 oleic acid (high concentration) group decreased,and the expression levels of ACE,AT1R and AngⅡ content and the ratio of ACE/ACE2 increased. 3) Spearman correlation analysis showed that the oleic acid content,blood-related biochemical indicators (TG content,ALT and AST activities) and lipid droplet number and lipid droplet area were negatively correlated with the expression level of ACE2 and Ang1-7 content,but positively correlated with the expression level of ACE and AngⅡ content.[Conclusions] All members of RAS exist in BRL-3A cells,and the two axes are involved in the development of NAFLD in oleic acid-induced BRL-3A cells. Moreover,the ACE2-mediated Ang1-7/MasR pathway dominates,with the low concentrations of oleic acid;the high concentration of oleic acid-treated cells aggravates the damage,and the ACE-mediated AngⅡ/AT1R pathway predominates. Therefore,there was a significant negative correlation between ACE2 and the extent of inflammatory damage induced by oleic acid.

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备注/Memo

备注/Memo:
收稿日期:2019-06-25。
基金项目:国家自然科学基金项目(30871838);江苏高校优势学科建设工程资助项目(PAPD)
作者简介:王换换,助理实验师。
通信作者:张源淑,教授,博士,主要从事动物营养和机能生物化学研究,E-mail:zhangyuanshu@njau.edu.cn。
更新日期/Last Update: 1900-01-01