阿米替林对非酒精性脂肪性肝病细胞模型脂质沉积及生化代谢的影响

刘勤; 张强; 吴方雄; 闫蓉; 李蓉; 王佳; 牛春燕

doi:10.3969/j.issn.1001-5256.2021.01.020

阿米替林对非酒精性脂肪性肝病细胞模型脂质沉积及生化代谢的影响

DOI: 10.3969/j.issn.1001-5256.2021.01.020

刘勤¹,
张强¹,
吴方雄²,
闫蓉²,
李蓉²,
王佳²,
牛春燕^{2, 3, ,}

1.
厦门大学医学院，福建厦门 361102
2.
西安医学院第一附属医院消化内科，西安 710077
3.
南京市溧水区人民医院，东南大学附属中大医院溧水分院消化内科，南京 211200

基金项目:

陕西省科技厅重点研发计划 (2017SF-274)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突，特此声明。

作者贡献声明：刘勤负责课题设计与实验实施，资料收集整理，数据分析，撰写论文；张强参与实验实施，资料收集整理；李蓉负责实验质量控制及论文修改；吴方雄、闫蓉、王佳负责文献收集与整理；牛春燕进行课题设计，审核论文，并对论文负责。

详细信息

作者简介:
刘勤(1995—)，女，主要从事肝脏疾病方面的研究

通信作者:
牛春燕，415606772@qq.com

西安医学院第一附属医院与南京市溧水区人民医院(东南大学附属中大医院溧水分院)为共同第一通信单位

中图分类号: R575.5
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出版历程
- 收稿日期: 2020-06-11
- 录用日期: 2020-08-28
- 出版日期: 2021-01-20

Effect of amitriptyline on lipid deposition and biochemical metabolism in a cell model of nonalcoholic fatty liver disease

1.
School of Medicine, Xiamen University, Xiamen, Fujian 361102, China
2.
Department of Gastroenterology, The First Affiliated Hospital of Xi′an Medical University, Xi′an 710077, China
3.
Nanjing Lishui People's Hospital; Department of Gastroenterology, Lishui Branch of Zhongda Hospital Affiliated to Southeast University, Nanjing 211200, China

摘要

摘要: 目的探讨阿米替林通过调节酸性鞘磷脂酶(ASM)/神经酰胺(CE)通路对非酒精性脂肪性肝病(NAFLD)细胞模型脂质及生化代谢的影响。方法体外培养HepG2和L02细胞构建NAFLD细胞模型，MTT比色法测定细胞增殖率，油红O染色观察细胞内脂滴变化。实验分组：正常对照组、模型组、Ami组、TNFα组、Ami+TNFα组。全自动生化分析仪检测细胞内TG、TC及细胞上清液ALT、AST水平，ELISA法检测细胞内总的CE、ASM水平，Western Blot检测细胞内ASM蛋白的表达；实时荧光定量PCR检测细胞内ASM mRNA水平的表达。计量资料多组间比较采用单因素方差分析，进一步两两比较采用Turkey检验。结果与正常对照组相比，NAFLD模型组ASM蛋白和mRNA表达量以及CE、TG、TC、ALT、AST水平明显升高(P值均＜0.05)；与模型组相比，Ami组ASM蛋白和mRNA表达量以及CE、TG、TC、ALT、AST水平明显降低(P值均＜0.05)，TNFα组ASM蛋白和mRNA表达量以及CE、TG、ALT、AST水平明显升高(P值均＜0.05)；与TNFα组比较，Ami+TNFα组ASM蛋白和mRNA表达量以及CE、TG、TC、ALT、AST水平明显降低(P值均＜0.05)。结论 ASM/CE通路促进脂质积聚、导致脂肪变，阿米替林可通过抑制该通路改善NAFLD肝细胞的脂质沉积。
- 非酒精性脂肪性肝病 /
- 阿米替林 /
- 磷脂酶类 /
- 神经酰胺类 /
- 脂质贮积病
Abstract: Objective To investigate the effect of amitriptyline on lipid deposition and biochemical metabolism in a cell model of nonalcoholic fatty liver disease (NAFLD) by regulating the acid sphingomyelinase (ASM)/ceramide (CE) pathway. Methods HepG2 and L02 cells were cultured in vitro to establish a cell model of NAFLD. MTT colorimetry was used to measure cell proliferation rate, and oil red O staining was used to observe the change of lipid droplets in cells. In the experiment, the cells were divided into normal control group, model group, Ami group, TNFα group, and Ami+TNFα group. An automatic biochemical analyzer was used to measure the levels of triglyceride (TG) and total cholesterol (TC) in cells and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in supernatant; ELISA was used to measure the levels of CE and ASM in cells; Western blot was used to measure the protein expression ASM in cells, and RT-PCR was used to measure the mRNA expression of ASM in cells. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Turkey test was used for further comparison between two groups. Results Compared with the normal control group, the NAFLD model group had significant increases in the protein and mRNA expression of ASM and the levels of CE, TG, TC, ALT, and AST (all P < 0.05). Compared with the model group, the Ami group had significant reductions in the protein and mRNA expression of ASM and the levels of CE, TG, TC, ALT, and AST (all P < 0.05), and the TNFα group had significant increases in the protein and mRNA expression of ASM and the levels of CE, TG, ALT, and AST (all P < 0.05). Compared with the TNFα group, the Ami+TNFα group had significant reductions in the protein and mRNA expression of ASM and the levels of CE, TG, TC, ALT, and AST (all P < 0.05). Conclusion The ASM/CE pathway promotes lipid accumulation and may lead to hepatocyte steatosis, and amitriptyline can alleviate lipid deposition in NAFLD hepatocytes by inhibiting the ASM/CE pathway.
- Non-alcoholic Fatty Liver Disease /
- Amitriptyline /
- PhospHolipases /
- Ceramides /
- Lipidoses

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图 1 油红O染色结果(×200)

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图 2 不同浓度Ami干预HepG2和L02细胞的油红O染色结果(×200)

下载: 全尺寸图片幻灯片

图 3 各组细胞ASM蛋白的表达水平

下载: 全尺寸图片幻灯片

表 1 不同浓度FFA干预细胞的OD值和细胞增殖率

FFA浓度(mmol/L)	HepG2		L02
FFA浓度(mmol/L)	OD值	增殖率(%)	OD值	增殖率(%)
0	0.755±0.038	100.00	0.861±0.046	100.00
0.125	0.744±0.038	98.45	0.872±0.028	101.37
0.25	0.727±0.030	95.90	0.838±0.027	97.05
0.5	0.676±0.038	88.20	0.839±0.032	97.17
1	0.645±0.044¹⁾	83.60	0.729±0.033	91.05
2	0.561±0.012¹⁾	71.00	0.740±0.025¹⁾	84.38
F值	13.472		6.930
P值	＜0.05		＜0.05
注：1)与空白组比较，P＜0.05。

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表 2 正常对照组和NAFLD模型组细胞内TG、TC、FFA含量比较

组别	HepG2			L02
组别	TG(mmol/g prot)	TC(mmol/g prot)	FFA(mmol/g prot)	TG(mmol/g prot)	TC(mmol/g prot)	FFA(mmol/g prot)
正常对照组	0.142±0.011	0.171±0.033	55.009±6.285	0.117±0.013	0.151±0.018	44.906±4.881
NAFLD模型组	0.541±0.056	0.633±0.045	224.585±25.600	0.370±0.055	0.458±0.052	196.491±14.846
t值	10.398	10.749	9.215	6.64	8.799	15.362
P值	0.009	0.009	0.012	0.022	0.013	0.004

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表 3 不同浓度阿米替林干预脂肪变细胞的OD值和增殖率

组别	HepG2		L02
组别	OD值	增殖率(%)	OD值	增殖率(%)
正常对照组	0.814±0.045	100.00	0.950±0.027	100.00
NAFLD模型组	0.727±0.040	88.11	0.892±0.043	93.34
Ami 25 μmol/L	0.741±0.040	90.08	0.887±0.026	92.80
Ami 50 μmol/L	0.755±0.036	91.91	0.907±0.031	95.07
Ami 100 μmol/L	0.778±0.040	95.06	0.930±0.023	97.73
F值	2.139		2.188
P值	＞0.05		＞0.05

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表 4 各组HepG2细胞内TG、TC、FFA水平比较

组别	TG(mmol/g prot)	TC(mmol/g prot)	FFA(mmol/g prot)
正常对照组	0.153±0.010	0.170±0.012	65.350±11.407
NAFLD模型组	0.566±0.030¹⁾	0.631±0.043¹⁾	266.266±18.112¹⁾
Ami 25 mmol/L	0.553±0.029	0.622±0.020	268.887±8.053
Ami 50 mmol/L	0.362±0.054²⁾	0.506±0.042²⁾	204.097±11.280²⁾
Ami 100 mmol/L	0.233±0.020²⁾	0.342±0.024²⁾	166.003±12.837²⁾
F值	99.124	124.967	125.931
P值	＜0.05	＜0.05	＜0.05
注：1)与正常对照组比较，P＜0.05；2)与NAFLD模型组比较，P＜0.05。

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表 5 各组L02细胞内TG、TC、FFA水平比较

组别	TG(mmol/g prot)	TC(mmol/g prot)	FFA(mmol/g prot)
正常对照组	0.127±0.015	0.139±0.012	48.600±6.942
NAFLD模型组	0.384±0.030¹⁾	0.504±0.044¹⁾	212.226±13.089¹⁾
Ami 25 mmol/L	0.363±0.016	0.458±0.016	210.774±14.166
Ami 50 mmol/L	0.254±0.019²⁾	0.380±0.026²⁾	152.150±8.900²⁾
Ami 100 mmol/L	0.205±0.030²⁾	0.282±0.025²⁾	122.543±11.284²⁾
F值	66.778	90.103	111.315
P值	＜0.05	＜0.05	＜0.05
注：1)与正常对照组比较，P＜0.05；2)与NAFLD模型组比较，P＜0.05。

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表 6 各组细胞ASM的mRNA相对表达量

组别	HepG2	L02
正常对照组	1	1
NAFLD模型组	2.069±0.124¹⁾	1.847±0.386¹⁾
Ami组	1.462±0.379²⁾	1.321±0.364
TNFα组	2.685±0.367²⁾	2.708±0.195²⁾
Ami+TNFα组	1.888±0.493³⁾	1.754±0.373³⁾
F值	11.28	13.653
P值	＜0.05	＜0.05
注：1)与正常对照组比较，P＜0.05；2)与NAFLD模型组比较，P＜0.05；3)与TNFα组比较，P＜0.05。

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表 7 各组细胞的ELISA检测结果(OD值)

组别	HepG2		L02
组别	ASM	CE	ASM	CE
正常对照组	0.338±0.004	0.104±0.003	0.312±0.005	0.100±0.002
NAFLD模型组	0.667±0.013¹⁾	0.174±0.003¹⁾	0.571±0.003¹⁾	0.157±0.003¹⁾
Ami组	0.343±0.006²⁾	0.116±0.006²⁾	0.331±0.005²⁾	0.114±0.005²⁾
TNFα组	0.791±0.004²⁾	0.220±0.004²⁾	0.670±0.002²⁾	0.187±0.004²⁾
Ami+TNFα组	0.453±0.003³⁾	0.130±0.003³⁾	0.393±0.004³⁾	0.120±0.007³⁾
F值	2594.967	450.127	5098.961	194.498
P值	＜0.05	＜0.05	＜0.05	＜0.05
注：1)与正常对照组比较，P＜0.05；2)与NAFLD模型组比较，P＜0.05；3)与TNFα组比较，P＜0.05。

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表 8 各组HepG2细胞的生化指标比较

组别	TG(mmol/g prot)	TC(mmol/g prot)	FFA(mmol/g prot)	ALT(U/L)	AST(U/L)
正常对照组	0.159±0.022	0.169±0.013	63.498±6.788	7.701±0.516	6.379±0.865
NAFLD模型组	0.534±0.071¹⁾	0.640±0.068¹⁾	255.506±28.882¹⁾	28.566±2.721¹⁾	23.558±4.083¹⁾
Ami组	0.209±0.217²⁾	0.194±0.021²⁾	123.956±12.264²⁾	9.247±1.513²⁾	8.068±1.623²⁾
TNFα组	0.720±0.055²⁾	0.729±0.033	302.303±17.738²⁾	34.552±3.905²⁾	29.409±3.022²⁾
Ami+TNFα组	0.262±0.041³⁾	0.303±0.066³⁾	166.610±17.608³⁾	14.604±3.507³⁾	10.272±1.686³⁾
F值	80.053	95.042	85.008	57.547	49.669
P值	＜0.05	＜0.05	＜0.05	＜0.05	＜0.05
注：1)与正常对照组比较，P＜0.05；2)与NAFLD模型组比较，P＜0.05；3)与TNFα组比较，P＜0.05。

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表 9 各组L02细胞的生化指标比较

组别	TG(mmol/g prot)	TC(mmol/g prot)	FFA(mmol/g prot)	ALT(U/L)	AST(U/L)
正常对照组	0.130±0.015	0.141±0.011	50.665±5.181	6.154±0.757	7.131±0.948
NAFLD模型组	0.402±0.040¹⁾	0.503±0.030¹⁾	211.240±17.370¹⁾	12.201±3.156¹⁾	18.214±2.153¹⁾
Ami组	0.157±0.020²⁾	0.171±0.018²⁾	106.681±11.549²⁾	6.231±0.857²⁾	8.274±1.439²⁾
TNFα组	0.553±0.038²⁾	0.615±0.064²⁾	271.230±12.798²⁾	18.152±1.806²⁾	25.549±2.992²⁾
Ami+TNFα组	0.213±0.023³⁾	0.234±0.025³⁾	145.189±18.377³⁾	7.917±1.538³⁾	9.271±1.694³⁾
F值	116.301	113.124	116.704	23.211	48.842
P值	＜0.05	＜0.05	＜0.05	＜0.05	＜0.05
注：1)与正常对照组比较，P＜0.05；2)与NAFLD模型组比较，P＜0.05；3)与TNFα组比较，P＜0.05。

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