NLRP3基因敲减对高脂高糖饮食诱导的非酒精性脂肪性肝炎小鼠模型的影响

黄倩; 王卓媛; 安梓铭; 辛鑫; 孙沁梅; 苟小军; 胡义扬; 冯琴

doi:10.12449/JCH240514

NLRP3基因敲减对高脂高糖饮食诱导的非酒精性脂肪性肝炎小鼠模型的影响

DOI: 10.12449/JCH240514

黄倩^{1, b, 3, 4},
王卓媛^{1, b, 3, 4},
安梓铭^{1, b, 3, 4},
辛鑫^{1, 3, 4},
孙沁梅^{1, 3, 4},
苟小军⁵,
胡义扬^{1, 3, 4},
冯琴^{1, b, 3, 4, , ,}

1a.
上海中医药大学附属曙光医院肝病研究所，上海 201203
1b.
上海中医药大学附属曙光医院科技实验中心，上海 201203
2.
上海市中医临床重点实验室，上海 201203
3.
肝肾疾病病证教育部重点实验室，上海 201203
4.
上海市宝山区中西医结合医院，上海 201900

基金项目:

国家自然科学基金面上项目 (82174040);

上海中医药大学关键领域优秀博士培育项目 (2-089);

上海市宝山区科学技术委员会医学卫生项目 (21-E-63)

伦理学声明： 本研究方案于2021年9月3日经由上海中医药大学实验动物伦理委员会审批，批号：PZSHUTCM210903007，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：黄倩负责设计论文框架，起草论文；黄倩、王卓媛负责实验操作，研究过程的实施；黄倩、安梓铭负责数据收集，统计学分析，绘制图表；冯琴、辛鑫、孙沁梅负责论文修改；冯琴、胡义扬、苟小军负责拟定写作思路；冯琴指导撰写文章并最后定稿。

详细信息

通信作者:
冯琴， fengqin@shutcm.edu.cn （ORCID： 0000-0002-4641-1636）

计量
- 文章访问数: 308
- HTML全文浏览量: 102
- PDF下载量: 40
- 被引次数: 0
出版历程
- 收稿日期: 2023-08-15
- 录用日期: 2023-08-31
- 出版日期: 2024-05-25

Effect of NOD‑like receptor family pyrin domain containing 3 knockdown on a mouse model of nonalcoholic steatohepatitis induced by high-fat high-carbohydrate diet

Qian HUANG^{1, b, 3, 4},
Zhuoyuan WANG^{1, b, 3, 4},
Ziming AN^{1, b, 3, 4},
Xin XIN^{1, 3, 4},
Qinmei SUN^{1, 3, 4},
Xiaojun GOU⁵,
Yiyang HU^{1, 3, 4},
Qin FENG^{1, b, 3, 4
, ,
,}

1a.
Institute of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine Shanghai 201203，China
1b.
Experimental Center for Science and Technology,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine，Shanghai 201203，China
2.
Shanghai Key Laboratory of Traditional Chinese Clinical Medicine，Shanghai 201203，China
3.
Key Laboratory of Liver and Kidney Diseases,Ministry of Education，Shanghai 201203，China
4.
Baoshan District Hospital of Integrated Traditional Chinese and Western Medicine，Shanghai 201900，China

Research funding:

National Natural Science Foundation of China (82174040);

The Excellent Doctoral Projects in Key Fields of Shanghai University of Traditional Chinese Medicine (2-089);

District level Medical and Health Key Project of Shanghai Baoshan District Science (21-E-63)

More Information

Corresponding author: FENG Qin， fengqin@shutcm.edu.cn （ORCID：0000-0002-4641-1636）

摘要

摘要: 目的探讨NOD样受体热蛋白结构域相关蛋白3（NLRP3）基因敲减对高脂高糖诱导的非酒精性脂肪性肝炎（NASH）小鼠模型的影响。方法将44只小鼠随机分为正常饮食组（CON）20只，高脂高糖造模组（HFHC）24只。造模14周末，随机选取4只HFHC组小鼠进行腺相关病毒9（AAV9）尾静脉注射预实验，4周后验证NLRP3敲减模型是否成功。18周末确认敲减成功后，对剩余40只小鼠进行AAV9一次性尾静脉注射，分为CON+NLRP3敲减阴性对照组（CON+NLRP3-NC）、CON+NLRP3敲减组（CON+NLRP3-KD）、HFHC+NLRP3-NC及HHFHC+NLRP3-KD组，每组10只，继续造模6周。24周末取材观察炎症小体活化效应，检测小鼠体质量、肝质量、肝指数及糖代谢（空腹血糖、空腹胰岛素及HOMA-IR指数）指标；检测小鼠肝脂质含量（肝组织TG及油红O染色）、肝脏炎症（血清ALT活性、HE染色及炎症相关基因）及肝纤维化（天狼星红染色及纤维化相关基因）指标。计量资料多组间比较使用单因素方差分析，进一步两两比较采用LSD-t检验。结果与CON+NLRP3-NC组相比，Western Blot结果提示，HFHC+NLRP3-NC组的NLRP3、pro-Caspase1、Caspase1、ASC及IL-1β蛋白水平均升高，HFHC+NLRP3-KD组均降低（P值均<0.05）；HFHC+NLRP3-NC组小鼠体质量、肝质量、肝指数及糖代谢指标均有不同程度升高，HFHC+NLRP3-KD组均显著改善（P值均<0.05）；在肝脂肪沉积方面，与CON+NLRP3-NC组相比，HFHC+NLRP3-NC组肝脏TG明显增高，油红O染色显示大量红色脂滴，HFHC+NLRP3-KD组肝脏TG及肝脂滴数量显著减少（P值均<0.01）；在肝脏炎症方面，HFHC+NLRP3-NC组血清ALT，非酒精性脂肪性肝病活动度（NAS）评分及炎症相关基因均较CON+NLRP3-NC组明显升高，HFHC+NLRP3-KD组均明显降低（P值均<0.01）；在肝纤维化方面，HFHC+NLRP3-NC组肝胶原纤维面积以及纤维化相关基因均较CON+NLRP3-NC组明显升高，HFHC+NLRP3-KD组纤维化相关基因均明显降低（P值均<0.05），胶原纤维面积虽有降低趋势但差异无统计学意义（P>0.05）。结论 NLRP3基因敲减可显著改善高脂高糖饮食诱导的NASH小鼠模型肝脂肪沉积及炎症。
- 非酒精性脂肪性肝病 /
- NLR家族，热蛋白结构域包含蛋白3 /
- 膳食，高脂 /
- 炎症 /
- 小鼠，近交C57BL
Abstract: Objective To investigate the effect of NOD-like receptor family pyrin domain containing 3 （NLRP3） knockdown on a mouse model of nonalcoholic steatohepatitis （NASH） induced by high-fat high-carbohydrate （HFHC） diet. Methods A total of 44 mice were randomly divided into normal diet group （CON group） with 20 mice and HFHC group with 24 mice. At the end of week 14 of modeling， 4 mice were randomly selected from the HFHC group for the pre-experiment of adeno-associated virus （AAV） by tail vein injection， and NLRP3 knockdown was verified after 4 weeks. After NLRP3 knockdown was verified at the end of week 18， the remaining 40 mice were given a single tail vein injection of AAV， and then they were divided into CON+NLRP3 knockdown negative control group （CON+NLRP3-NC group）， CON+NLRP3 knockdown group （CON+NLRP3-KD group）， HFHC+NLRP3-NC group， and HFHC+NLRP3-KD group， with 10 mice in each group. At the end of week 24， the activation of NLRP3 inflammasome was observed； related indicators were measured， including body weight， liver weight， liver index， and glucose metabolism （fasting blood glucose， fasting insulin， and Homeostasis Model Assessment of Insulin Resistance ［HOMA-IR］ index）； the indicators of liver lipid content （liver triglyceride ［TG］ and oil red O staining）， liver inflammation （serum alanine aminotransferase ［ALT］ activity， HE staining， and inflammation-related genes）， and liver fibrosis （Sirius Red staining and fibrosis-related genes） were measured. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results Compared with the CON+NLRP3-NC group based on the results of Western Blot， the HFHC+NLRP3-NC group had significant increases in the protein expression levels of NLRP3， pro-Caspase1， Caspase1， ASC， and IL-1β， while the HFHC+NLRP3-KD group had significant reductions in these levels （all P<0.05）. The HFHC+NLRP3-NC group showed varying degrees of increase in body weight， liver weight， liver index， and glucose metabolism indicators， while the HFHC+NLRP3-KD group showed significant improvements in these indicators （all P<0.05）. As for hepatic fat deposition， compared with the CON+NLRP3-NC group， the HFHC+NLRP3-NC group had a significant increase in liver TG， with a large number of red lipid droplets shown by oil red O staining， and the HFHC+NLRP3-KD group had significant reductions in liver TG and the number of lipid droplets in the liver （all P<0.01）. In terms of liver inflammation， compared with the CON+NLRP3-NC group， the HFHC+NLRP3-NC group had significant increases in serum ALT， NAFLD activity score， and inflammation-related genes， while the HFHC+NLRP3-KD group had significant reductions in these indicators （all P<0.01）. As for liver fibrosis， compared with the CON+NLRP3-NC group， the HFHC+NLRP3-NC group had significant increases in collagen fiber area and fibrosis-related genes， and the HFHC+NLRP3-KD group had significant reductions in fibrosis-related genes （all P<0.05） and a tendency of reduction in collagen fiber area （P>0.05）. Conclusion NLRP3 knockdown can significantly improve hepatic fat deposition and inflammation in a mouse model of HFHC-induced NASH.
- Non-alcoholic Fatty Liver Disease /
- NLR Family， Pyrin Domain-Containing 3 Protein /
- Diet， High-Fat /
- Inflammation /
- Mice， Inbred C57BL

HTML全文

图 1 动物造模及干预流程

Figure 1. Flow chart of animal modeling and intervention

下载: 全尺寸图片幻灯片

图 2 NLRP3基因敲减对肝脏NLRP3蛋白表达的影响

Figure 2. Effect of NLRP3 knockdown on liver NLRP3 protein expression

下载: 全尺寸图片幻灯片

图 3 NLRP3基因敲减对肝脏NLRP3转录和蛋白水平的影响

Figure 3. Effects of NLRP3 knockdown on liver NLRP3 transcription and protein levels

下载: 全尺寸图片幻灯片

图 4 NLRP3基因敲减对炎症小体相关指标及下游IL-1β蛋白的影响

Figure 4. Effects of NLRP3 knockdown on inflammatory-related proteins and IL-1β protein

下载: 全尺寸图片幻灯片

图 5 NLRP3基因敲减对小鼠体质量的影响

Figure 5. Effects of NLRP3 knockdown on body weight

下载: 全尺寸图片幻灯片

图 6 NLRP3基因敲减对小鼠肝脏形态及肝脏脂滴的影响（×200）

Figure 6. Effects of NLRP3 knockdown on hepatic morphology and lipid droplets（×200）

下载: 全尺寸图片幻灯片

图 7 NLRP3基因敲减对小鼠肝组织病理的影响（×200）

Figure 7. Effects of NLRP3 knockdown on hepatic histopathology （×200）

下载: 全尺寸图片幻灯片

图 8 NLRP3基因敲减对小鼠肝组织胶原沉积的影响（×200）

Figure 8. Effects of NLRP3 knockdown on hepatic collagen deposition （×200）

下载: 全尺寸图片幻灯片

表 1 PCR引物序列

Table 1. PCR Primer sequences

基因	上游（5'-3'）	下游（5'-3'）
NLRP3	AACGACCCCTTCATTGAC	GAGGAAGAGGAGGAAGGACA
CXCL10	TCTCCATCACTCCCCTTTAC	TTCGGCAGTTACTTTTGTCTC
IL-1β	TACATCAGCACCTCACAAGC	AGAAACAGTCCAGCCCATACT
CCL2	ATCGGAACCAAATGAGATCAG	GCTTCAGATTTACGGGTCAAC
COL1α1	TGACTGGAAGAGCGGAGAGTA	GACGGCTGAGTAGGGAACAC
COL3α1	CCTGGAGCCCCTGGACTAATA	TCACCTGAAGGACCTCGTGTT
α-SMA	CTCTGCCTCTAGCACACAACT	CCACGAGTAACAAATCAAAGC
β-actin	CCTCTATGCCAACACAGT	AGCCACCAATCCACACAG
注：CXCL10，CXC趋化因子配体10；CCL2，趋化因子2；α-SMA，α平滑肌肌动蛋白；COL3α1，Ⅲ型胶原α1；COL1α1，Ⅰ型胶原α1；β-actin，β-肌动蛋白。

下载: 导出CSV

表 2 NLRP3基因敲减对小鼠末次肝质量、体质量、肝体比、FBG、FIN及HOMA-IR的影响

Table 2. Effects of NLRP3 knockdown on body weight， liver weight， liver/body weight ratio， FBG， FIN， and HOMA-IR

组别	动物数（只）	体质量（g）	肝质量（g）	肝体比	FBG（mg/dL）	FIN（ng/mL）	HOMA-IR
CON+NLRP3-NC	10	26.29±1.50	0.96±0.06	0.036±0.002	4.61±0.80	0.31±0.14	0.06±0.02
CON+NLRP3-KD	10	27.27±1.42	0.98±0.07	0.038±0.004	4.13±0.64	0.41±0.16	0.07±0.02
HFHC+NLRP3-NC	10	47.09±4.40^1）	2.68±0.56^1）	0.055±0.010^1）	11.41±2.36^1）	2.86±0.37^1）	1.57±0.30^1）
HFHC+NLRP3-KD	10	43.40±5.72^2）	1.82±0.57^2）	0.041±0.009^2）	10.30±2.37	2.09±0.54^2）	0.98±0.41^3）
F值		70.03	32.89	11.46	41.91	80.50	52.33
P值		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
注：与CON+NLRP3-NC组相比，1） P<0.01；与HFHC+NLRP3-NC组相比，2） P<0.01，3） P<0.05。

下载: 导出CSV

表 3 NLRP3基因敲减对小鼠肝脏TG含量和油红O染色面积百分比的影响

Table 3. Effects of NLRP3 knockdown on hepatic TG content and Oil red O staining area

组别	动物数（只）	肝脏TG（mg/g）	油红O染色面积（%）
CON+NLRP3-NC	10	45.65±12.97	3.82±0.80
CON+NLRP3-KD	10	54.47±20.14	1.51±0.82
HFHC+NLRP3-NC	10	119.80±21.65^1）	33.36±2.96^1）
HFHC+NLRP3-KD	10	81.45±16.53^2）	20.72±0.90^2）
F值		25.82	248.6
P值		<0.001	<0.001
注：与CON+NLRP3-NC组相比，1） P<0.01；与HFHC+NLRP3-NC组相比，2） P<0.01。

下载: 导出CSV

表 4 NLRP3基因敲减对小鼠血清ALT水平和肝组织NAS评分的影响

Table 4. Effects of NLRP3 knockdown on serum ALT and hepatic NAS score

组别	动物数（只）	ALT（U/L）	肝细胞脂肪变性（分）	小叶内炎症（分）	肝细胞气球样变（分）	NAS总分（分）
CON+NLRP3-NC	10	22.03±5.56	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
CON+NLRP3-KD	10	14.81±1.17	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
HFHC+NLRP3-NC	10	174.20±71.66^1）	3.00±0.00^1）	2.67±0.52^1）	2.00±0.00^1）	7.67±0.52^1）
HFHC+NLRP3-KD	10	67.15±47.82^2）	1.83±0.75^3）	1.33±0.52^3）	1.00±0.63^3）	4.17±1.72^3）
F值		17.39	92.06	73.33	55.00	101.80
P值		<0.001	<0.001	<0.001	<0.001	<0.001
注：与CON+NLRP3-NC组相比，1） P<0.01；与HFHC+NLRP3-NC组相比，2） P<0.05，3） P<0.01。

下载: 导出CSV

表 5 NLRP3基因敲减对小鼠肝脏炎症相关基因表达的影响

Table 5. Effects of NLRP3 knockdown on the expression of hepatic inflammation-related genes

组别	动物数（只）	IL-1β mRNA	IL-6 mRNA	CXCL10 mRNA	CCL2 mRNA
CON+NLRP3-NC	10	1.16±0.56	0.95±0.58	1.10±0.47	1.01±0.45
CON+NLRP3-KD	10	1.29±0.45	0.57±0.39	1.01±0.35	0.81±0.35
HFHC+NLRP3-NC	10	5.61±2.56^1）	2.01±0.61^1）	2.52±2.01^1）	2.62±1.47^1）
HFHC+NLRP3-KD	10	3.19±1.00^2）	1.24±0.51^2）	1.32±0.41^2）	1.51±0.58^2）
F值		29.09	13.10	5.73	12.93
P值		<0.001	<0.001	0.002	<0.001
注：与CON+NLRP3-NC组相比，1） P<0.01；与HFHC+NLRP3-NC组相比， 2） P<0.01。

下载: 导出CSV

表 6 NLRP3基因敲减对小鼠纤维化相关基因表达和肝脏胶原纤维的影响

Table 6. Effects of NLRP3 knockdown on the expression of fibrosis-related genes and hepatic collagen fiber

组别	动物数（只）	胶原纤维阳性面积百分比（%）	mRNA 相对表达量
组别	动物数（只）	胶原纤维阳性面积百分比（%）	α-SMA mRNA	COL3α1 mRNA	COL1α1mRNA
CON+NLRP3-NC	10	0.13±0.07	0.61±0.16	0.95±0.23	1.00±0.06
CON+NLRP3-KD	10	0.16±0.06	0.75±0.37	0.91±0.44	1.00±0.12
HFHC+NLRP3-NC	10	0.97±0.28^1）	0.89±0.23^1）	3.63±0.98^2）	4.08±0.53^2）
HFHC+NLRP3-KD	10	0.61±0.16	0.56±0.18^3）	2.17±1.47^3）	2.22±0.68^3）
F值		5.65	4.53	23.51	9.23
P值		0.006	0.007	<0.001	<0.001
注：与CON+NLRP3-NC组相比，1） P<0.05，2） P<0.01；与HFHC+NLRP3-NC组相比， 3） P<0.01。

下载: 导出CSV

参考文献(20)

[1]	YOUNOSSI ZM, KOENIG AB, ABDELATIF D, et al. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes[J]. Hepatology, 2016, 64( 1): 73- 84. DOI: 10.1002/hep.28431.
[2]	YOUNOSSI Z, ANSTEE QM, MARIETTI M, et al. Global burden of NAFLD and NASH: Trends, predictions, risk factors and prevention[J]. Nat Rev Gastroenterol Hepatol, 2018, 15( 1): 11- 20. DOI: 10.1038/nrgastro.2017.109.
[3]	DAY CP, JAMES OF. Steatohepatitis: A tale of two“hits”?[J]. Gastroenterology, 1998, 114( 4): 842- 845. DOI: 10.1016/s0016-5085(98)70599-2.
[4]	PRÓCHNICKI T, MANGAN MS, LATZ E. Recent insights into the molecular mechanisms of the NLRP3 inflammasome activation[J]. F1000Res, 2016, 5: F1000 Faculty Rev-1469. DOI: 10.12688/f1000research.8614.1.
[5]	WU XF, ZHANG F, XIONG X, et al. Tetramethylpyrazine reduces inflammation in liver fibrosis and inhibits inflammatory cytokine expression in hepatic stellate cells by modulating NLRP3 inflammasome pathway[J]. IUBMB Life, 2015, 67( 4): 312- 321. DOI: 10.1002/iub.1348.
[6]	YANG G, LEE HE, LEE JY. A pharmacological inhibitor of NLRP3 inflammasome prevents non-alcoholic fatty liver disease in a mouse model induced by high fat diet[J]. Sci Rep, 2016, 6: 24399. DOI: 10.1038/srep24399.
[7]	WEI Q, ZHU R, ZHU JY, et al. E2-induced activation of the NLRP3 inflammasome triggers pyroptosis and inhibits autophagy in HCC cells[J]. Oncol Res, 2019, 27( 7): 827- 834. DOI: 10.3727/096504018X15462920753012.
[8]	WANG L, HAUENSTEIN AV. The NLRP3 inflammasome: Mechanism of action, role in disease and therapies[J]. Mol Aspects Med, 2020, 76: 100889. DOI: 10.1016/j.mam.2020.100889.
[9]	CALCAGNO DM, CHU A, GAUL S, et al. NOD-like receptor protein 3 activation causes spontaneous inflammation and fibrosis that mimics human NASH[J]. Hepatology, 2022, 76( 3): 727- 741. DOI: 10.1002/hep.32320.
[10]	MRIDHA AR, WREE A, ROBERTSON AAB, et al. NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice[J]. J Hepatol, 2017, 66( 5): 1037- 1046. DOI: 10.1016/j.jhep.2017.01.022.
[11]	QU JW, YUAN ZQ, WANG GY, et al. The selective NLRP3 inflammasome inhibitor MCC950 alleviates cholestatic liver injury and fibrosis in mice[J]. Int Immunopharmacol, 2019, 70: 147- 155. DOI: 10.1016/j.intimp.2019.02.016.
[12]	LATZ E, XIAO TS, STUTZ A. Activation and regulation of the inflammasomes[J]. Nat Rev Immunol, 2013, 13( 6): 397- 411. DOI: 10.1038/nri3452.
[13]	NEGRIN KA, ROTH FLACH RJ, DISTEFANO MT, et al. IL-1 signaling in obesity-induced hepatic lipogenesis and steatosis[J]. PLoS One, 2014, 9( 9): e107265. DOI: 10.1371/journal.pone.0107265.
[14]	QIN WW, WENG JP. Hepatocyte NLRP3 interacts with PKCε to drive hepatic insulin resistance and steatosis[J]. Sci Bull(Beijing), 2023, 68( 13): 1413- 1429. DOI: 10.1016/j.scib.2023.06.003.
[15]	KOHLI R, KIRBY M, XANTHAKOS SA, et al. High-fructose, medium chain trans fat diet induces liver fibrosis and elevates plasma coenzyme Q9 in a novel murine model of obesity and nonalcoholic steatohepatitis[J]. Hepatology, 2010, 52( 3): 934- 944. DOI: 10.1002/hep.23797.
[16]	KLEINER DE, BRUNT EM, VAN NATTA M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease[J]. Hepatology, 2005, 41( 6): 1313- 1321. DOI: 10.1002/hep.20701.
[17]	VANDANMAGSAR B, YOUM YH, RAVUSSIN A, et al. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance[J]. Nat Med, 2011, 17( 2): 179- 188. DOI: 10.1038/nm.2279.
[18]	HUANG SL, WU YW, ZHAO ZH, et al. A new mechanism of obeticholic acid on NASH treatment by inhibiting NLRP3 inflammasome activation in macrophage[J]. Metabolism, 2021, 120: 154797. DOI: 10.1016/j.metabol.2021.154797.
[19]	BOARU SG, BORKHAM-KAMPHORST E, TIHAA L, et al. Expression analysis of inflammasomes in experimental models of inflammatory and fibrotic liver disease[J]. J Inflamm(Lond), 2012, 9( 1): 49. DOI: 10.1186/1476-9255-9-49.
[20]	CSAK T, GANZ M, PESPISA J, et al. Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells[J]. Hepatology, 2011, 54( 1): 133- 144. DOI: 10.1002/hep.24341.