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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

引用本文:
Citation:

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
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)

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

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
  • 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。

     

  • 图  1  动物造模及干预流程

    Figure  1.  Flow chart of animal modeling and intervention

    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

    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

    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

    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

    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)

    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)

    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)

    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±4.401) 2.68±0.561) 0.055±0.0101) 11.41±2.361) 2.86±0.371) 1.57±0.301)
    HFHC+NLRP3-KD 10 43.40±5.722) 1.82±0.572) 0.041±0.0092) 10.30±2.37 2.09±0.542) 0.98±0.413)
    F 70.03 32.89 11.46 41.91 80.50 52.33
    P <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±21.651) 33.36±2.961)
    HFHC+NLRP3-KD 10 81.45±16.532) 20.72±0.902)
    F 25.82 248.6
    P <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±71.661) 3.00±0.001) 2.67±0.521) 2.00±0.001) 7.67±0.521)
    HFHC+NLRP3-KD 10 67.15±47.822) 1.83±0.753) 1.33±0.523) 1.00±0.633) 4.17±1.723)
    F 17.39 92.06 73.33 55.00 101.80
    P <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±2.561) 2.01±0.611) 2.52±2.011) 2.62±1.471)
    HFHC+NLRP3-KD 10 3.19±1.002) 1.24±0.512) 1.32±0.412) 1.51±0.582)
    F 29.09 13.10 5.73 12.93
    P <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
    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±0.281) 0.89±0.231) 3.63±0.982) 4.08±0.532)
    HFHC+NLRP3-KD 10 0.61±0.16 0.56±0.183) 2.17±1.473) 2.22±0.683)
    F 5.65 4.53 23.51 9.23
    P 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
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