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丹葛解酲汤对酒精性肝病大鼠模型的改善作用及其机制分析

郭苏军 周波 马丽

引用本文:
Citation:

丹葛解酲汤对酒精性肝病大鼠模型的改善作用及其机制分析

DOI: 10.12449/JCH251123
基金项目: 

宁夏自然科学基金 (2023AAC03210);

国家中医药管理局高水平中医药重点学科建设项目 (zyyzdxk-2023209);

2022年全国名老中医药专家传承工作室建设项目(国中医药人教函〔2022〕75号) 

伦理学声明:本研究方案经由宁夏医科大学实验动物伦理委员会审批,批号:IACUC-NYLAC-2023-288,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:郭苏军负责收集数据,资料分析,撰写论文;马丽参与课题设计,拟定写作思路;周波负责修改论文,指导撰写文章并最后定稿。
详细信息
    通信作者:

    马丽, mali3549@163.com (ORCID: 0000-0002-6646-3484)

The mechanism of Dange Jiecheng decoction improving alcoholic liver disease in a rat model

Research funding: 

Ningxia Natural Science Foundation (2023AAC03210);

Construction Project of High-Level Key Disciplines in Traditional Chinese Medicine by the National Administration of Traditional Chinese Medicine (zyyzdxk-2023209);

2022 National Famous Old Chinese Medicine Experts Inheritance Workstation Construction Project(Guo Zhongyiyao Renjiao Han [2022] No. 75) 

More Information
    Corresponding author: MA Li, mali3549@163.com (ORCID: 0000-0002-6646-3484)
  • 摘要:   目的  探讨丹葛解酲汤对酒精性肝病(ALD)大鼠的改善作用,并基于肿瘤蛋白P53/谷胱甘肽过氧化物酶4(GPX4)信号通路分析其机制。  方法  36只雄性SD大鼠随机分为正常组、模型组、丹葛解酲汤组和复方益肝灵片组,每组9只。采用梯度灌胃56%酒精建立模型,同时灌胃给药,持续12周。实验结束收集大鼠血清和肝组织,检测大鼠血清中ALT、AST和TG水平,肝组织中丙二醛(MDA)、活性氧(ROS)、谷胱甘肽(GSH)和Fe2+水平;HE染色观察病理组织变化;油红O染色和普鲁士蓝染色分别观察肝组织脂肪及铁沉积情况;采用RT-qPCR和Western Blot法检测肝组织中P53、GPX4、NOX1、PTGS2、FTH1 mRNA和蛋白表达。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LSD-t检验。  结果  与正常组相比,模型组肝细胞排列紊乱,结构不完整,有炎症细胞浸润和大量脂肪空泡;血清中AST、ALT、TG和肝组织MDA、ROS、Fe2+水平均升高(P值均<0.01),P53、NOX1、PTGS2 mRNA及蛋白表达均增加(P值均<0.01),血清GSH水平及肝组织GPX4、FTH1 mRNA及蛋白表达均降低(P值均<0.01)。与模型组相比,丹葛解酲汤组和复方益肝灵片组中大鼠肝细胞炎症减少,排列整齐,血清中AST、ALT、TG和肝组织MDA、ROS、Fe2+水平及P53、NOX1、PTGS2 mRNA及蛋白表达减少(P值均<0.05),GSH水平及肝组织GPX4、FTH1 mRNA和蛋白表达水平增加(P值均<0.05),肝组织脂肪变性改善和铁沉积减少(P值均<0.01)。  结论  丹葛解酲汤可有效改善ALD大鼠疾病进展,其机制可能与控调P53/GPX4通路抑制铁死亡有关。

     

  • 注: a,正常组;b,模型组;c,丹葛解酲汤组;d,复方益肝灵片组。

    图  1  ALD大鼠肝组织病理情况(HE染色,×100)

    Figure  1.  Liver tissue pathological sections in rats with alcoholic liver disease(HE,×100)

    图  2  ALD大鼠肝组织ROS水平(免疫荧光,×200)

    Figure  2.  ROS levels in liver tissue of rats with alcoholic liver disease (immunofluorescence,×200)

    注: a,正常组;b,模型组;c,丹葛解酲汤组;d,复方益肝灵片组。

    图  3  ALD大鼠肝组织脂质蓄积情况(油红O染色,×200)

    Figure  3.  Lipid accumulation in liver tissue of rats with alcoholic liver disease (oil red O staining, ×200)

    注: a,正常组;b,模型组;c,丹葛解酲汤组;d,复方益肝灵片组。

    图  4  ALD大鼠肝组织铁离子沉积情况(普鲁士蓝,×100)

    Figure  4.  Iron ion deposition in liver tissue of rats with alcoholic liver disease (prussian blue staining,×100)

    图  5  各组大鼠肝组织P53、GPX4、NOX1、PTGS2、FTH1蛋白表达情况

    Figure  5.  Protein expression of P53, GPX4, NOX1, PTGS2, and FTH1 in liver tissue of rats from each group

    表  1  引物序列

    Table  1.   Primer sequences

    基因 序列(5′→3′) 长度(bp)
    NOX1 F:TCCTAAACTACCGACTCTTCCTCA 141
    R:ACTCATTGTCCCACATTGGTCTC
    GPX4 F:AGGCAGGAGCCAGGAAGTAATC 212
    R:ACCACGCAGCCGTTCTTATC
    FTH1 F:CGCCAGAACTACCACCAGGACT 184
    R:TCAGTTTCTCAGCATGTTCCCTC
    PTGS2 F:CGTTTGAAGAACTTACAGGAGAGAA 94
    R:AGCAGGGCGGGATACAGTT
    P53 F:GGCTCCGACTATACCACTATCCACT 151
    R:CACAAACACGAACCTCAAAGCT
    GAPDH F:CTGGAGAAACCTGCCAAGTATG 138
    R:GGTGGAAGAATGGGAGTTGCT
    下载: 导出CSV

    表  2  ALD大鼠血脂和肝功能情况

    Table  2.   Blood lipids and liver function in rats with alcoholic liver disease

    组别 动物数(只) TG(mmoL/L) ALT(U/L) AST(U/L)
    正常组 6 0.04±0.01 10.24±1.35 33.23±0.92
    模型组 6 2.84±0.221) 43.05±1.131) 60.99±0.511)
    丹葛解酲汤组 6 1.29±0.012) 13.02±0.922) 35.93±0.872)
    复方益肝灵片组 6 2.24±0.022) 34.35±2.332) 35.97±3.682)
    F 356.20 440.70 132.70
    P <0.000 1 <0.000 1 <0.000 1

    注:与正常组比较,1)P<0.01;与模型组比较,2)P<0.01。

    下载: 导出CSV

    表  3  ALD大鼠肝组织MDA、ROS、GSH和Fe2+含量或活性情况

    Table  3.   The levels or activities of MDA, ROS, GSH, and Fe²+ in liver tissue of rats with alcoholic liver disease

    组别 动物数(只) Fe2+(μmol/gprot) GSH(μmol/gprot) MDA(nmoL/mL) ROS荧光强度(%)
    正常组 6 3.60±0.91 21.82±1.92 9.77±0.53 0.06±0.03
    模型组 6 10.67±0.771) 6.76±1.331) 20.38±0.381) 32.75±2.191)
    丹葛解酲汤组 6 5.74±0.322) 16.96±1.132) 10.77±0.382) 8.46±2.702)
    复方益肝灵片组 6 7.83±0.662) 11.70±1.633) 12.44±3.362) 20.58±1.592)
    F 55.63 54.03 23.52 167.30
    P <0.000 1 <0.000 1 0.000 3 <0.000 1

    注:与正常组比较,1)P<0.01;与模型组比较,2)P<0.01,3)P<0.05。

    下载: 导出CSV

    表  4  ALD大鼠肝组织脂质蓄积情况

    Table  4.   Lipid accumulation in liver tissue of rats with alcoholic liver disease

    组别 动物数(只) 油红O染色面积(%)
    正常组 6 1.05±0.11
    模型组 6 6.51±0.221)
    丹葛解酲汤组 6 1.77±0.112)
    复方益肝灵片组 6 3.22±0.042)
    F 988.70
    P <0.000 1

    注:与正常组比较,1)P<0.01;与模型组比较,2)P<0.01。

    下载: 导出CSV

    表  5  ALD大鼠肝组织铁沉积情况

    Table  5.   Iron deposition in liver tissue of rats with alcoholic liver disease

    组别 动物数(只) 普鲁士蓝染色面积(%)
    正常组 6 0.175±0.002
    模型组 6 0.201±0.0031)
    丹葛解酲汤组 6 0.176±0.0012)
    复方益肝灵片组 6 0.186±0.0082)
    F 24.06
    P 0.000 2

    注:与正常组比较,1)P<0.01;与模型组比较,2)P<0.01。

    下载: 导出CSV

    表  6  ALD大鼠P53、GPX4、NOX1、PTGS2、FTH1 mRNA相对表达量

    Table  6.   The mRNA expression of P53, GPX4, NOX1, PTGS2, and FTH1 in rats with alcoholic liver disease

    组别 P53 mRNA NOX1 mRNA PTGS2 mRNA GPX4 mRNA FTH1 mRNA
    正常组 1.02±0.24 1.01±0.13 1.01±0.17 1.01±0.13 1.01±0.13
    模型组 2.17±0.291) 1.93±0.061) 1.49±0.131) 0.60±0.061) 0.62±0.051)
    丹葛解酲汤组 1.34±0.252) 1.38±0.102) 1.08±0.082) 0.81±0.053) 0.83±0.023)
    复方益肝灵片组 1.63±0.093) 1.67±0.073) 1.19±0.083) 0.80±0.043) 0.79±0.023)
    F 13.68 54.49 8.96 13.35 15.54
    P 0.001 6 <0.000 1 0.006 2 0.001 8 0.001 1

    注:与正常组比较,1)P<0.01;与模型组比较,2)P<0.01,3)P<0.05。

    下载: 导出CSV

    表  7  ALD大鼠P53、GPX4、NOX1、PTGS2、FTH1蛋白表达情况

    Table  7.   The protein expression of P53, GPX4, NOX1, PTGS2, and FTH1 in rats with alcoholic liver disease

    组别 P53 NOX1 PTGS2 GPX4 FTH1
    正常组 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00
    模型组 6.31±1.191) 1.57±0.081) 1.99±0.011) 0.49±0.011) 0.16±0.041)
    丹葛解酲汤组 2.39±0.552) 1.20±0.042) 1.33±0.072) 0.72±0.032) 0.57±0.052)
    复方益肝灵片组 3.83±0.622) 1.29±0.163) 1.60±0.122) 0.58±0.012) 0.34±0.063)
    F 169.10 19.62 58.82 613.60 115.20
    P <0.000 1 0.000 5 <0.000 1 <0.000 1 <0.000 1

    注:与正常组比较,1)P<0.01;与模型组比较,2)P<0.01,3)P<0.05。

    下载: 导出CSV
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    返回文章
    返回