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Toll样受体4在对乙酰氨基酚致小鼠肝损伤过程中对肝脏再生的影响

陈明月 郑秀良 乔亚琴 沈海涛 路燕

引用本文:
Citation:

Toll样受体4在对乙酰氨基酚致小鼠肝损伤过程中对肝脏再生的影响

DOI: 10.3969/j.issn.1001-5256.2023.05.017
基金项目: 

国家自然科学基金青年科学基金项目 (81800524)

伦理学声明:本研究方案于2018年3月经由安徽医科大学实验动物伦理委员会审批,批号:LLSC20180080,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:陈明月负责分析数据,撰写论文和参与课题设计;郑秀良、乔亚琴、沈海涛参与实验操作和修改论文;路燕负责课题设计和修改论文。
详细信息
    通信作者:

    路燕,luyancathrine@163.com (ORCID: 0000-0001-5116-5145)

Effect of Toll-like receptor 4 on liver regeneration during acetaminophen-induced liver injury in mice

Research funding: 

National Natural Science Foundation of China Youth Science Fund Project (81800524)

More Information
  • 摘要:   目的  观察抑制Toll样受体4(TLR4)是否影响对乙酰氨基酚(APAP)致小鼠肝损伤过程中的肝脏再生,初步探讨TLR4参与肝脏再生的机制。  方法  将78只雄性CD-1小鼠采用随机数字表法分为9组,其中对照组(正常对照组、溶剂对照组、抑制剂对照组)每组6只,实验组(APAP 24 h组、TAK-242+APAP 24 h组、APAP 48 h组、TAK-242+APAP 48 h组、APAP 72 h组、TAK-242+APAP 72 h组)每组10只。实验组小鼠给予单剂量腹腔注射APAP(300 mg/kg),TAK-242在APAP注射前3 h以3 mg/kg剂量腹腔注射。在不同时间点收集各组小鼠血清和肝脏组织。采用生化方法检测小鼠血清ALT水平,HE染色检测肝脏组织病理改变。RT-PCR、Western blot、免疫组化方法检测Cyclin D1、PCNA、Ki-67、STAT3、p-STAT3的表达。正态分布的计量资料两组间比较采用成组t检验;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。非正态分布的计量资料两组间比较采用Mann-Whitney U检验;多组间比较及进一步两两比较均采用Kruskal-Wallis H检验。  结果  与正常对照组相比,APAP 24 h组和APAP 48 h组的血清ALT水平均明显较高(P值均<0.05);TAK-242+APAP 24 h和48 h组的血清ALT水平均明显高于同时间点APAP组(P值均<0.05)。HE染色结果显示,APAP处理的小鼠肝脏可见典型的小叶中心性坏死,TAK-242+APAP 24 h和48 h组小鼠肝脏的坏死面积均显著大于同时间点APAP组(P值均<0.05)。RT-PCR、Western blot、免疫组化结果显示,TAK-242+APAP 24 h、48 h和72 h组Cyclin D1 mRNA和蛋白表达水平均明显低于同时间点的APAP组(P值均<0.05);TAK-242+APAP 24 h、48 h和72 h组PCNA mRNA表达水平均明显低于同时间点的APAP组(P值均<0.05),TAK-242+APAP 24 h和48 h组PCNA蛋白表达水平均明显低于同时间点的APAP组(P值均<0.05);TAK-242+APAP 24 h和72 h组Ki-67 mRNA表达水平均明显低于同时间点的APAP组(P值均<0.05),TAK-242+APAP 24 h、48 h和72 h组Ki-67蛋白表达水平均明显低于同时间点的APAP组(P值均<0.05)。此外,TAK-242+APAP 24 h和48 h组STAT3磷酸化水平均显著低于同时点的APAP组(P值均<0.05)。  结论  TLR4可能通过提高STAT3磷酸化水平促进APAP诱导的小鼠肝损伤过程中的肝脏再生。

     

  • 图  1  小鼠血清ALT水平比较

    Figure  1.  Comparison of serum ALT levels in mice

    图  2  小鼠肝脏HE染色结果 (×100)

    注:a,正常对照组;b,溶剂对照组;c,抑制剂对照组。

    Figure  2.  HE staining results of mouse liver (×100)

    图  3  抑制TLR4对肝损伤恢复的影响(HE染色,×100)

    注:a,正常对照组;b,APAP 24 h组;c,TAK-242+APAP 24 h组;d,APAP 48 h组;e,TAK-242+APAP 48 h组;f,APAP 72 h组;g,TAK-242+APAP 72 h组。

    Figure  3.  Effect of inhibition of TLR4 on recovery from liver injury (HE staining, ×100)

    图  4  肝组织cyclin D1、PCNA的蛋白表达

    注:a,Cyclin D1蛋白表达水平;b,PCNA蛋白表达水平。

    Figure  4.  Cyclin D1, PCNA protein expressions in liver tissue

    图  5  免疫组化染色检测肝组织Ki-67蛋白表达(×100)

    注:a,正常对照组;b,APAP 24 h组;c,TAK-242+APAP 24 h组;d,APAP 48 h组;e,TAK-242+APAP 48 h组;f,APAP 72 h组;g,TAK-242+APAP 72 h组。

    Figure  5.  Ki-67 protein expression in liver tissue detected by Immunohistochemical staining (×100)

    图  6  各组Ki-67阳性细胞累积光密度值比较

    Figure  6.  Comparison of integrated option density of Ki-67 positive cells

    图  7  肝组织STAT3磷酸化蛋白的表达

    Figure  7.  Expression of phosphorylated STAT3 protein in liver tissue

    表  1  RT-PCR引物序列

    Table  1.   RT-PCR primer sequences

    基因 上游(5′-3′) 下游(5′-3′)
    Cyclin D1 AGGCGGATGAGAACAAGCAG CCTTGTTTAGCCAGAGGCCG
    Ki-67 CCATCATTGACCGCTCCTT CTGCCAGTGTGCTGTTCTAC
    PCNA GGGTTGGTAGTTGTCGCTGT CCAAGGAGACGTGAGACGAG
    GAPDH GACATGCCGCCTGGAGAAAC AGCCCAGGATGCCCTTTAGT
    下载: 导出CSV

    表  2  APAP对小鼠血清ALT及肝脏组织的影响

    Table  2.   Effects of APAP on serum ALT and liver tissue in mice

    组别 动物数(只) ALT(U/L) 肝脏坏死面积(%)
    正常对照组 6 12.339±4.245 0
    APAP 24 h组 10 1 449.848±209.4911) 41.600(38.617~46.502)1)
    APAP 48 h组 10 281.702±140.5921) 36.050(25.127~45.718)1)
    APAP 72 h组 10 45.251±4.298 0 (0~1.903)
    H 17.857 16.035
    P <0.001 0.001
    注:与正常对照组比较,1)P<0.05。
    下载: 导出CSV

    表  3  抑制TLR4在各时间点对APAP肝损伤小鼠血清ALT及肝脏组织的影响

    Table  3.   Effects of TLR4 inhibition on serum ALT and liver tissue of mice with APAP-induced liver injury at different time point

    组别 动物数(只) 24 h 48 h 72 h
    ALT(U/L)
       APAP组 10 1 449.848±209.491 281.702±140.592 45.251±4.298
       TAK-242+APAP组 10 3 484.212±960.336 1 151.505±656.722 56.995±24.763
       t -4.628 -2.896 -1.045
       P 0.002 0.040 0.352
    肝脏坏死面积(%)
       APAP组 10 41.600(38.617~46.502) 36.050(25.127~45.718) 0(0~1.903)
       TAK-242+APAP组 10 58.554(55.889~73.409) 59.558(54.856~60.591) 2.713(0~8.289)
       Z -2.610 -2.611 -1.294
       P 0.008 0.008 0.310
    下载: 导出CSV

    表  4  APAP对小鼠肝组织Ki-67 mRNA、Cyclin D1 mRNA、PCNA mRNA表达的影响

    Table  4.   Effects of APAP on the expression of Ki-67 mRNA, Cyclin D1 mRNA and PCNA mRNA in mouse liver

    组别 动物数(只) Ki-67 mRNA相对表达量 Cyclin D1 mRNA相对表达量 PCNA mRNA相对表达量
    正常对照组 6 1.002±0.072 1.034±0.325 1.009±0.162
    APAP 24 h组 10 1.027±0.184 1.061±0.102 1.347±0.1151)
    APAP 48 h组 10 83.566±23.3621) 3.707±0.2551) 5.327±0.1621)
    APAP 72 h组 10 39.316±10.9161) 2.417±0.3361) 1.739±0.0861)
    F 27.853 88.659 657.231
    P <0.001 <0.001 <0.001
    注:与正常对照组比较,1)P<0.05。
    下载: 导出CSV

    表  5  抑制TLR4对小鼠肝组织Ki-67 mRNA、Cyclin D1 mRNA、PCNA mRNA表达的影响

    Table  5.   Effects of TLR4 inhibition on the expression of Ki-67 mRNA, Cyclin D1 mRNA and PCNA mRNA in mouse liver

       组别 动物数(只) 24 h 48 h 72 h
    Ki-67 mRNA相对表达量
       APAP组 10 1.027±0.184 83.566±23.362 39.316±10.916
       TAK-242+APAP组 10 0.366±0.129 70.145±31.141 5.950±0.678
       t 5.107 0.597 5.284
       P 0.007 0.583 0.006
    Cyclin D1 mRNA相对表达量
       APAP组 10 1.061±0.102 3.707±0.255 2.417±0.336
       TAK-242+APAP组 10 0.678±0.073 2.918±0.487 1.837±0.202
       t 6.116 2.871 2.957
       P 0.001 0.028 0.025
    PCNA mRNA相对表达量
       APAP组 10 1.347±0.115 5.327±0.162 1.739±0.086
       TAK-242+APAP组 10 0.830±0.075 2.937±0.144 0.620±0.035
       t 6.529 19.074 20.909
       P 0.003 <0.001 <0.001
    下载: 导出CSV
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