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奥沙利铂对肝星状细胞活化的影响及机制探讨

王存凯 王一军 王丹丹 谢肖立 刘红玉 白云 姜慧卿 王玉珍

引用本文:
Citation:

奥沙利铂对肝星状细胞活化的影响及机制探讨

DOI: 10.12449/JCH240612
基金项目: 

河北省2023年度医学科学研究课题 (20230019)

利益冲突声明:本文不存在任何利益冲突。
作者贡献声明:王存凯负责设计论文框架,起草论文;王存凯、王一军、谢肖立负责实验操作,研究过程的实施;王丹丹、白云负责数据整理、统计学分析;刘红玉负责论文修改;王玉珍、姜慧卿负责拟定写作思路,指导撰写文章并最后定稿。
详细信息
    通信作者:

    王玉珍, wyzhen211@126.com (ORCID: 0009-0001-4437-4616)

Effect of oxaliplatin on the activation of hepatic stellate cells and its mechanism

Research funding: 

Medical Science Research Project of Hebei Province in 2023 (20230019)

More Information
    Corresponding author: WANG Yuzhen, wyzhen211@126.com (ORCID: 0009-0001-4437-4616)
  • 摘要:   目的  观察奥沙利铂对肝星状细胞(HSC)活化的影响,进一步探讨奥沙利铂与microRNA-30a-5p及自噬的关系。  方法  培养HSC-LX2并做如下分组:(1)对照组、PDGF处理组、奥沙利铂处理组、奥沙利铂+PDGF处理组;(2)对照组、microRNA-30a-5p转染组,PDGF处理组、microRNA-30a-5p转染+PDGF处理组;(3)对照组、3-MA组、microRNA-30a-5p抑制剂组、microRNA-30a-5p抑制剂+3-MA组。应用免疫蛋白印迹技术(Western Blot)分析HSC活化相关蛋白Ⅰ型胶原(Collagen-‍Ⅰ)、抗α-平滑肌肌动蛋白(α-SMA)及HSC自噬相关蛋白Beclin 1、p62、LC3B的表达;溶酶体示踪剂和免疫荧光检测LC3B自噬小体的表达。实时荧光定量PCR(RT-PCR)检测microRNA-30a-5p表达水平。应用生物信息学技术预测microRNA-30a-5p在HSC中的潜在靶点。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。  结果  应用奥沙利铂处理细胞时,RT-PCR结果显示奥沙利铂处理组microRNA-30a-5p的表达水平高于对照组(P<0.01);Western Blot结果显示奥沙利铂处理组的HSC活化相关蛋白α-SMA、Collagen-Ⅰ及自噬相关蛋白Beclin 1、LC3BⅡ/Ⅰ水平均降低(P值均<0.001);免疫荧光结果显示奥沙利铂处理组自噬小体低于对照组(P<0.05)。进一步应用microRNA-30a-5p模拟类似物(mimic)转染HSC-LX2结果显示,与对照组相比,microRNA-30a-5p mimic组自噬相关蛋白Beclin 1和LC3BⅡ/Ⅰ的表达水平均降低(P值均<0.05);HSC活化相关蛋白Collagen-Ⅰ的表达水平亦降低(P<0.001);应用microRNA-30a-5p抑制剂转染HSC-LX2,Western Blot结果显示,与对照组比较,microRNA-30a-5p抑制剂组HSC活化相关蛋白Collagen-Ⅰ、α-SMA,自噬相关蛋白Beclin 1的表达水平均升高(t值分别为2.412.324.57,P值均<0.05)。Western Blot结果显示,与对照组相比,microRNA-30a-5p抑制剂组HSC自噬相关蛋白Beclin 1及活化相关蛋白α-SMA表达水平均升高(P值均<0.05),应用自噬抑制剂3-MA处理后,两组间上述蛋白表达无显著差异(P>0.05)。通过TargetScan、PicTar和miRanda生物信息软件分析表明,自噬相关蛋白Beclin 1是microRNA-30a-5p的潜在靶标。  结论  奥沙利铂可通过上调microRNA-30a-5p表达从而抑制HSC激活,为肝纤维化的治疗提供新的思路和作用靶点。

     

  • 图  1  不同浓度的奥沙利铂对HSC-LX2活性的影响

    Figure  1.  The effect of different concentrations of oxaliplatin on the survival rate of HSC-LX2

    图  2  奥沙利铂抑制HSC自噬和活化相关蛋白的表达

    Figure  2.  Oxaliplatin inhibits the activation and autophagy of HSC

    图  3  LC3B 的Lysotracker red和免疫荧光(×200)

    Figure  3.  LC3B expression observed by Lysotracker red and immunofluorescence(×200)

    图  4  不同浓度的奥沙利铂对microRNA-30a-5p表达量的影响

    Figure  4.  The effect of different concentrations of oxaliplatin on the expression of microRNA-30a-5p

    图  5  HSC-LX2中的microRNA-30a-5p水平

    Figure  5.  The microRNA-30a-5p levels in LX-2

    图  6  PDGF处理后microRNA-30a-5p表达情况

    Figure  6.  microRNA-30a-5p levels in HSC-LX2 with PDGF

    图  7  microRNA-30a-5p抑制HSC-LX2自噬及活化

    Figure  7.  microRNA-30a-5p inhibits autophagy and activation of HSC-LX2

    图  8  microRNA-30a-5p抑制剂促进HSC-LX2自噬及活化

    Figure  8.  microRNA-30a-5p inhibitors promote autophagy and activation of HSC-LX2

    图  9  3-MA阻断microRNA-30a-5p抑制剂促进HSC活化的作用

    Figure  9.  3-MA blocks the effect of microRNA-30a-5p inhibitors in promoting HSC activation

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  • 收稿日期:  2023-09-06
  • 录用日期:  2023-10-26
  • 出版日期:  2024-06-25
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