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奥沙利铂对肝星状细胞活化的影响及机制探讨
DOI: 10.12449/JCH240612
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。
Effect of oxaliplatin on the activation of hepatic stellate cells and its mechanism
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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2.41、2.32、4.57，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。 Abstract:Objective To investigate the effect of oxaliplatin on the activation of hepatic stellate cells （HSCs）， as well as the association of oxaliplatin with microRNA-30a-5p and autophagy. Methods HSC-LX2 cells were cultured and divided into groups according to the following three protocols： control group， PDGF treatment group， oxaliplatin treatment group， oxaliplatin+PDGF treatment group； control group， microRNA-30a-5p transfection group， PDGF treatment group， microRNA-30a-5p transfection+PDGF treatment group； control group， 3-MA group， microRNA-30a-5p inhibitor group， microRNA-30a-5p inhibitor+3-MA group. Western Blot was used to measure the expression of HSC activation-related proteins （Collagen-I and alpha-smooth muscle actin ［α- SMA］） and HSC autophagy-related proteins （Beclin-1， P62， and LC3B）； LysoTracker staining and immunofluorescence assay were used to measure the expression of LC3B autophagosomes； RT-PCR was used to measure the expression level of microRNA-30a-5p； bioinformatics techniques were used to predict the potential targets of microRNA-30a-5p in HSCs. The independent-samples t test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results After the cells were treated with oxaliplatin， RT-PCR results showed that the oxaliplatin treatment group had a significantly higher expression level of microRNA-30a-5p than the control group （P<0.01）； Western Blot showed that the oxaliplatin treatment group had significant reductions in the expression levels of the HSC activation-related proteins α-SMA and Collagen-Ⅰ and the autophagy-related proteins Beclin 1 and LC3BⅡ/Ⅰ （all P<0.001）； immunofluorescence assay showed that the oxaliplatin treatment group had a significantly lower number of autophagosomes than the control group （P<0.05）. After HSC-LX2 cells were transfected with microRNA-30a-5p mimic， compared with the control group， the microRNA-30a-5p mimic group had significant reductions in the expression levels of the autophagy-related proteins Beclin 1 and LC3BⅡ/Ⅰ （P<0.05） and the HSC activation-related protein Collagen-Ⅰ （P<0.001）； after HSC-LX2 cells were transfected with microRNA-30a-5p inhibitor， Western Blot showed that compared with the control group， the microRNA-30a-5p inhibitor group had significant increases in the expression levels of the HSC activation-related proteins Collagen-Ⅰ and α-SMA and the autophagy-related protein Beclin 1 （t=2.41， 2.32， and 4.57， all P<0.05）. Western Blot showed that compared with the control group， the microRNA-30a-5p inhibitor group had significant increases in the expression levels of the HSC autophagy-related protein Beclin 1 and the HSC activation-related protein α-SMA （both P<0.05）， and after the treatment with the autophagy inhibitor 3-MA， there were no significant differences in the expression of these proteins between the two groups （P>0.05）. The bioinformatics analysis using TargetScan， PicTar， and miRanda databases showed that the autophagy-related protein Beclin-1 might be a potential target of miRNA-30a-5p. Conclusion Oxaliplatin can inhibit the activation of HSCs by upregulating the expression of microRNA-30a-5p， which provides new ideas and a new target for the treatment of liver fibrosis. -
Key words:
- Oxaliplatin /
- Hepatic Fibrosis /
- Hepatic Stellate Cells /
- MicroRNAs /
- Autophagy
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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 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
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