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咖啡酸苯乙酯对肝星状细胞的作用及机制分析
DOI: 10.3969/j.issn.1001-5256.2022.10.014
利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。
作者贡献声明：杨宁、石娟娟、党双锁负责课题设计，资料分析，撰写论文；邓江、王怡恺、王文俊参与收集数据，数据分析；杨宁、邓江、王怡恺、常莎、高宁负责课题实验，实验数据分析；杨宁、石娟娟、党双锁负责拟定写作思路，指导撰写文章并最后定稿。
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摘要:
目的 探讨咖啡酸苯乙酯(CAPE)对大鼠肝星状细胞(HSC)-T6的作用及作用机制。 方法 不同浓度CAPE(5、10、15 μmol/L) 分别作用于HSC-T6细胞，同时构建GFP-LC3质粒转染至HSC-T6细胞，自噬诱导剂雷帕霉素和自噬抑制剂3-Methyladenine(3-MA)进行干预，采用MTT方法检测CAPE对HSC-T6细胞增殖的影响，透射电镜观察CAPE对HSC-T6细胞超微结构的影响，细胞免疫荧光实验检测CAPE对HSC-T6细胞LC3表达的影响，qRT-PCR检测自噬相关基因ATG5、ATG7、ATG12、Beclin1和LC3 mRNA的表达情况，Western-Blot检测ATG7、Beclin1、LC3I/Ⅱ、p-AKT/AKT、p-mTOR蛋白的表达情况。计量资料组间比较采用单因素方差分析及Dunnett-t检验。 结果 CAPE干预组较对照组可抑制HSC-T6细胞增殖，且生长明显受到抑制，细胞质中可观察到脂滴和轮盘状自噬体散在分布。与对照组(13.34%±2.59%)相比，不同浓度CAPE 5 μmol/L (23.68%±3.76%，t=-5.553，P＜0.001)、10 μmol/L(43.47%±3.83%，t=-15.958，P＜0.001)、15 μmol/L(57.25%±2.78%，t=-28.334，P＜0.001)的LC3荧光染色均显著增强，且呈正相关。CAPE干预组(10、15 μmol/L)ATG5、ATG7、ATG12、Beclin1和LC3 mRNA水平明显增加(P值均＜0.05)。GFP-LC3质粒转染至HSC-T6细胞后，CAPE干预组(79.01%±6.69%)较对照组(67.06%±6.74%)LC3荧光染色明显增强(t=-3.083，P=0.012)；同时给予自噬诱导剂雷帕霉素(86.88%±5.42%)，LC3表达较CAPE组明显增强(t=-2.239，P=0.049)；而给予自噬抑制剂3-MA(71.22%±4.29%)，其表达则明显减低(t=-2.404，P=0.037)；CAPE干预组和诱导剂雷帕霉素组ATG7、Beclin1和LC3 I/Ⅱ的蛋白表达水平增加，AKT/p-AKT、p-mTOR蛋白的表达水平降低；而抑制剂3-MA组则得出相反的结果。 结论 CAPE可能通过抑制AKT/mTOR信号通路诱导自噬形成，进而抑制HSC-T6细胞增殖。 Abstract:Objective To assess the effect and underlying molecular events of caffeic acid phenethyl ester (CAPE) on rat hepatic stellate HSC-T6 cells. Methods HSC-T6 cells were grown and treated with different concentrations of CAPE (5, 10, or 15 μmol/L), transfected with or without LC3-GFP plasmid, and then treated with or without an autophagy inducer rapamycin or the autophagy inhibitor 3-methyladenine (3-MA). The changed cell viability and morphology were assessed by using cell viability MTT assay and Transmission electron microscope, respectively. The expression of LC3 protein in HSC-T6 cells was detected by immunofluorescence assay, the autophagy-related genes expression of ATG5, ATG7, ATG12, Beclin1 and LC3 were detected by qRT-PCR, and the expression of ATG7, Beclin1, LC3I/Ⅱ, p-AKT/AKT, p-mTOR protein was detected by Western-blot. Comparison between multiple groups was analyzed by one-way ANOVA with Dunnett t-test. Results Compared with the control, CAPE treatment significantly reduced cell viability but induced formation of lipid droplets and roulette-shaped autophagosomes. Compared with the control (13.34%±2.59), LC3 protein was significantly induced in HSC-T6 cells after CAPE treatment (5 μmol/L, 23.68%±3.76, t=-5.553, P < 0.001; 10 μmol/L, 43.47%±3.83, t=-15.958, P < 0.001; 15 μM, 57.25%±2.78, t=-28.334, P < 0.001), while levels of ATG5, ATG7, ATG12, Beclin 1, and LC3 mRNAs were all significantly increased in 10 μm and 15 μm CAPE treated cells vs the control (all P < 0.05). After LC3 overexpression in HSC-T6 cells, LC3 protein was induced vs the vector control (79.01%±6.69% vs 67.06%±6.74%, t=-3.083, P=0.012), while rapamycin treatment further increased LC3 expression (86.88%±5.42%, t=-2.239, P=0.049); however, 3-MA treatment significantly decreased LC3 expression in cells (71.22%±4.29%, t=-2.404, P=0.037). In addition, levels of ATG7, Beclin1, and LC3 Ⅰ/Ⅱ proteins were increased, whereas levels of AKT/p-AKT and p-mTOR were decreased in the CAPE and rapamycin groups vs controls. However, the 3-MA treatment had an opposite result, indicating that 3-MA reversed CAPE-induced effects in HSC-T6 cells. Conclusion Caffeic acid phenethyl ester may induce autophagy to reduce cell viability in hepatic stellate cells by inhibition of the AKT/mTOR signaling. -
Key words:
- Liver Cirrhosis /
- Caffeic Acids /
- Hepatic Stellate Cell
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图 1 不同浓度CAPE对HSC-T6细胞超微结构影响
注：黑色箭头，内质网；黄色箭头，线粒体；红色箭头，自噬体；最上方标尺5 μm，中间标尺1 μm，最下方标尺0.5 μm。
Figure 1. Transmission electron microscope (TEM) detection of ultrastructure in HSC-T6 cells with CAPE treatment
图 2 CAPE对LC3蛋白表达的影响
注：a, LC3蛋白萤光染色情况；b, LC3蛋白萤光的定量分析。
Figure 2. The expression levels of LC3 in HSC-T6 cells with CAPE treatment
图 3 CAPE下调AKT/mTOR信号通路诱导自噬形成
注：a, 不同干预情况下，LC3蛋白荧光染色情况；b，LC3蛋白荧光的定量分析；c，不同浓度CAPE对AKT/mTOR信号通路相关蛋白表达的影响。
Figure 3. CAPE induce autophagy to inhibit HSC-T6 cells proliferation by the AKT/mTOR signaling pathway
表 1 Real-time PCR引物序列
Table 1. Primes of Real-time PCR
基因 引物 温度(℃) 长度(bp) 来源 ATG5 F 5′-TCAGTGGAGGCAACAGAACC-3′ 55.49 20 大鼠 R 5′-TTCACTGAGCAAAAGCGTGC-3′ 55.71 20 大鼠 ATG7 F 5′-GAGACCGTCTGAGCAACCAC-3′ 56.38 20 大鼠 R 5′-GATGACACAGGAAAGGGTGC-3′ 54.5 20 大鼠 ATG12 F 5′-CGGACTGTCCAAGCACTCAT-3′ 55.68 20 大鼠 R 5′-CCCATGCCTGTGATTTGCAG-3′ 55.49 20 大鼠 LC3 F 5′-CGGGTTGAGGAGACACACAA -3′ 55.53 20 大鼠 R 5′-ATGAGCCGGACATCTTCCAC -3′ 55.44 20 大鼠 Beclin1 F 5′-GAATGGAGGGGTCTAAGGCG -3′ 55.52 20 大鼠 R 5′-GAATGGTCACTCGGTCCAGG -3′ 55.76 20 大鼠 β-actin F 5′-GGAGATTACTGCCCTGGCTCCTA-3′ 60.2 23 大鼠 R 5′-GACTCATCGTACTCCTGCTTGCTG-3′ 59.3 24 大鼠 
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表 2 CAPE对自噬相关的基因表达的影响
Table 2. The expression levels of autophagy-related genes in HSC-T6 cells with CAPE treatment
指标 对照组 CAPE F值 方差分析的P值 5 μmol/L 10 μmol/L 15 μmol/L ATG5 1.02±0.01 1.06±0.04 1.46±0.091) 2.43±0.421) 15.770 ＜0.05 ATG7 1.05±0.02 1.08±0.04 3.30±0.231) 5.44±0.151) 665.826 ＜0.01 ATG12 1.01±0.06 1.07±0.03 2.33±0.081) 3.80±0.171) 585.839 ＜0.01 Becline1 0.99±0.08 1.07±0.05 1.55±0.071) 2.44±0.131) 182.940 ＜0.01 LC3 1.08±0.04 1.12±0.10 1.55±0.111) 2.38±0.271) 44.963 ＜0.01 注：与对照组比较，1)P＜0.01。
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