Lv Jian, Bai Ruidan, Wang Li, Gao Jiefang, Zhang Hong
Department of Pharmacy, Renmin Hospital of Wuhan University, Zhang Zhidong Road, Wuhan, Hubei, 430060, People's Republic of China.
BMC Pharmacol Toxicol. 2018 Oct 16;19(1):64. doi: 10.1186/s40360-018-0255-9.
An increasing number of studies are investigating the effects of Chinese medicine on hepatic fibrosis, but only few studies have examined the anti-fibrogenic properties of Artesunate (ART). The aim of the present study was to explore the anti-fibrotic effects of ART on LX-2 cells, the human HSC cell line, and to determine potential molecular mechanisms via the focal adhesion kinase (FAK)/ protein kinase B (Akt)/ β-catenin pathway.
LX-2 cells were stimulated with different concentration of ART (0, 12.5, 25 and 50 μg/ml) for 12, 24, 48 or 72 h, their proliferation was analyzed using the Cell Counting Kit-8 (CCK-8) assay. LX-2 cells were treated with different doses of ART (0, 12.5, 25 and 50 μg/ml) for 24 h, their apoptosis was measured using flow cytometry, the levels of mRNAs encoding collagen I or α-smooth muscle actin (α-SMA) were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the levels of key proteins in the FAK/Akt/β-catenin signaling pathway were assessed by western blotting. Specific inhibitors of FAK were added to the LX-2 cells cultures to explore the potential signaling.
Exposing LX-2 cells to ART efficiently inhibited their proliferation, significantly promoted early apoptosis in a dose-dependent manner, and markedly downregulated the mRNA expression of α-SMA and collagen I. In addition, ART, similar to FAK inhibitor PF562271 significantly inhibited the FAK/Akt/β-catenin signaling pathway by reducing the levels of phosphorylated FAK, Akt and GSK-3β.
Our present study shows that ART could regulate the proliferation, apoptosis and activation of LX-2. Meanwhile, the anti-fibrogenic mechanisms of ART was correlated with FAK/Akt/β-catenin pathway. Future research should verify and extend these findings, as well as explore other molecules and therefore serve as useful therapeutic targets.
越来越多的研究正在调查中药对肝纤维化的影响，但仅有少数研究考察了青蒿琥酯（ART）的抗纤维化特性。本研究的目的是探讨ART对人肝星状细胞系LX-2细胞的抗纤维化作用，并通过粘着斑激酶（FAK）/蛋白激酶B（Akt）/β-连环蛋白途径确定潜在的分子机制。
用不同浓度的ART（0、12.5、25和50μg/ml）刺激LX-2细胞12、24、48或72小时，使用细胞计数试剂盒-8（CCK-8）检测法分析其增殖情况。用不同剂量的ART（0、12.5、25和50μg/ml）处理LX-2细胞24小时，使用流式细胞术检测其凋亡情况，使用逆转录-定量聚合酶链反应（RT-qPCR）测定编码I型胶原或α-平滑肌肌动蛋白（α-SMA）的mRNA水平，并通过蛋白质印迹法评估FAK/Akt/β-连环蛋白信号通路中关键蛋白的水平。将FAK特异性抑制剂加入LX-2细胞培养物中以探索潜在的信号传导。
将LX-2细胞暴露于ART可有效抑制其增殖，以剂量依赖性方式显著促进早期凋亡，并显著下调α-SMA和I型胶原的mRNA表达。此外，与FAK抑制剂PF562271类似，ART通过降低磷酸化FAK、Akt和GSK-3β的水平，显著抑制FAK/Akt/β-连环蛋白信号通路。
我们目前的研究表明，ART可调节LX-2细胞的增殖、凋亡和激活。同时，ART的抗纤维化机制与FAK/Akt/β-连环蛋白途径相关。未来的研究应验证并扩展这些发现，并探索其他分子，从而作为有用的治疗靶点。
