Enhanced effects of novel oridonin analog CYD0682 for hepatic fibrosis.
Enhanced effects of novel oridonin analog CYD0682 for hepatic fibrosis.
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DOI：
10.1016/j.jss.2015.07.042
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发表时间：
2015-12
期刊：
影响因子：
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通讯作者：
Radhakrishnan RS
中科院分区：
文献类型：
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作者：
Bohanon FJ;Wang X;Graham BM;Ding C;Ding Y;Radhakrishnan GL;Rastellini C;Zhou J;Radhakrishnan RS
激活的肝星状细胞（HSC）是导致肝纤维化过程中细胞外基质（ECM）蛋白沉积过多的原因。此前，我们课题组报道了天然化合物冬凌草甲素可诱导细胞凋亡，抑制细胞增殖，并下调活化HSC中的ECM蛋白。在本研究中，研究了冬凌草甲素衍生物CYD0682对活化的人LX-2和大鼠HSC-T6星状细胞系的抗纤维化作用。通过阿拉玛蓝测定法测量细胞增殖。通过细胞死亡ELISA以及Yo-Pro-1和碘化丙啶染色来检测细胞凋亡。通过流式细胞术测定细胞周期。对细胞蛋白表达进行免疫印迹和免疫荧光染色。 CYD0682 处理以剂量和时间依赖性方式显着抑制 LX-2 细胞增殖，48 小时内 IC50 值为 0.49 μM，效力比冬凌草甲素强约 10 倍。在 HSC-T6 细胞中观察到类似的结果。相比之下，2.5 μM CYD0682 处理对人肝细胞系 C3A 的增殖没有显着影响。 CYD0682 处理诱导 LX-2 细胞凋亡和 S 期细胞周期停滞，并与 p53、p21 和 cleaved-caspase-3 的激活相关。 CYD0682 以时间和剂量依赖性方式显着抑制肌成纤维细胞标记蛋白 α-平滑肌肌动蛋白和主要 ECM 蛋白 I 型胶原和纤连蛋白。此外，CYD0682 预处理可阻断 TGFβ 诱导的 I 型胶原和纤连蛋白的产生。与冬凌草甲素相比，其新型衍生物CYD0682可能作为更有效的抗肝纤维化药物。
Activated hepatic stellate cells (HSC) are responsible for excess extracellular matrix (ECM) protein deposition in liver fibrosis. Previously, our group reported that the natural compound oridonin induces apoptosis, inhibits cell proliferation, and down-regulates ECM proteins in activated HSC. In this study, the anti-fibrogenic effects of oridonin derivative CYD0682 on the activated human LX-2 and rat HSC-T6 stellate cell lines were investigated. Cell proliferation was measured by Alamar Blue assay. Apoptosis was detected by Cell Death ELISA and staining of Yo-Pro-1 and propidium iodide. Cell cycle was determined by flow cytometry. Immunoblot and Immunofluorescence staining were performed for cellular protein expression. CYD0682 treatment significantly inhibited LX-2 cells proliferation in a dose- and time-dependent manner with an IC50 value of 0.49 μM for 48 hours, ~10–fold greater potency than oridonin. Similar results were observed in HSC-T6 cells. In contrast, 2.5 μM of CYD0682 treatment had no significant effects on proliferation of the human hepatocyte cell line C3A. CYD0682 treatment induced LX-2 cell apoptosis and S-phase cell cycle arrest, and was associated with activation of p53, p21, and cleaved-caspase-3. The myofibroblast marker protein α-smooth muscle actin and major ECM proteins type I collagen and fibronectin were markedly suppressed in a time- and dose-dependent fashion by CYD0682. Furthermore, pre-treatment with CYD0682 blocked TGFβ-induced type I collagen and fibronectin production. In comparison with oridonin, its novel derivative CYD0682 may act as a more potent anti-hepatic fibrosis agent.