Department of Orthopaedic Surgery, Experimental Orthopaedics, Centre for Medical Biotechnology (ZMB/Biopark 1), University of Regensburg, Regensburg, Germany.
Department of Orthopaedic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
Stem Cell Res Ther. 2021 Apr 29;12(1):252. doi: 10.1186/s13287-021-02317-6.
Curcumin has anti-inflammatory effects and qualifies as a potential candidate for the treatment of osteoarthritis (OA). However, curcumin has limited bioavailability. Extracellular vesicles (EVs) are released by multiple cell types and act as molecule carrier during intercellular communication. We assume that EVs can maintain bioavailability and stability of curcumin after encapsulation. Here, we evaluated modulatory effects of curcumin-primed human (h)BMSC-derived EVs (Cur-EVs) on IL-1β stimulated human osteoarthritic chondrocytes (OA-CH).
CellTiter-Blue Viability- (CTB), Caspase 3/7-, and live/dead assays were used to determine range of cytotoxic curcumin concentrations for hBMSC and OA-CH. Cur-EVs and control EVs were harvested from cell culture supernatants of hBMSC by ultracentrifugation. Western blotting (WB), transmission electron microscopy, and nanoparticle tracking analysis were performed to characterize the EVs. The intracellular incorporation of EVs derived from PHK26 labeled and curcumin-primed or control hBMSC was tested by adding the labeled EVs to OA-CH cultures. OA-CH were pre-stimulated with IL-1β, followed by Cur-EV and control EV treatment for 24 h and subsequent analysis of viability, apoptosis, and migration (scratch assay). Relative expression of selected anabolic and catabolic genes was assessed with qRT-PCR. Furthermore, WB was performed to evaluate phosphorylation of Erk1/2, PI3K/Akt, and p38MAPK in OA-CH. The effect of hsa-miR-126-3p expression on IL-1β-induced OA-CH was determined using CTB-, Caspase 3/7-, live/dead assays, and WB.
Cur-EVs promoted viability and reduced apoptosis of IL-1β-stimulated OA-CH and attenuated IL-1β-induced inhibition of migration. Furthermore, Cur-EVs increased gene expression of BCL2, ACAN, SOX9, and COL2A1 and decreased gene expression of IL1B, IL6, MMP13, and COL10A1 in IL-1β-stimulated OA-CH. In addition, phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK, induced by IL-1β, is prevented by Cur-EVs. Cur-EVs increased IL-1β-reduced expression of hsa-miR-126-3p and hsa-miR-126-3p mimic reversed the effects of IL-1β.
Cur-EVs alleviated IL-1β-induced catabolic effects on OA-CH by promoting viability and migration, reducing apoptosis and phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK thereby modulating pro-inflammatory signaling pathways. Treatment of OA-CH with Cur-EVs is followed by upregulation of expression of hsa-miR-126-3p which is involved in modulation of anabolic response of OA-CH. EVs may be considered as promising drug delivery vehicles of curcumin helping to alleviate OA.
姜黄素具有抗炎作用，是治疗骨关节炎（OA）的潜在候选药物。然而，姜黄素的生物利用度有限。细胞外囊泡（EVs）由多种细胞类型释放，并在细胞间通讯中充当分子载体。我们假设 EVs 可以在封装后维持姜黄素的生物利用度和稳定性。在这里，我们评估了姜黄素预刺激的人（h）BMSC 衍生的 EVs（Cur-EVs）对 IL-1β 刺激的人 OA 软骨细胞（OA-CH）的调节作用。
使用细胞活力测定（CTB）、半胱天冬酶 3/7- 和死活测定来确定 hBMSC 和 OA-CH 的细胞毒性姜黄素浓度范围。通过超速离心从 hBMSC 细胞培养上清液中收获 Cur-EVs 和对照 EVs。进行 Western blot（WB）、透射电子显微镜和纳米颗粒跟踪分析以表征 EVs。通过向 OA-CH 培养物中添加标记的 EVs 来测试源自 PHK26 标记和姜黄素预刺激或对照 hBMSC 的 EVs 的细胞内掺入。用 IL-1β 预先刺激 OA-CH，然后用 Cur-EV 和对照 EV 处理 24 小时，随后分析活力、凋亡和迁移（划痕试验）。使用 qRT-PCR 评估选定的合成代谢和分解代谢基因的相对表达。此外，进行 WB 以评估 OA-CH 中 Erk1/2、PI3K/Akt 和 p38MAPK 的磷酸化。使用 CTB、半胱天冬酶 3/7- 和死活测定以及 WB 来确定 hsa-miR-126-3p 表达对 IL-1β 诱导的 OA-CH 的影响。
Cur-EVs 促进了 IL-1β 刺激的 OA-CH 的活力并减少了凋亡，并减轻了 IL-1β 诱导的迁移抑制。此外，Cur-EVs 增加了 IL-1β 刺激的 OA-CH 中 BCL2、ACAN、SOX9 和 COL2A1 的基因表达，并降低了 IL1B、IL6、MMP13 和 COL10A1 的基因表达。此外，Cur-EVs 可预防由 IL-1β 诱导的 Erk1/2、PI3K/Akt 和 p38 MAPK 的磷酸化。Cur-EVs 增加了 IL-1β 降低的 hsa-miR-126-3p 的表达，hsa-miR-126-3p 模拟物逆转了 IL-1β 的作用。
Cur-EVs 通过促进活力和迁移、减少凋亡和 Erk1/2、PI3K/Akt 和 p38 MAPK 的磷酸化，减轻了 IL-1β 对 OA-CH 的分解代谢作用，从而调节促炎信号通路。用 Cur-EVs 处理 OA-CH 会导致 hsa-miR-126-3p 的表达上调，这与 OA-CH 的合成代谢反应的调节有关。EVs 可被视为有前途的姜黄素药物递送载体，有助于缓解 OA。
