Shaanxi Clinical Research Center for Oral Disease & Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi'an, Shaanxi, People's Republic of China.
Air Force Medical Center, Fourth Military Medical University, Beijing, People's Republic of China.
Int J Nanomedicine. 2023 Mar 24;18:1469-1489. doi: 10.2147/IJN.S402170. eCollection 2023.
INTRODUCTION: Osteoarthritis (OA) is a chronic degenerative joint disease accompanied by an elevated macrophage proinflammatory phenotype, which is triggered by persistent pathologically elevated calcium ion levels in mitochondria. However, existing pharmacological compounds targeting the inhibition of mitochondrial calcium ion (m[Ca]) influx are currently limited in terms of plasma membrane permeability and low specificity for ion channels and transporters. In the present study, we synthesized mesoporous silica nanoparticle-amidated (MSN)-ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA)/triphenylphosphine (TPP)-polyethylene glycol (PEG) [METP] nanoparticles (NPs), which specifically target mitochondria and block excess calcium ion influx. METHODS: m[Ca] overload in OA mouse bone marrow-derived macrophages (BMDMs) was detected by a fluorescence probe. A tissue in situ fluorescence colocalization assay was used to evaluate METP NP uptake by macrophages. BMDMs from healthy mice were pretreated with a concentration gradient of METP NPs followed by lipopolysaccharide (LPS) stimulation and detection of m[Ca] levels in vitro. The optimal METP NP concentration was further applied, and the endoplasmic reticulum (ER) and cytoplasm calcium levels were detected. The inflammatory phenotype was measured by surface markers, cytokine secretion and intracellular inflammatory gene/protein expression. A Seahorse cell energy metabolism assay was performed to elucidate the mechanism by which METP NPs reverse the BMDM proinflammatory phenotype. RESULTS: The present study identified calcium overload in BMDM mitochondria of OA mice. We demonstrated that METP NPs reversed the increased m[Ca] levels in mitochondria and the proinflammatory phenotype of BMDMs, with both in vivo and in vitro experiments, via the inhibition of the mitochondrial aspartate-arginosuccinate shunt and ROS production. CONCLUSION: We demonstrated that METP NPs are effective and highly specific regulators of m[Ca] overload. In addition, we demonstrated that these METP NPs reverse the macrophage proinflammatory phenotype by restoring m[Ca] homeostasis, thereby inhibiting the tissue inflammatory response and achieving a therapeutic effect for OA.
简介：骨关节炎（OA）是一种慢性退行性关节疾病，伴有巨噬细胞促炎表型升高，这是由线粒体中持续病理性升高的钙离子水平引发的。然而，现有的靶向抑制线粒体钙离子（m[Ca]）内流的药理学化合物在质膜通透性和对离子通道和转运体的特异性方面受到限制。在本研究中，我们合成了介孔硅纳米粒子酰胺化（MSN）-乙二胺四乙酸（EGTA）/三苯基膦（TPP）-聚乙二醇（PEG）[METP]纳米粒子（NPs），这些 NPs 专门针对线粒体并阻断过量钙离子内流。 方法：通过荧光探针检测 OA 小鼠骨髓来源巨噬细胞（BMDM）中的 m[Ca]过载。使用组织原位荧光共定位测定法评估 METP NP 被巨噬细胞摄取的情况。用浓度梯度的 METP NPs 预处理健康小鼠的 BMDM，然后进行脂多糖（LPS）刺激并在体外检测 m[Ca]水平。进一步应用最佳 METP NP 浓度，检测内质网（ER）和细胞质钙水平。通过表面标志物、细胞因子分泌和细胞内炎症基因/蛋白表达来测量炎症表型。通过 Seahorse 细胞能量代谢测定法阐明 METP NPs 逆转 BMDM 促炎表型的机制。 结果：本研究鉴定出 OA 小鼠 BMDM 线粒体中的钙过载。我们通过抑制线粒体天冬氨酸-精氨酸穿梭和 ROS 产生，证明 METP NPs 逆转了体内和体外实验中 BMDM 线粒体中增加的 m[Ca]水平和促炎表型。 结论：我们证明 METP NPs 是 m[Ca]过载的有效且高度特异的调节剂。此外，我们证明这些 METP NPs 通过恢复 m[Ca]稳态来逆转巨噬细胞促炎表型，从而抑制组织炎症反应并实现 OA 的治疗效果。
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