Aim. The present study evaluated the therapeutic effects of luteolin in alleviating pulpitis of dental pulp- (DP-) derived microvesicles (MVs) via the inhibition of protein kinase R- (PKR-) mediated inflammation. Methodology. Proteomic analysis of immortalized human dental pulp (DP-1) cell-derived MVs was performed to identify PKR-associated molecules. The effect of luteolin on PKR phosphorylation in DP-1 cells and the expression of tumor necrosis factor-α (TNF-α) in THP-1 macrophage-like cells were validated. The effect of luteolin on cell proliferation was compared with that of chemical PKR inhibitors (C16 and 2-AP) and the unique commercially available sedative guaiacol-parachlorophenol. In the dog experimental pulpitis model, the pulps were treated with (1) saline, (2) guaiacol-parachlorophenol, and (3) luteolin. Sixteen teeth from four dogs were extracted, and the pulp tissues were analyzed using hematoxylin and eosin staining. Immunohistochemical staining was performed to analyze the expression of phosphorylated PKR (pPKR), myeloperoxidase (MPO), and CD68. Experimental endodontic-periodontal complex lesions were established in mouse molar through a silk ligature and simultaneous MV injection. MVs were prepared from DP-1 cells with or without pretreatment with 2-AP or luteolin. A three-dimensional microcomputed tomography analysis was performed on day 7 (). Periodontal bone resorption volumes were calculated for each group (nonligated–ligated), and the ratio of bone volume to tissue volume was measured. Results. Proteomic analysis identified an endogenous PKR activator, and a protein activator of interferon-induced PKR, also known as PACT, was included in MVs. Luteolin inhibited the expressions of pPKR in DP-1 cells and TNF-α in THP-1 cells with the lowest suppression of cell proliferation. In the dog model of experimental pulpitis, luteolin treatment suppressed the expression of pPKR-, MPO-, and CD68-positive cells in pulp tissues, whereas guaiacol-parachlorophenol treatment caused coagulative necrosis and disruption. In a mouse model of endodontic-periodontal complex lesions, luteolin treatment significantly decreased MV-induced alveolar bone resorption. Conclusion. Luteolin is an effective and safe compound that inhibits PKR activation in DP-derived MVs, enabling pulp preservation.

中文翻译：

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木犀草素是一种潜在的免疫调节天然化合物，可对抗牙髓炎症

目的。本研究评估了木犀草素通过抑制蛋白激酶 R- (PKR-) 介导的炎症来缓解牙髓 (DP-) 衍生微泡 (MV) 牙髓炎的治疗效果。方法。对永生化人牙髓 (DP-1) 细胞衍生的 MV 进行蛋白质组学分析，以鉴定 PKR 相关分子。验证了木犀草素对 DP-1 细胞中 PKR 磷酸化和 THP-1 巨噬细胞样细胞中肿瘤坏死因子-α（TNF- α ）表达的影响。将木犀草素对细胞增殖的影响与化学 PKR 抑制剂（C16 和 2-AP）以及独特的市售镇静剂愈创木酚-对氯苯酚进行了比较。在狗实验性牙髓炎模型中，用（1）盐水、（2）愈创木酚-对氯苯酚和（3）木犀草素处理牙髓。拔除四只狗的十六颗牙齿，并使用苏木精和伊红染色对牙髓组织进行分析。采用免疫组织化学染色分析磷酸化 PKR (pPKR)、髓过氧化物酶 (MPO) 和 CD68 的表达。通过丝结扎和同时注射 MV，在小鼠磨牙中建立了实验性牙髓-牙周复合体病变。MV 由 DP-1 细胞制备，无论是否经过 2-AP 或木犀草素预处理。第 7 天进行了三维显微计算机断层扫描分析（）。计算每组（未结扎-结扎）的牙周骨吸收体积，并测量骨体积与组织体积的比率。结果。蛋白质组学分析鉴定出一种内源性 PKR 激活剂，并且干扰素诱导的 PKR 的蛋白质激活剂（也称为 PACT）包含在 MV 中。木犀草素抑制 DP-1 细胞中 pPKR 和 THP-1 细胞中 TNF- α的表达，对细胞增殖的抑制作用最低。在实验性牙髓炎的狗模型中，木犀草素治疗抑制牙髓组织中 pPKR、MPO 和 CD68 阳性细胞的表达，而愈创木酚-对氯酚治疗则引起凝固性坏死和破坏。在牙髓-牙周复合体病变的小鼠模型中，木犀草素治疗显着降低了 MV 诱导的牙槽骨吸收。结论。木犀草素是一种有效且安全的化合物，可抑制 DP 衍生 MV 中的 PKR 激活，从而实现牙髓保存。