Department of Orthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Research Center of Dental Materials and Oral Tissue Regeneration & Shandong Provincial Clinical Research Center for Oral Diseases, Jinan, Shandong, People's Republic of China.
Drug Des Devel Ther. 2024 Nov 4;18:4943-4960. doi: 10.2147/DDDT.S482969. eCollection 2024.
This study examines the effects and mechanisms of catalpol (CAT) on the proliferation and osteogenic differentiation of cultured human periodontal ligament stem cells (hPDLSCs) in vitro and assesses the impact of CAT on periodontal remodeling in vivo using an orthodontic tooth movement (OTM) model in rats.
hPDLSCs were cultured in a laboratory setting, and their proliferation and osteogenic differentiation were assessed using the Cell-counting Kit-8 (CCK-8), Alizarin Red Staining (ARS), quantitative calcium assay, alkaline phosphatase (ALP) staining and activity assay, and immunofluorescence assay. Additionally, the expression of collagen type 1 (COL-1), ALP, and runt-related transcription factor-2 (RUNX-2) was evaluated through qRT-PCR and Western blot analysis. To verify the function of the estrogen receptor-α (ER-α)-mediated phosphatidylinositol-3-kinase-protein kinase B (PI3K/AKT) pathway in this mechanism, LY294002 (a PI3K signaling pathway inhibitor) and the ER-α specific inhibitor methyl-piperidine-pyrazole (MPP) were used. The osteogenic markers ER-α, AKT, and p-AKT (phosphoprotein kinase B) were identified through Western blot analysis. Eighteen male Sprague-Dawley rats were assigned to two groups randomly: a CAT group receiving CAT and a control group receiving an equivalent volume of saline. Micro-computed tomography (micro-CT) analysis was employed to evaluate tooth movement and changes in alveolar bone structure. Morphological changes in the periodontal tissues between the roots were investigated using hematoxylin and eosin (HE) staining and tartaric-resistant acid phosphatase (TRAP) staining. The expression of COL-1, RUNX-2, and nuclear factor-κB (NF-κB) ligand (RANKL) was assessed through immunohistochemical staining (IHC) to evaluate periodontal tissue remodeling. Tests were analyzed using GraphPad Prism 8 software. Differences among more than two groups were analyzed by one-way or two-way analysis of variance (ANOVA) followed by the Tukey's test. Values of < 0.05 were regarded as statistically significant.
In vitro experiments demonstrated that 10 μM CAT significantly promoted the proliferation, ALP activity, and calcium nodule formation of hPDLSCs, with a notable increase in the expression of COL-1, ALP, RUNX-2, ER-α, and p-AKT. The PI3K/AKT pathway was inhibited by LY294002, and further analysis using MPP suggested that ER-α mediated this effect. In vivo, experiments indicated that CAT enhanced the expression of COL-1 and RUNX-2 on the tension side of rat tooth roots, reduced the number of osteoclasts on the compression side, inhibited RANKL expression, and suppressed OTM.
CAT can promote hPDLSCs proliferation and osteogenic differentiation in vitro through the ER-α/PI3K/AKT pathway and enhance periodontal tissue remodeling in vivo using OTM models. These findings suggest the potential for the clinical application of catalpol in preventing relapse following OTM.
本研究旨在探讨梓醇（CAT）对体外培养的人牙周膜干细胞（hPDLSCs）增殖和成骨分化的影响及其作用机制，并通过大鼠正畸牙齿移动（OTM）模型评估 CAT 对体内牙周重塑的影响。
在实验室条件下培养 hPDLSCs，使用细胞计数试剂盒-8（CCK-8）、茜素红染色（ARS）、定量钙测定、碱性磷酸酶（ALP）染色和活性测定以及免疫荧光染色评估 hPDLSCs 的增殖和成骨分化。此外，通过 qRT-PCR 和 Western blot 分析评估胶原 I 型（COL-1）、ALP 和 runt 相关转录因子 2（RUNX-2）的表达。为了验证雌激素受体-α（ER-α）介导的磷脂酰肌醇 3-激酶蛋白激酶 B（PI3K/AKT）通路在该机制中的作用，使用了 LY294002（PI3K 信号通路抑制剂）和 ER-α 特异性抑制剂甲基-哌啶-吡唑（MPP）。通过 Western blot 分析鉴定成骨标志物 ER-α、AKT 和磷酸化蛋白激酶 B（p-AKT）。将 18 只雄性 Sprague-Dawley 大鼠随机分为两组：CAT 组给予 CAT，对照组给予等量生理盐水。采用微计算机断层扫描（micro-CT）分析评估牙齿移动和牙槽骨结构变化。使用苏木精和伊红（HE）染色和抗酒石酸酸性磷酸酶（TRAP）染色观察根间牙周组织的形态变化。通过免疫组织化学染色（IHC）评估 COL-1、RUNX-2 和核因子-κB 配体（RANKL）的表达，以评估牙周组织重塑。使用 GraphPad Prism 8 软件进行测试分析。采用单因素或双因素方差分析（ANOVA）加 Tukey 检验分析两组以上的差异。当 P 值<0.05 时，认为差异具有统计学意义。
体外实验表明，10 μM CAT 显著促进 hPDLSCs 的增殖、ALP 活性和钙结节形成，COL-1、ALP、RUNX-2、ER-α 和 p-AKT 的表达显著增加。LY294002 抑制 PI3K/AKT 通路，进一步使用 MPP 分析表明 ER-α 介导了这种作用。体内实验表明，CAT 增强了大鼠牙根张力侧 COL-1 和 RUNX-2 的表达，减少了压缩侧破骨细胞的数量，抑制了 RANKL 的表达，并抑制了 OTM。
CAT 可通过 ER-α/PI3K/AKT 通路促进体外 hPDLSCs 的增殖和成骨分化，并通过 OTM 模型增强体内牙周组织重塑。这些发现提示梓醇在预防 OTM 后复发方面具有临床应用潜力。
