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| 滋阴通络方含药血清经ERα/PI3K/Akt通路促进成骨机制 |
| Mechanism of Ziyin Tongluo Formula-containing serum inpromoting osteogenesis via the ERα/PI3K/Akt pathway |
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| DOI：10.3969/j.issn.1006-7108.2026.08.002 |
| 中文关键词: 滋阴通络方 MC3T3-E1 细胞 ERα/PI3K/Akt信号通路 成骨分化 |
| 英文关键词:Ziyin Tongluo formula MC3T3-E1 cells ERα/PI3K/Akt signaling pathway osteogenic differentiation |
| 基金项目:广东省中医药局面上项目（20251175）；广州市科技局市校联合青年项目（2025A03J1226） |
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| 中文摘要: |
| 目的 探讨滋阴通络方（Ziyin Tongluo Formula，ZYTLF）含药血清对小鼠成骨前体细胞 MC3T3-E1增殖、分化及雌激素受体α（estrogen receptor α，ERα）/磷脂酰肌醇-3-激酶（phosphatidylinositol 3-kinases，PI3K）/蛋白激酶B（protein kinase B，Akt）信号通路的影响。方法 采用CCK-8法检测5 %、10 %、15 %的ZYTLF含药血清作用6、12、24 h对MC3T3-E1细胞增殖的影响；通过碱性磷酸酶（alkaline phosphatase，ALP）染色（诱导第7天）和茜素红染色（诱导第21天）评估成骨分化能力。将细胞分为对照组、空白血清组、特立帕肽（Teriparatide, 10 nmol / L）组及ZYTLF含药血清组，Western Blot检测成骨蛋白Runt相关转录因子2（runt-related transcription factor 2，Runx2）、骨钙素（osteocalcin，OCN）、Ⅰ 型胶原蛋白（collagen Ⅰ，Col Ⅰ ）及ERα、磷酸化PI3K（p-PI3K）/PI3K、磷酸化Akt（p-Akt）/Akt的表达。设置ERα抑制剂ICI 182780( 1 μmol / L）干预组，通过RT-qPCR和Western Blot分析ZYTLF含药血清对Runx2、OCN和Col Ⅰ 等成骨相关指标、ERα 及 PI3K/Akt 通路的调控作用。结果 空白血清组与对照组相比无显著差异，而与空白血清组相比，10 % ZYTLF含药血清显著促进MC3T3-E1细胞增殖，增强ALP活性及矿化结节形成，并上调Runx2、OCN、Col Ⅰ及 ERα 蛋白表达（P<0.05），同时激活PI3K/Akt通路（p-PI3K/PI3K和p-Akt/Akt比值升高，P<0.05）。ERα 抑制剂干预后，上述成骨指标及通路的mRNA和蛋白表达均被显著抑制（P<0.05），而补充ZYTLF含药血清可逆转抑制效应（P<0.05）。结论 ZYTLF含药血清可以激活ERα/PI3K/Akt通路促进成骨细胞增殖分化，其机制可能由ERα/PI3K/Akt轴介导。 |
| 英文摘要: |
| Objective To investigate the effects of Ziyin Tongluo Formula (ZYTLF)-containing serum on the proliferation and differentiation of mouse osteoblastic precursor cells (MC3T3-E1) and the estrogen receptor α (ERα)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Methods Cell proliferation was assessed by CCK-8 assay after treatment with 5%, 10%, or 15% ZYTLF-containing serum for 6, 12, or 24 h. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) staining (day 7) and Alizarin Red S staining (day 21). Cells were divided into four groups: control, blank serum, teriparatide (10 nmol/L), and ZYTLF-containing serum. Western blotting was performed to detect osteogenic proteins (Runx2, OCN, Col I) and ERα, p-PI3K/PI3K, p-Akt/Akt expression. An ERα inhibitor (ICI 182780, 1 μmol/L) intervention group was established. RT-qPCR and Western blotting analyzed ZYTLF-containing serum’s regulatory effects on osteogenic markers (Runx2, OCN, Col I), ERα, and PI3K/Akt signaling. Results No significant differences were observed between the blank serum group and the control group. In contrast, compared with the blank serum group, treatment with 10% ZYTLF-containing serum significantly enhanced the proliferation of MC3T3-E1 cells (P<0.05), increased alkaline phosphatase (ALP) activity, and promoted mineralization nodule formation (P<0.05). Concurrently, this treatment upregulated protein expression of osteogenic markers including Runx2, osteocalcin (OCN), collagen type I (Col I), and ERα (P<0.05). Furthermore, the PI3K/Akt pathway was activated, as evidenced by elevated ratios of phosphorylated PI3K to total PI3K (p-PI3K/PI3K) and phosphorylated Akt to total Akt (p-Akt/Akt) (P<0.05). Following intervention with the ERα inhibitor ICI 182780 (1 μmol/L), mRNA and protein expression levels of the aforementioned osteogenic markers (Runx2, OCN, Col I) and pathway components (ERα, p-PI3K/PI3K, p-Akt/Akt) were markedly suppressed (P<0.05). Notably, supplementation with ZYTLF-containing serum effectively reversed these inhibitory effects (P<0.05). Conclusion ZYTLF-containing serum promotes osteoblast proliferation and differentiation by activating the ERα-PI3K-Akt signaling pathway, indicating mediation by the ERα/PI3K/Akt axis. |
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