骨碎补总黄酮通过miR-125b-2-3p调控COL2A1对大鼠生长板软骨细胞增殖的影响

Total flavonoids from Drynariae rhizome promote growth plate chondrocyte proliferation via miR-125b-2-3p-mediated COL2A1 regulation in rats

关键词:  骨碎补总黄酮  印度豪猪蛋白-甲状旁腺激素相关蛋白通路  COL2A1基因  微小RNA 125b-2-3p  大鼠  生长板软骨细胞

Key words:  Drynariae rhizome total flavonoids  Ihh-PTHrP pathway  COL2A1  miR-125b-2-3p  rat  growth plate chondrocyte

摘要:

[目的] 研究旨在探讨骨碎补总黄酮通过调控大鼠生长板软骨细胞微小RNA 125b-2-3p（miR-125b-2-3p）表达对Ⅱ型胶原基因（COL2A1）和印度刺猬蛋白-甲状旁腺激素相关蛋白（Ihh-PTHrP）信号通路的调节作用及对细胞增殖的促进效果。[方法] 选取2周龄的SD大鼠胫骨近端生长板软骨细胞进行体外培养并完成细胞鉴定。采用不同浓度的骨碎补总黄酮对细胞进行处理，通过噻唑蓝染色（MTT）实验筛选出最能促进细胞生长的药物浓度。利用实时定量逆转录聚合酶链式反应（RT-qPCR）技术筛选出能够抑制甲状旁腺激素相关蛋白（PTHrP）表达的质粒。在后续实验中，设置多个实验组，包括空白对照组、骨碎补总黄酮组、Ihh-PTHrP通路激活组、PTHrP抑制剂组、骨碎补总黄酮+PTHrP组以及骨碎补总黄酮+PTHrP抑制剂组。通过MTT法评估骨碎补总黄酮和PTHrP对细胞增殖的影响，并通过酶联免疫吸附（ELISA）法测定细胞中COL2A1的表达水平。进一步利用生物信息学方法筛选可能调控COL2A1表达的微小核糖核酸（miRNAs），提取细胞培养上清液中的总RNA，并通过RT-qPCR分析筛选出的miRNAs在不同实验组中的表达差异。通过双荧光素酶报告基因实验验证miRNA对COL2A1表达的调控作用。最后，通过miRNA抑制剂和模拟物对COL2A1表达的影响，证实骨碎补总黄酮通过miRNA调控促进COL2A1的表达和细胞增殖。[结果] 发现250 mg/L的骨碎补总黄酮在促进细胞增殖方面效果最佳，骨碎补总黄酮组和骨碎补总黄酮+PTHrP组均能显著促进细胞增殖，而PTHrP抑制剂则抑制了细胞增殖。双荧光素酶报告基因实验确认miR-125b-2-3p能够调控COL2A1的表达，且在空白对照组和骨碎补总黄酮组中miR-125b-2-3p表达存在显著差异。进一步实验表明，骨碎补总黄酮和miR-125b-2-3p抑制剂均能提高COL2A1的表达，而miR-125b-2-3p模拟物则降低其表达。[结论] 骨碎补总黄酮通过降低miR-125b-2-3p的表达，增强Ihh-PTHrP通路中COL2A1的蛋白表达，从而促进生长板软骨细胞的增殖，对治疗与生长板软骨细胞增殖相关的疾病具有潜在的应用价值。

Abstract:

[Objective] To investigate how total flavonoids from Drynariae rhizome（TFD） promote rat growth plate chondrocyte proliferation through miR-125b-2-3p-mediated regulation of COL2A1 and the Ihh-PTHrP signaling pathway. [Methods] Growth plate chondrocytes from 2-week-old SD rats were isolated and cultured. After identification：optimal TFD concentration（250 mg/L） was determined by MTT assay. PTHrP-inhibiting plasmids were screened via qRT-PCR. Six experimental groups were established：blank control，TFD treatment，Ihh-PTHrP activation，PTHrP inhibition，TFD+PTHrP，TFD+PTHrP inhibitor. Cell proliferation（MTT），COL2A1 expression（ELISA），and miRNA screening（bioinformatics+qRT-PCR） were performed. miRNA-COL2A1 interactions were validated by dual-luciferase reporter assays. [Results] TFD（250 mg/L） maximally promoted proliferation（P<0.05）. Compared with the control group，the total flavonoids from TFD treatment group showed：significantly enhanced cell proliferation capacity，markedly downregulated miR-125b-2-3p expression，substantially upregulated COL2A1 gene expression. Dual-luciferase confirmed miR-125b-2-3p directly targets COL2A1，miR-125b-2-3p inhibitor mimicked TFD effects，while mimics antagonized them. [Conclusion] TFD enhances chondrocyte proliferation by downregulating miR-125b-2-3p to increase COL2A1 expression in the Ihh-PTHrP pathway，suggesting therapeutic potential for growth plate disorders.