背景:先前的研究表明,中药可以通过增强间充质干细胞的功能来治疗炎症和缺氧疾病。然而,其机制尚不清楚。本研究旨在探讨中药四君子汤及其单体人参皂苷Rg1对人脐带间充质干细胞(hUC-MSCs)增殖和分化的影响及其作用机制。
方法:应用不同浓度的四君子汤和Rg1对诱导的hUC-MSCs进行分化。CCK-8测试用于评估细胞增殖活性并鉴定合适的药物浓度。采用茜素红染色法检测钙结节的形成,和油红O染色用于评估脂滴的形成。PCR用于检测与成骨分化相关的基因表达,成脂分化,和HIF-1α信号通路在hUC-MSCs中的作用。进行蛋白质印迹分析以评估成骨分化和HIF-1α中的蛋白表达。进行ELISA以测量HIF-1α信号传导因子和炎性细胞因子表达。生化测定用于评估氧化应激指标的变化。
结果:四君子汤和Rg1均显示出剂量依赖性的促进hUC-MSC增殖。四君子汤可显著提高成骨相关基因和蛋白的表达,比如Osterix,骨钙蛋白,RUNX2和骨桥蛋白,并激活hUC-MSCs中的HIF-1α通路。(P<0.05)。用Rg1处理后在基因水平观察到类似的效果。同时,四君子汤能显著降低促炎细胞因子TNF-α的分泌,IL-6和IL-1β,同时增加了成骨分化过程中抗炎细胞因子IL-10的分泌(P<0.05)。此外,四君子汤可降低hUC-MSCs成骨分化过程中的氧化应激水平,增强其抗氧化能力(P<0.05)。然而,四君子汤对hUC-MSCs成脂分化的影响不显著(P>.05)。
结论:四君子汤可促进hUC-MSCs的增殖和成骨分化,可能通过激活HIF-1α信号通路和通过降低炎症和氧化应激水平调节微环境。Rg1可能参与此过程。
BACKGROUND: Previous researches have demonstrated that the traditional Chinese medicine could therapeutically treat inflammatory and hypoxic diseases by enhancing the functionality of mesenchymal stem cells. However, its mechanism was not yet clear. This research aimed to investigate the impact of the traditional Chinese medicine Sijunzi decoction and its herb monomer ginsenoside Rg1 on the proliferation and differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) and explore the underlying mechanisms.
METHODS: Different concentrations of Sijunzi decoction and Rg1 were applied to differentiating induced hUC-MSCs. The CCK-8 test was utilized to evaluate cell proliferation activity and identify suitable drug concentrations. Alizarin Red staining was employed to detect the formation of calcium nodules, and Oil Red O staining was used to assess the formation of lipid droplets. PCR was utilized to examine gene expression related to osteogenic differentiation, adipogenic differentiation, and the HIF-1α signaling pathway in hUC-MSCs. Western blot analysis was conducted to evaluate protein expression in osteogenic differentiation and HIF-1α. ELISA was performed to measure HIF-1α signaling factors and inflammatory cytokine expression. Biochemical assays were used to assess changes in oxidative stress indicators.
RESULTS: The Sijunzi decoction and Rg1 both demonstrated a dose-dependent promotion of hUC-MSC proliferation. The Sijunzi decoction significantly increased the expression of genes and proteins relevant to osteogenesis, such as osterix, osteocalcin, RUNX2, and osteopontin, and activated the HIF-1α pathway in hUC-MSCs. (P < .05). Similar effects were observed at the gene level after treatment with Rg1. Simultaneously, Sijunzi decoction significantly reduced the secretion of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, while increasing the secretion of the anti-inflammatory cytokine IL-10 during osteogenic differentiation (P < .05). Moreover, Sijunzi decoction lowered oxidative stress levels and enhanced the antioxidant capacity of hUC-MSCs during osteogenic differentiation (P < .05). However, the impact of Sijunzi decoction on hUC-MSCs toward adipogenic differentiation was not significant (P > .05).
CONCLUSIONS: Sijunzi decoction promotes the proliferation and osteogenic differentiation of hUC-MSCs, potentially through the activation of the HIF-1α signaling pathway and by modulating the microenvironment via reducing inflammation and oxidative stress levels. Rg1 might be involved in this process.