Effects and Mechanism of Quercetin on Osteogenic Differentiation of BMSCs
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摘要:
目的 探究槲皮素对骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）成骨分化的影响及其作用机制。 方法 将BMSCs分为空白对照组（Control）和槲皮素（Quercetin）低剂量组（4.8 mL/kg）、中剂量组（9.6 mL/kg）、高剂量组（19.2 mL/kg）分别用含药血清进行干预，而阳性对照组用成骨分化培养基（Osteogenic differentiation medium）处理，采用流式细胞仪分析细胞周期，MTT法评估细胞增殖水平，碱性磷酸酶（alkaline phosphatase，ALP）检测试剂盒测定细胞活性，茜素红染色观察钙化结节形成。通过Real-time PCR和Western Blot检测β-连环蛋白（β-catenin）及成骨分化关键因子Runt相关转录因子2（runt-related transcription factor 2，RUNX2）和骨钙素（osteocalcin，OCN）的表达水平。 结果 与对照组相比，槲皮素含药血清促进BMSCs增殖（P = 0.000205 ，P =0.000063 ），并增强成骨分化后的钙结节形成，成骨活性和ALP活性。Western Blot和PCR结果显示，槲皮素调成骨分化过程中β-catenin （P <0.0001 ）、RUNX2 （P <0.0001 ）及OCN （P <0.0001 ）的mRNA和蛋白表达。结论 槲皮素能有效促进BMSCs成骨分化，其机制可能通过激活Wnt/β-catenin信号通路，上调成骨相关转录因子RUNX2的表达来实现。 -
关键词:
- 槲皮素 /
- 骨髓间充质干细胞 /
- 成骨分化 /
- Wnt/β-catenin
Abstract:Objective To explore the effect and mechanism of quercetin on osteogenic differentiation of bone marrow mesenchymal stem cells （BMSCs）. Methods BMSCs were divided into a blank control group （Control） and quercetin （Quercetin） low-dose group （4.8 mL/kg）, medium-dose group （9.6 mL/kg）, and high-dose group （19.2 mL/kg） intervened with drug-containing serum, while the positive control group was treated with osteogenic differentiation medium, respectively. The cell cycle was analysed by flow cytometry, cell proliferation was detected by MTT assay, cell activity was determined by Alkaline phosphatase （ALP） assay kit, and calcified nodule formation was observed by alizarin red staining. The expression of β-catenin and the key factors of osteogenic differentiation, runt-related transcription factor 2 （RUNX2） and osteocalcin （OCN）, were detected by qPCR and Western blot, respectively. Results Compared with the control group, quercetin-containing serum significantly promoted the proliferation of BMSCs （P = 0.000205 , P =0.000063 ） and enhanced the formation of calcium nodules, and increased osteogenic and ALP activity after osteogenic differentiation. The results of qPCR and Western blot showed that the quercetin group significantly up-regulated the mRNA and protein expression of β-catenin （P <0.0001 ）, RUNX2 （P <0.0001 ） and OCN （P <0.0001 ） during osteogenic differentiation.Conclusion Quercetin can effectively promote the osteogenic differentiation of BMSCs, and its mechanism is achieved by activating the Wnt/β-catenin signaling pathway and up-regulating the expression of the osteogenesis-related transcription factor RUNX2. -
图 1 槲皮素对BMSCs细胞活力的影响（$ \bar x \pm s $，n = 3）
与Control组比较，*P < 0.05，**P < 0.01，***P < 0.001。
Figure 1. Effect of quercetin on cell viability of BMSCs （$ \bar x \pm s $，n = 3）
图 2 槲皮素对BMSCs细胞周期的影响（$ \bar x \pm s $，n = 3）
与Control组比较，*P < 0.05，**P < 0.01，***P < 0.001。
Figure 2. Effect of quercetin on the cell cycle of BMSCs （$ \bar x \pm s $，n = 3）
图 3 槲皮素对BMSCs中ALP活性的影响（$ \bar x \pm s $，n = 3）
与Control组比较，**P < 0.01，***P < 0.001。
Figure 3. Effect of quercetin on ALP activity in BMSCs （$ \bar x \pm s $，n = 3）
图 4 各试验组茜素红染色（200×，Scale bar = 100 μm）
Figure 4. Alizarin red staining for each test group （200×，Scale bar = 100 μm）
图 5 槲皮素对β-catenin、RUNX2和OCN mRNA表达的影响（$ \bar x \pm s $，n = 3）
A：BMSCs中β-catenin的mRNA表达量；B：BMSCs中RUNX2的mRNA表达量；C：BMSCs中OCN的mRNA表达量；与Control组比较，**P < 0.01，***P < 0.001。
Figure 5. Effect of quercetin on mRNA expression of β-catenin，RUNX2 and OCN （$ \bar x \pm s $，n = 3）
图 6 槲皮素对β-catenin、RUNX2和OCN蛋白表达的影响（$ \bar x \pm s $，n = 3）
A：BMSCs中β-catenin、RUNX2和OCN的蛋白表达电泳图；B：BMSCs中β-catenin的蛋白表达量；C：BMSCs中RUNX2的蛋白表达量；D：BMSCs中OCN的蛋白表达量；与Control组比较，*P < 0.05，**P < 0.01，***P < 0.001。
Figure 6. Effect of quercetin on the expression of β-catenin，RUNX2 and OCN proteins （$ \bar x \pm s $，n = 3）
表 1 BMSCs的细胞周期分布（$\bar x \pm s $，%，n = 3）
Table 1. Cell cycle distribution of BMSCs （$\bar x \pm s $，%，n = 3）
实验分组 G1（%） S（%） G2（%） Control 71.4 ± 0.91 7.87 ± 1.44 16.85 ± 1.08 quercetin+4.8 mL/kg 65.41 ± 3.47* 7.97 ± 1.26 22.9 ± 5.29 quercetin+9.6 mL/kg 60.11 ± 1.92** 11.73 ± 4.15 22.27 ± 4.36 quercetin+19.2 mL/kg 54.33 ± 2.96** 12.14 ± 4.52 28.48 ± 3.7** Osteogenic differentiation medium 48.66 ± 3.03*** 12.81 ± 2.99 38.53 ± 3.62*** 多组间F 34.81 1.709 13.52 多组间P < 0.0001 ***0.2241 0.0005 ***与Control组比较，*P < 0.05，**P < 0.01，***P < 0.001。 
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