7,8-二羟基黄酮通过Wnt/β-catenin信号通路调控人牙周膜干细胞成骨分化

7,8-dihydroxyflavone regulates osteogenic differentiation of human periodontal ligament stem cells through Wnt/β-catenin signaling pathway

目的 探究7,8-二羟基黄酮(7,8-DHF)对人牙周膜干细胞(hPDLSCs)成骨分化的影响和机制。方法 将从健康人牙周膜组织中分离培养的hPDLSCs用不同浓度的7,8-二羟基黄酮(0,0.5,1,5,10,20,40μmol/L)处理,通过MTT法检测hPDLSCs活性。将hPDLSCs分为5组:对照组、低剂量组、中剂量组、高剂量组和XAV939(Wnt/β-catenin信号通路抑制剂)组。各组hPDLSCs均用成骨诱导液培养,对照组的成骨诱导液不添加药物,低剂量组、中剂量组和高剂量组的成骨诱导液中分别添加0.5,1,5μmol/L的7,8-二羟基黄酮,XAV939组的成骨诱导液中同时添加5μmol/L的7,8-二羟基黄酮和10μmol/L的XAV939。各组hPDLSCs共培养21 d,每2 d更换培养基,检测碱性磷酸酶(ALP)活性。通过茜素红染色观察钙化结节形成(OD_(590 nm))。通过qRT-PCR检测Runt相关转录因子2(Runx2)、骨钙素(OCN)、骨桥蛋白(OPN)、骨形态发生蛋白-2(BMP-2)、Wnt-3a、β-catenin和糖原合成酶激酶-3β(GSK-3β)的mRNA表达水平。通过Western blot检测Wnt-3a、β-catenin和GSK-3β的蛋白表达水平。结果 不同浓度的7,8-二羟基黄酮处理后,hPDLSCs的相对活力差异无统计学意义(F=1.693,P=0.152)。与对照组相比,低剂量组、中剂量组和高剂量组hPDLSCs的ALP相对活性升高,钙化结节形成量升高,Runx2、OCN、OPN和BMP-2的mRNA相对表达量升高,Wnt-3a和β-catenin蛋白相对表达量升高,GSK-3β蛋白相对表达量降低(均P<0.05)。与低剂量组相比,中剂量组和高剂量组hPDLSCs中ALP相对活性升高,钙化结节形成量升高,Runx2、OCN、OPN和BMP-2的mRNA相对表达量升高,Wnt-3a和β-catenin蛋白相对表达量升高,GSK-3β蛋白相对表达量降低(均P<0.05)。与高剂量组相比,XAV939组hPDLSCs中ALP相对活性降低,钙化结节形成量降低,Runx2、OCN、OPN和BMP-2的mRNA相对表达量降低,Wnt-3a和β-catenin蛋白相对表达量降低,GSK-3β蛋白相对表达量升高(均P<0.05)。结论 7,8-二羟基黄酮通过激活Wnt/β-catenin信号通路促进hPDLSCs成骨分化。

Objective To investigate the effect of 7,8-dihydroxyflavone(7,8-DHF) on the osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs) and its mechanism.Methods The hPDLSCs isolated from the periodontal tissue of healthy human were treated with different concentrations of 7,8-dihydroxyflavone(0,0.5,1,5,10,20,40 μmol/L),and then the activity of hPDLSCs was detected by MTT assay.The hPDLSCs were divided into 5 groups:control group,low-dose group,medium-dose group,high-dose group,and XAV939(a Wnt/β-catenin signaling pathway inhibitor) group.The hPDLSCs in control group were only cultured with osteogenic induction solution.The hPDLSCs in low-dose group,medium-dose group and high-dose group were cultured with osteogenic induction solutions containing 0.5,1,5 μmol/L 7,8-dihydroxyflavone,respectively.The hPDLSCs in XAV939 group were cultured with osteogenic induction solution containing 5 μmol/L 7,8-dihydroxyflavone and 10 μmol/L XAV939 at the same time.The hPDLSCs were cultured for 21 d in each group,and the medium was changed every two days.Alkaline phosphatase(ALP) activity was detected and calcified nodule formation was detected by Alizarin red staining.The mRNA expression levels of Runt-associated transcription factor 2(Runx2),osteocalcin(OCN),osteopontin(OPN),bone morphogenetic protein-2(BMP-2),Wnt-3a,β-catenin and glycogen synthase kinase-3β(GSK-3β) were detected by qRT-PCR.The protein expression levels of Wnt-3a,β-catenin and GSK-3β were detected by Western blot.Results There was no significant difference in the relative activity of hPDLSCs after treated with different concentrations of 7,8-dihydroxyflavone(F=1.693,P=0.152).Compared with control group,the relative ALP activity of hPDLSCs in low-dose group,medium-dose group and high-dose group increased,the formation of calcified nodule increased,the relative mRNA levels of Runx2,OCN,OPN and BMP-2 increased,and the relative expression levels of Wnt-3a and β-catenin increased,while the relative expression level of GSK-3β decreased(P<0.05).Compared with low-dose group,the relative ALP activity of hPDLSCs in medium-dose group and high-dose group increased,the formation of calcified nodule increased,the relative mRNA levels of Runx2,OCN,OPN and BMP-2 increased,the relative expression levels of Wnt-3a and β-catenin increased,while the relative expression level of GSK-3β decreased(P<0.05).Compared with high-dose group,the relative ALP activity of hPDLSCs decreased in XAV939 group,the formation of calcified nodule decreased,the relative mRNA expression levels of Runx2,OCN,OPN and BMP-2 decreased,the relative expression levels of Wnt-3a and β-catenin decreased,while the relative expression level of GSK-3β increased(P<0.05).Conclusion 7,8-dihydroxyflavone can promote the osteogenic differentiation of hPDLSCs by activating Wnt/β-catenin signaling pathway.