血根碱通过NF-κB信号通路调控牙周膜干细胞成骨分化

Sanguinarine Regulates Osteogenic Differentiation of Periodontal Ligament Stem Cells through NF-κB Signal Pathway

目的:探究血根碱(SAN)对肿瘤坏死因子-α(TNF-α)处理的人牙周膜干细胞(hPDLSCs)成骨分化的影响及机制。方法:将h PDLSCs分为6组:Control组、TNF-α组、0.1SAN组、1SAN组、10SAN组和100SAN组,所有hPDLSCs均用成骨诱导培养液培养。除Control组外,其他组细胞培养液中均添加10 ng/mL的TNF-α。0.1SAN组、1SAN组、10SAN组和100SAN组细胞培养液中分别添加0、0.1、1、10和100μmol/L的血根碱。各组hPDLSCs均在37℃、5%CO_(2)条件下培养21 d。通过可见光比色法检测碱性磷酸酶(ALP)活性。通过茜素红染色观察钙化结节形成,并统计OD_(562 nm)(代表钙化结节形成量)。通过qRT-PCR检测Runt相关转录因子2(RUNX2)、骨钙素(OCN)、osterix(OSX)、牙骨质附着蛋白(CAP)、Smad4转录水平。通过Western blot检测核因子-κB(NF-κB)p65磷酸化水平。结果:与Control组比较,TNF-α组细胞的相对ALP活性降低和钙化结节形成量以及RUNX2、OCN、OSX、CAP和Smad4的m RNA相对表达量降低(P<0.05),p-NF-κB p65/NF-κB p65升高(P<0.05)。与TNF-α组比较,1SAN组、10SAN组和100SAN组的相对ALP活性和钙化结节形成量以及RUNX2、OCN、OSX、CAP和Smad4的m RNA相对表达量升高(P<0.05),p-NF-κB p65/NF-κB p65降低(P<0.05)。结论:血根碱可促进TNF-α处理的hPDLSCs的成骨分化,其机制可能与抑制NF-κB的激活有关,血根碱可能是促进炎性微环境中hPDLSCs成骨分化的候选药物。

Objective:To investigate the effect and mechanism of sanguinarine(SAN)on osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs)treated with tumor necrosis factor-α(TNF-α).Methods:hPDLSCs were divided into 6 groups:Control group,TNF-αgroup,TNF-α+0.1SAN group,TNF-α+1SAN group,TNF-α+10SAN group and TNF-α+100SAN group.All hPDLSCs were cultured in osteogenic induction medium.Except Control group,10 ng/mL TNF-αwas added to the culture medium of other groups.0,0.1,1,10,100μmol/L sanguinarine were added to the culture medium of TNF-α+0.1SAN group,TNF-α+1SAN group,TNF-α+10SAN group and TNF-α+100SAN group,respectively.HPDLSCs of all groups were cultured at 37℃and 5%CO_(2) for 21 days.The activity of alkaline phosphatase(ALP)was detected by visible light colorimetry.The formation of calcified nodules was observed by alizarin red staining,and OD_(562 nm)(representing the amount of calcified nodules)was counted.The transcription levels of Runt-related transcription factor 2(RUNX2),osteocalcin(OCN),osterix(OSX),cementum attachment protein(CAP)and Smad4 were detected by qRT-PCR.The phosphorylation level of NF-kappa B(NF-κB)p65 was detected by Western blot.Results:Compared with that in the Control group,the relative ALP activity,amount of calcified nodules,and the relative expression of RUNX2,OCN,OSX,CAP and Smad4 mRNA in TNF-αgroup decreased(P<0.05),while p-NF-κB p65/NF-κB p65 increased(P<0.05).Compared with that in the TNF-αgroup,the relative ALP activity,amount of calcified nodules,RUNX2,OCN,OSX,CAP and Smad4 mRNA expression of TNF-α+1SAN group,TNF-α+10SAN group and TNF-α+100SAN group increased(P<0.05),while p-NF-κB p65/NF-κB p65 decreased(P<0.05).Conclusion:Sanguinarine can promote the osteogenic differentiation of hPDLSCs treated with TNF-α,and the mechanism may be related to the inhibition of the activation of NF-κB.Sanguinarine may be a candidate drug to promote the osteogenic differentiation of hPDLSCs in inflammatory microenvironment.