炎症微环境作用下P38 MAPK调控Wnt/Ca^(2+)信号通路对牙周膜干细胞成骨分化的影响研究

Effect of P38 MAPK on Osteogenic Differentiation of Periodontal Ligament Stem Cells by Regulating Wnt/Ca^(2+) Signaling Pathways under Inflammatory Microenvironment

目的探讨炎症微环境作用下P38丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)对牙周膜干细胞(periodontal ligament stem cell,PDLSC)成骨分化和Wnt/Ca^(2+)信号通路的影响。方法将人PDLSC分为对照组、TNF-α组、TNF-α+SB203580组(20μmol/L SB20358)、TNF-α+SB203580+XAV939组(20μmol/L SB20358+10μmol/L XAV939),除对照组外其余各组细胞给予10 ng/mL TNF-α模拟炎症微环境。茜素红染色检测成骨分化能力,比较各组细胞碱性磷酸酶(alkaline phosphatase,ALP)活性,RT-qPCR检测成骨关键基因Runt相关转录因子2(Runx2)、骨钙蛋白(osteocalcin,OCN)、Osterix(OSX)蛋白、骨桥蛋白(osteopontin,OPN)mRNA水平,蛋白质印迹检测P38 MAPK、β-catenin、钙调蛋白依赖性激酶Ⅱ(CaMKⅡ)、Nemo样激酶(Nemo-like kinase,NLK)蛋白质表达。结果TNF-α组细胞的染色强度明显减弱且钙化结节的数量明显减少,TNF-α+SB203580组的染色强度、钙化结节数量较TNF-α组得到明显改善,TNF-α+SB203580+XAV939组细胞的染色强度、钙化结节数量与TNF-α+SB203580组差别不明显。与对照组比较,TNF-α组ALP活性和Runx2、OCN、OSX、OPN mRNA水平降低(P<0.05),P38 MAPK蛋白质表达(P<0.05),β-catenin、CaMKⅡ、NLK蛋白质表达降低(P<0.05)。与TNF-α组比较,TNF-α+SB203580组ALP活性和Runx2、OCN、OSX、OPN mRNA水平升高(P<0.05),P38 MAPK蛋白表达质降低(P<0.05),β-catenin、CaMKⅡ、NLK蛋白质表达升高(P<0.05)。与TNF-α+SB203580组,TNF-α+SB203580+XAV939组β-catenin蛋白质表达降低(P<0.05),但ALP活性、Runx2、OCN、OSX、OPN mRNA水平和P38 MAPK、CaMKⅡ、NLK蛋白质表达比较无统计差异(P>0.05)。结论炎症微环境下条件下P38 MAPK抑制剂可提高PDLSC成骨分化能力,可能与激活Wnt/Ca^(2+)信号通路有关。

Objective To explore the effect of P38 mitogen-activated protein kinase(MAPK)on osteogenic differentiation and Wnt/Ca^(2+)signaling pathways in periodontal ligament stem cells(PDLSC)under microinflammatory environment.Methods Human PDLSC were divided into 4 groups:control group,TNF-αgroup,TNF-α+SB203580 group(20μmol/L SB20358)and TNF-α+SB203580+XAV939 group(20μmol/L SB20358+10μmol/L XAV939).Except for cells in the control group,cells in the other groups were given 10 ng/mL TNF-αto simulate inflammatory microenvironment.The osteogenic differentiation ability was detected by alizarin red staining.The activity of alkaline phosphatase(ALP)in each group was compared.The expression levels of RUNt-related transcription factor 2(Runx2),osteocalcin(OCN),Osterix(OSX)and osteopontin(OPN)were detected by qRT-PCR,and the expression levels of P38 MAPK,β-catenin,calmodulin dependent protein kinaseⅡ(CaMKⅡ)and Nemo-like(NLK)were detected by Western blotting.Results The staining intensity and number of calcification nodules in the TNF-αgroup were significantly decreased,and in comparison of those in the TNF-αgroup,the staining intensity and number of calcification nodules were significantly improved in the TNF-α+SB203580 group,but there was no significant difference in staining intensity or number of calcification nodules between the TNF-α+SB203580+XAV939 group and the TNF-α+SB203580 group.Compared with those in the control group,the activity of ALP and mRNA expression levels of Runx2,OCN,OSX and OPN were decreased in the TNF-αgroup(P<0.05),the expression level of P38 MAPK protein was increased(P<0.05),and the expression levels ofβ-catenin,CaMKⅡand NLK proteins were decreased(P<0.05).Compared with those in the TNF-αgroup,the activity of ALP and mRNA expression levels of Runx2,OCN,OSX and OPN were increased in the TNF-α+SB203580 group(P<0.05),the expression level of P38 MAPK protein was decreased(P<0.05),and the expression levels ofβ-catenin,CaMKⅡand NLK proteins were increased(P<0.05).Compared with those in the TNF-α+SB203580 group,the expression level ofβ-catenin protein was decreased in the TNF-α+SB203580+XAV939 group(P<0.05),but there was no significant difference in ALP activity,mRNA expression levels of Runx2,OCN,OSX and OPN or expression levels of P38 MAPK,CaMKⅡand NLK proteins between the two groups(P>0.05).Conclusion P38 MAPK inhibitor can improve osteogenic differentiation of PDLSC under inflammatory microenvironment,which may be related to activating Wnt/Ca^(2+)signaling pathways.