LED红光上调MAPK信号促进炎性环境中人牙髓干细胞成骨/成牙本质分化

LED red light up⁃regulates MAPK signal to promote osteogenic/odontogenic differentiation of human dental pulp stem cells in an inflammatory environment

目的探讨发光二极管(light-emitting diode,LED)红光对人牙髓干细胞(human dental pulp stem cells,hDPSCs)成骨/成牙本质分化的影响及机制。方法本实验研究已通过单位伦理委员会审查批准。组织块酶消化法培养hDPSCs,在0、1、5、10μg/mL脂多糖(lipopolysaccharide,LPS)刺激下,CCK-8法检测hDPSCs增殖,筛选LPS刺激浓度。设置CG组(矿化诱导)、LPS+CG组、LPS+CG+LED光照组(能量分别为2、4、6、8、10 J/cm^(2))。第7天进行碱性磷酸酶(alkaline phosphatase,ALP)染色及活性测定,实时荧光定量PCR检测ALP、成骨细胞特异性转录因子(osterix,OSX)、牙本质基质蛋白-1(dentin matrix protein-1,DMP-1)、牙本质涎磷蛋白(dentin sialo-phosphoprotein,DSPP)基因表达情况,第21天进行茜素红染色及钙结节定量分析,筛选最佳光照能量。设置LPS+CG组、LPS+CG+LED组(最佳能量),ELISA法检测第1、3、5、7天肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及白介素-1β(interlenkin-1β,IL-1β)表达量。Western blot法检测细胞外调节蛋白激酶1/2(extracellular regulated protein kinases 1/2,ERK1/2)、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、p38、细胞外调节蛋白激酶5(extracellular regulated protein kinases 5,ERK5)及磷酸化蛋白表达。分别阻断ERK1/2、JNK、ERK5、p38通路后,Western blot法检测第7天ALP、OSX、DMP-1、DSPP蛋白表达。结果CCK-8结果显示10μg/mL LPS诱导下,在第5、7天hDPSCs增殖低于0、1、5μg/mL LPS组(P<0.05),后续选择10μg/mL作为LPS刺激浓度。ALP染色及活性,ALP、OSX、DMP-1、DSPP的基因表达水平及钙结节定量结果示LPS+CG+4 J/cm^(2)组均高于其他处理组(P<0.05)。4 J/cmLED红光上调MAPK信号促进炎性环境中人牙髓干细胞成骨/成牙本质分化LED红光促成骨/成牙本质分化能力最强,作为后续实验光照能量。ELISA结果显示第5、7天,LPS+CG+LED组的TNF-α与IL-1β的分泌表达量低于LPS+CG组(P<0.05)。Western blot结果显示4 J/cm^(2) LED红光促进p-ERK1/2、p-p38、p-JNK、p-ERK5蛋白表达;分别阻断通路后,LPS+CG+LED+U0126(抑制ERK1/2)、SP600125(抑制JNK)、BIX02189(抑制ERK5)组ALP、OSX、DMP-1、DSPP蛋白表达量低于LPS+CG+LED组(P<0.001),LPS+CG+LED+SB203580(抑制p38)组ALP、OSX、DMP-1蛋白表达量与LPS+CG+LED组比较无显著差异(P>0.05)。结论LED红光促进炎症环境下hDPSCs成骨/成牙本质分化,其作用机制可能为通过上调ERK1/2、JNK、ERK5信号减少炎症因子TNF-α与IL-1β释放有关。

Objective To study the effect of light-emitting diode(LED)red light on the osteogenic/odontogenic differentiation of human dental pulp stem cells(hDPSCs)and its mechanism were discussed.Methods This study has been reviewed and approved by the Ethics Committee.hDPSCs were cultured by tissue block enzyme digestion.The proliferative capacity of hDPSCs was detected by the CCK-8 at days 1,3,5 and 7 under stimulation with 0,1,5 and 10μg/mL lipopolysaccharide(LPS),and the LPS stimulatory concentration was screened.The CG group(mineralization induction),LPS+CG group,and LPS+CG+(2,4,6,8,and 10 J/cm^(2))LED red light groups were set.On day 7,alkaline phosphatase(ALP)staining and ALP activity were determined.Relative expression levels of the ALP,osterix(OSX),dentin matrix protein-1(DMP-1)and dentin sialophosphoprotein(DSPP)genes were measured by qRT-PCR.On day 21,alizarin red staining and calcium nodule quantitative determination were performed to screen the best light energy.The LPS+CG group and LPS+CG+LED group(optimal energy)were set up,and the secretion and expression levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were detected by ELISAs on days 1,3,5 and 7.The relative expression levels of the extracellular regulated protein kinases 1/2(ERK1/2),p38,c-Jun N-terminal kinase(JNK),and extracellular regulated protein kinases 5(ERK5)proteins and their phosphorylated proteins in the MAPK signaling path-way were detected by Western blots.After the pathway was blocked,the relative expression levels of the ALP,OSX,DMP-1,and DSPP proteins after LED red light irradiation on day 7 were detected by Western blots.Results CCK-8 assays showed that the proliferation of hDPSCs induced by 10μg/mL LPS was lower than that of the 0,1,and 5μg/mL groups on the 5th and 7th days(P<0.05),and 10μg/mL was selected as the LPS stimulatory concentration in the follow-up experiment.ALP staining,ALP activity,gene expression levels of ALP,OSX,DMP-1 and DSPP and calcium nodule quantification in the LPS+CG+4 J/cm^(2) group were higher than those in the other treatment groups(P<0.05).4 J/cm^(2) LED red light had the strongest ability to promote osteogenic/odontogenic differentiation and was used as the LED light energy density in subsequent experiments.ELISA showed that the secretion and expression levels of TNF-αand IL-1βin the LPS+CG+LED group were lower than those in the LPS+CG group on the 5th and 7th days(P<0.05).Western blot analysis showed that 4 J/cm^(2) LED red light promoted the expression levels of the p-ERK1/2,p-p38,p-JNK and p-ERK5 proteins.After the MAPK pathway was blocked,the expression levels of the ALP,OSX,DMP-1,and DSPP proteins in the LPS+CG+LED+U0126(ERK1/2 inhibitor),SP600125(JNK inhibitor),and BIX02189(ERK5 inhibitor)groups were lower than those in the LPS+CG+LED group(P<0.001).The protein expression levels of ALP,OSX and DMP-1 in the LPS+CG+LED+SB203580(p38 inhibitor)group were not significantly different from those in the LPS+CG+LED group(P>0.05).Conclusion In inflammatory conditions,LED red light promotes osteogenic/odontogenic differentiation of hDPSCs.This effect may be attributed to enhancement of the ERK1/2,JNK,and ERK5 signaling path-ways,which reduces the production of the inflammatory cytokines TNF-αand IL-1β.