龟甲水提液调控NF-κB炎症微环境对MC3T3-E1成骨分化的影响

Effect of Testudinis Carapax et Plastrum Aqueous Extract Regulating NF-κB Inflammation Microenvironment on Osteogenic Differentiation of MC3T3-E1

目的:探讨龟甲水提液调控核转录因子-κB(NF-κB)炎症微环境促小鼠前成骨细胞系(MC3T3-E1)成骨分化的作用及机制。方法:体外培养MC3T3-E1细胞,并进行成骨诱导,龟甲水提液干预。实验分为空白组、成骨诱导组、龟甲水提液(20 mg·L^-1)联合成骨诱导组。细胞增殖毒性检测试剂盒(CCK-8)法检测MC3T3-E1增殖能力,并确定最佳干预浓度;碱性磷酸酶(ALP)染色和茜素红(ARS)染色检测MC3T3-E1成骨分化能力;实时荧光定量PCR(Real-time PCR)检测NF-κB p65,NF-κB p105,白细胞介素-6(IL-6),ALP和Ⅰ型胶原(COL-Ⅰ)mRNA的表达。结果:CCK-8结果显示,随着时间的增长,MC3T3-E1增殖虽无统计学差异,但呈上升趋势,而在20 mg·L^-1质量浓度时增殖较其他组明显;ALP和ARS染色结果显示,成骨诱导组和龟甲水提液联合成骨诱导组染色阳性率较空白组明显升高;Real-time PCR结果显示,龟甲干预第7天,与空白组比较,龟甲水提液联合成骨诱导组NF-κB p105和IL-6 mRNA的表达明显降低(P<0.01),ALP和COL-ⅠmRNA的表达明显升高(P<0.05);龟甲干预第14天,与空白组比较,龟甲水提液联合成骨诱导组NF-κB p65,NF-κB p105和IL-6 mRNA的表达明显降低(P<0.01),ALP和COL-ⅠmRNA的表达明显升高(P<0.05,P<0.01)。结论:龟甲水提液能够促进MC3T3-E1成骨分化,其机制可能与抑制NF-κB炎性微环境有关。

Objective:To investigate the role and mechanism of Testudinis Carapax et Plastrum aqueous extract in promoting osteogenic differentiation of mouse preosteoblast cell line(MC3T3-E1)by regulating nuclear transcription factor-κB(NF-κB)inflammation microenvironment.Method:MC3T3-E1 cells were cultured in vitro,and osteogenic induction(OI)was performed.Testudinis Carapax et Plastrum was prepared and treated the cells.Cells were devided into control group,osteogenic induction group and Testudinis Carapax et Plastrum(20 mg·L^-1)with osteogenic induction group.The proliferation of MC3T3-E1 was detected by cell counting kit-8(CCK-8),and the optimum concentration of intervention was determined.MC3T3-E1 differentiation and osteogenic mineralization were assayed using alkaline phosphatase(ALP)and Alizarin red staining(ARS),respectively.The expressions of NF-κB p65,NF-κB p105,interleukin-6(IL-6),ALP and Collagen-Ⅰ(COL-Ⅰ)mRNA were detected by Real-time PCR.Result:The results of CCK-8 showed that the proliferation of MC3T3-E1 did not change statistically with time,but it showed an upward trend,while the proliferation at 20 mg·L^-1 was more obvious than other groups.The ALP and ARS showed that the positive staining rate of osteogenic induction group and Testudinis Carapax et Plastrum with osteogenic induction group were higher than control group.Real-time PCR results showed that on the 7th day in culture,the expression of NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group(P<0.01),and the expression of ALP and COL-ⅠmRNA was significantly upregulated(P<0.05),on the 14th day,the expression of NF-κB p65,NF-κB p105 and IL-6 mRNA in Testudinis Carapax et Plastrum with osteogenic induction group was significantly lower than that in control group(P<0.01).The expression of ALP and COL-ⅠmRNA was significantly increased(P<0.05,P<0.01).Conclusion:Testudinis Carapax et Plastrum aqueous extract can promote osteogenic differentiation of MC3T3-E1 via a mechanism associated with the regulation of inhibition of NF-κB inflammatory microenvironment.