姜黄素通过Nrf2信号通路促进炎症状态下牙周膜干细胞的成骨分化

Curcumin upregulates osteogenesis under inflammatory condition in periodontal ligament stem cells via Nrf2 signaling pathway

目的观察姜黄素对炎症状态下的牙周膜干细胞(PDLSCs)成骨分化的影响,并初步探讨这一影响是否与Nrf2信号通路有关。方法通过组织块法分离培养牙周膜干细胞,脂多糖(LPS)建立体外炎症微环境模型,使用不同浓度姜黄素处理细胞,CCK8、ALP定量分析筛选出姜黄素的最适作用浓度进行后续实验。设置分组:空白对照组、LPS组、LPS+姜黄素组,采用Western blotting、RT-PCR、ALP定量分析、ALP染色、茜素红染色检测成骨相关指标。Western blotting检测Nrf2水平,进一步构建Nrf2小干扰模型并检测干扰效率。设置分组:LPS+姜黄素组、LPS+姜黄素+siNrf2组,并检测相关成骨指标。结果成功分离培养牙周膜干细胞,并建立牙周膜干细胞体外炎症微环境。CCK8、ALP定量分析结果显示,低浓度的姜黄素无细胞毒性(F1d=6.08,F3d=9.404,F5d=166.289,P<0.001),且能上调ALP活性,0.1μmol/L为最适浓度(F=314.269,P<0.001),进一步实验结果显示,姜黄素可以增高炎症状态下ALP活性以及成骨相关蛋白和基因ALP、COL1、RUNX2的表达(FALP活性=120.401,FALP蛋白=48.505,FCOL1蛋白=473.372,FRUNX2蛋白=363.425,FALP基因=75.652,FCOL1基因=454.257,FRUNX2基因=98.761,P<0.001)。且ALP染色加深,矿化结节增多。并且Nrf2蛋白水平的变化与成骨变化趋势一致(F=27.323,P<0.001)。之后,成功构建Nrf2小干扰模型,Nrf2蛋白水平降低(t=9.910,P=0.001),相对于LPS+姜黄素组,LPS+姜黄素+siNrf2组的成骨相关指标均下调且ALP染色变浅(tALP活性=10.027,tALP蛋白=15.641,tCOL1蛋白=10.357,tRUNX2蛋白=13.305,tALP基因=19.965,tCOL1基因=12.590,tRUNX2基因=9.027,P<0.001)。结论姜黄素可以逆转炎症状态下牙周膜干细胞下降的成骨分化能力,这一逆转作用与Nrf2信号通路有关。

Objective To investigate the effects of curcumin on osteogenic differentiation of periodontal ligament stem cells(PDLSCs) in inflammatory microenvironment and the role of Nrf2 signaling pathway. Methods PDLSCs were isolated and cultured with tissue block method in vitro. Lipopolysaccharide(LPS) was used to establish an inflammatory microenvironment model in vitro. PDLSCs were treated with different concentrations of curcumin. The optimal curcumin concentration was determined with CCK8 and ALP activity assay. The PDLSCs were divided into 3 groups: control group, LPS group and LPS + curcumin group. The osteogenic indexes were evaluated with Western blotting, real-time PCR, ALP activity analysis, ALP staining and Alizarin Red staining. The protein level of Nrf2 was detected with Western blotting. Next, siNrf2 was constructed to transfect the PDLSCs and the interference efficiency was tested with Western blotting. The PDLSCs were divided into 2 groups: LPS + curcumin group and LPS + curcumin + siNrf2 group, and the osteogenic indexes were tested. Results PDLSCs were isolated and cultured and the inflammatory microenvironment in vitro was established successfully. CCK8 and ALP activity results showed that curcumin at low concentration was non-toxic to PDLSCs in inflammatory state(F1 d=6.08, F3 d=9.404, F5 d=166.289, P<0.001), was able to increase ALP activity, and the optimal concentration was 0.1 μmol/L(F=314.269, P<0.001). Further study showed that curcumin increased the ALP activity, and protein and mRNA levels of ALP, COL1, RUNX2(FALP activity=120.401;the protein levels: FALP=48.505, FCOL1=473.372, FRUNX2=363.425;the mRNA levels: FALP=75.652, FCOL1=454.257, FRUNX2=98.761, P<0.001). Besides, ALP staining became darker and the calcium content increased. The change of Nrf2 level showed similar trend as osteogenesis(F=27.323, P<0.001). In the siNrf2 model, the protein level of Nrf2 decreased(t=9.910, P=0.001). Compared with LPS + curcumin group, the LPS+curcumin+siNrf2 group had decreased osteogenic indexes(tALP activity=10.027;the protein levels: tALP=15.641, tCOL1=10.357, tRUNX2=13.305;the mRNA levels: tALP=19.965, tCOL1=12.590, tRUNX2=9.027, P<0.001) and ALP staining became lighter. Conclusion Curcumin can reverse the osteogenic differentiation of periodontal ligament stem cells in inflammatory state, which is related to Nrf2 signaling pathway.