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

doi:10.6040/j.issn.1671-7554.0.2019.1187

Abstract

Abstract: 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(F_1d=6.08, F_3d=9.404, F_5d=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(F_{ALP activity}=120.401; the protein levels: F_ALP=48.505, F_COL1=473.372, F_RUNX2=363.425; the mRNA levels: F_ALP=75.652, F_COL1=454.257, F_RUNX2=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(t_{ALP activity}=10.027; the protein levels: t_ALP=15.641, t_COL1=10.357, t_RUNX2=13.305; the mRNA levels: t_ALP=19.965, t_COL1=12.590, t_RUNX2=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.

Key words: Periodontal ligament stem cells, Osteogenic differentiation, Curcumin, Inflammatory environment, Nuclear factor erythroid-2-related factor

CLC Number:

R329.28

XIONG Yixuan, ZHAO Bin, JIA Linglu, ZHANG Wenjing, XU Xin. Curcumin upregulates osteogenesis under inflammatory condition in periodontal ligament stem cells via Nrf2 signaling pathway[J].Journal of Shandong University (Health Sciences), 2020, 58(5): 19-26.

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