[1]刘丹,黄思柔,刘璞,等.基于Wnt/β-连环蛋白信号通路探究金天格胶囊对肿瘤坏死因子α干预下前成骨细胞生物学功能的影响[J].中国中医骨伤科杂志,2023,31(06):1-7+14.[doi:10.20085/j.cnki.issn1005-0205.230601]
　LIU Dan,HUANG Sirou,LIU Pu,et al.Efficacy of Jintiange on the Biological Function of Preosteoblasts under Tumor Necrosis Factor-α Intervention Based on Wnt/β-catenin Signal Pathway[J].Chinese Journal of Traditional Medical Traumatology & Orthopedics,2023,31(06):1-7+14.[doi:10.20085/j.cnki.issn1005-0205.230601]
点击复制

基于Wnt/β-连环蛋白信号通路探究金天格胶囊对肿瘤坏死因子α干预下前成骨细胞生物学功能的影响()
分享到：

《中国中医骨伤科杂志》[ISSN:1005-0205/CN:42-1340/R]

卷:
第31卷
期数:
2023年06期
页码:
1-7+14
栏目:
实验研究
出版日期:
2023-06-15

文章信息/Info

Title:
Efficacy of Jintiange on the Biological Function of Preosteoblasts under Tumor Necrosis Factor-α Intervention Based on Wnt/β-catenin Signal Pathway
文章编号:
1005-0205(2023)06-0001-07
作者:
刘丹1; 黄思柔2; 刘璞1; 李娟3; 陈琳4△
1西安医学院第一附属医院风湿免疫科(西安,710077)
2西安医学院研究生工作部
3西安市第五医院风湿免疫科
4西安医学院第一附属医院药学部
Author(s):
LIU Dan1; HUANG Sirou2; LIU Pu1; LI Juan3; CHEN Lin4△
1Department of Rheumatology and Immunology,the First Affiliated Hospital of Xi'an Medical University,Xi'an 710077,China;
2Department of Graduate Work,Xi'an Medical University,Xi'an 710021,China;
3Department of Rheumatology and Immunology,Xi'an Fifth Hospital,Xi'an 710082,China;
4Department of Pharmacy,the First Affiliated Hospital of Xi'an Medical University,Xi'an 710077,China.
关键词:
Wnt/β-连环蛋白信号通路;  金天格胶囊;  前成骨细胞;  生物学功能
Keywords:
Wnt/β-catenin signal pathway;  Jintiange;  preosteoblasts;  biological function
分类号:
R-33
DOI:
10.20085/j.cnki.issn1005-0205.230601
文献标志码:
A
摘要:
目的:探讨基于Wnt/β-连环蛋白(Wnt/β-catenin)信号通路对金天格胶囊在肿瘤坏死因子α(TNF-α)干预下前成骨细胞生物学功能的影响。方法:取第4代MC3T3-E1细胞,将细胞分为正常组、TNF-α组(TNF-α 15 ng/mL)、TNF-α+10 μg/mL金天格组(TNF-α 15 ng/mL+10 μg/mL金天格胶囊)、TNF-α+20 μg/mL金天格组(TNF-α 15 ng/mL+20 μg/mL金天格胶囊)、TNF-α+40 μg/mL金天格组(TNF-α 15 ng/mL+40 μg/mL金天格胶囊)、Dickkopf相关蛋白1(DKK1)组(TNF-α 15 ng/mL+40 μg/mL金天格胶囊+0.1 μg/mL Wnt/β-catenin信号通路抑制剂DKK1)、40 μg/mL金天格组(40 μg/mL金天格胶囊),各组细胞均使用相应药物共培养24 h。CCK8法检测细胞增殖; 流式细胞术检测细胞凋亡; ELISA法检测细胞上清白介素-6(IL-6)、IL-1β、IL-10水平; ALP试剂盒法检测细胞ALP活性; 茜素红染色观察细胞矿化分化情况; qRT-PCR法和Western-Blot法检测细胞β-catenin、糖原合成酶激酶3β(GSK-3β)、Runt相关转录因子2(RUNX2)、核因子-κB受体活化因子配体(RANKL)、骨保护素(OPG)表达情况。结果:与正常组相比,TNF-α组细胞存活率、ALP活性、相对吸光度值、钙结节数量、上清IL-10水平、p-β-catenin、p-GSK-3β、RUNX2、OPG表达显著下降,细胞凋亡率、上清IL-6、IL-1β水平、RANKL表达升高,差异有统计学意义(P<0.05); 40 μg/mL金天格组细胞存活率、ALP活性、相对吸光度值、钙结节数量、p-β-catenin、p-GSK-3β、RUNX2、OPG表达升高,细胞凋亡率、RANKL表达降低,差异有统计学意义(P<0.05); 与TNF-α组相比,TNF-α+10 μg/mL金天格组、TNF-α+20 μg/mL金天格组、TNF-α+40 μg/mL金天格组细胞存活率、ALP活性、相对吸光度值、钙结节数量、上清IL-10水平、p-β-catenin、p-GSK-3β、RUNX2、OPG表达显著升高,细胞凋亡率、上清IL-6、IL-1β水平、RANKL表达下降,差异有统计学意义(P<0.05); Wnt/β-catenin信号通路抑制剂DKK1逆转了金天格胶囊对TNF-α刺激的前成骨细胞促增值和分化作用,并增加细胞凋亡和炎症反应。结论:金天格胶囊可通过Wnt/β-catenin信号通路抑制TNF-α诱导的前成骨细胞的凋亡和炎症,促进细胞增殖和分化。
Abstract:
Objective:To explore the efficacy of Jintiange on the biological function of preosteoblasts under the intervention of tumor necrosis factor-α(TNF-α)based on Wnt/β-catenin signaling pathway.Methods:The fourth generation MC3T3-E1 cells were divided into normal group,TNF-α group(TNF-α 15 ng/mL),TNF-α+10 μg/mL Jintiange group(TNF-α 15 ng/mL+10 μg/mL Jintiange),TNF-α+20 μg/mL Jintiange group(TNF-α 15 ng/mL+20 μg/mL Jintiange),TNF-α+40 μg/mL Jintiange group(TNF-α 15 ng/mL+40 μg/mL Jintiange),Dickkopf related protein 1(DKK1)group(TNF-α 15 ng/mL+40 μg/mL Jintiange+0.1 μg/mL Wnt/β-catenin signal pathway inhibitor DKK1),and 40 μg/mL Jintiange group(40 μg/mL Jintiange),and all cells were cultured for 24 h with corresponding drugs.Cell proliferation was detected by CCK8 method.Cell apoptosis was detected by flow cytometry.The levels of interleukin-6(IL-6),interleukin-1β(IL-1β),and interleukin-10(IL-10)in cell supernatant were detected by ELISA.Cell ALP activity was detected by Kit assay.The Alizarin red staining was used to observe the mineralization and differentiation of cells.The expression of β-catenin,glycogen synthase kinase 3β(GSK-3β),run related transcription factor 2(RUNX2),nuclear factor-κB receptor activating factor ligand(RANKL),and osteoprotegerin(OPG)were detected by qRT-PCR and Western Blot.Results:Compared with the normal group,the cell survival rate,ALP activity,relative absorbance value,number of calcium nodules,the level of IL-10 in the supernatant,and the expression of p-β-catenin,p-GSK-3β,RUNX2,and OPG in the TNF-α group decreased obviously,while the apoptosis rate,the levels of IL-6 and IL-1β in supernatant,and the expression of RANKL increased(P<0.05).The cell survival rate,ALP activity,relative absorbance value,number of calcium nodules,and the expression of p-β-catenin,p-GSK-3β,RUNX2,and OPG in the 40 μg/mL Jintiange group increased,while the cell apoptosis rate,and the expression of RANKL decreased(P<0.05).Compared with TNF-α group,the cell survival rate,ALP activity,relative absorbance value,number of calcium nodules,the level of IL-10 in the supernatant,and the expression of p-β-catenin,p-GSK-3β,RUNX2,and OPG in the TNF-α+10 μg/mL Jintiange group,TNF-α+20 μg/mL Jintiange group,TNF-α+40 μg/mL Jintiange group decreased obviously,while the apoptosis rate,the levels of IL-6 and IL-1β in supernatant,and the expression of RANKL increased(P<0.05).DKK1,an inhibitor of Wnt/β-catenin signaling pathway,reversed the effects of Jintiange on TNF-α stimulated osteoblast proliferation and differentiation,and increased cell apoptosis and inflammatory response.Conclusion:Jintiange can inhibit TNF-α-induced apoptosis and inflammation of osteoblasts through Wnt/β-catenin signaling pathway,and promote cell proliferation and differentiation.

参考文献/References:

[1] HUANG C C,TSENG T T,LIU S C,et al.S1P increases VEGF production in osteoblasts and facilitates endothelial progenitor cell angiogenesis by inhibiting miR-16-5p expression via the c-Src/FAK signaling pathway in rheumatoid arthritis[J].Cells,2021,10(8):2168.
[2] BERARDI S,CORRADO A,MARUOTTI N,et al.Osteoblast role in the pathogenesis of rheumatoid arthritis[J].Mol Biol Rep,2021,48(3):2843-2852.
[3] LI Y,ZHANG Z,CUI F,et al.Traditional Chinese medicine bionic tiger bone powder for the treatment of AI-associated musculoskeletal symptoms[J].Evid Based Complement Alternat Med,2017:2478565.
[4] SHEN Y,WANG N,ZHANG Q,et al.Jin-Tian-Ge ameliorates ovariectomy-induced bone loss in rats and modulates osteoblastogenesis and osteoclastogenesis in vitro[J].Chin Med,2022,17(1):78.
[5] MAEDA K,KOBAYASHI Y,KOIDE M,et al.The regulation of bone metabolism and disorders by Wnt signaling[J].Int J Mol Sci,2019,20(22):5525.
[6] LI S,YIN Y,YAO L,et al.TNF-α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p[J].Mol Med Rep,2020,22(2):1017-1025.
[7] HONG W,ZHANG W.Hesperidin promotes differentiation of alveolar osteoblasts via Wnt/β-catenin signaling pathway[J].J Recept Signal Transduct Res,2020,40(5):442-448.
[8] HAMEISTER R,LOHMANN C H,DHEEN S T,et al.The effect of TNF-α on osteoblasts in metal wear-induced periprosthetic bone loss[J].Bone Joint Res,2020,9(11):827-839.
[9] HIOKI T,TOKUDA H,KUROYANAGI G,et al.Olive polyphenols attenuate TNF-α-stimulated M-CSF and IL-6 synthesis in osteoblasts:suppression of Akt and p44/p42 MAP kinase signaling pathways[J].Biomed Pharmacother,2021,141:111816.
[10] JEONG M J,LIM D S,KIM S O,et al.Effect of rosmarinic acid on differentiation and mineralization of MC3T3-E1 osteoblastic cells on titanium surface[J].Anim Cells Syst(Seoul),2021,25(1):46-55.
[11] YEOM J,MA S,LIM Y H.Probiotic propionibacterium freudenreichii MJ2 enhances osteoblast differentiation and mineralization by increasing the OPG/RANKL ratio[J].Microorganisms,2021,9(4):673.
[12] AMARASEKARA D S,KIM S,RHO J.Regulation of osteoblast differentiation by cytokine networks[J].Int J Mol Sci,2021,22(6):2851.
[13] KOVÁCS B,VAJDA E,NAGY E E.Regulatory effects and interactions of the Wnt and OPG-RANKL-RANK signaling at the bone-cartilage interface in osteoarthritis[J].Int J Mol Sci,2019,20(18):4653.
[14] GAO X,WU Q,ZHANG X,et al.Salvianolate ameliorates osteopenia and improves bone quality in prednisone-treated rheumatoid arthritis rats by regulating RANKL/RANK/OPG signaling[J].Front Pharmacol,2021,12:710169.
[15] HOUSCHYAR K S,TAPKING C,BORRELLI M R,et al.Wnt pathway in bone repair and regeneration-what do we know so far[J].Front Cell Dev Biol,2019,6:170.
[16] SANG C,ZHANG Y,CHEN F,et al.Tumor necrosis factor alpha suppresses osteogenic differentiation of MSCs by inhibiting semaphorin 3B via Wnt/β-catenin signaling in estrogen-deficiency induced osteoporosis[J].Bone,2016,84:78-87.
[17] LI Y,YUAN L,JIANG S,et al.Interleukin-35 stimulates tumor necrosis factor-α activated osteoblasts differentiation through Wnt/β-catenin signaling pathway in rheumatoid arthritis[J].Int Immunopharmacol,2019,75:105810.
[18] SHEN W,GUAN Y Y,WU R M,et al.Protective effects of Wang-Bi tablet on bone destruction in collagen-induced arthritis by regulating osteoclast-osteoblast functions[J].J Ethnopharmacol,2019,238:111861.

备注/Memo

备注/Memo:
基金项目:西安医学院第一附属医院横向基金项目(HX-2022-01)
陕西省自然科学基础研究计划项目(2021JZ-59)
陕西省重点研发计划项目(2021SF-259)
西安市科技计划项目(2021JH-03-0427)
△通信作者 E-mail:Cl13379295579@163.com
更新日期/Last Update: 2023-06-10