中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (23): 3691-3699.doi: 10.12307/2022.669
• 骨组织构建 bone tissue construction • 上一篇 下一篇
杨锐娟1,李阳阳1,蔡瑞艳1,刘慧兵1,郭 春1,2
收稿日期:
2021-09-20
接受日期:
2021-11-29
出版日期:
2022-08-18
发布日期:
2022-02-11
通讯作者:
郭春,博士,教授,新乡医学院第一附属医院河南省神经修复重点实验室,河南省卫辉市 453100;新乡医学院第一附属医院骨科,河南省卫辉市 453100
作者简介:
杨锐娟,女,1991年生,河南省卫辉市人,汉族,2016年福建医科大学毕业,硕士,主要从事感染性骨疾病骨再生机制研究。
基金资助:
Yang Ruijuan1, Li Yangyang1, Cai Ruiyan1, Liu Huibin1, Guo Chun1, 2
Received:
2021-09-20
Accepted:
2021-11-29
Online:
2022-08-18
Published:
2022-02-11
Contact:
Guo Chun, MD, Professor, Henan Key Laboratory of Neural Regeneration, First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan Province, China; Department of Orthopedics, First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan Province, China
About author:
Yang Ruijuan, Master, Henan Key Laboratory of Neural Regeneration, First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan Province, China
Supported by:
摘要:
文题释义:
核因子κB受体活化因子配体(receptor activator of nuclear ractor-kB ligand,RANKL):又称骨保护素配体,是一种激活破骨细胞的破骨细胞分化因子。RANKL与破骨细胞表面的RANK结合引起信号级联反应,最终激活NFATc1,NFATc1的激活导致下游破骨细胞分化的标记基因TRAP,CK和基质金属蛋白酶的表达。RANKL的过度表达会导致一系列骨疾病。
抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP):是一种在破骨细胞中特异表达的骨吸收和破骨细胞活性因子。血清TRAP浓度与骨密度呈负相关,血清和骨中TRAP的高表达提示骨疾病的发生。
背景:白细胞介素1是一种重要的促炎细胞因子,已被证实在调节骨炎症和骨重建中发挥重要作用。有研究表明,白细胞介素1α可诱导MC3T3-E1细胞凋亡,同时抑制成骨细胞分化。
目的:探讨白细胞介素1α在小鼠破骨细胞活化和骨流失中的作用及机制。
方法:①细胞实验:分别以白细胞介素1α单独或与核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)联合作用于RAW264.7细胞1 d和4 d。CCK8检测细胞活力,抗酒石酸酸性磷酸酶染色检测多核破骨细胞,实时荧光定量PCR、免疫荧光染色及Western blot检测破骨形成相关的特异基因及核转录因子κB和Wnt/β-catenin信号通路相关基因的mRNA和蛋白表达情况;分别以白细胞介素1α单独或与RANKL和巨噬细胞集落刺激因子联合作用于骨髓源性巨噬细胞7 d,抗酒石酸酸性磷酸酶染色检测多核破骨细胞,Western blot检测破骨形成相关的特异基因的蛋白水平的表达情况。②动物实验:将小鼠随机分为2组:对照组腹腔注射PBS,实验组腹腔注射白细胞介素1α溶液,每周2次,5周后取材,采用μCT、苏木精-伊红染色、抗酒石酸酸性磷酸酶染色和免疫荧光分析小鼠股骨骨组织变化及相关基因的表达情况。
结果与结论:①细胞实验结果显示,白细胞介素1α单独干预可显著促进RAW264.7细胞增殖,而白细胞介素1α与RANKL联合作用可刺激RAW264.7细胞向破骨细胞分化(P < 0.05);在RANKL或RANKL+巨噬细胞集落刺激因子存在的情况下,白细胞介素1α明显上调RAW264.7细胞和骨髓源性巨噬细胞中破骨细胞相关标志物的表达(P < 0.05),并增加抗酒石酸酸性磷酸酶阳性多核破骨细胞的数量(P < 0.05);白细胞介素1α显著激活RAW264.7细胞的核转录因子κB和Wnt/β-catenin信号通路(P < 0.05);阻断核转录因子κB或Wnt3信号通路不仅逆转了白细胞介素1α引起的RAW264.7细胞的核转录因子κB和Wnt3信号通路的激活,而且减弱了白细胞介素1α诱导的破骨细胞特异性基因的上调(P < 0.05)。②动物实验结果显示,与体外细胞实验结果一致,白细胞介素1α可诱导C57BL/6J小鼠骨流失并上调破骨特异基因TRAF6,RANK,p65和Wnt3的表达(P < 0.05)。③上述数据证实,白细胞介素1α通过激活核转录因子κB和Wnt信号通路诱导破骨细胞活化和骨丢失。
https://orcid.org/0000-0002-6852-7277 (郭春)
中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程
中图分类号:
杨锐娟, 李阳阳, 蔡瑞艳, 刘慧兵, 郭 春. 白细胞介素1α诱导破骨细胞活化和骨流失[J]. 中国组织工程研究, 2022, 26(23): 3691-3699.
Yang Ruijuan, Li Yangyang, Cai Ruiyan, Liu Huibin, Guo Chun. Interleukin-1 alpha induces osteoclast activation and bone loss[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(23): 3691-3699.
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Western blot assay
To identify the function of IL-1α in the differentiation of pre-osteoclasts, RAW264.7 cells were administered with IL-1α (0, 1.0, 5.0, and 10 ng/mL) for 3 days. After treatment for 3 days, the osteoclast-specific proteins including RANK, TRAF6, COX-2, CA2, TRAP, MMP9 and CK were measured by western blot assay.
To explore the function of IL-1α in the differentiation of mature osteoclasts, RAW264.7 cells were induced by 50 ng/mL RANKL for 2 days and then cultured with IL-1α and RANKL for another 1 day. After treatment for 3 days, the osteoclast-specific proteins including RANK, TRAF6, COX-2, CA2, TRAP, MMP9, and CK were measured by western blot assay. BMMs were induced by 30 ng/mL M-CSF and 50 ng/mL RANKL for 3 days before being administered with varied dosages of IL-1α with M-CSF and RANKL for another 3 days. After treatment for 6 days, the osteoclast-specific proteins including RANK, TRAF6, TRAP, and CK were measured by western blot assay.
RAW264.7 cells were intervened with IL-1α at 10 ng/mL from 0 to 2 hours and at 0, 1.0, 2.5, 5.0, 10 ng/mL for 1 hour. After treatment for 0 to 2 hours or 1 hour, the expression of NF-κB signal proteins including phosphorylated inhibitor of nuclear factor-κB α (p-IκB α), phosphorylated inhibitor of nuclear factor-κB kinase α/β (p-IKK α/β) and phosphorylated p65 (p-p65) were measured by western blot assay.
After pretreatment with BAY 11-7082 (1 or 5 μmol/L, Selleck Chemicals, Houston, Texas, USA), an NF-κB signal pathway inhibitor, for 2 hours, RAW264.7 cells were administered with IL-1α for 1 hour or 3 days. After treatment with IL-1α for 1 hour or 3 days, the protein levels of NF-κB signaling (p-IκB α, p-IKK α/β, p-p65) and osteoclast-specific proteins (RANK, TRAF6, COX-2, CA2, TRAP, MMP9, CK) were measured by western blot assay.
RAW264.7 cells were intervened with IL-1α at 10 ng/mL from 0 to 2 hours or at 0, 1.0, 2.5, 5.0, 10 ng/mL for 1 hour. After treatment for 0 to 2 hours or 1 hour, the levels of Wnt signaling proteins including β-catenin, Wnt3 and GSK3β were tested by western blot assay.
After pretreatment with XAV-939 (10 μmol/L or 25 μmol/L, Selleck Chemicals, Houston, Texas, USA), a Wnt/β-catenin signal pathway inhibitor, for 2 hours, RAW264.7 cells were intervened with 10 ng/mL
IL-1α for 1 hour. After treatment with IL-1α for 1 hour, the level of the Wnt pathway proteins including β-catenin, Wnt3, and GSK3β was tested by western blot assay. After pretreatment with XAV-939 for 2 hours, RAW264.7 cells were intervened with 10 ng/mL IL-1α for 3 days. After treatment with IL-1α for 3 days, the level of osteoclast-specific proteins (RANK, TRAF6, COX-2, CA2, TRAP, MMP9, and CK) was measured using western blot assay.
Total protein lysates were obtained by Cytobuster™ protein extraction reagent (cat. no. 71009-3; EMD Millipore) supplemented with protease inhibitor cocktail, 1 mmol/L Na3VO4 and 25 mmol/L NaF. An equal amount of protein samples (40 μg) were performed by gel electrophoresis, transferred to PVDF membranes (EMD Millipore) and blocked with 10% non-fat milk. Then the membranes were incubated with specific primary antibodies overnight at 4˚C against TRAF6 (sc-8409), p65 (sc-8008), p-p65 (sc-136548), MMP-9 (sc-13520), CK (sc-48353), CA2 (sc-48351), β-catenin (sc-7963), GSK3β (sc-377213), IκBα (sc-1643), p-IκBα (sc-8404), Wnt3 (sc-74537) or β-actin (sc-47778) at dilutions of 1:300 or primary antibodies against RANK (ab13918), p-IKKα/β (ab194528), IKKβ (ab178870), TRAP (ab191406) and COX-2 (ab15191) at dilutions of 1:500. Horseradish peroxidase-conjugated anti-rabbit IgG (1:2000, A0216, Beyotime, Shanghai, China) and anti-mouse IgG (1:2000, A0208, Beyotime) and were used as the secondary antibodies for detection. The protein signals were detected using chemiluminescence (ECL, CoWin Biosciences, Beijing, China) and the images were obtained using the MiniChemi™ III chemiluminescence apparatus (Beijing Sage Creation Science, Beijing, China). The gray value was analyzed by the ImageJ software version 1.46 (NIH, USA). The protein expression was evaluated relative to that of β-actin.
IL-1α treatment in vivo studies
In total, 24 male mice were assigned into two groups at random: (1) PBS (control); and (2) IL-1α. Mice were then treated with 100 μL
of IL-1α solution (5 μg/kg) dissolved in PBS or 100 μL of PBS by intraperitoneal injection twice a week for 5 weeks. After intervention for 5 weeks, all animals were euthanized using a 30% volume displacement rate per minute of CO2 in an induction chamber, following which the femurs were harvested for histological analysis and micro-computed tomography (μCT).
μCT analyses
After administration of IL-1α by intraperitoneal injection (5 μg/kg, twice a week) for 5 weeks, the effect of IL-1α on bone microstructure in vivo was analyzed by μCT. The dissected femurs were analyzed by a Micro-CT Skyscan 1 276 system (Bruker Corporation, USA). Specimens were scanned using 85 kV (200 μA) X-ray energy at 9 μm isometric resolution and 400-millisecond integration time. The cylindrical area of cortical bone beginning from the mid-diaphysis with a 2 mm extension was collected and analyzed. At the same gray value, the range of interest was set to a region with a height of 500 μm and length of 2 000 μm from the growth-plate. The region beginning from the growth-plate of the center metaphysis at 2 mm (220 slices) proximal length was used to analyze the trabecular bone. The following properties of the trabecular and cortical bone were calculated: (1) bone mineral density (BMD); (2) trabecular number (Tb.N); (3) bone volume/total volume (BV/TV); (4) trabecular separation (Tb.Sp); (5) trabecular thickness (Tb.Th); and (6) total porosity (%).
Histological analysis
After administration of IL-1α by intraperitoneal injection (5 μg/kg, twice a week) for 5 weeks, the effect of IL-1α on osteoclast activation and bone loss in vivo was demonstrated by hematoxylin-eosin (H&E) and TRAP staining.
The dissected femurs were fixed with 4% PFA for 2 days, decalcified for 21 days with a 10% EDTA solution, dehydrated using a gradient ascension ethanol, transparentized twice in dimethyl benzene and embedded by paraffin. The slices (5 µm) were prepared for TRAP staining or in accordance with the standard steps. Photomicrographs were obtained by a Nikon 55i microscope (Nikon, Japan) under a light microscope (magnification, ×20). Sections were evaluated for each femur from n ≥3 mice per group.
Immunofluorescence assay
After pretreatment with BAY11-7082 (10 µM) for 2 hours, RAW264.7 cells were intervened with 10 ng/mL IL-1α for 1 hour. After treatment with IL-1α for 1 hour, the expression of p65 was measured using immunofluorescence.
After pretreatment with XAV-939 (15 µM) for 2 hours, RAW264.7 cells were intervened with 10 ng/mL IL-1α for 1 hour. After treatment with IL-1α for 1 hour, the level of β-catenin was measured using immunofluorescence.
After administration of IL-1α by intraperitoneal injection (5 μg/kg, twice a week) for 5 weeks, the effect of IL-1α on the expression of RANK, TRAF6, p65 and Wnt3 in the decalcified bone was detected by immunofluorescence.
After treatment, cells and slices were fixed with 4% PFA for 15 minutes, permeabilized for 10 minutes with 0.1% Triton X-100 solution and blocked for 1 hour with 5% BSA, added a primary antibody overnight at 4˚C, washed 3-5 times with TBS, stained using the corresponding secondary antibodies, namely Alexa Fluor® 488-labeled goat anti-mouse IgG (1:400, P0188; Beyotime) or Cy3-labelled goat anti-rabbit IgG (1:400, P0183, Beyotime). The nuclei were counterstained using 4,6-diamino-2-phenyl indole (DAPI) (10 μg/mL; Solarbio, Beijing, China). The images of samples were obtained using an inverted fluorescence microscope (magnification, ×40; Zeiss AG, Germany), and analyzed by the ImageJ (version 1.46, NIH, USA).
Main outcome measures
The effect of IL-1α on cell viability, osteoclast differentiation, the expression of osteoclastogenesis-related genes and genes related to the NF-κB and Wnt/β-catenin pathways in RAW264.7 cells or BMMs; changes in bone morphology, bone resorption, bone microarchitecture, and bone resorption markers induced by IL-1α in mice.
Statistical analysis
All statistical results were expressed as the means±SD of at least three individual experiments. Student’s t-test, repeated measures analysis of variance and one-way analysis of variance followed by SNK post hoc test using the SPSS 22.0 software (IBM Corp., USA) were performed to analyze the experimental data between groups. The value of P < 0.05 was regarded as statistical significance. The statistical methods of this study were reviewed by the biostatistician of the First Affiliated Hospital of Xinxiang Medical University.
文题释义:
核因子κB受体活化因子配体(receptor activator of nuclear ractor-kB ligand,RANKL):又称骨保护素配体,是一种激活破骨细胞的破骨细胞分化因子。RANKL与破骨细胞表面的RANK结合引起信号级联反应,最终激活NFATc1,NFATc1的激活导致下游破骨细胞分化的标记基因TRAP,CK和基质金属蛋白酶的表达。RANKL的过度表达会导致一系列骨疾病。
抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP):是一种在破骨细胞中特异表达的骨吸收和破骨细胞活性因子。血清TRAP浓度与骨密度呈负相关,血清和骨中TRAP的高表达提示骨疾病的发生。
有研究表明,白细胞介素1α可诱导MC3T3-E1细胞凋亡,同时抑制成骨细胞分化。
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