吉林大学学报(医学版)  2018, Vol. 44 Issue (02): 260-264

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李希宁, 陈晓春, 朱云飞, 曹泾楠, 邹忆婷, 杜霄, 王旗春
LI Xining, CHEN Xiaochun, ZHU Yunfei, CAO Jingnan, ZOU Yiting, DU Xiao, WANG Qichun
PERK信号通路对实验性绝经后骨质疏松雌性大鼠成骨细胞分化的影响
Effect of PERK signaling pathway on osteoblast differentiation of female rats with experimental postmenopausal osteoporosis
吉林大学学报(医学版), 2018, 44(02): 260-264
Journal of Jilin University (Medicine Edition), 2018, 44(02): 260-264
10.13481/j.1671-587x.20180210

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收稿日期: 2017-07-27
引用本文 0
李希宁, 陈晓春, 朱云飞, 曹泾楠, 邹忆婷, 杜霄, 王旗春. PERK信号通路对实验性绝经后骨质疏松雌性大鼠成骨细胞分化的影响[J]. 吉林大学学报(医学版), 2018, 44(02): 260-264.
LI Xining, CHEN Xiaochun, ZHU Yunfei, CAO Jingnan, ZOU Yiting, DU Xiao, WANG Qichun. Effect of PERK signaling pathway on osteoblast differentiation of female rats with experimental postmenopausal osteoporosis[J]. Journal of Jilin University (Medicine Edition), 2018, 44(02): 260-264.
PERK信号通路对实验性绝经后骨质疏松雌性大鼠成骨细胞分化的影响
李希宁 , 陈晓春 , 朱云飞 , 曹泾楠 , 邹忆婷 , 杜霄 , 王旗春     
湖州师范学院医学院病理学教研室, 浙江 湖州 313000
收稿日期: 2017-07-27
基金项目: 国家自然科学基金青年基金资助课题（81602805）；浙江省大学生科技创新计划资助课题（2016R427011）
作者简介: 李希宁(1986-), 男, 山东省泰安市人, 讲师, 医学博士, 主要从事代谢性骨病发病机制方面的研究。
通信作者: 王旗春, 教授, 硕士研究生导师(Tel:0572-2322569, E-mail:wangqc@zjhu.edu.cn)
[摘要]: 目的: 探讨实验性雌性大鼠绝经后骨质疏松（PMOP）治疗前后骨组织中PERK、ATF4、Runx2、osterix、RANKL和OPG的变化，阐明PERK信号通路在PMOP发生中的作用。方法: 复制雌性大鼠卵巢去势骨质疏松模型。45只大鼠分为正常对照组（大鼠不做处理，15只）、骨质疏松组（雌性大鼠卵巢摘除，15只）和骨质疏松治疗组（大鼠卵巢摘除后尾静脉注射雌激素，15只）。观察各组大鼠血清中Ⅰ型胶原（ColⅠ）、碱性磷酸酶（ALP）和骨钙素（OCN）水平。饲养3个月后取各组大鼠股骨骨干做病理切片，RT-PCR法检测各组大鼠骨组织中PERK、ATF4、Runx2、osterix、RANKL和OPG基因表达水平，Westernblotting法分析PERK、ATF4、Runx2、osterix、RANKL和OPG蛋白表达水平。结果: 与对照组比较，骨质疏松组大鼠血清中ColⅠ、ALP和OCN水平明显降低（P < 0.05或P < 0.01）；与骨质疏松组比较，骨质疏松治疗组大鼠血清中ColⅠ、ALP和OCN水平明显升高（P < 0.01）。与对照组比较，骨质疏松组大鼠骨组织中PERK、ATF4、Runx2和osterix基因表达水平明显下降（P < 0.05或P < 0.01），RANKL基因表达水平明显升高（P < 0.01）；与骨质疏松组比较，骨质疏松治疗组大鼠骨组织中PERK、ATF4、Runx2和osterix基因表达水平明显升高（P < 0.05或P < 0.01），RANKL基因表达水平明显降低（P < 0.01）。与对照组比较，骨质疏松组大鼠骨组织中PERK、Runx2和osterix蛋白表达水平明显下降（P < 0.05或P < 0.01），RANKL蛋白表达水平明显升高（P < 0.05或P < 0.01）；与骨质疏松组比较，骨质疏松治疗组大鼠骨组织中PERK、Runx2和osterix蛋白表达水平明显上升（P < 0.05或P < 0.01），RANKL蛋白表达水平明显降低（P < 0.01）。HE染色，与对照组比较，骨质疏松组大鼠骨组织中骨吸收陷窝变大，骨吸收增强，导致骨质流失；与骨质疏松组比较，骨质疏松治疗组大鼠骨质吸收减弱，骨骼恢复正常结构。结论: 雌性大鼠卵巢切除及注射雌激素治疗后，其骨组织中PERK表达趋势与成骨细胞转录因子Runx2和osterix变化一致，与破骨细胞转录因子RANKL表达相反，提示PMOP发病时成骨细胞功能减弱与PERK表达下降有关。
关键词: 绝经后骨质疏松    PERK    成骨细胞    破骨细胞    转录因子    
Effect of PERK signaling pathway on osteoblast differentiation of female rats with experimental postmenopausal osteoporosis
LI Xining, CHEN Xiaochun, ZHU Yunfei, CAO Jingnan, ZOU Yiting, DU Xiao, WANG Qichun     
Department of Pathology, School of Medical Sciences, Huzhou Normal University, Huzhou 313000, China
[Abstract]: Objective: To investigate the changes of PERK, Runx2, osterix, RANKL and OPG in bone tissue of the female rats with experimental postmenopausal osteoporosis (PMOP) before and after treatment, and to elucidate the role of PERK signaling pathway in PMOP. Methods: The ovariectomized rats were reproduced to osteoporosis models.A total of 45 rats were divided into normal control group (the rats didn't receive any treatment, n=15), osteoporosis group (the rats were ovariectomized, n=15) and osteoporosis treatment group (the ovariectomized rats were injected with estrogen through caudal vein, n=15).The changes of serum collagen Ⅰ (Col Ⅰ), alkaline phosphatase (ALP) and osteocalcin (OCN) of the rats in various groups were observed.Three months after feeding, the femoral shaft of the rats in various groups were taken for pathological section.The gene expression levels of PERK, ATF4, Runx2, osterix, RANKL and OPG in bone tissue of the rats in various groups were detected by RT-PCR; the protein expression levels of PERK, ATF4, Runx2, osterix, RANKL and OPG were detected by Western blotting method. Results: Compared with control group, the levels of serum Col Ⅰ, ALP and OCN in the rats in osteoporosis group were significantly decreased (P < 0.05 or P < 0.01); compared with osteoporosis group, the levels of serum Col Ⅰ, ALP and OCN of the rats in osteoporosis treatment group were significantly increased (P < 0.01).Compared with control group, the gene expression levels of PERK, ATF4, Runx2 and osterix in bone tissue of the rats in osteoporosis group were significantly decreased (P < 0.01), and the gene expression level of RANKL was increased(P < 0.01); compared with osteoporosis group, the gene expression levels of PERK, ATF4 Runx2 and osterix in bone tissue of the rats in osteoporosis treatment group were significantly increased (P < 0.01), and the gene expression level of RANKL was significantly decreased (P < 0.01).Compared with control group, the protein expression levels of PERK, Runx2 and osterix in bone tissue of the rats in osteoporosis group were significantly decreased (P < 0.05 or P < 0.01), and the protein expression level of RANKL were increased(P < 0.05 or P < 0.01); compared with osteoporosis group, the protein expression levels of PERK, Runx2 and osterix in bone tissue of the rats in osteoporosis treatment group were significantly increased (P < 0.05 or P < 0.01), and the protein expression level of RANKL was significantly decreased (P < 0.01).The HE staining results showed that compared with control group, the bone resorption pits in bone tissue of the rats in osteoporosis group became large with the increased bone absorption, which caused bone loss; compared with osteoporosis group, the resorption in bone tissue of the rats in osteoporosis treatment group was decreased, and the bone structure returned to normal. Conclusion: After the female rats are ovariectomized and injected with estrogen, the expression trends of PERK and osteoblast transcription factors Runx2 and osterix are consistent, in contrast with the osteoclast transcription factor RANKL expression, suggesting that the osteoblast function is reduced and it is related to the decreased expression of PERK in PMOP onset.
Key words: postmenopausal osteoporosis     PERK     osteoblast     osteoclast     transcription factor    

绝经后骨质疏松(postmennopausal osteoporosis, PMOP)是一种与衰老有关的常见病，主要发生在绝经后妇女[1]。PMOP核心发病机制为雌激素下降之后导致骨重建与骨吸收失衡，即成骨细胞的骨形成能力下降而破骨细胞的骨吸收能力增强，从而导致骨组织微细结构破坏、骨脆性增加以及易于骨折[2-3]。因此，PMOP的防治已成为目前迫切需要解决的公共健康问题。目前PMOP发病机制研究主要集中在成骨细胞和破骨细胞功能不平衡，导致骨质流失过多[4-6]，但成骨细胞功能减弱和破骨细胞功能增强的诱导机制并未给予深入研究[7]。PERK是一种丝氨酸/苏氨酸的蛋白激酶, 内质网上错误或未折叠蛋白的聚集可引起PERK的活化, 是内质网应激的直接结果[8]。PERK基因敲除的小鼠中，成骨细胞的分化和成熟障碍并伴有Ⅰ型胶原(typeⅠcollogen，ColⅠ)分泌减少，导致骨骼发育受损，同时成骨细胞内核心结合因子1(core-binding factor a1，cbfa1)和osterix的表达下调[9-10]，提示PERK很可能是一种新的调节骨骼发育和成骨细胞活动的因子。本实验通过动物实验观察PERK信号通路与成骨细胞分化和凋亡有关的转录因子、细胞因子和其他重要信息分子之间的联系，探讨PERK信号通路在PMOP发生时在成骨细胞凋亡和骨转换加速中的作用，阐明PMOP的发生机制，为制定骨质疏松的防治策略提供新的思路和理论依据。

1 材料与方法 1.1 实验动物、主要试剂和仪器

雌性Wistar大鼠(浙江大学实验动物中心)，实验动物生产许可证号：SYXK(浙)2012-0178。TRIzol试剂盒(Invitrogen公司, 美国)，High-Capacity cDNA ReverseTranscription Kit和Power SYBR® Green PCRMaster Mix(Applied Biosystems公司, 美国)，SMARTpool reagents Nrf2和Transfection Reagent(ThermoFisher公司, 美国)，Primary antibody(Santa Cruz公司, 美国)，RIPA裂解液(Transgen Biotech公司, 中国)。凝胶电泳仪、电泳槽和转膜槽(Bio-Rad公司，美国)，PCR仪(ThermoFisher公司，美国)。

1.2 大鼠PMOP模型复制及实验分组

雌性Wistar大鼠适应饲养1周后，分为3组，每组15只：①正常对照组(大鼠不作处理)；②骨质疏松组(雌性大鼠卵巢摘除)；③骨质疏松治疗组(大鼠卵巢摘除后尾静脉注射雌激素)。

1.3 血液分析仪检测大鼠血清中蛋白水平

大鼠饲养3个月后，采用眼球摘除法取大鼠全血样本，室温静置2h，1000r·min-1离心5 min，取上层血清样本，经血液分析仪检测各种大鼠血清中ColⅠ、碱性磷酸酶(alkaline phosphatase, ALP)和骨钙素(osteoclcin, OCN)水平。

1.4 RT-PCR法检测大鼠骨组织中PERK、ATF4、Runx2、osterix、RANKL和OPG基因表达

取大鼠股骨，经液氮反复冷冻变脆敲碎，按照TRIzol试剂盒说明书提取总RNA，并用Nano-Drop分光光度计测定RNA浓度。按照High-Capacity cDNA Reverse Transcription Kit和Power SYBR® Green PCR Master Mix说明书合成cDNA和PCR扩增，GAPDH作为内参。使用StepOnePlus system software导出Ct值，基因相对表达水平采用2-(△Ct)计算。

1.5 Westernblotting法检测大鼠骨组织中PERK、ATF4、Runx2、osterix、RANKL和OPG蛋白表达

取大鼠股骨，经液氮反复冷冻变脆敲碎，RIPA裂解液提取总蛋白，BCA法测定蛋白质浓度，每个泳道加入15 μg蛋白后进行SDS-PAGE电泳，电泳后转到PVDF膜，使用5%脱脂奶粉封闭PVDF膜2 h后，分别加入稀释后的抗小鼠一抗4℃孵育过夜，辣根过氧化物酶标记的二抗37℃孵育1 h，ECL显影，应用Gel Image system-1600凝胶成像系统采集图像。

1.6 HE染色观察骨组织形态学

取大鼠股骨，4%多聚甲醛固定24 h，20%甲酸和15%乙二胺四乙酸脱钙14 d，各级乙醇脱水、浸蜡、包埋、切片，HE染色观察。

1.7 统计学分析

采用SPSS13.0统计软件进行统计学分析。各组大鼠血清中成骨转换因子水平、骨组织中PERK、ATF4、Runx2、osterix、RANKL和OPG基因和蛋白表达水平均以x±s表示，多组间样本均数比较采用单因素方差分析。以P < 0.05为差异有统计学意义。

2 结果 2.1 各组大鼠血清中ColⅠ、ALP和OCN水平

与对照组比较，骨质疏松组大鼠血清中ColⅠ、ALP和OCN水平明显降低(P < 0.05或P < 0.01)；与骨质疏松组比较，骨质疏松治疗组大鼠血清ColⅠ、ALP和OCN水平明显升高(P < 0.01)。见表 1。

表 1 各组大鼠血清ColⅠ、ALP和OCN水平 Table 1 Levels of serum ColⅠ, ALP and OCN of rats in various groups
[n=15, x±s, ρB/(μg·L-1)]
Group Col Ⅰ ALP OCN
Control 10.16±1.56 7.83±1.74 8.77±2.96
Osteoporosis 4.99±1.62** 3.01±1.24* 2.06±0.37**
Osteoporosis treatment 9.02±2.13△ 7.50±1.38△ 8.45±1.37△
*P < 0.05, * *P < 0.01 compared with control group; △P < 0.01 compared with osteoporosis group.
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2.2 各组大鼠骨组织中PERK、ATF4、Runx2、osterix、RANKL和OPG基因表达水平

与对照组比较，骨质疏松组大鼠骨组织中PERK和ATF4基因表达水平明显降低(P < 0.05或P < 0.01)，成骨细胞转录因子Runx2和osterix基因表达水平明显降低(P < 0.05或P < 0.01)，而诱导破骨细胞分化促进骨质吸收因子RANKL基因表达水平明显升高(P < 0.01)；与骨质疏松组比较，骨质疏松治疗组骨组织中PERK、ATF4、Runx2和osterix基因表达水平均明显升高(P < 0.05或P < 0.01)，而RANKL表达水平明显下降(P < 0.01)。见表 2。

表 2 各组大鼠骨组织中成骨标志物基因表达水平 Table 2 Gene expression levels of osteogenic markers in bone tissue of rats in various groups
(n=15, x±s)
Group PERK ATF4 Runx2 osterix RANKL OPG
Control 3.24±2.13 6.12±1.65 4.17±0.74 8.65±2.95 5.87±1.85 6.10±0.98
Osteoporosis 0.80±1.62** 2.51±1.40* 1.66±0.48** 1.72±1.56** 14.62±3.01** 4.74±1.52
Osteoporosis treatment 3.65±2.68△△ 6.01±0.37△△ 3.82±1.17△ 7.41±2.51△△ 6.37±1.74△△ 5.89±2.09
*P < 0.05, * *P < 0.01 compared with control group; △P < 0.05, △△P < 0.01 compared with osteoporosis group.
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2.3 各组大鼠骨组织中PERK、ATF4、Runx2、osterix、RANKL和OPG蛋白表达

与对照组比较，骨质疏松组大鼠骨组织中PERK、Runx2和osterix蛋白表达水平明显降低(P < 0.05或P < 0.01)，ATF4和RANKL蛋白表达水平明显升高(P < 0.05或P < 0.01)；与骨质疏松组比较，骨质疏松治疗组大鼠骨组织中PERK和Runx2蛋白表达水平均明显升高(P < 0.05或P < 0.01)，而RANKL蛋白表达水平明显降低(P < 0.01)。见图 1和表 3。

Lane1: Control group; Lane 2: Osteoporosis treatment group; Lane 3: Osteoporosis group. 图 1 各组大鼠骨组织中成骨标志物蛋白表达电泳图 Figure 1 Electrophoregram of protein expressions of osteogenic markers in bone tissue of rats in various groups
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2.4 各组大鼠骨组织形态表现
表 3 各组大鼠骨组织中成骨标志物蛋白表达水平 Table 3 Levels of protein expressions of osteogenic markers in bone tissue of rats in various groups
(n=15, x±s)
Group PERK ATF4 Runx2 Osterix RANKL OPG
Control 0.87±0.54 1.24±0.65 1.17±0.74 0.25±0.13 0.07±0.03 0.40±0.74
Osteoporosis 0.06±0.02** 1.51±0.34* 0.66±0.13** 0.72±0.39** 0.69±0.49** 0.54±0.22
Osteoporosis treatment 0.38±0.15△△ 1.61±0.57 1.12±0.68△ 0.96±0.51△△ 0.15±0.38△△ 0.49±0.09
*P < 0.05, * * P < 0.01 compared with control group; △P < 0.05, △△ P < 0.01 compared with osteoporosis group.
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与对照组比较，骨质疏松组大鼠股骨骨干中破骨细胞数量增多，功能活跃，骨干中出现多个骨质吸收形成的骨陷窝；与骨质疏松组比较，骨质疏松治疗组大鼠股骨骨干结构恢复正常，破骨细胞功能减弱，骨质吸收陷窝消失。见图 2(插页四)。

A:Control group; B:Osteoporosis group; C:Osteoporosis treatment group. 图 2 各组大鼠骨组织形态学表现(HE, ×200) Figure 2 Morphology of bone tissue of rats in various groups(HE, ×200)
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3 讨论

PMOP的核心发病机制为雌激素下降后导致骨重建与骨吸收失衡[11]。目前对于PMOP发病机制的研究[12]主要集中在成骨细胞的活性下降和破骨细胞的活性增强，但内在的调控机制尚缺乏研究。本研究通过动物体内实验探讨PMOP发生时分子水平调控机制的异常改变，为PMOP的防治提供新的分子靶点和理论依据。

正常情况下，骨骼的结构完整和功能保持取决于破骨细胞和成骨细胞协同作用，维持骨重建与骨吸收的平衡[13]。成骨细胞作为骨形成的主要功能细胞，在分化成熟过程中会逐步表达特异性标志物，如ALP、OCN和ColⅠ等[14-15]。同时，在此过程中也会受到一系列转录因子的调控，如Runx2、Osterix/Sp7、β-catenin、ATF4、核转录因子激活蛋白1和Smads等[16-17]，它们彼此协同作用，共同参与调节成骨细胞分化及骨形成。成骨细胞功能下降，导致骨转录因子和成骨因子表达减少，是导致骨质疏松形成的主要因素。PERK基因敲除的小鼠中，成骨细胞分化和成熟障碍，并伴有ColⅠ分泌减少，导致骨骼发育受损。ATF4是PERK通路下游靶分子，同时也是较早受到关注的与成骨细胞分化和骨形成相关的转录因子[18]。ATF4可协同cbfa1刺激成骨细胞内OCN的表达，提示ATF4在成骨细胞分化、成熟和骨骼发育中有重要作用[18]。研究[19]显示：体外培养功能活跃的成骨细胞中BiP和PDI表达水平明显升高，而骨质疏松症患者功能不活跃的成骨细胞中BiP和PDI表达水平明显下降，由此推测内质网应激很可能刺激成骨细胞的成骨作用。

本研究结果显示：雌性大鼠卵巢切除后，PERK和ATF4表达水平明显降低，成骨细胞转录因子Runx2和osterix表达水平明显降低，而诱导破骨细胞分化促进骨质吸收的因子RANKL表达明显升高，提示雌性大鼠卵巢切除后成骨细胞分化功能减弱而破骨细胞成熟增强引起骨质吸收增强导致出现PMOP，通过外源补充雌激素能有效缓解此种症状。本研究结果为PMOP发生机制研究提供了科学依据，为制定骨质疏松的防治策略提供了新的思路和理论依据。

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