陈永锋,冯亚非,宋和强,王 鹏,燕 明.神经生长因子抗体在小鼠膝关节炎疼痛模型中的作用研究[J].,2021,(21):4038-4044 |
神经生长因子抗体在小鼠膝关节炎疼痛模型中的作用研究 |
The Effect of Nerve Growth Factor Antibody in Mouse Knee Arthritis Pain Model |
投稿时间:2021-03-02 修订日期:2021-03-25 |
DOI:10.13241/j.cnki.pmb.2021.21.008 |
中文关键词: 膝关节炎 神经生长因子抗体 软骨基质 血管形成 |
英文关键词: Knee arthritis Nerve growth factor antibody Cartilage matrix Angiogenesis |
基金项目:国家自然科学基金面上项目(81871799) |
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中文摘要: |
摘要 目的:探究神经生长因子(Nerve growth factor,NGF)抗体L148M在碘乙酸(Monoiodoacetate,MIA)诱导的膝关节炎(Kee osteoarthritis,KOA)小鼠模型中的作用机制。方法:随机将8周龄C57BL/6雄性小鼠分为对照组、MIA组和L148M组。采用关节腔注射20 mg/mL MIA诱导KOA小鼠模型。手术后2周,L148M组小鼠给予腹腔注射L148M(10 mg/kg)处理。通过苏木精-伊红(HE)染色、番红O染色、OARSI评分和Micro-CT分析评估小鼠膝关节软骨及软骨下骨组织形态学变化。通过qRT-PCR检测CCR2、MCP1、MMP-1、MMP-3、MMP-13、COL10、IL-1β和TNF-α的mRNA表达水平。通过Western blotting检测INOS、COX-2、Collagen II和Aggrecan的蛋白表达水平。通过免疫组织化学法分析VEGFA和Ang-1的蛋白表达水平。通过Micro-CT血管造影分析软骨下骨血管形成数量。结果:HE染色结果显示,L148M组小苏软骨细胞数和关节软骨厚度均高于MIA组。番红O染色显示,L148M组小鼠基质降解小于MIA组。L148M组OARSI评分显著低于MIA组(P<0.05)。micro-CT扫描结果表明,L148M组小鼠软骨和软骨下骨结构完整,没有明显病理损伤。qRT-PCR检测结果显示,与MIA组相比,L148M组小鼠COL10、MMP1、MMP3和MMP-13表达水平均显著降低(P<0.05)。Western blotting检测结果表明,与MIA组相比,L148M组小鼠Collagen II和Aggrecan表达水平升高,INOS和COX-2表达水平降低(P<0.05)。疼痛行为学分析显示,与MIA组相比,L148M组小鼠行进距离和机械刺激反应阈值均降低(P<0.05)。micro-CT血管造影分析显示,L148M小鼠组微血管数量和体积明显低于MIA组(P<0.05)。免疫组化检测显示,L148M组小鼠VEGFA和Ang-1蛋白表达水平均显著低于MIA组(P<0.05)。结论:L148M通过抑制软骨下骨异常血管生成,缓解关节炎症疼痛;并通过抑制炎症细胞因子表达,减轻软骨和软骨下骨病理损伤。 |
英文摘要: |
ABSTRACT Objective: To explore the molecular mechanism of the protective effect of Nerve growth factor (NGF) antibody L148M in a mouse model of Kio osteoarthritis (KOA) induced by iodoacetic acid (MIA). Methods: Eight-week-old male C57BL/6 male mice were randomly divided into a control group, MIA group, and L148M group. KOA mouse model was induced by injecting 20 mg/mL MIA into joint cavity. Two weeks after the operation, mice in the L148M group were intraperitoneally injected with L148M (10 mg/kg). Hematoxylin-eosin (HE) staining, safranin O staining, OARSI score, and Micro-CT analysis were used to observe the morphological changes of knee joint cartilage and subchondral bone in mice. The expression levels of CCR2, MCP1, MMP-1, MMP-3, MMP-13, COL10, IL-1β and TNF-α mRNA were detected by qRT-PCR. The expression levels of INOS, COX-2, Collagen II and Aggrecan were detected by Western blotting. The expression of VEGFA and Ang-1 protein was analyzed by immunohistochemistry. Micro-CT angiography analyzes the number of new blood vessels in subchondral bone. Results: HE staining results showed that the number of chondrocytes and cartilage thickness in the L148M group were higher than those in the MIA group. Safranin O staining showed that the matrix degradation of the L148M group was less than that of the MIA group. The OARSI score of the L148M group was significantly lower than that of the MIA group(P<0.05). The results of micro-CT scan showed that the cartilage and subchondral bone of the L148M group were intact and there was no obvious pathological damage. The results of qRT-PCR showed that compared with the MIA group, the mRNA expression level of COL10, MMP1, MMP3 and MMP-13 in the L148M group were significantly reduced (P<0.05). Western blotting results showed that compared with the MIA group, the protein expression levels of Collagen II and Aggrecan were increased in the L148M group, and the protein expressions level of INOS and COX-2 were decreased(P<0.05). Pain behavior analysis showed that compared with the MIA group, the travel distance and mechanical stimulus response threshold were decreased in the L148M group (P<0.05). Micro-CT angiography analysis showed that the number and volume of microvessels in the L148M group were significantly lower than those in the MIA group (P<0.05). Immunohistochemical examination showed that the protein expression level of VEGFA and Ang-1 in L148M group were significantly lower than those in MIA group (P<0.05). Conclusion: L148M can alleviate the joint pain response by inhibiting abnormal angiogenesis of subchondral bone; and reduce the pathological damage of cartilage and subchondral bone by inhibiting the expression of inflammatory cytokines. |
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