Artificial tiger bone meal improves degeneration of spinal facet joints and immune microenvironment of articular cartilage in rats with spine osteoarthritis

doi:10.12307/2025.778

Abstract

Abstract: BACKGROUND: Spine osteoarthritis is a chronic inflammatory disease of the spine, articular ligaments, and tendons, and is the main cause of chronic low back pain. Artificial tiger bone powder has anti-inflammatory and pain-relieving biological effects, so it is expected to provide a new strategy for targeted therapy of spinal osteoarthritis.
OBJECTIVE: To investigate the mechanism of artificial tiger bone meal in regulating the expression level of interleukin-23/interleukin-17 immune axis in improving spine osteoarthritis in rats.
METHODS: Sixty SD rats were randomly divided into blank control group, model group, artificial tiger bone meal low-, medium- and high-dose groups, with 12 rats in each group. Except for the blank control group, osteoarthritis models were established in the other groups by injecting 0.1 mL of iodoacetic acid into the joint cavity of the lumbar vertebral joint capsule of the rats. 15 days after modeling, the artificial tiger bone meal low, medium and high dose groups were treated by intragastric administration of 120, 240 and 480 mg/kg per day respectively. The blank control group and model group were given the same volume of normal saline by gavage. After 21 days of treatment, the expression levels of interleukin-17, interleukin-23, matrix metalloproteinase-3, and chemokine receptor-6 in rat spinal articular cartilage were measured by western blot assay. The expression levels of related inflammatory factors in rat serum were detected by ELISA. The degree of joint injury and degeneration in rats was observed by hematoxylin-eosin staining and safranin O-fast green staining.
RESULTS AND CONCLUSION: (1) The results of western blot assay showed that the expression levels of interleukin 17, interleukin 23, matrix metalloproteinase 3, and chemokine receptor 6 in the articular cartilage of rats in the artificial tiger bone meal high-dose group were significantly lower than those in the model group (P < 0.05). (2) ELISA results showed that compared with the model group, the artificial tiger bone meal medium- and high-dose groups had a significant down-regulating effect on the levels of serum interleukin 17, tumor necrosis factor α, interleukin 1β, and matrix metalloproteinase 3 (P < 0.05). (3) Hematoxylin-eosin staining and saffron O-solid green staining showed that the infiltration and proliferation of synovial inflammatory cells in each treatment group of artificial tiger bone meal were significantly improved compared with the model group, and the pathological inflammation Osteoarthritis Research Society International score and Mankin’s score in the medium- and high-dose groups of artificial tiger bone meal were significantly reduced (P < 0.05). (4) It is suggested that artificial tiger bone meal could significantly improve the inflammation and degeneration of spinal facet joints caused by iodoacetic acid chemical stimulation. Among them, the effect of the high-dose group was more significant. The mechanism may be to inhibit the expression of interleukin-23/interleukin-17 immune axis-related inflammatory factors and improve the immune microenvironment of articular cartilage.

Key words: spine osteoarthritis, artificial tiger bone meal, interleukin-23/interleukin-17 immune axis, inflammation, rat, engineered tissue construction

CLC Number:

Li Wen, Zhao Jingyi, Hu Wen, Xiong Bin, Tang Chao. Artificial tiger bone meal improves degeneration of spinal facet joints and immune microenvironment of articular cartilage in rats with spine osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(26): 5511-5519.

Figures/Tables 8

2.1 实验动物数量分析共纳入60只大鼠，48只大鼠经脊柱骨关节炎造模后均存活，其中43只大鼠攀爬跳跃活动减退，造模15 d后通过小动物高分辨率CT下可见骨关节炎改变，造模成功率为90%，5只大鼠未见明确脊柱骨关节炎表现(模型组2只、低剂量组1只、中剂量组1只、高剂量组1只)。空白对照组12只完全存活。 2.2 人工虎骨粉对大鼠脊柱关节软骨中炎症因子表达水平的影响与空白对照组相比，模型组大鼠软骨组织IL-17、IL-23、MMP-3、CCR-6水平显著升高(P < 0.05)，人工虎骨粉低剂量组大鼠软骨组织IL-23和MMP-3水平显著升高(P < 0.05)，中剂量组大鼠软骨组织MMP-3水平显著升高(P < 0.05)，高剂量组大鼠与空白对照组无统计学差异(P > 0.05)；与模型组相比，人工虎骨粉低剂量组大鼠软骨组织CCR-6水平显著降低(P < 0.05)，中、高剂量组大鼠软骨组织IL-17、IL-23、CCR-6显著降低(P < 0.05)，高剂量组MMP-3显著降低(P < 0.05)；与人工虎骨粉低剂量组相比，高剂量组大鼠软骨组织IL-23、MMP-3水平显著降低(P < 0.05)，中剂量组IL-17、IL-23、MMP-3、CCR-6无明显统计学差异(P > 0.05)，高剂量组IL-17、CCR-6无统计学差异(P > 0.05)；与人工虎骨粉中剂量组相比，高剂量组IL-17、IL-23、MMP-3、CCR-6无统计学差异(P > 0.05)，见图3，4。 2.3 人工虎骨粉对骨关节炎大鼠血清细胞因子的影响如图5所示，与空白对照组比较，模型组和人工虎骨粉不同剂量组大鼠血清TNF-α、IL-1β和IL-17水平均显著升高(P < 0.05)。与模型组比较，人工虎骨粉低剂量组血清TNF-α、IL-1β水平显著下调(P < 0.05)，人工虎骨粉中、高剂量组血清TNF-α、IL-1β和IL-17水平显著下调(P < 0.05)，而人工虎骨粉低剂量组血清IL-17相较模型组无统计学差异(P > 0.05)。与人工虎骨粉低剂量组比较，中、高剂量组血清TNF-α、IL-1β和IL-17水平显著下调(P < 0.05)。与人工虎骨粉中剂量组比较，高剂量组TNF-α、IL-1β水平显著下调(P < 0.05)，而高剂量组血清IL-17相较中剂量组无统计学差异(P > 0.05)。 2.4 人工虎骨粉对骨关节炎大鼠血清MMP-3的影响如图6所示，MMP-3在各造模组大鼠中高表达，与空白对照组相比差异有显著性意义(P < 0.05)。与模型组比较，人工虎骨粉中、高剂量组血清MMP-3水平显著下调(P < 0.05)，而低剂量组效果有限，相较模型组未见统计学差异(P > 0.05)。与低剂量组比较，人工虎骨粉中、高剂量组血清MMP-3水平显著下调(P < 0.05)。与中剂量组比较，高剂量组血清MMP-3水平显著下调(P < 0.05)。 2.5 人工虎骨粉对大鼠脊柱关节病理变化的影响 2.5.1 大鼠脊柱关节苏木精-伊红染色所有最终进入研究的大鼠采用0.3%戊巴比妥钠腹腔麻醉后脱臼处死后，取出各组大鼠腰椎及小关节组织，观察腰椎关节突的关节面退变情况，见图7。组织切片行苏木精-伊红染色，光镜下观察滑膜增生和炎症细胞浸润情况、骨和软骨破坏情况等病理学变化。如图8所示，在空白对照组中，大鼠关节腔面光滑，软骨表面平滑光整，结构清晰可见，滑膜组织形态正常，滑膜组织细胞排列较整齐，滑膜中没有观察到炎症细胞的浸润；而模型组关节结构和形态严重受损，关节表面凹凸不平，软骨细胞排列混乱，存在明显的软骨和骨破坏，滑膜中有大量炎症细胞聚集和浸润，上皮表面粗糙；人工虎骨粉低剂量组中，滑膜中炎症细胞的浸润及滑膜增生情况较模型组有所改善，但仍存在较为明显的软骨破坏现象；人工虎骨粉中、高剂量组较模型组关节炎症状改善显著，关节结构和形态较模型组正常，滑膜中存较少炎症细胞浸润，滑膜增生轻微，无明显的软骨损伤现象。大鼠脊柱关节组织病理炎症国际骨关节炎研究学会评分显示：与模型组相比，人工虎骨粉各剂量组的病理炎症评分均有下降，人工虎骨粉各剂量组对大鼠脊柱关节炎症具有显著的改善作用，相较模型组差异有显著性意义(P < 0.05)。 2.5.2 大鼠脊柱关节番红O-固绿染色对各组大鼠脊柱关节组织切片行番红O-固绿染色，光镜下观察大鼠脊柱关节软骨损伤情况和软骨下骨形态，红染部分为软骨基质，蓝绿色为骨组织。如图9所示，在空白对照组中大鼠脊柱关节软骨结构正常，表面光整，软骨细胞排列整齐，"

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