Effect of fibroblast growth factor receptor 1 inhibitor on bone destruction in rats with collagen-induced arthritis

doi:10.12307/2025.293

Abstract

Abstract: BACKGROUND: Preliminary research by our group suggests that targeting fibroblast growth factor receptor 1 (FGFR1) may be an effective strategy for treating RA.
OBJECTIVE: To investigate the effects of an FGFR1 inhibitor (PD173074) on bone destruction in rats with collagen-induced arthritis.
METHODS: Twenty-five female Sprague-Dawley rats were randomly divided into five groups: normal control group, model group, methotrexate group, low-dose PD173074 group, and high-dose PD173074 group. Except for the normal control group, rat models of type II collagen-induced arthritis were made in each group. After successful modeling, rats were injected intraperitoneally with sterile PBS in the normal and model groups, 1.04 mg/kg methotrexate in the methotrexate group, and 5 and 20 mg/kg in the low-dose group and high-dose PD173074 groups, once a week. After 4 weeks of drug administration, clinical symptoms and joint swelling in rats were observed. Micro-CT was used for three-dimensional reconstruction and analysis of the ankle joints. Pathological changes in the ankle joints were observed. Periarticular angiogenesis and the expression of receptor activator of nuclear factor-κB ligand were detected. The expression levels of p-FGFR1, vascular endothelial growth factor A, and tartrate-resistant acid phosphatase in the synovial membrane were measured. Pathological changes in the liver, spleen, and kidney were observed and liver, spleen, and kidney indices were calculated.
RESULTS AND CONCLUSION: PD173074 could alleviate clinical symptoms and joint swelling, delay bone loss, improve bone structure, reduce synovial invasion and cartilage bone erosion, reduce the number of periarticular osteoclasts, inhibit angiogenesis in synovial tissues, reduce the expression of receptor activator of nuclear factor-κB ligand, and inhibit the expression of FGFR1 phosphorylated protein, tartrate-resistant acid phosphatase and vascular endothelial growth factor A. Pathologic observation of the liver, spleen and kidney in rats showed no obvious toxic side effects after PD173074 treatment. To conclude, the FGFR1 inhibitor can delay the progression of joint inflammation and bone destruction and inhibit angiogenesis in the rat model of type II collagen-induced arthritis. The therapeutic effect of PD173074 has been preliminarily validated in the type II collagen-induced arthritis model and may act by inhibiting FGFR1 phosphorylation, which provides a direction for the search of new therapeutic targets for rheumatoid arthritis.

Key words: rheumatoid arthritis, PD173074, fibroblast growth factor receptor 1, collagen-induced arthritis, animal model, bone destruction, angiogenesis

CLC Number:

Han Haihui, Meng Xiaohu, Xu Bo, Ran Le, Shi Qi, Xiao Lianbo. Effect of fibroblast growth factor receptor 1 inhibitor on bone destruction in rats with collagen-induced arthritis [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(5): 968-977.

Figures/Tables 6

2.1 实验动物数量分析此次实验选用大鼠25只，每组5只，实验过程中气体吸入麻醉稳定可控，Ⅱ型胶原诱导关节炎模型相对稳定，腹腔注射给药方式安全、操作规范，实验期间无动物死亡，最终分析纳入分析25只。 2.2 PD173074减轻了胶原诱导性关节炎大鼠临床症状以及关节肿胀为了研究PD173074对胶原诱导性关节炎大鼠的治疗效果，设计了如图所示的实验流程(图1)。从二次免疫后开始评估，发现经过胶原诱导性关节炎造模的大鼠临床评分和踝关节厚度随着时间变化显著升高。治疗前3周，各组之间的临床症状评分、踝关节厚度无明显差异。 2.2.1 临床症状评分在治疗3周后，即第32天时，统计分析表明，与模型组症状评分(6.80±0.45)相比，PD173074低剂量组(5.60±0.55)和PD173074高剂量组(4.60±0.55)评分显著降低(P < 0.05，P < 0.001)，而两个PD173074剂量组之间差异无显著性意义。在第36天时，模型组评分(6.60±0.55)显著高于甲氨蝶呤组(4.80±0.84)(P < 0.05)。 2.2.2 踝关节厚度在第32天时，PD173074高剂量组踝关节厚度(5.54±0.12) mm显著小于模型组(6.45±0.22) mm (P < 0.01)，而其他治疗组与模型组均未见明显差异。在第36天时，踝关节厚度甲氨蝶呤组为(5.60±0.25) mm、PD173074低剂量组为(5.28±0.32) mm、PD173074高剂量组为(5.13±0.16) mm，均显著小于模型组(6.29±0.23) mm(P < 0.05，P < 0.01，P < 0.001)，PD173074高剂量组小于甲氨蝶呤组(P < 0.05)。 2.3 PD173074延缓了Ⅱ型胶原诱导关节炎模型大鼠踝关节骨破坏，减少了骨质丢失，具有较好的骨保护作用实验采用micro-CT三维重建了大鼠踝关节并进一步重建了距骨的三维图像(图2)。与对照的模型组相比差异显著，这说明大鼠的骨破坏模型已经成功建立。在模型组中，可以清楚地看到胫距关节、跖跗关节、近端和远端趾间关节均发生了显著的关节破坏，这种骨破坏表现为关节间隙的消失、明显的骨刺形成以及远端趾间关节的畸形，尤其是在胫距关节区域。图2显示了大鼠踝关节距骨骨小梁的数据分析(n=5)。骨显微结构分析显示PD173074对骨破坏有保护作用。 2.3.1 骨小梁骨体积分数是指骨小梁体积与组织体积的比值，较低的数值表明骨小梁减少，这是骨质疏松症的特征之一。未经过治疗的模型组(64.88±6.45)%显示骨小梁骨体积分数百分比显著低于正常对照组(93.38±3.15)%(P < 0.000 1)；而经过甲氨蝶呤、PD173074治疗后，骨小梁骨体积分数相对模型组显著升高，并且PD173074高剂量组(87.41±4.33)%要优于甲氨蝶呤组(75.94±4.88)%(P < 0.05)。 2.3.2 骨小梁骨密度代表松质骨骨密度，是指在松质骨，即充满骨小梁的骨组织区域内，骨矿物质的密度。模型组骨密度中羟基磷灰石(524.63±37.71) mg/cm3较正常对照组(868.43±13.96) mg/cm3显著降低，PD173074低剂量组(685.04±88.2) mg/cm3和PD173074高剂量组(789.45±80.94) mg/cm3显著高于模型组(P < 0.05，P < 0.000 1)，并且PD173074高剂量组优于甲氨蝶呤组(636.5±88.77) mg/cm3(P < 0.05)。 2.4 PD173074减轻了Ⅱ型胶原诱导关节炎模型大鼠踝关节病理损伤，减少破骨细胞形成见图3。 2.4.1 苏木精-伊红染色分析显示，正常对照组的大鼠踝关节滑膜细胞保持了正常形态，细胞核清楚可辨，细胞质染色均匀，没有显著的炎症细胞积聚或渗透，血管增生不明显，软骨边缘平滑，结构正常，骨结构未见损坏，骨小梁紧凑有序。而在模型组中，大鼠踝关节滑膜显示出侵蚀现象，伴随大量炎症细胞的积聚和渗透，滑膜细胞染色呈深蓝色，关节结构遭到严重破坏。在药物干预各组的关节都表现出了不同程度的结构变化。踝关节滑膜炎症评分发现，PD173074低剂量组(5.6±1.1)和PD173074高剂量组(4.0±1.0)明显低于模型组(7.8±0.8)(P < 0.05，P < 0.000 1)。 2.4.2 Safranin O/Fast Green染色结果发现，模型组软骨大量丢失，提示明显的软骨损伤；骨小梁排列稀疏，可见明显的骨量流失。经过治疗后，甲氨蝶呤组及PD173074低剂量组与模型组相比，软骨侵蚀评分无明显差异，而PD173074高剂量组(1.6±0.3)与模型组(2.8±0.3)相比显著下降(P < 0.001)。此外，PD173074高剂量组软骨侵蚀评分显著低于甲氨蝶呤组(2.4±0.5)及PD173074低剂量组(2.27±0.43)(P < 0.01，P < 0.05)。 2.4.3 抗酒石酸酸性磷酸酶染色用抗酒石酸酸性磷酸酶染色标记破骨细胞，对各组大鼠踝关节的阳性染色数量进行统计分析。模型组阳性染色数量(52.6±9.0)明显多于正常对照组，而经过治疗后PD173074高剂量组(30.6±6.2)阳性染色细胞数量有明显下降(P < 0.01)。 2.5 PD173074抑制了关节周围血管生成，并降低了RANKL的表达通过免疫荧光染色，用vWF标记血管内皮细胞，用RANKL标记表达破骨细胞前体的细胞，DAPI标记细胞核，见图4。对vWF和RANKL进行双色免疫荧光共染，分析vWF阳性染色和RANKL阳性染色在滑膜感兴趣区域ROI(Region of Interest)中的比例。发现模型组vWF和RANKL表达要明显高于正常对照组，而经过治疗，各组均能降低vWF的表达。并且PD173074高剂量组vWF和RANKL阳性比例要低于甲氨蝶呤组(P < 0.05，P < 0.01)，但与PD173074低剂量组无明显统计学差异。 2.6 PD173074治疗后大鼠滑膜中p-FGFR1、血管内皮生长因子A、抗酒石酸酸性磷酸酶表达情况 Western blot检测结果发现，PD173074能够抑制FGFR1磷酸化蛋白表达(P < 0.05)，并且抑制了滑膜组织中血管内皮生长因子A和抗酒石酸酸性磷酸酶蛋白的表达(P < 0.05)，见图5。 2.7 PD173074治疗后大鼠肝、脾、肾无明显变化对大鼠肝、脾、肾内脏进行苏木精-伊红染色，发现各组内脏均无明显的病理改变，未发现有明显的炎性细胞聚集，组织浸润，空泡形成，大量纤维连接等表现。计算每只大鼠的肝脏、脾脏、肾脏的质量与各自体质量的比值，分别得到肝脏指数、脾脏指数和肾脏指数。研究中发现肝脏指数中PD173074高剂量组高于正常组(P < 0.05)，而甲氨蝶呤与PD173074低剂量组没有明显差异，见图6。"

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