Objective: To systematically explore the intervention efficacy of annexin A5 (AnxA5) on temporomandibular joint osteoarthritis (TMJOA) induced by complete Freund's adjuvant (CFA) in rats. Methods: Thirty-two 6-week-old male SD rats (weighing 200-250 g) were assigned to histological analysis (n=20) or micro-CT analysis (n=12). The TMJOA model was established by bilateral intra-articular injection of CFA, while the control group received saline. Three days later, TMJOA rats were randomly divided into three groups (n=8 each): CFA model group (saline injection), CFA+HA group [hyaluronic acid (HA), once weekly for 2 weeks], and CFA+AnxA5 group (AnxA5 injection every 3 days for 4 injections). The saline group was injected with the same frequency and dosage of saline as that of AnxA5. The changes in the structure of the temporomandibular joint were observed through HE and safranin O-eosin green staining, and the total thickness of the cartilage (TC), the thickness of the hypertrophic chondrocyte layer (HL), the thickness of the fibrocartilage layer (FL), as well as the thickness of the anterior band (A), Intermediate zone (In) and posterior bands (P) of the articular disc were measured.The changes in the content of glycosaminoglycans in the condylar cartilage matrix were observed by Alcian blue and toluidine blue staining. The contents of MMP 13, collagen Ⅱ and TNF-α in the condylar cartilage matrix were observed by Immunohistochemical staining. The infiltration quantity of M1-type macrophages was observed by immunofluorescence staining. The situation of subchondral bone remodeling of the condyle was observed by micro-CT, data such as bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were collected and calculated. Results: The results of HE and safinin-O fast green staining showed that compared with the thickness of each layer of cartilage and the thickness of the articular disc in the saline group [TC: (306.85±39.87) μm, HL: (123.16±27.19) μm, FL: (43.87±7.01) μm, A: (263.55±13.55) μm, In: (155.91±26.80) μm, P: (263.12±36.21) μm], the TC and HL in the CFA model group were significantly thinner [(218.30±38.32) and (79.90±18.54) μm ](P<0.001), FL was significantly thicker [(67.69±13.46) μm] (P<0.001), and the A, In and P of the articular disc were significantly thicker [respectively (305.27±23.37), (200.96±18.75) and (321.92±16.86) μm] (P<0.001). Compared with the CFA model group, the TC [(252.76±32.23) and (270.37±21.87) μm] and HL [(108.15±11.13) and (108.41±17.30) μm] in the CFA+HA treatment group and the CFA+AnxA5 intervention group were significantly thicker (P<0.05), while FL [(46.08±5.99) and (45.58±5.27) μm] was significantly thinner (P<0.01). The thickness of the A, In and P of the articular disc in the CFA+HA treatment group [(272.54±19.66), (180.24±14.47) and (273.79±29.28) μm] and in the CFA+AnxA5 intervention group [(263.66±10.68), (168.60±9.87) and (279.31±25.79) μm] were significantly thinner (P<0.05). The results of Alcian blue and toluidine blue staining showed that in the saline group, the staining of the condylar hypertrophic layer was obvious and uniform, while in the CFA model group, it was lightly stained, and in some areas, it was even unstained. After treatment with HA and AnxA5, the staining became relatively intense, and the loss of glycosaminoglycans slowed down.The IHC staining results showed that compared with the saline group (MMP13: 0.24±0.03,COL-Ⅱ:0.36±0.12), the content of MMP13 increased (0.37±0.07), and the content of COL-Ⅱ decreased (0.16±0.03) (both P<0.001). Compared with the CFA model group, the CFA+HA treatment group and the CFA+AnxA5 intervention group showed significantly decreased MMP13 levels (0.29±0.04 and 0.29±0.08) (both P<0.05) and significantly increased COL-Ⅱ levels (0.21±0.04 and 0.22±0.03, both P<0.01).The results of HE and IF staining showed that compared with the saline group, there was a large infiltration of inflammatory cells in the synovium of rats in the CFA model group and the number of M1-type macrophages increased (P<0.001). Compared with the CFA model group, both the CFA+HA treatment group and the CFA+AnxA5 intervention group showed a significant reduction in M1 macrophage numbers (P<0.05, P<0.01). The IHC staining results showed that compared with the saline group, the content of TNF-α in the CFA model group increased (P<0.001), while in the CFA+HA treatment group and CFA+AnxA5 intervention group, the content of TNF-α decreased (P<0.05;P<0.001).The Micro-CT results showed that compared with the saline group, the CFA model group exhibited significant decreases in condylar BV/TV, Tb.N, and Tb.Th (P<0.001, P<0.01, P<0.001).The difference between the CFA+HA treatment group and the CFA model group was not statistically significant. Compared with the CFA model group, the CFA+AnxA5 intervention group showed significantly higher BV/TV, Tb.N and Tb.Th (P<0.01, P<0.05, P<0.05). Conclusions: In the CFA-induced inflammation-dominated rat TMJOA model, AnxA5 can protect cartilage homeostasis, inhibit synovial inflammatory response, and simultaneously improve abnormal subchondral bone remodeling. 目的： 系统探究膜联蛋白A5（AnxA5）对弗式完全佐剂（CFA）诱导的大鼠颞下颌关节骨关节炎（TMJOA）的干预疗效。 方法： 本实验选取6周龄雄性SD大鼠（体重200~250 g）32只（其中20只用于组织学检测，12只用于显微CT检测），采用双侧关节腔内注射生理盐水或CFA法分别建立大鼠对照模型（生理盐水组8只）和TMJOA模型（24只）。3天后，将TMJOA模型大鼠按照随机数字表法分为3组，每组8只；注射与AnxA5频次及用量一致的生理盐水为CFA模型组；每周注射1次透明质酸（HA），连续注射2次为CFA+HA治疗组；每隔3天注射1次AnxA5，连续注射4次，为CFA+AnxA5干预组；生理盐水组注射与AnxA5频次及用量一致的生理盐水。通过HE和番红O-固绿染色观察颞下颌关节结构的改变，并测量软骨总厚度（TC）、肥大软骨细胞层（HL）、纤维软骨层（FL）的厚度，以及关节盘前带、中带、后带的厚度；通过阿利新蓝和甲苯胺蓝染色观察髁突软骨基质中糖胺聚糖含量的变化；通过免疫组织化学染色（IHC）观察髁突软骨基质中基质金属蛋白酶13（MMP13）、Ⅱ型胶原蛋白（COL-Ⅱ）和肿瘤坏死因子α（TNF-α）含量的变化；通过免疫荧光染色（IF）观察M1型巨噬细胞的浸润数量；通过显微CT观察髁突软骨下骨重塑的情况，采集计算骨体积分数（BV/TV）、骨小梁数量（Tb.N）和骨小梁厚度（Tb.Th）数据。 结果： HE染色和番红O-固绿染色结果显示，与生理盐水组的软骨各层厚度［TC、HL、FL分别为（306.85±39.87）、（123.16±27.19）和（43.87±7.01）μm］及关节盘厚度［前带、中带、后带分别为（263.55±13.55）、（155.91±26.80）和（263.12±36.21）μm］相比，CFA模型组大鼠的TC和HL［（218.30±38.32）和（79.90±18.54）μm］均显著变薄（均P<0.001），FL［（67.69±13.46）μm］显著增厚（P<0.001），关节盘前带、中带、后带［分别为（305.27±23.37）、（200.96±18.75）和（321.92±16.86）μm］均显著增厚（P<0.001）；与CFA模型组相比，CFA+HA治疗组和CFA+AnxA5干预组的TC［（252.76±32.23）和（270.37±21.87）μm］及HL［（108.15±11.13）和（108.41±17.30）μm］均显著增厚（P<0.05），FL［（46.08±5.99）和（45.58±5.27）μm］均显著变薄（P<0.01），关节盘前带、中带和后带厚度［CFA+HA治疗组分别为（272.54±19.66）、（180.24±14.47）和（273.79±29.28）μm，CFA+AnxA5干预组分别为（263.66±10.68）、（168.60±9.87）和（279.31±25.79）μm］均显著变薄（P<0.05）。阿利新蓝和甲苯胺蓝染色结果显示，生理盐水组髁突肥大层染色明显且均匀，CFA模型组淡染，部分区域甚至失染，CFA+HA治疗组和CFA+AnxA5干预组染色相对浓郁，糖胺聚糖流失减缓。IHC结果显示，与生理盐水组的MMP13（0.24±0.03）和COL-Ⅱ（0.36±0.12）相比，CFA模型组的MMP13含量（0.37±0.07）显著升高，COL-Ⅱ含量（0.16±0.03）显著降低（均P<0.001）；与CFA模型组相比，CFA+HA治疗组和CFA+AnxA5干预组的MMP13含量（0.29±0.04和0.29±0.08）均显著降低（均P<0.05），COL-Ⅱ含量（0.21±0.04和0.22±0.03）均显著升高（均P<0.01）。HE和IF染色结果显示，与生理盐水组相比，CFA模型组大鼠滑膜大量炎症细胞浸润，M1型巨噬细胞数量增多（P<0.001），与CFA模型组相比，CFA+HA治疗组和CFA+AnxA5干预组的M1型巨噬细胞数量均显著减少（P<0.05，P<0.01）。IHC染色结果显示，相比于生理盐水组，CFA模型组TNF-α含量显著升高（P<0.001）；与CFA模型组相比，CFA+HA治疗组和CFA+AnxA5干预组中TNF-α的含量显著降低（P<0.05；P<0.001）。显微CT结果显示，与生理盐水组相比，CFA模型组大鼠髁突的BV/TV、Tb.N、Tb.Th均显著降低（P<0.001，P<0.01，P<0.001）；CFA+HA治疗组与CFA模型组的差异无统计学意义（P>0.05）；CFA+AnxA5干预组的BV/TV、Tb.N和Tb.Th均显著高于CFA模型组（P<0.01，P<0.05，P<0.05）。 结论： 在CFA诱导的炎症主导型大鼠TMJOA模型中，AnxA5可保护软骨稳态、抑制滑膜炎症反应，同时改善软骨下骨异常重塑。.
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