背景:牛膝(A.bidentata)是一种常见的中草药,用于治疗骨关节炎(OA)。鼠尾草苷D(Ach-D)是分离自双歧杆菌的葡糖苷酸皂苷。
目的:评估Ach-D的作用机制及其对OA的影响。
方法:在进行前交叉韧带横断(ACLT)和内侧半月板切除术(MMx)的大鼠和白细胞介素(IL)-1β诱导的软骨细胞中评估了Ach-D的作用。使用SafraninO-Fast绿色和苏木精-曙红染色检测大鼠软骨组织的组织学变化。免疫组织化学染色,qRT-PCR,ELISA,免疫印迹,和免疫荧光检测软骨退变相关因子和炎症相关因子的表达。CCK-8,LDH测定,进行EdU染色以检测软骨细胞死亡。
结果:Ach-D剂量依赖性地降低了国际骨关节炎研究学会(OARSI)评分,减轻软骨损伤,并降低ACLT-MMx模型中CTX-II和COMP的血清浓度。Ach-D增加了胶原II和聚集蛋白聚糖的表达水平,降低了软骨退变相关蛋白的水平,大鼠软骨组织中的ADAMTS-5、MMP13和MMP3。此外,节点样受体蛋白3(NLRP3)相关的炎症被Ach-D减少,如NLRP3、ASC、GSDMD,IL-6,TNF-α,IL-1β,和大鼠软骨组织中的IL-18。在原代大鼠软骨细胞中,Ach-D保护免受IL-1β诱导的活力损失和LDH释放。Wnt3a是Ach-D的靶蛋白。机械上,Ach-D通过抑制Wnt信号传导减轻OA。
结论:ACH-D可能通过抑制Wnt信号通路减轻炎症和软骨退化,从而减少OA。
BACKGROUND: Achyranthes bidentata Blume (A. bidentata) is a common Chinese herb used to treat osteoarthritis (OA). Achyranthoside D (Ach-D) is a glucuronide saponin isolated from A. bidentata.
OBJECTIVE: To assess the mechanisms of action of Ach-D and its effects on OA.
METHODS: The effects of Ach-D were evaluated in rats underwent anterior cruciate ligament transection (ACLT) with medial meniscectomy (MMx) and in interleukin (IL)-1β-induced chondrocytes. Histological changes in rat cartilage tissues were detected using Safranin O-Fast green and haematoxylin-eosin staining. Immunohistochemical staining, qRT-PCR, ELISA, immunoblotting, and immunofluorescence were conducted to examine cartilage degeneration-related and inflammation-related factor expression. CCK-8, LDH assay, and EdU staining were performed to detect chondrocyte death.
RESULTS: Ach-D dose-dependently reduced the Osteoarthritis Research Society International (OARSI) scores, alleviated cartilage injury, and decreased the serum concentrations of CTX-II and COMP in ACLT-MMx models. Ach-D increased the expression levels of collagen II and aggrecan and decreased the levels of cartilage degeneration-related proteins, ADAMTS-5, MMP13, and MMP3, in rat cartilage tissues. Additionally, nod-like receptor protein 3 (NLRP3)-related inflammation was reduced by Ach-D, as shown by the significantly inhibited expression levels of NLRP3, ASC, GSDMD, IL-6, TNF-α, IL-1β, and IL-18 in rat cartilage tissues. In primary rat chondrocytes, Ach-D protected against IL-1β-induced viability loss and LDH release. Wnt3a is the target protein of Ach-D. Mechanistically, Ach-D alleviated OA by inhibiting Wnt signalling.
CONCLUSIONS: ACH-D may reduce inflammation and cartilage degeneration by inhibiting the Wnt signalling pathway, thereby reducing OA.