黑麦花粉对软骨细胞炎症因子表达的调控及大鼠膝骨关节炎的干预效果

Regulation of Secale cereale pollen on inflammatory cytokine expression in chondrogenic cells and its intervention effects on knee osteoarthritis in rats

中文关键词:  黑麦花粉  骨关节炎  软骨细胞  抗炎  软骨基质代谢

英文关键词:Secale cereale pollen  osteoarthritis  chondrogenic cells  anti-inflammatory  cartilage matrix metabolism

中文摘要:

      目的 探讨黑麦花粉对软骨细胞炎症及大鼠膝骨关节炎的干预保护作用。方法 体外实验以10 μg/mL脂多糖(lipopolysaccharide, LPS)诱导小鼠软骨细胞ATDC5构建炎症模型, 设0、0.25、0.50、1.00 μg/mL黑麦花粉处理组, 将实验分为 3 组, 即空白对照组、模型组(LPS)和实验组(LPS+黑麦花粉), 采用优化后的逆转录-实时荧光定量聚合酶链式反应技术, 检测白细胞介素-1β (interleukin-1β, IL-1β)、白细胞介素-6 (interleukin-6, IL-6)、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)、神经生长因子(nerve growth factor, NGF)、前列腺素E2 (prostaglandin E2, PGE2)、环氧合酶-2 (cyclooxygenase-2, COX-2)炎症因子及软骨降解相关基质金属蛋白酶(matrix metalloproteinases-1/-2/-3, MMP-1、MMP-2、MMP-3)的相对表达水平; 体内实验以Ⅱ型胶原酶诱导建立SD大鼠膝骨关节炎模型, 设空白对照组、模型组、18 mg/(kg·d)塞来昔布阳性对照组及0.167 g/(kg·d)黑麦花粉组, 灌胃给药4周。通过测量关节周长评估关节肿胀, 组织学染色观察关节病变, 用酶联免疫吸附(enzyme-linked immunosorbent assay, ELISA)测定大鼠滑膜组织中IL-2、TNF-α、COX的水平和免疫组化检测Ⅱ型胶原α1链(collagen type Ⅱ alpha 1 chain, COL2A1)及基质金属蛋白酶-13 (matrix metalloproteinase-13, MMP-13)的表达。结果 体外结果显示: 0.2500~1.0000 μg/mL黑麦花粉可促进软骨细胞增殖, 且呈质量浓度依赖性降低LPS诱导的促炎因子(IL-1β、IL-6、TNF-α、PGE2)及基质金属蛋白酶(MMP-1/2/3)基因表达, 1 μg/mL时上述基因表达可恢复至阴性对照水平; 而COX-2基因表达无显著变化(P>0.05)。体内结果表明: 黑麦花粉组大鼠相对关节周长差从模型组(5.84±0.60) mm降至(3.12±0.46) mm (P<0.001), 能够改善关节肿胀度; 膝关节病理切片显示软骨表面粗糙与褶皱改善、滑膜增生面积降低、炎性浸润呈散在分布, Mankin评分显著低于模型组(P<0.05); 血清COX水平从1.30±0.01至1.19±0.01 (P<0.05), IL-2、TNF-α水平略有下降, 但是无显著性差异(P>0.05); 免疫组化显示软骨基质合成相关蛋白COL2A1阳性染色面积增加, 降解相关蛋白MMP13阳性染色面积减少, 实现软骨基质代谢“促合成-抑降解”双向调节。结论 黑麦花粉可抑制LPS诱导的软骨细胞炎症反应与基质降解, 减轻大鼠膝骨关节炎的病变程度, 调节软骨基质代谢平衡, 发挥对骨关节炎的干预保护作用。

英文摘要:

      Objective To investigate the interventional and protective effects of Secale cereale pollen on chondrocyte inflammation and knee osteoarthritis in rats. Methods In vitro experiment, a chondrocyte inflammation model was established by inducing mouse chondrocyte line ATDC5 with 10 μg/mL lipopolysaccharide (LPS). The experiment was divided into 3 groups: Blank control group, model group (LPS-induced) and experimental groups (LPS+Secale cereale pollen at concentrations of 0, 0.25, 0.50 and 1.00 μg/mL). Reverse transcription-quantitative polymerase chain reaction was performed to detect the gene expressions of inflammatory factors including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), nerve growth factor (NGF), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), as well as cartilage matrix metalloproteinases-1/-2/-3 (MMP-1, MMP-2 and MMP-3). In vivo experiment, a knee osteoarthritis model was constructed in SD rats via intra-articular injection of type Ⅱ collagenase. The rats were randomly assigned to 4 groups: Blank control group, model group, positive control group treated with celecoxib at 18 mg/(kg·d), and Secale cereale pollen group treated with 0.167 g/(kg·d). All treatments were administered via gavage for 4 consecutive weeks. Joint swelling was evaluated by measuring the joint circumference. Histopathological changes of the knee joint were observed using histological staining. The levels of IL-2, TNF-α and COX in the synovial tissue of rats were determined by enzyme-linked immunosorbent assay (ELISA), and the expressions of collagen type Ⅱ alpha 1 chain (COL2A1) and matrix metalloproteinase-13 (MMP-13) were detected by immunohistochemistry. Results The in vitro results showed that Secale cereale pollen at mass concentrations ranging from 0.2500 to 1.0000 μg/mL promoted chondrocyte proliferation, and dose-dependently down-regulated the gene expressions of LPS-induced pro-inflammatory factors (IL-1β, IL-6, TNF-α, PGE2) and matrix metalloproteinases (MMP-1/2/3). At the mass concentration of 1 μg/mL, the expressions of the aforementioned genes were restored to the level of the negative control group, while the COX-2 gene expression showed no significant change (P>0.05). The in vivo results demonstrated that the relative difference in joint circumference of rats in the Secale cereale pollen group decreased significantly from (5.84±0.60) mm in the model group to (3.12±0.46) mm (P<0.001), indicating an obvious improvement in joint swelling. Pathological sections of the knee joint revealed ameliorated roughness and folds of the cartilage surface, reduced synovial hyperplasia area, and scattered inflammatory cell infiltration. The Mankin score of the Secale cereale pollen group was significantly lower than that of the model group (P<0.05). The serum COX level decreased from 1.30±0.01 to 1.19±0.01 (P<0.05), whereas the levels of IL-2 and TNF-α showed a slight downward trend without statistical significance (P>0.05). Immunohistochemical results indicated that the positive staining area of COL2A1, a protein related to cartilage matrix synthesis, was increased, while that of MMP13, a protein associated with cartilage matrix degradation, was reduced, thus achieving a bidirectional regulation of cartilage matrix metabolism characterized by “promoting synthesis and inhibiting degradation”. Conclusion Secale cereale pollen can inhibit LPS-induced chondrocyte inflammation and matrix degradation, alleviate the pathological severity of knee osteoarthritis in rats, and regulate the metabolic balance of cartilage matrix, thereby exerting interventional and protective effects against osteoarthritis.