伊班膦酸钠对膝骨关节炎大鼠自噬相关蛋白LC3 Ⅱ的影响*

EFFRCT OF IBANDRONTE SODIUM ON AUTOPHAGY RELATED PROTEIN LC3Ⅱ IN RAT MODEL OF KNEE OSTEOARTHRITIS

摘要:

目的 研究伊班膦酸钠对膝骨关节炎大鼠自噬相关蛋白LC3 Ⅱ的表达变化,初步探讨软骨组织中自噬水平与骨关节炎发生发展的关系。方法 将30只3月龄雌性SD大鼠随机分为假手术组(Sham)、生理盐水治疗组(ACLT+NS)和伊班膦酸钠治疗组(ACLT+IB),每组10只。ACLT+NS组、ACLT+IB组行膝关节前交叉韧带切断术(ACLT)建立膝骨关节炎模型。术后1周ACLT+IB组予以伊班膦酸钠干预。检测指标：番红染色、Mankin`s评分；关节软骨下骨形态学检测；大鼠血I型胶原C端肽(CTX-I)、II型胶原C端肽(CTX-II)及IL-1、IL-6水平；基质金属蛋白酶-13(MMP-13)及自噬相关蛋白LC3Ⅱ表达。结果 番红染色ACLT+NS组软骨结构破坏明显,Mankin`s评分显著高于Sham组和ACLT+IB组(P＜0.001)； ACLT+IB组相对骨体积(BV/TV)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)等均明显高于ACLT+NS组(P＜0.001),骨小梁分离度(Tb.Sp)则显著低于ACLT+NS(P＜0.001)； ACLT+NS组血清CTX-I、CTX-II、IL-1、IL-6水平高于Sham组和ACLT+IB组(P＜0.05)；相比较于Sham组和ACLT+IB组,ACLT+NS组的MMP-13表达水平增高(P＜0.001),LC3Ⅱ表达降低(P＜0.001)。结论 伊班膦酸钠能够下调MMP-13蛋白表达,并减少炎性因子IL-1和IL-6分泌,进而保护关节软骨,改善软骨下骨小梁微观结构,可能与其促进细胞自噬水平有关。

Abstract:

Objective To access the effect of ibandronate sodium on cartilage and subchondral bone in knee osteoarthritis rat induced anterior cruciate ligament transaction(ACLT). To explore the role of autophagy in the development of osteoarthritis through the observation of expression of autophagy related protein LC3Ⅱin cartilage and subchondral bone. Methods Thirty 3-month-old female SD rats were randomly divided into 3 groups (n = 10): sham operation group (SHAM), ACLT+NS group, ACLT+IB group. Rats in ACLT+NS and ACLT+IB groups were undergone ACLT on the right knee. The rats of sham group were undergone sham operation. After one week the rats of ACLT+IB group were given intraperitoneal injection of ibandronate sodium at a dosage of 10μg/kg weekly for 12weeks. In contrast, ACLT+NS group was given NS at the same dose. All rats were sacrificed at 13weeks post surgery. Safranin staining were used to observe the the structure of articular cartilage. The degeneration of cartilage was graded by Mankin' s scale. The subchondral bone changes were quantitatively analyzed with Micro-CT. Secretion of IL-1 (Interleukin-1), IL-6 (Interleukin-6), C-terminal cross-linking telopeptide of type I collagen(CTX-I) and C-terminal cross-linking telopeptide of type I collagen(CTX-II) in serum was examined by ELISA. The protein expression level of matrix metalloproteinase-13(MMP-13) and autophagy related protein LC3-Ⅱwas detected by western blotting. Results ① The destruction of articular cartilage in ACLT+NS was observed, mankin’s scores in ACLT+IB and Sham groups were significantly lower than that in ACLT+NS group (P＜0.001). ② Compared with that of ACLT+NS group, the rats in ACLT+IB group significantly increased the trabecular bone relative volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N) (P＜0.001), and significantly decreased trabecular bone separation (Tb.Sp) (P＜0.001). ③ The serum levels of CTX-I、CTX-II、IL-1、IL-6 in ACLT+IB and Sham groups were significantly lower than that in ACLT+NS group (P＜0.05). Compared with that of ACLT+NS group, the rats in ACLT+IB group significantly decreased the expression of MMP-13. The expression of LC3Ⅱ in ACLT+NS group was significantly lower than that of ACLT+IB and Sham groups (P＜0.001). Conclusion Sodium ibandronate can down-regulate the expression of MMP-13, reduce the secretion of inflammatory factors IL-1 and IL-6, promote the level of autophagy, protect the articular cartilage and improve the microstructure of bone trabeculae under cartilage.