“杜仲-当归”介导Wnt/β-catenin信号转导调控MMP-13表达防治关节炎的作用机制研究

“Eucommia Ulmoides-Angelica Sinensis” Ameliorate Osteoarthritis Through the Regulation of MMP-13 Via Wnt/β-catenin Signaling

关键词:  骨性关节炎  软骨细胞  杜仲-当归  基质金属蛋白酶  Wnt/β-catenin信号

Key words:  osteoarthritis  chondrocyte  Eucommia ulmoides-Angelica sinensis  matrix metallo proteinase  Wnt/β-catenin signaling

摘要:

目的 研究“杜仲-当归”介导Wnt/β-catenin信号转导调控MMP13表达防治骨性关节炎（osteoarthritis，OA）的作用机制。方法 取SD大鼠，随机分为Sham组、OA组、OA+补肾组（杜仲40 mg·kg^-1）、OA+活血组（当归40 mg·kg^-1）、OA+补肾活血组（杜仲-当归药液40 mg·kg^-1）、OA+阳性药物组（西乐葆10 mg·kg^-1+奥泰灵1.2 mg·kg^-1）共6组。OA模型制备成功后，各组大鼠给予对应药物治疗，连续给药8周。番红O染色检测软骨组织变化，免疫组织化学检测软骨组织中MMP-13表达，ELISA检测大鼠血清IL-6、IL-8含量，Western blotting检测软骨组织中MMP-3、MMP-9、MMP-13、β-catenin蛋白表达。分离正常SD大鼠软骨细胞，IL-1β（10 ng·mL^-1）干预诱导体外OA炎症模型并进行药物干预。番红O染色检测软骨细胞表型变化，ELISA检测软骨细胞IL-6、IL-8含量，免疫荧光染色检测软骨细胞β-catenin表达，qRT-PCR检测软骨细胞中IL-6、IL-8、MMP-3、MMP-9和MMP-13的mRNA表达，Western blotting检测软骨细胞中MMP-3、MMP-9、MMP-13、β-catenin蛋白表达。结果 药物处理对OA导致的软骨组织损伤有不同程度的修复作用；杜仲-当归可下调血清中IL-6、IL-8水平，下调软骨组织中MMP-3、MMP-9、MMP-13、b-catenin的蛋白表达。与IL-1b组相比，杜仲-当归可提高软骨细胞存活率，改善细胞形态，下调软骨细胞IL-6、IL-8水平，降低软骨细胞中b-catenin荧光强度，并下调软骨细胞中IL-6、IL-8、MMP-3、MMP-9、MMP-13的mRNA表达和MMP-3、MMP-9、MMP-13、b-catenin的蛋白表达。结论 体内、体外试验表明补肾活血中药“杜仲-当归”可以通过促进软骨细胞增殖、调控炎症因子水平、改善软骨基质降解、改善软骨病理损伤起到对OA的防治作用，且作用机制可能与介导Wnt/β-catenin信号转导调控MMP-13表达有关。

Abstract:

OBJECTIVE To study the mechanism of “Eucommia ulmoides(EU)-Angelica sinensis(AS)” mediated Wnt/β-catenin signal in regulating the expression of MMP13 in the prevention and treatment of osteoarthritis(OA). METHODS SD rats were randomly divided into six groups: sham group, OA group, OA+kidney tonifying group(EU 40 mg·kg^-1), OA+blood activating group(AS 40 mg·kg^-1), OA+kidney tonifying and blood activating group(EU-AS solution 40 mg·kg^-1), OA+positive conrol group(celebrex 10 mg·kg^-1+otailin 1.2 mg·kg^-1), after inducing of OA model, rats in each group were administrated for 8 weeks. Safranin O(SO) staining was then used to evaluate the injury of cartilage, expression of MMP-13 in cartilage was detected by immunohistochemical levels of IL-6 and IL-8 in rats serum were detected by ELISA, the expression of MMP-3, MMP-9, MMP-13 and β-catenin in cartilage was analyzed by Western blotting. The chondrocyte isolated from normal SD rats were incubated with IL-1β(10 ng·mL^-1) to induce inflammation model of OA. SO staining was used to detect the phenotype of chondrocytes, levels of IL-6 and IL-8 in chondrocytes were detected by ELISA, β-catenin expression in chondrocytes was detected by using immunofluorescence staining, mRNA expression of IL-6, IL-8, MMP-3, MMP-9 and MMP-13 in chondrocytes was analyzed by qRT-PCR, protein expression of MMP-3, MMP-9, MMP-13 and β-catenin in chondrocytes was analyzed by Western blotting. RESULTS There were different degrees of repair of cartilage injury in the administration groups, the levels of IL-6 and IL-8 in serum, the expression of MMP-3, MMP-9, MMP-13 and β-catenin in cartilage tissues were also down-regulated. Compared with IL-1β group, EU-AS could improve chondrocyte cell survival rate, improve cell morphology, reduce the levels of IL-6 and IL-8 in chondrocytes, reduce the fluorescence intensity of β-catenin in chondrocytes, down regulate the mRNA expression of IL-6, IL-8, MMP-3, MMP-9 and MMP-13 in chondrocytes and the protein expression of MMP-3, MMP-9, MMP-13 and β-catenin. CONCLUSION EU-AS, a traditional Chinese medicine for invigorating the kidney and promoting blood circulation, can ameliorate OA by promoting the chondrocyte proliferation, regulating the level of inflammatory factors, inhibiting cartilage matrix degradation and cartilage pathological damage, and the mechanism may be related to the regulation of MMP-13 expression by Wnt/β-catenin signal transduction.