芦荟苷体内外对大鼠骨关节炎模型的炎症抑制效应

Anti-inflammation effects of aloin in rat osteoarthritis in vivo and in vitro

目的探讨芦荟苷对SD大鼠骨关节炎保护作用机制。方法体外用LPS诱导关节软骨细胞损伤模型,分为模型组、低浓度芦荟苷组、中浓度芦荟苷组、高浓度芦荟苷组,并设对照组。通过CCK-8法检测芦荟苷的细胞毒性,筛选最适试验浓度,通过Calcein-AM/PI染色、HE及番红O染色检测软骨细胞增殖、形态及蛋白多糖的分泌,Hoechst 33258检软骨细胞测DNA含量,DMMB测定糖胺聚糖含量,实时q PCR检测相关基因的表达,ELISA法检测IL-1β、IL-6、TNF-α和iNOS等炎性因子的水平。手术离断SD大鼠前交叉韧带建立骨关节炎模型,隔日关节腔分别注射50μmol/L芦荟苷和生理盐水100μl,连续1月,HE及番红固绿染色显示病理损伤。结果芦荟苷可以促进软骨细胞增殖,且在50μmol/L时增殖作用最明显(P<0.01)。模型组糖胺聚糖分泌减少(P<0.001),软骨细胞活力明显降低(P<0.01),细胞形态呈细长的类成纤维细胞样,蛋白多糖分泌量降低,软骨特异性基因ACAN和COLA2A1的表达量明显下调,而MMP-3、MMP-13、ADAMTS-4、ADAMTS-5、IL-1β、IL-6、环氧化酶-2(COX-2)的m RNA表达显著上调(均P<0.01),IL-1β、IL-6、TNF-α、i NOS水平显著增高(均P<0.01)。动物试验表明,与模型组相比,芦荟苷组关节软病理损伤程度明显减轻。结论芦荟苷可抑制LPS所致的炎症因子的表达和释放,减轻软骨细胞外基质降解,缓解骨关节炎的进展。

This study was designed to investigate the protective mechanism of aloin against osteoarthritis in SD rats.Articular chondrocytes were divided into control group,model group,low-dose aloin group and medium-dose aloin group,while the articular chondrocyte injury model was constructed by LPS induction in vitro.CCK-8 method was used to evaluate the cytotoxicity and theoptimal test concentration of aloin;the proliferation,morphology and proteoglycans production of cartilage were detected by Calcein-AM/PI staining,HE staining and Saffron O staining.Hoechst 33258 was used to detect the DNA content of cartilage cell;DMMB was used to detect the content of glycosaminoglycans;realtime qPCR was used to detect the expression of related genes;while ELISA was used to detect the levels of inflammatory factors IL-1β,IL-6,IL-1 TNF-αand iNOS.At the same time,osteoarthritis model was constructed by cutting the anterior cruciate ligament in SD rat,and the articular cavity was injected with 50μmol/L aloin or 100μl normal saline for one month.Then the pathological injury of articular cartilage was observed by HE and Saffron solid green staining.Data showed that aloin could promote the proliferation of chondrocytes,and the proliferative effect was most obvious at 50μmol/L(P<0.01).Compared with control group,model group demonstrated glycosaminoglycans secretion decline(P<0.001),cartilage cell viability decrease(P<0.01),fibroblast-like thin cells form,polysaccharide protein secretion reduce,cartilage specific genes ACAN and COLA2 A1 down-regulation,but a raise in the mRNA expression levels of MMP-3,MMP-13,ADAMTS-4,ADAMTS-5,beta,IL-6,IL-1-2 ring oxidase(cox-2)(P<0.01),as well as increase in the levels of IL-1,IL-6,TNF-αand iNOS levels(P<0.01).The animal experiment showed that the degree of pathological damage of joint soft tissue in aloin group was significantly reduced,as compared with the model group.In conclusion,aloin can inhibit the expression and release of inflammatory cytokines induced by LPS,reduce the degradation of chondrocytes extracellular matrix,and alleviate the development of osteoarthritis.