芦荟多糖对大鼠骨关节炎治疗作用的体内和体外研究

In vivo and in vitro study on the therapeutic effects of aloe polysaccharides on osteoarthritis in rats

目的通过体外和体内实验探究芦荟多糖(APS)对大鼠骨关节炎(OA)的治疗作用。方法体外分离SD乳鼠的关节软骨细胞,培养至2代后,通过CCK-8法筛选实验组APS安全剂量。后续通过检测DNA含量和糖胺聚糖(GAG)分泌量评估软骨细胞的增殖和基质降解情况,实时荧光定量PCR检测OA相关基因表达情况,酶联免疫吸附测定(ELISA)法检测炎症因子水平;通过HE染色、番红O和免疫荧光染色等来观察其抗炎和软骨保护效果;并进一步在SD大鼠骨关节炎模型中探究其作用效果。结果通过CCK-8法筛选出剂量为5、10、20 g/L的APS作为低、中、高剂量组。各剂量组的DNA含量均显著高于模型组(P<0.05);高剂量组GAG分泌量高于模型组(P<0.05)。高剂量组的蛋白聚糖(ACAN)和Ⅱ型胶原(COL2A1)基因表达水平相对模型组显著上调(P<0.05),而肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、基质金属蛋白酶(MMP)-3和MMP-13基因表达显著下调(P<0.05)。低、中、高剂量组TNF-α和IL-6的分泌量显著低于模型组(P<0.05),HE和番红O染色也显示APS实验组的细胞形态较模型组好,MMP-13免疫荧光显示高剂量组的表达量显著低于模型组(P<0.05)。体内实验软骨组织番红O染色及国际骨关节炎研究学会(OARSI)软骨损伤评分结果显示APS干预的实验组关节软骨损伤程度明显低于模型组(P<0.05)。结论APS对OA具有一定的抗炎和软骨保护作用。

Objective To explore the therapeutic effects of aloe polysaccharides(APS)on osteoarthritis(OA)in rats through in vitro and in vivo experiments.Methods The articular chondrocytes of SD suckling mice were isolated in vitro and cultured to 2 generations.The concentration of APS in the experimental group was screened by CCK-8.The chondrocyte proliferation and matrix degradation were assessed by detecting DNA content and proteoglycan(GAG)secretion.The real time fluorescent quantitative PCR was used to detect osteoarthritis-related gene expression,and enzyme-linked immunosorbent assay(ELISA)method was used to detect inflammatory factor levels.Hematoxylin-Eosin(HE)staining,Safranin O and immunofluorescence staining were used to observe the anti-inflammatory and cartilage protection effects of APS.Its effects on osteoarthritis were further explored in SD rat model.Results The APS concentrations of 5 g/L,10 g/L and 20 g/L were selected by CCK-8 as the low,medium and high dose experimental groups.It was found that DNA contents were significantly higher in each experimental group than those of the model group(P<0.05).The detection showed that of GAG secretion was significantly higher in the high-dose experimental group than that of the model group(P<0.05).PCR results showed that the expression levels of aggrecan(ACAN)and typeⅡcollagen(COL2A1)were significantly up-regulated in the high-dose experimental group compared with those of the model group(P<0.05),while tumor necrosis factor(TNF)-α,interleukin(IL)-6,the expression of matrix metalloproteinase(MMP)-3 and MMP-13 were significantly down-regulated(P<0.05).The secretion levels of TNF-αand IL-6 were significantly lower in the high-dose experimental group than those of the model group(P<0.05).HE and Safranin O staining also showed that the cell morphology was better in the APS experimental group than that of the model group.The immunofluorescence assay showed that the expression level of MMP 13 was significantly lower in the high-dose experimental group than that of the model group(P<0.05).In vivo articular cartilage tissue Safranin O staining and the International Osteoarthritis Research Society(OARSI)cartilage damage score also showed that the cartilage damage was significantly lower in the experimental group than that in the model group(P<0.05).Conclusion APS has certain anti-inflammatory and cartilage protective effects on OA.