改性柑橘果胶对软骨细胞的影响

Effect of modified citrus pectin on chondrocytes

背景:半乳糖苷凝集素3是关节软骨损伤和骨关节炎发生、发展中重要的前炎性因子,也是该类疾病治疗的潜在靶点。改性柑橘果胶是半乳糖苷凝集素3的竞争性抑制剂,但目前改性柑橘果胶对关节软骨的作用尚不清楚。目的:考察改性柑橘果胶对体外培养软骨细胞及白细胞介素1β诱导的骨性关节炎软骨细胞代谢活性及相关基因表达的影响。方法:①收集兔膝关节软骨细胞,分别以0(正常对照),250,500,750 mg/L的改性柑橘果胶进行处理,处理1,3,5 d;②改性柑橘果胶后处理实验:收集兔膝关节软骨细胞,首先以白细胞介素1β处理24 h,再分别以0,250,500,750 mg/L的改性柑橘果胶处理1,3,5 d,同时设置正常对照;③改性柑橘果胶预处理实验:收集兔膝关节软骨细胞,首先分别以0,250,500,750 mg/L的改性柑橘果胶预处理6 h,再以白细胞介素1β处理1,3,5 d,同时设置正常对照。于对应的时间点,采用RT-qPCR检测软骨细胞Ⅱ型胶原、蛋白聚糖、SOX9、Ⅰ型胶原、基质金属蛋白酶13和半乳糖苷凝集素3的mRNA相对表达,进一步通过免疫荧光染色考察软骨细胞Ⅱ型胶原的合成。结果与结论:①与正常软骨细胞相比,不同质量浓度的改性柑橘果胶处理可以增加软骨细胞的相对增殖活性,提高SOX9和Ⅱ型胶原的表达水平、降低Ⅰ型胶原、基质金属蛋白酶13和半乳糖苷凝集素3表达水平;②与正常对照组相比,两种白细胞介素1β处理后均会下调软骨细胞的蛋白聚糖、SOX9、Ⅱ型胶原的表达水平,上调半乳糖苷凝集素3、基质金属蛋白酶13的表达水平;③与单纯白细胞介素1β处理组比较,白细胞介素1β先处理+改性柑橘果胶处理可上调Ⅱ型胶原、蛋白聚糖和SOX9的表达水平,降低半乳糖苷凝集素3、基质金属蛋白酶13和I型胶原的表达水平;改性柑橘果胶预处理+白细胞介素1β后处理可上调Ⅱ型胶原、蛋白聚糖和SOX9的表达,降低Ⅰ型胶原、半乳糖苷凝集素3和基质金属蛋白酶13的表达,但上调的Ⅱ型胶原和蛋白聚糖表达水平未能达到正常软骨水平;④结果表明在此次实验条件下,改性柑橘果胶具有增强软骨细胞增殖活性、维持其表型的作用;同时,改性柑橘果胶干预可改变白细胞介素1β诱导软骨细胞的基因表达,上调软骨细胞外基质合成及软骨形成转录因子相关基因的表达水平,下调分解代谢、炎性因子及去分化相关基因的表达水平。

BACKGROUND:Galectin-3 is an important proinflammatory factor during articular cartilage injury and the development of osteoarthritis.Therefore,it could be a potential target for the treatment of osteoarthritis.Modified citrus pectin is a competitive inhibitor of galectin-3.However,the effect of modified citrus pectin on cartilage is not unclear.OBJECTIVE:To investigate the effect of modified citrus pectin on the metabolic activity and genes expression of in vitro cultured chondrocytes and interleukin-1β induced osteoarthritis chondrocytes.METHODS:(1)Chondrocytes from rabbit knee were collected and subjected to the treatment of modified citrus pectin of 0(normal control),250,500 or 750 mg/L respectively for 1,3,and 5 days.(2)Posttreatment experiment of modified citrus pectin:Chondrocytes of rabbit knee were collected.Chondrocytes were pretreated by interleukin-1β for 24 hours,then posttreated with modified citrus pectin of 0(control),250,500 or 750 mg/L respectively for 1,3,and 5 days.At the same time,a normal control group was set up.(3)Pretreatment experiment of modified citrus pectin:Chondrocytes of rabbit knee were collected.Chondrocytes were pretreating with modified citrus pectin of 0(control),250,500 or 750 mg/L respectively for 6 hours and following by posttreating by interleukin-1β for 1,3,and 5 days.At the same time,a normal control group was set up.At the corresponding time point,the mRNA levels of type Ⅱ collagen,aggrecan,SOX9,type Ⅰ collagen,matrix metalloproteinase 13 and galectin-3 were determined via RT-qPCR.Furthermore,immunofluorescence staining was conducted to examine the synthesis of type Ⅱ collagen of chondrocytes.RESULTS AND CONCLUSION:(1)Compared with normal control group,modified citrus pectin treatment with different mass concentrations significantly enhanced chondrocyte viability,upregulated mRNA of SOX9 and type Ⅱ collagen,and downregulated mRNA of type Ⅰ collagen,matrix metalloproteinase 13 and galectin-3.(2)Compared with normal control group,interleukin-1β treatment induced downregulation of expression levels of aggrecan,SOX9,and type Ⅱ collagen,and upregulation of expression levels of galectin-3 and matrix metalloproteinase 13.(3)Compared with interleukin-1β treated groups,interleukin 1βpretreatment+modified citrus pectin treatment up-regulated the expression levels of type Ⅱ collagen,aggrecan and SOX9,and decreased the expression levels of galectin-3,matrix metalloproteinase 13 and type Ⅰ collagen.Modified citrus pectin+interleukin 1β posttreatment up-regulated the expression of type Ⅱ collagen,aggrecan and SOX9,and decreased the expression of type Ⅰ collagen,galectin-3 and matrix metalloproteinase 13,but up-regulated type Ⅱ collagen and aggrecan expression levels failed to reach normal cartilage levels.(4)In summary,under the experimental conditions,modified citrus pectin could enhance the proliferation activity of chondrocytes and maintain their phenotype.At the same time,the intervention of modified citrus pectin can change the gene expression of interleukin 1β-induced chondrocytes,and up-regulate the expression of cartilage cell extracellular matrix synthesis and cartilage formation transcription factor-related genes,and down-regulate the expression levels of catabolism,inflammatory factors and dedifferentiation related genes.