摘要
目的:探讨相容性溶质四氢嘧啶(ectoine)在体外对大鼠关节软骨细胞的影响。方法:取4周龄Sprague-Dawley大鼠进行原代软骨细胞培养，配置0.5%、1.0%、1.5%(w/v)三种质量浓度的四氢嘧啶溶液。以不同浓度四氢嘧啶处理软骨细胞，观察胰酶刺激2 min后软骨细胞形态上的变化及50℃高温刺激下软骨细胞的存活率。用活性氧(ROS)实验检测软骨细胞用四氢嘧啶预处理后以H2O2刺激下细胞内ROS水平并检测细胞色素C氧化酶1(MTCO1)基因的表达。以四氢嘧啶预处理后，用白细胞介素(IL)-1β刺激软骨细胞建立骨关节炎模型，用实时定量PCR(RT-qPCR)检测软骨细胞环氧化酶-2(COX-2)、金属基质蛋白酶-3，-9(MMP-3，MMP-9)、Ⅱ型胶原(Col2A1)mRNA的表达，并进行Ⅱ型胶原的免疫荧光染色。用单因素方差分析统计各组间差异。结果:四氢嘧啶可明显增加软骨细胞耐消化性，在胰酶刺激2 min后保持软骨细胞无明显形态变化；以未经四氢嘧啶预处理的软骨细胞作为对照组，在50 ℃高温下不同浓度四氢嘧啶组的软骨细胞存活率分别为(68±5)%、(83±7)%及(89±4)%，高于对照组(38±7)%的存活率(F＝77.16，P<0.001)；另外，四氢嘧啶还能抵抗细胞氧化，经四氢嘧啶预处理的软骨细胞内ROS水平均低于对照组(F＝157.2，P<0.05)。与单纯IL-1β处理相比，经四氢嘧啶预处理后可降低软骨细胞COX-2(F＝110.4)和MMP-3(F＝154.4)、MMP-9(F＝125.5)的表达水平(均为P<0.001)，并维持Ⅱ型胶原的生成。结论:四氢嘧啶对关节软骨细胞具有多种保护作用，可作为治疗骨关节炎的一种潜在药物。
Abstract
Objective:To explore the protective effects of Ectoine, a compatible solute in nature, on articular chondrocytes in rats through in vitro study.Methods:Four-week-old Sprague-Dawley rats were used for primary chondrocyte culture. Ectoine solutions were prepared at 0.5%, 1.0%, 1.5% (w/v) concentrations. After treated with ectoine, the morphology of chondrocytes after trypsin digestion for two minutes and the viability of chondrocytes at 50°C were observed. Reactive oxygen species (ROS) assay was used to detect the ROS lever and the expression of cytochrome C oxidase one (MTCO1) gene in chondrocytes pre-treated with ectoine and post-stimulated with H2O2. Chondrocytes were pre-treated with ectoine and stimulated with interleukin (IL)-1β to establish a model of osteoarthritis (OA). Cells not treated with ectoine were used as a control group. mRNA of cyclooxygenase-2 (COX-2), matrix metalloproteinase (MMP)-3, MMP-9 and collagen type Ⅱ alpha-1 (Col2A1)were measured by real-time quantitative PCR (RT-qPCR). Immunofluorescence was used to assess the expression of Col2A1. Data were analyzed by one-way ANOVA.Results:Ectoine significantly increased the digestive tolerance of chondrocytes. There was no obvious morphological change of chondrocytes after trypsin treatment for two minutes. The viability of chondrocytes in different concentrations of ectoine groups was (68±5)%, (83±7)% and (89±4)%, respectively, which was significantly higher than that in the control group (38±7)% at 50 ℃ (F=77.16, P<0.001). In addition, ectoine also resisted cell oxidation, and the level of ROS in ectoine pretreated chondrocytes was significantly lower than that in control group (F=157.2, P<0.05). Compared with only IL-1β treatment, ectoine pre-treatment significantly reduced the expression of COX-2, MMP-3, MMP-9 and maintained the synthesis of type Ⅱcollagen in chondrocytes (F=110.4, 154.4, both P<0.001).Conclusion:The results of this study provide potent evidence that ectoine has the protective effects on chondrocytes, and could be utilized as a potentially therapeutic agent in the treatment of OA.
基金项目
深圳市龙岗区医疗卫生科技计划项目(LGW2021-037)
万方数据