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中华关节外科杂志(电子版) ›› 2024, Vol. 18 ›› Issue (01) : 69 -77. doi: 10.3877/cma.j.issn.1674-134X.2024.01.010

基础论著

四氢嘧啶对大鼠关节软骨细胞的保护作用
李朋1,†(), 苗立帅1, 朱智奇1   
  1. 1. 518000　香港中文大学（深圳）附属第二医院，深圳市龙岗区人民医院骨科
  • 收稿日期:2023-04-07 出版日期:2024-02-01
  • 通信作者: 李朋
  • 基金资助:
    深圳市龙岗区医疗卫生科技计划项目(LGW2021-037)

Protective effects of ectoine on articular chondrocytes in rats

Peng Li1,†(), Lishuai Miao1, Zhiqi Zhu1   

  1. 1. Orthopedic Surgery Department of The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong(Shenzhen), Longgang District People’ s Hospital of Shenzhen, Shenzhen 518000, China
  • Received:2023-04-07 Published:2024-02-01
  • Corresponding author: Peng Li
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引用本文：

李朋, 苗立帅, 朱智奇. 四氢嘧啶对大鼠关节软骨细胞的保护作用[J]. 中华关节外科杂志(电子版), 2024, 18(01): 69-77.

Peng Li, Lishuai Miao, Zhiqi Zhu. Protective effects of ectoine on articular chondrocytes in rats[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2024, 18(01): 69-77.

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。

关键词: 大鼠, 骨关节炎, 软骨细胞, 炎症
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.

Key words: Rats, Osteoarthritis, Chondrocytes, Inflammation
图1 四氢嘧啶(ectoine)的结构及作用机制。图A为四氢嘧啶的化学结构；图B为四氢嘧啶的作用机制，示四氢嘧啶能改善细胞表面的水合作用、增加分子间的间距、提高细胞膜脂质头部基团的流动性
Figure 1 Structure and mechanism of ectoine. A is structure of ectoine; B is the mechanism of ectoine, that improves the fluidity of the lipid head groups in the cell membraneand stabilizes macromolecules
图2 RT-qPCR(实时定量PCR)的引物序列注：COX-2-炎症因子环氧化酶-2；MMP-金属基质蛋白酶；MTCO1-细胞色素C氧化酶1；Col2A1-Ⅱ型胶原；β-actin-β-肌动蛋白
Figure 2 Primer sequences for RT-qPCR Note: COX-2- cyclooxygenase-2；MMP- matrix metalloproteinase；MTCO1- cytochrome C oxidase 1；Col2A1- collagen type II alpha-1
表2 高温刺激下各组细胞存活率之间比较[%，(±s)]
Table 2 Comparison of viability among groups at high temperature
组别Groups 样本数Samples 存活率Viability
对照组Control group 6 38±7
0.5%四氢嘧啶组0.5% Ectoine group 6 68±5a
1.0%四氢嘧啶1.0% Ectoine group 6 83±7ab
1.5%四氢嘧啶组1.5% Ectoine group 6 89±4ac
F值   77.16
P值   <0.001
图3 胰酶消化2 min后软骨细胞形态变化注：图中标尺为10 μm；Ectoine-四氢嘧啶；对照组软骨细胞明显收缩成点状，随着四氢嘧啶浓度增加，收缩成点状的软骨细胞逐渐变少
Figure 3 Morphologicl changes of chondrocytes after trypsin treatment for two minutes Note: the scale bar is 10 μm；chondrocytes in the control group had contracted and turned into spot-like cells; with increasing ectoine concentration, fewer chondrocytes contracted into spot-like cells after treatment with trypsin
图4 软骨细胞ROS(活性氧)荧光染色及MTCO1(细胞色素C氧化酶1)表达。图A为四氢嘧啶或DEX(地塞米松)预处理＋H2O2刺激软骨细胞后ROS实验荧光图片，示DEX组及不同浓度四氢嘧啶组软骨细胞内的荧光强度均低于H2O2对照组；图B为不同浓度四氢嘧啶组软骨细胞内的荧光强度值；图C为MTCO1基因的表达在各组间的比较注：图中标尺为10 μm；Ec-四氢嘧啶；DEX-地塞米松；*-P<0.05，**-P<0.01，***-P<0.001
Figure 4 ROS assay and MTCO1 relative expression in chondrocytes. A are fluorescent images of ROS in chondrocytes pretreated with ectoine or DEX and stimulated with H2O2, showing that fluorescence intensities in different concentration ectoine groups were lower than that in the H2O2 control group；B is the average fluorescence intensity in chondrocytes from different concentration ectoine groups; C is the expression of MTCO1 in different groups Note：scale bar is 10 μm；Ec- ectoine; DEX- dexamethasone；*-P<0.05，**-P<0.01，***-P<0.001
图5 软骨细胞COX(环氧化酶)-2、MMP(基质金属蛋白酶)-3和MMP-9的相对表达量。图A为COX-2的相对表达量；图B为MMP-3的相对表达量；图C为MMP-9的相对表达量注：Ec-四氢嘧啶；IL-白介素；DEX-地塞米松；*-P<0.05，**-P<0.01，***-P<0.001
Figure 5 Relative expression of COX-2, MMP-3 and MMP-9 in chondrocytes. A is the relative expression of COX-2; B is the relative expression of MMP-3；C is the relative expression of MMP-9 Note：Ec- ectoine; IL-interlukin; DEX- dexamethasone; *-P<0.05，**-P<0.01，***-P<0.001
图6 软骨细胞COL2A1(Ⅱ型胶原)免疫荧光染色及qPCR。图A为四氢嘧啶或DEX(地塞米松)预处理＋IL(白介素)-1β刺激后，软骨细胞的COL2A1免疫荧光染色图像，示DEX组及不同浓度四氢嘧啶组软骨细胞内的荧光强度均高于IL-1β组；图B为不同浓度四氢嘧啶组软骨细胞内的荧光强度值；图C为各组间Col2A1基因表达的比较注：图中标尺为10 μm；Ec-四氢嘧啶；IL-白介素；DEX-地塞米松；*-P<0.05，**-P<0.01，***-P<0.001
Figure 6 Immunofluorescence staining and qPCR analysis of COL2A1 in chondrocytes. A are immunofluorescence staining images of chondrocytes after pretreated with ectoine or DEX and stimulated with IL-1β，showing that fluorescence intensity of COL2A1 in chondrocytes in different ectoine concentration groups was greater than that in IL-1β group; B is the average fluorescence intensity value in chondrocytes from different ectoine concentration groups; C is Col2A1 expression in different groups Note：scale bar is 10 μm；Ec- ectoine; IL-interlukin; DEX- dexamethasone；*-P<0.05，**-P<0.01，***-P<0.001
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