Protective effects of ectoine on articular chondrocytes in rats

doi:10.3877/cma.j.issn.1674-134X.2024.01.010

Abstract

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.

Key words: Rats, Osteoarthritis, Chondrocytes, Inflammation

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.

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Figures/Tables 7

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

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

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±5^a
1.0%四氢嘧啶1.0% Ectoine group	6	83±7^ab
1.5%四氢嘧啶组1.5% Ectoine group	6	89±4^ac
F值		77.16
P值		<0.001

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

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

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

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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