Expression of mitochondrial creatine kinase 2 in a rat model of temporomandibular joint osteoarthritis and its role in inflammation progression

doi:10.12307/2025.921

Abstract

Abstract: BACKGROUND: Due to its unknown etiology and the lack of definitive curative treatments, management of temporomandibular joint osteoarthritis primarily focuses on symptom relief. Therefore, the identification of effective early molecular diagnostic biomarkers or potential therapeutic targets holds great significance.
OBJECTIVE: To investigate the expression of mitochondrial creatine kinase 2 in temporomandibular joint osteoarthritis in rats and its role in the progression of inflammation.
METHODS: (1) Animal experiment: Twenty Sprague-Dawley rats were randomly divided into control and unilateral anterior crossbite groups (n=10). A rat model of temporomandibular joint osteoarthritis was made in the unilateral anterior crossbite group. Four weeks after modeling, histological evaluations, including hematoxylin-eosin and Safranin O-fast green staining, were performed to assess pathological changes in the cartilage and subchondral bone of the mandibular condyle. Quantitative real-time PCR and immunohistochemical staining were utilized to detect the mRNA and protein expression levels of interleukin-1β, matrix metalloproteinase 13, type II collagen, aggrecan, and mitochondrial creatine kinase 2 in condylar cartilage. (2) Cell experiment: Passage 3 condylar cartilage cells from Sprague-Dawley rats were divided into control group and model group. Cells in the control group were routinely cultured, while an inflammation model of condylar cartilage cells was established with interleukin 1β in the model group were treated with interleukin-1β to induce inflammatory cell models. After 24 hours of interleukin-1β treatment, western blot was used to evaluate the expression of matrix metalloproteinase 13, type II collagen proteins in chondrocytes. Western blot was also used to detect the protein expression of mitochondrial creatine kinase 2 in the model group at 0, 12, 24, 48, and 72 hours after treatment with interleukin-1β.
RESULTS AND CONCLUSION: (1) Animal experiment: The results of hematoxylin-eosin staining showed that the unilateral anterior crossbite group exhibited disintegration of the fibrous layer in the cartilage of the mandibular condyle, disorganization of chondrocyte hierarchy, dense cellularity of the proliferative layer, obvious cell clustering, and infiltration of inflammatory cells in the subchondral bone. The results of Safranin O-fast green staining showed that in the cartilage matrix in the unilateral anterior crossbite group, the red stain became less extensive and lighter in color, and the tidal line was discontinuous. The mRNA and protein expression levels of interleukin-1β and matrix metalloproteinase 13 were elevated in the unilateral anterior crossbite group compared with the control group (P < 0.05). Conversely, the relative expression of type II collagen, aggrecan, and mitochondrial creatine kinase 2 mRNA and protein decreased (P < 0.05). (2) Cell experiment: Compared with the control group, the protein expression of matrix metalloproteinase 13 in the experimental group was significantly increased (P < 0.05), while the protein expression of type II collagen was decreased (P < 0.05). With prolonged interleukin-1β treatment, the protein expression of mitochondrial creatine kinase 2 in condylar chondrocytes of the model group gradually decreased. All the results indicate that the relative expression level of mitochondrial creatine kinase 2 was negatively correlated with the degree of chondrocyte inflammation in the rat model of temporomandibular joint osteoarthritis. Therefore, it is reasonable to infer that mitochondrial creatine kinase 2 is an important indicator for assessing the progression of inflammation in temporomandibular joint osteoarthritis.

Key words: temporomandibular joint, osteoarthritis, mitochondrial creatine kinase 2, condylar cartilage, interleukin-1β, engineered tissue construction

CLC Number:

Nigeayi · Aihemaiti, Yilidanna · Dilixiati, An Wei, Maimaitituxun · Tuerdi. Expression of mitochondrial creatine kinase 2 in a rat model of temporomandibular joint osteoarthritis and its role in inflammation progression[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(32): 6877-6884.

Figures/Tables 4

2.1 动物实验结果 2.1.1 实验动物数量分析 20只SD大鼠全部进入结果分析。 2.1.2 两组大鼠髁突软骨炎症情况苏木精-伊红染色结果显示，对照组髁突软骨表面平整，不存在裂隙，分为纤维层、增殖层、肥大层、钙化软骨层4层；单侧前牙反牙合组髁突软骨纤维层崩解、细胞排列无序，增殖层细胞密集，细胞簇集明显，软骨下骨有炎症细胞浸润，见图1。番红O-固绿染色结果显示，相对于对照组，单侧前牙反牙合组软骨基质红染范围变小、颜色变浅，潮线不连续，见图1。单侧前牙反牙合组髁突软骨苏木精-伊红染色与番红O-固绿染色Mankin评分均高于对照组(P < 0.000 1)，见图1。 2.1.3 两组大鼠髁突软骨中炎症基因及骨分解代谢基因表达比较 qRT-PCR检测结果显示，与对照组比较，单侧前牙反牙合组大鼠髁突软骨中白细胞介素1β、基质金属蛋白酶13 mRNA表达升高(P < 0.01，P < 0.05)，Ⅱ型胶原、蛋白聚糖mRNA表达降低(P < 0.001，P < 0.05)，见图2。免疫组化染色结果显示，与对照组比较，单侧前牙反牙合组大鼠髁突软骨中白细胞介素1β、基质金属蛋白酶13染色明显变深，Ⅱ型胶原、蛋白聚糖染色明显变浅，定量分析结果显示单侧前牙反牙合组髁突软骨中白细胞介素1β、基质金属蛋白酶13阳性细胞率均高于对照组(P < 0.01，P < 0.05)，Ⅱ型胶原、蛋白聚糖阳性细胞率均低于对照组(P < 0.05，P < 0.01)，见图3。 2.1.4 两组大鼠髁突软骨中线粒体肌酸激酶2表达比较 qRT-PCR检测结果显示，单侧前牙反牙合组大鼠髁突软骨中线粒体肌酸激酶2 mRNA表达低于对照组(P < 0.01)，见图2。免疫组化染色结果显示，单侧前牙反牙合组大鼠髁突软骨中肥大浅层的线粒体肌酸激酶2着色不如对照组，定量分析结果显示单侧前牙反牙合组大鼠髁突软骨中线粒体肌酸激酶2阳性细胞率低于对照组(P < 0.05)，见图3。 2.2 细胞实验结果 2.2.1 髁突软骨细胞的鉴定经过四五天培养后，髁突软骨细胞进入对数生长期，待培养至第2代时细胞多呈圆形、多边形或星形，像铺路石样结构，细胞之间接触紧密，与正常的髁突软骨细胞形态相同。免疫荧光染色结果显示，超90%的细胞Ⅱ型胶原免疫荧光染色呈阳性(图4)。 2.2.2 髁突软骨细胞炎症模型鉴定 Western blot检测结果显示，模型组软骨细胞中基质金属蛋白酶13蛋白表达高于对照组(P < 0.05)，Ⅱ型胶原蛋白表达低于对照组(P < 0.01)，见图5A。由此确定软骨细胞炎症模型建立成功。 2.2.3 模型组髁突软骨细胞中线粒体肌酸激酶2蛋白表达变化 Western blot检测结果显示，随着白细胞介素1β处理时间的延长，髁突软骨细胞中线粒体肌酸激酶2蛋白表达逐渐降低，与处理0 h比较，处理24，48，72 h的线粒体肌酸激酶2蛋白表达降低(P < 0.05，P < 0.001，P < 0.000 1)；与处理12 h比较，处理24，48，72 h的线粒体肌酸激酶2蛋白表达降低(P < 0.05，P < 0.001)；与处理24 h比较，处理48，72 h的线粒体肌酸激酶2蛋白表达降低(P < 0.05，P < 0.01)；与处理48 h比较，处理72 h的线粒体肌酸激酶2蛋白表达降低(P < 0.05)，见图5B。"

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