引用本文:王哲纬,周安远,王勇麟,裴晓敏,钟秋玲,杨 渊.地榆皂苷Ⅰ对大鼠膝骨关节炎的作用机制研究[J].中国临床新医学,2024,17(6):639-644.
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地榆皂苷Ⅰ对大鼠膝骨关节炎的作用机制研究
王哲纬1,周安远1,王勇麟1,裴晓敏1,钟秋玲1,杨 渊1,2
1.广西医科大学再生医学与医用生物资源开发应用省部共建协同创新中心,南宁 530021;2.广西医大开元埌东医院骨科,南宁 530028
摘要:
[摘要] 目的 研究地榆皂苷Ⅰ对大鼠膝骨关节炎的作用机制。方法 选择SD雄性乳鼠8只,提取乳鼠软骨细胞,采用CCK-8实验检测细胞增殖率以确定最佳药物浓度。将软骨细胞随机分为对照组、炎症组和治疗组,对照组在完全培养基中进行培养,炎症组在含有10 μg/mL白细胞介素-1β(IL-1β)的培养基中培养,治疗组在含有10 μg/mL IL-1β和最佳浓度地榆皂苷Ⅰ的培养基中培养。采用Calcein-AM/PI染色观察各组软骨细胞生长情况。采用实时荧光定量聚合酶链式反应(RT-qPCR)检测各组软骨细胞中白细胞介素-6(IL-6)、基质金属蛋白酶13(MMP13)mRNA相对表达量。取8周龄左右的SD雄性大鼠24只随机分为对照组、炎症组和治疗组,每组8只,对炎症组和治疗组建立骨关节炎模型,建模完成4周后,炎症组和对照组的关节腔内注射等体积生理盐水,治疗组的关节腔内注射100 μL最佳浓度的地榆皂苷Ⅰ溶液,1次/周,连续给药4周。采用HE染色和番红O-固绿染色观察各组膝关节组织结构。采用免疫组化染色观察各组膝关节组织中IL-6、MMP13蛋白表达情况。结果  CCK-8实验结果显示,各实验浓度均未表现明显细胞毒性,当地榆皂苷Ⅰ的浓度为20 mg/L时,软骨细胞增殖率最高。Calcein-AM/PI染色结果显示,炎症组和治疗组细胞增殖率低于对照组,治疗组细胞增殖率高于炎症组,差异有统计学意义(P<0.05)。RT-qPCR检测结果显示,炎症组和治疗组IL-6、MMP13 mRNA相对表达量高于对照组,治疗组IL-6、MMP13 mRNA相对表达量低于炎症组,差异有统计学意义(P<0.05)。HE染色和番红O-固绿染色结果显示,对照组的膝关节组织结构比炎症组和治疗组完整,与炎症组相比,治疗组膝关节组织损伤有所减轻。免疫组化染色结果显示,炎症组IL-6、MMP13蛋白表达水平最高,治疗组IL-6、MMP13蛋白表达水平高于对照组。结论 地榆皂苷Ⅰ可通过维持软骨细胞活力、降低炎症因子的释放以及减少软骨细胞外基质(ECM)的降解发挥抗炎作用,对膝骨关节炎有一定的药用潜力。
关键词:  地榆皂苷Ⅰ  骨关节炎  抗炎  白细胞介素-6  基质金属蛋白酶13  膝骨关节炎大鼠模型
DOI:10.3969/j.issn.1674-3806.2024.06.09
分类号:
基金项目:国家重点研发计划课题(编号:2020YFB1313801);国家自然科学基金地区科学基金项目(编号:82360425);南宁市青秀区科技计划项目(编号:2020014)
A study on mechanism of action of Ziyuglycoside Ⅰ on knee osteoarthritis in rats
WANG Zhewei1, ZHOU Anyuan1,WANG Yonglin1, PEI Xiaomin1, ZHONG Qiuling1, YANG Yuan1,2
1.Collaborative Innovation Centre of Regenerative Medicine and Medical Bioresource Development and Application Co-constructed by the Province and Ministry, Guangxi Medical University, Nanning 530021, China; 2.Department of Orthopedics, Guangxi Medical University Kaiyuan Langdong Hospital, Nanning 530028, China
Abstract:
[Abstract] Objective To study the mechanism of action of Ziyuglycoside Ⅰ on knee osteoarthritis in rats. Methods Eight male Sprague-Dawley(SD) suckling rats were selected to extract chondrocytes, and the cell proliferation rate was detected by CCK-8 assay to determine the optimal drug concentration. The chondrocytes were randomly divided into control group, inflammation group and treatment group. The control group was cultured in complete medium, and the inflammation group was cultured in medium containing 10 μg/mL interleukin-1β(IL-1β), and the treatment group was cultured in medium containing 10 μg/mL IL-1β and the optimal concentration of Ziyuglycoside Ⅰ . Calcein-AM/PI staining was used to observe the growth of chondrocytes in each group. Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR) was used to detect the relative expressions of interleukin-6(IL-6) and matrix metalloproteinase 13(MMP13) mRNA in chondrocytes in each group. Twenty-four male SD rats about 8 weeks old were randomly divided into control group, inflammation group and treatment group, with 8 rats in each group. Osteoarthritis models were established for the inflammation group and the treatment group. After 4 weeks of completing the osteoarthritis models, the inflammation group and the control group were injected with equal volume of normal saline into the joint cavity, and the treatment group was injected with the optimal concentration of Ziyuglycoside Ⅰ solution into the joint cavity, and the drugs were administered once a week for 4 weeks. The tissue structure of the knee joints in each group was observed by using hematoxylin and eosin(HE) staining and safranine O-solid green staining. Immunohistochemical staining was used to observe the expressions of IL-6 and MMP13 proteins in the knee joint tissues of each group. Results The results of CCK-8 assay showed that no obvious cytotoxicity was observed in all the experimental concentrations, and the chondrocyte proliferation rate was the highest when the concentration of Ziyuglycoside Ⅰ was 20 mg/L. The results of Calcein-AM/PI staining showed that the cell proliferation rate in the inflammation group and the treatment group was lower than that in the control group, and the cell proliferation rate in the treatment group was higher than that in the inflammation group, and the differences were statistically significant(P<0.05). The results of RT-qPCR detection showed that the relative expression levels of IL-6 and MMP13 mRNA in the inflammation group and the treatment group were higher than those in the control group, and the relative expression levels of IL-6 and MMP13 mRNA in the treatment group were lower than those in the inflammation group, and the differences were statistically significant(P<0.05). The results of HE staining and safranine O-solid green staining showed that the tissue structure of the knee joints in the control group was more complete than that in the inflammation group and the treatment group, and compared with the inflammation group, the treatment group showed a reduction in knee joint tissue damage. The results of immunohistochemical staining showed that the inflammation group had the highest expression levels of IL-6 and MMP13 proteins and the treatment group had higher expression levels of IL-6 and MMP13 proteins than the control group. Conclusion Ziyuglycoside Ⅰ can play a role in anti-inflammatory effects by maintaining chondrocyte vitality, reducing the release of inflammatory factors and reducing the degradation of extracellular matrix(ECM) of chondrocytes, and has certain medicinal potential in treatment of knee osteoarthritis.
Key words:  Ziyuglycoside Ⅰ  Osteoarthritis  Anti-inflammation  Interleukin-6(IL-6)  Matrix metalloproteinase 13(MMP13)  Rat model of knee osteoarthritis