A study on mechanism of action of Ziyuglycoside Ⅰ on knee osteoarthritis in rats

WANG Zhewei; ZHOU Anyuan; WANG Yonglin; PEI Xiaomin; ZHONG Qiuling; YANG Yuan

doi:10.3969/j.issn.1674-3806.2024.06.09

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 inflam-mation group was cultured in medium containing 10 pig/mL interleukin-1β(IL-1β),and the treatment group was cultured in medium containing 10 μg/mL IL-1 β 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,inflam-mation 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 extra-cellular matrix(ECM)of chondrocytes,and has certain medicinal potential in treatment of knee osteoarthritis.