Qiu Lu, Zhang Min, Li Chenghao, Hou Yehu, Liu Hao, Lin Jia, Yao Juan, Duan Dong Zhu, Zhang Yi Xi, Li Mi, Li Ya Ling, Wang Peng, Li Jin Tian, Jin Xiao Jie, Liu Yong Qi
Gansu University Key Laboratory for Molecular Medicine and Chinese Medicine Prevention and Treatment of Major Diseases, Gansu University of Chinese Medicine, Lanzhou, China.
Key Laboratory of Dunhuang Medicine, Ministry of Education, Gansu University of Chinese Medicine, Lanzhou, China.
Front Pharmacol. 2022 Nov 23;13:993498. doi: 10.3389/fphar.2022.993498. eCollection 2022.
Osteoarthritis (OA) is a multifactorial and chronic degenerative joint disease. Due to the adverse effects of currently used drugs, a safer and more effective therapy for treating OA is needed. Peroxisome proliferator-activated receptor-γ (PPARγ) is a key protein protecting cartilage. DNMT1-mediated hypermethylation of PPARγ promoter leads to its suppression. Therefore, DNMT1 might be an effective target for exerting cartilage protective effects by regulating the epigenetic expression of PPARγ. Dabushen decoction (DD) is a representative prescription of Dunhuang ancient medical prescription, which has a potential therapeutic effect on OA. So far, the research of the efficacy and material basis of DD in the treatment of OA remains unclear. In this study, Micro-CT, HE staining, S-O staining, and immunohistochemistry analysis were used to demonstrate that DD increased the expression of PPARγ and collagen synthesis in an OA rat model. Next, the structure of DNMT1 was used to screen the active constituents of DD by molecular docking method for treatment OA. Seven potential active constituents, including isoliquiritigenin, emodin, taxifolin, catalpol, alisol A, zingerone, and schisandrin C were hited. The protective effect of the potential active constituents to chondrocytes were evaluated by protein capillary electrophoresis, immunofluorescence assays, and culture of rat knee cartilage. The five constituents, such as alisol A, emodin, taxifolin, isoliquiritigenin, and schisandrin C could promote the expression of PPARγ and ameliorate IL-1β-induced downregulation of collagen II and the production of MMP-13. Alisol A and Emodin could effectively mitigate cartilage damage. At last, molecular dynamics simulations with MM-GBSA method was applied to investigate the interaction pattern of the active constituents and DNMT1 complexes. The five constituents, such as alisol A, emodin, taxifolin, isoliquiritigenin, and schisandrin C achieved a stable binding pattern with DNMT1, in which alisol A has a relatively high binding free energy. In conclusion, this study elucidates that the active constituents of DD (alisol A, emodin, taxifolin, isoliquiritigenin, and schisandrin C) could ameliorate osteoarthritis PPARγ preservation by targeting DNMT1.These findings facilitated clinical use of DD and provided a valuable strategy for developing natural epigenetic modulators from Chinese herbal formula.
骨关节炎（OA）是一种多因素慢性退行性关节疾病。由于目前使用的药物存在不良反应，因此需要一种更安全、有效的OA治疗方法。过氧化物酶体增殖物激活受体γ（PPARγ）是保护软骨的关键蛋白。DNMT1介导的PPARγ启动子高甲基化导致其受到抑制。因此，DNMT1可能是通过调节PPARγ的表观遗传表达发挥软骨保护作用的有效靶点。达布深汤（DD）是敦煌古医方的代表性方剂，对OA具有潜在治疗作用。目前，DD治疗OA的疗效及物质基础研究尚不清楚。本研究采用Micro-CT、HE染色、S-O染色及免疫组化分析，证实DD可增加OA大鼠模型中PPARγ的表达及胶原蛋白合成。接下来，利用DNMT1的结构，通过分子对接方法筛选DD治疗OA的活性成分。筛选出7种潜在活性成分，包括异甘草素、大黄素、紫杉叶素、梓醇、泽泻醇A、姜辣素和五味子丙素。通过蛋白质毛细管电泳、免疫荧光分析及大鼠膝关节软骨培养，评估潜在活性成分对软骨细胞的保护作用。泽泻醇A、大黄素、紫杉叶素、异甘草素和五味子丙素这5种成分可促进PPARγ表达，改善IL-1β诱导的Ⅱ型胶原蛋白下调及MMP-13的产生。泽泻醇A和大黄素可有效减轻软骨损伤。最后，应用MM-GBSA方法进行分子动力学模拟，研究活性成分与DNMT1复合物的相互作用模式。泽泻醇A、大黄素、紫杉叶素、异甘草素和五味子丙素这5种成分与DNMT1形成稳定的结合模式，其中泽泻醇A具有相对较高的结合自由能。总之，本研究阐明了DD的活性成分（泽泻醇A、大黄素、紫杉叶素、异甘草素和五味子丙素）可通过靶向DNMT1改善骨关节炎并保护PPARγ。这些发现有助于DD的临床应用，并为从中药方剂中开发天然表观遗传调节剂提供了有价值的策略。
