Key Laboratory of Chinese Medicine Rheumatology of Zhejiang Province, School of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China.
Department of Nephrology, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, Wenzhou, Zhejiang, China.
J Ethnopharmacol. 2023 Dec 5;317:116776. doi: 10.1016/j.jep.2023.116776. Epub 2023 Jun 19.
Jieduquyuziyin prescription (JP) is a traditional Chinese medicine utilized to treat systemic lupus erythematosus (SLE). Its efficacy has been confirmed through clinical trials and empirical evidence, leading to its authorized use in Chinese hospitals. The development of JP exemplifies the integration of traditional wisdom and scientific approaches, demonstrating the interdisciplinary essence of ethnopharmacology. These results emphasize the potential value of traditional medicine in addressing autoimmune disorders.
This study aims to address the effect of JP in MRL/lpr mice and elucidate the pharmacological mechanism by which JP targets CD11a and CD70 DNA methylation via the miR-29b-sp1/DNMT1 pathway.
MRL/lpr mice were divided into three groups: the model group (received distilled water), the positive group (administered AAV/miR-29b-3p inhibitor), and the JP group (treated with JP decoction). C57BL/6 mice were constituted as a control group. Through ELISA assay, serum and urine samples were assessed for anti-dsDNA, TNF-α, TGF-β, IL-2, and UP. HE and Masson staining were conducted to reveal renal pathology. Genome DNA was extracted from CD4 T cells of mice spleens to evaluate methylation level. The methylation of CD11a, CD70, and CD40L promoter regions was analyzed by targeted bisulfate sequencing. Their expression at the mRNA and protein levels was examined using quantitative real-time PCR, western blot analysis, immunohistochemistry, and immunofluorescence staining of kidney tissues. Furthermore, the molecular mechanisms underlying the regulation of the miR-29b-sp1/DNMT1 pathway by JP were explored with Jurkat cells transfected with miR-inhibitors or miR-mimics.
Mice treated with JP exhibited a significant decrease in anti-dsDNA, TNF-α, TGF-β, and UP, accompanied by a significant increase in IL-2. HE staining revealed JP effectively mitigated renal inflammatory response, while Masson staining indicated a reduction in collagen fiber content. In addition, JP exhibited a significant impact on the global hypomethylation of SLE, as evidenced by the induction of high methylation levels of CD11a and CD70 promoter regions, mediated through the miR-29b-sp1/DNMT1 pathway.
Our findings demonstrate JP exerts a protective effect against spontaneous SLE development, attenuates renal pathological changes, and functions as a miRNA inhibitor to enhance CD11a and CD70 DNA methylation through the modulation of the miR-29b-sp1/DNMT1 pathway.
解毒祛瘀滋阴方（JP）是一种用于治疗系统性红斑狼疮（SLE）的中药。其疗效已通过临床试验和经验证据得到证实，因此被授权在中国医院使用。JP 的开发体现了传统智慧与科学方法的融合，展示了民族药理学的跨学科本质。这些结果强调了传统医学在治疗自身免疫性疾病方面的潜在价值。
本研究旨在探讨 JP 在 MRL/lpr 小鼠中的作用，并通过 miR-29b-sp1/DNMT1 通路阐明 JP 通过靶向 CD11a 和 CD70 的 DNA 甲基化来发挥作用的药理学机制。
将 MRL/lpr 小鼠分为三组：模型组（给予蒸馏水）、阳性组（给予 AAV/miR-29b-3p 抑制剂）和 JP 组（给予 JP 煎剂）。C57BL/6 小鼠作为对照组。通过 ELISA 检测试剂盒，评估血清和尿液样本中的抗 dsDNA、TNF-α、TGF-β、IL-2 和 UP。进行 HE 和 Masson 染色以揭示肾脏病理。从小鼠脾脏的 CD4 T 细胞中提取基因组 DNA，以评估甲基化水平。通过靶向亚硫酸氢盐测序分析 CD11a、CD70 和 CD40L 启动子区域的甲基化。使用定量实时 PCR、western blot 分析、免疫组织化学和肾脏组织的免疫荧光染色检测其在 mRNA 和蛋白水平的表达。此外，通过转染 miR-抑制剂或 miR-模拟物的 Jurkat 细胞探索 JP 调节 miR-29b-sp1/DNMT1 通路的分子机制。
JP 治疗的小鼠抗 dsDNA、TNF-α、TGF-β 和 UP 显著降低，而 IL-2 显著升高。HE 染色显示 JP 有效减轻了肾脏炎症反应，而 Masson 染色显示胶原纤维含量减少。此外，JP 对 SLE 的整体低甲基化具有显著影响，表现为 CD11a 和 CD70 启动子区域的高甲基化水平诱导，这是通过 miR-29b-sp1/DNMT1 通路介导的。
我们的研究结果表明，JP 对自发性 SLE 发展具有保护作用，减轻肾脏病理变化，并作为 miRNA 抑制剂通过调节 miR-29b-sp1/DNMT1 通路增强 CD11a 和 CD70 的 DNA 甲基化。
