三黄消岩方通过激活 AMPK/Nrf2 信号通路调节高迁移率族蛋白 B1 介导的异常炎症微环境改善糖尿病足。
San Huang Xiao Yan recipe modulates the HMGB1-mediated abnormal inflammatory microenvironment and ameliorates diabetic foot by activating the AMPK/Nrf2 signalling pathway.
机构信息
Institute of Vascular Anomalies, Shanghai TCM-Integrated Hospital, Shanghai University of Traditional Chinese Medicine, 303 Changyang Road, Shanghai 200082, China.
Institute of Vascular Anomalies, Shanghai TCM-Integrated Hospital, Shanghai University of Traditional Chinese Medicine, 303 Changyang Road, Shanghai 200082, China.
出版信息
Phytomedicine. 2023 Sep;118:154931. doi: 10.1016/j.phymed.2023.154931. Epub 2023 Jun 20.
BACKGROUND
Diabetic foot (DF) is one of the serious complications of diabetes and lacks of therapeutic drugs. Abnormal and chronic inflammation promoting foot infection and wound healing delay are the main pathogenesis of DF. The traditional prescription San Huang Xiao Yan Recipe (SHXY) has been used in the clinical treatment of DF for several decades as approved hospital experience prescription and showed remarkable therapeutic effect, but the mechanisms by which SHXY treats DF are still unclear.
PURPOSE
Objectives of this study were to investigate SHXY anti-inflammatory effect on DF and explore the molecular mechanism for SHXY.
METHODS
We detected the effects of SHXY on DF in C57 mouse and SD rat DF models. Animal blood glucose, weight and wound area were detected every week. Serum inflammatory factors were detected by ELISA. H&E and Masson's trichrome were used to observe tissue pathology. Single-cell sequencing data reanalysis revealed the role of M1 macrophages in DF. Venn analysis showed the co-target genes between DF M1 macrophages and compound-disease network pharmacology. Western blotting was used to explored target protein expression. Meanwhile, RAW264.7 cells were treated with drug-containing serum of SHXY to further unravel the roles of target proteins during high glucose-induced inflammation in vitro. The Nrf2 inhibitor ML385 was used on RAW 264.7 cells to further explore the relationship between Nrf2, AMPK and HMGB1. The main components of SHXY were analysed by HPLC. Finally, the treatment effect of SHXY on DF were detected on rat DF model.
RESULTS
In vivo, SHXY can ameliorate inflammatory, accelerate wound healing and upregulate expression of Nrf2, AMPK and downregulate of HMGB1. Bioinformatic analysis showed that M1 macrophages were the main inflammatory cell population in DF. Moreover, the Nrf2 downstream proteins HO-1 and HMGB1 were potential DF therapeutic targets for SHXY. In vitro, we also found that SHXY increased AMPK and Nrf2 protein levels and downregulated HMGB1 expression in RAW264.7 cells. Inhibiting the expression of Nrf2 impaired the inhibition effect of SHXY on HMGB1. SHXY promoted Nrf2 translocation into the nucleus and increased the phosphorylation of Nrf2. SHXY also inhibited HMGB1 extracelluar release under high glucose. In rat DF models, SHXY also exhibited significant anti-inflammatory effect.
CONCLUSION
The SHXY activated AMPK/Nrf2 pathway to suppress abnormal inflammation on DF via inhibiting HMGB1 expression. These findings provide novel insight into the mechanisms by which SHXY treats DF.
背景
糖尿病足（DF）是糖尿病的严重并发症之一，缺乏治疗药物。异常和慢性炎症促进足部感染和伤口愈合延迟是 DF 的主要发病机制。传统处方三黄消岩方（SHXY）已作为经验处方在 DF 的临床治疗中使用了几十年，具有显著的治疗效果，但 SHXY 治疗 DF 的机制仍不清楚。
目的
本研究旨在探讨 SHXY 对 DF 的抗炎作用及其分子机制。
方法
我们在 C57 小鼠和 SD 大鼠 DF 模型中检测了 SHXY 对 DF 的影响。每周检测动物血糖、体重和伤口面积。通过 ELISA 检测血清炎症因子。H&E 和 Masson 三色染色观察组织病理学变化。单细胞测序数据再分析揭示了 M1 巨噬细胞在 DF 中的作用。Venn 分析显示了 DF M1 巨噬细胞与化合物疾病网络药理学的共同靶基因。Western blot 用于探索靶蛋白的表达。同时，用 SHXY 含药血清处理 RAW264.7 细胞，进一步揭示靶蛋白在高糖诱导的体外炎症中的作用。用 Nrf2 抑制剂 ML385 处理 RAW264.7 细胞，进一步探讨 Nrf2、AMPK 和 HMGB1 之间的关系。用 HPLC 分析 SHXY 的主要成分。最后，在大鼠 DF 模型上检测 SHXY 的治疗效果。
结果
体内，SHXY 可改善炎症，加速伤口愈合，上调 Nrf2、AMPK 的表达，下调 HMGB1 的表达。生物信息学分析显示，M1 巨噬细胞是 DF 中的主要炎症细胞群。此外，Nrf2 下游蛋白 HO-1 和 HMGB1 是 SHXY 治疗 DF 的潜在靶点。体外，我们还发现 SHXY 增加了 RAW264.7 细胞中 AMPK 和 Nrf2 蛋白水平，并下调了 HMGB1 的表达。抑制 Nrf2 表达可损害 SHXY 对 HMGB1 的抑制作用。SHXY 促进 Nrf2 向核内转位，并增加 Nrf2 的磷酸化。SHXY 还可抑制高糖下 HMGB1 的细胞外释放。在大鼠 DF 模型中，SHXY 也表现出明显的抗炎作用。
结论
SHXY 通过抑制 HMGB1 表达激活 AMPK/Nrf2 通路抑制 DF 异常炎症。这些发现为 SHXY 治疗 DF 的机制提供了新的见解。
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