Changchun University of Chinese Medicine, Changchun, 130117, China.
State Key Laboratory of Integration and Innovation of Classic Formula and Modern Chinese Medicine, Lunan Pharmaceutical Group Co. Ltd., Linyi, 276005, China.
J Ethnopharmacol. 2024 Dec 5;335:118621. doi: 10.1016/j.jep.2024.118621. Epub 2024 Jul 23.
Mailuo Shutong Pill (MLST), a traditional Chinese medicine (TCM), has been widely used for clearing heat and detoxifying, eliminating stasis and dredging meridians, dispelling dampness and diminishing swelling. Earlier study found that MLST could improve cerebral ischemic-reperfusion injury, however, the potential mechanism has not been well evaluated.
In this study, a well established and widely used mice model of middle cerebral artery occlusion/reperfusion (MCAO/R) was preformed to evaluate the protective function of MLST on cerebral ischemic-reperfusion injury and further discuss the potential pharmacological mechanisms.
Chemical profiling of MLST was analyzed based on Ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry. ICR mice were challenged by MCAO/R surgery. The protective effect of MLST on MCAO/R injury was evaluated by neurological deficit score, cerebral infarct rate, brain water content, H&E and nissl staining. The blood-brain barrier (BBB) integrity was detected by Evans blue staining. The potential pharmacological mechanism of MLST in treating MCAO/R injury was further elucidated by the methods of proteomics, central carbon targeted metabolomics, as well as Western blot. Immunohistochemistry was used to detect the microglia infiltration, enzyme linked immunosorbent assay (ELISA) kit was explored to evaluate the content of IL-1β, TNF-α and IL-6 in brain tissue, and Western blot was used to detect proteins expression in brain tissue.
A total of 76 chemical compounds have been determined in MLST. MLST effectively protected mice from MCAO/R injury, which was confirmed by lower neurological deficit score, cerebral infarct rate, brain water content and nissl body loss, and improved brain pathology. Meanwhile, MLST upregulated the expression of ZO-1, Occludin and Claudin 5 by downregulating the ratio of TIMP1/MMP9 to suppress the entrance of Evans blue to brain tissue, indicating that MLST maintained the integrity of BBB. Further studies indicated that MLST inhibited the inflammatory level of brain tissue by inhibiting microglia infiltration and downregulating NLRP3 inflammasome signaling pathway. The results of proteomics, Western blot, and central carbon targeted metabolomics confirmed that MLST regulated Glycolysis/Gluconogenesis, Pyruvate metabolism and TCA cycle in brain tissue of mice with MCAO/R.
MLST inhibits neuroinflammation by regulating glucose metabolism disorders to interfere with immune metabolism reprogramming and inhibit the NLRP3 inflammasome signaling pathway, and finally improve cerebral ischemia-reperfusion injury. This study confirms that MLST is a potential drug for treating Cerebral ischemic stroke.
脉络舒通丸（MLST）是一种传统的中药，已广泛用于清热解毒、活血化瘀、祛湿消肿。早期研究发现，MLST 可以改善脑缺血再灌注损伤，但其潜在机制尚未得到很好的评估。
本研究采用经典且广泛应用的大脑中动脉闭塞/再灌注（MCAO/R）小鼠模型，评估 MLST 对脑缺血再灌注损伤的保护作用，并进一步探讨其潜在的药理机制。
采用超高效液相色谱-电喷雾离子轨道阱串联质谱法分析 MLST 的化学成分谱。采用 MCAO/R 手术对 ICR 小鼠进行处理。通过神经功能缺损评分、脑梗死率、脑水含量、H&E 和尼氏染色评估 MLST 对 MCAO/R 损伤的保护作用。通过伊文思蓝染色检测血脑屏障（BBB）的完整性。通过蛋白质组学、中枢碳靶向代谢组学以及 Western blot 进一步阐明 MLST 治疗 MCAO/R 损伤的潜在药理机制。免疫组织化学检测小胶质细胞浸润，酶联免疫吸附试验（ELISA）试剂盒评估脑组织中 IL-1β、TNF-α 和 IL-6 的含量，Western blot 检测脑组织中蛋白质的表达。
共鉴定出 MLST 中的 76 种化合物。MLST 能有效保护小鼠免受 MCAO/R 损伤，表现在神经功能缺损评分、脑梗死率、脑水含量和尼氏小体丢失降低，以及脑组织病理改善。同时，MLST 通过下调 TIMP1/MMP9 的比值，上调 ZO-1、Occludin 和 Claudin 5 的表达，抑制 Evans 蓝进入脑组织，从而维持 BBB 的完整性。进一步的研究表明，MLST 通过抑制小胶质细胞浸润和下调 NLRP3 炎性小体信号通路，抑制脑组织的炎症水平。蛋白质组学、Western blot 和中枢碳靶向代谢组学的结果证实，MLST 调节了大脑组织中糖酵解/葡糖生成、丙酮酸代谢和 TCA 循环。
MLST 通过调节葡萄糖代谢紊乱抑制神经炎症，干扰免疫代谢重编程，抑制 NLRP3 炎性小体信号通路，从而改善脑缺血再灌注损伤。本研究证实 MLST 是一种治疗缺血性脑卒中的潜在药物。
