背景:知龙活血通瘀胶囊(ZL)是临床上治疗急性缺血性脑卒中(AIS)的药物。然而,只有少数研究探讨了ZL治疗AIS的机制。
目的:探讨ZL介导的巨噬细胞极化和炎症的潜在机制,为AIS的治疗提供参考。
方法:16只SD大鼠饲喂不同剂量的ZL(0、0.4、0.8和1.6g/kg/d)4天,制备ZL血清。在500ng/mL脂多糖(LPS)刺激后,向RAW264.7细胞施用ZL血清。然后,实验包括ELISA,流式细胞术,实时定量PCR和Westernblot验证ZL对巨噬细胞极化和炎症的影响。接下来,用LPS和ZL血清处理后,将let-7i抑制剂转染到RAW264.7细胞中,以验证ZL对let-7i/TLR9/MyD88信号通路的调节作用。此外,let-7i与TLR9之间的相互作用通过双荧光素酶试验得到证实.
结果:ZL血清中白细胞介素(IL)-6和肿瘤坏死因子-α(TNF-α)的表达明显降低,并增加了LPS刺激的巨噬细胞中IL-10和转化生长因子β1(TGF-β1)的表达。此外,ZL血清向M2极化巨噬细胞,降低TLR9,MyD88和iNOS的表达,以及增加let-7i的表达式,CHIL3和精氨酸酶-1。值得一提的是ZL血清的效应是剂量依赖性的。然而,let-7i抑制剂在LPS刺激的巨噬细胞中恢复了所有上述作用。此外,TLR9是let-7i的靶标。
结论:ZL靶向let-7i抑制TLR9表达,从而抑制TLR9/MyD88通路的激活,促进M2极化,并抑制AIS中炎症的发展。
BACKGROUND: Zhilong Huoxue Tongyu Capsule (ZL) is clinically prescribed for acute ischemic stroke (AIS). However, only a few studies have addressed the mechanisms of ZL in treating AIS.
OBJECTIVE: To explore the underlying mechanism of macrophage polarization and inflammation mediated by ZL, and to provide a reference for AIS treatment.
METHODS: Sixteen SD rats were fed with different dose of ZL (0, 0.4, 0.8, and 1.6 g/kg/d) for 4 days to prepare ZL serum. After 500 ng/mL lipopolysaccharide (LPS) stimulation, RAW264.7 cells were administrated with ZL serum. Then, experiments including ELISA, flow cytometry, real-time quantitative PCR and Western blot were performed to verify the effects of ZL on macrophage polarization and inflammation. Next, let-7i inhibitor was transfected in RAW264.7 cells when treated with LPS and ZL serum to verify the regulation of ZL on the let-7i/TLR9/MyD88 signaling pathway. Moreover, the interaction between let-7i and TLR9 was confirmed by the dual-luciferase assay.
RESULTS: ZL serum significantly decreased the expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), and increased the expression of IL-10 and transforming growth factor β1 (TGF-β1) of LPS stimulated-macrophages. Furthermore, ZL serum polarized macrophages toward M2, decreased the expressions of TLR9, MyD88, and iNOS, as well as increased the expressions of let-7i, CHIL3, and Arginase-1. It is worth mentioning that the effect of ZL serum is dose-dependent. However, let-7i inhibitor restored all the above effects in LPS stimulated-macrophages. In addition, TLR9 was the target of let-7i.
CONCLUSIONS: ZL targeted let-7i to inhibit TLR9 expression, thereby inhibiting the activation of the TLR9/MyD88 pathway, promoting the M2 polarization, and inhibiting the development of inflammation in AIS.