小檗碱通过干扰 TLR4 和 MyD88 之间的相互作用抑制促炎 M1 巨噬细胞极化。
Inhibitory effects of berberine on proinflammatory M1 macrophage polarization through interfering with the interaction between TLR4 and MyD88.
机构信息
Department of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
Institute of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
出版信息
BMC Complement Altern Med. 2019 Nov 19;19(1):314. doi: 10.1186/s12906-019-2710-6.
BACKGROUNDS
Inflammation is recognized as the key pathological mechanism of type 2 diabetes. The hypoglyceamic effects of berberine (BBR) are related to the inhibition of the inflammatory response, but the mechanism is not completely clear.
METHODS
The inflammatory polarization of Raw264.7 cells and primary peritoneal macrophages were induced by LPS, and then effects and underlying mechanisms of BBR were explored. An inflammatory model was established by LPS treatment at different concentrations for different treatment time. An ELISA assay was used to detect the secretions of TNF-α. RT-PCR was applied to detect M1 inflammatory factors. The F4/80 ratio and CD11c ratio of primary peritoneal macrophages were determined by flow cytometry. The expressions of p-AMPK and TLR4 were detected by Western blot. The cytoplasmic and nuclear distributions of NFκB p65 were observed by confocal microscopy. The binding of TLR4 to MyD88 was tested by CoIP, and the affinity of BBR for TLR4 was assessed by molecular docking.
RESULTS
Upon exposure to LPS, the secretion of TNF-α and transcription of inflammatory factors in macrophages increased, cell morphology changed and protrusions appeared gradually, the proportion of F4/80CD11c M1 macrophages increased, and the nuclear distribution of NFκB p65 increased. BBR pretreatment partially inhibited the changes mentioned above. However, the expression of TLR4 and p-AMPK did not change significantly after LPS intervention for 3 h. Meanwhile, CoIP showed that the interaction between TLR4 and MyD88 increased, and BBR inhibited the binding. Molecular docking suggested that BBR might interact with TLR4.
CONCLUSIONS
Inflammatory changes were induced in macrophages after LPS stimulation for 3 h, and BBR pretreatment inhibited inflammatory polarization. BBR might interact with TLR4 and disturb TLR4/MyD88/NFκB signalling pathway, and it might be the mechanism by which BBR attenuated inflammation in the early phase.
背景
炎症被认为是 2 型糖尿病的关键病理机制。小檗碱（BBR）的降血糖作用与抑制炎症反应有关，但机制尚不完全清楚。
方法
用 LPS 诱导 Raw264.7 细胞和原代腹腔巨噬细胞的炎症极化，然后探讨 BBR 的作用及机制。用不同浓度 LPS 处理不同时间建立炎症模型。用 ELISA 法检测 TNF-α分泌，用 RT-PCR 法检测 M1 炎症因子，用流式细胞术检测原代腹腔巨噬细胞 F4/80 比值和 CD11c 比值，用 Western blot 法检测 p-AMPK 和 TLR4 的表达，用共聚焦显微镜观察 NFκB p65 的胞质和核分布，用 CoIP 法检测 TLR4 与 MyD88 的结合，用分子对接法评估 BBR 与 TLR4 的亲和力。
结果
LPS 作用后，巨噬细胞 TNF-α分泌和炎症因子转录增加，细胞形态改变，逐渐出现突起，F4/80CD11c M1 巨噬细胞比例增加，NFκB p65 核分布增加。BBR 预处理部分抑制了上述变化。但 LPS 干预 3 h 后 TLR4 和 p-AMPK 的表达无明显变化。同时，CoIP 显示 TLR4 与 MyD88 的相互作用增加，BBR 抑制结合。分子对接表明 BBR 可能与 TLR4 相互作用。
结论
LPS 刺激 3 h 后巨噬细胞发生炎症变化，BBR 预处理抑制炎症极化。BBR 可能与 TLR4 相互作用，干扰 TLR4/MyD88/NFκB 信号通路，这可能是 BBR 在早期减轻炎症的机制。
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