高迁移率族蛋白 B1/Toll 样受体 4 诱导脑出血后自噬并促进神经炎症。
HMGB1/TLR4 induces autophagy and promotes neuroinflammation after intracerebral hemorrhage.
机构信息
From the Department of Neurology (C.L., Y.L., X. Z., H.L., X. C.), First Affiliated Hospital of Kunming Medical University, PR China.
From the Department of Neurology (C.L., Y.L., X. Z., H.L., X. C.), First Affiliated Hospital of Kunming Medical University, PR China.
出版信息
Brain Res. 2022 Oct 1;1792:148003. doi: 10.1016/j.brainres.2022.148003. Epub 2022 Jul 9.
BACKGROUND AND PURPOSE
Intracerebral hemorrhage (ICH) causes autophagy as well as inflammation; the latter is known to involve the high-mobility group box 1 protein (HMGB1)/Toll-like receptor 4 (TLR4) axis. Here we investigated whether this axis may help mediate both the autophagy and inflammation associated with ICH.
METHODS
ICH was induced by injecting autologous blood into Sprague-Dawley rats, followed in some cases by intracerebroventricular injection of short interfering RNA (siRNA) against HMGB1 or TLR4 at 6 h after ICH induction or by intraperitoneal injection of the autophagy inhibitor 3-methyladenine (3-MA) or autophagy activator rapamycin at 6, 24, and 48 h after ICH induction. Western blotting, immunohistochemistry or immunofluorescence was used to assess levels of HMGB1/TLR4 signaling pathway proteins as well as markers of autophagy (LC3B, Beclin1, Atg5) or inflammation (IL-1 beta, TNF-α). Numbers of apoptotic cells were determined using TUNEL staining. Changes in levels of these proteins were correlated with neurological deficits measured using the modified Neurological Severity Score.
RESULTS
ICH caused HMGB1 to translocate from the nucleus into the cytoplasm, and it up-regulated expression of TLR4 and myeloid differentiation factor 88 (MyD88), and induced neurological deficits. Administering siRNA against HMGB1 or TLR4 reversed this up-regulation. Levels of markers of autophagy (LC3B, Beclin1, Atg5) or inflammation (IL-1 beta, TNF-α) were significantly higher 72 h after ICH than at baseline, as were the numbers of TUNEL-positive cells. Administering siRNA against HMGB1 or TLR4 markedly alleviated inflammation, and autophagy, apoptosis, and neurological deficits. Similarly, administering autophagy inhibitor 3-MA alleviated inflammation, apoptosis, and neurological deficits. Conversely, autophagy activator rapamycin exacerbated these effects of ICH.
CONCLUSIONS
During the acute phase of ICH, the HMGB1/TLR4/MyD88 axis acts via autophagy to promote inflammation.
背景与目的
脑出血（ICH）可引起自噬和炎症；后者已知涉及高迁移率族蛋白 B1（HMGB1）/Toll 样受体 4（TLR4）轴。在这里，我们研究了该轴是否可能有助于介导与 ICH 相关的自噬和炎症。
方法
通过向 Sprague-Dawley 大鼠脑内注射自体血来诱导 ICH，在 ICH 诱导后 6 小时，在某些情况下通过脑室内注射针对 HMGB1 或 TLR4 的短发夹 RNA（siRNA），或通过腹腔内注射自噬抑制剂 3-甲基腺嘌呤（3-MA）或自噬激活剂雷帕霉素，在 ICH 诱导后 6、24 和 48 小时，通过 Western blot、免疫组织化学或免疫荧光检测 HMGB1/TLR4 信号通路蛋白以及自噬标志物（LC3B、Beclin1、Atg5）或炎症标志物（IL-1β、TNF-α）的水平。通过 TUNEL 染色确定凋亡细胞的数量。使用改良的神经严重程度评分测量神经功能缺损，将这些蛋白水平的变化与神经功能缺损相关联。
结果
ICH 导致 HMGB1 从核内转移到细胞质，上调 TLR4 和髓样分化因子 88（MyD88）的表达，并引起神经功能缺损。给予 HMGB1 或 TLR4 的 siRNA 可逆转这种上调。ICH 后 72 小时，自噬标志物（LC3B、Beclin1、Atg5）或炎症标志物（IL-1β、TNF-α）的水平以及 TUNEL 阳性细胞的数量均显著高于基线。给予 HMGB1 或 TLR4 的 siRNA 可显著减轻炎症、自噬、凋亡和神经功能缺损。同样，给予自噬抑制剂 3-MA 可减轻炎症、凋亡和神经功能缺损。相反，自噬激活剂雷帕霉素加剧了 ICH 的这些作用。
结论
在 ICH 的急性期，HMGB1/TLR4/MyD88 轴通过自噬作用促进炎症。
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