HMGB1-TLRs/RAGE信号通路在急性脑出血后通过细胞自噬调控炎症损伤的机制研究

Stroke is one of the leading causes of mortality and disability in the world. Intracerebral hemorrhage (ICH) is one of the most dangerous subtypes of the stroke, which bring higher burden of individual, family, and society. Cell autophagy is ubiquitous stability mechanism in eukaryotic cells, which play key roles in embryonic development, cell self-protection, and survival. The effects of autophagy are both advantageous and disadvantageous in various diseases. Our previous studies showed that the expression of Beclin-1 had increased after ICH, which indicated that cell autophagy was activated after ICH. But cell autophagy is a protective mechanism or damage mechanism, which is not clear. Our previous study found that HMGB1 participated in secondary brain injury mainly by RAGE/TLR4 signaling pathway after ICH. HMGB1 could activate cell autophagy by RAGE/TLR4 signaling pathway. Cell autophagy has dual function, which can promote inflammation and also can suppress the inflammatory response. Based on our previous studies, this study will further explore that how HMGB1 regulate cell autophagy by TLRs/RAGE signaling pathways; and activation of autophagy whether impact inflammation or not, which participate n the early brain injury. In order to confirm the predictive value of HMGB1-LRs/RAGE signaling pathway, case-control study and prospective cohort study were used after ICH which provide a new way of the treatment of ICH, and discovery of new therapeutic targets and strategies.

脑出血病死率和致残率居各型脑卒中之首，给个人、家庭和社会造成沉重的负担。细胞自噬是真核细胞普遍存在的自稳机制，在胚胎发育和细胞自我保护等过程中发挥关键作用。自噬在各种疾病的作用存在一定的分歧，既有利又有弊。我们研究发现脑出血后自噬因子Beclin-1表达增加，说明在脑出血后自噬被激活。脑出血后激活的自噬是一种保护机制还是损伤机制，目前还不清楚。我们的研究发现脑出血后HMGB1通过RAGE和TLR4参与继发性脑损伤。HMGB1通过RAGE或TLR4激活自噬。自噬具体双重作用，既能促进炎症反应，也能抑制炎症反应。本研究将进一步探讨HMGB1-TLRs/RAGE信号通路是否能通过细胞自噬调控炎症反应继而参与脑出血后的早期损伤及其分子机制。同时采用病例对照研究和前瞻性队列研究确定HMGB1-TLRs/RAGE信号通路对脑出血患者预后的预测价值，从而提供脑出血的治疗方式，发现新的治疗靶点和策略。

脑出血病死率和致残率居各型脑卒中之首，给个人、家庭和社会造成沉重的负担。细胞自噬是真核细胞普遍存在的自稳机制，在胚胎发育和细胞自我保护等过程中发挥关键作用。自噬具体双重作用，既能促进炎症反应，也能抑制炎症反应。本项目探讨HMGB1-TLRs信号通路通过细胞自噬调控炎症反应继而参与脑出血后的早期损伤及其分子机制。同时采用病例对照研究和前瞻性队列研究确定HMGB1-TLRs信号通路对脑出血患者预后的预测价值，从而提供脑出血的治疗方式，发现新的治疗靶点和策略。.我们成功制作大鼠自体脑出血模型，运用免疫组化及荧光技术观察到正常的大鼠脑组织中HMGB1位于细胞核中，当脑出血后即从细胞核分泌到细胞浆及细胞外，其含量在24和72小时明显地高于假手术组（图1）。同时采用Western blot技术进一步证实脑出血后HMGB1的含量较前显著地增多。HMGB1下游受体TLR4及MyD88在脑出血后也明显增多（图2）。采用免疫荧光技术发现HMGB1与TLR4共表达，TLR4与MyD88共表达（图2），其说明在脑出血后激活了HMGB1/TLR4信号通路。脑出血后，自噬因子Beclin1、Atg5、LC3B的表达量明显地增多，采用电镜观察到在血肿周围存在大量的自噬体（图3和图4）。侧脑室注射HMGB1siRNA和TLR4siRNA后Beclin1、Atg5、LC3B的表达明显下降，但高于假手术组。上述实验表明急性脑出血后，HMGB1-TLR4/ MyD88信号通路调节细胞自噬。同时研究还发现在脑出血后，炎症因子IL-1和TNF-a及凋亡细胞表达明显地增多，侧脑室注射HMGB1siRNA和TLR4 siRNA后IL-1和TNF-a（图5和图6）及凋亡细胞（图7）表达明显减少。腹腔注射自噬抑制剂3-methyladenine后IL-1和TNF-a及凋亡细胞表达量明显减少，而注射自噬激动剂rapamycin后IL-1和TNF-a及凋亡细胞表达量明显增多。上述实验说明在急性脑出血后HMGB1/TLR4信号通路通过细胞自噬调节炎症反应。.收集脑出血患者的血标本，HMGB1和TLR4含量较正常人明显地升高，与患者三月的不良神经功能结局及病死率正相关同时也和血肿体积正相关。HMGB1和TLR4等生物标志物可以预测脑出血患者的预后，其存在一定的临床价值和转化医学价值。.