脊髓小胶质细胞ProS/Mer系统调控TLR4/MyD88/NF-κB信号通路在脓毒症神经肌肉功能障碍中的作用及机制研究

The sepsis not only induces the multiple organs dysfunction, but also induces the neuromuscular dysfunction (NMD) as well. The occurrence rate of NMD is from 56 % to 100 %, which is a threat to the patients. The pathogenesis of NMD induced by sepsis is not entirely clear. Our previous results found that the sepsis-induced the activation of TLR4/MyD88/NF-κB pathway of spinal microglia could aggravate the NMD, and the inhibition of spinal inflammation could alleviate the NMD. The other results found that the ProS/Mer system of microglia has the negative regulation to the TLR4/MyD88/NF-κB pathway in the spinal inflammation. Thus, we supposed that the sepsis activated the TLR4/MyD88/NF-κB pathway of spinal microglia, which aggravated the sepsis-induced the NMD; however, the ProS/Mer system of spinal microglia inhibited the spinal inflammation to alleviate the NMD in sepsis. The animal model of sepsis will be built by cecal ligation and perforation procedure. The objective is to clarify the relationship between ProS/Mer system and TLR4/MyD88/NF-κB pathway of spinal microglia in the regulation of the heterogeneity of nicotinic acetylcholine receptors in septic NMD by stereoscopy combined with immunofluorescence staining and microbiology. This study could bring a new breakthrough of prevention and treatment for critically ill patients with NMD.

脓毒症不仅引起多器官功能障碍，而且会导致神经肌肉功能障碍（NMD），其发生率高达56%~100%，已成为临床上亟待解决的重要问题，但其发病机制仍未明确。本项目前期研究发现，脓毒症激活脊髓小胶质细胞TLR4/MyD88/NF-κB通路引起的脊髓炎症参与NMD的发生；抑制TLR4通路的激活、减轻脊髓炎症可改善NMD。而脊髓小胶质细胞ProS/Mer系统对TLR4通路的激活具有重要的负调控作用。因此，我们假设脓毒症激活脊髓小胶质细胞TLR4通路引起脊髓炎症，参与脓毒症NMD的发生；ProS/Mer系统通过负调控TLR4通路改善脓毒症NMD。本项目拟采用脓毒症大鼠模型，应用体视学联合免疫荧光、分子生物学和细胞生物学等技术，研究脊髓小胶质细胞ProS/Mer系统调控TLR4通路在脓毒症NMD中的作用和机制，为临床上防治脓毒症NMD探寻新的研究方法和治疗方案。

脓毒症不仅引起多器官功能障碍，而且会导致神经肌肉功能障碍，已成为临床上亟待解决的问题之一。而脓毒症不仅对外周器官和组织造成损伤，也会引起中枢神经炎症，进而加重外周骨骼肌功能障碍。脊髓作为运动传导的“承上启下”部分，其功能越来越得到重视，此外，小胶质细胞作为中枢神经的免疫细胞，在脊髓炎症的发生发展过程中参与了重要作用；其中，小胶质细胞ProS/Mer在调控炎症过程中发挥了关键作用。因此，探寻脊髓小胶质细胞在脓毒症引起的骨骼肌功能障碍中的作用十分必要。在前期研究工作的基础上，本课题组使用了野生型（WT）和Mer-/-大鼠建立了脓毒症骨骼肌障碍模型（CLP），并分别培养了原代脊髓小胶质细胞用于体外实验。通过本课题的研究，取得如下结果：1.相比于WT大鼠，CLP加重了Mer-/-大鼠肌肉萎缩和功能；2. Mer-/-大鼠CLP后神经元损伤和神经肌肉接头（NMJ）分解；3. Mer-/-大鼠CLP 后脊髓炎症明显增加。4. . Mer-/-大鼠CLP后TLR4信号通路的表达增加， STAT1和SOCS1/3表达降低；5. ProS/Mer通过激活STAT1和SOCS信号通路并抑制TLR4/MyD88/NF-κB信号通路减轻CLP后骨骼肌功能障碍。上述实验结果证明了敲除Mer显着增加炎性细胞因子的释放，加重脓毒症引起的肌肉萎缩和神经肌肉功能障碍。激活Mer通过上调STAT/SOCS信号通路和抑制TLR4/MyD88/NF-κB信号通路抑制脓毒症引起的炎症反应，为临床上防治脓毒症NMD探寻新的研究方法和治疗方案。