网状蛋白Nogo-B通过调控巨噬细胞自噬参与炎症调节的机理研究

Autophagy is a cell housekeeping mechanism that controls inflammatory response.Endoplasmic reticulum (ER) is believed to be the membrane source and the scaffoldfor autophagosome formation and, takes an important role in regulating autophagicflux. The precise mechanisms of ER stress-mediated inflammation andautophagy are yet fully understood. Nogo-B is a reticulon family protein mainly localizedto ER membrane. It is highly expressed in monocytes/macrophages. Previously, wehave shown that Nogo-B regulates ER morphology, vesicle formation and inflammation. The objective of this study is to investigate whether Nogo-B regulates sepsis induced inflammation via controlling autophagy. Our preliminary data show that genetic loss of Nogo-B (Nogo KO) protected against LPS-mediated sepsis in vivo by increasing autophagic flux and decreasing proinflammatory cytokine (IL-1beta) production. Nogo-B deficiency led to enhanced starvation- and LPS-induced autophagy, decreased IL-1beta production and sustained NF-kapaB signaling. Thus, we hypothesize that Nogo-B, a functional ER protein,controls autophagic flux to regulate inflammation. To test the hypotheses, we will (1)elucidatethe molecular mechanisms of Nogo-B in regulating autophagy and inflammasome in macrophages in vitro; (2) define the role of Nogo-B-mediated autophagy in acute inflammation regulation in vivo. Collectively, results from these experiments will lead to a great understanding on the mechanisms of autophagosome formation and the regulation of inflammation. Our study may shed light in developing new strategies in treating inflammatory diseases.

自噬在炎症发生与发展中起至关重要作用，但其发生及对炎症病理过程调控的分子机制尚不明确。内质网（ER）为自噬调控分子提供作用场所，也为自噬小体的形成及延伸提供膜来源和模板。定位于ER的网状蛋白Nogo-B普遍存在且高表达于巨噬细胞等炎性细胞；参与内质网形态和钙信号的调控。前期研究表明，Nogo-B缺失可显著增加自噬通量、降低促炎因子，从而抵抗LPS所致的败血症，但不影响LPS介导NFkB信号通路。据此，我们推论Nogo-B作为重要ER功能蛋白，经控制自噬而限制炎症小体加工促炎因子，进而调节炎症。本课题将运用新建的细胞特异性Nogo-B敲除小鼠在在体内明确Nogo-B介导自噬调控急性炎症的机制；体外阐明Nogo-B调控巨噬细胞自噬和炎症小体的分子机理。本研究将使我们进一步揭示自噬发生之分子机制，并验证通过促进自噬来控制炎症的手段。研究结果也将为我们治疗炎症相关性疾病提供理论基础和新的治疗靶点。

自噬在炎症发生与发展中起至关重要作用，但其发生及对炎症病理过程调控的分子机制尚不明确。内质网（ER）为自噬调控分子提供作用场所，也为自噬小体的形成及延伸提供膜来源和模板。定位于ER的网状蛋白Nogo-B普遍存在且高表达于巨噬细胞等炎性细胞；参与内质网形态和钙信号的调控。本课题研究首次1）完成并验证了Nogo-B对自噬小体形成，自噬小体形成数量和形态，自噬小体与溶酶体结合以及Nogo-B对自噬通量的影响；2）完成Nogo-B与自噬相关调控蛋白相互作用的研究；3）验证了Nogo-B通过调控与BCL2结合、BCL2内质网定位进而调节自噬启动蛋白Beclin1 的研究；4）验证了Nogo-B通过自噬调控炎性小体，进而影响炎症因子IL-b成熟与分泌的研究；5）成功构建Nogo-B基因敲除（Nogo-/-）小鼠和髓性细胞特异性Nogo基因敲除（NogomacKO）小鼠；6）通过运用Nogo-/- NogomacKO 基因修饰小鼠验证了巨噬细胞Nogo-B对于LPS诱导的败血症的小鼠的生存率和炎症水平；7）通过运用Nogo-/- NogomacKO 基因修饰小鼠验证了巨噬细胞Nogo-B对于盲肠结扎穿孔诱导的急性肺损伤模型中发挥的作用。本研究将使我们进一步揭示自噬发生之分子机制，并验证通过促进自噬来控制炎症的手段。研究结果也将为我们治疗炎症相关性疾病提供理论基础和新的治疗靶点。

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