PI3K/Akt/mTOR信号通路对小胶质细胞表达细胞因子的调节及神经节细胞生存的影响

Microglia are the immune cells in central nervous system（CNS）. The activation of microglia may lead to the neuronal cell death with the secretion of cytokines, such as IL-1, IL-6, TNF-α etc. However, activated microglia also produce neurotrophic factors (NF-3, BDGF, NGF etc.)，which are crucial to neuronal protection and axonal regeneration. PI3K/AKT/mTOR is a novel signaling pathway discovered in recent years. It has comprehensive function in cell proliferation, growth and secretion. Previous researches showed the production of NGF, BDGF and other neurotrophic factors may be regulated by PI3K/Akt/mTOR pathway. In the study, small interfering RNA and other methods will be used to block or activate PI3K/AKT/mTOR signaling pathway in vitro and vivo. After the success of both in vitro and in vivo animal model, the cytokine expression will be analyzed by RNA microarray and immunocytochemistry, including NGF, BDGF, IL-1, IL-6 etc. The phosphorylation level of PI3K, Akt, JAK, JNK, MAPK will be evaluated by western blot, and the number of RGCs (retinal ganglion cells) will be counted with the blue-light confocal scanning laser ophthalmoscope (bCSLO). With these studies, the regulation of PI3K/AKT/mTOR signal pathway on neurotrophic factors and cytokines, the phosphorylation level of PI3K, Akt, JAK, JNK, MAPK, the relationship between PI3K/Akt/mTOR and other signal pathway will be elucidated. The relationship between PI3K/Akt/mTOR and survival of RGCs will also be observed. The study plays a significant role in understanding the effect of PI3K/Akt/mTOR pathway on neurotrophic effects and RGCs survival, and possibly lead to new treatment of RGCs protection.

小胶质细胞（MG）是中枢神经系统（CNS）的免疫细胞，活化后分泌多种细胞因子，炎症因子可导致受损神经元死亡，而神经营养因子对神经元保护具有重要意义。PI3K/Akt/mTOR是近年来新发现的一条信号转导通路，对细胞增殖、分化、分泌等有广泛作用。初步研究表明，小胶质细胞产生NGF、BDGF等神经营养因子可能受该通路的调控。 本项目拟应用siRNA等技术对培养细胞及转基因小鼠的PI3K/Akt/mTOR通路进行激活、阻断，运用RNA微阵列、western blot、激光共焦显微镜分别对MG的细胞因子表达差异、PI3K/Akt等的磷酸化水平及视网膜神经节细胞（RGCs）存活数量进行观察，研究PI3K/Akt/mTOR信号转导通路对小胶质细胞产生细胞因子的调控及RGCs生存的影响。 研究将丰富我们对小胶质细胞功能的认识，拓宽神经元保护的思路，同时也将为中枢神经系统相关疾病的诊疗提供科学依据。

小胶质细胞（MC）是中枢神经系统的免疫细胞。慢性神经退行性疾病中，持续活化的MC是造成神经元损伤和机体功能障碍的原因之一。抑制MC过度活化是神经保护的潜在靶点。.本课题主要探讨：第一、两种中药单体（汉防己甲素Tet和罂粟碱PA）对MC活化的抑制及机制。第二、这两种药物对RGCs的保护及机制。.我们发现：.（一）Tet能抑制LPS诱导的MC活化。⑴ 在1μM的浓度下，Tet能够减少活化的MC比例，降低CD11b的表达。⑵ Tet能剂量依赖性的抑制小胶质细胞TNF-a和IL-1b转录、翻译、释放。.（二）Tet通过NF-kB和ERK通路抑制LPS诱导的MC活化。我们进行了Western blot检测，发现p-P65, p-IKK以及p-ERK1/2的表达随Tet浓度加大呈剂量依赖性的下调。提示NF-Kb、ERK信号通路参与Tet对MC的调控。我们还联合使用ERK的抑制剂PD98059和NF-kB的抑制剂BAY作用于LPS诱导的MC，发现Tet没有进一步的抑制作用。因此，Tet是通过NF-kB和ERK通路抑制MC活化。.（三）PA在正常血药浓度下，剂量依赖性的抑制LPS诱导的MC活化。Tet虽然能抑制MC活化，但正常血药浓度（10ng）作用并不明显。我们发现PA能在极低浓度下显著抑制MC活化。在0.4ug/ml, 2ug/ml, 10ug/ml下(正常血药浓度50ug/ml)，PA能够梯度抑制TNF-a, IL-1b, iNOS, COX-2等基因表达，并显著降低IL-1b, TNF-a释放。我们进一步应用PD98059、BAY以及PI3K抑制剂Wortmannin、STAT3的抑制剂AG490联合PA来筛选其作用机制，发现：NF-kB可能是PA作用通路。Western blot也进一步验证了这个猜想。.（四）PA能在低剂量下抑制RGCs凋亡。我们建立了原代大鼠RGCs培养体系，发现：1）2ug/ml PA能够显著降低TUNEL阳性的RGC数量；2）2ug/ml PA组在1周、2周、3周、4周时，CCK8值均高于正常对照组；3）2ug/ml PA组RGC的线粒体膜电位也较正常对照组高。.本研究首次发现低剂量PA对MC活化的抑制及RGCs的保护，为体外实验及临床应用奠定了前期基础。本课题培养博士生2名，硕士生1名，发表SCI论文5篇，申请国家发明专利1项。