利用条件基因敲除技术研究内源性H2S调控Shh通路对HIBD后新生小鼠海马神经再生的影响

Studies have demonstrated that the neurogenesis after hypoxic-ischemic brain damage (HIBD) contributes to functional recovery. Endogenous H2S is mainly generated in brain by cystathionine β synthase (CBS) from L-cysteine. We have reported that NSCs expressed CBS and produced H2S. And addition of endogenous H2S donor, L-cysteine, stimulated proliferation and increased the differentiation potential of NSCs to neurons. However, the role of endogenous H2S in HIBD is unclear. Recently, we found that L-cysteine increased the Shh expression in NSC. Additionally, L-cysteine promoted proliferation of NSC in the DG of hippocampus, and improved the learning and memory performance in HIBD mice. Moreover, it has been demonstrated that the proliferation of NSC in the hippocampus was decreased, and L-cysteine has no obvious effect on proliferation of NSC in the same brain region in the NSC special Shh-deficient mice (NestinCre/Shhflox/flox) following HIBD. Arising from this, we hypothesized that endogenous H2S promotes hippocampal neurogenesis in neonatal mouse following HIBD by activating Shh pathway, and played a neuroprotective role in HIBD. To test the relationship of endogenous H2S and hippocampus neurogenesis in neonatal mouse following HIBD, we would set up the inducible NSC special CBS-deficient mice (NestinCreERT2/CBSflox/flox ) and investigate the underlying mechanisms of L-cysteine in three steps of preventive strategy of HIBD, via Shh pathway. This project will disclose new insights into therapeutic method in HIBD and provide new animal models.

研究表明促进神经再生可有效改善缺氧缺血性脑损伤（HIBD）后神经功能。我们曾率先报导左旋半胱氨酸（经CBS催化生成内源性H2S）能促进神经干细胞（NSC）增殖分化，但其在HIBD中的作用及机制不详。最近我们发现左旋半胱氨酸可上调NSC中Shh表达，并促进HIBD新生小鼠海马NSC增殖，改善其认知功能；而我们建立的NSC特异性Shh基因敲除(NestinCre/Shhflox/flox)小鼠HIBD后海马NSC增殖降低，且左旋半胱氨酸对其疗效不佳。由此我们推断内源性H2S通过激活Shh通路诱导海马神经再生，促进HIBD后神经功能恢复。本课题拟首次构建NSC特异性CBS基因敲除小鼠(NestinCreERT2/CBSflox/flox)，观察内源性H2S对HIBD后神经再生的影响及其与Shh通路的关系，阐明左旋半胱氨酸在HIBD中的三级预防策略，为探讨HIBD治疗新策略提供实验依据和动物模型。

本项目按照计划进行，经费预算进行，安排合理。利用流式、免疫组化、蛋白印迹等分子生物学方法，结合行为学等方法，探讨内源性H2S供体-左旋半胱氨酸（L-Cysteine）在新生小鼠缺氧缺血性（hypoxia-ischemia, HI）脑损害中的作用及其机制，结果如下：（1）L-Cys可提高受损皮层的CBS表达和H2S水平；（2）L-Cys明显抑制HI诱导后急性脑损伤，包括神经元凋亡，脑水肿及脑组织缺失，可能是其通过Akt和/或ERK通路调节Bax/Bcl-2和活性caspase-3的表达；（3）L-Cys调节HI诱导内质网应激发挥神经保护作用；（4）L-Cys抑制HI后受损皮层区神经胶质细胞的激活，炎症因子的分泌及外源性免疫细胞的入侵。（5）L-Cys促进HI后受损皮层区小胶质细胞/巨噬细胞从炎性M1向抗炎性M2的转化表型。（6）MAPK（p38和/或JNK）、NF-κB等信号途径参与了L-Cys抑制小胶质细胞的活化过程。（7）L-Cys促进AMP激活蛋白激酶（AMPK）的激活和AMPK抑制剂消除了这些半胱氨酸的抗凋亡和抗炎作用。（8）L-Cys抑制HI后减轻突触损伤，促进突触重构，并有效地减轻HI脑损伤及改善行为缺陷。（9）同时H2S合成酶抑制剂可抑制L-Cys这些神经保护作用。由此，我们推测L-Cys可以通过生成H2S为HIE的治疗提供新的治疗方法（详见代表论著1,2,3）。申请人近年来发现H2S在新生小鼠HI的多个病理过程中均有干预作用，如凋亡、炎症、自噬、氧化应激、突触重构、内质网应激等，提示H2S的神经保护作用是多途径多靶点的。.在本项目资助下，共发表SCI论文20篇，培养博士研究生２名，硕士研究生7名，并以此为支撑获得2018年和2020年国家基金面上项目两项（项目号为82072535，81873768）。