FHC调控氧化应激诱导的鹅卵泡颗粒细胞凋亡的作用机制

Oxidative stress induces the apoptosis of follicular granulosa cells, resulting in follicular atresia. Ferritin heavy chain (FHC) plays an important role in mediating oxidative stress and apoptosis. Our previous studies showed that the level of FHC mRNA expression in the ovary of the laying goose was 13.25 folds higher than that of the prelaying goose. The amounts of FHC mRNA and protein expression in atresia follicles were 2.78 folds and 1.45 folds higher than F1 follicles, respectively. Oxidative stress of follicular granulosa cells induced by 3-nitropropionic acid (3-NPA) reduced granulosa cell viability and enhanced the level of reactive oxygen species, Caspase 3 and FHC gene expression in the goose. We hypothesize that FHC can mediate the follicular granulosa cell apoptosis induced by oxidative stress through c-Jun N-terminal kinase (JNK) and B-cell lymphoma 2 (Bcl-2) pathways. However, the molecular mechanism remains to be investigated. Based on our previous studies, the overexpression/interference vectors with FHC gene will be transfected into granulosa cells from follicles of the Sichuan white goose. The effects of high and low expression of FHC on the activities of antioxidant enzymes and the expression level of apoptotic genes and proteins will be determined. The functions of FHC regulating the oxidative stress and the action pathway of FHC mediating apoptosis in granulosa cells will be clarified. Furthermore, granulosa cells transfected with overexpression/interference FHC gene vectors will be treated by 3-NPA combined with JNK and Bcl-2 inhibitors. The action of FHC regulating the follicular granulosa cell apoptosis induced by oxidative stress will be assessed. Therefore, the action mechanism of FHC regulating the apoptosis induced by oxidative stress in the granulosa cell will be uncovered in the Sichuan white goose. These results should provide the theoretical basis in the molecular regulation mechanism of the apoptosis in follicular granulosa cells and follicular atresia, and a novel approach for the research on improving egg-laying performance in avian.

氧化应激能诱导颗粒细胞凋亡，导致卵泡闭锁。铁蛋白重链（FHC）在氧化应激和细胞凋亡过程中具有重要调控作用。我们前期研究表明闭锁卵泡FHC基因和蛋白表达量分别是F1卵泡的2.78和1.45倍；3-硝基丙酸（3-NPA）能显著降低鹅卵泡颗粒细胞活性，增加ROS水平，促进Caspase 3和FHC基因表达。推测FHC可通过JNK和Bcl-2途径调控氧化应激诱导的颗粒细胞凋亡，其机制仍不清楚。项目将FHC过表达和干扰载体转染至鹅卵泡颗粒细胞，研究FHC对颗粒细胞氧化应激及JNK和Bcl-2凋亡途径基因和蛋白表达的影响，明确FHC调控颗粒细胞氧化应激和细胞凋亡的作用途径；用3-NPA联合JNK和Bcl-2抑制剂处理已转染FHC载体的颗粒细胞，研究并揭示FHC调控氧化应激诱导的鹅卵泡颗粒细胞凋亡的作用机制，为阐明颗粒细胞凋亡和卵泡闭锁机制的研究奠定理论基础，同时为提高家禽产蛋性能研究提供新的切入点。

机体内过量的铁离子可通过芬顿反应产生活性氧自由基（ROS），并最终损伤细胞。铁蛋白重链（FHC）是构成铁蛋白的一个主要亚基，具有亚铁氧化酶活性，在氧化应激和细胞凋亡过程中具有重要调控作用。研究表明，氧化应激能诱导颗粒细胞凋亡，导致卵泡闭锁。我们前期研究结果发现，四川白鹅闭锁卵泡Fhc基因和蛋白表达量显著高于等级卵泡，提示FHC在鹅颗粒细胞凋亡和卵泡闭锁过程中具有重要的调控作用，然而其具体调控机制仍不清楚。3-硝基丙酸（3-NPA）能显著降低鹅卵泡颗粒细胞活性，增加ROS水平，促进Caspase 3和Fhc基因表达。我们推测FHC可通过JNK和BCL-2途径调控氧化应激诱导的颗粒细胞凋亡。因此，本项目用3-NPA联合JNK和BCL-2抑制剂处理已转染Fhc载体的颗粒细胞，研究并揭示FHC调控氧化应激诱导的鹅卵泡颗粒细胞凋亡的作用机制。项目主要获得以下结果：（1）FHC可通过下调颗粒细胞ROS生成来发挥抗3-NPA诱导的氧化应激的作用。（2）FHC通过介导BCL-2线粒体凋亡途径发挥抗颗粒细胞凋亡作用。（3）FHC能通过介导JNK和BCL-2信号转导途径抗氧化应激诱导的颗粒细胞细胞凋亡。项目实施期间，发表学术论文4篇，其中SCI收录论文2篇；获授权实用新型专利2项，获授权国际专利2项；参加学术会议3次，做学术报告2次，发表会议论文4篇。培养硕士研究生2名。