ALOX15B-JNK在热应激诱导支持细胞氧化应激和凋亡中的作用

doi:10.11843/j.issn.0366-6964.2023.12.016

摘要/Abstract

摘要： 旨在研究花生四烯酸脂氧合酶15B(arachidonate lipoxygenase type B, ALOX15B)是否参与了热应激条件下丙二醛(malondialdehyde, MDA)与活性氧(reactive oxygen species, ROS)的生成,进而调控睾丸支持细胞凋亡,以及JNK信号通路(jun amino terminal kinase, JNK)是否在这一过程中发挥了作用。本研究从20只3周龄的三元杂交仔猪的睾丸组织中分离出支持细胞,随机分为3组,每组3个重复,将体外培养的仔猪睾丸支持细胞在44℃条件下处理30 min作为热应激模型,分别转染ALOX15B的siRNA、添加ALOX15B抑制剂(黄芩素)或JNK抑制剂后,运用流式细胞术检测细胞的凋亡率,使用Western blotting检测磷酸化JNK、ALOX15B和p53的蛋白表达,利用ELISA检测8-HETE与15-HETE的含量,采用试剂盒检测MDA和ROS的水平。结果显示,与单独的热应激组相比,黄芩素和热应激联合处理减少了8-HETE和15-HETE的含量(P<0.01),降低了MDA和ROS的水平(P<0.01),抑制了热应激诱导的细胞凋亡率(P<0.01);在热应激条件下,黄芩素和ALOX15B的siRNA均抑制了热应激诱导的JNK的磷酸化(P<0.01);JNK抑制剂SP600125可抑制热应激条件下p53的表达水平(P<0.01),降低细胞凋亡率(P<0.01)。与单独的热应激组相比,SP600125和热应激联合处理反馈抑制了ALOX15B的表达(P<0.01),减少了脂质过氧化物8-HETE(P<0.01)与15-HETE的含量(P<0.05),降低了MDA和ROS的水平(P<0.01)。这表明,在热应激条件下,ALOX15B使MDA和ROS的水平升高,诱导了支持细胞凋亡,JNK通过激活p53参与了ALOX15B调节的支持细胞凋亡;JNK可反馈调节ALOX15B、8-HETE、15-HETE、MDA和ROS的水平。

关键词: 热应激, 睾丸支持细胞, JNK, 细胞凋亡, ALOX15B

Abstract: The objective of this study was to investigate whether arachidonate lipoxygenase type 15B (ALOX15B) participates in the production of malondialdehyde (MDA) as well as reactive oxygen species (ROS), regulates the apoptosis of porcine sertoli cells and determines the role of JNK in the process. In this experiment, porcine sertoli cells from the testicular tissues of 20 3-week-old three-way hybrid piglets was isolated and randomly divided into 3 groups with 3 replicates in each group. Porcine sertoli cells were treated at 44℃ for 30 min as the heat stress model. After siRNA transfection of ALOX15B and addition of ALOX15B inhabitor (baicalein) or JNK inhibitor, the apoptosis rates of the cells were detected by flow cytometry, the expressions of phosphorylated JNK, ALOX15B and p53 were detected by Western blotting, and the contents of 8-HETE and 15-HETE were analyzed by ELISA. The levels of MDA and ROS were detected by the kit. The results showed that the combined treatment of baicalein and heat stress decreased the content of 8-HETE and 15-HETE (P<0.01), reduced the levels of MDA and ROS(P<0.01), and inhibited the apoptosis rate induced by heat stress (P<0.01). Under heat stress, bai-calein and ALOX15B siRNA inhibited JNK phosphorylation induced by heat stress (P<0.01). SP600125 inhibited the expression of p53 downstream by decreasing the phosphorylation of JNK (P<0.01), and reduced the apoptosis induced by heat stress (P<0.01). Meanwhile, compared to the heat stress group, SP600125 combined with heat stress also gave negative feedback on the protein level of ALOX15B (P<0.01), decreased the content of lipid peroxides 8-HETE (P<0.01) and 15-HETE (P<0.05), and reduced the level of MDA and ROS (P<0.01). In conclusion, ALOX15B regulates apoptosis of porcine sertoli cells under heat stress; JNK participated in the effect of ALOX15B, and regulated apoptosis of porcine sertoli cells through p53, and could have the feedback on the levels of ALOX15B, 8-HETE, 15-HETE, MDA and ROS.

Key words: heat stress, sertoli cells, JNK, apoptosis, ALOX15B

中图分类号:

S828.3

薛鸿雁, 杨孟雨, 杨欢, 董丽君, 蔡霞清, 赵泽民, 王鲜忠. ALOX15B-JNK在热应激诱导支持细胞氧化应激和凋亡中的作用[J]. 畜牧兽医学报, 2023, 54(12): 5056-5065.

XUE Hongyan, YANG Mengyu, YANG Huan, DONG Lijun, CAI Xiaqing, ZHAO Zemin, WANG Xianzhong. The Role of ALOX15B-JNK in Heat Stress-induced Oxidative Stress and Apoptosis of Sertoli Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5056-5065.

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