谷氨酸通过T1R1/T1R3-ERK1/2通路调节香猪睾丸间质细胞自噬相关基因表达  

Glutamate regulating autophagy-related gene expression in Leydig cells of Xiang pig through T1R1/T1R3-ERK1/2 pathway

作　　者：王涵 徐永健 蒙利洁 龚婷 WANG Han;XU Yong-jian;MENG Li-jie;GONG Ting(College of Animal Science,Guizhou University/Key Laboratory of Plateau Mountain Animal Genetics,Breeding and Reproduction,Ministry of Education/Guizhou Key Laboratory of Animal Genetics,Breeding and Reproduction,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州大学动物科学学院/高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室,贵州贵阳550025

出　　处：《南方农业学报》2023年第6期1829-1836,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31702117);贵州省科学技术项目(黔科合基础〔2020〕1Y135号,黔科合基础〔2018〕1404号)。

摘　　要：【目的】探究L-谷氨酸刺激对香猪睾丸间质细胞自噬的影响,为进一步研究味觉受体T1R1/T1R3通过雷帕霉素靶蛋白(mTOR)调控下自噬对雄性生殖的影响提供理论依据。【方法】选取30日龄的健康从江香猪分离培养睾丸间质细胞,采用不同浓度L-谷氨酸刺激睾丸间质细胞T1R1/T1R3,探究激活T1R1/T1R3对睾丸间质细胞自噬相关基因(TAS1R1、TAS1R3、ERK1、ERK2、mTOR、Beclin 1和MAP1LC3B)表达的影响。【结果】从江香猪睾丸间质细胞在培养72~96 h增殖最快,于培养96 h时达对数生长期。10 mmol/L是L-谷氨酸刺激睾丸间质细胞味觉受体T1R1/T1R3的有效浓度。经10 mmol/L的L-谷氨酸刺激后,睾丸间质细胞的mTOR、ERK1和ERK2基因相对表达量极显著高于对照组(P<0.01,下同),而Beclin 1基因相对表达量极显著低于对照组,MAP1LC3B基因相对表达量显著低于对照组(P<0.05,下同);ERK1/2和p-S6K1蛋白相对表达量显著高于对照组,而Beclin 1蛋白相对表达量显著低于对照组。单丹磺尸酰胺(MDC)染色结果显示,经10 mmol/L的L-谷氨酸刺激后,睾丸间质细胞内的酸性自噬泡荧光强度明显低于对照组,表明胞内自噬受抑制。【结论】10 mmol/L的L-谷氨酸可激活从江香猪睾丸间质细胞T1R1/T1R3,且自噬相关基因TAS1R1、TAS1R3、ERK1、ERK2和mTOR及相关蛋白T1R1、T1R3、ERK1/2、p-S6K1和p-mTOR表达升高,而Beclin 1和MAP1LC3B基因及Beclin 1蛋白表达降低,故推测谷氨酸可通过激活味觉受体T1R1/T1R3参与雄性生殖自噬的负调控。【Objective】This paper explored the effect of L-glutamate stimulation on autophagy in Xiang pig Leydig cells,providing a theoretical basis for further research on the effects of taste receptor T1R1/T1R3 on male reproduction under the regulation of rapamycin target protein(mTOR)through autophagy.【Method】Selecting Leydig cells isolated and cultured from healthy 30 days old Congjiang Xiang pigs,different concentrations of L-glutamate were used to stimulate Leydig cells T1R1/T1R3,to investigate the effect of activating T1R1/T1R3 on expression of autophagy-related genes of Leydig cell(TAS1R1,TAS1R3,ERK1,ERK2,mTOR,Beclin 1 and MAP1LC3B).【Result】The proliferation of Leydig cells from Congjiang Xiang pig was the fastest during 72-96 h of cultivation,and reached a logarithmic growth phase at 96 h of cultivation.10 mmol/L was the effective concentration of L-glutamate to stimulate the taste receptor T1R1/T1R3 in Leydig cells.After stimulation with 10 mmol/L L-glutamate,the relative expression levels of mTOR,ERK1,and ERK2 genes in Leydig cells were extremely significantly higher than those in the control group(P<0.01,the same below).The relative expression levels of Beclin 1 gene were extremely significantly lower than those in the control group.And the relative expression levels of MAP1LC3B gene were significantly lower than those in the control group(P<0.05,the same below).The relative expression levels of ERK1/2 and p-S6K1 proteins were significantly higher than those in the control group,while the relative expression levels of Beclin 1 protein were significantly lower than those in the control group.The staining results of dansylcadaverine(MDC)showed that after stimulation with 10 mmol/L L-glutamate,the fluorescence intensity of acidic autophagic vesicles in Leydig cells was significantly lower than that of the control group,indicating that intracellular autophagy was inhibited.【Conclusion】10 mmol/L of L-glutamate can activate the Leydig cells T1R1/T1R3 of Congjiang Xiang pig,and the expression of autopha

关 键 词：从江香猪 睾丸间质细胞 味觉受体T1R1/T1R3 自噬 谷氨酸 

分 类 号：S828.89[农业科学—畜牧学]