GnIH通过p38MAPK信号通路对猪卵巢颗粒细胞自噬与凋亡的影响  

Effects of GnIH on Autophagy and Apoptosis of Porcine Ovarian Granulosa Cells via p38MAPK Signaling Pathway

作　　者：张鑫 霍孔林 宋星星 张多妮 胡文 胡传活[1] 李珣[1] ZHANG Xin;HUO KongLin;SONG XingXing;ZHANG DuoNi;HU Wen;HU ChuanHuo;LI Xun(College of Animal Science and Technology,Guangxi University,Nanning 530004)

机构地区：[1]广西大学动物科学技术学院,南宁530004

出　　处：《中国农业科学》2020年第9期1904-1912,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金青年基金项目(31402151)、广西自然科学基金面上项目(2017GXNSFAA198086)、广西自然科学基金青年基金项目(2015GXNSFBA139077)。

摘　　要：【目的】细胞自噬和凋亡存在着相互制约,p38MAPK信号通路作为细胞凋亡的主要调控通路之一,也对细胞自噬存在促进和抑制的双重作用。已有研究表明,促性腺激素抑制激素(gonadotropin-inhibitory hormone,GnIH)对细胞自噬与凋亡均有影响,但作用机制尚不明确。故探究GnIH通过p38MAPK信号通路对猪卵巢颗粒细胞(pGCs)自噬与凋亡的影响及其机理,为解决母猪的产子率以及同期发情等问题提供参考。【方法】从猪卵巢中提取卵巢颗粒细胞,进行体外培养。1、探究GnIH对p38MAPK信号通路的最佳作用时间:按孵育GnIH时间梯度(0 min、10 min、30 min、60 min、90 min)分组,用Western blot检测猪卵巢颗粒细胞p38与p-p38的蛋白表达量变化;2、验证GnIH对p38MAPK信号通路的影响:按(空白对照、GnIH、p38激活剂(U-46619)、U-46619+GnIH)分组,用Western blot检测p38与p-p38的蛋白表达量变化;3、探究不同浓度GnIH对自噬和凋亡的影响:按(空白对照、10^-6 mol·L^-1 GnIH、10^^-8 mol·L^-1 GnIH、10^-10 mol·L^-1 GnIH、10^-12 mol·L^-1 GnIH)分组,用Western blot检测自噬与凋亡标志性蛋白的表达量变化;4、验证不同浓度GnIH通过p38信号通路对自噬和凋亡的影响:将细胞分成6组(空白对照、U-46619、U-46619+10^-6 mol·L^-1 GnIH、U-46619+10^-8 mol·L^-1 GnIH、U-46619+10^-10 mol·L^-1 GnIH、U-46619+10^-12 mol·L^-1 GnIH),用Western blot检测自噬与凋亡标志性蛋白的表达量变化。【结果】1.GnIH孵育10 min后,显著降低p38与p-p38的蛋白表达量(P<0.05),提示,GnIH对p38MAPK信号通路的最佳作用时间为10 min;2.U-46619显著促进pGCs的p38磷酸化水平(P<0.05),GnIH显著抑制pGCs的p38磷酸化水平(P<0.05),提示,U-46619使p38MAPK信号通路活化,GnIH对p38MAPK信号通路的活化有抑制作用;3.当GnIH的浓度为10^-6 mol·L^-1时,pGCs的自噬和凋亡水平显著升高(P<0.05),随着GnIH浓度的降低,pGCs的自噬水平逐渐升高(P<0.05),pGCs的凋亡水平逐渐�【Objective】Studies have shown that autophagy and apoptosis restrict each other.As one of the main regulatory pathways of apoptosis,p38MAPK signaling pathway also has the dual effects of promoting and inhibiting autophagy.It has been proved that gonadotropin inhibitory hormone(gonadotropin-inhibitory hormone,GnIH)has effects on autophagy and apoptosis,but the mechanism of action is not clear.This experiment was conducted to study the effects of GnIH on autophagy and apoptosis of porcine ovarian granulosa cells via p38MAPK signaling pathway and its mechanism.【Method】Oval granulosa cells were extracted from pig ovaries and cultured in vitro.To explore the best time of GnIH on p38MAPK signaling pathway,according to the time gradient of incubation GnIH(0,10,30,60,and 90 min),Western blot was used to detect the protein expression of p38 and p-p38 in pGCs.To verify the effect of GnIH on p38MAPK signaling pathway,the cells were divided into 4 groups(control,GnIH,p38 activating agent(U-46619),and U-46619+GnIH),Western blot was used to detect the protein expression of p38 and p-p38.To investigate the effects of different concentrations of GnIH on autophagy and apoptosis:the cells were divided into 5 groups(control,10^-6 mol·L^-1 GnIH,10^-8 mol·L^-1 GnIH,10^-10 mol·L^-1 GnIH,and 10^-12 mol·L^-1 GnIH),Western blot was used to detect the protein expression of autophagy and apoptosis.To verify the effects of different concentrations of GnIH on autophagy and apoptosis through p38 signaling pathway:the cells were divided into 6 groups(control,U-46619,U-46619+10^-6 mol·L^-1 GnIH,U-46619+10^-8 mol·L^-1 GnIH,U-46619+10^-10 mol·L^-1 GnIH,and U-46619+10^-12 mol·L^-1 GnIH),Western blot was used to detect the protein expression of autophagy and apoptosis.【Result】After incubation with GnIH for 10 min,the protein expression of p38 and p-p38 was significantly decreased(P<0.05).The results suggested that the optimum action time of GnIH on p38MAPK signaling pathway was 10 min;U-46619 significantly promoted the phosphoryl

关 键 词：自噬 凋亡 GNIH P38MAPK 猪 卵巢颗粒细胞 

分 类 号：S828[农业科学—畜牧学]