γ-氨基丁酸对绵羊卵巢颗粒细胞凋亡及类固醇激素分泌的影响

doi:10.13560/j.cnki.biotech.bull.1985.2023-0784

摘要/Abstract

摘要：

【目的】 该文章旨在探究γ-氨基丁酸（GABA）对绵羊卵巢颗粒细胞（GCs）凋亡及类固醇激素分泌的影响。【方法】 卵巢GCs培养液中分别添加0、10^-8、10^-7、10^-6和10^-5 mol/L的GABA处理24 h后，采用CCK-8法和流式细胞术分别检测GCs增殖及凋亡情况，筛选出GABA适宜培养浓度用于后续试验，采用ELISA检测GABA对GCs雌激素（E₂）及孕酮（P₄）分泌的影响，RT-qPCR及Western blot技术检测促GCs增殖（PCNA、CCNB1、CCND1）、促凋亡（Caspase-3、Bax）、抗凋亡（Bcl-2）和类固醇激素合成相关基因（CYP11A1、StAR、3β-HSD、CYP19A1）mRNA及蛋白的表达。【结果】 添加GABA处理绵羊卵巢GCs 24 h，各组均可显著促进GCs增殖（P<0.05），抑制其凋亡（P<0.05）。GABA可显著上调PCNA、CCNB1、CCND1、Bcl-2 mRNA及蛋白表达量（P<0.05），显著下调Bax、Caspase-3 mRNA及蛋白表达量（P<0.05）和Bax/Bcl-2比值（P<0.05）。GABA可显著促进P₄的分泌（P<0.05），显著抑制E₂的分泌（P<0.05）。显著上调StAR、3β-HSD、CYP11A1的mRNA及蛋白表达水平（P<0.05），显著下调CYP19A1的mRNA及蛋白水平（P<0.05）。【结论】 GABA通过调节CCNB1、CCND1、PCNA、Caspase-3、Bcl-2、Bax基因表达促进GCs的增殖，抑制其凋亡；通过调节StAR、3β-HSD、CYP11A1、CYP19A1基因表达促进GCs的P₄分泌，抑制E₂生成。

关键词: γ-氨基丁酸, 绵羊颗粒细胞, 凋亡, 类固醇激素

Abstract:

【Objective】 This study was conducted to investigate the effects of γ-aminobutyric acid（GABA）on the apoptosis and the secretion of steroid hormones in ovine ovarian granulosa cells（GCs）. 【Method】 The GCs were treated with 0, 10^-8, 10^-7, 10^-6 and 10^-5 mol/L GABA for 24 h, respectively. Then, the cell proliferation and apoptosis were detected by CCK-8 and flow cytometry. And the appropriate culture concentration of GABA was screened for subsequent experiments. ELISA was used to detect the secretion levels of estrogen（E₂）and progesterone（P₄）in the culture supernatant after GCs were treated with the appropriate concentration. The qRT-PCR and Western blot were to detect the mRNA and protein expressions of GCs proliferation-promoting genes（PCNA, CCNB1, and CCND1）, anti-apoptosis genes（Bcl-2）, apoptosis-promoting genes（Bax, Caspase-3）and steroid hormone synthesis-related genes（StAR, 3β-HSD, CYP11A1, CYP19A1）.【Result】 The 10^-6 mol/L GABA treatment for 24 h significantly promoted GCs proliferation（P < 0.05）and significantly inhibited its apoptosis（P < 0.05）. The mRNA and protein expressions of PCNA, CCNB1, CCND1 and Bcl-2 in the experimental group were higher than those in the control group（P < 0.05）. Compared with the control group, the mRNA and protein expressions of Bax and Caspase-3 in the experimental group decreased significantly（P < 0.05）, as well as Bax/Bcl-2 decreased significantly（P < 0.05）. Treatment of ovine ovarian GCs with 10^-6 mol/L GABA for 24 h significantly promoted the synthesis of P₄（P < 0.05）and significantly inhibited the synthesis of E₂（P < 0.05）. Compared with the control group, the mRNA and protein expressions of StAR, 3β-HSD and CYP11A1 in the experimental group increased significantly（P < 0.05）and the mRNA and protein expressions of CYP19A1 decreased significantly（P < 0.05）. 【Conclusion】 The results demonstrate that GABA treatment could promote GCs proliferation as well as inhibit GCs apoptosis by regulating the expression of CCNB1, CCND1, PCNA, Caspase-3, Bax and Bcl-2, and promote the synthesis of P₄ as well as inhibit the synthesis of E₂ by regulating the expressions of StAR, 3β-HSD, CYP11A1 and CYP19A1.

Key words: γ-aminobutyric acid, ovine granulosa cells, apoptosis, steroid hormone

单新雨, 李太春, 杨若晨, 段香茹, 康佳, 张英杰, 刘月琴. γ-氨基丁酸对绵羊卵巢颗粒细胞凋亡及类固醇激素分泌的影响[J]. 生物技术通报, 2024, 40(3): 312-321.

SHAN Xin-yu, LI Tai-chun, YANG Ruo-chen, DUAN Xiang-ru, KANG Jia, ZHANG Ying-jie, LIU Yue-qin. Effect of γ-aminobutyric Acid on Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells[J]. Biotechnology Bulletin, 2024, 40(3): 312-321.

图/表 10

表1 引物序列

Table 1 Primer sequence

基因Gene	引物序列Primer sequence（5'-3'）	退火温度Annealing temperature/℃	产物长度Size/bp
GAPDH	F: GGTCGGAGTGAACGGATTTG	60	222
GAPDH	R: CTTGACTGTGCCGTGGAACTT	60	222
CCND1	F：GGCGTTCGTAATCGTGTTTTG	60	185
CCND1	R：CAGGTAGAAGGAGTGAGGAATGTGT	60	185
PCNA	F：GCCGAGGAGAACAAGCAGA	60	123
PCNA	R：GAGGGTGGGTTGGAAATGAA	60	123
CCNB1	F：GGCAGGAGGACTATGACATT	60	222
CCNB1	R：TCAGGCACCCACTTCTCTTTC	60	222
Bax	F：CGAGACCTGAAGCCAGAGAC	60	183
Bax	R：GCAAAGATACAGCCAACACC	60	183
Caspase-3	F：CCCATCCTCACCATCATCAC	60	275
Caspase-3	R：GCACAAACACGCACCTCAA	60	275
Bcl-2	F：ACTTGGACCTGTCGCTGTCC	60	120
Bcl-2	R：CCTGCGTTTGGAGTGGTAGA	60	120
CYP11A1	F：AACATGGAGCTGCAGAGGAT	60	120
CYP11A1	R：CCAATGTCCAGCCCATGATG	60	120
StAR	F：TCTTTGAGTTCGGAGGGGTC	60	270
StAR	R：GGAGAAATCAAACAGGGGCC	60	270
3β-HSD	F：TGGACCCGTCGATCTGAAAA	60	283
3β-HSD	R：GCGTACAAGAAGTCTGCCTC	60	283
CYP19A1	F：AATGCTGCCCCAGGTGTT	60	379
CYP19A1	R：CGGCGTCTCTGCGATTTT	60	379

表2 一抗信息

Table 2 Primary antibody information

一抗名称Name of primary antibody	品牌及货号Brand and coale	稀释度Proportion of dilution	分子量Molecular weight/kD
Caspase-3	Abcam ab13847	1∶1 000	34
StAR	Abcam ab133657	1∶1 000	32
3β-HSD	Abcam ab55268	1∶1 000	42
Bax	Abcam ab32503	1∶1 000	21
Bcl-2	Abcam ab182858	1∶1 500	26
CCNB1	Abcam ab181593	1∶1 500	55
CCND1	Abcam ab16663	1∶200	33
CYP11A1	Abcam ab272494	1∶1 000	60
CYP19A1	Abcam ab18995	1∶800	55
PCNA	Abcam ab29	1∶800	30
β-actin（C4）（内参）	Santa Cruz SC-47778	1∶1 500	43

图1 绵羊卵巢颗粒细胞鉴定（400×） A：FSHR染色绵羊卵巢颗粒细胞；B：DAPI染色绵羊卵巢颗粒细胞细胞核；C：叠加染色效果

Fig. 1 Identification of ovine ovarian granulosa cells（400×） A: FSHR staining of ovine ovarian granulosa cells. B: DAPI staining of ovine ovarian granulosa cell nucleus. C: Superimposed dyeing effect

图2 不同浓度GABA对绵羊颗粒细胞增殖的影响柱状图标注不同字母表示显著差异（P<0.05），下同

Fig. 2 Effects of different concentrations of GABA on proliferation in ovine granulosa cells The different letters indicate the significant difference（P<0.05）, the same below

图3 GABA处理绵羊颗粒细胞凋亡结果图 A、B、C、D、E分别为 0、10-8、10-7、10-6、10-5 mol/L GABA对绵羊颗粒细胞凋亡的影响； Q1为机械损伤致死细胞，Q2为晚期凋亡，Q3为早期凋亡，Q4为活细胞

Fig. 3 Results of apoptosis in ovine granulosa cells treated with GABA A、B、C、D、E is the effects of 0, 10-8, 10-7, 10-6, 10-5 mol/L GABA on the apoptosis of ovine granulosa cells. Q1 was dead cells caused by mechanical injury; Q2 was late apoptosis; Q3 was early apoptosis; Q4 was living cells

表3 不同浓度GABA对绵羊颗粒细胞凋亡率的影响

Table 3 Effect of different concentrations of GABA on apoptosis rate in ovine granulosa cells

项目Item	正常细胞率Normal cell rate/%	早期凋亡率Early apoptosis rate/%	晚期凋亡率Late apoptosis rate/%	总凋亡率Total apoptosis rate/%
Control	81.97±0.61^a	1.58±0.22^ab	13.43±0.15^a	15.02±0.26^a
10^-8	84.97±0.49^b	1.42±0.27^a	11.43±0.38^b	12.85±0.56^b
10^-7	87.63±0.24^c	1.63±0.21^ab	9.16±0.52^c	10.78±0.31^c
10^-6	89.27±0.03^d	1.21±0.09^a	7.76±0.24^d	8.97±0.16^d
10^-5	86.80±0.58^c	2.12±0.16^b	9.70±0.17^c	11.82±0.04^e

图4 GABA对绵羊颗粒细胞增殖相关基因及蛋白表达的影响 A：GABA对增殖相关基因mRNA相对表达量的影响结果统计；B：Western blot结果图；C：GABA对增殖相关基因蛋白表达量的影响结果统计

Fig. 4 Effects of GABA on the expressions of proliferation-related genes mRNA and proteins in ovine granulosa cells A: Statistics of mRNA expression results of proliferation-related genes after GABA treatment. B: Western blot results. C: Statistics of protein expression results of proliferation-related genes after GABA treatment

图5 GABA对绵羊颗粒细胞凋亡相关基因及蛋白表达的影响 A：GABA对凋亡相关基因mRNA相对表达量的影响结果统计；B：GABA对Bax/Bcl-2比值的影响；C：Western blot结果图；D：GABA对凋亡相关基因蛋白表达量的影响结果统计

Fig. 5 Effects of GABA on the expressions of apoptosis-related genes mRNA and proteins in ovine granulosa cells A: Statistics of mRNA expression results of apoptosis-related genes after GABA treatment. B: Effect of GABA on ratio of mRNA expression of Bax/Bcl-2. C: Western blot results. D: Statistics of protein expression results of apoptosis-related genes after GABA treatment

表4 10-6 mol/L GABA对绵羊GCs类固醇激素分泌的影响

Table 4 Effects of 10-6 mol/L GABA on steroid hormone secretion of GCs in ovine granulosa cells

项目 Item	对照组 Control group	试验组 Test group
雌二醇（E₂）/（pg·mL^-1）	77.25±1.79^a	61.30±0.74^b
孕酮（P₄）/（pmol·L^-1）	344.45±7.03^a	400.66±14.78^b

图6 GABA对绵羊GCs类固醇合成相关基因和蛋白表达的影响 A：GABA对类固醇激素合成相关基因mRNA相对表达量的影响结果统计；B：Western blot结果图；C：GABA对类固醇激素合成相关基因蛋白表达量的影响结果统计

Fig. 6 Effects of GABA on the expressions of steroid hormone synthesis-related genes and proteins in ovine granulosa cells A: Statistics of mRNA expression results of steroid hormone synthesis-related genes after GABA treatment. B: Western blot results. C: Statistics of protein expression results of steroid hormone synthesis-related genes after GABA treatment

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