Page 41 - 《中国药房》2021年5期

苍附导痰丸含药血清调控大鼠卵巢颗粒细胞自噬的作用 Δ

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谢蓬蓬 ，刘佳玥，曾 蕾（广州中医药大学第一临床医学院，广州 510405）
中图分类号 R285.5 文献标志码 A 文章编号 1001-0408（2021）05-0547-05
DOI 10.6039/j.issn.1001-0408.2021.05.07

摘 要 目的：研究苍附导痰丸含药血清调控大鼠卵巢颗粒细胞（GCs）自噬的作用。方法：将3月龄SD大鼠分为生理盐水组（生
理盐水，灌胃）、卵巢刺激素（FSH）注射组（10.71 IU/kg，皮下注射）和苍附导痰丸高、中、低剂量灌胃组[0.5、1、2 mg/g（以生药量计），
灌胃]，每组6只，每天皮下注射/灌胃给药1次，连续3天；末次给药后，于腹主动脉采血，获取各组大鼠的含药血清。将大鼠GCs分
为空白对照组、模型组、FSH组（阳性对照）和苍附导痰丸高、中、低剂量组，除空白对照组直接加入生理盐水组大鼠血清100 μL外，
其余各组均先给予丙酸睾丸酮诱导以复制自噬模型，然后模型组加入生理盐水组大鼠血清100 μL，各给药组分别加入对应药物组
含药血清100 μL。采用酶联免疫吸附法（ELISA）检测各组细胞上清液中雌二醇（E2 ）、孕激素（P）的含量，采用Western blot法检测
各组GCs中磷脂酰肌醇3激酶（PI3K）、蛋白激酶B（Akt）、哺乳动物雷帕霉素靶蛋白（mTOR）蛋白的相对表达量；采用实时荧光定
量聚合酶链式反应法检测各组细胞中 PI3K、Akt、mTOR 和自噬相关因子 Beclin 1、LC3Ⅰ、LC3Ⅱ、p62 mRNA 的相对表达量。结
果：与空白对照组比较，模型组细胞上清液中E2含量以及细胞中PI3K、Akt、mTOR mRNA和蛋白的相对表达量均显著降低（P＜
0.01），Beclin 1、LC3Ⅰ、LC3Ⅱ mRNA的相对表达量均显著升高（P＜0.01）。与模型组比较，FSH组和苍附导痰丸中、高剂量组细
胞上清液中E2含量显著升高，细胞中Beclin 1、LC3Ⅰ、LC3Ⅱ、p62 mRNA的相对表达量均显著降低（P＜0.05或P＜0.01）；各给药
组细胞中 PI3K、Akt、mTOR mRNA 和蛋白的相对表达量均显著升高（P＜0.05 或 P＜0.01）。结论：苍附导痰丸可能是通过激活
PI3K/Akt/mTOR通路相关蛋白及其mRNA的表达，从而抑制GCs的自噬。
关键词 苍附导痰丸；卵巢颗粒细胞；自噬；PI3K/Akt/mTOR通路

Effect of Cangfu Daotan Pill Containing Serum on Autophagy of Ovarian Granulosa Cells of Rats
XIE Pengpeng，LIU Jiayue，ZENG Lei（The First Clinical Medical College，Guangzhou University of TCM，
Guangzhou 510405，China）

ABSTRACT OBJECTIVE：To study the effect of Cangfu daotan pill（CDP）containing serum on autophagy of ovarian granulosa
cells（GCs）of rat. METHODS：Three-month-old SD rat were divided into normal saline group（normal saline，ig），FSH injection
group（10.71 IU/kg，ih），CDP irrigation high-dose，medium-dose and low-dse groups [0.5，1，2 mg/g（by crude drug），ig]，with 6
rats in each group. They were given relevant medicine subcutaneously/intragastrically，once a day，for consecutive 3 days. After
last medication，blood sample was collected from the abdominal aorta to obtain drug-containing serum. GCs of rat were divided
into blank control group，model group，FSH group（positive control）and CDP high-dose，medium-dose and low-dose groups. The
autophagy model was induced by giving testosterone propionate，except that the blank control group was directly added with 100
μL serum of normal saline group. Then model group was given 100 μL serum of normal saline group，and administration groups
were given 100 μL drug-containing serum of corresponding drug group. The contents of estradiol（E2 ）and progesterone（P）in
supernatant of cells were determined by ELISA. Western blot assay was used to detect protein expression of PI3K，Akt and mTOR
in cells. The mRNA expression of PI3K，Akt，mTOR，Beclin 1，LC3Ⅰ，LC3Ⅱ and p62 were detected by RT-PCR. RESULTS：
Compared with blank control group，the content of E2 in supernatant，relative mRNA and protein expression of PI3K，Akt and
mTOR were decreased significantly in model group（P＜0.01），while relative mRNA expression of Beclin 1，LC3Ⅰ and LC3Ⅱ
were increased significantly（P＜0.01）. Compared with model group，the content of E2 in supernatant were significantly increased
in FSH group，CDP medium-dose and high-dose groups，while relative mRNA expression of Beclin 1，LC3Ⅰ，LC3Ⅱ and p62
were decreased significantly （P＜0.05 or P＜0.01）；relative mRNA and protein expression of PI3K，Akt and mTOR were
increased significantly in administration groups（P＜0.05 or P＜0.01）. CONCLUSIONS：CDP can inhibit autophagy of GCs by
activating related protein and mRNA expression of PI3K/Akt/
Δ 基金项目：国家自然科学基金资助项目（No.81804135）
＊主治中医师，硕士。研究方向：中医药防治妇科疾病。电话： mTOR signaling pathway.
020-38076311。E-mail：LXPP4239@163.com KEYWORDS Cangfu daotan pill；Ovarian granulosa cells；
# 通信作者：副主任中医师，博士。研究方向：生殖健康与生殖障 Autophagy；PI3K/Akt/mTOR signaling pathway
碍。电话：020-38076311。E-mail：407687448@qq.com

中国药房 2021年第32卷第5期 China Pharmacy 2021 Vol. 32 No. 5 ·547 ·