林蛙油对雷公藤所致大鼠卵泡发育障碍的影响

孙雪缘; 王玉; 何星瑶; 邸琳; 刘新宇; 赵宏宇; 张凤清

doi:10.13386/j.issn1002-0306.2022090023

林蛙油对雷公藤所致大鼠卵泡发育障碍的影响

doi: 10.13386/j.issn1002-0306.2022090023

孙雪缘^{1, 2,},
王玉²,
何星瑶^{1, 2},
邸琳²,
刘新宇²,
赵宏宇^2, ,,
张凤清^1, ,

1.
长春工业大学，化学与生命科学学院，吉林长春 130012
2.
吉林省中医药科学院药效毒理评价中心，吉林长春 130012

基金项目: 长春市科技发展计划项目（21ZY62）；吉林省中医药科技项目（2022073）。

详细信息

作者简介:
孙雪缘（1998−），女，硕士，研究方向：天然产物与功能食品开发，E-mail：sunxueyuanaaa@163.com

通讯作者:
赵宏宇（1987−），男，硕士，助理研究员，研究方向：中药药理、功能食品功能评价研究，E-mail：169080112@qq.com

张凤清（1967−），女，硕士，教授，研究方向：天然产物及功能食品开发，E-mail：1044424827@qq.com

中图分类号: R285.5
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- 文章访问数: 34
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- 被引次数: 0
出版历程
- 收稿日期: 2022-09-05
- 网络出版日期: 2023-05-22
- 刊出日期: 2023-07-01

Effects of Oviductus Ranae on Follicular Developmental Dysfunction by Tripterygium in Rats

Xueyuan SUN^{1, 2
,},
Yu WANG²,
Xingyao HE^{1, 2},
Lin DI²,
Xinyu LIU²,
Hongyu ZHAO^{2
, ,},
Fengqing ZHANG^{1
, ,}

1.
College of Chemistry and Life Sciences, Changchun University of Technology, Changchun 130012, China
2.
Pharmacodynamic and Toxicological Evaluation Center, Jilin Academy of Traditional Chinese Medicine Sciences, Changchun 130012, China

摘要

摘要: 目的：研究林蛙油对卵泡发育障碍模型大鼠卵巢中的卵泡和磷脂酰肌醇受体3-酪氨酸激酶（phosphatidylinositol 3-kinase, PI3K）/卵蛋白激酶受体B（protein kinase B, Akt）的信号通路的影响。方法：筛选后的雌性WISTAR大鼠40只，随机分为对照组、模型组、阳性药组和林蛙油高、低剂量组；除对照组外其余各组大鼠每日灌胃雷公藤多苷溶液（40 mg/kg），阳性药组大鼠灌胃戊酸雌二醇溶液（0.1 mg/kg）和醋酸甲地孕酮溶液（0.8 mg/kg），林蛙油高、低剂量组大鼠分别灌胃林蛙油溶液（400、200 mg/kg）。第8周测定大鼠血清雌二醇（estradiol, E2）、孕酮（progesterone, P）、促黄体生成素（Luteinizing hormone, LH）、促卵泡生成素（Follicle-stimulating hormone, FSH）和睾酮（testosterone, T）含量；摘取卵巢和子宫称量记录，计算器官指数；一侧卵巢做HE染色和TUNEL染色计算各级卵泡数量。另外一侧卵巢测定其中PI3K、AKT、人第10号染色体缺失的磷酸酶（phosphatase and tensin homolog deleted on chromosome ten, PTEN）和哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）的mRNA相对表达量以及PI3K、磷酸化蛋白激酶B（phosphorylated protein kinase B, p-Akt）的蛋白含量。结果：大鼠卵泡发育障碍模型成功建立。与模型组大鼠相比，林蛙油高剂量组大鼠子宫湿重和子宫指数显著升高（P<0.05）；林蛙油高、低剂量组大鼠发情周期显著缩短（P<0.05），次级卵泡数量显著升高（P<0.05），卵泡闭锁率显著降低（P<0.05），血清FSH含量显著减少（P<0.05）、T和E2含量显著升高（P<0.05），PI3K蛋白含量显著增多（P<0.05），Akt蛋白磷酸化水平显著提高（P<0.05），PTEN mRNA相对表达量显著减少（P<0.05）；林蛙油高剂量组大鼠卵巢中mTOR mRNA相对表达量显著低于模型组大鼠（P<0.05）。结论：林蛙油对雷公藤多苷所致大鼠卵泡发育障碍有明显的改善作用，林蛙油可以上调PI3K/Akt信号通路促进卵泡发育障碍的大鼠卵泡的生长和发育。
- 林蛙油 /
- 卵泡发育障碍 /
- 卵巢 /
- 雷公藤多苷 /
- 磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路
Abstract: Objective: To study the effect of oviductus ranae (OR) on follicle development and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in the ovaries of follicular developmental dysfunction rats. Methods: Randomly divide the screened 40 female WISTAR rats into control group (CG), model group (MG), positive qroup (PG), OR high-dose (ORHG) and low-dose group (ORLG). Except the CG rats, the rats in other groups were treated with triptolide solution (40 mg/kg) once a day. The PG were treated with estradiol valerate solution (0.1 mg/kg) and megestrol acetate solution (0.8 mg/kg). The ORHG and ORLG were treated with OR solution (400、200 mg/kg). The rat serum was collected to determine the contents of estradiol (E2), progesterone (P), Luteinizing hormone (LH), Follicle-stimulating hormone (FSH) and testosterone (T) at 8th week . The ovaries and uterus of rats were harvested and weighed, and the organ index was calculated. HE staining and TUNEL staining were performed on one ovary to calculate the number of follicles at all levels. The relative expression of PI3K, AKT, phosphatase and tensin homolog deleted on chromosome ten (PTEN) mammalian target of rapamycin (mTOR) mRNA and the contents of PI3K, Akt, phosphorylated protein kinase B(p-Akt) protein in the other ovary was measured. Result: The results indicated that the follicular development dysfunction model was successfully established. Compared with the MG, the wet weight and index of uterus of the ORHG was increased significantly (P<0.05). The estrous cycle of rats in the ORHG and ORLG was significantly shortened (P<0.05), the number of secondary follicles was significantly increased (P<0.05), the follicular atresia rate was significantly reduced (P<0.05), the serum FSH and LH content was decreased significantly (P<0.05), the serum T and E2 content was increased significantly (P<0.05), PI3K protein content was increased significantly (P<0.05), Akt protein phosphorylation level was increased significantly (P<0.05), the relative expression of PTEN mRNA was decreased significantly (P<0.05). The relative expression of mTOR mRNA in ORHG was significantly lower than that of MG (P<0.05). Conclusion: OR can obviously improve the follicular development dysfunction of rats induced by triptolide, and it can up-regulate PI3K/Akt signal pathway to promote the growth and development of follicles in rats with follicular development dysfunction.
- oviductus ranae(OR) /
- follicular developmental dysfunction /
- ovary /
- triptolide /
- phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway

HTML全文

图 1 各组大鼠卵巢切片HE染色（40×）

Figure 1. HE staining of ovarian sections of rats by group （40×）

注：图中A为初级卵泡，B为次级卵泡，C为黄体，D为闭锁初级卵泡，E为闭锁次级卵泡。

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图 2 各组大鼠卵巢切片TUNEL染色（200×）

Figure 2. TUNEL staining of ovarian sections of rats in each group （200×）

注: a颗粒细胞发育正常的卵泡，b颗粒细胞凋亡即将闭锁的卵泡。

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图 3 大鼠卵巢中PI3K、Akt、 p-Akt蛋白表达电泳图

Figure 3. Electrophoretogram of PI3K, Akt, p-Akt protein expression in rat ovaries

注: A、B对照组；C、D模型组；E、F阳性药组；G、H林蛙油高剂量组；I、J林蛙油低剂量组。

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图 4 大鼠卵巢PI3K、Akt及p-Akt蛋白含量和Akt蛋白磷酸化水平

Figure 4. The contents of PI3K, Akt and p-Akt protein and the phosphorylation level of Akt protein in rat ovary

注：#表示与对照组相比差异显著P<0.05；*表示与模型组比差异显著P<0.05；图5同。

下载: 全尺寸图片幻灯片

图 5 各组大鼠mRNA相对表达水平

Figure 5. mRNA expression levels of rats in each group

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequences

引物	序列（5’-3’）	长度（bp）
PTEN	上游 TGTAAAGCTGGAAAGGGACG 下游 CCTCTGACTGGGAATTGTGAC	20 21
PI3K	上游 GGATGCTGAATGGTACTGGG 下游 TGTAAGAGTGTAATCGCCGTG	20 21
Akt	上游 GCCCTCAAGTACTCATTCCAG 下游 ACACAATCTCCGCACCATAG	21 20
mTOR^*	上游 ATTCAATCCATAGCCCCGTC 下游 TGCATCACTCGTTCATCCTG	20 20
β-actin	上游 ACCTTCTACAATGAGCTGCG 下游 CTGGATGGCTACGTACATGG	20 20

下载: 导出CSV

表 2 各组大鼠器官指数以及器官湿重（$\bar {\rm{x}} $±s, n=8）

Table 2. The organ index and organ wet weight of each group of rats ($\bar {\rm{x}} $±s, n=8)

组别	卵巢湿重（mg）	卵巢指数（mg·g⁻¹）	子宫湿重（mg）	子宫指数（mg·g⁻¹）
对照组	100.00±16.84	0.41±0.08	426.00±41.04	1.72±0.17
模型组	84.00±12.58^#	0.33±0.05^#	400.62±29.45	1.56±0.13^#
阳性药组	95.25±13.71	0.38±0.05^*	423.00±31.22	1.71±0.15^*
林蛙油高剂量	90.75±11.29	0.36±0.04	432.12±28.77^*	1.72±0.11^*
林蛙油低剂量	91.50±8.99	0.37±003	418.75±58.01	1.67±0.17
注：#表示与对照组相比差异显著P<0.05，*表示与模型组比差异显著P<0.05；表3~表5同。

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表 3 各组大鼠发情周期（$\bar {\rm{x}} $±s, n=8）

Table 3. Estrous cycle of rats by group ($\bar {\rm{x}} $±s, n=8)

组别	发情周期（d）
对照组	4.39±0.67
模型组	5.50±1.31^#
阳性药组	4.67±0.69
林蛙油高剂量组	4.00±0.00^*
林蛙油低剂量组	4.25±0.45^*

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表 4 各组大鼠血清激素水平（$\bar {\rm{x}} $±s, n=8）

Table 4. Serum hormone levels in rat groups ($\bar {\rm{x}} $±s, n=8)

	FSH（mIU·mL⁻¹）	LH （mIU·mL⁻¹）	P （ng·mL⁻¹）	T （ng·mL⁻¹）	E2 （pg·mL⁻¹）
对照组	2.92±0.82	4.29±0.87	11.08±3.31	0.050±0.03	7.16±2.47
模型组	4.08±0.71^#	5.13±0.44^#	7.95±1.64^#	0.022±0.01^#	4.38±1.48^#
阳性药组	2.72±0.85^*	3.41±1.10^*	11.71±3.98^*	0.046±0.02^*	7.48±1.98^*
林蛙油高剂量组	2.72±0.46^*	4.37±1.22	8.85±2.62	0.045±0.02^*	6.64±2.40^*
林蛙油低剂量组	2.90±0.55^*	4.65±1.04	9.20±2.57	0.048±0.02^*	6.93±2.34^*

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表 5 各组大鼠各级卵泡数量（$\bar {\rm{x}} $±s, n=8）

Table 5. Number of follicles at all levels in each group of rats ($\bar {\rm{x}} $±s, n=8)

组别	初级卵泡（个）	闭锁初级卵泡（个）	次级卵泡（个）	闭锁次级卵泡（个）	黄体（个）	总闭锁率（%）
对照组	6.25±1.67	7.50±1.93	9.62±3.16	8.00±1.85	3.38±1.06	49.61±2.72
模型组	4.38±1.30	6.25±1.49	5.13±1.81^#	8.25±1.85	1.63±0.52^#	61.08±3.07^#
阳性药组	4.00±1.07^#	5.25±0.71^#	8.50±1.69^*	7.50±1.41	2.38±0.74^*#	50.76±4.73^*
林蛙油高剂量组	3.88±2.47^#	4.75±1.98^#	8.00±2.33^*	7.75±3.20	2.38±1.60	52.51±3.94^*
林蛙油低剂量组	3.88±1.64^#	4.88±2.17^#	7.50±2.33^*	8.00±2.14	2.00±1.31^#	53.13±6.29^*

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