肝豆补肾汤通过激活ERK信号通路减少Wilson病TX小鼠异常精子生成并促进生精细胞增殖

doi:10.12122/j.issn.1673-4254.2024.11.02

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (11): 2063-2073.doi: 10.12122/j.issn.1673-4254.2024.11.02

• • 上一篇

肝豆补肾汤通过激活ERK信号通路减少Wilson病TX小鼠异常精子生成并促进生精细胞增殖

银苗朱¹(), 陈奎玉¹, 吴丽敏²^,³, 江鹏宇¹, 籍志慧¹, 张念¹, 周欢¹, 韩辉¹()

^1.安徽中医药大学第一附属医院脑病中心，安徽合肥 230031
^2.中国科学技术大学生命科学与医学部，安徽合肥 230001
^3.中国科学技术大学附属第一医院生殖与遗传分院，安徽合肥 230001

收稿日期:2024-05-06 出版日期:2024-11-20 发布日期:2024-11-29
通讯作者: 韩辉 E-mail:1492657304@qq.com;hanhuidoctor2022@163.com
作者简介:银苗朱，在读硕士研究生，E-mail: 1492657304@qq.com
基金资助:
国家自然科学基金(82374212);安徽省科技厅临床专项转化项目(202204295107020044);安徽省教育厅重点项目(2022AH050523)

Gandou Bushen Decoction improves spermatogenesis and promotes spermatogenic cell proliferation in Wilson disease TX mice by activating testicular ERK signaling pathway

Miaozhu YIN¹(), Kuiyu CHEN¹, Limin WU²^,³, Pengyu JIANG¹, Zhihui JI¹, Nian ZHANG¹, Huan ZHOU¹, Hui HAN¹()

^1.Brain Disease Center, First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230031, China
^2.Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230001, China
^3.Reproductive and Genetic Branch, First Affiliated Hospital of University of Science and Technology of China, Hefei 230001, China

Received:2024-05-06 Online:2024-11-20 Published:2024-11-29
Contact: Hui HAN E-mail:1492657304@qq.com;hanhuidoctor2022@163.com
Supported by:
National Natural Science Foundation of China(82374212)

摘要/Abstract

摘要：

目的探究肝豆补肾汤（GDBSD）影响Wilson病（WD）模型TX雄性小鼠生殖障碍的机制。方法以15只正常雄性DL小鼠为对照组，将60只纯合雄性TX小鼠随机分为4组（n=15）：WD模型组、青霉胺组、GDBSD组和GDBSD+U0126组（U0126为一种特异性MAPK/ERK激酶抑制剂），其中青霉胺组给予0.09 g/kg青霉胺药液灌胃；GDBSD组给予GDBSD药液灌胃，0.2 mL/（10 g·d）；GDBSD+U0126组给予同样GDBSD灌胃另外每日下午同一时间腹腔注射U0126（20 mg/kg）。干预4周后检测各组睾丸组织铜含量；利用HE染色法观察小鼠睾丸与附睾的深层次病理结构及精子构造，通过电子显微镜技术探究睾丸中生精细胞与精子的超微结构特点；TUNEL法标记睾丸组织凋亡细胞；免疫印迹检测B淋巴细胞瘤-2基因（Bcl-2）、细胞色素C（Cytc）、半胱天冬蛋白酶-3（Caspase-3）、细胞外信号调节激酶（ERK）和磷酸化激活的细胞外信号调节激酶（p-ERK）蛋白表达；免疫组化标记Brdu阳性细胞；精液质量检测精子密度、活力及畸形率。通过雌鼠的妊娠率和产仔数评估雄性TX小鼠的生育力水平。结果与WD模型组相比，青霉胺组、GDBSD组改善了小鼠的睾丸的组织和细胞病理学变化以及精子密度、活力程度和畸形率和生育力水平，并增加了睾丸JOHNSEN评分（P<0.05）；Cytc、Caspase-3蛋白表达水平下调，Bcl-2表达上调（P<0.05），GDBSD+U0126组逆转了GDBSD治疗后Bcl-2表达的上调（P<0.05）；ERK1/2信号通路研究结果显示，5组之间ERK1/2总蛋白表达水平差异无统计学意义（P>0.05）。与对照组相比，WD模型组p-ERK蛋白表达含量明显减少；与WD模型组相比，青霉胺组、GDBSD组p-ERK蛋白表达含量增加；GDBSD+U0126组p-ERK蛋白表达含量低于GDBSD组（P<0.05）；在施用U0126后，生精细胞增殖减少，生精细胞凋亡数量增加（P<0.05）。结论 GDBSD可以改善Wilson病模型TX小鼠异常精子发生和生育力下降，其机制可能通过激活ERK信号通路从而改善生精细胞的增殖和凋亡状况。

关键词: Wilson病, 肝豆补肾汤, 铜沉积, ERK信号通路, 精子发生

Abstract:

Objective To investigate the therapeutic mechanism of Gandou Bushen Decoction (GDBSD) for improving reproductive disorders in male mouse models of Wilson disease (WD). Methods Sixty male homozygous TX mice were randomized equally into 4 groups and treated with daily gavage of saline (WD model group), penicillamine (0.09 g/kg), or GDBSD (0.2 mL/10 g), or with intraperitoneal injection of U0126 (20 mg/kg) in addition to GDBSD gavage, with 15 male DL mice as control. After 4 weeks of treatment, copper content in testicular tissue of the mice was detected, and histopathology of the testes and epididymis was examined using HE staining and electron microscopy. TUNEL staining was used to identify apoptotic cells in the testes. The protein expressions of Bcl-2, Cytc, caspase-3, ERK, and p-ERK in the testicular tissue were evaluated with Western blotting, and BrdU-positive cells were detected with immunohistochemical labeling. Sperm density, viability, malformation rate and fertility levels of male mice were studied. Results Treatment with penicillamine and GDBSD obviously improved pathological changes of the testis, increased sperm density and motility, lowered sperm abnormality rate,fertility levels and increased testicular JOHNSEN score of TK mice, but the therapeutic effect of GDBSD was blocked by U0126. GDBSD treatment significantly lowered Cytc and caspase-3 expressions and increased Bcl-2 expression in the testicular tissue of TX mice (P<0.05), while U0126 treatment significantly lowered testicular Bcl-2 expression level. No significant differences were found in total protein expression levels of ERK1/2 among the 5 groups, but p-ERK protein expression was significantly reduced in WD and U0126 groups and increased in penicillamine and GDBSD groups. Conclusion GDBSD can improve spermatogenesis and enhance fertility of male TX mice with WD possibly by activating the ERK signaling pathway to enhance proliferation and reduce apoptosis of the spermatogenic cells.

Key words: Wilson disease, Gandou Bushen Decoction, copper deposition, ERK signaling pathway, spermatogenesis

银苗朱, 陈奎玉, 吴丽敏, 江鹏宇, 籍志慧, 张念, 周欢, 韩辉. 肝豆补肾汤通过激活ERK信号通路减少Wilson病TX小鼠异常精子生成并促进生精细胞增殖[J]. 南方医科大学学报, 2024, 44(11): 2063-2073.

Miaozhu YIN, Kuiyu CHEN, Limin WU, Pengyu JIANG, Zhihui JI, Nian ZHANG, Huan ZHOU, Hui HAN. Gandou Bushen Decoction improves spermatogenesis and promotes spermatogenic cell proliferation in Wilson disease TX mice by activating testicular ERK signaling pathway[J]. Journal of Southern Medical University, 2024, 44(11): 2063-2073.

图/表 12

表1 小鼠睾丸组织铜含量的比较

Tab.1 Comparison of testicular copper levels among the 5 groups (μg/g, Mean±SD, n=5)

Group	Testicular copper level (μg/g)
Control	2.772±0.306
WD	9.355±0.425*
Penicillamine	4.129±0.550^#
GDBSD	4.952±0.264 *^#
GDBSD+U0126	5.597±0.678^#

图1 小鼠睾丸组织中ERK、p-ERK蛋白表达

Fig.1 Western blotting for detecting expressions of ERK signaling pathway proteins in mouse testicular tissue. A: ERK. B: p-ERK (Mean±SD, n=5). C: Original protein bands. *P<0.05 vs Control group; #P<0.05 vs WD group; &P<0.05 vs GDBSD group.

图2 小鼠睾丸组织HE染色结果

Fig.2 HE staining of testicular tissues of the mice. A: Control group. B: WD group. C: Penicillamine group. D: GDBSD group. E: GDBSD+U0126 group. F: Johnsen scores of the germinal tubules in the testicular tissue. *P<0.05 vs control group; #P<0.05 vs WD group; &P<0.05 vs GDBSD group.

图3 小鼠附睾组织HE染色结果

Fig.3 HE staining of mouse epididymal tissues. A: Control group. B: WD group. C: Penicillamine group. D: GDBSD group. E: GDBSD+U0126 group.

表2 小鼠精子质量比较

Tab.2 Comparison of sperm quality of the mice (Mean±SD, n=5)

Group	Sperm density (10⁷/mL)	Sperm survival rate (%)	Sperm malformation rate (%)
Control	4.73±0.17	71.51±1.65	14.97±2.35
WD	2.20±0.22*	20.59±1.81*	79.62±4.44*
Penicillamine	3.50±0.16*^#	61.72±1.12*^#	26.96±3.60*^#
GDBSD	3.43±0.50*^#	66.53±1.72*^#	24.84±1.67*^#
GDBSD+U0126	2.67±0.12^&	52.50±2.25 ^&	77.74±3.39^&

图4 小鼠附睾内精液的HE染色结果

Fig.4 Representative images of HE staining of mouse spermatozoa. A: Control group. B: WD group. C: penicillamine group. D: GDBSD group. E: GDBSD+U0126 group. Red arrows: Sperms with tail curl; Orange arrows: Sperms with a head without a tail; Green arrows: Sperms with head deformity.

图5 小鼠附睾内精子的超微结构

Fig. 5 Ultrastructural changes of mouse spermatozoa in each group observed with transmission electron microscope (scale bar=500 nm). A: Control group. B: WD group. C: Penicillamine group. D: GDBSD group. E: GDBSD+U0126 group.

图6 各组小鼠睾丸组织中生精细胞的超微结构

Fig.6 Ultrastructure of mouse spermatocytes in each group observed with transmission electron microscope. A, B: Control group. C, D: WD group. E, F: Penicillamine group. G, H: GDBSD group. I, J: GDBSD+U0126 group. Black arrows indicate mitochondrial structures.

图7 小鼠睾丸组织的 TUNEL 染色结果

Fig. 7 TUNEL staining of mouse testicular tissue. A-C: Control group. D-F: WD group. G-I: Penicillamine group. J-L: GDBSD group. M-O: GDBSD+U0126 group. P: Apoptosis rate of testicular tissue in each group. Apoptotic cells are stained green and nuclei are stained blue (Mean±SD,n=5). *P<0.05 vs Control group; #P<0.05 vs WD group; &P<0.05 vs GDBSD group.

图8 小鼠睾丸组织中Caspase-3、Cytc、Bcl-2的蛋白表达

Fig. 8 Western blotting for detecting expressions of apoptosis-related proteins in mouse testis. A: Caspase-3. B: Cytc. C: Bcl-2. D: Original protein bands. *P<0.05 vs Control group, #P<0.05 vs WD group, &P<0.05 vs GDBSD group.

图9 小鼠睾丸组织中免疫组化标记的BrdU

Fig.9 Immunohistochemically labeled BrdU-positive cells in mouse testicular tissue. A: Control group. B: WD group. C: Penicillamine group. D: GDBSD group. E: GDBSD+U0126 group. F: Percentages of BrdU-positive cells in each group. *P<0.05 vs Control group; #P<0.05 vs WD group; &P<0.05 vs GDBSD group.

表3 5组雄性小鼠生育能力比较

Tab.3 Comparison of fertility of male mice among the 5 groups (Mean±SD, n=5)

Group	Fertility rate (%)	Pups per litter
Control	80.00	10.11±1.73
WD	37.50	4.00±0.82*
Penicillamine	62.50	7.40±1.04^#
GDBSD	66.67	8.14±1.55^#
GDBSD+U0126	42.86	5.00±0.81^&

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肝豆补肾汤通过激活ERK信号通路减少Wilson病TX小鼠异常精子生成并促进生精细胞增殖

Gandou Bushen Decoction improves spermatogenesis and promotes spermatogenic cell proliferation in Wilson disease TX mice by activating testicular ERK signaling pathway

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