摘要
目的通过网络药理学和体内外实验研究,探讨铁死亡在龟鹿二仙胶治疗少弱精子症中的作用。方法通过TCMSP数据库、HERB数据库、PubChem数据库和Swiss数据库获取龟鹿二仙胶的活性成分和作用靶点,Genecard数据库和FerrDb数据库检索铁死亡基因,GEO数据库获取少弱精子症差异基因,三者取交集构建蛋白互作网络,进行基因本体(gene ontology,GO)功能和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。使用H2O2刺激小鼠睾丸支持细胞构建氧化应激损伤模型,并用龟鹿二仙胶含药血清干预损伤模型细胞,观察细胞形态,检测细胞活性氧(reactive oxygen species,ROS)、乳酸脱氢酶(lactate dehydrogenase,LDH)、总铁和亚铁离子含量,透射电镜观察细胞超微结构,qRT-PCR和Western blotting检测细胞谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)和酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long chain family member 4,ACSL4)的mRNA和蛋白表达。使用环磷酰胺构建小鼠少弱精子症模型,ELISA检测小鼠性激素水平,苏木素-伊红(HE)染色法观察小鼠睾丸组织病理学改变,免疫组化检测小鼠睾丸组织GPX4和ACSL4蛋白表达。结果共获取龟鹿二仙胶81个活性成分和190个作用靶点,686个铁死亡基因和4777个少弱精子症差异基因,龟鹿二仙胶干预少弱精子症铁死亡靶点共11个,GO富集分析共检测到869个条目,KEGG共有35条。体外实验显示,龟鹿二仙胶含药血清能显著降低细胞内ROS、LDH、总铁和亚铁离子含量(P<0.05、0.01),修复损伤线粒体,显著降低ACSL4的mRNA和蛋白表达(P<0.01),显著促进GPX4的mRNA和蛋白表达(P<0.01)。体内实验发现,龟鹿二仙胶能改善少弱精子症小鼠血清中性激素水平和睾丸组织病理损伤,显著降低小鼠睾丸组织中ACSL4蛋白表达(P<0.01),显著促进睾丸组织中GPX4蛋白表达(P<0.01)。结论龟鹿二仙胶能有效治疗少弱精子症,拮抗铁死亡的发生可能是龟鹿二仙胶治疗少弱精子症潜在的作用机制。
Objective To investigate the role of ferroptosis in the treatment of oligospermia with Guilu Erxian Gel(龟鹿二仙胶,GLEXG)through network pharmacology and in vivo,in vitro experiments.Methods The active ingredients and targets of GLEXG were obtained from TCMSP,HERB,PubChem and Swiss databases,ferroptosis-related genes were retrieved from Genecard and FerrDb databases.The differentially expressed genes in oligospermia were obtained from GEO database,and protein interaction network was constructed by taking the intersection of the three datasets.Gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analyses were performed.In vitro,an oxidative stress injury model was constructed in H2O2-induced mouse testicular sertoli cells,and cells were treated with GLEJ-containing serum.Cell morphology,reactive oxygen species(ROS),lactate dehydrogenase(LDH),total iron and ferrous ion were measured.The ultrastructure of cells was observed by transmission electron microscopy.The mRNA and protein expressions of glutathione peroxidase 4(GPX4)and acyl-CoA synthetase long chain family member 4(ACSL4)were measured by qRT-PCR and Western blotting.In vivo,a mouse model of oligospermia was constructed using cyclophosphamide.The levels of sex hormones were measured by ELISA,and pathological changes in testicular tissue were observed by hematoxylin-eosin(HE)staining.The protein expressions of GPX4 and ACSL4 in testicular tissue were detected by immunohistochemistry.Results A total of 81 active ingredients and 190 targets of GLEXG were obtained,along with 686 ferroptosis-related genes and 4777 differentially expressed genes in oligospermia.A total of 11 shared targets were identified for GLEXG and ferroptosis in oligospermia by network pharmacology,and a total of 869 items and 35 KEGG pathways were enriched.In vitro experiments showed that GLEXG-containing serum significantly reduced ROS,LDH,total iron,and ferrous ion levels(P<0.05,0.01),repaired damaged mitochondria,and significantly decreased the mRNA and protein expressions of ACSL4(P<0.01),significantly promoted the mRNA and protein expressions of GPX4(P<0.01).In vivo experiments showed that GLEXG improved sex hormone levels in serum and pathological damage of testicular tissue,significantly decreased the expression of ACSL4 protein in testicular tissue(P<0.01),and significantly promoted the expression of GPX4 protein in testicular tissue(P<0.01).Conclusion GLEXG can effectively treat oligospermia,and antagonising the occurrence of iron death may be a potential mechanism of action of GLEXG in treatment of oligospermia.
作者
李玲
黄家望
冯芷莹
刘卓琳
王康宇
王磊
何清湖
LI Ling;HUANG Jia-wang;FENG Zhi-ying;LIU Zhuo-lin;WANG Kang-yu;WANG Lei;HE Qing-hu(School of Traditional Chinese Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;School of Integrated Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;Hunan Hengyang Dongjian Pharmaceutical Co.,Ltd.,Hengyang 421411,China;Department of Rehabilitation and Healthcare,Hunan University of Medicine,Huaihua 418000,China;Hunan Engineering Research Center of Internet-Chinese and Western Medicine Collaboration-Health Service,Huaihua 418000,China)
出处
《中草药》
CAS
CSCD
北大核心
2023年第20期6722-6733,共12页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(81973863)
国家级大学生创新创业训练计划项目(S202010541105,S202210541108)。
关键词
龟鹿二仙胶
少弱精子症
铁死亡
网络药理学
实验验证
Guilu Erxian Gel
oligospermia
ferroptosis
network pharmacology
experimental verification
