Makoolati Zohreh, Bahrami Hasan, Zamanzadeh Zahra, Mahaldashtian Maryam, Moulazadeh Alireza, Ebrahimi Lida, Naghdi Majid
Department of Anatomical Sciences, Faculty of Medicine, Fasa University of Medical Sciences, Fasa, Iran.
Department of Genetic, Basic Sciences Faculty, Nour Danesh Institute of higher Education, Isfahan, Iran.
Anim Reprod. 2022 Aug 19;19(2):e20220036. doi: 10.1590/1984-3143-AR2022-0036. eCollection 2022.
Infertility is one of the most prevalent health disorders in reproductive-age males and females. (Fc), an herbal plant, has been used traditionally for the treatment of different diseases such as infertility especially in Iranian folk medicine. This study examined the effects of Fc leaf extract on the proliferation of mice spermatogonial stem cells (SSCs). Phenolic, flavonoid content, major polyphenolic compounds and antioxidant activity of the extract was evaluated respectively by Folin-Ciocateu, aluminum chloride, HPLC and the FRAP and DPPH methods. Testicular cells of neonate mice were extracted and their identity was confirmed using cytokeratin for Sertoli and Oct-4, CDHI and PLZF for SSCs. Effects of Fc (0.0875, 0.175, 0.35, 0.71 and 1.42 mg/ml) was evaluated at third, 7th, 9th and 14th days of culture by colony assay. The expression of the Mvh, GFRα1 and Oct-4 genes and the viability and proliferation of cultured cells was assessed at the end of the culture period. The extract has a rich phenolic and flavonoid content such as Rutin, Psoralen, Bergapten and Caffeoylmalic acid using HPLC analysis. It also had a potent reducing and radical scavenging activity. Morphology of colonies was similar in all groups. Higher viability, proliferation, colony number and diameter of SSCs was seen in the presence of Fc leaf extract in a dose-dependent manner so that higher number and diameter of colonies were observed in two higher doses of 0.71 and 1.42 mg/ml, separately for each time point relative to other groups. The Mvh, Oct-4 and GFRα1 genes expression had no significant differences between groups. It seems that Fc leaf extract not only had no any cytotoxic effects on the viability and proliferation of SSCs but also support their stemness state. So, this culture system can be employed for enrichment of germ stem cells for use in clinical applications.
不孕不育是育龄男性和女性中最普遍的健康问题之一。紫花苜蓿（Fc）是一种草本植物，在传统医学中，尤其是在伊朗民间医学中，一直被用于治疗包括不孕不育在内的各种疾病。本研究检测了Fc叶提取物对小鼠精原干细胞（SSCs）增殖的影响。分别采用福林-酚试剂法、氯化铝法、高效液相色谱法以及铁离子还原抗氧化能力（FRAP）法和二苯基苦味酰基自由基（DPPH）法评估提取物中的酚类、黄酮类含量、主要多酚化合物和抗氧化活性。提取新生小鼠的睾丸细胞，并用细胞角蛋白鉴定支持细胞，用八聚体结合转录因子4（Oct-4）、细胞分化相关蛋白1（CDHI）和锌指蛋白8（PLZF）鉴定精原干细胞。在培养的第3、7、9和14天，通过集落试验评估Fc（0.0875、0.175、0.35、0.71和1.42 mg/ml）的作用。在培养期结束时，评估小鼠Vasa同源物（Mvh）、胶质细胞源性神经营养因子受体α1（GFRα1）和Oct-4基因的表达以及培养细胞的活力和增殖情况。通过高效液相色谱分析发现，该提取物含有丰富的酚类和黄酮类物质，如芦丁、补骨脂素、佛手柑内酯和咖啡酰苹果酸。它还具有较强的还原和自由基清除活性。所有组的集落形态相似。在Fc叶提取物存在的情况下，精原干细胞的活力、增殖、集落数量和直径均呈剂量依赖性增加，因此，在每个时间点，相对于其他组，在0.71和1.42 mg/ml这两个较高剂量下观察到的集落数量和直径更高。各组之间Mvh、Oct-4和GFRα1基因表达无显著差异。Fc叶提取物似乎不仅对精原干细胞的活力和增殖没有任何细胞毒性作用，而且还能维持其干细胞状态。因此，这种培养系统可用于富集生殖干细胞，以供临床应用。
