First Clinical Medical College, Beijing University of Chinese Medicine, Beijing, 100029, China; Andrology Department, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.
Andrology Department, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.
J Ethnopharmacol. 2021 Apr 24;270:113790. doi: 10.1016/j.jep.2021.113790. Epub 2021 Jan 15.
Cuscuta chinensis Lam. and Lycium barbarum L. (SC-FL) is a commonly used kidney tonic Chinese medicine combination that is widely used in the clinical treatment of oligoasthenospermia.However, its specific mechanism remains unclear and requires in-depth study.
To explore the potential targets of SC-FL in the treatment of oligoasthenospermia using network pharmacology, and to verify the results with in vivo and in vitro experiments.
A herb-compound-target-disease network and PPI network were constructed with Cytoscape software. The targets of SC-FL for the treatment of male sterility were introduced into a bioinformatics annotation database, and the GO and KEGG databases were used for pathway enrichment analysis. Subsequently, Tripterygium wilfordii Hook. f. (GTW) polyglycoside was used to induce a spermatogenic dysfunction model in GC-1 spg cells and SD male rats in in vitro and in vivo experiments, respectively. The SC-FL and PI3K pathway inhibitor LY294002 was used to intervene in the spermatogenic dysfunction model to detect the expression of proteins and mRNA related to the PI3K pathway and to detect the indicators related to proliferation and apoptosis.
In in vitro experiments, the percentage of spermatogenic cells and the proportion of GC-1 spg cells at G0/G1 and G2/M stages in the model group (GTW group) and the inhibitor group (LY group) were significantly decreased (P < 0.01) compared with the blank control group (NC group). The apoptosis rate of the GTW group was significantly increased (P < 0.01). The ultrastructures of GC-1 spg cells in the GTW group and LY group were obviously destroyed. Compared with the GTW group, the SC-FL group had a significantly reduced apoptosis rate of GC-1 spg cells, reduced percentage of cells in S phase, and a significantly improved mitochondrial membrane potential. SC-FL can repair the ultrastructure of GC-1 spg cells damaged by GTW. The above effects of SC-FL are closely related to up-regulation of GFRa1, RET, PI3K, p-AKT, and Bcl-2 and down-regulation of BAD and BAX proteins and mRNA expression. In vivo, compared with the GTW group, the body mass, testicular mass, and epididymal weight of the GTW + SC-FL group were significantly increased (P < 0.01). Sperm concentrations and the PR + NP of GTW + SC-FL were significantly higher than in the GTW group (P < 0.01 or P < 0.05). FSH, LH, and T levels in the GTW + SC-FL and LY + SC-FL groups were significantly higher than those in the GTW and LY group (P < 0.01 or P < 0.05). HE staining results showed that the morphology of testicular tissue in the GTW + SC-FL and LY + SC-FL groups was superior to that in the GTW and LY group. The above effects of SC-FL are closely related to the up-regulation of proteins and mRNA expression of PI3K, p-AKT, and Bcl-2.
Through the PI3K/Akt signaling pathway, SC-FL up-regulates GFRa1, RET, PI3K, p-AKT, and Bcl-2, and down-regulates the expression of BAD and BAX proteins and mRNA, thus reducing the percentage of GC-1 spg cells in S-phase, significantly increasing the mitochondrial membrane potential, significantly reducing cell apoptosis, and improving sperm counts and viability.
草药组合 SC-FL（菟丝子和枸杞）治疗少精子症的作用机制：基于网络药理学的研究与验证
基于网络药理学探讨 SC-FL 治疗少精子症的潜在靶点，并通过体内和体外实验进行验证。
采用 Cytoscape 软件构建草药-化合物-靶标-疾病网络和 PPI 网络，将 SC-FL 治疗男性不育的靶标导入生物信息学注释数据库，利用 GO 和 KEGG 数据库进行通路富集分析。随后，采用雷公藤多苷（GTW）诱导 GC-1 spg 细胞和 SD 雄性大鼠建立生精功能障碍模型，分别在体内和体外实验中，采用 SC-FL 和 PI3K 通路抑制剂 LY294002 干预生精功能障碍模型，检测与 PI3K 通路相关的蛋白和 mRNA 表达情况，并检测与增殖和凋亡相关的指标。
在体外实验中，与空白对照组（NC 组）相比，模型组（GTW 组）和抑制剂组（LY 组）的生精细胞比例和 GC-1 spg 细胞 G0/G1 和 G2/M 期比例明显降低（P<0.01），GTW 组的细胞凋亡率明显升高（P<0.01）。GTW 组和 LY 组的 GC-1 spg 细胞超微结构明显受损。与 GTW 组相比，SC-FL 组 GC-1 spg 细胞的凋亡率明显降低，S 期细胞比例明显降低，线粒体膜电位明显升高，SC-FL 可修复 GTW 损伤的 GC-1 spg 细胞超微结构。SC-FL 的上述作用与 GFRa1、RET、PI3K、p-AKT 和 Bcl-2 蛋白和 mRNA 表达上调以及 BAD 和 BAX 蛋白和 mRNA 表达下调密切相关。在体内实验中，与 GTW 组相比，GTW+SC-FL 组的体重、睾丸质量和附睾质量明显增加（P<0.01）。GTW+SC-FL 组的精子浓度和 PR+NP 明显高于 GTW 组（P<0.01 或 P<0.05）。GTW+SC-FL 和 LY+SC-FL 组的 FSH、LH 和 T 水平明显高于 GTW 组和 LY 组（P<0.01 或 P<0.05）。HE 染色结果显示，GTW+SC-FL 和 LY+SC-FL 组睾丸组织形态明显优于 GTW 组和 LY 组。SC-FL 的上述作用与 PI3K、p-AKT 和 Bcl-2 蛋白和 mRNA 表达上调密切相关。
通过 PI3K/Akt 信号通路，SC-FL 上调 GFRa1、RET、PI3K、p-AKT 和 Bcl-2，下调 BAD 和 BAX 蛋白和 mRNA 的表达，从而降低 GC-1 spg 细胞 S 期比例，显著增加线粒体膜电位，显著降低细胞凋亡，提高精子计数和活力。
