甜菜碱通过影响Bcl-2-Pi3k-Akt系列基因的甲基化改善少弱精子症的机制研究

Mechanism Research on Betaine Improveing Oligoasthenozoospermia by Influencing the Methylation of the Bcl-2-Pi3k-Akt Series Genes

关键词:  甜菜碱  少弱精子症  甲基化  BCL-2-PI3K-AKT通路

Key words:  betaine  oligoasthenozoospermia  methylation  BCL-2-PI3K-AKT pathway

摘要:

摘要：目的 探究甜菜碱（Betaine；BET）通过调控Bcl-2、Pi3k、Akt系列基因甲基化改善少弱精子症（Oligoasthenozoospermia；OLI）的作用机制。方法 将60只SD雄性大鼠随机分为空白组、模型组（雷公藤多苷，Tripterygium wilfordii glycosides；TWGs 60 mg·kg-1·d-1）、阳性药物组（左卡尼汀，300 mg·kg-1·d-1）及BET低、中、高剂量组（200、400、800 mg·kg-1·d-1）。采用HE染色观察大鼠睾丸组织病理变化；ELISA法检测大鼠血清中T（睾酮）、LH（促黄体生成素）、FSH（促卵泡激素）水平；免疫荧光染色法分析大鼠睾丸组织中Caspase-3、BCL-2、PI3K和AKT的表达水平；Western blotting检测大鼠睾丸组织BCL-2和PI3K的表达水平；甲基化捕获测序检测Bcl-2、Pi3k和Akt系列靶基因甲基化水平；pi-RNA测序用于分析BET对Bcl-2、Pi3k、Akt系列靶基因的调控作用；对潜在作用靶基因进行GO功能和KEGG通路富集分析。结果 与空白组相比，模型组大鼠睾丸曲细精管的生精细胞大量脱落（P < 0.001），排列紊乱，管腔扩张；T和FSH水平降低（P < 0.01）、LH水平升高（P < 0.01）；Caspase-3表达显著升高（P < 0.001）；BCL-2、PI3K和AKT表达显著降低（P < 0.001）。与模型组相比，BET中、高剂量组睾丸曲细精管生精细胞数量显著增加（P < 0.001），生精细胞形态改善；T和FSH水平升高（P < 0.01），LH水平降低（P < 0.01）；Caspase-3表达显著降低（P < 0.001）；BCL-2、PI3K和AKT表达显著升高（P < 0.001)。甲基化捕获测序表明，BET可以显著提高Bcl-2、Pi3k、Akt系列靶基因甲基化水平；pi-RNA测序显示BET通过pi-RNA/PIWI复合物调控Bcl-2、Pi3k、Akt系列靶基因的甲基化。GO和KEGG分析提示其作用与PI3K-AKT通路相关。结论 BET对TWGs所致OLI大鼠模型具有治疗作用。其机制可能与BET通过提供甲基基团促进Bcl-2、Pi3k、Akt系列靶基因甲基化，以及激活BCL-2-PI3K-AKT信号通路上调相关蛋白表达有关。

Abstract:

ABSTRACT: OBJECTIVE To investigate the mechanism of betaine (BET) in ameliorating oligoasthenozoospermia (OLI) through regulating the methylation of Bcl-2, Pi3k, and Akt series genes. METHODS Sixty male Sprague-Dawley rats were randomly divided into blank control group, model group (TWGs, 60 mg·kg?1·d?1), positive control group (L-carnitine, 300 mg·kg?1·d?1), and BET treatment groups (200, 400, 800 mg·kg?1·d?1). Testicular histopathology was examined by HE staining. Serum testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels were measured using ELISA. Immunofluorescence staining was employed to analyze the expression of Caspase-3, BCL-2, PI3K, and AKT in testicular tissue. Western blotting was employed to determine the expression of BCL-2 and PI3K in testicular tissue. Methylation capture sequencing was performed to detect methylation levels of Bcl-2, Pi3k, and Akt series genes. Meanwhile, pi-RNA sequencing was used to assess the regulatory effects of BET on target genes. GO functional and KEGG pathway enrichment analyses were performed on potential target genes. RESULTS Compared with the blank control group, the model group exhibited significant pathological changes in the testicular seminiferous tubules, including massive exfoliation of spermatogenic cells (P < 0.001), disorganized arrangement, and tubular dilation; hormonal analysis showed decreased levels of testosterone (T) and follicle-stimulating hormone (FSH) (P < 0.01) and increased luteinizing hormone (LH) levels (P < 0.01); the expression of Caspase-3 was markedly up-regulated (P < 0.001); additionally, the expression of BCL-2, PI3K, and AKT was significantly reduced (P < 0.001). Compared with the model group, the medium- and high-dose BET treatment groups demonstrated notable improvements: the number of spermatogenic cells in the seminiferous tubules significantly increased (P < 0.001), and cellular morphology improved; T and FSH levels rose (P < 0.01), while LH levels decreased (P < 0.01); the expression of Caspase-3 was significantly reduced (P < 0.001); furthermore, the expression of BCL-2, PI3K, and AKT was markedly up-regulated (P < 0.001). Methylation capture sequencing revealed that BET significantly enhanced the methylation levels of target genes in the Bcl-2, Pi3k, and Akt families. pi-RNA sequencing further indicated that BET regulates the methylation of these target genes via the pi-RNA/PIWI complex. GO and KEGG pathway analysis suggested that this mechanism is associated with the PI3K-AKT signaling pathway. CONCLUSION BET exerts therapeutic effects on the TWGs-induced OLI rat model. The underlying mechanism may involve BET promoting methylation of target genes (Bcl-2, Pi3k, and Akt) by supplying methyl groups, as well as activating the BCL-2-PI3K-AKT signaling pathway to up-regulate the expression of associated proteins.