背景:少弱精子症是男性不育患者中最常见的精液异常类型。甜菜碱(BET)已被证明具有改善精液质量的药理作用。BET也属于睾丸中的内源性生理活性物质。然而,BET在大鼠睾丸中的生理功能及其抗少弱精子症的药理机制尚不清楚。
目的:本研究旨在证明BET对雷公藤多苷(TWG)诱导的少弱精子症大鼠模型的治疗作用和潜在机制。
方法:通过连续灌胃TWG(60mg/kg)28天建立少弱精子症大鼠模型。阴性对照组,少弱精子症组,阳性药物组(左卡尼汀,300mg/kg),和200毫克/千克,400mg/kg,建立800mg/kgBET组,探讨BET对少弱精子症大鼠模型的治疗作用。通过HE和TUNEL染色评价治疗效果。DNMT3A的免疫荧光分析,PIWIL1,PRMT5,SETDB1,BHMT2和METTL3,甲基化捕获测序,Pi-RNA测序,和分子对接用于阐明潜在的药理机制。
结果:证明BET能显著恢复TWG引起的睾丸病理损伤,也能显著逆转生精细胞的凋亡。生精细胞中DNMT3A蛋白的表达水平,少弱精子症组PIWIL1、PRMT5、SETDB1、BHMT2和METTL3显著下降。400mg/kg和800mg/kgBET组可显著增加上述蛋白的表达水平。甲基化捕获测序表明BET能显著提高Spata的5mC甲基化水平,Spag,和Specc精子发生相关基因。Pi-RNA测序证明,上述基因在BET干预下产生大量的Pi-RNA。Pi-RNA可与PIWI蛋白形成复合物,参与靶基因的DNA甲基化。分子对接表明BET可能不直接充当甲基转移酶的底物,而是通过促进甲硫氨酸循环和增加S-腺苷甲硫氨酸合成来参与DNA甲基化。
结论:BET对TWPs诱导的少弱精子症大鼠模型有明显的治疗作用。其机制主要涉及BET可以增加Spata的甲基化水平,规格,和Spag靶基因通过PIWI/Pi-RNA途径和甲基转移酶(包括DNA甲基转移酶和组蛋白甲基转移酶)的上调。
BACKGROUND: Oligoasthenozoospermia is the most common type of semen abnormality in male infertile patients. Betaine (BET) has been proved to have pharmacological effects on improving semen quality. BET also belongs to endogenous physiological active substances in the testis. However, the physiological function of BET in rat testis and its pharmacological mechanism against
oligoasthenozoospermia remain unclear.
OBJECTIVE: This research aims to prove the therapeutic effect and potential mechanism of BET on oligoasthenozoospermia rat model induced by Tripterygium wilfordii glycosides (TWGs).
METHODS: The
oligoasthenozoospermia rat model was established by a continuous gavage of TWGs (60 mg/kg) for 28 days. Negative control group,
oligoasthenozoospermia group, positive drug group (levocarnitine, 300 mg/kg), and 200 mg/kg, 400 mg/kg, and 800 mg/kg BET groups were created for exploring the therapeutic effect of BET on the oligoasthenozoospermia rat model. The therapeutic effect was evaluated by HE and TUNEL staining. Immunofluorescence assay of DNMT3A, PIWIL1, PRMT5, SETDB1, BHMT2, and METTL3, methylation capture sequencing, Pi-RNA sequencing, and molecular docking were used to elucidate potential pharmacological mechanisms.
RESULTS: It is proved that BET can significantly restore testicular pathological damage induced by TWGs, which also can significantly reverse the apoptosis of spermatogenic cells. The spermatogenic cell protein expression levels of DNMT3A, PIWIL1, PRMT5, SETDB1, BHMT2, and METTL3 significantly decreased in oligoasthenozoospermia group. 400 mg/kg and 800 mg/kg BET groups can significantly increase expression level of the above-mentioned proteins. Methylation capture sequencing showed that BET can significantly increase the 5mC methylation level of Spata, Spag, and Specc spermatogenesis-related genes. Pi-RNA sequencing proved that the above-mentioned genes produce a large number of Pi-RNA under BET intervention. Pi-RNA can form complexes with PIWI proteins to participate in DNA methylation of target genes. Molecular docking indicated that BET may not directly act as substrate for methyltransferase and instead participates in DNA methylation by promoting the methionine cycle and increasing S-adenosylmethionine synthesis.
CONCLUSIONS: BET has a significant therapeutic effect on
oligoasthenozoospermia rat model induced by TWPs. The mechanism mainly involves that BET can increase the methylation level of Spata, Specc, and Spag target genes through the PIWI/Pi-RNA pathway and up-regulation of methyltransferases (including DNA methyltransferases and histone methyltransferases).