Tian Jia, Xie Zhi-Yuan, Pei Cheng-Bin, Song Ai-Hua, Zhou Yue, Ma Liang-Hong
Ningxia Human Sperm Bank, Research Institute of Medical Sciences, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750001, China.
Key Laboratory of the Ministry of Education for Fertility Preservation and Maintenance, Ningxia Medical University, Yinchuan, Ningxia 750001, China.
Zhonghua Nan Ke Xue. 2024 Jun;30(6):483-492.
To investigate the effect of exosomes loaded with Lycium barbarum miRNA (Lb-miR2911) on spermatogenic function recovery in non-obstructive azoospermia (NOA) rats through cross-regulation of the Wnt/β-catenin signaling pathways.
We established an NOA model in 30 four-week-old male SD rats by intraperitoneal injection of busulfan. At 5 weeks after modeling, we equally randomized the rats into a model control group (MC，untreated), an Lb-miR2911EXO group (Lb-miR2911EXO ，treated by intratesticular injection of Lb-miR2911-loaded exosomes), and a sham group (Shame，treated by intratesticular injection of exosomes-empty drug), with another 10 male SD rats taken as normal controls（NC）. We observed the uptake and metabolic changes of Lb-miR2911 in the testis tissue of the rats by RNA FISH at 2 and 6 weeks after treatment, detected cell proliferation, spermatogenesis and gene expressions of the Wnt/β-catenin signaling pathways in the testis tissue by Transcriptome sequencing analysis combined with Western blot and RT-PCR at 12 weeks, evaluated the recovery of the spermatogenic function based on the testis tissue morphology and sperm quality, and assessed the organ toxicity of Lb-miR2911 in the tissue and organs of the rats based on histomorphological analysis and the levels of serum TNF-α, IL-1β, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and other relevant indicators.
After 12 weeks of treatment, histomorphological analysis showed regular arrangement of spermatogenic cells at all levels in the testis tissue, with a large number of mature sperm in the tubular lumen, and with significantly higher Johnsen scores, testis weight, testicular index, sperm concentration and sperm motility in the Lb-miR2911EXO than in the sham group (all P< 0.05). Compared with the model controls, the Lb-miR2911EXO group exhibited remarkably down-regulated gene expression of DACT3 (P< 0.05), up-regulated expressions of DVL2 and β-catenin (P< 0.05), elevated levels of p-DVL2 and β-catenin (nucleus) proteins (P< 0.05), increased expressions of cell proliferation-related genes CCND1, CCNE1 and CCNE2 (P< 0.05) and spermatogenesis-related genes DMC1, CCR6, JAM2 and KLC3 (P< 0.05). No pathological changes were observed in the lung, liver and kidney tissues of the rats, or in the levels of serum TNF-α, IL-1β, AST, ALT, creatinine and urea nitrogen in the rats treated with Lb-miR2911EXO compared with the normal controls (P > 0.05).
Lb-miR2911-loaded exosomes promote spermatogenic function recovery in NOA rats through cross-regulation of the DACT3, Wnt and β-catenin signaling pathways.
通过Wnt/β-连环蛋白信号通路的交叉调节，研究负载枸杞微小RNA（Lb-miR2911）的外泌体对非梗阻性无精子症（NOA）大鼠生精功能恢复的影响。
通过腹腔注射白消安，在30只4周龄雄性SD大鼠中建立NOA模型。建模后5周，将大鼠随机分为模型对照组（MC，未治疗）、Lb-miR2911外泌体组（Lb-miR2911EXO，经睾丸内注射负载Lb-miR2911的外泌体治疗）和假手术组（Shame，经睾丸内注射空载体外泌体治疗），另取10只雄性SD大鼠作为正常对照组（NC）。治疗后2周和6周，通过RNA FISH观察大鼠睾丸组织中Lb-miR2911的摄取和代谢变化；治疗12周时，通过转录组测序分析结合蛋白质免疫印迹法和逆转录-聚合酶链反应，检测睾丸组织中的细胞增殖、生精情况以及Wnt/β-连环蛋白信号通路的基因表达；根据睾丸组织形态和精子质量评估生精功能的恢复情况；基于组织形态学分析以及血清肿瘤坏死因子-α、白细胞介素-1β、天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）等相关指标，评估Lb-miR2911对大鼠组织和器官的器官毒性。
治疗12周后，组织形态学分析显示，Lb-miR2911EXO组大鼠睾丸组织各级生精细胞排列规则，管腔内有大量成熟精子，Johnsen评分、睾丸重量、睾丸指数、精子浓度和精子活力均显著高于假手术组（均P<0.05）。与模型对照组相比，Lb-miR2911EXO组DACT3基因表达显著下调（P<0.05），DVL2和β-连环蛋白表达上调（P<0.05），p-DVL2和β-连环蛋白（细胞核）蛋白水平升高（P<0.05），细胞增殖相关基因CCND1、CCNE1和CCNE2表达增加（P<0.05），生精相关基因DMC1、CCR6、JAM2和KLC3表达增加（P<0.05）。与正常对照组相比，Lb-miR2911EXO组大鼠肺、肝、肾组织未观察到病理变化，血清肿瘤坏死因子-α、白细胞介素-1β、AST、ALT、肌酐和尿素氮水平也无明显变化（P>0.05）。
负载Lb-miR2911的外泌体通过对DACT3、Wnt和β-连环蛋白信号通路的交叉调节，促进NOA大鼠生精功能的恢复。
