基于转录组测序研究丹参酮ⅡA抑制小鼠睾丸细胞凋亡的作用

Mechanism Study on Tanshinone ⅡA Alleviating Heat Stress-Induced Testicular Cell Apoptosis in Mice Through Transcriptome Sequencing

[目的]研究丹参酮ⅡA(TSA)对热应激导致的小鼠睾丸损伤的影响及其作用机制。[方法]取60只C57小鼠随机分为空白对照组、热应激(HS)组和TSA低、中、高剂量组,每组12只。除空白对照组外,其余小鼠使用单次睾丸热应激处理构建睾丸损伤模型,分别于造模后第2天开始,TSA低、中、高剂量组小鼠分别按10μg/g、20μg/g、40μg/g的剂量腹腔注射溶解于二甲基亚砜(DMSO)的TSA溶液,HS组小鼠注射给予等容积的DMSO溶剂,连续干预7 d。利用病理学、高通量测序及生物信息学分析、分子生物学技术等多种手段,研究TSA对热应激致小鼠睾丸损伤的影响及作用机制。[结果]与空白对照组比较,HS组小鼠睾丸曲细精管直径显著减小,睾丸组织内细胞凋亡显著增加,睾酮含量显著降低(P<0.05);与HS组比较,TSA干预后,小鼠睾丸曲细精管直径显著增大,睾丸组织内细胞凋亡显著减少,睾酮含量显著上升(P<0.05)。通过高通量转录组测序,HS组与空白对照组、TSA中剂量组间的关键差异表达基因聚类于细胞凋亡信号通路;Western blot结果表明HS组通过上调细胞凋亡通路内蛋白表达导致细胞凋亡的发生,引起睾丸组织器质性病变,进而影响睾丸分泌功能;TSA干预后能够抑制细胞凋亡通路内蛋白表达,缓解由热应激导致的睾丸损伤。[结论]TSA能够有效缓解由热应激引起的小鼠睾丸损伤,其机制可能是通过抑制细胞凋亡而发挥作用。

[Objective]To study the effect of tanshinone ⅡA(TSA) on testicular impairment in mice induced by heat stress and its mechanism of action.[Methods]Sixty C57 mice were randomly divided into control group,heat stress(HS) group,and TSA-low,TSA-medium,TSA-high dose group,with 12 mice in each group.Except for those in control group,the mice were treated with a single testicular heat stress treatment to build the testicular impairment model.Starting from the next day of modeling,7-day intraperitoneal injection of 10 μg/g,20 μg/g,and 40 μg/g TSA DMSO solution was respectively performed in TSA-low,TSA-medium,and TSA-high dose group.Within the same period of time,the intraperitoneal injection of same volume of DMSO was performed in control group.The effect and its mechanism of TSA were evaluated by the techniques of pathology,high-throughput sequencing,bioinformatic analysis and molecular biology.[Results] Compared with control group,the diameter of testicular seminiferous tubules of mice in the HS group was significantly reduced,apoptosis in testicular tissue was significantly increased,and testosterone level was significantly decreased(P<0.05).In the TSA groups,the diameter of testicular seminiferous tubules was significantly increased,apoptosis in testicular tissue was significantly reduced,and testosterone level was significantly increased(P<0.05),compared with HS group.In high-throughput transcriptome sequencing,the key differentially expressed genes of the mice in the HS group,the control group and the TSA-medium group were mainly found in the cell apoptosis signaling pathway.The results of Western blot showed that the cell apoptosis in the HS group,which led to organic lesions of testicular tissue and testicular secretory function impairment,was induced by the up-regulating the protein expression in the apoptosis pathway.TSA treatment can inhibit the expression of proteins in the apoptosis pathway and alleviate the testicular impairment caused by heat stress.[Conclusion]TSA can alleviate the mice testicular impairment induced by heat stress,and its mechanism may relate to inhibiting cell apoptosis.