益肾通络方对苯并(a)芘致精子DNA损伤模型小鼠睾丸靶基因影响的多组学研究

Multi-omic Study on Effect of Yishen Tongluo Formula(益肾通络方)on Testicular Target Gene in Benzo(a)pyrene-induced Sperm DNA Damage Model Mice

目的探讨益肾通络方对精子DNA损伤的影响及可能作用机制。方法40只ICR小鼠随机分为空白组,模型组和益肾通络方低、中、高剂量组,每组8只。除空白组外其余各组按100 mg/(kg·d)灌胃苯并(a)芘复制精子DNA损伤模型,益肾通络方低、中、高剂量组上午造模,下午分别给予益肾通络方水煎液5.95、11.89、23.78 g/(kg·d)灌胃,空白组和模型组同时灌胃0.1 ml/10 g生理盐水,各组均持续8周。检测各组小鼠精子DNA碎片指数(DFI),根据DFI结果进行睾丸转录组学和蛋白组学分析,对两组学的结果进行趋势分析、GO分析和KEGG富集通路分析。采用九象限法筛选益肾通络方作用靶基因,并采用Western blot法进行验证。结果模型组小鼠精子DFI明显高于空白组(P<0.01);与模型组比较,益肾通络方高剂量组小鼠精子DFI显著下降(P<0.01),而益肾通络方低、中剂量组差异无统计学意义(P>0.05),故对空白组、模型组、益肾通络方高剂量组进行转录组学和蛋白组学分析。结果显示,共筛选出1272个差异基因(DEGs)和1309个差异蛋白(DEPs)。趋势分析筛选出8种趋势类型,其中谱2为模型组表达下调,益肾通络方干预后恢复的基因/蛋白;谱5为模型组表达上调,益肾通络方干预后恢复的基因/蛋白。因此,选择对谱2和谱5中的基因或蛋白进一步分析。GO分析显示,在生物学过程、细胞成分、分子功能类别中排第一位分别是发育过程、细胞内部分、分子功能监管。谱2和谱5中基因KEGG富集通路前五位分别为铂类药物抗药性、破骨细胞分化、多种细胞凋亡、凋亡及非酒精性脂肪肝疾病,蛋白KEGG通路前五个通路分别为系统性红斑狼疮、氮代谢、蛋白消化吸收、非洲锥虫病和B细胞受体信号通路。九象限法筛选出X线交叉互补修复基因1(Xrcc1)、细胞色素b5还原酶3(Cyb5r3)、血红素结合蛋白1(Hebp1)、丝氨酸蛋白酶45(Prss45)为益肾通络方靶基因。经Western blot验证,与空白组比较,模型组小鼠睾丸组织Xrcc1及Prss45蛋白表达显著下调(P<0.05)。益肾通络方高剂量组Xrcc1蛋白表达较模型组显著上调(P<0.01)。结论益肾通络方可修复由苯并(a)芘染毒引起的精子DNA损伤,其机制可能与耐药、凋亡等通路及Xrcc1有关。

Objective To investigate the effect of Yishen Tongluo Formula(益肾通络方,YSTL)on sperm DNA damage and the possible mechanism.Methods Forty ICR mice were randomly divided into control group,model group,YSTL low-dose(YSTLL),medium-dose(YSTLM)and high-dose(YSTLH)group,with 8 mice in each group.Except for the control group,mice were given 100 mg/(kg·d)of Benzo(a)pyrene(BaP)to replicate the sperm DNA damage model.The three YSTL groups were modeled in the morning,while received the decoction of YSTL at the dose of 5.95,11.89 and 23.78 g/(kg·d)by gavage respectively in YSTLL,YSTLM and YSTLH groups;the control group and model group received 0.1 ml/10 g of normal saline by gavage;the administration lasted for 8 weeks in all gorups.The sperm DNA fragmentation index(DFI)was detected.Transcriptome and proteome analysis were performed according to DFI results.Trend analysis,GO and KEGG analysis were performed based on the two omics results.The target genes of YSTL were screened out by nine quadrant method and verified by Western blotting.Results The DFI of the model group was significantly higher than that of the control group(P<0.01).Compared to that in the model group,the DFI significantly decreased in the YSTLH group(P<0.01),while there was not significant difference in the YSTHL and YSTHM groups(P>0.05).Therefore,the transcriptome and proteome analysis were performed in the control group,model group,and YSTLH group.A total of 1272 differential expressed genes(DEGs)and 1309 differential expressed proteins(DEPs)were screened out.Eight trend types were screened out;as profile 2 was the gene/protein which was down-regulated in the model group and restored after the intervention of YSTH,and profile 5 was the one up-regulated in the model group and restored after the intervention of YSTH.Profile 2 and 5 were selected for further analysis.GO analysis showed that development process,intracellular part and molecular function regulation ranked first in the biological processes,cellular components and molecular function categories,respectively.The top five KEGG-enriched pathways of genes of profile 2 and profile 5 were platinum drug resistance,osteoclast differentiation,various cell apoptosis,apoptosis,and nonalcoholic fatty liver disease,while proteins were mainly enriched in systemic lupus erythematosus,nitrogen metabolism,protein digestion and absorption,African trypanosomiasis and B cell receptor signaling pathway.X-ray cross complementation repair gene 1(Xrcc1),cytochrome b5 reductase 3(Cyb5r3),hemopexin 1(Hebp1),and serine protease 45(Prss45)were screened out by the nine-quadrant method,and significant decreases were found of Xrcc1 and the model group than the control group through Western blotting verification(P<0.05).The expression of Xrcc1 in the YSTLH group was significantly up-regulated compared to the model group(P<0.01).Conclusion YSTL can repair sperm DNA damage caused by BaP exposure.The mechanism may be related to the pathways of drug resistance and apoptosis,as well as Xrcc1.