寒证ED大鼠模型动脉差异表达蛋白质的TMT技术筛查与生物信息学分析

Screenings and bio-informatics analysis of differently expressed proteins in arteria of excessive cold syndrome ED rat model by TMT technology

目的筛查寒证勃起功能障碍(ED)大鼠模型动脉差异表达蛋白质,并进行生物信息学分析,为研究寒证ED发病机制提供新的思路与实验依据。方法将100只雄性性成熟SD大鼠随机分为正常对照组(N组,15只)、寒证造模组(M组,85只),分别在正常环境(22±2)℃及冷环境(4±0.5)℃饲养,干预24周并建立稳定的寒证模型后,通过APO勃起实验筛选出ED大鼠并分为寒证ED组(ED组)和寒证ED干预组(用于其它研究),剩余大鼠为寒证证候组(S组)。采用TMT技术筛查并鉴定寒证ED大鼠动脉差异表达蛋白质,并进行功能富集分析和KEGG通路富集分析。结果(1)模型的建立:1)生物学表征结果为:与N组相比,M组大鼠反应迟钝,舌苔苍白,大便湿软,小便色淡,体重增长显著减缓(P<0.05),饮食量、饮水量显著升高(P<0.05);2)APO实验结果为:与N组相比,M组大鼠勃起潜伏期显著延长,勃起次数显著减少(P<0.05),ED成模率为47.95%;(2)蛋白质组学:通过TMT技术共鉴定出差异蛋白质364种,其中上调蛋白281种,下调蛋白83种。GO分析显示差异蛋白质主要参与小分子代谢,氧化还原过程,在线粒体和线粒体外膜均有分布,且具有催化活性、氧化还原酶活性等。KEGG信号通路分析发现差异蛋白质涉及氧化磷酸化通路、PPAR信号通路、铁死亡通路等152条信号通路。结论(1)长期冷刺激可成功诱导建立寒证ED证候与病证结合大鼠模型,其生物学表征符合中医临床证候特点;(2)寒证ED大鼠动脉差异表达蛋白质涉及到复杂的生物学过程、分子功能与信号通路,为进一步基于动脉病理改变层面开展寒证ED发病机制研究奠定了基础。

Objective To comparatively analyze differentially expressed proteins in arteria of erectile dysfunction(ED)rat model with excessive cold syndrome by proteomics technology and bioinformation analysis for better understand the pathogenesis of ED with excessive cold syndrome.Methods Total of 100 sexually mature male SD rats were randomly divided into two groups such as normal control group(group N,15 rats)and excessive cold syndrome model group(group M,85 rats).The rats in group N were fed in normal environment(22±2)℃and the rats in group M were rasied in cold environment(4±0.5)℃.After 24 weeks of intervention,rats with stable excessive cold syndrome were established.ED rats in group M were screened by APO penile erection test and defined as group ED,and the rest were defined as excessive cold syndrome model group(group S).TMT technology was used to screen and identify differentially expressed proteins in aortic of ED rats,and diferentilly expressed proteins were clustered by gene ontology(CO)functional enrichment analysis and KEGG pathway enrichment analysis.Results(1)Establishment of the model:1)The rats in group M presented slow response,pale tongue,loose stool,light-colored urine,significant slow down in weight gain(P<0.05),and significantly increased food intake and water intake(P<0.05),compared with that of the rats in group N;2)The erection latency period of the rats in group M was significantly prolonged,the number of erections was significantly reduced,compared with the rats in group N,(P<0.05),and the occurrence rate of ED in animal model accounted for 47.95%;(2)Proteomics:A total of 364 differentially expressed proteins were identified by TMT technology,including 281 upregulated proteins and 83 down regulated proteins.CO analysis showed that the differential proteins were mainly involved in small molecule metabolism,oxidation-reduction process.They distributed at mitochondrial and mitochondrial outer membrane,and had catalytic activity and oxidoreductase activity.KEGG signaling pathway analysis demonstrated that the diferential proteins involved 152 signaling pathways,including oxidative phosphorylation pathway,PPAR signaling pathway,and ferroptosis pathway.Conclusions(1)The cold environment can successfully induce the erectile dysfunction with excessive cold syndrome in rats,and its biological characteristics are consistent with the characteristics of clinical syndrome in traditional Chinese medicine;(2)Differentially expressed proteins identified in arterial of ED rats are involved in complex biological processes,molecular functions and signaling pathways,which laid a foundation for studying the pathogenesis of ED from the perspective of arterial pathological changes.