摘要
目前,病毒感染对宿主代谢的重塑已在多种病毒中得到研究,猪流行性腹泻病毒(PEDV)作为一种猪肠道致病性冠状病毒,其与宿主代谢系统之间的相互作用尚未见报道。有研究表明血管紧张素转化酶2(ACE2)参与机体病理生理状态下的糖代谢调控,且在抗PEDV感染中发挥作用。作者拟探究PEDV感染对仔猪肠道葡萄糖代谢的调节作用以及ACE2在其中的可能作用,明确ACE2、PEDV感染与仔猪肠道葡萄糖代谢的相互关系。本试验以健康及PED仔猪为研究对象,通过收集血液,分离截取十二指肠、空肠和回肠组织样品,进行如下试验:1)检测小肠各段组织中PEDV M基因及N基因和蛋白表达;2)血液中葡萄糖和乳酸等含量的检测;Western blot检测葡萄糖转运载体SGLT-1和GLUT-2蛋白表达变化;3)RT-qPCR检测小肠各段组织中糖酵解及TCA循环途径关键酶的基因转录变化;4)ELISA和Western blot分析小肠各段组织中Ang1-7、AngII的含量及ACE2、MasR的蛋白表达;5)Spearman法分析ACE2与PEDV感染及糖代谢相关酶表达变化间的相关性。结果表明:1)腹泻仔猪空肠组织中存在M基因表达,在500~600 bp之间出现了预期的阳性条带;PEDV-N蛋白在仔猪十二指肠、空肠和回肠组织中均有表达,以空肠组织中表达最高(P<0.01);2)与正常健康组相比,PEDV感染仔猪血液中葡萄糖(P<0.05)和乳酸(P<0.01)含量显著或极显著升高;PEDV感染显著或极显著上调了十二指肠和空肠组织中SGLT-1和GLUT-2的蛋白表达(P<0.05&P<0.01);3)PEDV感染仔猪小肠组织中糖酵解关键酶HK2、PFK、PKM和LDHA基因转录水平显著升高(P<0.01),而TCA循环途径关键酶CS、OGDH、IDH的基因转录水平显著降低(P<0.05&P<0.01);4)PEDV感染仔猪小肠组织中ACE2和MasR蛋白表达极显著下调(P<0.01);Ang1-7含量极显著降低(P<0.01),AngII含量极显著升高(P<0.01);5)PEDV感染与仔猪小肠组织中的SGLT-1、GLUT-2、HK2、PKM、PFK均呈正相关关,与ACE2-Ang1-7-MasR轴呈负相关关系。结果提示:PEDV感染肠道组织中糖酵解途径被激活,TCA循环途径被抑制,同时弱化了ACE2介导的ACE2-Ang1-7-MasR通路。表现出以葡萄糖摄入和乳酸生成增加为特征的Warburg效应,且PEDV感染诱导的仔猪肠道葡萄糖代谢重塑与ACE2的表达呈负相关关系。
At present,the remodeling of host metabolism by viral infection has been studied in a variety of viruses.As a porcine intestinal pathogenic coronavirus,the interaction between porcine epidemic diarrhea virus(PEDV)and host metabolic system has not been reported.Studies have shown that angiotensin-converting enzyme 2(ACE2)is involved in the regulation of glucose metabolism in the pathophysiological state of the body and plays a role in anti-PEDV infection.In order to explore the regulatory effect of PEDV infection on intestinal glucose metabolism in piglets and the possible role of ACE2,the relationship between ACE2,PEDV infection and intestinal glucose metabolism in piglets was clarified.In this experiment,healthy and PED piglets were used as the research objects.By collecting blood,duodenum,jejunum and ileum tissue samples were isolated and intercepted for the following experiments:1)The expression of PEDV M gene and N gene and protein in each segment of small intestine was detected.2)Detection of glucose and lactic acid in blood;western blot was used to detect the expression of glucose transporter SGLT-1 and GLUT-2protein.3)RT-qPCR was used to detect the gene transcription of key enzymes in glycolysis and TCA cycle pathway in each intestinal segment;4)ELISA and Western blot were used to analyze the content of Ang1-7,AngII and the protein expression of ACE2 and MasR in each intestinal segment;5)Spearman method was used to analyze the correlation between ACE2 and PEDV infection and glucose metabolism-related enzyme expression changes.The results showed that:1)There was M gene expression in the jejunum tissue of diarrhea piglets,and a positive band appeared at about 552 bp.PEDV-N protein was expressed in duodenum,jejunum and ileum of piglets,with the highest expression in jejunum(P<0.01).2)Compared with the normal healthy group,the contents of glucose(P<0.05)and lactic acid(P<0.01)in the blood of PEDV-infected piglets were significantly or extremely significantly increased;the protein expressions of SGLT-1 and GLUT-2 in duodenum and jejunum were significantly or extremely significantly up-regulated(P<0.05&P<O.O1).3)The results demonstrated that the transcriptional levels of key glycolytic enzymes(HK2,PFK,PKM,and LDHA)were markedly elevated(P<0.O1)in the small intestinal tissues of PEDV-infected piglets,whereas those of key TCA cycle enzymes(CS,OGDH,and IDH)were significantly suppressed(P<0.05&P<0.01).;4)In the small intestinal tissues of PEDV-infected piglets,the protein expressions of ACE2 and MasR were significantly down-regulated(P<0.O1).In conjunction with these changes,the level of Angl-7 was markedly decreased(P<0.O1),whereas that of AngII was considerably increased(P<0.O1);5)PEDV infection was positively correlated with SGLT-1,GLUT-2,HK2,PKM and PFK in the small intestine of piglets,and negatively correlated with ACE2-Ang1-7-MasR axis.The results suggest that PEDV infection in intestinal tissues activates glycolysis and suppresses the TCA cycle,concomitant with a weakened ACE2mediated ACE2-Ang1-7-MasR pathway.This metabolic rewiring manifests as a Warburg effect,characterized by increased glucose uptake and lactate production.Furthermore,this PEDV-induced remodeling of intestinal glucose metabolism is negatively correlated with ACE2 expression.
作者
陈雪清
马畅
王公民
王换换
张源淑
CHEN Xueqing;MA Chang;WANG Gongmin;WANG Huanhuan;ZHANG Yuanshu(Key Laboratory of Animal Physiology and Biochemistry,Ministry of Agriculture,Nanjing Agricultural University,Nanjing 210095,China)
出处
《畜牧兽医学报》
北大核心
2026年第3期1661-1672,共12页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31972640)。
