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旨在利用HIF-1α激动剂二甲基乙二酰甘氨酸(DMOG)上调HIF-1α的表达水平，研究HIF-1α对猪肠道上皮细胞糖酵解相关酶基因及β防御素表达的调节作用.以猪空肠上皮细胞系IPEC-J2细胞为研究对象，分为对照组和DMOG处理组，试验设置6个处理浓度(0、 0.25、0.5、1、2、4 mM)和2个处理时间(4 h和24 h),采用CCK8检测细胞活力确定后续试验的培养基是否含血清及DMOG的浓度范围，基于上述试验结果，CCK8检测不同作用时间(0、1、2、4、8、24 h)下，低剂量(0.25 mM)和高剂量(2 mM)DMOG对细胞活力的影响，酶联免疫吸附实验(ELISA)和蛋白免疫印迹(WB)检测HIF-1α的蛋白表达水平，实时荧光定量PCR(qPCR)检测HIF-1α及其下游靶基因mRNA的表达情况，比色法检测糖酵解途径关键酶活性的变化.结果发现，2 mM的DMOG处理4、8和24 h时能显著抑制IPEC-J2细胞活力(P<0.05).WB结果表明，2 mM DMOG能显著上调细胞内总HIF-1α的蛋白表达(P<0.05).2 mM DMOG处理4 h能显著上调HIF-1α下游靶基因葡萄糖转运蛋白1(GLUT-1)、乳酸脱氢酶A(LDHA)、6-磷酸果糖-2-激酶(PFK-3)、己糖激酶2(HK2)的mRNA表达水平(P<0.05),同时上调糖酵解关键限速酶丙酮酸激酶(PK)、磷酸果糖激酶(PFK)、己糖激酶(HK)的活性(P<0.05),2 mM DMOG处理24 h则显著上调细胞内PK活性(P<0.05),表明DMOG可能通过上调HIF-1α途径，促进IPEC-J2细胞的代谢类型转向糖酵解.ELISA结果发现，2 mM DMOG处理4 h能够促进猪β防御素1(PBD1)和猪β防御素2(PBD2)的蛋白表达水平(P<0.05).综上所述，DMOG能够上调猪IPEC-J2细胞中HIF-1α及其下游糖酵解相关酶基因和β防御素的表达，初步表明低氧诱导因子HIF-1α可能对猪IPEC-J2细胞β防御素表达具有调节作用.
Abstract：The aim of this experiment was to use the HIF-1α agonist Dimethyloxalyl glycine(DMOG)to upregulate the expression level of HIF-1α,and to study the regulatory effect of HIF-1α on the expression of glycolysis-related enzyme genes and β-defensins in porcine intestinal epithelial cells.IPEC-J2 cells were treated with different concentrations of DMOG(0,0.25,0.5,1,2 mM)and different treatment times(0,1,2,4,8,24 h).The results of the CCK8 assay showed that under the action of 2 mM DMOG,cell viability was inhibited at 4 h, 8 h and 24 H.IPEC-J2 cells were treated with different concentrations of DMOG(0,0.25,0.5,1,2 mM)and two treatment times(4 h and 24 h).The results of Western blotting indicated that 2 mM DMOG could significantly upregulate the protein expression of total HIF-1α in cells(P< 0.05).Treatment with 2 mM DMOG for 4 h could significantly upregulate the mRNA expression levels of HIF-1α downstream target genes, including Glucose transporter 1(GLUT-1),Lactate dehydrogenase A(LDHA),6-phosphofructo-2-kinase(PFK-3),and Hexokinase 2(HK2)(P< 0.05).At the same time, it also upregulated the activities of key rate-limiting enzymes in glycolysis, namely Pyruvate kinase(PK),Phosphofructokinase(PFK),and Hexokinase(HK)(P<0.05).After treatment with 2 mM DMOG for 24 h, the PK activity in cells was significantly upregulated(P< 0.05),indicating that DMOG might promote the metabolic pathway of IPEC-J2 cells to shift towards glycolysis through the upregulation of the HIF-1α pathway.The results of the enzyme-linked immunosorbent assay(ELISA)showed that treatment with 2 mM DMOG for 4 h could promote the protein expression levels of Porcine beta defensin 1(PBD1)and Porcine beta defensin 2(PBD2)(P<0.05).In conclusion, DMOG could upregulate the expression of HIF-1α,its downstream glycolysis-related enzyme genes, and β-defensins in porcine IPEC-J2 cells, preliminarily indicating that the hypoxia-inducible factor HIF-1α might have a regulatory effect on the expression of β-defensins in porcine IPEC-J2 cells.
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基本信息:
DOI：10.26978/j.cnki.xnmdzk.2026.01.02
中图分类号:S828
引用信息:
[1]李明月,张佳雯,李愿,等.低氧诱导因子HIF-1α激动剂对猪IPEC-J2小肠上皮细胞糖酵解相关酶基因及β防御素表达的影响[J].西南民族大学学报(自然科学版),2026,52(01):9-19.DOI:10.26978/j.cnki.xnmdzk.2026.01.02.
基金信息:
四川省科技计划项目(2023NSFSC1148); 西南民族大学中央高校基本科研业务专项资金优秀学生培养工程项目(ZYN2024171)