摘要
目的:研究糖尿病胃轻瘫(Diabetic gastroparesis,DGP)幽门括约肌组织中氮能神经元损伤的关键基因靶点及其相关机制。方法:采用SD大鼠腹腔注射链脲佐菌素构建I型糖尿病胃轻瘫模型。通过大鼠幽门括约肌组织转录组测序筛选DGP的关键差异性表达基因,并利用免疫组化、免疫印迹等方法验证其在组织中的表达情况。随后,通过小干扰RNA敲低PC12细胞中该基因的表达,从而探索其具体的作用机制。结果:转录组测序分析提示DGP大鼠幽门括约肌组织共存在808个差异基因,其中247个基因上调,561个基因下调。其中,"神经活性配体-受体相互作用"通路中的早期生长应答基因1(Early growth response 1,EGR1)的表达发生了显著下调,对照组比造模组的表达量为1.02±0.03 vs 0.13±0.07(P<0.01)。并且,大鼠DGP模型的免疫组化、免疫印记等方法均成功验证了其表达的下调。PC12细胞敲低EGR1后,神经型一氧化氮合酶(Neuronal nitric oxide synthase,nNOS)及细胞凋亡相关基因Bcl-2的表达发生了显著下调。结论:DGP中EGR1可能是引起氮能神经元nNOS表达降低和凋亡的关键基因,该靶点的发现将为DGP的治疗提供新的思路。
Objective:To study the key genetic targets and related mechanisms of nitrergic neuron injury in pyloric sphincter of diabetic gastroparesis(DGP).Methods:The Type I diabetic gastroparesis models were established in SD rats through intraperitoneal injection of streptozotocin.Key differentially expressed genes in DGP were screened through transcriptome sequencing of rat pyloric sphincter tissue,and their expression in tissues was verified using qRT-PCR,western blot,and immunofluorescence.Further,the specific mechanism of action of the gene was explored by knocking down their expression in PC12 cells using small interfering RNA(siRNA).Results:Transcriptome sequencing analysis indicated the presence of 808 differentially expressed genes(DEGs)in the pyloric sphincter tissues of DGP rats,with 247 genes upregulated and 561 downregulated.Among them,the expression of early growth response 1(EGR1)in the'neuroactive ligand-receptor interaction'pathway was significantly downregulated in the pyloric sphincter,with expression levels of 1.02±0.03 in the control group versus 0.13±0.07 in the model group(P<0.01).In the DGP models,a significant decrease in the expression of neuronal nitric oxide synthase(nNOS)and EGR1 protein were validated in the pyloric sphincter muscle using qRT-PCR,western blot,and immunofluorescence.In vitro experiments demonstrated that the knocking down of EGR1 in PC12 cells led to a significant downregulation expression of nNOS and Bcl-2,which would further lead to functional impairment and apoptosis of nitrergic neurons.Conclusions:EGR1 may be a key gene in DGP that causes reduced expression of nNOS and apoptosis in nitrergic neurons.The identification of this target provides new insights for the treatment of DGP.
作者
谢旎
谢浩然
余兰婷
陆嘉伟
梅竹
吴颖
苏冠豪
李百文
XIE Ni;XIE Hao-ran;YU Lan-ting;LU Jia-wei;MEI Zhu;WU Ying;SU Guan-hao;LI Bai-wen(Shanghai Key Laboratory of Pancreatic Diseases,Department of Gastroenterology,Shanghai General Hospital,Shanghai Jiaotong University School of Medicine,Shanghai,200080,China;Hepatobiliary and Pancreatic Center,Tsinghua Changgung Hospital,Beijing,100084,China)
出处
《现代生物医学进展》
CAS
2024年第12期2201-2206,2273,共7页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(82270568)
上海市科技创新计划项目(22Y11907800)
上海市第一人民医院特色临床研究项目(CTCCR-2021B02)。
