摘要
目的探究益景汤对早期糖尿病视网膜病变(DR)大鼠视网膜组织中微小RNA(miRNA)表达的影响,筛选出益景汤干预早期DR的靶点miRNA。方法将20只2月龄雄性SD大鼠使用链脲佐菌素建立糖尿病大鼠模型,随机分为模型组(MG)和益景汤组(YJG),另将常规喂养的大鼠设为对照组(CG),每组10只。YJG组大鼠每日灌胃益景汤12.50 g/kg,CG、MG组大鼠灌胃等体积0.9%氯化钠注射液,均干预16周。给药完成后记录大鼠体质量,摘除双侧眼球剥离视网膜,提取总RNA,PCR扩增构建小RNA文库,采用高通量测序技术对该文库进行测序。对差异miRNA进行靶基因预测,并对靶基因进行基因本体论(GO)、Pathway分析,选择差异倍数较大的miRNA进行PCR验证。结果(1)已知miRNA差异:筛选出644个已知miRNA,其中差异miRNA共59个。与CG组比较,MG组35个miRNA上调、6个miRNA下调,YJG组20个miRNA上调、4个miRNA下调;与MG组比较,YJG组6个miRNA上调、13个miRNA下调,差异均有统计学意义(均P<0.05);其中,1个上调miRNA,11个下调miRNA为益景汤干预DM大鼠后,3组共同差异性表达miRNA。(2)新发现miRNA差异:3组筛选出242个新发现的miRNA,其中差异miRNA共105个。与CG组比较,MG组50个miRNA上调,15个miRNA下调;与CG组比较,YJG组55个miRNA上调,20个miRNA下调;与MG组比较,YJG组16个miRNA上调,6个miRNA下调,差异均有统计学意义(均P<0.05)。(3)差异miRNA生物学功能和通路:差异基因主要涉及生物学过程方面的多种蛋白的转录调节;靶基因关联的通路主要是肿瘤相关的信号通路和糖脂代谢相关通路。(4)PCR验证:MG组大鼠视网膜miR-673-3p表达低于CG组,余11个miRNA表达均高于CG组;YJG组大鼠视网膜miR-673-3p、miR-23b-5p表达均高于MG组,余10个miRNA表达均低于MG组,差异均有统计学意义(均P<0.05)。除miR-23b-5p外,其余11个miRNA与上述RNA检测结果一致。结论miR-673、miR-1、miR-133、miR-214、miR-23b、miR-31a、miR-451等可能是益景汤干预早期DR微血管损害的靶点miRNA。
OBJECTIVE Yijing(miRNA)expression in the retinal tissues of rats with early diabetic retinopathy(DR)and identify the target miRNA for Yijing Decoction intervention in early DR.METHODS A total of 20 male SD rats aged two months were used to establish a diabetic rat model with streptozotocin and randomly divided into the model group(MG)and Yijing Decoction group(YJG).Another group of conventionally fed rats served as the control group(CG).YJG rats were orally administered Yijing Decoction at 12.50 g/kg daily,while CG and MG rats were orally administered an equal volume of 0.9%sodium chloride injection.The intervention lasted for 16 weeks.After completing the administration,the body weight of the rats was recorded,and the retinas were removed from both eyes.Total RNA was extracted,and a small RNA library was constructed by PCR amplification for high-throughput sequencing.Differential miRNAs were predicted for target genes,and gene ontology(GO)and pathway analyses were performed on target genes.Differential miRNAs with significant fold changes were selected for PCR validation.RESULTS(1)Known miRNA differences:A total of 644 known miRNAs were screened,with 59 differential miRNAs identified.Compared to the CG group,the MG group had 35 upregulated miRNAs and six downregulated miRNAs,while the YJG group had 20 upregulated miRNAs and fourdownregulated miRNAs.Compared to the MG group,the YJG group had six upregulated miRNAs and 13 downregulated miRNAs,the differences were statistically significant(all P<0.05).Among them,one upregulated miRNA and 11 downregulated miRNAs were commonly differentially expressed in the three groups after Yijing Decoction intervention in diabetic rats.(2)Newly discovered miRNA differences:A total of 242 newly discovered miRNAs were screened,with 105 differential miRNAs identified.Compared to the CG group,the MG group had 50 upregulated miRNAs and 15 downregulated miRNAs,while the YJG group had 55 upregulated miRNAs and 20 downregulated miRNAs.Compared to the MG group,the YJG group had 16 upregulated miRNAs and six downregulated miRNAs,and the differences were statistically significant(all P<0.05).(3)Biological functions and pathways of differential miRNAs:Differential genes were mainly involved in the transcription regulation of various proteins in biological processes.The pathways associated with target genes were mainly tumor-related signaling pathways and lipid metabolism-related pathways.(4)PCR validation:In the MG group,the expression of miR-673-3p in the retina was lower than that in the CG group,and the expression of the remaining 11 miRNAs was higher than that in the CG group.In the YJG group,the expression of miR-673-3p and miR-23b-5p in the retina was higher than that in the MG group,and the expression of the remaining 10 miRNAs was lower than that in the MG group,and the differences were statistically significant(all P<0.05).Except for miR-23b-5p,the other 11 miRNAs were consistent with the results of the above RNA detection.CONCLUSIONS The miR-673,miR-1,miR-133,miR-214,miR-23b,miR-31a,miR-451,etc.,may be target miRNAs for Yijing Decoction intervention in early microvascular damage in DR.
作者
王丽
邱心悦
孟春
刘光辉
WANG Li;QIU Xinyue;MENG Chun;LIU Guanghui(The People's Hospital Affiliated to Fujian University of Traditional Chinese Medicine,Fuzhou 350004,China)
出处
《中国中医眼科杂志》
2024年第3期205-213,共9页
China Journal of Chinese Ophthalmology
基金
福建省自然科学基金项目(2023J02840,2023J01852)
福建省卫生健康委员会中青年骨干项目(2019-ZQNB-12)。
