荔枝核总黄酮对肝纤维化大鼠lncRNAs、mRNAs表达的影响和生物学功能分析

Analysis of the effects of total flavonoids on differential lncRNAs and mRNAs based on high-throughput sequencing technology

目的基于高通量测序技术探讨荔枝核总黄酮(TFL)对肝纤维化大鼠差异lncRNAs、mRNAs表达的影响及其生物学功能分析。方法2021年4—7月于广西中医药大学实验动物中心进行实验,建立肝纤维化大鼠模型,将大鼠随机分为对照组(Control,n=6)、模型组(Model,n=7)和荔枝核总黄酮组(TFL,n=7),TFL组大鼠给予TFL 50 mg·kg-1·d-1干预6周。采用HE和Masson染色观察肝脏组织纤维化改变,ELISA法测定血清透明质酸酶(HA)、Ⅳ型胶原(Ⅳ-C)、层黏连蛋白(LN)、Ⅲ型前胶原(PC-Ⅲ),RNA-seq高通量测序技术筛选Model和TFL组差异表达lncRNAs和mRNAs,cis方式预测差异表达lncRNAs靶基因,GO和KEGG对各差异表达lncRNAs靶基因和mRNAs进行生物学功能富集分析。结果纤维化评分比较,Model组>TFL组>Control组(F=14.420,P<0.001),大鼠血清HA、LN、Ⅳ-C和PCⅢ水平比较,Model组>TFL组>Control组(F=47.055、74.655、177.328、54.445,P均<0.001)。共筛选出Model组与TFL组差异表达lncRNAs 73个(上调43个,下调30个),Model组与TFL组差异表达mRNAs 261个(上调150个,下调111个),采用cis方式共预测到Model与TFL组24个靶基因;差异表达lncRNAs靶基因和mRNAs的生物学富集功能分析显示TFL通过参与昼夜节律、谷胱甘肽代谢泛醌、戊糖磷酸途径等信号通路发挥抗肝纤维化的作用。结论TFL能够明显改善大鼠纤维化组织病理学形态,降低血清HA、Ⅳ-C、LN、PC-Ⅲ含量,其作用机制可能与调控特定差异lncRNAs和mRNAs表达,参与昼夜节律、谷胱甘肽代谢泛醌、戊糖磷酸途径等信号通路有关。

Objective To explore the effects of Total Flavonoids from Litchi Seed on the expression of differential lncRNAs and mRNAs in liver fibrosis rats using high-throughput sequencing technology and to analyze its biological functions.Methods Experiments was conducted at the experimental animal center of Guangxi University of Chinese Medicine from April 2021 to July 2021.A rat model of liver fibrosis was established,with rats randomly assigned to the control group(Control),model group(Model),and Total Flavonoids from Litchi Seed group(TFL).The TFL group was administered 50mg·kg-1·d-1 of TFL for a total of 6 weeks.HE and Masson staining were used to observe liver tissue fibrosis changes.Serum levels of hyaluronidase(HA),type IV collagen(Ⅳ-C),laminin(LN),and type III procollagen(PC III)were measured using ELISA.RNA-seq high-throughput sequencing technology was used to identify differentially expressed lncRNAs and mRNAs between the Model and TFL groups.The cis method was used to predict target genes of differentially expressed lncRNAs.GO and KEGG analyses were conducted for biological function enrichment of the differentially expressed lncRNAs target genes and mRNAs.Results Compared with the Model group,the TFL group exhibited a significant decrease in liver fibrosis score(F=14.420,P<0.001)and serum levels of HA,Ⅳ-C,LN,and PC-Ⅲ(F=47.055,74.655,177.328,54.445,P<0.001).A total of 73 differentially expressed lncRNAs(43 up-regulated,30 down-regulated)were identified between the Model and TFL groups,along with 261 differentially expressed mRNAs(150 up-regulated,111 down-regulated).Using the cis method,24 target genes were predicted between the Model and TFL groups.Biological function enrichment analysis indicated that TFL plays an anti-liver fibrosis role by participating in signaling pathways such as circadian rhythm,glutathione metabolism,ubiquinone,and pentose phosphate.Conclusion TFL significantly improved the pathological morphology of fibrotic tissue in rats,reduced serum levels of HA,Ⅳ-C,LN,and PC-III,and exerted an anti-liver fibrosis effect.The mechanism of action may involve regulating the expression of specific differential lncRNAs and mRNAs and participating in signaling pathways such as circadian rhythms,glutathione metabolism,ubiquinone,and pentose phosphate.