Key Genes of ASTRAGALI RADIX and NOTOGINSENG RADIX ET RHIZOMA Compounds(AN)in Treating IRI-Induced AKI through RNA-Seq and WGCNA Analysis
Objective:This study aimed to study the key target genes of ASTRAGALI RADIX and NOTOGINSENG RADIX ET RHIZOMA Com-pounds(AN)in treating renal ischemia-reperfusion(IRI)-induced acute kidney injury(AKI)through high-throughput transcriptome se-quencing(RNA-Seq)and weighted gene co-expression network(WGCNA).Methods:A total of 27 C57BL/6 mice were randomly divided into the normal group,model control group,and treatment group(AN of 3.9 g/kg).The AKI model was established by blocking the renal blood supply for 45 minutes followed by reperfusion.After modeling,AN was administrated for three days.The renal function,renal pathologi-cal changes,and contents of inflammatory factors were determined to evaluate the therapeutic effect of AN on IRI-induced AKI.In addition,three randomly selected kidney tissue samples were selected in each group,and total RNA was extracted for high-throughput RNA-Seq.For the gene expression data obtained by RNA-Seq,the DESeq2 package in R was used to analyze the differentially expressed genes(DEGs),and the WGCNA package was used to determine the co-expression modules and key genes closely related to AN treatment of IRI-induced AKI.The screened key genes were verified by real-time polymerase chain reaction(PCR).The key genes in the co-expression module were further subjected to gene ontology(GO)and KEGG orthology-based annotation system(KOBAS)signaling pathway enrichment analysis,so as to de-scribe the biological functions of key genes and reveal the possible mechanism of AN in treating AKI.Results:Compared with the model con-trol group,the treatment group(AN of 3.9 g/kg)significantly reduced the serum levels of creatinine and urea nitrogen and the mRNA expres-sions of interleukin-1 β(Il1β),Il6,and tumor necrosis factor(Tnfα)in renal tissue of mice with IRI-induced AKI(P＜0.01)and improved the renal tubular injury.RNA-Seq results showed that compared with the model control group,259 genes changed significantly in the treatment group(AN of 3.9 g/kg).A total of 23 co-expression modules were obtained by WGCNA analysis,among which the black co-expression mod-ule genes were significantly negatively correlated with IRI-induced AKI treated by AN(r=0.78,P=7×10 4).The core genes in the 84 black co-expression modules were intersected with the DEGs of the treatment group(AN of 3.9 g/kg)and the model control group to obtain the key gene,namely B4galt5.The protein-protein interaction(PPI)network analysis of the core protein of the black co-expression modules obtained the key genes including Adrm1,Ubqln1,Psmd4,and Psmc2.The five key genes verified by RT-PCR were consistent with the result of RNA-Seq.GO and KOBAS analysis showed that the key genes were mainly related to protein glycation modification and proteasome-mediated regulated metabolic processes.Conclusion:AN may regulate glycation modification or proteasome-related metabolic pathways to ameliorate IRI-induced AKI by targeting key genes such as B4galt5,Adrm1,Ubqln1,Psmd4,and Psmc2.
ASTRAGALI RADIX and NOTOGINSENG RADIX ET RHIZOMA CompoundsAcute kidney injury(AKI)Renal ischemia-reperfusionRNA-SeqWeighted gene co-expression network(WGCNA)
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