数字中医药（英文）2026，Vol.9Issue(3)：442-457,16.DOI:10.1016/j.dcmed.2026.08.009
基于lncRNA-miRNA-mRNA 网络分析的六味地黄汤改善D-半乳糖诱导衰老小鼠认知功能障碍的分子机制
The molecular mechanisms of Liuwei Dihuang Decoction in improving cognitive dysfunction in D-galactose-induced aged mice revealed by an lncRNA‒miRNA‒mRNA network analysis
摘要
Abstract
Objective To elucidate the potential mechanisms by which Liuwei Dihuang Decoction(六味地 黄 汤,LWDHD)ameliorates D-galactose(D-gal)-induced cognitive impairment in aged mice through a long noncoding RNA(lncRNA)-micro RNA(miRNA)-messenger RNA(mRNA)regulatory network analysis. Methods A total of 70 specific pathogen-free(SPF)male C57BL/6 mice(weighing 18-22 g)were randomly divided into control,model,low-dose LWDHD(LWDHD-L),high-dose LWD-HD(LWDHD-H),and positive control(vitamin E)groups(n=14 per group).An aged mouse model was established by subcutaneous injection of D-gal(100 mg/kg)once daily for 6 weeks.During this period,the LWDHD-L and LWDHD-H groups were administered LWDHD at dos-es of 10 and 20 g/kg,respectively,while the vitamin E group received 200 mg/kg of vitamin E by gavage at 10 mL/kg once daily.The Morris water maze(MWM)test was used to assess be-havioral changes in mice.A navigation test was conducted on day 5 to record escape latency,and a probe trial was performed on day 6 to record the number of platform crossings and the time spent in the target quadrant.Hematoxylin and eosin(HE)staining was used to observe pathological changes in the hippocampus.Immunohistochemistry was used to assess the ex-pression levels of glial fibrillary acidic protein(GFAP)and ionized calcium-binding adaptor molecule 1(IBA1)in the hippocampus to evaluate the efficacy of LWDHD treatment.Based on the efficacy results,the control,model,and LWDHD-H groups were selected for microar-ray analysis.Differentially expressed lncRNAs,miRNAs,and mRNAs were screened accord-ing to predefined fold-change and statistical significance criteria.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were subsequently performed.Three LWDHD-H-regulated miRNAs were selected as core nodes.Their up-stream lncRNAs and downstream mRNAs were predicted using starBase/miRcode and Tar-getScan/miRDB/miRTarBase,respectively,and candidate interactions were further screened by expression-correlation analysis and the Multiple Target MiRNA(MuTaMe)method.Significant lncRNA-miRNA-mRNA regulatory relationships were integrated and visualized using Cytoscape.The relative expression levels of three lncRNAs(NONMMUT096619.1,NON-MMUT108543.1,and NONMMUT051216.2),three miRNAs(mmu-miR-764-5p,mmu-miR-300-5p,and mmu-miR-8102),and three mRNAs(Itgb4,Dnmt3a,and Abca8a)in the control,model,and LWDHD-H groups were measured by reverse transcription quantitative poly-merase chain reaction(RT-qPCR)to validate the microarray results.In addition,the levels of tumor necrosis factor(TNF)-α,interleukin(IL)-1β,and IL-6 in the hippocampus and serum of aged mice were measured by enzyme-linked immunosorbent assay(ELISA)to evaluate the inhibitory effects of LWDHD on inflammatory cytokines. Results For behavioral assessment,mice in the model group exhibited prolonged escape la-tency,fewer platform crossings,and less time spent in the target quadrant.For the hippocam-pal pathological and glial activation assessment,the expression levels of GFAP and IBA1 in the dentate gyrus(DG)and cornu ammonis 3(CA3)regions of the hippocampus were signifi-cantly increased(P<0.01),and HE staining revealed marked pathological damage in the hip-pocampus in the model group.The LWDHD-L,LWDHD-H,and vitamin E groups effectively shortened the escape latency,increased the number of platform crossings,decreased the ex-pression levels of GFAP and IBA1 in the DG and CA3 regions of the hippocampus(P<0.05 or P<0.01),and ameliorated hippocampal pathological damage.The time spent in the target quadrant was significantly increased in the LWDHD-H and vitamin E groups compared with the model group(P<0.01).Compared with the LWDHD-L group,the LWDHD-H group showed a shorter escape latency and a longer time spent in the target quadrant(P<0.05 or P<0.01).Subsequently,99 lncRNAs,3 miRNAs,and 23 mRNAs whose dysregulated expres-sion was reversed by LWDHD-H treatment were identified.RT-qPCR showed that in the mod-el group,the expression levels of NONMMUT096619.1,NONMMUT108543.1,NONMMUT-051216.2,mmu-miR-764-5p,Itgb4,Dnmt3a,and Abca8a were upregulated,whereas that of mmu-miR-300-5p and mmu-miR-8102 were downregulated(P<0.01).In the LWDHD-H group,the expression levels of NONMMUT096619.1,NONMMUT108543.1,NONMMUT-051216.2,mmu-miR-764-5p,Itgb4,Dnmt3a,and Abca8a were downregulated,while the ex-pression levels of mmu-miR-300-5p and mmu-miR-8102 were upregulated.These expression trends were generally consistent with the microarray findings,and mmu-miR-8102 expres-sion level was significantly elevated in the LWDHD-H group compared with the model group(P<0.01).An LWDHD-H-associated hippocampal lncRNA-miRNA-mRNA regulatory net-work comprising 191 lncRNAs,3 miRNAs,and 68 mRNAs was constructed,with mmu-miR-764-5p,mmu-miR-300-5p,and mmu-miR-8102 serving as core regulatory nodes.KEGG en-richment analysis revealed significant enrichment in leukocyte transendothelial migration,mitophagy,and steroid hormone biosynthesis pathways.GO functional analysis revealed en-riched biological processes,including the I-κB kinase(IKK)/nuclear factor(NF)-κB signaling pathway.For inflammatory cytokine assessment,the levels of TNF-α,IL-1β,and IL-6 in the hippocampus and serum of the model group were significantly elevated(P<0.01),while the LWDHD-L,LWDHD-H,and vitamin E groups effectively reduced the levels of TNF-α,IL-1β,and IL-6 in the hippocampus and serum(P<0.05 or P<0.01).Compared with the LWDHD-L group,the LWDHD-H group showed a significant reduction in the levels of IL-1β and IL-6 in the hippocampus and serum(P<0.05 or P<0.01). Conclusion LWDHD effectively ameliorates D-gal-induced cognitive dysfunction in aged mice,and its mechanism may be associated with attenuation of neuroinflammation and modulation of the hippocampal lncRNA-miRNA-mRNA regulatory network.关键词
六味地黄汤/衰老小鼠/lncRNA-miRNA-mRNA 网络/海马体/认知功能/炎症Key words
Liuwei Dihuang Decoction/Aging mice/lncRNA-miRNA-mRNA network/Hippocampus/Cognitive function/Inflammation引用本文复制引用
李力松,易健,陈博威,欧阳银,曾繁佐,徐雅倩,刘柏炎..基于lncRNA-miRNA-mRNA 网络分析的六味地黄汤改善D-半乳糖诱导衰老小鼠认知功能障碍的分子机制[J].数字中医药（英文）,2026,9(3):442-457,16.基金项目
Hunan Provincial Natural Science Foundation Project(2025JJ80185),and Hunan Provincial Department of Edu-cation Scientific Research Project(23A0274). （2025JJ80185）
