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 LWDHD (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 doses 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 behavioral 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 expression 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 microarray analysis. Differentially expressed lncRNAs, miRNAs, and mRNAs were screened according 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 upstream lncRNAs and downstream mRNAs were predicted using starBase/miRcode and TargetScan/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, NONMMUT108543.1, 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 polymerase 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 latency, fewer platform crossings, and less time spent in the target quadrant. For the hippocampal 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 significantly increased (P < 0.01), and HE staining revealed marked pathological damage in the hippocampus 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 expression 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 expression was reversed by LWDHD-H treatment were identified. RT-qPCR showed that in the model 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 expression 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 expression 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 network 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 enrichment analysis revealed significant enrichment in leukocyte transendothelial migration, mitophagy, and steroid hormone biosynthesis pathways. GO functional analysis revealed enriched 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.