Zhi-zi-chi decoction mitigates depression by enhancing lncRNA Six3os1 expression and promoting histone H3K4 methylation at the BDNF promoter

Abstract

Traditional Chinese medicine, particularly Zhi-zi-chi decoction (ZZCD), is gaining recognition as a potential treatment for depression. This study aimed to uncover the molecular mechanisms behind ZZCD's antidepressant effects, focusing on lncRNA Six3os1 and histone H3K4 methylation at the BDNF promoter. Network pharmacology and in vivo experiments were conducted to identify ZZCD targets and evaluate its impact on depression-related behaviours and neuron injury. The role of Six3os1 in recruiting KMT2A to the BDNF promoter and its effects on oxidative stress and neuron injury were investigated. ZZCD reduced depression-like behaviours and neuron injury in mice subjected to chronic stress. It upregulated Six3os1, which facilitated KMT2A recruitment to the BDNF promoter, leading to increased histone H3K4 methylation and enhanced BDNF expression. ZZCD also inhibited CORT-induced neuron injury, inflammatory response and oxidative stress in vitro. ZZCD's antidepressant properties involve Six3os1 upregulation, which exerts neuroprotective effects by inhibiting oxidative stress and neuron injury, thereby alleviating depressive symptoms. Targeting Six3os1 upregulation may offer a potential therapeutic intervention for depression.

Keywords: BDNF; KMT2A; LncRNA Six3os1; Zhi‐zi‐chi decoction; anti‐depressive mechanism; histone methylation; neuron injury; traditional Chinese medicine.

FIGURE 1

ZZCD ameliorates depression‐like behaviours and neuron injury in CUMS mice. (A) Sucrose preference of CUMS mice treated with low‐, medium‐ and high‐dose ZZCD tested by SPT. (B) Retention time in the open field centre of CUMS mice treated with low‐, medium‐ and high‐dose ZZCD tested by OFT. (C) Immobility time of tail suspension of CUMS mice treated with low‐, medium‐ and high‐dose ZZCD tested by TST. (D) Immobility time of forced swimming of CUMS mice treated with low‐, medium‐ and high‐dose ZZCD tested by FST. (E) Body weight of CUMS mice treated with low‐, medium‐ and high‐dose ZZCD. (F) Neuron injury in hippocampal tissues of CUMS mice treated with low‐, medium‐ and high‐dose ZZCD analysed by HE, Nissl and TUNEL staining. (G) Statistical analysis of pathological scores of hippocampal tissues of CUMS mice treated with low‐, medium‐ and high‐dose ZZCD. (H) Statistical analysis of Nissl‐positive neurons in hippocampal tissues of CUMS mice treated with low‐, medium‐ and high‐dose ZZCD. (I) Statistical analysis of TUNEL‐positive neurons in the hippocampal tissue of CUMS mice treated with low‐, medium‐ and high‐dose ZZCD. n = 12 mice for each treatment. *p < 0.05 compared with control mice, #p < 0.05 compared with untreated CUMS mice.

FIGURE 2

Network pharmacology and bioinformatics analysis of BDNF significance in ZZCD treating depression. (A) Venn diagram of the target genes related to depression retrieved from the GeneCards and DisGeNET databases. (B) Venn diagram of the depression‐related target genes with the target genes of active ingredients. (C) A network diagram of active ingredients and target genes. The left circle is the active ingredients of ZZ (blue) and DDC (red), and the right node is the target gene. (D) PPI network of the 40 candidate target genes constructed by STRING database. Different colour nodes represent proteins encoded by genes, and the connection between nodes represents the interaction between proteins. (E) Interaction network of 40 candidate target genes drawn by Cytoscape software. The yellow gene represents the candidate target gene filtered by the CytoNCA plug‐in. (F) Interaction network of 12 candidate target genes filtered by CytoNCA plug‐in. (G) KEGG pathway enrichment analysis of 40 candidate target genes. The abscissa represents Gene ratio, and different colours represent the p value, with redder colour reflecting lower p values and more significant difference. The dot size indicates the number of entry identifiers of genes, with larger dot representing more entry identifiers. (H) A box plot of the differential expression of BDNF gene in eight normal samples and eight disease samples in the GSE84183 dataset. (I) BDNF expression in the hippocampal tissue of CUMS mice and those treated with low‐, medium‐ and high‐dose ZZCD examined by RT‐qPCR. (J) Western blot of BDNF protein in the hippocampal tissue of CUMS mice and those treated with low‐, medium‐ and high‐dose ZZCD. n = 12 mice for each treatment. *p < 0.05 compared with control mice, #p < 0.05 compared with untreated CUMS mice.

FIGURE 3

ZZCD represses CORT‐induced hippocampal neuron injury and upregulates BDNF expression. (A) Viability of hippocampal neurons treated with ZZCD at varied concentrations detected by CCK‐8 assay. (B) Viability of CORT‐stimulated hippocampal neurons treated with ZZCD at varied concentrations detected by CCK‐8 assay. (C) BDNF expression in CORT‐stimulated hippocampal neurons treated with ZZCD detected by RT‐qPCR. (D) Western blot of BDNF protein in the CORT‐stimulated hippocampal neurons treated with ZZCD. (E) Viability of CORT‐stimulated hippocampal neurons treated with ZZCD detected by CCK‐8 assay. (F) Apoptosis of CORT‐stimulated hippocampal neurons treated with ZZCD detected by flow cytometry. (G) Levels of TNF‐α, IL‐1β and IL‐6 in the supernatant of CORT‐stimulated hippocampal neurons treated with ZZCD detected by ELISA. (H), SOD, GSH and CAT activities and MDA production in the supernatant of CORT‐stimulated hippocampal neurons treated with ZZCD detected by ELISA. *p < 0.05 compared with control neurons, #p < 0.05 compared with untreated CORT‐stimulated neurons. The cell experiment was repeated three times.

FIGURE 4

Six3os1 increases BDNF expression by recruiting KMT2A to the BDNF promoter and promoting histone H3K4me3. (A) Six3os1 expression in the hippocampal tissue of control and CUMS mice detected by RT‐qPCR. *p < 0.05 compared with control neurons, # p < 0.05 compared with untreated CORT‐stimulated neurons, (B) Correlation analysis of Six3os1 and BDNF expression, with 12 mice in each group. (C) H3K4me3 modification in the BDNF promoter region analysed by UCSC database. (D) Venn diagram of genes encoding H3K4me3‐associated transferases in the GSEA‐MSigDB database. (E) The binding between KMT2A and Six3os1 predicted by catRAPID website. (F) Six3os1 expression in primary hippocampal neurons treated with oe‐Six3os1 or sh‐Six3os1 detected by RT‐qPCR. *p < 0.05 compared with neurons transduced with oe‐NC, #p < 0.05 compared with neurons transduced with sh‐NC. (G) Western blot of BDNF and H3K4me3 proteins in primary hippocampal neurons treated with sh‐Six3os1.*p < 0.05 compared with neurons transduced with oe‐NC, #p < 0.05 compared with neurons transduced with sh‐NC. (H) RIP assay of Six3os1 binding to KMT2A protein in primary hippocampal neurons. *p < 0.05 compared with IgG. (I) ChIP assay of KMT2A and H3K4me3 enrichment in the BDNF promoter region in primary hippocampal neurons. (J) RNA pull‐down experiment to detect the binding of Six3os1 and KMT2A promoter in primary hippocampal neurons. *p < 0.05 compared with neurons transduced with oe‐NC or Bio‐probe‐NC, #p < 0.05 compared with neurons transduced with sh‐NC. The cell experiment was repeated three times.

FIGURE 5

Six3os1 elevates BDNF expression and alleviates CORT‐induced hippocampal neuron injury. (A) BDNF knockdown efficiency in hippocampal neurons detected by RT‐qPCR. *p < 0.05 compared with neurons transduced with sh‐NC. (B) Six3os1 and BDNF expression in CORT‐stimulated hippocampal neurons treated with oe‐Six3os1 or combined with sh‐BDNF detected by RT‐qPCR. (C) Western blot of BDNF protein in CORT‐stimulated hippocampal neurons treated with oe‐Six3os1 or combined with sh‐BDNF. (D) CCK‐8 assay of cell viability of CORT‐stimulated hippocampal neurons treated with oe‐Six3os1 or combined with sh‐BDNF. (E) Apoptosis of CORT‐stimulated hippocampal neurons treated with oe‐Six3os1 or combined with sh‐BDNF measured by flow cytometry. (F) Levels of TNF‐α, IL‐1β and IL‐6 in the supernatant of CORT‐stimulated hippocampal neurons treated with oe‐Six3os1 or combined with sh‐BDNF detected by ELISA. (G) SOD, GSH and CAT activities and MDA production in the supernatant of CORT‐stimulated hippocampal neurons treated with oe‐Six3os1 or combined with sh‐BDNF detected by ELISA. *p < 0.05 compared with CORT‐stimulated hippocampal neurons treated with oe‐NC + sh‐NC, #p < 0.05 compared with neurons transduced with CORT‐stimulated hippocampal neurons treated with oe‐Six3os1 + sh‐NC. The cell experiment was repeated three times.

FIGURE 6

ZZCD represses CORT‐induced hippocampal neuron injury by upregulating the Six3os1/BDNF axis. CORT‐stimulated hippocampal neurons were treated with ZZCD + sh‐NC + oe‐NC, ZZCD + sh‐Six3os1 + oe‐NC or ZZCD + sh‐Six3os1 + oe‐BDNF. (A) Expression of Six3os1 and BDNF in hippocampal neurons detected by RT‐qPCR. (B) Western blot of BDNF and H3K4me3 proteins in hippocampal neurons. (C) Viability of hippocampal neurons detected by CCK‐8 assay. (D) Apoptosis of hippocampal neurons detected by flow cytometry. (E) Levels of TNF‐α, IL‐1β and IL‐6 in the supernatant of hippocampal neurons detected by ELISA. (F) SOD, GSH and CAT activities and MDA production in the supernatant of hippocampal neurons detected by ELISA. *p < 0.05 compared with CORT‐stimulated hippocampal neurons treated with oe‐NC + sh‐NC, #p < 0.05 compared with CORT‐stimulated hippocampal neurons treated with ZZCD + oe‐NC + sh‐NC, &p < 0.05 compared with CORT‐stimulated hippocampal neurons treated with. ZZCD + oe‐Six3os1 + sh‐NC. The cell experiment was repeated three times.

FIGURE 7

ZZCD alleviates depression‐like behaviours and neuron injury in mice by upregulating the Six3os1/BDNF axis. CUMS mice were treated with ZZCD + sh‐NC + oe‐NC, ZZCD + sh‐Six3os1 + oe‐NC or ZZCD + sh‐Six3os1 + oe‐BDNF. (A) Sucrose preference of CUMS mice tested by SPT. (B) Retention time in the open field centre of CUMS mice tested by OFT. (C) Immobility time of tail suspension of CUMS mice tested by TST. (D) Immobility time of forced swimming of CUMS mice tested by FST. (E) Body weight of CUMS mice. (F) Neuron injury in hippocampal tissues of CUMS mice analysed by HE, Nissl and TUNEL staining. (G) Statistical analysis of pathological scores of hippocampal tissues of CUMS mice. (H) Statistical analysis of Nissl‐positive neurons in hippocampal tissues of CUMS mice. (I) Statistical analysis of TUNEL‐positive neurons in the hippocampal tissue of CUMS mice. (J) Expression of Six3os1 and BDNF in hippocampal tissues of CUMS mice detected by RT‐qPCR. (K) Western blot of BDNF and H3K4me3 proteins in hippocampal tissues of CUMS mice. (L) Levels of TNF‐α, IL‐1β and IL‐6 in mouse serum detected by ELISA. (M) SOD, GSH and CAT activities and MDA production in mouse serum detected by ELISA. n = 12 mice for each treatment. *p < 0.05 compared with control mice treated with oe‐NC + sh‐NC, #p < 0.05 compared with CUMS mice treated with oe‐NC + sh‐NC, &p < 0.05 compared with CUMS mice treated with ZZCD + oe‐NC + sh‐NC, ^p < 0.05 compared with CUMS mice treated with. ZZCD + oe‐Six3os1 + sh‐NC.