KLF10 overexpression alleviates neuroinflammation and anxiety-depressive disorder in rats by downregulating the NF-κB/NLRP3 pathway

doi:10.12122/j.issn.1673-4254.2026.05.03

Abstract

Abstract:

Objective To elucidate the neuroimmune regulatory mechanism of the circadian rhythm gene KLF10 as a biomarker for anxiety depressive disorder (ADD). Methods The differentially expressed circadian rhythm genes were screened using human peripheral blood gene chip data from the GEO database (including 64 healthy individuals, 62 patients with major depressive disorder [MDD], and 59 with ADD) in conjunction with the MSigDB database. Weighted gene co-expression network analysis and machine learning models were employed to identify the core genes, followed by KEGG pathway enrichment analysis and evaluation of their diagnostic efficacy using ROC curves. In a male SD rat model of ADD induced by chronic restraint and corticosterone stress, the changes in depressive-like behaviors, hippocampal and amygdala pathologies, levels of inflammatory and pro-inflammatory cytokines, co-localization of KLF10 and p-p65 expression, and expression levels of NF‑κB/NLRP3 pathway molecules were examined following stereotactic AAV virus injection into the lateral ventricle for KLF10 overexpression. Results Compared with healthy individuals, the depressive patients showed differential expressions of 9 circadian rhythm genes. Compared with the MDD patients, the patients with ADD had significantly higher immune infiltration scores with upregulated NOD-like receptor and NF‑κB signaling pathways. The specific biomarker KLF10 demonstrated a diagnostic efficacy of 0.885. In the rat models of AOD, KLF10 overexpression significantly ameliorated depression- and anxiety-like behaviors, restored the balance between the pro- and anti-inflammatory cytokines, and improved hippocampal and amygdala pathologies. KLF10 overexpression also markedly upregulated NFKBIA mRNA, downregulated NLRP3 and RELA mRNAs, and reduced protein expressions of p-IκB‑α, p-p65, and NLRP3 in the brain tissues of the rats. Conclusion KLF10 overexpression ameliorates ADD behaviors in rats by inhibiting hippocampal-amygdala inflammation via downregulating the NF‑κB/NLRP3 pathway, suggesting the potential of KLF10 as a diagnostic biomarker and therapeutic target for AOD.

Key words: anxiety-depressive disorder, neuro-inflammation, NF?κB/NLRP3 pathway, circadian rhythm genes

Anlan LIU, Weifeng GUO, Jianxiang LI, Tiange ZHANG, Dan XU. KLF10 overexpression alleviates neuroinflammation and anxiety-depressive disorder in rats by downregulating the NF-κB/NLRP3 pathway[J]. Journal of Southern Medical University, 2026, 46(5): 994-1005.

Figures/Tables 7

Tab.1 Primer sequencs for qRT-PCR

Primer	Sequence (5'→3')	Length (bp)	Tm (℃)
m-β-actin-F	TGCTATGTTGCCCTAGACTTCG	22	61.4
m-β-actin-R	GTTGGCATAGAGGTCTTTACGG	22	60.8
m-NFKBIA-F	CTCCGAGACTTTCGAGGAAATAC	23	60.2
m-NFKBIA-R	GCCATTGTAGTTGGTAGCCTTCA	23	61.4
m-RELA-F	ATGTGGAGATCATTGAGCAGC	21	60.2
m-RELA-R	CCTGGTCCTGTGTAGCCATT	20	60.3
m-NLRP3-F	ATCAACAGGCGAGACCTCTG	20	61.3
m-NLRP3-R	GTCCTCCTGGCATACCATAGA	21	60.2
m-CASP1-F	TTTCCGCAAGGTTCGATTTTCA	22	61.0
m-CASP1-R	GGCATCTGCGCTCTACCATC	20	61.8
m-IL1B-F	GAAATGCCACCTTTTGACAGTG	22	60.2
m-IL1B-R	TGGATGCTCTCATCAGGACAG	21	61.0

Tab.2 Demographic data of the healthy individuals and patients with depression included in this study ［n (%)］

Item	Control	MDD	ADD
Gender
Male	33 (51.56)	24 (37.50)	19 (29.69)
Female	31 (48.44)	40 (62.50)	45 (70.31)
Age (year)
18-30	20 (31.25)	26 (40.63)	28 (43.75)
30-45	23 (35.94)	19 (29.69)	21 (32.81)
45-60	21 (32.81)	17 (26.56)	10 (15.63)
Unknown	0 (0.00)	2 (3.13)	5 (7.81)
Disease duration (month)
<6	-	41 (64.06)	36 (56.25)
6-12	-	10 (15.63)	22 (34.38)
13-24	-	9 (14.06)	5 (7.81)
>24	-	4 (6.25)	1 (1.56)

Fig.1 Differences in expressions of circadian genes between ADD and MDD patients, and WGCNA of ADD. A: Volcano map of the differential genes (DEGs) between ADD and MDD patients. B: Venn diagram of CCGs and the DEGS between ADD and MDD patients. C: Circos plot showing DE-CCGs expression patterns and the distribution of their chromosomal locations. D: Heat map of CCG expression levels. E: Heat map of correlation analysis of DE-CCGs. F: Analysis of the scale-free fit index (left) and the mean connectivity (right) for selecting various soft-thresholding powers (β). G: Clustering dendrogram for genes in burn traits; each color below represents one co-expression gene module. H: Scatter plot of the key module. Each point in the scatter plot represents one gene. I: Heat map depicting correlations between modules, phenotypes and immune cells. *P<0.05, **P<0.01. Control group (n=64), MDD group (n=62), ADD group (n=59).

Fig.2 Relationship between circadian genes and immunity and functional enrichment pathways. A: Bar plot of the proportion of 22 types of immune cells in ADD patients. Column names are sample ID. B: Box plot of ImmuneScore and ESTIMATEScore in ADD and MDD patients. C: Box plot for the composition of immune cells in ADD and MDD patients. D: Heat map for correlation between crucial CCGs and 22 immune cells. E,F: KEGG pathway enrichment analysis of the up (E)/down (F) intersection genes in ADD patients compared with the healthy control (the red lines show the significance). *P<0.05, **P<0.01, ***P<0.001. MDD group (n=62), ADD group (n=59).

Fig.3 ADD-specific biomarkers and their diagnostic efficacy. A: Top key genes random forest importance sorting lollipop chart. B: Violin plots showing expression levels of the 25 key module genes in different groups. C: Venn diagram showing the intersection of diagnostic markers obtained using 3 algorithms (Wilcox-test was used to compare the differences). D: Validation of the prognostic model: ROC curve of two risk genes and their combination in ADD. E: Expression correlation of two key genes in ADD patients (P=8.06e-09, rpearson=0.65, 95% CI : 0.48-0.77). F: Relationship between two key CCGs and gene ontology of the key nodes in the core pathways. *P<0.05, **P<0.01, ***P<0.001. MDD group (n=62), ADD group (n=59).

Fig.4 Overexpression of KLF10 improves ADD behaviors and restors neuronal function and the pro-inflammatory/anti-inflammatory balance of the rat models. A: Timeline of SD rat modeling, brain stereotactic injection of AVV, and behavioral tests. B: Proportion of immobility time in forced swimming test. C: Total number of entries in the elevated plus maze test. D: Proportion of open arm activity time in elevated plus maze test. E: Percentage of open arm entries in the elevated plus maze test. F: Representative images of rat activity in the elevated plus maze. G: Representative images of Nissl staining in different groups (Scale bar=50 μm). H: ELISA of pro-inflammatory/anti-inflammatory cytokines in the serum. I: ELISA of pro-inflammatory/anti-inflammatory cytokines in the brain tissue. *P<0.05, **P<0.01, ***P<0.001 (n=10).

Fig.5 Effect of KLF10 overexpression on expressions of NF-κB/NLRP3 pathway signaling molecules in the hippocampus and amygdala of the rats. A: Immunofluorescence staining showing the expressions of KLF10 and p-p65 in the hippocampus and amygdala of the rats (Scale bar=50 μm). B: Average fluorescence intensity and proportions of KLF10- and p-p65-positive cells in the hippocampus and amygdala. C: mRNA expressions of key node molecules of the NF-κB/NLRP3 pathway. D: Western blotting of NF-κB/NLRP3 pathway key proteins. E: p-IκB, IκB, NF-κB p65, p-p65, NLRP3 protein expression levels. F: Relative quantification of p-IκB and IκB ontology, p-p65 and NF-κB p65 ontology. *P<0.05, **P<0.01, ***P<0.001, n=6.

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