BMAL1 downregulation under aging and HFD conditions promotes NASH progression by binding to HIF-1α and modulating the glycolysis-NLRP3 inflammasome axis.
Key Findings
Results
Aged HFD-fed mice exhibited more severe NASH phenotypes than young HFD-fed mice.
Aged mice were 18 months old and young mice were 6 weeks old; both groups were fed a high-fat diet (HFD) for 16 weeks to induce NASH.
Aged HFD-fed mice showed elevated NLRP3 inflammasome activity, enhanced glycolysis, and reduced BMAL1 expression compared to young HFD-fed mice.
Aged mice displayed more pronounced lipid accumulation, inflammation, oxidative stress, and fibrosis in liver tissue.
Results
Transcriptomic analysis identified NLRP3-related signaling and circadian rhythm pathways as central contributors to age-specific NASH pathogenesis.
Gene expression profiling of liver tissue was performed using RNA sequencing to identify molecular signatures.
Both NLRP3-related signaling pathways and circadian rhythm pathways were highlighted as key differentially regulated pathways in aged versus young HFD-fed mice.
BMAL1, a core circadian rhythm gene, was identified as downregulated in aged HFD-fed mice.
Results
BMAL1 directly bound to HIF-1α, thereby suppressing glycolysis in hepatocytes.
The interaction between BMAL1 and HIF-1α was validated using molecular docking and co-immunoprecipitation (Co-IP) assays.
Treatment with the HIF-1α inhibitor 2-methoxyestradiol (2-ME2) phenocopied the effects of BMAL1 overexpression on glycolysis suppression.
Results
BMAL1 overexpression combined with glycolysis or HIF-1α inhibition significantly attenuated NLRP3 inflammasome activation and NASH-related pathologies in senescent hepatocytes.
In vitro, BMAL1 overexpression plasmids were transfected into AML-12 cells co-treated with H2O2 and free fatty acid (FFA) to model senescent NASH.
Treatment with 2-deoxy-D-glucose (2-DG, a glycolysis inhibitor) or 2-methoxyestradiol (2-ME2, a HIF-1α inhibitor) alongside BMAL1 overexpression significantly downregulated NLRP3 expression.
Combined interventions attenuated lipid accumulation, inflammation, oxidative stress, and fibrosis in senescent NASH cells.
Results
Hepatocyte-specific BMAL1 overexpression in aged HFD-fed mice inhibited glycolysis and NLRP3 activation, resulting in improvement of NASH-related pathologies.
Hepatocyte-specific BMAL1 overexpression was achieved in aged HFD-fed mice through adeno-associated virus serotype 8 (AAV8) delivery.
AAV8-mediated BMAL1 overexpression markedly inhibited glycolysis in liver tissue of aged HFD-fed mice.
NLRP3 inflammasome activation was reduced following hepatocyte-specific BMAL1 restoration, and NASH-related pathologies including lipid accumulation, inflammation, and fibrosis were improved.
Methods
A senescence-associated cellular model of NASH was established using co-treatment of murine hepatocyte AML-12 cells with H2O2 and free fatty acid (FFA).
AML-12 murine hepatocytes were used as the in vitro model.
H2O2 was used to induce cellular senescence and FFA was used to induce steatosis, together replicating an aged NASH cellular environment.
This model was used to test the effects of BMAL1 overexpression and pharmacological inhibition of glycolysis and HIF-1α.
Background
Epidemiological evidence indicates higher incidence and mortality rate of NASH in the elderly population compared to younger groups.
The study cites epidemiological studies demonstrating age-related differences in NASH incidence and mortality.
The mechanisms underlying age-related exacerbation of NASH were described as poorly understood prior to this study.