Objective:

2

To investigate the therapeutic potential of pseudolaric acid B (PAB) on non-alcoholic fatty liver disease (NAFLD) and its underlying molecular mechanism

in vitro

and

in vivo

.

Methods:

2

Eight-week-old male C57BL/6J mice (

n

=32) were fed either a normal chow diet (NCD) or a high-fat diet (HFD) for 8 weeks. The HFD mice were divided into 3 groups according to a simple random method

including HFD

PAB low-dose [10 mg/(kg•d)

PAB-L

]

and PAB high-dose [20 mg/(kg•d)

PAB-H

]

groups. After 8 weeks of treatment

glucose metabolism and insulin resistance were assessed by oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). Biochemical assays were used to measure the serum and cellular levels of total cholesterol (TC)

triglycerides (TG)

aspartate aminotransferase (AST)

alanine aminotransferase (ALT)

low-density lipoprotein cholesterol (LDL-C)

and high-density lipoprotein cholesterol (HDL-C). White adipose tissue (WAT)

brown adipose tissue (BAT) and liver tissue were subjected to hematoxylin and eosin (H

&

E) staining or Oil Red O staining to observe the alterations in adipose tissue and liver injury. PharmMapper and DisGeNet were used to predict the NAFLD-related PAB targets. Peroxisome proliferator-activated receptor alpha (PPARα) pathway involvement was suggested by Kyoto Encyclopedia of Genes and Genomes (KEGG) and search tool Retrieval of Interacting Genes (STRING) analyses. Luciferase reporter assay

cellular thermal shift assay (CETSA)

and drug affinity responsive target stability assay (DARTS) were conducted to confirm direct binding of PAB with PPARα. Molecular dynamics simulations were

applied to further validate target engagement. RT-qPCR and Western blot were performed to assess the downstream genes and proteins expression

and validated by PPARα inhibitor MK886.

Results:

2

PAB significantly reduced serum TC

TG

LDL-C

AST

and ALT levels

and increased HDL-C level in HFD mice (

P

<

0.01). Target prediction analysis indicated a significant correlation between PAB and PPARα pathway. PAB direct target binding with PPARα was confirmed through luciferase reporter assay

CETSA

and DARTS (

P

<

0.05 or

P

<

0.01). The target engagement between PAB and PPARα protein was further confirmed by molecular dynamics simulations and the top 3 amino acid residues

LEU321

MET355

and PHE273 showed the most significant changes in mutational energy. Subsequently

PAB upregulated the genes expressions involved in lipid metabolism and mitochondrial biogenesis downstream of PPARα (

P

<

0.05 or

P

<

0.01). Significantly

the PPARα inhibitor MK886 effectively reversed the lipid-lowering and PPARα activation properties of PAB (

P

<

0.05 or

P

<

0.01).

Conclusion:

2

PAB mitigates lipid accumulation

ameliorates liver damage

and improves mitochondrial biogenesis by binding with PPARα

thus presenting a potential candidate for pharmaceutical development in the treatment of NAFLD.