
==== Front
Acta Pharm Sin B
Acta Pharm Sin B
Acta Pharmaceutica Sinica. B
2211-3835
2211-3843
Elsevier

S2211-3835(24)00191-6
10.1016/j.apsb.2024.05.014
Original Article
Schisanhenol ameliorates non-alcoholic fatty liver disease via inhibiting miR-802 activation of AMPK-mediated modulation of hepatic lipid metabolism
Li Bin a†
Xiao Qi a†
Zhao Hongmei b†
Zhang Jianuo a
Yang Chunyan a
Zou Yucen a
Zhang Bengang a
Liu Jiushi a
Sun Haitao htsun@implad.ac.cn
a⁎
Liu Haitao htliu@implad.ac.cn
a⁎
a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, China
b Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Department of Pathophysiology, Peking Union Medical College, Beijing 100005, China
⁎ Corresponding authors. htsun@implad.ac.cnhtliu@implad.ac.cn
† These authors made equal contributions to this work.

17 5 2024
9 2024
17 5 2024
14 9 39493963
14 1 2024
25 3 2024
29 4 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Non-alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, is a common metabolic liver disease worldwide. Currently, satisfactory drugs for NAFLD treatment remain lacking. Obesity and diabetes are the leading causes of NAFLD, and compounds with anti-obesity and anti-diabetic activities are considered suitable candidates for treating NAFLD. In this study, biochemical and histological assays revealed that a natural lignan schisanhenol (SAL) effectively decreased lipid accumulation and improved hepatic steatosis in free fatty acid (FFA)-treated HepG2 cells and high-fat diet (HFD)-induced NAFLD mice. Further, molecular analyses, microRNA (miRNA)-seq, and bioinformatics analyses revealed that SAL may improve NAFLD by targeting the miR-802/adenosine monophosphate-activated protein kinase (AMPK) pathway. Liver-specific overexpression of miR-802 in NAFLD mice significantly impaired SAL-mediated liver protection and decreased the protein levels of phosphorylated (p)-AMPK and PRKAB1. Dual-luciferase assay analysis further confirmed that miR-802 inhibits hepatic AMPK expression by binding to the 3' untranslated region of mouse Prkab1 or human PRKAA1. Additionally, genetic silencing of PRKAA1 blocked SAL-induced AMPK pathway activation in FFA-treated HepG2 cells. The results demonstrate that SAL is an effective drug candidate for treating NAFLD through regulating miR-802/AMPK-mediated lipid metabolism.

Graphical abstract

A natural compound, schisanhenol (SAL), exerts anti-NAFLD effects by inhibiting miR-802 and activating the AMPK signaling pathway to regulate lipid homeostasis.Image 1

KEY WORDS

Schisanhenol
Non-alcoholic fatty liver disease
Hepatic steatosis
Lipid metabolism
miR-802
AMPK
SREBP-1c
PPARα
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pmc1 Introduction

Non-alcoholic fatty liver disease (NAFLD), the most common chronic liver disease, is characterized by excessive lipid accumulation in hepatocytes1. NAFLD is a progressive spectrum of diseases, ranging from simple hepatic steatosis to nonalcoholic steatohepatitis, fibrosis, cirrhosis, and hepatocarcinoma2. Obesity and type 2 diabetes are the leading risk factors for NAFLD, and their increased prevalence has resulted in the NAFLD epidemic3. Currently, NAFLD affects more than 25% of the world population and is a global health concern4. Owing to the complex pathogenesis of NAFLD, currently satisfactory drug therapies for NAFLD remain lacking. Notably, therapeutic options are solely limited to lifestyle adjustments and indirect control drugs such as hypolipidaemic agents, insulin sensitizers, and hepatoprotectors5. However, prolonged adherence to these drugs is difficult for most patients and most indirect drugs are not always effective in preventing deterioration6. Consequently, developing new therapeutic drugs for NAFLD is urgently needed.

Although NAFLD pathogenesis remains unclear, lipid metabolism disorders are commonly involved initially and critically in its progression7. Increased de novo synthesis of fatty acids, the influx of lipids from adipose tissues, and reduced lipid decomposition can promote excessive lipid deposition in hepatocytes, resulting in hepatosteatosis8. Therefore, reversing hepatic lipid metabolism disorders may be an effective strategy for improving NALFD9. Adenosine monophosphate-activated protein kinase (AMPK), an important metabolic regulator, regulates glycolipid metabolism in hepatocytes10. Notably, hepatic AMPK, a factor that mainly contributes to excessive lipid deposition in the NAFLD, is attenuated in high-fat diet (HFD)-fed mice11. AMPK activation prevents hepatic steatosis by inhibiting fatty acid synthesis and enhancing fatty acid oxidation8. Furthermore, the loss of AMPK exacerbates the pathological features of NAFLD in liver-specific AMPK knockout mice12. Therefore, AMPK plays a key role in reducing lipid deposition and ameliorating hepatic steatosis.

Aberrantly expressed microRNAs (miRNAs) are associated with impaired AMPK function in NAFLD13. miRNAs are small noncoding RNAs that regulate post-transcriptional gene expression by promoting messenger RNA cleavage or inhibiting protein translation14. The microRNA expression profiles of patients with NAFLD and healthy individuals differ significantly, and aberrant microRNA expression critically contributes to NAFLD development and progression14, 15, 16. Further, clinical studies have suggested that certain miRNAs could be used as biomarkers for NAFLD diagnosis, prognosis, and treatment17,18. Notably, dysregulated miRNAs inhibit AMPK expression and activity in the liver, promoting NAFLD13. For instance, hepatic miR-33, miR-34a, miR-1224-5p, and miR-291b-3p are upregulated in NAFLD, resulting in deranged hepatic lipid metabolism and hepatic steatosis by repressing AMPK activity13,19, 20, 21. Conversely, inhibiting their overexpression improves AMPK activity, resulting in reduced lipid accumulation in the liver13. Therefore, the miRNA/AMPK axis may represent a potential therapeutic target for NAFLD treatment.

Natural products are important sources of new drugs, and compounds with anti-obesity and anti-diabetic effects may have therapeutic potential for NAFLD3. Schisanhenol (SAL), a biphenylcyclooctene-type lignan, is a main active ingredient of the Schisandraceous family and exhibits hepatoprotective, anti-obesity, anti-diabetic, neuroprotective, and anti-oxidant activities22, 23, 24, 25. SAL exhibits a good protective effect against CCl4-induced liver injury in vitro and ameliorates liver fibrosis by inhibiting hepatic stellate cell activation26,27. In addition, SAL analogues, such as deoxyschizandrin, schizandrin B, and gomisin J, exhibit good anti-NAFLD effects, and its analogue, gomisin M1, exerts hepatoprotective effects in hepatocellular carcinoma by modulating miR-2128, 29, 30, 31, 32. However, whether and how SAL ameliorates NAFLD remains unknown. The present study demonstrated that SAL administration effectively improved lipid accumulation and hepatic steatosis by activating the AMPK pathway in free fatty acid (FFA)-treated HepG2 cells and HFD-fed mice. Mechanistically, SAL downregulates hepatic miR-802 expression, which directly targets mouse Prkab1 or human PRKAA1, resulting in AMPK pathway activation and subsequent attenuation of hepatic lipogenesis. To our knowledge, SAL may be a novel therapeutic candidate for NAFLD.

2 Materials and methods

2.1 Chemicals and reagents

SAL (≥98% purity) was isolated from Schisandra chinensis fruit by our group (Supporting Information Figs. S1 and S2)22. Pioglitazone hydrochloride (PGZ) tablets were provided by Jiangsu Deyuan Pharmaceutical Co., Ltd. (Jiangsu, China). Alanine aminotransferase (ALT, #100020000) and aspartate aminotransferase (AST, #100020010) assay kits were purchased from Biosino Biotechnology and Science Inc. (Beijing, China). The kits used to detect triglycerides (TG, #BC0625), total cholesterol (TC, #BC1985), low-density lipoprotein cholesterol (LDL-C, #BC5335), and high-density lipoprotein cholesterol (HDL-C, #BC5325), and for Oil red O staining (#G1263) were provided by Beijing Solarbio Science Technology Co., Ltd. (Beijing, China). Dulbecco’s modified Eagle’s medium (#C11995500BT) and fetal bovine serum (FBS, #10099141) were purchased from Gibco (Carlsbad, CA, USA). The Cell Counting Kit-8 (CCK-8, #C0037) was purchased from Beyotime Biotechnology (Shanghai, China). The Bicinchoninic Acid (BCA) Protein Assay Kit (#23227) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The antibody specific for phospho-AMP-activated protein kinase alpha (p-AMPKThr172) was obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies specific for AMP-activated alpha 1 (PRKAA1, #10929-2-AP), PRKAA2 (#18167-1-AP), sterol regulatory element binding protein-1c (SREBP-1c, #14088-1-AP), acetyl-CoA carboxylase (ACC, #21923-1-AP), p-ACC (#29119-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #10494-1-AP) were purchased from Proteintech Biotechnology (Wuhan, China). AMP-activated beta 1 (PRKAB1, #ab32112) and peroxisome proliferator-activated receptor α (PPARα, #ab227074) were obtained from Abcam (Cambridge, UK). Adeno-associated virus 8 overexpressing miR-802-5p (AAV8-pre-miR-802-5p, represented as miR-802), AAV8 overexpressing miR-802 control (AAV8-GFP), AAV8 encoding miR-802-RNA interference (AAV8-anti-miR-802), and AAV8 encoding miR-802-RNA interference control (AAV8-ctrl) were synthesized by GeneChem (Shanghai, China). miR-802 and NC inhibitors were purchased from General Biosystems (Anhui, China).

2.2 Animals

Male C57BL/6J mice (18–22 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and housed under controlled temperature (22 ± 2 °C) and humidity (60 ± 5%) with a 12-h light/dark cycle. Food and water were freely available to all animals. Animal care and experimental protocols were conducted in compliance with the principles of the Institutional Animal Care and Use Committee at the Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China) in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).

2.3 Establishment and treatment of the NAFLD mouse model

The NAFLD mouse model was established after 1 week of acclimatization using a HFD (10% lard, 1% cholesterol, 10% egg yolk powder, 0.2% bile salts, and 78.8% basal feed). Mice in the control group were fed a normal diet. After 10 weeks, mice were randomly assigned to six groups (n = 10): control group, model group (HFD), PGZ group (HFD + 10 mg/kg PGZ), low-dose SAL group (SAL-L, HFD + 5 mg/kg), medium-dose SAL group (SAL-M, HFD + 10 mg/kg), and high-dose SAL group (SAL-H, HFD + 20 mg/kg). Mice in the treatment groups were gavaged daily with PGZ or SAL, whereas the control and model groups were administered the same volume of 0.5% CMC-Na for 4 weeks. After treatment, the mice were euthanized under anaesthesia (pentobarbital sodium) after overnight fasting. Serum was obtained by centrifuging blood samples at 6000 rpm for 15 min at 4 °C using a TLD-5013 centrifuge (Anke, Shanghai, China). The liver tissues were collected and divided into two parts. One part of the liver was fixed in 4% paraformaldehyde for histopathological examination, and the other was stored in liquid nitrogen for subsequent experiments.

2.4 AAV8 injection mouse model

After adaptive feeding, 70 mice were randomly divided into seven groups (n = 10): control group (normal diet), AAV8-control vectors (AAV8-NC) group (AAV8-ctrl + HFD or AAV8-GFP + HFD), SAL-H group (AAV8-NC + SAL-H + HFD), AAV8-anti-miR-802 group (AAV8-anti-miR-802 + HFD), AAV8-anti-miR-802 + SAL-H group (AAV8-anti-miR-802 + SAL-H + HFD), AAV8-pre-miR-802 group (AAV8-pre-miR-802 + HFD), AAV8-pre-miR-802 + SAL-H group (AAV8-pre-miR-802 + SAL-H + HFD). After 8 weeks of feeding a normal diet or HFD, HFD-fed mice were injected with AAV8-NC or AAV8-miR-802 (1.5 × 1012 v.g.) in 200 μL phosphate-buffered saline (PBS) via tail vein. Two weeks later, mice in the SAL-H, AAV8-anti-miR-802 + SAL-H, and AAV8-pre-miR-802 + SAL groups were administered SAL-H (20 mg/kg) by gavage once daily for 4 weeks, whereas the other groups were administered the same volume of 0.5% CMC-Na. Thereafter, the mice were sacrificed, and serum and liver samples were collected. Frozen sections were prepared from each liver tissue sample for fluorescence microscopy to determine the transfection efficiency.

2.5 Serum/hepatic biochemical assays

Serum levels of ALT and AST were determined using an AU480 fully automatic biochemical analyzer (Beckman). TG, TC, LDL-C, and HDL-C levels in the liver were measured using commercially available kits according to the manufacturer’s protocols.

2.6 Histopathological examination

A part of the harvested liver tissues was fixed in a 4% paraformaldehyde solution and embedded in paraffin. Paraffin-embedded liver sections were stained with hematoxylin and eosin (H&E) for pathological evaluation. Frozen liver sections were stained with Oil red O and Nile red to evaluate lipid droplet accumulation. The histological features of the liver tissues were observed and captured using the CaseViewer software (3DHISTECH, Hungary).

2.7 Small RNA high-throughput sequencing

Total RNA was extracted from the hepatic tissues of the mice from the control, HFD, and SAL-H groups (n = 3 per group) using TRIzol reagent (Invitrogen, #15596018CN). RNA quality was assessed using a NanoPhotometer spectrophotometer (Implen) and an Agilent Bioanalyzer 2100 System (Agilent Technologies). Library for miRNA sequencing was generated using the NEBNext Ultra™ small RNA Sample Library Prep Kit for Illumina (NEB) following the manufacturer’s instructions, and library quality was assessed using the Agilent Bioanalyzer 2100 System. Clustering of the index-coded samples was performed using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Thereafter, the library preparations were sequenced on the Illumina sequencing platform, and 50-bp single-end reads were generated. Raw data in fastQ were processed using custom Perl and Python scripts and then filtered based on Q20, Q30, and GC content. Next, the obtained clean reads were mapped to the mouse genome and aligned against miRBase to identify the mature miRNAs. The miRNA expression profiles of each sample were analyzed and normalized to transcripts per million using previously described methods33. DESeq2 (v1.16.1) was employed to determine differentially expressed miRNAs, with a significance threshold of P < 0.05 and absolute foldchange ≥2.

2.8 Cell culture and viability

The HepG2, LO2, and Hepa1c1c7 cells were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 1% penicillin/streptomycin, and cultured in an incubator at 37 °C and 5% CO2. The cells were seeded in 96-well plates and cultured overnight. Then, SAL (5, 10, and 20 μmol/L) was added, and plates were incubated again for 24 h. Three independent replicates were employed for each concentration in each group. Cell viability was determined using a CCK-8 assay.

2.9 Cell treatment and Oil red O staining

HepG2 cells were seeded on glass coverslips in 6-well plates and cultured to 80% confluence. Then, cells were exposed to 1 mmol/L FFA containing sodium oleate and sodium palmitate (2:1) in the presence or absence of SAL (5, 10, and 20 μmol/L). After 24 h, the cells were rinsed twice with PBS and fixed with 4% paraformaldehyde. Next, the cells were soaked in 60% isopropanol and stained with an Oil red O working solution. Stained cells were observed and photographed using an optical microscope. Semi-quantitative analysis was carried out using the ImageJ software. All experiments were repeated thrice.

2.10 Cellular TG and TC activity assays

Following treatment, the primary medium was discarded, and the cells were washed with PBS thrice. Cells were digested with trypsin and centrifuged to obtain the supernatant. The intracellular TG and TC contents in each well were measured using commercial kits following the manufacturer’s instructions. The protein levels in each well were quantified using a BCA protein assay kit. All experiments were repeated thrice.

2.11 Cell immunofluorescence assay

For immunofluorescence staining, HepG2 cells were fixed with 4% paraformaldehyde for 15 min and washed thrice with PBS. Subsequently, fixed cells were permeated with 0.3% Triton X-100 for 10 min. After rinsing, 10% goat serum in PBS was added, and the cells were blocked at room temperature for 1 h. Thereafter, the cells were incubated with p-AMPK antibody (1:200, Abcam, #ab32047) at 4 °C overnight. After washing with PBS, the cells were incubated with an Alexa Fluor 488-conjugated anti-rabbit IgG antibody for 1 h at room temperature. Next, the nuclei were stained with 4′,6-diamidino-2-phenylindole for 10 min. After rinsing thrice with PBS, the cells were stained with phalloidin (UElandy, #YP0052S) for 20 min. After washing with PBS, the cells were observed under a fluorescence microscope (Olympus), and the results were analyzed using ImageJ software. All experiments were repeated thrice.

2.12 Dual-luciferase reporter assay

The 3′ untranslated region (UTR) of mouse Prkab1 from the cDNA of mouse liver was amplified via PCR using the primers: forward, 5′-GGACTAGTTGTTGCTTGTTCCAAAAGAAGAGCTC-3′ and reverse, 5′-CCCAAGCTTCAAGTCAGGGTTTTGAAAACAGTAACAAAAG-3′. The Prkab1 3′ UTR mRNA was then inserted into the pMIR-Report Luciferase vector to generate the luciferase reporter vector. The Prkab1 3′ UTR mut plasmids (forward, 5′-GGACTAGTTGTTGCTTGTTCCAAAAGAAGAGCTC-3′ and mutation reverse, 5′-CCCAAGCTTCAAGTCAGGGTTTTGAAAAGACTTAGAAAAG-3′) were constructed through amplifying and inserting the 3′ UTR mut of the Prkab1 mRNA into the Luciferase vector. The human PRKAA1 3′ UTR and 3′ UTR mut plasmids (forward, 5′-GGACTAGTATAATGTTCCTGATGTTAACAGAAGACTG-3′; reverse, 5′-CCCAAGCTTGCAATATTTAAATATTTTCAAAATAAAACACAGTAACTAAAATG-3′ and mutation reverse, 5′-CCCAAGCTTGCAATATTTAAATATTTTCAAAATAAAACTCTGAATCTAAAATG-3′) were constructed through amplification using the human cDNA and inserting the 3′ UTR and 3′ UTR mut of the PRKAA1 mRNA into the Luciferase vector, respectively. Reporter assays were performed using mouse Hepa1c1c7 and human HepG2 cells. Cells were seeded in a 24-well plate and cultured for 24 h. Then, cells were transfected with 40 nmol/L miR-802 mimic or mimic control and co-transfected with 0.8 μg of wild-type (WT) 3′ UTR-luc or mutated-type (MUT) 3′ UTR-luc per well using the Lipofectamine 2000 System (Thermo Fisher Scientific). The pGL-4.74 vectors (0.1 μg/well) were co-transfected as the endogenous control for luciferase activity. The luciferase activity was determined after 24 h of transfection using the Dual Luciferase Reporter Gene Assay Kit (Beyotime, #RG029M).

2.13 Small interfering RNA (siRNA) transfection in cells

HepG2 cells (2 × 105 cells/well) were seeded in 6-well plates and classified into seven groups: control, FFA, FFA + SAL, FFA + siPRKAA1, FFA + siPRKAA1 + SAL, FFA + miR-802 inhibitor, and FFA + miR-802 inhibitor + SAL. Non-targeting siRNA and siRNA targeting PRKAA1 were transiently transfected into HepG2 cells using the EZ Cell Transfection Reagent (Life-iLab, #AC04L092) according to the manufacturer’s instructions. Transfecting miR-802 inhibitor or NC inhibitor into HepG2 cell was performed following the manufacturer’s protocol. Thereafter, cells were treated with 1 mmol/L FFA and/or SAL (20 μmol/L) for 24 h. Proteins were extracted from the cells for further analysis.

2.14 Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA from liver tissues or HepG2 cells was extracted using TRIzol reagent (Sigma, #T9424), and RNA purity was determined using a nanodrop (Allsheng). The RNA was reverse transcribed into cDNA using UEIris II RT-PCR System for First-Strand cDNA Synthesis Kit (US Everbright, #R2028). For miRNA, RNA was reverse transcribed using a miRNA 1st Strand cDNA Synthesis Kit (Vazyme, #MR101-02). qRT-PCR was performed using the Hieff® qPCR SYBR Green Master Mix (YEASEN, #11203ES03) on the QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific). Primer sequences for the mRNAs and miRNAs are listed in Supporting Information Table S1. Gapdh and U6 were used as internal reference genes. Relative transcript levels of the test genes were calculated using the 2−ΔΔCT method.

2.15 Protein extraction and Western blotting

Proteins were extracted from mouse liver and cells by RIPA lysis buffer (Beyotime Biotechnology) containing protease and phosphatase inhibitors. The BCA protein assay kit was used to measure protein concentration. Protein samples were separated using 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Subsequently, the membrane was blocked with 5% fat-free milk and incubated with primary antibodies against GAPDH (1:40,000), ACC (1:4000), p-ACC (1:1500), PPARα (1:2000), SREBP-1c (1:2500), PRKAA1 (1:6000), PRKAA2 (1:1200), and PRKAB1 (1:3000) overnight at 4 °C. Membranes were incubated with the corresponding secondary antibodies (1:5000) for 2 h at room temperature. Specific bands were visualized using an enhanced chemiluminescence reagent (Thermo Scientific) and captured using a BioSpectrum Gel Imaging System (UVP). Protein expression was normalized to that of GAPDH.

2.16 Statistical analysis

Experimental data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8.0. Statistical comparisons between different groups were performed using a one-way analysis of variance. P < 0.05 was considered statistically significant.

3 Results

3.1 SAL alleviates lipid accumulation in FFA-treated HepG2 cells

To evaluate the therapeutic effects of SAL on NAFLD, an FFA-treated HepG2 cell model was established. First, a CCK-8 assay was used to evaluate the cell viability of SAL treatment (5, 10, and 20 μmol/L) in the absence or presence of FFA-treated HepG2 cells and LO2 cells. The results indicated that the concentrations used in this study were safe (Fig. 1A and Supporting Information Fig. S3). Oil red O staining showed that SAL significantly decreased the FFA-treated cellular lipid accumulation in a dose-dependent manner (Fig. 1B). The lipid-lowering effect of SAL was further confirmed by measuring the TG levels in HepG2 cells (Fig. 1C). Notably, SAL dramatically decreased TC content at high doses (Fig. 1D). Therefore, these results suggested that SAL markedly attenuated FFA-treated lipid accumulation in HepG2 cells.Figure 1 Effect of SAL on lipid accumulation in FFA-treated HepG2 cells. (A) Effects of SAL (5, 10, and 20 μmol/L) on HepG2 cell viability with or without FFA. (B) Lipid droplet deposition in FFA-treated HepG2 cells was assessed using Oil red O staining. Effects of SAL (5, 10, and 20 μmol/L) on TG (C) and TC (D). The data are presented as the mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group.

Figure 1

3.2 SAL regulates lipid metabolism in FFA-treated HepG2 cells

Owing to the inhibitory effect of SAL on lipid accumulation, we assessed whether SAL affects lipid metabolism-related proteins and gene expression in FFA-treated HepG2 cells. Western blotting analysis showed that SAL treatment significantly decreased SREBP-1c expression and increased PPARα levels compared to those in FFA-treated HepG2 cells (Fig. 2A). Further, qRT-PCR analysis indicated that the upregulation of fat-related synthase genes (ACC, SCD1, FAS, DGAT1, and DGAT2) and the downregulation of lipolysis-related gene (CPT-1) in FFA-treated HepG2 cells were restored following SAL treatment (Fig. 2B). As AMPK is an important cellular energy sensor that regulates hepatic lipogenesis10, we further assessed p-AMPK and its downstream protein p-ACC expression to investigate whether SAL exerts its metabolic regulatory activity via the AMPK pathway. Immunofluorescence results showed that the fluorescence intensity of p-AMPK in FFA-treated HepG2 cells was significantly decreased, whereas SAL treatment dramatically recovered it (Fig. 2C). Western blotting revealed that FFA exposure reduced p-ACC levels, and this effect was significantly enhanced by SAL (Fig. 2D). Together, these data strongly indicate that SAL inhibits lipid accumulation and promotes β-oxidation via activating the AMPK pathway in FFA-treated hepatocytes.Figure 2 Effect of SAL on lipid accumulation through the AMPK pathway in FFA-treated HepG2 cells. (A) Representative Western blotting results and densitometric analysis of SREBP-1c and PPARα. (B) qRT-PCR analysis of ACC, SCD1, FAS, DGAT1, DGAT2, and CPT-1 gene expression. (C) Representative immunofluorescence images of p-AMPK (green) expression and Western blotting analysis of AMPK. Phalloidin (red) and 4ʹ,6-diamidino-2-phenylindole (DAPI, blue) were used as cell skeleton and nuclear counterstaining probes, respectively. (D) Representative Western blotting results and densitometric analysis of p-ACC. The results are expressed as the mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group.

Figure 2

3.3 SAL alleviates HFD-induced NAFLD in mice

To examine the effects of SAL on NAFLD, a mouse model was established by feeding mice an HFD for 10 weeks. Subsequently, SAL (5, 10, and 20 mg/kg) was administered orally for another 4 weeks. PGZ was used as a positive control. The histological staining results of H&E, Oil red O, and Nile red showed excessive lipid droplet accumulation, balloon-like changes, hepatocyte swelling and necrosis, and slight inflammatory cell infiltration in the liver of the model group (Fig. 3A). Notably, SAL administration improved these histological changes dose-dependently. In addition, serum ALT and AST and hepatic TG, TC, and LDL-C levels in the model group were remarkably increased, whereas the hepatic HDL-C content was considerably decreased (Fig. 3B). SAL treatment significantly decreased serum ALT and AST and hepatic TG, TC, and LDL-C levels and increased hepatic HDL-C levels compared to those in the model group. In addition, SAL treatment dose-dependently improved the HFD-induced impairment of insulin action and reduced serum IL-6 levels in NAFLD mice (Supporting Information Figs. S4 and S5). Therefore, SAL effectively improved lipid metabolism and liver function in NAFLD mice.Figure 3 SAL ameliorates hepatic steatosis in HFD-induced NAFLD mice. (A) Representative images of H&E, Oil red O, and Nile red staining of liver sections. (B) Serum ALT and AST and hepatic TG, TC, LDL-C, and HDL-C levels. The results are expressed as the mean ± SD (n = 6). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group.

Figure 3

3.4 SAL regulates lipid metabolism via activating the AMPK signaling pathway in NAFLD mice

The cell experiment results indicated AMPK as a key element in SAL, affecting lipid accumulation. Subsequently, in vivo experiments were conducted to confirm whether SAL attenuates hepatic steatosis through the AMPK pathway. Western blotting results showed that the protein levels of hepatic p-AMPK were significantly lower in the model group than in the control group (Fig. 4A). In contrast, SAL treatment considerably increased p-AMPK expression dose-dependently. The protein expression of p-ACC, SREBP-1c, and PPARα in the liver was determined to further explore the effect of SAL on the downstream signaling of AMPK. Consistent with the p-AMPK results, a significant reduction in p-ACC and PPARα levels was observed in the model group compared with the control group, whereas their levels were remarkably increased in SAL-treated mice. Conversely, hepatic SREBP-1c expression was significantly increased in the model group, whereas it was notably reduced following SAL treatment. Next, we evaluated the expression of the genes involved in lipogenesis and lipolysis. Notably, the expression of lipogenesis genes (Acc, Scd1, Fas, Dgat1, and Dgat2) was enhanced, and that of the lipolysis gene (Cpt-1) was dramatically decreased in the model group compared with that in the control group (Fig. 4B). SAL treatment inhibited Acc, Scd1, Fas, Dgat1, and Dgat2 and increased Cpt-1 expression compared with the model group, indicating that it regulates lipid metabolism by activating the AMPK pathway.Figure 4 SAL modulates lipid metabolism via the AMPK signaling pathway in HFD-induced NAFLD mice. (A) Representative Western blotting results and densitometric analysis of p-AMPK, AMPK, p-ACC, SREBP-1c, and PPARα (n = 3). (B) qRT-PCR analysis of Acc, Scd1, Fas, Dgat1, Dgat2, and Cpt-1 gene expression (n = 5). The results are expressed as the mean ± SD. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group.

Figure 4

3.5 SAL modulates miR-802 expression to improve lipid metabolism

To investigate whether miRNAs are involved in the regulatory effects of SAL on lipid accumulation in NAFLD mice, we compared the miRNA profiles of livers from the control, model, and SAL-H groups using miRNA-seq analysis. The replications of the control, model, and SAL-H groups showed satisfactory clustering within their groups, as demonstrated using the principal component analysis (Supporting Information Fig. S6). As shown in Fig. 5A, 51 miRNAs were differentially expressed in the livers of the model and control groups and 55 were differentially expressed between the SAL-H and model groups (Supporting Information Tables S2 and S3). A Venn diagram was used to summarise the differentially expressed miRNAs among groups, and 32 overlapping miRNAs were identified (Fig. 5B). Among them, 20 known mature miRNAs with the opposite expression trends were identified, including seven upregulated and 13 downregulated miRNAs. To determine the potential roles of these 20 miRNAs in ameliorating NAFLD, we predicted the target genes of each miRNA using the TargetScan database and enriched their signaling pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. As shown in Fig. 5C, the target genes of miR-802 were highly involved in lipid metabolism and the AMPK signaling pathway, indicating the role of miR-802 in alleviating hepatic lipid metabolism disorder in NAFLD mice via SAL. Subsequently, qRT-PCR was performed to determine hepatic miR-802 expression in each group. miR-802 expression was dramatically increased in the model group, and SAL-H treatment significantly reversed this increase (Fig. 5D), which was consistent with the detected and predicted data. Therefore, miR-802 may be an important target of SAL to improve lipid metabolism in NAFLD mice.Figure 5 SAL decreases hepatic miR-802 expression in HFD-induced NAFLD mice. (A) Volcanic plots showing the analysis of different genes. (B) Venn diagram verifying different genes among groups, including known mature miRNAs with the opposite expression trend. (C) KEGG signaling pathway analysis of miR-802 target genes. (D) qRT-PCR analysis of miR-802 expression in the livers of mice from the control, HFD, and SAL-H groups (n = 3 per group). The results are expressed as the mean ± SD. ###P < 0.001 vs. control group; ∗∗P < 0.01 vs. model group.

Figure 5

3.6 SAL alleviates HFD-induced NAFLD through miR-802 down-regulation

To verify the role of miR-802 in SAL-ameliorated lipid accumulation and hepatic steatosis, NAFLD mice were injected with AAV8 expressing or inhibiting miR-802 via the tail vein. Immunohistological analysis showed that more than 80% of hepatocytes of AAV8-anti-miR-802 and AAV8-pre-miR-802 transduced liver tissues display GFP positivity (Supporting Information Fig. S7A), suggesting high efficiency of transduction. Consistently, analysis of miR-802 expression showed that AAV8-anti-miR-802 or AAV8-pre-miR-802 effectively knocked down or overexpressed the miR-802 (Fig. S7B). H&E, Oil red O, and Nile red staining showed that miR-802 suppression in the liver reduced lipid droplet accumulation, balloon-like changes, hepatocyte swelling, and necrosis in NAFLD mice, whereas its overexpression exacerbated these pathological changes (Fig. 6A). Notably, hepatic miR-802 overexpression in NAFLD mice almost abolished the SAL-H-induced improvement in lipid accumulation and hepatic steatosis. Consequently, we examined the effects of miR-802 expression on hepatic TG, TC, LDL-C, and HDL-C levels in NAFLD mice. Consistent with the histological staining results, the abnormal levels of hepatic TG, TC, LDL-C, and HDL-C were effectively reversed by AAV8-anti-miR-802 treatment in NAFLD mice relative to AAV8-NC mice, whereas AAV8-pre-miR-802 treatment dramatically worsened these lipid metabolism-related indicators compared with AAV8-NC mice (Fig. 6B). Compared with the AAV8-NC group, SAL-H treatment resulted in significantly decreased levels of hepatic TG, TC, and LDL-C and increased hepatic HDL-C levels. Expectedly, miR-802 overexpression in the liver almost completely abolished these effects. Therefore, miR-802 downregulation is critical for alleviating HFD-induced NAFLD via SAL.Figure 6 SAL improves hepatic steatosis in HFD-treated mice through downregulating miR-802. (A) Representative images of H&E, Oil red O, and Nile red staining of liver sections. (B) Hepatic TG, TC, LDL-C, and HDL-C levels. The results are expressed as the mean ± SD (n = 6). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group; NS, no significance.

Figure 6

3.7 SAL promotes AMPK pathway activation by inhibiting miR-802 expression

Ppp2ca, Prkab1, and Pafah1b1, predicted miR-802 target genes, play important roles in lipid metabolism. qRT-PCR results showed that SAL-H treatment significantly decreased hepatic Prkab1 expression in HFD-induced NAFLD mice but did not affect Ppp2ca and Pafah1b1 expression (Supporting Information Fig. S8), suggesting that SAL activating the AMPK pathway may be achieved by attenuating miR-802-regulated Prkab1 expression. To further verify whether SAL promotes AMPK pathway activation by inhibiting miR-802 expression, we examined the expression of AMPK signaling pathway-related genes and proteins by transfecting NAFLD mice with AAV8-pre-miR-802 or AAV8-anti-miR-802. PRKAB1, also called AMPKβ1, is a β subunit of AMPK. miR-802 overexpression notably decreased Prkab1 expression but increased Acc and Fas expression in the livers of NAFLD mice (Fig. 7A). In contrast, miR-802 suppression significantly increased Prkab1 expression and decreased Acc and Fas expression. Similarly, SAL-H treatment significantly increased Prkab1 expression and decreased Acc and Fas expression, whereas miR-802 overexpression almost completely abolished these effects. Therefore, SAL potentially functions upstream of miR-802 and regulates Prkab1 expression. As we all know, PRKAB1 is a critical bioenergetic sensor that maintains energetic homeostasis via activating the α subunits34. We speculated that miR-802 regulates Prkab1 expression and activates PRKAA1 and PRKAA2 in hepatocytes. As expected, hepatic miR-802 knockdown remarkably increased PRKAB1, PRKAA1, and PRKAA2 levels in NAFLD mice, whereas their levels were decreased in the mice in the AAV8-pre-miR-802 group (Fig. 7B). Further, miR-802 overexpression dramatically decreased p-AMPK, p-ACC, and PPARα levels and enhanced SREBP-1c levels in NAFLD mice, whereas miR-802 suppression reversed these changes. Therefore, miR-802 plays a critical role in regulating hepatic lipid metabolism in NAFLD mice through AMPK pathway.Figure 7 SAL activates the AMPK pathway through miR-802 downregulation. (A) qRT-PCR analysis of Prkab1, Acc, and Fas genes expression (n = 5). (B) Representative Western blotting results and densitometric analysis of PRKAB1, PRKAA1, PRKAA2, p-AMPK, p-ACC, SREBP-1c, and PPARα (n = 3). The results are expressed as the mean ± SD. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group; NS, no significance.

Figure 7

3.8 miR-802 binds to Prkab1 and regulates its expression

To determine whether Prkab1 is a potential target of miR-802, we performed bioinformatics predictions of miRNA targets. Notably, mouse Prkab1 and human PRKAA1 exhibit putative miR-802 binding sites in 3' UTR. To confirm miR-802 and Prkab1/PRKAA1 binding, we conducted a luciferase reporter assay using WT or MUT 3' UTR of Prkab1/PRKAA1 (Fig. 8A and B). miR-802 overexpression dramatically decreased luciferase activity in Prkab1-WT, whereas no change was observed in Hepa1c1c7 cells stimulated with mutant 3' UTR. Similarly, in HepG2 cells, miR-802 mimic treatment resulted in the suppression of luciferase intensity in PRKAA1-WT, but this effect was not observed in PRKAA1-MUT, indicating that miR-802 directly targets mouse Prkab1 and human PRKAA1 and suppresses their expression.Figure 8 SAL activates AMPK via inhibiting miR-802 and its binding to PRKAA1 or Prkab1. (A) Targeted miR-802 and Prkab1/PRKAA1 WT or Prkab1/PRKAA1 Mut sequences. (B) Dual-luciferase reporter assay. (C) Representative Western blotting results and densitometric analysis of PRKAA1, p-AMPK, p-ACC, SREBP-1c, and PPARα. The results are expressed as the mean ± SD (n = 3–5). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group; NS, no significance.

Figure 8

3.9 SAL activates the AMPK signaling pathway by inhibiting miR-802 and its binding to PRKAA1

To assess whether SAL activates the AMPK signaling pathway through miR-802 modulation of PRKAA1, PRKAA1 was knocked down in HepG2 cells using siPRKAA1 transfection. Further, PRKAA1 knockdown was verified using Western blotting (Supporting Information Fig. S9). A significant reduction in PRKAA1 expression was observed in the model group compared with the control group, whereas SAL or/and miR-802 inhibitor treatment remarkably increased PRKAA1 expression (Fig. 8C). In contrast, siPRKAA1 treatment almost completely abolished the enhancement of SAL-induced PRKAA1 expression. Similarly, SAL treatment significantly increased p-AMPK, p-ACC, and PPARα expression and decreased SREBP-1c expression in FFA-treated HepG2 cells, whereas PRKAA1 knockdown reversed these changes. Therefore, PRKAA1 is crucial for AMPK pathway activation by SAL.

4 Discussion

Lipid metabolism dysfunction is the core pathological process involved in NAFLD occurrence and development8. Therefore, improving lipid metabolism disorders is an effective strategy for blocking or delaying disease progression35. Natural substances exert beneficial effects on hepatic steatosis36. In particular, bioactive molecules with low toxicity and lipid-lowering properties have received increasing attention for effectively alleviating NAFLD37. In this study, we demonstrated the excellent therapeutic effects of SAL against lipid accumulation and hepatic steatosis in in vitro and in vivo NAFLD models for the first time. Additionally, we clarified the mechanism underlying the involvement of the miR-802/AMPK axis.

Hepatocyte regulates the primary metabolic and physiological functions of the liver38. HepG2 cell line is commonly used to establish the in vitro NAFLD model 38. Although it is a tumor cell type, it possesses the same characteristics as normal human hepatocytes and is easier to culture than primary human hepatocytes39. Therefore, the HepG2 cell line was used to study the effects of SAL on lipid metabolism. SAL treatment notably reduced intracellular oil droplets and TG and TC levels in an FFA-treated HepG2 cell model. Further, immunofluorescence and Western blotting showed that SAL significantly up-regulated p-AMPK, p-ACC, and PPARα expression and decreased SREBP-1c expression, suggesting that SAL can improve lipid deposition and activate the AMPK pathway.

The effects of SAL on hepatic steatosis were examined using an HFD-induced NAFLD mouse model. Consistent with previous reports, HFD-fed mice showed notable liver steatosis and damage, accompanied by increased serum AST and ALT and hepatic TG, TC, and LDL-C levels and decreased hepatic HDL-C compared with mice fed a standard chow diet3,40. Following SAL intervention, serum AST and ALT and hepatic TG, TC, and LDL-C levels decreased, whereas HDL-C levels increased. In addition, SAL significantly ameliorated HFD-induced hepatic lipid accumulation and steatosis. Therefore, SAL effectively alleviated hepatic steatosis, hepatocyte ballooning, and dyslipidemia and improved liver function in HFD-induced NAFLD mice. Consequently, we explored the underlying therapeutic mechanisms.

A previous study reported that SAL alleviates oxidized LDL-induced endothelial dysfunction via an AMPK-dependent mechanism41. Notably, AMPK is the master cellular energy sensor that regulates many metabolic processes42. Our results revealed that SAL activates the AMPK signaling pathway in an in vitro NAFLD model. AMPK is a heterotrimer complex that comprises a catalytic α subunit and two regulatory β and γ subunits43. AMPK activation ameliorates NAFLD through multiple mechanisms, including lipid catabolism and lipogenesis inhibition44. AMPK phosphorylation inhibits lipid synthesis by inactivating SREBP-1c and ACC11. ACC, a crucial lipogenic enzyme, stimulates de novo lipogenesis and inhibits fatty acid oxidation45. SREBP-1c, a pivotal transcription factor, regulates fatty acid desaturation and lipogenesis-related enzyme expression46. In the present study, SAL treatment significantly increased p-AMPK and ACC expression and decreased the expression of SREBP-1c and its target gene, Fas, in HFD-induced NAFLD mice. Further, p-AMPK down-regulates SREBP-1c and its target gene, Fas, in the NAFLD model47, which was also observed in the present study. In addition to inhibiting hepatic lipogenesis, p-AMPK also enhances fatty acid β-oxidation by upregulating PPARα-related fatty acid oxidation genes like Cpt-143. Fatty acid oxidation is another metabolic pathway that regulates liver lipid homeostasis, and its imbalance results in abnormal TG accumulation and subsequent hepatic steatosis11. PPARα is a master factor that controls the β-oxidation of fatty acids, preventing hepatic lipid accumulation by promoting fatty acid catabolism48. CPT-1, encoded by Cpt1, a target gene of PPARα, is the rate-limiting enzyme in fatty acid oxidation49. In this study, SAL treatment significantly increased PPARα and Cpt-1 levels in mice in the model group. These findings suggested that SAL ameliorates NAFLD by activating the AMPK pathway with subsequent inhibition of SREBP-1c-mediated lipogenesis and the enhancement of PPARα-mediated fatty acid β-oxidation.

Aberrantly expressed miRNAs in the liver are associated with impaired AMPK function in NAFLD13. Dysregulated miRNAs in NAFLD inhibit hepatic AMPK expression and activity50. As miRNAs are crucial regulators of lipid metabolism, we further explored whether miRNAs are involved in the anti-NAFLD mechanism of SAL using miRNA-seq and bioinformatics analyses. Our study revealed that miR-802 is involved in the AMPK pathway and lipid metabolism, suggesting that miR-802 plays a potential role in the regulation of hepatic lipid metabolism by SAL. Consistent with previous reports, miR-802 expression was dramatically increased in HFD-induced NAFLD mice34, and SAL treatment significantly decreased its expression. Notably, miR-802 overexpression in NAFLD mice almost completely abolished the SAL-induced improvement in hepatic steatosis. Further, miR-802 knockdown notably improved hepatic steatosis and increased p-AMPK, p-ACC, and PPARα expression, and decreased SREBP-1c expression in NAFLD mice. Collectively, these results revealed that SAL exerted protective effects against NAFLD via the miR-802/AMPK axis. miRNAs bind to many mRNAs and modulate their expression51. In this study, three target mRNAs (Ppp2ca, Prkab1, and Pafah1b1) of miR-802 were predicted to be involved in lipid metabolism. qRT-PCR results showed that SAL treatment significantly decreased hepatic Prkab1 expression in HFD-induced NAFLD mice but did not affect Ppp2ca and Pafah1b1 expression. This implies that SAL activates the AMPK pathway by attenuating miR-802-regulated Prkab1 expression. Further, miR-802 can bind the 3' UTRs of both mouse Prkab1 and human PRKAA1, resulting in decreased AMPK expression and activity34. To further examine whether SAL function in lipid accumulation in hepatocytes involves miR-802/PRKAA1, PRKAA1 was knocked down in HepG2 cells using siPRKAA1. PRKAA1 knockdown significantly reversed the effects of SAL on p-AMPK, p-ACC, PPARα, and SREBP-1c levels in hepatocytes, suggesting that PRKAA1 is essential for AMPK pathway activation by SAL. These results suggested that SAL exerts protective effects against NAFLD by modulating miR-802/AMPK-mediated lipid metabolism. In addition, we found that SAL dose-dependently improved the HFD-induced impairment of insulin action in mice. In addition, SAL treatment also reduced serum IL-6 levels in HFD-induced NAFLD mice, suggesting that insulin resistance and pro-inflammatory signaling pathways may also be involved in the anti-NAFLD mechanism of SAL. However, the precise mechanism requires further investigation.

5 Conclusions

This is the first study to demonstrate SAL as an effective therapeutic agent for NAFLD prevention and treatment. The therapeutic effect of SAL was attributed to enhanced lipolysis and fatty acid oxidation, along with its anti-lipogenic activity. Further, SAL exerted protective effects against NAFLD by modulating AMPK-mediated lipid metabolism by inhibiting miR-802. Our findings revealed that, owing to its modulatory effects on the miR-802/AMPK axis, SAL may be a potential leading compound for anti-NAFLD drug development.

Acknowledgments

This work was supported by 10.13039/501100001809 National Natural Science Foundation of China (Nos. 82304708 and U23A20511 ); the CAMS Innovation Fund for Medical Sciences (2023-I2M-QJ-013 , 2021-I2M-1-031 , 2022-I2M-2-002 , and 2023-I2M-3-006 , China); the 10.13039/501100005089 Beijing Natural Science Foundation (No. Z220019 , China).

Author contributions

Bin Li: Writing – original draft, Methodology, Investigation, Funding acquisition. Qi Xiao: Writing – original draft, Methodology, Investigation. Hongmei Zhao: Methodology, Investigation, Formal analysis. Jianuo Zhang: Investigation. Chunyan Yang: Software, Formal analysis. Yucen Zou: Investigation, Formal analysis. Bengang Zhang: Supervision. Jiushi Liu: Software, Formal analysis. Haitao Sun: Writing – review & editing, Validation, Funding acquisition. Haitao Liu: Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis.

Conflicts of interest

The authors declare no conflicts of interest.

Appendix A Supporting information

The following is the Supporting information to this article:Multimedia component 1

Multimedia component 1

Peer review under the responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Appendix A Supporting information to this article can be found online at https://doi.org/10.1016/j.apsb.2024.05.014.
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