Lygodium microphyllum Inhibits de Novo Lipogenesis Activity in the Hepatocytes of High-Fat High-Fructose-Induced Rats by Increasing the Levels of SIRT1 and AMPK

Introduction

Non-alcoholic liver disease (NAFLD) is a condition of hepatic steatosis (when approximately 5% of hepatocytes contain large lipid droplets or when intrahepatic triglyceride content exceeds 5.6%) that is generated by high-fat or high-sugar dietary intake. The development of NAFLD may be initiated by de novo lipogenesis (DNL) in the hepatocytes.^1,2 DNL converts excessive glucose or fructose into fatty acid and triglycerides, thus inhibiting DNL is strongly strived for as a therapeutic target for lipid metabolism-related disease. Numerous proteins are responsible for reducing DNL, including AMP-activated protein kinase (AMPK) and Sirtuin1 (SIRT1).³ AMPK phosphorylates and inactivates acetyl CoA carboxylase (ACC), the enzyme that catalyzes the first and rate-limiting step in DNL.^4,5 Sirtuin 1 (SIRT1) is also well-known for its role in DNL by regulating hepatocyte lipid metabolism via AMP-activated protein kinase activation. AMPK activation by SIRT1 shelters against the induction of fatty acid synthase and lipid accumulation promoted by high glucose.⁶ SIRT1 contributes to the deacetylation and inhibition of SREBP-1C activity in the regulation of hepatic lipid metabolism.⁷

NAFLD affects approximately 20–30% of the global population⁸ and reaches 27.4% in Asia.⁹ However, there is no specific FDA-approved medication for NAFLD, with several agents being studied in Phase III clinical trials, and others in Phase II clinical trials.¹⁰ Individuals diagnosed with NAFLD are advised to undergo diet and exercise as non-pharmacological therapy, while pharmacological therapy generally involves medications such as antidiabetic agents (metformin, pioglitazone), anti-hyperlipidemic drugs (statins), or anti-obesity drugs (orlistat) depending on the patient’s condition.^11,12 These medications are non-specific to NAFLD and may carry significant risks of side effects. Therapies targeting lipogenesis are considered preferable as they specifically inhibit fat formation, however, these drugs are being studied in humans, such as Firsocostat and ASC40,¹³ thus indicating that anti-lipogenesis drug discovery remains a challenge. Plant sterols have been FDA-approved as lipid-lowering drugs that work to reduce cholesterols, low-density lipoproteins (LDL), and triglycerides, and increase high-density lipoproteins (HDL) in both human and animal models.^14–18

The fern plant Lygodium microphyllum (Cav). R.Br. (synonym Lygodium scandens var. microphyllum (Cav). Luerss., listed in https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:17143140-1) of family Lygodiaceae, grows in abundance on Kalimantan island. This plant has been reported to possess hepatoprotective activity in carbon tetrachloride-induced hepatotoxicity mice.¹⁹ Several compounds have been identified and isolated from L. microphyllum, of which are the phytosterols, namely beta-sitosterol, a major compound in the plant.²⁰ The ethanol extract of this plant is practically not toxic as evidenced by the high LC₅₀ value towards Artemia salina larvae (203.704 ppm) and female mice (LD₅₀ > 5000 mg/kg BW).^21,22 However, an in vivo study is needed to confirm the effect of L. microphyllum on SIRT1 and AMPK. Considering this, our study aims to investigate the anti-lipogenesis activity of the ethanol extract of L. microphyllum herbs and beta-sitosterol in the liver tissue of rats through the levels of SIRT1 and AMPK.

Material and Methods

Chemicals

Chemicals, standard drugs, and kit reagents were ethanol 70% technical grade (Bratachem Indonesia), chloroform (CAS No. 67–66-3; Merck Millipore https://www.merckmillipore.com/), beta-Sitosterol (CAS No. 83–46-5; Sigma-Aldrich https://www.sigmaaldrich.com/ID/en/), resveratrol (CAS No. 501–36-0; Sigma-Aldrich https://www.sigmaaldrich.com/ID/en/product/sigma/r5010), metformin 500 mg/tablet (PT Hexpharm Jaya Laboratories Indonesia http://en.hexpharmjayalaboratories.web.indotrading.com/about), regular chow BR II (PT. Wonokoyo Jaya Corporindo Indonesia https://www.wonokoyo.co.id/), home-made beef tallow, Sania superfry palm oil (PT Wilmar Cahaya Indonesia https://www.wilmarcahayaindonesia.com/product-detail/MzQ=), chicken egg yolk and egg white, liquid fructose (Rose Brand Indonesia https://rosebrand.co.id/), D-fructose powder (Pudak Scientific https://www.pudak-scientific.com/), SIRT1 Rabbit Polyclonal Antibody (Catalog No. A17307; Abclonal https://abclonal.com/catalog-antibodies/SIRT1RabbitpAb/A17307), AMPKa1/AMPKa2 Rabbit Polyclonal Antibody (Catalog No. A17289; Abclonal https://abclonal.com/catalog-antibodies/AMPKa1AMPKa2RabbitpAb/A17289), beta-Actin Rabbit Monoclonal Antibody (Catalog No. AC038; Abclonal https://abclonal.com/catalog-antibodies/ActinRabbitmAb/AC038), PRO-PREP™ Protein Extraction Solution (Catalog No. 17081; iNtRON Bio), acrylamide (CAS No. 79–06-1; Sigma-Aldrich https://www.sigmaaldrich.com/ID/en/product/sial/01696), GangNam-STAIN™ Prestained Protein Ladder (iNtRONbio Cat. No. 24052), N,N′-methylenebisacrylamide (CAS No. 110–26-9; Sigma-Aldrich https://www.sigmaaldrich.com/ID/en/product/sigma/m7279), Hematoxylin (CAS No. 517–28-2; Sigma-Aldrich https://www.sigmaaldrich.com/ID/en/product/sigma/h9627), Eosin Y (https://www.sigmaaldrich.id/id_en/e4009-5g-eosin-y-free-acid).

Plant Collection and Preparation of the Extract

The climbing fern herbs were found in abundance in a tropical rainforest in Samarinda, East Kalimantan, Indonesia. The herbs were identified by Dr. Atik Retnowati (Scopus ID 6507457958), a certified botanist at Herbarium Bogoriense, the Research Center for Biology, the Indonesian Institute of Sciences, Indonesia. The herbs were confirmed as Lygodium microphyllum (Cav). R.Br. of family Lygodiaceae (voucher specimen number 750IPH101), classified as a Least Concern species with no requirements for approval from the local government for its utilization, despite its correspondence with those described in The IUCN Red List of Threatened Species (https://www.iucnredlist.org/species/194153/8883960).

Extraction was carried out by following a previous method using approximately 3.6 kg of the herbs in 3 L of technical grade ethanol 70% for 3×24 hours at room temperature (26 ± 2 °C), which yielded 15.86% w/w a viscous extract of L. microphyllum (abbreviated as ELM).²¹

Animals and Ethical Approval

This animal study was conducted by following the 3Rs (Replacement, Reduction, and Refinement) Principles of the ARRIVE (Animal Research Reporting of In Vivo Experiments) Guidelines (https://arriveguidelines.org/), and was approved by the Research Ethics Committee of Padjadjaran University (No. 376/UN6.KEP/EC/2023), which strictly follows The Guide for the Care and Use of Laboratory Animals (NRC 2011; eighth edition) (https://grants.nih.gov/grants/olaw/guide-for-The-care-and-use-of-laboratory-animals.pdf).²³ Procedures were conducted at the Laboratory of Pharmaceutical Research & Development for Pharmaca Tropicals of Mulawarman University, Samarinda, Indonesia (https://ff.unmul.ac.id/). Forty male Wistar rats (weighing 200 ± 10 g; age of 8 to 12 weeks) were housed in close-house cages, in a room equipped with an air-handling unit, regulated humidity (60 ± 10%), and temperature (25 ± 2 °C), in a 12:12 hour light-dark circle. The rats were acclimatized for 5 days and given standard rodent feed containing 18% protein and free access to drinking water.

HFHFD Preparation and Proximate Analysis

The high-fat high-fructose diet (HFHFD) chow was prepared by utilizing beef tallow (100 g) added to the regular chow BR II (100 g), egg yolk (25 g), egg white (8 g), palm oil (7 mL), and liquid fructose (25 g). The ingredients were mixed to homogenous, manually formed into pellets, and kept stored at 4–8 °C. Beef tallow was used as the primary fat source, due to its high content of saturated fatty acids (such as palmitic acid and stearic acid) and monounsaturated fatty acids (such as oleic acid).²⁴ The HFHFD chow was analyzed for its nutritional composition, eg carbohydrate, protein, and fat, by following the Official Methods of Analysis,²⁵ and resulted in a carbohydrate content of 23.2%, protein content of 21.5%, fat content of 41.8%, ash content of 0.5%, and moisture content of 12.8%.

Animal Modeling for NAFLD

Modeling for NAFLD was carried out on adult Wistar rats (6–8 weeks old; weighing 200–250 g), which were randomly caged into (1) the normal group (n = 3) and (2) the HFHFD group (n = 3), in a controlled environment of 25 ± 2 °C and 12:12 hour light-dark cycle. Rats in the normal group received a standard rodent feed containing 18% protein, while the HFHFD group received the best HFHFD (containing standard rodent feed, mixed with beef tallow 55% and fructose 60%), for 35 consecutive days. On day 15 and day 35, the rats were measured for their serum triglyceride, and on day 35 the rats were sacrificed. The livers were collected for histology analysis, by hematoxylin-eosin (H&E) staining. The NAFLD activity score (NAS) was determined using a Pro-Histo Microscope at 5 fields of view at 400x magnification. Ballooning degeneration, inflammation, and necrosis were scored 0: if absent; scored 1 (minimal): if < 25%; scored 2 (mild): if 25–50%; scored 3 (moderate): if 50–75%; and scored 4 (severe): if >75%.²⁶

Experimental Design

Male Wistar rats (n = 40) were randomly divided into 8 groups: (1) normal control group; (2) high-fat high-fructose diet (HFHFD) rats; (3) HFHFD rats treated with metformin (200 mg/kg BW);²⁷ (4) HFHFD rats treated with resveratrol (50 mg/kg BW);²⁸ (5) HFHFD rats treated with beta-sitosterol (20 mg/kg BW); (6) HFHFD rats treated with ELM dose of 200 mg/kg BW; (7) HFHFD rats treated with ELM dose of 400 mg/kg BW; and (8) HFHFD rats treated with ELM dose of 600 mg/kg BW. The dose of the standard drugs followed previous studies with modification.^27,28

Rats in the normal control group (group 1) were fed regular chow composed of ground corn, soybean, a protein source such as fish, and vegetable oil,²⁹ while other groups of rats were given a high-fat high-fructose diet (HFHFD) for 35 days. All drugs and ELMs were given orally on day 15 (D15) till the last day of the experiment (D35). On the last day (D35), rats were sacrificed using CO₂ euthanasia for 2 minutes by trained personnel. Death was confirmed by determining cardiac and respiratory arrest,³⁰ and the liver organs were harvested for further examination.

Liver Index and Macroscopic Assessments

The liver index and macroscopic assessments of the liver were conducted following a previous procedure described elsewhere with a few modifications.³¹ The livers of the rats were rinsed with saline solution (0.9% sodium chloride), weighed, examined for morphology, and documented by ensuring consistent lighting for each image. The liver index was obtained by calculating the rats’ liver and body weight.³²

Histopathology of the Liver Tissue and NAFLD Activity Score (NAS) Assessment

The liver of each rat was cut in halves, a half portion of the liver was analyzed for its protein content (SIRT1 and AMPK), while the other half portion was histologically analyzed. For histological analysis, the liver was immersed in 10% formalin buffer, then washed with running tap water, dehydrated with ethanol, cleaned with xylene, and embedded in paraffin. The liver was then sliced to a thickness of 5 μm and stained using hematoxylin and eosin. Histological changes were observed using a microscope (Pro Histo Biological Microscope Pro A31). NAFLD activity score (NAS) was conducted based on the previously established methods.^26,33–35 The NAS can range from 0 to 8 and is calculated by the sum of scores of steatosis (0–3), lobular inflammation (0–3), and hepatocyte ballooning degeneration (0–2).³⁵

Western Blot Analysis

The half part of the liver was put in a protein extraction solution and homogenized. The lysate was vortexed and the supernatant was collected for protein concentration measurement using the bicinchoninic acid assay (BCA) method, which is based on the reaction of sodium bicinchoninate with the cuprous ion generated by the biuret reaction at basic pH (pH > 8.0). The BCA assay method is commonly employed because of its simplicity, sensitivity, repeatability, and reproducibility.³⁶ The protein level was measured using a spectrophotometer until a concentration of 350 ppm was obtained. Subsequently, a total of 5 μL of the protein ladder was loaded into the left well, followed by 10 μL of protein sample into each well. The protein was separated using 12% SDS-PAGE followed by its transfer onto a nitrocellulose membrane. The membrane was then blocked using 1% skim milk for 30 minutes, and incubated with primary AMPKa1/AMPKa2 rabbit polyclonal antibody (1:1000), SIRT1 rabbit polyclonal antibody (1:1000), and beta-actin rabbit monoclonal antibody (1:500) for 24 hours at 4°C. Eventually, the membrane was washed with 1× TBST four times for 15 minutes each on a shaker and then incubated with a secondary antibody donkey anti-rabbit (1:15000) for 90 minutes at room temperature (25 ± 2 °C). The membrane was washed again with 1× TBST four times for 15 minutes each on a shaker and then prepared for detection. Beta-actin was used as a loading control to normalize the levels of protein. Protein bands were visualized, and the membrane was then scanned using a LI-COR Odyssey CLx scanner. Band intensities were measured using ImageJ (https://imagej.net/ij/).

Statistical Analysis

The data is presented in the form of mean ± standard deviation. Statistical analysis using a one-way ANOVA test followed by Tukey’s multiple comparison test. The statistical analysis and graph creation were performed using GraphPad Prism version 7 (https://www.graphpad.com/support/prism-7-updates/).

Results

Animal Modeling for NAFLD

Modeling for NAFLD was carried out on adult Wistar rats (6–8 weeks old; weighing 200–250 g), which were randomly caged into (1) the normal group (n = 3) and (2) the HFHFD group (n = 3), in a controlled environment of 25 ± 2 °C and 12:12 hour light-dark cycle. Rats in the normal group received a standard rodent feed containing 18% protein, while the HFHFD group received the best HFHFD (containing standard rodent feed, mixed with beef tallow 55% and fructose 60%), for 35 consecutive days. Rats fed with HFHFD revealed the pathogenesis of NAFLD, characterized by a significant increase in triglyceride levels compared to the normal group (Figure 1). Moreover, a histology examination of the liver tissue showed the presence of ballooning degeneration and necrosis, compared to those in the normal group (Figure 2).

Figure 1 NAFLD optimization in Wistar rats by measuring serum triglyceride levels of HFHFD-induced rats (205.11 ± 15.35 mg/dL at day 15 and 179.98 ± 51.69 mg/dL at day 35) compared to the normal group (81.78 ± 3.88 mg/dL at day 15 and 67.87 ± 9.86 mg/dL at day 35). Data are presented as mean ± SEM of 3 rats. A significant difference is marked by ** indicating p < 0.01. Abbreviation: HFHFD, high-fat high-fructose diet; NAFLD, non-alcoholic fatty liver disease; SEM, standard error mean.

Figure 2 NAFLD optimization in Wistar rats by histology examination of (A) the normal group and (B) HFHFD-induced rats at day 35 with 400x magnification. Red arrows, ballooning degeneration; blue arrows, Kupffer cell. Abbreviation: NAFLD, non-alcoholic fatty liver disease; HFHFD, high-fat high-fructose diet.

The Effects of ELM on the Liver Index and Morphology of the Liver of HFHFD-Induced NAFLD Rats

The effects of ELM on the morphology of the rat liver are depicted in Figure 3. Macroscopically, the morphology of the livers of all groups reveals no difference, showing a normal wedge shape. The rats in the normal control group exhibited the darkest reddish-brown color compared to those induced with HFHFD, indicating a healthy liver. Treating the rats with ELMs resulted in a darker reddish-brown color compared to that without treatment (the HFHFD group). Similarly, the treatment using metformin, resveratrol, or beta-sitosterol showed a darker reddish-brown color compared to that without treatment. The liver index of the HFHFD group was significantly higher than that of the normal control group. Rats treated with ELM showed a lower liver index than the HFHFD group (Figure 4).

Figure 3 Effects of ELM on the morphology of the liver of HFHFD-induced NAFLD rats: (A) the normal control group; (B) HFHFD group without treatment: (C) HFHFD rats treated with metformin (200 mg/kg BW); (D) HFHFD rats treated with resveratrol (50 mg/kg BW); (E) HFHFD rats treated with beta-sitosterol (20 mg/kg BW); (F) HFHFD rats treated with ELM dose of 200 mg/kg BW; (G) HFHFD rats treated with ELM dose of 400 mg/kg BW; (H) HFHFD rats treated with ELM dose of 600 mg/kg BW, with 400x magnification. Abbreviation: BW, body weight; ELM, the ethanol extract of Lygodium microphyllum; HFHFD, high-fat high-fructose diet; NAFLD, non-alcoholic fatty liver disease.

Figure 4 Effects of ELM on the liver index of HFHFD-induced NAFLD rats. The liver index score was analyzed using a one-way ANOVA. Each bar represents the mean + SEM of 3 rats in a group. *** indicating p < 0.001 vs the normal control group; # indicating p < 0.05 vs the HFHFD group; ## indicating p < 0.01 vs the HFHFD group. Abbreviation: ELM, the ethanol extract of Lygodium microphyllum; HFHFD, high-fat high-fructose diet; NAFLD, non-alcoholic fatty liver disease; SEM, standard error mean; BW, body weight.

Effects of ELM on the Histopathology and NAS Score of the Liver Tissue of HFHFD-Induced NAFLD Rats

The effects of ELM on the histopathology of the rat liver are depicted in Figure 5. Rats in the normal control group exhibited neatly arranged hepatocyte cells. Meanwhile, the HFHFD group shows ballooning degeneration (indicated by the red arrows) and steatosis (indicated by black arrows). Treating the rats with ELM slightly reduced the ballooning degeneration and steatosis. To determine the extent of liver damage, the NAS score was calculated, and confirmed that ELM significantly reduced the NAS score of the rats compared to the HFHFD group. Similarly, the treatment using metformin or resveratrol, not beta-sitosterol, also significantly reduced the NAS score compared to the HFHFD group (Figure 6).

Figure 5 Effects of ELM on the histopathology of the liver tissue of HFHFD-induced NAFLD rats: (A) the normal control group; (B) HFHFD group without treatment: (C) HFHFD rats treated with metformin (200 mg/kg BW); (D) HFHFD rats treated with resveratrol (50 mg/kg BW); (E) HFHFD rats treated with beta-sitosterol (20 mg/kg BW); (F) HFHFD rats treated with ELM dose of 200 mg/kg BW; (G) HFHFD rats treated with ELM dose of 400 mg/kg BW; (H) HFHFD rats treated with ELM dose of 600 mg/kg BW, with 400x magnificent. The red arrows indicate the ballooning cell, the blue arrows show the steatosis and the black arrows show the inflammation. Abbreviation: BW, body weight; ELM, the ethanol extract of Lygodium microphyllum; HFHFD, high-fat high-fructose diet; NAFLD, non-alcoholic fatty liver disease.

Figure 6 Effects of ELM on the NAS score of the liver tissue of HFHFD-induced NAFLD rats. The NAFLD activity score (NAS) was analyzed using a one-way ANOVA. Each bar represents the mean + SEM of 3 rats in a group. **** indicating p < 0.0001 vs the normal control group; # indicating p < 0.05 vs the HFHFD group; ## indicating p < 0.01 vs the HFHFD group. Abbreviation: ELM, the ethanol extract of Lygodium microphyllum; HFHFD, high-fat high-fructose diet; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; BW, body weight; SEM, standard error mean.

Effects of ELM on the AMPK/SIRT1 Levels in the Liver Tissue of HFHFD-Induced NAFLD Rats

The two isoforms of AMPK were separated as twin bands at a molecular weight of 62.8 kDa for AMPKα1 and a molecular weight of 62.3 kDa for AMPKα2 as depicted in Figure 7, while the bands of SIRT1 were observed at 110–130 kDa as portrayed in Figure 8.

Figure 7 Effects of ELM on the AMPKα1 (observed molecular weight of 62.8 kDa; calculated molecular weight of 64 kDa)/AMPKα2 (observed molecular weight of 62.3 kDa; calculated molecular weight of 65 kDa) levels in the liver tissue of HFHFD-induced NAFLD rats. The upper figure portrays the separated protein bands by Western blotting and the bar diagram of AMPK levels normalized to beta-actin is presented in the lower figure. No significant differences were observed. Higher doses of ELM increased the levels of AMPK in the liver tissue of HFHFD-induced NAFLD rats. The blue box indicates the bands of AMPKα1/AMPKα2 at 62.8 and 62.3 kDa. Abbreviation: AMPK, adenosine monophosphate-activated protein kinase; BW, body weight; ELM, the ethanol extract of Lygodium microphyllum; HFHFD, high-fat high-fructose diet; NAFLD, non-alcoholic fatty liver disease.

Figure 8 Effects of ELM on the SIRT1 (observed molecular weight of 110–130 kDa; calculated molecular weight of 61/81 kDa) levels in the liver tissue of HFHFD-induced NAFLD rats. The upper figure portrays the separated protein bands by Western blotting and the bar diagram of SIRT levels normalized to beta-actin is presented in the lower figure. Higher doses of ELM significantly increased the levels of SIRT1 in the liver tissue of HFHFD-induced NAFLD rats. ** indicating p < 0.001 vs the normal control group; # indicating p < 0.05 vs the HFHFD group; #### indicating p < 0.0001 vs the HFHFD group. The blue box indicates the band of SIRT1 at 130 kDa. Abbreviation: BW, body weight; ELM, the ethanol extract of Lygodium microphyllum; HFHFD, high-fat high-fructose diet; NAFLD, non-alcoholic fatty liver disease; SIRT1, Sirtuin1.

The effects of ELM on the AMPK/SIRT1 levels in the liver tissue of HFHFD-induced NAFLD rats are depicted in Figures 7 and 8, respectively. Inducing the rats with HFHFD lowered the levels of AMPK and SIRT1 compared to rats in the normal control group. Interestingly, treating the rats with ELM resulted in an increase in the levels of both AMPK and SIRT1 in the liver tissue of HFHFD-induced NAFLD rats. Metformin and resveratrol did not significantly alter AMPK levels, but beta-sitosterol reduced AMPK levels. Metformin, resveratrol, and beta-sitosterol could increase the levels of SIRT1 although not significant.

Discussion

The fern plant Lygodium microphyllum (Cav). R.Br. (Lygodiaceae) may have the potential to be utilized as a medicinal plant. In this study we confirm that (1) the ethanol extract of L. microphyllum (ELM) improves the morphology and the liver index of HFHFD-induced NAFLD rats comparably to those of metformin, resveratrol, or beta-sitosterol; (2) ELM improves the steatosis condition and NAS score of HFHFD-induced NAFLD rats comparably to those of metformin, resveratrol, or beta-sitosterol; (3) ELM may inhibit de novo lipogenesis by increasing the levels of both AMPK and SIRT1 in the liver tissue of HFHFD-induced NAFLD rats, which is better than metformin, resveratrol, or beta-sitosterol.

In a previous study, beta-sitosterol was reported as the major compound in L. microphyllum,²⁰ with total sterol levels of 954.04 µg/g, and the beta-sitosterol level of 283.55 µg/g,²¹ thus arising our interest in exploring the anti-lipogenesis activity of this fern plant. As formerly reported, plant sterols reduced cholesterols, low-density lipoproteins, and triglycerides, and increased high-density lipoproteins in both humans and animal models.^14–18

The inducement with HFHFD was administered for 35 days to stimulate de novo lipogenesis (DNL) in the rats. It was delineated by Softic and colleagues (2016) that high-fat high-sugar intake correlates with obesity and the development of NAFLD.³⁷ When a person consumes sucrose, it will undergo degradation to produce six-carbon monosaccharides, glucose, or fructose, which metabolism converges into glyceraldehyde-3 phosphate and dihydroxyacetone phosphate as intermediate products, and is further converted to pyruvate. Pyruvate will enter mitochondria, where it is changed to acetyl-CoA (Ac-CoA), to be utilized in the tricarboxylic acid (TCA) or citric acid cycle. When energy stores are sufficient, citric acid is transported back into the cytoplasm by the mitochondrial tricarboxylate transport system.³⁸ Citric acid is eventually converted to acetyl-CoA (Ac-CoA), by the action of adenosine triphosphate citrate lyase (ACL), which is the first step of endogenous fatty acid synthesis. Citric acid is an allosteric activator of cytoplasmic Ac-CoA carboxylase (ACC), which works to transform Ac-CoA to malonyl-CoA, thus initiating de novo lipogenesis (DNL).^4,39 Moreover, it was narrated that fructose could rapidly bind into both glycerol and free fatty acids, thus reinforcing DNL, a major contributor to NAFLD pathogenesis.⁴⁰

A study in humans treated with excess intake of carbohydrates for three weeks resulted in an increase of liver fat by 27%, while the increase of total body weight (BW) was only 2%. The same participants were eventually advised to take 24 weeks of a hypocaloric diet, and in consequence, resulted in a loss of 25% liver fat and 4% of BW.⁴¹ High fructose corn syrup (HFCS)-containing drinks were confirmed for their contribution to the development of NAFLD in humans due to their major role in hepatic DNL.⁴² In 358 participants with type 2 DM, higher carbohydrate intake correlates with the occurrence of liver steatosis in patients aged < 50 years old.⁴³

In our study, the inducement of the rats with HFHFD resulted in lower hepatic AMPK and SIRT1 levels, while treatment with ELM increased the levels of both proteins in the liver tissue. We compared this result to a former study by Lindholm et al (2013).⁴⁴

Lindholm and colleagues concluded that a high-fat diet (HFD) reduced the activities of both AMPKα1/AMPKα2 in white adipose tissue, heart, and liver of male Sprague–Dawley rats.⁴⁵ The effect of an HFD on AMPK has most frequently been studied in a single tissue type, usually in liver tissue,^44–46 heart,^44,47 and/or skeletal muscle.^48,49 The factors that contribute to the activation of AMPK are exercise, caloric restriction, and anti-DM drugs such as metformin and thiazolidinediones.^50–52 AMPK can be activated by SIRT1 which shelters against the induction of fatty acid synthase and lipid accumulation promoted by high glucose.⁶ AMPK mediates the metabolic hormones such as leptin, ghrelin, adiponectin, and glucocorticoids, thus activating glycolysis, fatty acid oxidation, and mitochondrial biogenesis, and reducing gluconeogenesis, glycogen, fatty acid, and protein synthesis.⁵¹ Moreover, 2 to 8 weeks of HFD feeding to male ddY mice revealed an alteration of hepatic LKB1-AMPK signaling and SIRT1 expression.⁵³

Metformin, an anti-diabetes mellitus biguanide class drug, was reported to activate AMPK in liver cells, thus reducing acetyl-CoA carboxylase (ACC) activity, stimulating fatty acid oxidation, and suppressing the expression of lipogenic enzymes.⁵⁴ Metformin effectively decreases blood glucose levels by inhibiting hepatic glucose synthesis, reducing intestinal absorption, and increasing insulin sensitivity. A combination of N-acetyl cysteine and metformin treatment for 48 weeks to 53 male and female patients with non-alcoholic hepatosteatosis showed remarkable improvements in the steatosis degree, ballooning, and NAS score.⁵⁵

Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a polyphenol compound found in abundant levels in grapes (Vitis vinifera), can inhibit adenosine triphosphate (ATP) synthase thus activating AMPK without affecting the serine/threonine kinase liver kinase B1 (abbreviated as LKB1, also known as serine/threonine kinase 11, abbreviated as STK11). This polyphenol compound binds to SIRT1 but does not change its catalytic activity.⁵⁶ Furthermore, a recent study by Afshari and co-workers (2023) confirmed that a combination of metformin and resveratrol diminishes liver steatosis by stimulating autophagy via the cAMP/AMPK/SIRT1 signaling pathway.⁵⁷ Hepatic steatosis is also attenuated by a combination of metformin and quercetin via the same pathway as reported by Afshari and co-workers.⁵⁸ Moreover, resveratrol and metformin could stimulate serum SIRT1 and improve insulin resistance in high fructose-fed animal models.⁵⁹

AMPK activation by phytosterols was limitedly described, thus unlocking further explorations. An article by Jie and co-workers reported that stigmasterol improved neuroinflammation in APP/PS1 mice, and inhibited the inflammatory response of microglia to Aβ42 oligomers via AMPK/NF-kappaB signaling.⁶⁰ Conversely, in our study, beta-sitosterol reduces the levels of AMPK in the liver tissue of NAFLD rats, which needs further exploration. It is evidence that beta-sitosterol could lessen the mass of adipose tissue and inhibit the proliferation of preadipocytes as proven by the in vitro study of Awad and colleagues (2000) in 3LT-L1 cells.⁶¹ It is believed that phytosterols may compete with free cholesterol for the binding with the micellar structure, increase cholesterol excretion via the feces, and lower plasma cholesterol levels,⁶² thus raising their use as complementary or add-on therapy for patients with obesity and diabetes mellitus.⁶³

Although we confirmed that ELM improves the morphology and the liver index of HFHFD-induced NAFLD rats, improves the steatosis condition and NAS score of HFHFD-induced NAFLD rats comparably to those of metformin, resveratrol, or beta-sitosterol, and may inhibit de novo lipogenesis by increasing the levels of both AMPK and SIRT1 in the liver tissue of HFHFD-induced NAFLD rats, our work did not evaluate the levels of the liver enzymes and the lipid profile of the rats., which may be listed as limitations of this study. Nevertheless, this study lays a solid foundation for future investigations into ELM as a promising natural remedy for NAFLD, opening new avenues for the development of alternative treatments for this increasingly prevalent liver condition. Understanding the proteins involved in the upstream and downstream pathways of SIRT1 and AMPK is still challenging and may corroborate the whole framework of the mechanism of this plant in inhibiting lipogenesis.

Conclusion

The present work studied the antilipogenesis activity of the ethanol extract of Lygodium microphyllum (Cav). R.Br., a fern plant found abundantly in a rainforest of Kalimantan island, Indonesia. Our study revealed that the ethanol extract of L. microphyllum improves the morphology, the liver index, the steatosis condition, and NAS score of high-fat high-fructose-induced NAFLD rats comparably to those of metformin, resveratrol, or beta-sitosterol. We confirm that this is the first report on the anti-lipogenesis activity of L. microphyllum by significantly increasing the levels of AMPK and SIRT1 in the liver tissue of NAFLD rats, better than that of metformin and resveratrol. It is suggested to further explore L. microphyllum for its anti-lipogenesis activity in patients with NAFLD.