Amelioration of the Lipogenesis, Oxidative Stress and Apoptosis of Hepatocytes by a Novel Proteoglycan from Ganoderma lucidum

Abstract

The steatosis and resultant oxidative stress and apoptosis play the important roles in the progression of nonalcoholic fatty liver disease (NAFLD), therefore, searching for the effective drugs against NAFLD has been a hot topic. In this work, we investigated a hyperbranched proteoglycan, namely FYGL extracted from Ganoderma lucidum, inhibiting the palmitic acid (PA)-induced steatosis in HepG2 hepatocytes. FYGL compose of hydrophilic polysaccharide and lipophilic protein. Both moieties conclude the reductive residues, such as glucose and cystine, making FYGL capable of anti-oxidation. Herein, we demonstrated that FYGL can significantly inhibit the steatosis, i.e., decrease the contents of triglycerides (TG) and total cholesterol (TC) in hepatic cells on the mechanism of increasing the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), therefore inhibiting the expressions of sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FASN), furthermore leading to the carnitine palmitoyl transferase-1 (CPT-1) expression increased against steatosis induced by fatty acids oxidation. Meanwhile, FYGL can alleviate reactive oxygen species (ROS) and malondialdehyde (MDA), promote superoxide dismutase (SOD) and total antioxidant capacity (T-AOC). Moreover, FYGL can prevent the cells from apoptosis by regulating the apoptosis-related protein expressions and alleviating oxidative stress. Notably, FYGL could significantly recover the cells activity and inhibit lactate dehydrogenase (LDH) release which were negatively induced by high concentration PA. These results demonstrated that FYGL has the potential functions to prevent the hepatocytes from lipid accumulation, oxidative stress and apoptosis, therefore against NAFLD.

INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) has attracted considerable attention due to its high global prevalence rate up to 25%.¹⁾ As the most common form of chronic liver disease, NAFLD characterized by the accumulation of fat in liver, is associated with many diseases such as obesity, cardiovascular disease and type 2 diabetes mellitus.²⁾ The NAFLD and related metabolic diseases are greatly harmful for human health. An in-depth understanding of the pathogenesis for NAFLD is quite imperative, and searching for a safe and effective treatment method has become a public health goal.

The “two-hit” theory proposed by Day and James in 1998 has been generally accepted to elucidate the pathogenesis of the disease.³⁾ Jin et al. reported that the lipid accumulation produced by dysregulation of hepatic lipid metabolism induces the sensitivity of endogenous damage factors increased in the body, thus forming the “first hit” of NAFLD.⁴⁾ On this basis, the activated factors further accelerate and intensify liver cell injury through oxidative stress and lipid peroxidation, and develop into an important “secondary attack,” which eventually leads to the liver inflammation, injury and even necrosis and fibrosis.⁵⁾

AMP-activated protein kinase (AMPK) has been extensively attended and researched because of its core role in regulating biological energy metabolism.⁶⁾ Both sterol regulatory element-binding protein 1 (SREBP1) and acetyl-CoA carboxylase (ACC) are downstream factors of AMPK, and play a regulatory role in hepatic lipid synthesis. The phosphorylation of AMPK (p-AMPK) inhibits the expression of SREBP1, and increases the phosphorylation of ACC (p-ACC).^7,8) The negative feedback of ACC enhances the activity of carnitine palmitoyl transferase-1 (CPT-1) which associates the oxidation of fatty acids.⁹⁾ In the NAFLD cells model, Zhou et al. found that downregulation of p-AMPK, p-ACC and upregulation of SREBP1 are the causes of lipid accumulation, which indicate that AMPK activation is beneficial in the treatment of NAFLD.¹⁰⁾ Oxidative stress and apoptosis also play an important role in the progression of NAFLD. The oxidative stress is resulted from the excessive accumulation of the reactive oxygen species (ROS), including superoxide anions, hydroxyl radicals and their derivatives.¹¹⁾ High concentration ROS damages cellular macromolecular (DNA, lipid, proteins, etc.), and leads to cell injury.¹²⁾ Apoptosis induced by oxidative stress controls the cellular process. Relevant studies have found that in nonalcoholic steatohepatitis (NASH) animal models, the apoptosis rate of liver cells increases significantly, also the liver fibrosis rate does.^13,14)

A lot of drugs, such as statins, obeticholic acid, etc. have been used for NAFLD treatment in clinic. However, there are still some issues considered for maximizing therapeutic effects and minimizing adverse reactions.¹⁵⁾ Therefore, searching for good antihyperlipidemics with low side effects has been a hot topic. Polysaccharides have been attended in natural medicine because of its various biological activities and extensive uses in functional food.¹⁶⁾ Many plants contain polysaccharides with biological activity, such as Ginkgo biloba,¹⁷⁾ Cichorium⁹⁾ and Lycium barbarum.¹⁸⁾

As a traditional Chinese medicine, Ganoderma lucidum has been used for thousands of years in Asia for its broad medicinal treatment,¹⁹⁾ specifically for metabolic diseases such as diabetes,²⁰⁾ obesity,²¹⁾ and cardiovascular syndromes.²²⁾ Previously, we extracted a hyperbranched proteoglycan, namely Fudan-Yueyang Ganoderma lucidum (FYGL), from Ganoderma lucidum. Figure 1 shows the dominant component structure of FYGL, which is a biomacromolecule with a molecular weight of 2.6 × 10⁵. FYGL is amphiphilic and compose of hydrophilic polysaccharides and lipophilic protein moieties with the ratio of saccharide : proteins of 77 : 17.²³⁾ The polysaccharide moiety compose dominantly of the reductive residues of glucose, galactose, rhamnose and arabinose, and the protein moiety is covalently bound by -O- linkages between Glc in saccharide and serine (Ser) or threonine (Thr) in protein including reductive residues of cystine and tyrosine. Those reductive residues make FYGL capable of anti-oxidation.²³⁾ It has been demonstrated that FYGL has efficient effect on anti-diabetes and anti-adiposity with high safety in vivo.²⁴⁾ In present work, we are going to further investigate FYGL function against NAFLD induced by free fatty acids in vitro. The palmitic acid (PA), which is generally used as a trigger to make a model of NAFLD, will be used to induce HepG2 cells to form NAFLD model.²⁵⁾ Hopefully FYGL would be used as a potential candidate drug for NAFLD treatment.

Fig. 1. Structure of FYGL Characterized by Chemical Analysis and NMR Spectroscopy

Rs represent the carbohydrate residues of →2,4)-α-L-Rhap-(1→, →6-β-D-Galp-1→, Araf-(1→ or →3,6)-β-D-Galp-(1→. Protein moieties are covalently bonded with carbohydrate moieties by Ser and Thr residues in -O- linkage.

MATERIALS AND METHODS

Materials

The FYGL used in this study was extracted from Ganoderma lucidum according to our previous work.²⁶⁾ We purchased Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) from Gibco Co. Ltd. (U.S.A.). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit was provided by Dojindo Co., Ltd. (Shanghai, China). PA and oil red O were provided by Sigma-Aldrich (Shanghai, China). Cell counting kit-8 (CCK-8), hematoxylin and ROS assay kit were provided by Yeasen Co., Ltd. (Shanghai, China). We purchased triglyceride (TG), total cholesterol (TC), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) assay kits from Jiancheng Bioengineering Institute (Nanjing, China). AMPK (#2757) and p-AMPK (#2531) antibody were obtained from Cell Signaling Technology (Danvers, MA, U.S.A.). SREBP1 (D151451) and fatty acid synthase (FASN) (D162701) were purchased from Sangon Co., Ltd. (Shanghai, China). ACC (ab45174), p-ACC (ab68191), CPT-1 (ab234111), cleaved caspase-3 (ab32042), Bax (ab32503) and Bcl-2 (ab32124) were purchased from Abcam (Cambridge, MA, U.S.A.). HepG2 cells were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Cell Culture and Treatment

DMEM contained 10% FBS and 1% penicillin–streptomycin was used to culture HepG2 cells in cell culture bottles or plates. All HepG2 cells were placed in an incubator with 5% CO₂ at a constant temperature of 37°C. PA solution was prepared as described previously.²⁷⁾ Briefly, PA was firstly dissolved in a 100 mM NaOH solution at 70°C and then mixed with 40% (w/v) bovine serum albumin (BSA) at 55°C. PA which concentrations range from 50 to 400 µM were used to treat HepG2 cells for 24 h. The optimum dosage was evaluated by both triglyceride content and cell activity of each concentration. For the following experimental analysis, the cultured HepG2 cells were treated by selected PA solutions mixed with different concentration of FYGL (0, 50, 100, 200, and 300 µg/mL) for 24 h. Cells cultured in normal medium were the control group and those treated by PA (200 or 300 µM) but without FYGL were model groups.

Cytotoxicity

The cell activity and LDH activity of HepG2 were measured by the CCK-8 assay and LDH tests, respectively. After the cells were treated in the way described in 2.2, the old medium was discarded and new medium containing CCK-8 solution was added into 96-well plates. About 1 h later, a multi-functional microplate reader (Cytation3, BioTek, U.S.A.) was used to measure the optical density (OD) at 450 nm. The LDH activity was determined by OD value at 490 nm using a commercial kit on the manufacturer’s instructions.

Cell Apoptosis

After the designed treatments, cells were stained with annexin V-FITC/PI apoptosis detection kit on the manufacturer’s instructions to detect early apoptotic cells (FITC stained) and late apoptotic cells (PI stained). All groups were detected by flow cytometry (Gallios, Beckman Coulter, U.S.A.).

Oil Red O Staining

Lipid accumulated in cells was measured by oil red O staining. HepG2 cells were cultured in a 6-well plate for 24 h, and then washed three times with phosphate buffered saline (PBS). Four percent paraformaldehyde was used to fix the cells on the plates, and punched with 0.2% PBS-tritonx-100 for 5 min. After the PBS-tritonx-100 were removed, the cells were washed with PBS three times and then incubated with 0.5 mL oil red O solution (0.35%) in dark conditions for another 30 min. After washed with 60% isopropyl alcohol and ultra-pure water, the cytoplasmic matrix was dyed blue with hematoxylin dye for 1 min. Excess dye were rinsed off with water, and the images of cells stained with oil red O and hematoxylin were viewed under a 100× optical microscope. For lipid quantification, the lipid-stained red dye was dissolved with 60% isopropanol solution and the absorbance was determined at 490 nm.

Measurement of Biochemical Parameters

A multi-functional microplate reader (Cytation3, BioTek) was used to measure the levels of TG, TC, MDA, SOD, and T-AOC in HepG2 cells.

Determination of ROS

A reactive oxygen assay kit of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to determine the intracellular ROS level. DCFH-DA itself does not have fluorescence. When DCFH-DA diffuses freely into cells, it is hydrolyzed by intracellular ester enzyme to form DCFH, and then the DCFH reacts with intracellular ROS to produce a green fluorescence of DCF, therefore the ROS level is indirectly determined by DCF fluorescence intensity. Flow cytometry (Gallios, Beckman Coulter) and laser confocal microscope (C2+, Nikon, Japan) were used to analyze the fluorescence intensity of DCF according to the manufacturer’s instructions.

Western Blot Analysis

Denatured protein samples carried a negative charge when they were treated with sodium dodecyl sulfate (SDS). Proteins were separated according to molecular weight in the polyacrylamide gel under the action of electric field. Protein bands that had been separated were transferred to the polyvinylidene difluoride (PVDF) membranes and then PVDF membranes were incubated with specific antibody and immunoglobulin G (IgG) peroxidase conjugated anti-rabbit secondary antibody in turn. Finally, the enhanced chemiluminescence solution (ECL) was used to detect the proteins on the membranes. The luminescent signals were recorded with X-ray film (Bio-Rad ChemiDoc MP, CA, U.S.A.).

Statistical Analysis

All data were analyzed by SPSS 19.0 (SPSS, Inc., Chicago, IL, U.S.A.), and presented as mean ± standard deviation (S.D.). One-way ANOVA test followed by the Bonferroni posttest was used for multiple comparisons to analyze the statistical significance. A difference with p value <0.05 is statistically significant.

RESULTS

Safety of FYGL on HepG2 Cells

HepG2 cells were cultured in DMEM contained different concentrations of FYGL for 24 h. The result of cell activity is shown in Fig. 2, when the concentration of FYGL was not higher than 500 µg/mL, there was no significant difference in cell activity between the FYGL treatment group and the blank group. Upon increasing FYGL concentration to 1000 µg/mL, the cell activity decreased, but still remained about 90%. The above results indicated that FYGL has almost no cytotoxicity when its concentration lower than 1000 µg/mL, suitable for the following experiments.

Fig. 2. In Vitro Cell Activity of HepG2 Cells Incubated with Different Concentrations of FYGL for 24 h at 37°C

Data were presented as mean ± S.D. (n = 5). * p < 0.05 versus control group.

PA-Induced TG Accumulation in HepG2 Cells

In order to establish the cell model of NAFLD, we first selected the appropriate PA concentration. Zero–400 µM PA were used to treat HepG2 cells to induce steatosis. The optimal PA concentration was required for both non-cytotoxicity and the maximum TG content to make the cell model of NAFLD. From Figs. 3A and B, the cell activity decreased as PA increased, while triglyceride content increased significantly. The cells were considered alive when the cell activity was higher than 90%. Therefore, 200 µM PA was selected as a proper concentration to make the cell model of NAFLD, and 300 µM PA was selected to explore FYGL effect on the lipotoxicity.

Fig. 3. (A) Effect of PA on Cell Activity of HepG2 Cells; (B) Effect of PA on TG Accumulation of HepG2 Cells

Mean ± S.D. was used to present date (n = 3). * p < 0.05, ** p < 0.01 versus control group.

FYGL Inhibit Lipid Accumulation in PA-Induced NAFLD Cells

To reveal FYGL effect on the lipid accumulation in NAFLD model cells, HepG2 cells were co-treated with PA (200 μΜ) and FYGL (0–300 µg/mL) for 24 h. Oil red O widely used to detect lipid accumulation is a fat-soluble dye that can specifically stain neutral fats such as triglycerides in tissues. In Fig. 4A, it can be intuitively seen that lots of red lipid droplets existed in the HepG2 cells induced by PA, while FYGL significantly reduced the accumulation of hepatic lipid. In Fig. 4B, lipid content significantly reduced by FYGL. In addition, TC and TG content were shown in Figs. 4C and D, respectively. Compared with model cells, FYGL decreased the level of TG and TC. All results demonstrated the inhibitory effect of FYGL on PA-induced lipid accumulation in HepG2 cells.

Fig. 4. Effect of FYGL on Inhibition of PA-Induced Lipid Accumulation in HepG2 Cells

HepG2 cells were incubated with FYGL in concentration range of 0–300 µg/mL and PA (200 µM) for 24 h. (A) Intracellular lipid droplets accumulation stained by oil red O and viewed by an inversion microscope (100 ×). (B) Intracellular lipid accumulation was quantitatively measured using a microplate reader at absorbance of 490 nm. (C, D) Intracellular TG and TC in HepG2 cells, respectively. Mean ± S.D. was used to present date (n = 3). ^## p < 0.01 versus control group, * p < 0.05, ** p < 0.01 versus model group. (Color figure can be accessed in the online version.)

FYGL Attenuate Oxidative Stress Induced by PA in HepG2 Cells

Braud et al. demonstrated that excessive oxidative stress can lead to cell damage and NAFLD.¹¹⁾ Oxidative stress has been extensively investigated for treatment of NAFLD. The quantitative evaluation methods of oxidative stress include the determination of ROS, oxidative damage to biomolecules and antioxidant status. For this purpose, we measured the level of ROS, MDA, SOD, and T-AOC. MDA is a product of lipid peroxidation and the primary marker of oxidative injury.²⁸⁾ SOD is an important component of antioxidant enzyme system which plays a key role in the balance between oxidation and oxidation resistance. T-AOC including various antioxidant substances and antioxidant enzymes is also an important indicator of antioxidant level.⁴⁾

Figure 5A showed that FYGL (100 and 300 µg/mL) could reduce the PA induced intracellular ROS level determined by DCF green fluorescence intensity, viewed by laser confocal fluorescence microscopy. Similarly, as shown in Figs. 5B and C, FYGL also decreased dose-dependently the PA induced ROS level measured by flow cytometry. In Fig. 5D, MDA content increased in PA model group and was reduced by FYGL treatment. Concomitantly, PA decreased SOD (Fig. 5E) and T-AOC (Fig. 5F) contents in HepG2 cells, while FYGL increased those antioxidant contents in dose-dependent manner. Based on these results, FYGL might have a positive effect on reducing the oxidative stress in HepG2 cells.

Fig. 5. Effect of FYGL on Attenuation of Oxidative Stress in Hepatic Steatosis

(A–C) ROS level determined by DCF green fluorescence intensity using laser scanning confocal microscope (200 ×), flow cytometer, and the digitalized DCF fluorescence intensity, respectively. (D–F) MDA, SOD and T-AOC contents in HepG2 cells, respectively. Data were presented as mean ± S.D. (n = 3). ^## p < 0.01, ^### p < 0.001 versus control group, * p < 0.05, ** p < 0.01 versus model group. (Color figure can be accessed in the online version.)

FYGL Ameliorate Steatosis through AMPK Pathway

As shown in Figs. 6A and B, the phosphorylation of AMPK (p-AMPK) was inhibited by PA, leading to the expression of phosphorylation of ACC (p-ACC) down-regulated, however, FYGL increased p-AMPK and p-ACC, leading to the inhibition of ACC activity and subsequently the fatty acid synthesis. In addition, we also found the expression of CPT-1 increased under the FYGL treatment, which imply that the negative feedback of ACC activity enhanced the activity of CPT-1, beneficial for the inhibition of steatosis accumulation. It was also found that the levels of SREBP1 and FASN in FYGL groups were decreased (Fig. 6C), compared with PA group. These results proved that FYGL inhibited PA triggered lipogenesis through AMPK pathway.

Fig. 6. Effect of FYGL on the Expressions of Proteins Involved in Steatosis

(A) p-AMPK, AMPK, p-ACC, ACC, CPT-1, SREBP1 and FASN proteins were analyzed by Western blot. (B, C) Ratio of p-AMPK/AMPK, p-ACC/ACC, CPT-1/GAPDH, SREBP1/GAPDH, FASN/GAPDH, respectively. Data were presented as mean ± S.D. (n = 3). ^## p < 0.01 versus control group, * p < 0.05, ** p < 0.01, *** p < 0.001 versus model group.

FYGL Recovered the Cell Activity of PA Induced HepG2 Cells

We found that when PA concentration was higher than 300 µM, PA had a significant cytotoxicity for HepG2 cells, leading to the activity decreased and the LDH increased. Therefore, we investigated FYGL effect on the lipotoxicity induced by 300 µM PA in HepG2 hepatocytes. From Fig. 7, FYGL recovered the cell activity and decreased LDH release in a dose-response manner in the HepG2 cells which were induced lipotoxic by 300 µM PA for 24 h. Moreover, the morphology which was damaged by PA was also recovered by FYGL.

Fig. 7. FYGL Effect on the Cell Activity (A) and the LDH Release (B) in PA Induced HepG2 Hepatocytes, Determined by the CCK-8 Assays and the LDH Tests, Respectively; (C) Images of HepG2 Cells Morphologies Affected by PA and PA + FYGL Co-culture for 24 h, Viewed by an Optical Microscope (100 ×)

Data were presented as mean ± S.D. (n = 3). ^## p < 0.01 versus control group. * p < 0.05, ** p < 0.01 versus model group.

FYGL Decreased the PA-Induced Apoptosis in HepG2 Cells

Caspase-3 proteins plays an irreplaceable role in apoptosis. Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) proteins, two representative members of the Bcl-2 family, are currently recognized as positive and negative regulators of mammalian apoptosis, respectively.¹⁴⁾ As showed in Figs. 8A and B, 300 µM PA induced cell apoptosis, but when FYGL and PA co-cultured with the cells for 24 h, FYGL reduced the PA-induced HepG2 cell apoptosis with dose-dependent manner, which were analyzed by flow cytometer. Western blot results showed that PA promoted the expression of cleaved caspase-3 and Bax, and inhibited the expression of Bcl-2. However, FYGL reverse the expressions of those proteins (Figs. 8C, D). These results strongly suggested that FYGL could protect the cells against PA-induced apoptosis.

Fig. 8. FYGL Effects on PA-Induced Apoptosis of HepG2 Hepatocytes Treated with 300 µM PA and 50, 100, 200, and 300 µg/mL FYGL

(A, B) Apoptosis determined by annexin V-FITC/PI kit using flow cytometer. (C) Cleaved caspase-3, Bax and Bcl-2 protein expressions analyzed by Western blot. (D) Ratio of cleaved caspase-3/β-actin, Bax/GAPDH, and Bcl-2/GAPDH, respectively. Data were presented as mean ± S.D. (n = 3). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus control group. * p < 0.05, ** p < 0.01 versus model group.

DISCUSSION AND CONCLUSION

Lipotoxicity is a complex process involved in many cellular dysfunction. High saturated fatty acids can lead to the fat accumulation in the liver, resulting in the incomplete fatty acid metabolism and the lipotoxic metabolites such as diacylglycerin, which can enhance the oxidative stress and endoplasmic reticulum response in the liver, induce the inflammatory response, and promote the cell apoptosis.²⁹⁾ Lowering lipid is considered as an effective strategy to ameliorate NAFLD.³⁰⁾

As above investigation, we found that after FYGL treated the PA-induced steatosis HepG2 cells, the intracellular lipid droplets were decreased, TG and TC contents were also decreased. These results suggested that FYGL treated NAFLD possibly by reducing the lipid accumulation. Similar studies had shown that Lycium barbarum polysaccharide (LBP) can suppress PA-induced hepatic triglyceride formation in vitro.¹⁸⁾ Zhong et al. reported that the Ganoderma lucidum polysaccharide peptide (GLPP) can alleviate hepatoteatosis.³¹⁾ Our previous study demonstrated that FYGL had an antihyperlipidemic effect in addition to antidiabetic in vivo.²⁴⁾

Our studies on the mechanism found that FYGL improved the p-AMPK level along with the p-ACC and CPT-1 expressions increased, inhibited the expressions of fat-synthesis-related proteins FASN and SREBP1, leading to the steatosis inhibition, which implied that FYGL inhibit the lipid synthesis through AMPK pathway. Our previous work also found FYGL up-regulating expression of p-AMPK in ob/ob mice.³²⁾

As an endogenous injury factor, ROS is involved in the symptoms of NAFLD, and has been widely concerned. Physiological ROS regulates cellular signal transduction for cell proliferation. But excessive ROS reacts with polyunsaturated fatty acids, leading to lipid peroxidation. Under normal circumstances, the antioxidant defense system protects the body from ROS damage. However, abnormal accumulation of lipid in the liver increases the productions of ROS and lipid peroxidation, which damage mitochondrial function. In our current study, FYGL significantly reduced the levels of ROS and MDA, increased SOD and T-AOC levels. FYGL contain reductive residues of glucose, galactose, rhamnose and arabinose in saccharide moiety, and cystine and tyrosine in protein moiety, those residues are antioxidants. Our previous in vivo studies also found that FYGL showed antioxidant activity in db/db mice. FYGL orally administrated decreased significantly the levels of MDA, 8-hydroxy-2′-deoxyguanosine (8-OHdG) and PC, and increased the activities of antioxidant enzymes such as SOD, catalase (CAT) and glutathione peroxidase (GSH-PX) in serum and liver.²⁴⁾ Wu et al. reported that oxidative stress could be attenuated by activating the AMPK signaling pathway. AMPK phosphorylation could prevent ROS production and enhance antioxidant enzyme activity.^33,34)

Moreover, the apoptosis also plays an important role in NAFLD. Our results demonstrated that FYGL decreased cells apoptosis by decreasing the cleaved caspase-3 and Bax expressions, and increasing Bcl-2 expressions. Similarly study has found that Ganoderma lucidum polysaccharides can prevent apoptosis and autophagy of intestinal epithelial cells caused by palmitic acid through the recovery of mitochondrial function.³⁵⁾

In summary, based on above results, FYGL has the ability to reduce fat accumulation, intracellular oxidative stress and apoptosis in PA-induced steatosis HepG2 cells. We proposed a mechanism of FYGL treating NAFLD, as shown in Fig. 9. FYGL can attenuate the hepatic steatosis, i.e., TG and TC in hepatic cells through up-regulation of AMPK and ACC phosphorylation, and down-regulation of SREBP1 and FASN expressions. Moreover, FYGL alleviated oxidative stress against apoptosis by decreasing ROS and MDA, as well as promoting T-AOC and SOD. Meanwhile, FYGL also increased Bcl-2 expression and inhibited cleaved caspase-3 and Bax expressions against hepatocyte apoptosis. Importantly, Ganoderma lucidum is both medicine and food in China. In general, the dose of Ganoderma lucidum is 30 g daily. FYGL content is only 1% in Ganoderma lucidum, so its equivalent human daily dose is roughly 0.015 g/kg/d, if human weight of 60 kg supposed. Our previous toxicology experiment showed that the LD50 of FYGL is 6 g/kg in vivo, highly safe.²⁶⁾ Therefore, FYGL, a natural compound, could be potentially used to treat NAFLD.

Fig. 9. Schematic Diagram of the Mechanism of FYGL Preventing Hepatic Cells from Lipid Accumulation, Oxidative Stress and Apoptosis by Up-Regulation of AMPK Signaling Pathway, Decrease of ROS and MDA Productions as Well as Increase of T-AOC and SOD, and Regulation of Cleaved Caspase-3, Bax and Bcl-2 Expressions

(Color figure can be accessed in the online version.)

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 21374022, 81374032), National Health and Family Planning Commission of the People’s Republic of China (No. 2017ZX09301006), Science and Technology Commission of Shanghai Municipality (No. 17401902700).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, Harrison SA, Brunt EM, Sanyal AJ. The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases. Hepatology, 67, 328–357 (2018).
• 2) Marjot T, Moolla A, Cobbold JF, Hodson L, Tomlinson JW. Nonalcoholic fatty liver disease in adults: current concepts in etiology, outcomes, and management. Endocr. Rev., 41, 66–117 (2020).
• 3) Day CP, James OFW. Steatohepatitis: a tale of two “hits”? Gastroenterology, 114, 842–845 (1998).
• 4) Jin XL, Wang K, Li QQ, Tian WL, Xue XF, Wu LM, Hu FL. Antioxidant and anti-inflammatory effects of chinese propolis during palmitic acid-induced lipotoxicity in cultured hepatocytes. J. Funct. Foods., 34, 216–223 (2017).
• 5) Vanni E, Bugianesi E, Kotronen A, De Minicis S, Yki-Jarvinen H, Svegliati-Baroni G. From the metabolic syndrome to nafld or vice versa? Dig. Liver Dis., 42, 320–330 (2010).
• 6) Song M, Yang G, Hoa TQ, Hieu HD, Amin ASM, Choe W, Kang I, Kim SS, Ha J. Anti-obesity effect of fermented persimmon extracts via activation of AMP-activated protein kinase. Biol. Pharm. Bull., 43, 440–449 (2020).
• 7) Li Y, Xu SQ, Mihaylova MM, Zheng B, Hou XY, Jiang BB, Park O, Luo ZJ, Lefai E, Shyy JYJ, Gao B, Wierzbicki M, Verbeuren TJ, Shaw RJ, Cohen RA, Zang MW. AMPK phosphorylates and inhibits srebp activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice. Cell Metab., 13, 376–388 (2011).
• 8) Zhang D, Xie T, Leung PS. Irisin ameliorates glucolipotoxicity-associated -cell dysfunction and apoptosis via AMPK signaling and anti-inflammatory actions. Cell. Physiol. Biochem., 51, 924–937 (2018).
• 9) Wu YL, Zhou F, Jiang HT, Wang ZJ, Hua C, Zhang YS. Chicory (cichorium intybus l.) polysaccharides attenuate high-fat diet induced non-alcoholic fatty liver disease via AMPK activation. Int. J. Biol. Macromol., 118 (Pt A), 886–895 (2018).
• 10) Zhou B, Zhou DL, Wei XH, Zhong RY, Xu J, Sun L. Astragaloside IV attenuates free fatty acid-induced er stress and lipid accumulation in hepatocytes via AMPK activation. Acta Pharmacol. Sin., 38, 998–1008 (2017).
• 11) Braud L, Battault S, Meyer G, Nascimento A, Gaillard S, de Sousa G, Rahmani R, Riva C, Armand M, Maixent JM, Reboul C. Antioxidant properties of tea blunt ROS-dependent lipogenesis: beneficial effect on hepatic steatosis in a high fat-high sucrose diet NAFLD obese rat model. J. Nutr. Biochem., 40, 95–104 (2017).
• 12) Baeza G, Amigo-Benavent M, Sarria B, Goya L, Mateos R, Bravo L. Green coffee hydroxycinnamic acids but not caffeine protect human HepG2 cells against oxidative stress. Food Res. Int., 62, 1038–1046 (2014).
• 13) Tian XF, Ji FJ, Zang HL, Cao H. Activation of the miR-34a/SIRT1/p53 signaling pathway contributes to the progress of liver fibrosis via inducing apoptosis in hepatocytes but not in HSCs. PLOS ONE, 11, e0158657 (2016).
• 14) Afonso MB, Castro RE, Rodrigues CMP. Processes exacerbating apoptosis in non-alcoholic steatohepatitis. Clin. Sci., 133, 2245–2264 (2019).
• 15) Sumida Y, Yoneda M. Current and future pharmacological therapies for NAFLD/NASH. J. Gastroenterol., 53, 362–376 (2018).
• 16) Kakar MU, Naveed M, Saeed M, Zhao SC, Rasheed M, Firdoos S, Manzoor R, Deng YL, Dai RJ. A review on structure, extraction, and biological activities of polysaccharides isolated from Cyclocarya paliurus (Batalin) Iljinskaja. Int. J. Biol. Macromol., 156, 420–429 (2020).
• 17) Yan ZG, Fan RF, Yin SJ, Zhao XN, Liu JZ, Li LH, Zhang WQ, Ge LJ. Protective effects of Ginkgo biloba leaf polysaccharide on nonalcoholic fatty liver disease and its mechanisms. Int. J. Biol. Macromol., 80, 573–580 (2015).
• 18) Jia L, Li W, Li JN, Li Y, Song H, Luan YS, Qi H, Ma LR, Lu XH, Yang Y. Lycium barbarum polysaccharide attenuates high-fat diet-induced hepatic steatosis by up-regulating SIRT1 expression and deacetylase activity. Sci. Rep., 6, 36209 (2016).
• 19) Klupp NL, Chang D, Hawke F, Kiat H, Cao HJ, Grant SJ, Bensoussan A. Ganoderma lucidum mushroom for the treatment of cardiovascular risk factors. Cochrane Database Syst. Rev., 2015, CD007259 (2015).
• 20) Ma HT, Hsieh JF, Chen ST. Anti-diabetic effects of Ganoderma lucidum. Phytochemistry, 114, 109–113 (2015).
• 21) Chang CJ, Lin CS, Lu CC, Martel J, Ko YF, Ojcius DM, Tseng SF, Wu TR, Chen YYM, Young JD, Lai HC. Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota. Nat. Commun., 6, 7489 (2015).
• 22) Tran HB, Yamamoto A, Matsumoto S, Ito H, Igami K, Miyazaki T, Kondo R, Shimizu K. Hypotensive effects and angiotensin-converting enzyme inhibitory peptides of reishi (Ganoderma lingzhi) auto-digested extract. Molecules, 19, 13473–13485 (2014).
• 23) Pan D, Wang LQ, Chen CH, Hu BW, Zhou P. Isolation and characterization of a hyperbranched proteoglycan from Ganoderma lucidum for anti-diabetes. Carbohydr. Polym., 117, 106–114 (2015).
• 24) Pan D, Zhang D, Wu JS, Chen CH, Xu ZX, Yang HJ, Zhou P. Antidiabetic, antihyperlipidemic and antioxidant activities of a novel proteoglycan from Ganoderma lucidum fruiting bodies on db/db mice and the possible mechanism. PLOS ONE, 8, e68332 (2013).
• 25) Ogawa Y, Imajo K, Honda Y, Kessoku T, Tomeno W, Kato S, Fujita K, Yoneda M, Saito S, Saigusa Y, Hyogo H, Sumida Y, Itoh Y, Eguchi K, Yamanaka T, Wada K, Nakajima A. Palmitate-induced lipotoxicity is crucial for the pathogenesis of nonalcoholic fatty liver disease in cooperation with gut-derived endotoxin. Sci. Rep., 8, 11365 (2018).
• 26) Teng BS, Wang CD, Yang HJ, Wu JS, Zhang D, Zheng M, Fan ZH, Pan D, Zhou P. A protein tyrosine phosphatase 1B activity inhibitor from the fruiting bodies of Ganoderma lucidum (Fr.) Karst and its hypoglycemic potency on streptozotocin-induced type 2 diabetic mice. J. Agric. Food Chem., 59, 6492–6500 (2011).
• 27) Qin H, Liu Y, Lu N, Li Y, Sun CH. cis-9,trans-11-Conjugated linoleic acid activates AMP-activated protein kinase in attenuation of insulin resistance in C2C12 myotubes. J. Agric. Food Chem., 57, 4452–4458 (2009).
• 28) Rolo AP, Teodoro JS, Palmeira CM. Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis. Free Radic. Biol. Med., 52, 59–69 (2012).
• 29) Benhamed F, Postic C. A new pathway to escape lipotoxicity. Clin. Res. Hepatol. Gastroenterol., 42, 3–5 (2018).
• 30) Wang PX, Zhang XJ, Luo PC, Jiang X, Zhang P, Guo JH, Zhao GN, Zhu XY, Zhang Y, Yang SJ, Li HL. Hepatocyte TRAF3 promotes liver steatosis and systemic insulin resistance through targeting TAK1-dependent signalling. Nat. Commun., 7, 10592 (2016).
• 31) Zhong DD, Xie ZW, Huang BY, Zhu S, Wang GQ, Zhou H, Lin SQ, Lin ZB, Yang BX. Ganoderma lucidum polysaccharide peptide alleviates hepatoteatosis via modulating bile acid metabolism dependent on FXR-SHP/FGF. Cell. Physiol. Biochem., 49, 1204–1220 (2018).
• 32) Yang Z, Chen CH, Zhao J, Xu WJ, He YM, Yang HJ, Zhou P. Hypoglycemic mechanism of a novel proteoglycan, extracted from Ganoderma lucidum, in hepatocytes. Eur. J. Pharmacol., 820, 77–85 (2018).
• 33) Wu SB, Wu YT, Wu TP, Wei YH. Role of AMPK-mediated adaptive responses in human cells with mitochondrial dysfunction to oxidative stress. Biochim. Biophys. Acta, 1840, 1331–1344 (2014).
• 34) Dong KL, Wu ML, Liu XM, Huang YJ, Zhang DY, Wang YT, Yan LJ, Shi DY. Glutaredoxins concomitant with optimal ROS activate AMPK through S-glutathionylation to improve glucose metabolism in type 2 diabetes. Free Radic. Biol. Med., 101, 334–347 (2016).
• 35) Liang ZEN, Yuan ZH, Guo JJ, Wu J, Yi JN, Deng J, Shan Y. Ganoderma lucidum polysaccharides prevent palmitic acid-evoked apoptosis and autophagy in intestinal porcine epithelial cell line via restoration of mitochondrial function and regulation of MAPK and AMPK/Akt/mTOR signaling pathway. Int. J. Mol. Sci., 20, 478 (2019).