Metabolic regulation of Ganoderma lucidum extracts in high sugar and fat diet-induced obese mice by regulating the gut-brain axis



In this study, an obese mouse model was developed by administering a high sugar and fat diet for 60 days, followed by administration of a special dietary supplement of Ganoderma lucidum extract for another 35 days. Then, the changes in histopathology of the liver, adipose, colon, intestines, spleen, renal and brain tissues; metabolism and transcription; and gut microbiota were monitored. The results showed that alcohol extracts of the G. lucidum fruit body could reduce body weight; change the serum levels of lipid; ameliorate the damage to the gut microbiota, colon, liver, brain and other organs induced by the high sugar and fat diet; and activate the leptin regulatory pathways in the hypothalamus to improve metabolism. These findings indicate that administration of the alcohol extracts of the G. lucidum fruit body has beneficial effects on the microbiome-gut-liver and microbiome-gut-brain axes, and activates leptin-mediated signaling to improve metabolic regulation.

1. Introduction

Owing to changes in lifestyle and eating habits, metabolic diseases, such as obesity, diabetes, hypertension, hyperlipemia, hyperuricemia, and nonalcoholic fatty liver disease (Non-AFLD), have become a pandemic in China (Long et al., 2019, Yuan et al., 2019, Zhang et al., 2016). Reports showed that there were about more than 450 million metabolic disease patients in China today (Swinburn et al., 2019). Although some risk factors, such as smoking, drinking, exercise, meditation, and genetics, are known to be associated with metabolic disorders (Chassaing et al., 2015, Sonnenburg and Bäckhed, 2016), scholars believe these factors do not explain the higher prevalence and variety of metabolic diseases in China relative to that in developed countries. Therefore, the development of prevention and treatment strategies is urgently needed.

One study showed that the gut microbiota are involved in the pathogenesis of obesity (Sonnenburg & Bäckhed, 2016), particularly the metabolites produced by the symbiotic microbes (e.g., short-chain fatty acids, lipopolysaccharides, methane, and trimethylamine oxide). These metabolites can cause chronic inflammatory reactions by increasing energy intake, slowing down bowel movement, and accelerating the accumulation of intracellular cholesterol, thus inducing obesity and insulin resistance and promoting atherosclerosis (De Vadder et al., 2014, Perry et al., 2016). Central obesity is the typical characteristic of metabolic syndrome, and diet is the key cause of obesity. Diet is one of the main factors influencing the gut microbiota composition; therefore, dietary supplements are important for the maintenance of homeostasis of gut microbiota. Modulation of gut-brain signaling was demonstrated to have unprecedented potential for treating obesity (Clemmensen et al., 2017, Solas et al., 2017, Torres-Fuentes et al., 2017) and type 2 diabetes (Grasset et al., 2017, Slyepchenko et al., 2016). Ganoderma lucidum is widely used as a medicinal and edible powder food for promoting health in Southeast Asia for a long history. Traditional Chinese medicine records that ganoderma lucidum has effects on delaying aging, while the mode of action are not very clear. In this study, an obese mouse model was prepared by administering a high sugar and fat diet for 60 days, followed by dietary supplementation of Ganoderma lucidum extracts for another 35 days. Then, changes in the histopathology of liver, adipose, colon, intestinal, spleen, renal, and brain tissues; metabolism and transcription; and gut microbiota were monitored, with the aim of elucidating the prebiotic effects of G. lucidum extracts on high-sugar-and-fat-diet-induced obese mice via regulation of the gut-brain axis.

2. Methods

2.1. Animal model preparation and treatments

Adult male KM mice (18–22 g, 9 months) obtained from the Center of Laboratory Animal of Guangdong Province (SCXK [Yue] 2008–0020, SYXK [Yue] 2008–0085) were pair-housed in plastic cages in a temperature-controlled (25 ± 2 °C) colony room under a 12/12-h light/dark cycle. Food and water were available ad libitum. All experimental protocols were approved by the Center of Laboratory Animals of the Guangdong Institute of Microbiology. All efforts were made to minimize the number of animals used.

The mice in the control group were fed a standard diet (Control or Normal), and the mice in the model groups were fed a high sugar and fat diet (Model). Water was freely available, and these treatments were administered for 3 months. After the mice were fed a high sugar and fat diet for one month, various doses of G. lucidum extract, GH (high-dose of G. lucidum extract) of 200 mg/kg/d, GM (middle-dose of G. lucidum extract) of 100 mg/kg/d, GL (low-dose of G. lucidum extract) of 50 mg/kg/d, extracted with ethanol were delivered by intragastric administration. The preparation and constituents of alcohol extracts of the G. lucidum fruit body (AGL) were indicated in our recently published paper . The high sugar and fat diet was continued for another two months.

The components of the high sugar and fat diet included 20% sucrose, 15% fat, 1.2% cholesterol, 0.2% bile acid sodium, 10% casein, 0.6% calcium hydrogen phosphate, 0.4% stone powder, 0.4% premix, and 52.2% basic feed. The heat ratio was as follows: protein 17%, fat 17%, and carbohydrate 46%.

2.2. Obesity related-parameter measurement

The appearance, behavior, and fur color of the animals were observed and documented every day. Animal weight was measured every 3 days during the drug administration period. Following the water maze testing, blood and serum samples were acquired. And cytokines were measured, and the brains of the animals were dissected. A total of 4 brains from each group were fixed in 4% paraformaldehyde solution and prepared as paraffinized sections. The sections were stained with hematoxylin-eosin (H&E) and immunohistochemistry staining and observed under light microscopy .

2.3. Microbiome analysis

Fresh intestinal content samples were collected before the fasting of the mice and stored at −80 °C. Frozen microbial DNA isolated from mice intestinal content samples with total masses ranging from 1.2 to 20.0 ng were stored at −20 °C. The microbial 16S rRNA genes were amplified using the forward primer 5′-ACTCCTACG GGAGGCAGCA-3′ and the reverse primer 5′-GGACTACHVGGGTWTCTAAT-3′, as well as the forward primer 5′-CCTAYGGGRBG CASCAG-3′ and reverse primer 5′-GGACTACNNGGGTATCTAAT-3′, for rats. Each amplified product was concentrated via solid-phase reversible immobilization and quantified by electrophoresis using an Agilent 2100 Bioanalyzer (Agilent). After quantification of DNA concentration by NanoDrop, each sample was diluted to a concentration of 1 × 109 mol/μl in TE buffer and pooled. A total of 20 μL of the pooled mixture was used for sequencing with the Illumina MiSeq sequencing system according to the manufacturer’s instructions. The resulting reads were analyzed as described previously .

2.4. Metabolomic analysis

A 40-mg feces sample was homogenized in 400 μL of deionized water containing 10 μg/mL of L-norvaline as an internal standard. Following centrifugation at 14,000g and 4 °C for 15 min, a total of 300 μL of the supernatant was transferred. The extraction was repeated by adding 600 μL of ice-cold methanol to the residue. The supernatants from the two extractions were combined. A total of 400 μL of the combined supernatants and 10 μL of the internal standard solution (50 μg/mL of l-norleucine) were combined and evaporated to dryness under a nitrogen stream. The residue was reconstituted in 30 μL of 20 mg/mL methoxyamine hydrochloride in pyridine, and the resulting mixture was incubated at 37 °C for 90 min. Then, 30 μL of BSTFA (with 1% TMCS) was added to the mixture and derivatized at 70 °C for 60 min prior to gas chromatography-mass spectrometer (GC–MS) metabolomics analysis.

Metabolomics instrumental analysis was performed on an Agilent 7890A gas chromatography system coupled to an Agilent 5975C inert MSD system (Agilent Technologies Inc., CA, USA). An OPTIMA® 5 MS Accent fused-silica capillary column (30 m × 0.25 mm × 0.25 μm; MACHEREY-NAGEL, Düren, GERMAN) was utilized to separate the derivatives. Helium (>99.999%) was used as a carrier gas at a constant flow rate of 1 mL/min through the column. The injection volume was 1 μL in split mode (2:1), and the solvent delay time was 6 min. The initial oven temperature was held at 70 °C for 2 min, ramped up to 160 °C at a rate of 6 °C/min, to 240 °C at a rate of 10 °C/min, to 300 °C at a rate of 20 °C/min, and finally held at 300 °C for 6 min. The temperatures of the injector, transfer line, and electron impact ion source were set to 250 °C, 260 °C, and 230 °C, respectively. The electron ionization (EI) energy was 70 eV, and data were collected in full scan mode (m/z 50–600).

The typical total ion current (TIC) chromatograms are illustrated in . Information on the peak picking, alignment, deconvolution, and further processing of raw GC–MS data can be found in previously published protocols. The final data were exported as a peak table file, including observations (sample name), variables (rt_mz), and peak areas. The data were normalized against total peak abundances before performing univariate and multivariate statistical analyses.

For the multivariate statistical analysis, the normalized data were imported into SIMCA software (version 14.1, Umetrics, Umeå, Sweden), where the data were preprocessed by unit variance (UV) scaling and mean centering before performing principle component analysis (PCA), partial least squares – discriminant analysis (PLS-DA), and orthogonal filter partial least-squares discriminant analysis (OPLS-DA). The model quality is described by the R2X or R2Y and Q2 values. R2X (PCA) or R2Y (PLS-DA and OPLS-DA) is defined as the proportion of variance in the data explained by the models and indicates the goodness of fit. Q2 is defined as the proportion of variance in the data that is predictable by the model and indicates the predictability of the current model, calculated by a cross-validation procedure . To avoid model over-fitting, a default 7-round cross-validation in SIMCA software was performed throughout to determine the optimal number of principal components .

For univariate statistical analysis, the normalized data were analyzed in the R platform (version 3.3.0), where parametric testing was performed on the normally distributed data by Welch’s t test, while nonparametric testing was conducted on the abnormally distributed data by the Wilcoxon Mann-Whitney test.

The variables with VIP (Variable importance in the projection) values in the OPLS-DA model larger than 1 and p values from the univariate statistical analysis lower than 0.05 were identified as potential differential metabolites . Fold change was calculated as the binary logarithm of the average normalized peak intensity ratio between Group 1 and Group 2, where a positive value indicates that the average mass response of Group 1 is higher than that of Group 2.

2.5. RNA sequencing

Total RNA was isolated using Trizol Reagent (Invitrogen Life Technologies), followed by determination of the concentration, quality, and integrity using a NanoDrop spectrophotometer (Thermo Scientific). Three global brain samples of RNA were used as input material for the RNA sample preparations. Sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First-strand cDNA was synthesized using random oligonucleotides and SuperScript II. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities, and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 200-bp length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15-cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high-sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on a Hiseq platform (Illumina) by Shanghai Personal Biotechnology Co., Ltd.

2.6. Histopathology and immunostaining

The liver, adipose, colon, intestinal, spleen, and renal tissues of the animals were dissected. A total of four samples from each group were fixed in 4% paraformaldehyde solution and prepared as paraffin sections. Sections were stained with hematoxylin-eosin (H&E), and TUNEL staining were performed. Immunostaining was performed using paraffin-embedded, 3-μm-thick sections and a two-step peroxidase-  technique (DAKO Envision kit, DAKO, Carpinteria, CA). Slides were observed by light microscopy and immunostained (IF) for antibodies against GAFP and IBA-I.

2.7. Western bolting

Briefly, global brain tissue from treated mice (purchased from the Beijing HFK Bioscience Co., LTD [Certificate No: SCXK (Jing) 2014-0004], was dissected, and proteins were extracted with (RIPA) lysis buffer (Thermo ScientificTM T-PERTM Tissue Protein Extraction Reagent, 78510). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5%  in Tris-buffered saline (20 mM Tris-HCl, 500 mM NaCl, pH 7.4) with 0.2% Tween-20 (Aladdin, T104863), the membranes were probed with antibodies overnight at 4 °C, followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse (Servicebio, G2211-1-A) or goat anti-rabbit (Servicebio, G2210-2-A) IgG secondary antibody (1:2000). The antibodies were as follows: anti- LEPR, AGPR, POMC and GABAergic (obtained from Affinity) as well as GAPDH (CST, 2118L) and β-Actin (CST, 4970S). Band intensity was quantified using ImageJ software (NIH).

2.8. Statistical analysis

All data are described as the means ± standard deviations (SD) of at least three independent experiments. Significant differences between treatments were assessed by one-way analysis of variance (ANOVA) with a significance level of p < 0.05 using the Statistical Package for the Social Sciences (SPSS; Abacus Concepts, Berkeley, CA, USA) and  5 (GraphPad, San Diego, CA, USA) software.