The incidence of overweight and obesity is increasing around the world. Epidemiological studies have identified a high body mass index as a risk factor for a range of chronic diseases, including cardiovascular disease, type 2 diabetes, chronic kidney disease, multiple cancers, and a range of musculoskeletal disorders [1
]. The prevention of obesity has become among the most challenging concerns for modern society. A growing amount of evidences suggest that the gut microbiota may serve as an important modulator of obesity by affecting the absorption of nutrients in the intestine [3
Recent studies have shown that gut microorganisms function similarly to endocrine organs, because they produce biologically active metabolites that affect the host. Intestinal microorganisms are in a position to produce large amounts of metabolites—some of which are absorbed directly into the systemic circulation and others are processed by the host enzymes [5
]. The intestinal microbiota of healthy people is mainly composed of Bacteroidetes and Firmicutes. It has been found that in obese individuals, the abundance of Bacteroidetes is decreased and the abundance of Firmicutes is increased [6
]. This suggests that Bacteroidetes and Firmicutes are essential for regulating obesity and can serve as obesity markers.
Allicin and its derivative, diallyl disulfide (DADS), are trithioallyl ether compounds that naturally occur in the bulbs of the lily family member, garlic, and are produced by alliinase, which produces allicin [7
]. Allicin is highly reactive with thiol groups and, under certain conditions, it also reacts with itself, forming further compounds that are also bioactive, such as vinyl-dithiins, ajoene and polysulfanes [8
]. Furthermore, allicin is an active sulfur (RSS) with oxidative properties that can regulate oxidative metabolism in cells. [9
Allicin’s structure determines many of its biological functions. For example, Feldberg et al. have found that allicin affects the synthesis of macromolecules such as DNA, RNA and protein [10
], and garlic consumption or the inclusion of garlic oils in the diet selectively reduces the concentration of blood triglycerides (TGs), total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) without affecting high-density lipoprotein-cholesterol (HDL-C) levels [11
]. The latest research has found that allicin promotes white adipose tissue browning through krüppel-like factor (KLF)15 [13
]. Some studies have also found that allicin has several biological functions, including antibacterial, blood pressure lowering and antioxidation [14
]. In addition, DADS and diallyl polysulfone, directly formed by the decomposition of allicin, have exhibited remarkable antibacterial effects. Koch and Lawson have demonstrated that allicin inhibits the growth of Escherichia coli
and Staphylococcus aureus
]. These studies indicate that allicin affects both fat deposition and microorganisms. Hence, we wondered whether these physiological functions of allicin are achieved by regulating the gut microbiota. However, we could not find data about allicin regulating the gut microbiota.
In this study, we explored the potential effects of allicin on mice with high-fat diet-induced obesity. We found that allicin suppressed body weight gain by regulating the gut microbiota.
2. Materials and Methods
2.1. Animal Experiment
Six-week-old C57BL/6 male mice, weaned from 4 weeks, were purchased from the Medical Laboratory Animal Center of Xi’an Jiaotong University (Xi’an, China; approval XJTULAC-2013-024). The animals were housed in stainless steel cages at room temperature (25 ± 2 °C), with a 12 h light/dark cycle. They were fed a commercial chow for a week to acclimatize to animal facilities and then weighed and randomly divided into two groups. One group was fed regular chow (control group, NFD, n = 6) and the other group received a high-fat diet (HFD, n = 12). We began the experiments once there was a significant difference in body weight between the HFD and NFD groups. The HFD group was randomly divided into two groups, which continued to receive a high-fat diet: one group was given normal saline (negative control, NC) and the other group was given 100 mg/kg/d allicin (Allicin) (S25256, Source Leaf Biological, Shanghai, China). The HFD in this study contained 60% fat and the NFD contained 10% fat (TrophicDiet, Nantong, China). During the experiments, body weight and feed intake were measured weekly. Mice were fasted overnight before being sacrificed; body weight was measured and tissues (inguinal white adipose tissue (iWAT), epididymal WAT (eWAT), brown adipose tissue (BAT) and liver) were excised, weighed and stored at −80 °C. In addition, the small intestine was immediately ligatured, and the contents were collected under aseptic conditions and frozen in liquid nitrogen for 16S rDNA sequencing. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Northwest A&F University and were approved by the Animal Ethics Committee of Northwest A&F University (approval number NWAFU-314020038). The animal experiments were confirmed by the Guide for the Care and Use of Laboratory Animals of China.
2.2. Glucose Tolerance Tests
After six weeks of allicin administration, obese mice were fasted overnight. Tail vein blood was used to measure glucose levels using a YUWELL 560 glucometer (Jiangsu, China). Glucose levels were measured twice at every time point (0, 15, 30, 60 and 120 min) after intraperitoneal injection of 1 g of glucose (Cat. No. XK 13-201-00310, Tianjin, China) per kg body weight dissolved in saline.
2.3. Serum Analysis
The mice were treated with ether and the heart blood was collected and centrifuged at 13,680× g for 10 min. The collected serum was used to determine the concentrations of serum cholesterol (TC), serum triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate amino transaminase (AST) and alanine amino transaminase (ALT).
2.4. Haematoxylin and Eosin (H&E) Staining
The iWAT, eWAT, BAT and small intestine tissue from representative mice of each group were fixed with 4% paraformaldehyde. After samples were dehydrated and embedded in paraffin, sections were cut using a Leica RM22559 microtome (Leica, Shanghai, China) and standard H&E staining was performed.
2.5. PCR Real-Time Quantitative PCR (RT-qPCR)
Total RNA was extracted using TRIzol reagent (TaKaRa, Otsu, Japan) following the manufacturer’s instructions. The mRNA was reverse transcribed with transcription kits (TaKaRa) to synthesise cDNA, and the cDNA was amplified using SYBR Green kits on a Stepone plus™ system (Thermo Fisher, Waltham, MA, USA). The primer sequences used are shown in Supplementary Table S1
, and the data were processed using the 2−ΔΔCT
2.6. Enzyme Activity Evaluation
The intestinal content of the enzymes, trypsin, amylase and lipase was determined by enzymatic activity kits (Institute of Bioengineering, Nanjing, China), according to the manufacturer’s instructions.
2.7. 16S rRNA Sequencing with Illumina MiSeq Sequencing
DNA was extracted from the intestinal contents using the Qiagen QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s directions. Illumina MiSeq sequencing and general data analyses were performed by Novogene, Beijing, China. The DNA (regions V3 and V4 of the bacterial 16S rRNA gene) was amplified with barcoded specific bacterial primers using PCR. The primers used were 338F: 5′-ATCCTACGGGAGGCAGCA-3′ and 806R: 5′-ggactachvgggtwtctaat-3′. Amplification was performed with the DNA template (50 ng) in a 25 μL reaction for 25–35 cycles with Phusion DNA Polymerase.
2.8. Bioinformatic Analysis
MiSeq sequencing results in double-ended sequence data. First, we filtered the measured fq data, then we filtered bases with a read-tail mass value of 20 or less, and then set a 50 bp window. If the average mass value in the window was lower than 20, the window began to intercept the back-end base and filtered the read below 50 bp after the quality control; then, the paired sequences were merged into a sequence according to the overlap relationship of the Paired-End (PE) sequencing. Then, the sequences were grouped into operational taxonomy units (OTUs) at 97% similarity. Basically, there was less than 3% sequence dissimilarity in all reads of the same OTU. The Ribosomal Database Project (RPD) was applied to classify the OTU sequences and identify the bacterial species.
2.9. Statistical Analysis
All data are expressed as the mean ± SD, and statistical analysis was performed with GraphPad Prism 7.0. Data were analyzed by Student’s t-test. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly statistically significant (* p < 0.05; ** p < 0.01).
Studies have shown that the gut microbiota is an important environmental factor that contributes to the development of obesity, insulin resistance and inflammation [22
]. The gut microbiota also promotes energy storage by suppressing thermogenesis in brown adipose tissues and promoting WAT expansion. Intestinal microorganisms produce many small, soluble metabolites, such as lipopolysaccharide, that induce proinflammatory cytokines, insulin resistance and WAT inflammation. These substances are absorbed by the intestines, transported to tissues and organs through the blood, and modulate metabolism and inflammation by regulating gene expression [23
]. Allicin enhances insulin activity [24
], and reduces hypertension and hyperlipidemia in diabetic patients [25
]. Herein, we found that allicin decreased body weight gain and fat accumulation by inhibiting adipogenesis and promoting lipolysis. The GTT and RT-qPCR results of the insulin signaling pathway-related genes, IRS1
, suggested that allicin significantly enhanced insulin sensitivity. Emerging evidence has demonstrated that allicin induces white adipocyte browning and reduces high-fat-induced obesity via the KLF15 signaling cascade [13
]. In this study, we found that allicin increased the expression of brown adipocyte-related genes and thermogenic genes. These results suggest that allicin reduced the body weight gain of obese mice through white adipose browning.
Obesity is accompanied by an expansion in the volume of adipose tissues, causing an increase in mechanical stress by contact with neighboring cells and extracellular matrix components. When adipocytes spread to near the oxygen diffusion limit, they experience hypoxia, resulting in dysregulation of adipokine production and inflammation [27
]. Under these circumstances, the secretion of leptin and resistin increases. In the current study, allicin reduced the expression of the adipokine-related genes, leptin, adipoq and resistin, and the area of fat cells decreased. These results indicated that allicin reduced fat deposition and inhibited inflammation in obese mice. Furthermore, we found that allicin reduced the LDL-C level in obese mice and increased the HLD-C and TG levels, which is consistent with previous research [12
The intestine morphology directly reflects the health state of the intestine [29
], which is mainly determined by the villi length, the crypt depth and their ratio. It is generally accepted that the ratio of villus to crypt is large, which is more conducive to the absorption of nutrients [30
]. In this study, allicin significantly increased the villi length and the ratio of villus length to crypt depth, demonstrating that allicin improved intestinal function. It has been shown that intestinal microorganisms affect intestinal morphology [31
], for example, the cecal Bacillus fragilis
population negatively correlated with crypt depth, while the abundance of cecal C. leptum positively correlated with the villus height [32
]. This indicated that allicin may affect intestinal morphology through gut microorganisms.
Recent research has revealed that a variety of small molecules substances, such as Cordycepin
, Fuzhuan brick tea
polysaccharides and Vanillin
, can reduce fat deposition by altering intestinal microbial composition [6
]. However, allicin-induced reduction in body weight gain of high-fat diet-induced obese mice through the gut microbiota has not been reported. In this study, allicin affected the intestinal microbial composition in obese mice induced by high-fat diet.
Gut microorganisms perform many functions that the body cannot perform, hence, they form a symbiotic relationship with the body. In 1983, Wostmann first discovered that in a germ-free (GF) environment, the rate of weight gain was slower than in a normal environment [35
]. Bäckhed et al. have further demonstrated that intestinal microorganisms suppressed the intestinal expression of angiopoietin-like 4 (ANGPTL4), a circulating inhibitor of lipoprotein lipase (LPL). This increased the LPL activity in adipocytes, then increased the absorption of fatty acids by the cells and the accumulation of TG in the adipocytes [36
]. Under GF conditions, the expression level of ANGPTL4 was higher, and LPL was inhibited, resulting in a slower rate of weight gain in mice. These findings showed that the intestinal microbiota plays an important role in fat formation.
In the intestinal microbiota, Firmicutes, Bacteroidetes and Actinobacteria account for more than 90% of all of the bacteria. It has been shown that obese mice were associated with gut microbiota changes; in ob/ob mice, the abundance of Firmicutes was increased, while the Bacteroidetes abundance was decreased. In contrast, in lean mice, the Firmicutes abundance was decreased, while the Bacteroidetes abundance was increased [37
]. This is consistent with the intestinal microbial differences observed between lean and obese humans [6
]. Our research revealed that allicin significantly increased the abundance of Bacteroidetes and decreased the abundance of Firmicutes in obese mice induced by a high-fat diet. This indicates that allicin affects intestinal microbial composition at the phylum level. Thus, the ratio of Firmicutes and Bacteroidetes can be used as a biomarker for obesity. However, several studies have found that the Firmicutes and Bacteroidetes ratio increased in obese individuals [38
]. Hence, a lower level analysis is necessary to detect changes in gut microorganisms. Therefore, we chose to use the order and a lower level of genus for comparison and analysis.
At the order level, Lactobacillus and Bifidobacterium are typically used as beneficial bacteria, and studies have found that the abundance of both was increased in obese individuals [40
]. In this study, we found that Bifidobacterium was significantly increased in allicin-treated mice. This was consistent with the findings of Lecomte et al. [41
]. Namely, the abundance of two bacterial species in obese mice depends on the experimental model. At the genus level, we analyzed the abundance of Akkermansia
XIVb and Eubacterium
Studies have shown that Akkermansia
colonizes the mucosal layer of the human intestine and increases mucosal thickness and gut barrier function. Akkermansia
also sends a signal directly to immune receptors, causing a host immune response, and at the same time it produces short-chain fatty acids that are beneficial for the host and for microbiota members [42
XIVb and Eubacterium
had an anti-obesity effect by producing butyrate, which is a source of energy for the colonocytes [43
]. Our results showed that at the genus level allicin significantly increased the abundance of Akkermansia
; however, it had no effect on Clostridium
XIVb and Eubacterium
. These results indicated that allicin reduced body weight gain and fat accumulation by increasing the abundance of beneficial species [5
Allicin has been widely studied for its anti-inflammatory, anti-cancer, anti-hypertensive and anti-obesity effects [44
]. Other study have found that allicin can alleviate the learning and memory disorders caused by exposure to lead during development [45
]. Interestingly, Cai et al. found that Flammulina velutipes
polysaccharides improved scopolamine-induced learning and memory impairment in mice by regulating the composition of intestinal microbes [46
]. In this study, we found that allicin can induce weight loss in obese mice induced by high-fat diet by regulating intestinal microbes. Therefore, we suspect that the improvement of memory and learning ability of allicin may also be achieved by intestinal microbes. But further research is needed.