If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, ChinaJiangsu Yuhang Food Technology Co., Ltd., Yancheng 224200, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, ChinaJiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou 225127, China
A growing stream of research suggests that probiotic fermented milk has a good effect on nonalcoholic fatty liver disease. This work aimed to study the beneficial effects of Lactobacillus rhamnosus hsryfm 1301 fermented milk (fermented milk) on rats with nonalcoholic fatty liver disease induced by a high-fat diet. The results showed that the body weight and the serum levels of total cholesterol, total glyceride, low-density lipoprotein, alanine transaminase, aspartate aminotransferase, free fatty acid, and reactive oxygen species were significantly increased in rats fed a high-fat diet (M) for 8 wk, whereas high-density lipoprotein cholesterol and superoxide dismutase were significantly decreased. However, the body weight and the serum levels of total cholesterol, total glyceride, alanine transaminase, aspartate aminotransferase, free fatty acid, reactive oxygen species, interleukin-8, tumor necrosis factor-α, and interleukin-6 were significantly decreased with fermented milk (T) for 8 wk, and the number of fat vacuoles in hepatocytes was lower than that in the M group. There were significant differences in 19 metabolites in serum between the M group and the C group (administration of nonfermented milk) and in 17 metabolites between the T group and the M group. The contents of 7 different metabolites, glycine, glycerophosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, thioetheramide-PC, d-aspartic acid, oleic acid, and l-glutamate, were significantly increased in the M group rat serum, and l-palmitoyl carnitine, N6-methyl-l-lysine, thymine, and 2-oxadipic acid were significantly decreased. In the T group rat serum, the contents of 8 different metabolites—1-O-(cis-9-octadecenyl)-2-O-acetyl-sn-glycero-3-phosphocholine, acetylcarnitine, glycine, glycerophosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, d-aspartic acid, oleic acid, and l-glutamate were significantly decreased, whereas creatinine and thymine were significantly increased. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that 50 metabolic pathways were enriched in the M/C group and T/M group rat serum, of which 12 metabolic pathways were significantly different, mainly distributed in lipid metabolism, amino acid, and endocrine system metabolic pathways. Fermented milk ameliorated inflammation, oxygenation, and hepatocyte injury by regulating lipid metabolism, amino acid metabolic pathways, and related metabolites in the serum of rats with nonalcoholic fatty liver disease.
With the rapid development of the economy and changes in lifestyle, nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease, affecting millions of people worldwide. It is caused by high cholesterol, high calorie intake, and a lack of exercise and physical activity (
Excess dietary cholesterol promotes free fatty acid (FFA) overload and increases levels of intracellular free cholesterol that can be cytotoxic in hepatocytes (
). Free fatty acids are either oxidized or esterified to produce triglycerides, and the ability of the liver to handle energy substrates is overwhelmed, which results in the accumulation of lipids and stimulates reactive oxygen species (ROS), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) overproduction (
). With the deepening of gut microbiota research, NAFLD has also been shown to be closely related to the dysbiosis of gut microbiota in several studies (
). Vitamin E, insulin sensitizers, pentoxifylline, and obeticholic acid are the main drugs that are widely used to treat NAFLD, but there are some side effects (
). However, there is growing evidence showing that Lactobacillus bulgaricus, Lactobacillus acidophilus, Bifidobacterium lactis, and milk fermented with these bacteria can alleviate NAFLD and have no adverse effects when used in patients with NAFLD (
). Lactobacillus not only decreased the levels of ALT, AST, total cholesterol (TC), and triglyceride (TG) in the serum of children with NAFLD but also improved gut dysbiosis by correcting the gut microbiota of mice with NAFLD and inhibiting hepatic lipid deposition (
Oral administration of compound probiotics ameliorates HFD-induced gut microbe dysbiosis and chronic metabolic inflammation via the G protein-coupled receptor 43 in non-alcoholic fatty liver disease rats.
). Moreover, evidence has also suggested that Lactobacillus could exert beneficial effects, including inhibition of gut pathogens, enhancement of mucosal barrier integrity, and immune modulation of NAFLD (
). Goat milk fermented with L. bulgaricus ssp. delbrueckii and Streptococcus thermophilus has a beneficial effect on cardiovascular health shown to lessen the inflammatory response, macrophages activation, and atherosclerosis development (
). Camel milk fermented with S. thermophilus, L. acidophilus, and Bifidobacterium bifidum not only was profitable for enhancing immunity and protecting against oxidative stress but also improved the lipid levels and lipid accumulation in hyperlipidemic rats (
Evaluating the nutritional and immune potentiating characteristics of unfermented and fermented turmeric camel milk in cyclophosphamide-induced immunosuppression in rats.
). Buffalo milk and soy milk fermented with B. lactis and Bifidobacterium longum also contributed to lowering the levels of plasma and liver lipids in rats, and they could promote the excretion of bile acids in the feces at the same time (
). Furthermore, by improving the gut microbiota and feces metabolites associated with lipid transport and reduction, koumiss (a kind of mare milk fermented with Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactobacillus kefiri, and Streptococcus parauberis) was effective in lowering the lipid levels in hyperlipidemia patients (
). Nevertheless, the specific mechanism has not yet been fully revealed.
Our previous research found that Lactobacillus rhamnosus hsryfm 1301 fermented milk can not only improve the levels of TC, TG, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in hyperlipidemic rats' serum but also improve the gut microbiota related to lipid metabolism (
); therefore, the effect of L. rhamnosus hsryfm 1301 fermented milk on serum metabolism, which is closely related to liver steatosis, was evaluated to explore the mechanism by which probiotics alleviate the effects of NAFLD.
MATERIALS AND METHODS
Bacteria and Culture
Lactobacillus rhamnosus hsryfm 1301, which was isolated and screened from the gut of subjects from Bama longevity in Bama Yao Autonomous County in Guangxi, China (
), was obtained from Jiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou University. The isolate was grown in de Man, Rogosa, and Sharpe medium at 37°C in an anaerobic jar (Ruskinn Technologies Ltd.) for 24 h.
Preparation of Fermented Milk
Activated L. rhamnosus hsryfm 1301 was inoculated into 12% (wt/vol) sterilized skim milk (Fonterra Co-operative Group LTD) with 3% (wt/vol) inoculum, fermented at 42°C for 21 h until the viable count was 109 cfu/mL, and then preserved at 4°C. The viable count of L. rhamnosus hsryfm 1301 was calculated by the plate counting method with de Man, Rogosa, and Sharpe agar (pH 6.8 ± 0.2) in triplicate, and plates were placed in an anaerobic jar at 37°C for 48 h.
Animal Trial
Animal Groups and Diets
Thirty 6-wk-old male Wistar rats (200 ± 20 g) of specific-pathogen-free grade obtained from Jinan Langyue Experimental Animal Breeding Co., Ltd., Shandong Province, China [License SCXK (Su) 2017-0044], were reared in ventilated, transparent, clean, and sanitary animal houses with room temperature of 23.0°C ± 1.0°C and humidity of 50% ± 5% and were fed basic feed. The rats were randomly divided into a control group (C), model group (M), and L. rhamnosus hsryfm 1301 fermented milk administration group (T), with 10 rats in each group after adaptive feeding of basic feed for 1 wk, with 12-h alternating periods of dark and light.
Studies have found that fermented milk could improve the lipid metabolism, oxidation injury, and inflammation in obese rats when the intragastric dose of the lactic acid bacteria in the fermented milk was 0.1 to 1 × 109 cfu/100 g BW (
Consumption of probiotic Lactobacillus fermentum MTCC: 5898-Fermented milk attenuates dyslipidemia, oxidative stress, and inflammation in male rats fed on cholesterol-enriched diet.
Effect of yogurt fermented by Lactobacillus Fermentum TSI and L. Fermentum S2 derived from a Mongolian traditional dairy product on rats with high-fat-diet-induced obesity.
). Meanwhile, in our previous study, fermented milk exerted beneficial effects on serum lipid levels in hyperlipidemic rats when the intragastric dose of L. rhamnosus hsryfm 1301 in the fermented milk was higher than 1 × 109 cfu/100 g BW (
). Therefore, the dose of 1 mL/100 g BW (equated with 1 × 109 cfu/100 g BW) was chosen in this study to explore the beneficial effects of L. rhamnosus hsryfm 1301 fermented milk on NAFLD rats.
The T group was administered intragastrically with L. rhamnosus hsryfm 1301 fermented milk at a dose of 1 mL/100 g BW every day, while the C group and the M group were administered intragastrically with 12% skim milk at equivalent doses every day throughout the trial. The C group was fed a basic diet [20% (wt/wt) flour, 10% rice flour, 20% corn, 26% drum head, 20% bean, 2% fish powder, and 2% bone powder; XieTong, Organism Inc.], and the M group and the T group were fed a high-fat diet [10% (wt/wt) lard oil, 1% cholesterol, 0.2% sodium cholate, and 78.8% basic diet; XieTong, Organism Inc.] to establish an NAFLD rat model within 56 d from the beginning to the end of the trial. The rats had free access to water and their diet, and their BW and diet consumption were measured weekly and daily, respectively. The care and use of rats in this study followed our institutional and national guidelines, and all rat experimentation was approved by the Ethics Committee of Yang Zhou University.
BW, Liver Index, and Histopathology Observation
The rats were fasted for 12 h and euthanized on d 56. The livers were removed and washed with phosphate-buffered saline before blotting and weighing. A 0.5-cm3 sample of the left lobe of the liver was fixed in 4% paraformaldehyde and stored at 4°C (
). Tubes were initially held stationary at 0°C for 30 min, and then serum was separated from the blood by centrifugation at 3,500 × g for 20 min at 4°C. The levels of TC, TG, HDL-C, LDL-C, ALT, AST, and superoxide dismutase (SOD) in serum were determined by a test kit (Sichuan Maker Biotechnology Inc.; Nanjing Jiancheng Bioengineering Institute) according to the biochemical analysis (
Live yeast supplementation during late gestation and lactation affects reproductive performance, colostrum and milk composition, blood biochemical and immunological parameters of sows.
), and the levels of FFA, ROS, TNF-α, IL-6, IL-8, TGF-β1, NF-κB, and IL-1β were determined by an ELISA kit (Nanjing Jiancheng Bioengineering Institute; R&D Systems) (
Serum levels of TSP-1, NF-κB and TGF-β1 in polycystic ovarian syndrome (PCOS) patients in northern China suggest PCOS is associated with chronic inflammation.
One hundred microliters of serum from each rat was added to the tube with 400 µL of precooled methanol/acetonitrile and vortexed and mixed before being placed at −20°C for 30 min, followed by centrifugation at 14,000 × g for 20 min at 4°C to precipitate protein (
). The supernatant was extracted and evaporated to dryness in a vacuum centrifuge. Then 100 µL of acetonitrile solution was added to redissolve the supernatant, and the solution was vortexed before centrifugation at 14,000 × g for 15 min at 4°C, and the supernatant was used for MS testing and analysis (
The hydrophilic interaction chromatography column of a 1290 Infinity ultra-high-performance liquid chromatography system (Agilent Technologies) was used to separate serum, and the column temperature was set to 25°C, with a flow rate of 0.3 mL/min and injection volume of 2 µL (
). The mobile phase consisted of (A) water containing 25 mmol/L aqueous ammonium acetate and 25 mmol/L ammonia and (B) acetonitrile. The following gradient program was used: 85% B at 0 to 1 min, linear change from 85% to 65% B at 1 to 12 min, linear change from 65% to 40% B at 12 to 12.1 min, 40% B at 12.1 to 15 min, linear change from 40% to 85% B at 15 to 15.1 min, and 85% B at 15.1 to 20 min (
). The serum samples were placed in a 4°C autosampler, and the random sequence was used for continuous analysis of serum samples to avoid the influence of the fluctuation of the instrument detection signal. Quality-control samples were prepared by pooling and mixing the same volume of each sample, which was inserted into the serum sample queue to monitor and check for system stability and the reliability of the experimental data (
The samples were ionized by electrospray ionization, in both positive ion and negative ion mode, and were analyzed by a Triple TOF 6600 mass spectrometer after ultra-high-performance liquid chromatography separation (
): Ion Source Gas1 (Gas1) at 60 units, Ion Source Gas2 (Gas2) at 60 units, curtain gas at 30 units, a source temperature of 600°C, and IonSpray Voltage Floating ± 5,500 V (positive ion and negative ion mode) after hydrophilic interaction chromatography separation. The m/z range of time-of-flight MS was set to 60 to 1,000 Da, and the accumulation time was set to 0.20 s/spectra; the product ion scanning was set to 25 to 1,000 Da, and the accumulation time was set to 0.05 s/spectra (
). Two-stage MS was performed by information-dependent acquisition with high-sensitivity mode selected; the declustering potential was set to ± 60 V (positive and negative mode), and the collision energy was fixed at 35 V ± 15 eV with exclusion of isotopes within 4 Da and 6 candidate ions to monitor each cycle (
The Kyoto Encyclopedia of Genes and Genomes (KEGG) C number corresponding to metabolites in the online KEGG database (http://geneontology.org/) was matched, all metabolic pathways involved in target metabolites were extracted according to the C number of target metabolites in the KEGG database, and target metabolites in metabolic pathways were marked by KEGG Mapper software (
In the enrichment analysis of the KEGG pathway annotation of the target metabolite set, considering the whole metabolites of each pathway as the background data set, the distribution of each KEGG pathway in the target metabolite set and the overall metabolite set were compared, and the significance level of metabolite enrichment of a KEGG pathway was evaluated by Fisher's exact test. Only pathways with P-values under a threshold of 0.05 were considered significant (
). Then, peak alignment, retention time correction, and peak area extraction were performed by the XCMS program (Scripps Research Institute). The data obtained by XCMS were processed to delete the ion peaks of the group sum >50%. The SIMCA-P 14.1 (Umetrics) software was used for pattern recognition. After Pareto-scaling pretreatment, the data were analyzed by multidimensional statistics, and orthogonal partial least-squares discriminant analysis was performed (
All data are presented as the mean ± standard deviation, and SPSS software version 20.0 was used for the statistical analysis. Statistical differences between experimental groups were determined by 2-tailed analysis, and the significance level was set at P < 0.05 for all comparisons. The serum metabolomic data were analyzed using the SIMCA-P 14.1 software.
RESULTS
Effects of L. rhamnosus hsryfm 1301 Fermented Milk on the BW and Liver Index of Rats
As shown in Figure 1A, the BW of rats in the C group, the M group, and the T group were between 214.67 g and 216.67 g at the beginning of the trial and had a tendency to increase during the experimental period; however, the BW of the M group was increased by 90.86%, whereas the BW of the C group and the T group was increased by 61.99% and 72.27%, respectively, which indicated that L. rhamnosus hsryfm 1301 fermented milk (fermented milk) could inhibit the BW gain of rats. Moreover, the liver index of rats in the T group was significantly lower than that of the M group (P < 0.05, Figure 1B), which further suggested that fermented milk may be conducive to improving the BW of NAFLD rats.
Figure 1The BW and liver index of rats. (A) Body weight of rats; (B) liver index of rats. C = control group; M = model group; T = the administration of fermented milk group. The data are shown as the mean ± SD (n = 10), and different letters for the index indicate significant differences (P < 0.05).
Effects of Fermented Milk on Biochemical Profiles in Rat Serum
Our study revealed that the levels of TC, TG, LDL-C, ALT, and AST in rat serum of the M group were significantly higher than those in the C group (P < 0.05, Figure 2), and HDL-C and SOD were significantly lower (P < 0.05, Figure 2), while the levels of TC, TG, LDL-C, ALT, and AST in rat serum of the T group were significantly lower (P < 0.05, Figure 2), and HDL-C and SOD were significantly higher (P < 0.05, Figure 2), which indicated that a high-fat diet increased lipid levels and decreased transaminase activities of the rats' serum and that fermented milk could effectively alleviate the lipid levels and transaminase activities of the rats' serum.
Figure 2The lipids, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and superoxide dismutase (SOD) levels in rat serum. (A) Total cholesterol (TC) and triglyceride (TG); (B) high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C); (C) ALT and AST; (D) SOD. C = control group; M = model group; T = the administration of fermented milk group. The data are shown as the mean ± SD (n = 10), and different letters for the index indicate significant differences (P < 0.05).
Compared with the C group, the levels of FFA and ROS in rat serum of the M group were significantly increased after feeding a high-fat diet for 8 wk (P < 0.05, Figure 3). Because FFA and ROS are the most intuitive markers of NAFLD symptoms, and combined with the test results of rats' liver and serum biochemical indices discussed earlier, these findings suggested that the rat model of NAFLD was successfully established. In contrast, the levels of FFA and ROS in rat serum of the T group were significantly decreased after 8 wk of intragastrically administered fermented milk (P < 0.05, Figure 3). Because serum with excess FFA and ROS causes an inflammatory reaction (
), the effect of fermented milk on the level of inflammatory factors in rat serum was researched in this study.
Figure 3The free fatty acid (FFA) and reactive oxygen species (ROS) levels in rat serum. (A) FFA; (B) ROS. C = control group; M = model group; T = the administration of fermented milk group. The data are shown as the mean ± SD (n = 10), and different letters for the index indicate significant differences (P < 0.05).
From Table 1, we can see that the levels of TNF-α, IL-6, IL-8, TGF-β1, NF-κB, and IL-1β in rat serum of the M group were significantly higher than those in the C group (P < 0.05), and these inflammation factors in the T group were significantly decreased (P < 0.05), which indicated that a high-fat diet could also result in inflammation in NAFLD rats, whereas fermented milk could inhibit inflammation.
TNF-α = tumor necrosis factor-α; TGF-β1 = transforming growth factor-β1; NF-κB = nuclear factor kappa-B; C = control group; M = model group; T = the administration of fermented milk group. The data are shown as the mean ± SD (n = 10).
Different superscript letters for the column indicate significant differences (P < 0.05).
a–c Different superscript letters for the column indicate significant differences (P < 0.05).
1 TNF-α = tumor necrosis factor-α; TGF-β1 = transforming growth factor-β1; NF-κB = nuclear factor kappa-B; C = control group; M = model group; T = the administration of fermented milk group. The data are shown as the mean ± SD (n = 10).
Effects of Fermented Milk on the Morphology and Structure of Rat Hepatocytes
Optical microscope observation showed that there was no steatosis in the livers of the C group rats, and hepatocytes had complete structures, clear boundaries, and obvious nuclei at wk 8 (Figure 4A). However, the livers in rats suffered from severe steatosis, the structure of hepatocytes was seriously damaged, and the boundary was blurred and had many fat vacuoles caused by feeding a high-fat diet for 8 wk (Figure 4B). Although there was steatosis in the rats' liver after intragastric administration of fermented milk for 8 wk, the symptoms were alleviated, and the number of fat vacuoles was decreased in hepatocytes and had clear boundaries when compared with the M group rats (Figure 4C).
Figure 4The hematoxylin-eosin staining of rat liver (magnification × 100). (A) Control group; (B) model group; (C) the administration of fermented milk group.
Effects of Fermented Milk on Rat Serum Metabolites
The influence intensity and interpretation ability of the expression pattern of each metabolite on the classification and discrimination of each group of samples were measured according to the variable importance for the projection obtained by the orthogonal partial least-squares discriminant analysis model to identify the differential metabolites with biological relevance. Metabolites with variable importance for the projection >1 and 0.05 < P < 0.1 were regarded as differential metabolites, and those with variable importance for the projection >1 and P < 0.05 were regarded as significant differential metabolites.
Serum metabolism analysis showed that 30 differential metabolites in rat serum were identified by positive ion and negative ion modes in the M/C group and T/M group (Table 2). There were 19 significant differential metabolites between the M group and the C group: 1-palmitoyl-lysophosphatidylcholine, PC(16:0/16:0), creatinine, glycine, glycerophosphocholine (GPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine, l-palmitoylcarnitine, thioetheramide-PC, N6-methyl-l-lysine, 15-deoxy-delta-12,14-prostaglandinJ2 (15-deoxy-delta-12,14-PGJ2), indoleacrylic acid, d-aspartic acid, oleic acid (OA), l-glutamate, thymine, 2-oxoadipic acid, arachidonic acid, uracil, and cholic acid (Table 2, P < 0.05). And between the T group and the M group, there were 17 significant differential metabolites, which were 1-palmitoyl-lysophosphatidylcholine, 1-O-(cis-9-octadecenyl)-2-O-acetyl-sn-glycero-3-phosphocholine, deoxycytidine, acetylcarnitine, PC(16:0/16:0), creatinine, glycine, GPC, 1-myristoyl-sn-glycero-3-phosphorylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, chenodeoxycholate, indoleacrylic acid, linoleic acid, d-aspartic acid, OA, l-glutamate, and thymine (Table 2, P < 0.05).
Table 2Common metabolites in positive and negative ion mode of M/C group and T/M groups
T = the administration of fermented milk group; M = model group; C = control group; VIP = variable importance for the projection; ESI = electrospray ionization; FC = fold change.
1 T = the administration of fermented milk group; M = model group; C = control group; VIP = variable importance for the projection; ESI = electrospray ionization; FC = fold change.
There were 19 differential metabolites identified by screening differential metabolites with opposite trends in the M/C and T/M comparison groups. The contents of glycine, GPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine, thioetheramide-PC, d-aspartic acid, OA, and l-glutamate in rat serum were significantly increased by feeding a high-fat diet for 8 wk (Table 2, P < 0.05), whereas creatinine, l-palmitoylcarnitine, N6-methyl-l-lysine, 15-deoxy-delta-12,14-PGJ2, thymine, and 2-oxadipic acid were significantly decreased (Table 2, P < 0.05), and 1-O-(cis-9-octadecenyl)-2-o-acetyl-sn-glycerol-3-phosphatecholine, acetylcarnitine, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine, 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine, and choline were nonsignificantly increased, and taurocholate was nonsignificantly decreased (Table 2, P > 0.05).
Nevertheless, after intragastric administration of fermented milk for 8 wk, the contents of 1-O-(cis-9-octadecenyl)-2-O-acetyl-sn-glycero-3-phosphocholine, acetylcarnitine, glycine, GPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine, d-aspartic acid, OA, and l-glutamate in rat serum were significantly decreased (Table 2, P < 0.05), and creatinine and thymine were significantly increased (Table 2, P < 0.05). In addition, thioetheramide-PC, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and choline were decreased, and l-palmitoylcarnitine, N6-methyl-l-lysine, 15-deoxy-delta-12,14-PGJ2, 2-ooxoadipic acid, and taurocholate were nonsignificantly decreased (Table 2, P > 0.05).
Analysis of KEGG Pathways of Differential Metabolites
The KEGG was used to further understand the effect of fermented milk on the serum metabolites of NAFLD rats. Compared with the C group (M/C), there were 37 significant metabolic pathways in the M group (Table 3, P < 0.05), which were mainly concentrated in the digestive system, nervous system, lipid metabolism, immune system, and amino acid metabolism, and the significant metabolic pathway of insulin resistance had a direct influence on NAFLD. However, 20 metabolic pathways were significantly changed by the administration of fermented milk when compared with the M group (T/M) (Table 3, P < 0.05), which were mainly concentrated in lipid metabolism, the nervous system, the endocrine system, amino acid metabolism, and neurodegenerative diseases. The results indicated that fermented milk could improve NAFLD by regulating the serum metabolites in these metabolic pathways.
Table 3Significant metabolic pathways in M/C and T/M groups
M = model group; C = control group; T = the administration of fermented milk group; OA = oleic acid; SOPC = 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine; PC = phosphatidylcholine.
1 M = model group; C = control group; T = the administration of fermented milk group; OA = oleic acid; SOPC = 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine; PC = phosphatidylcholine.
The results showed that administration of the fermented milk containing L. rhamnosus hsryfm 1301 for 8 wk had beneficial effects on NAFLD rats' serum, which is in agreement with the findings of some human studies (
), and ROS in serum can regulate secretion of IL-1β by stimulating the formation of the NLRP3 inflammasome, thus promoting TG accumulation in hepatocytes (
). However, studies have found that short-chain fatty acids (SCFA) could not only ameliorate hepatic steatosis by inhibiting the synthesis of FFA and cholesterol in the liver (
A search for synbiotics: Effects of enzymatically modified arabinoxylan and Butyrivibrio fibrisolvens on short-chain fatty acids in the cecum content and plasma of rats.
Short-chain fatty acids produced by synbiotic mixtures in skim milk differentially regulate proliferation and cytokine production in peripheral blood mononuclear cells.
Int. J. Food Sci. Nutr.2015; 66 (26398897): 755-765
), and our previous study also found that L. rhamnosus hsryfm 1301 fermented milk helps to increase the abundance of Lactobacillus and Bacteroidetes in the intestine (
) that produce SCFA. Therefore, improvement of the liver index by L. rhamnosus hsryfm 1301 fermented milk may be attributed to the related serum biochemical profile regulations of SCFA.
Lactobacillus rhamnosus hsryfm 1301 fermented milk not only significantly decreased the levels of TC, TG, and LDL-C in rat serum (P < 0.05) but also significantly decreased the levels of TNF-α, IL-6, NF-κB, and IL-1β in this study (P < 0.05), which may indicate that fermented milk is effective against BW augmentation via the inhibition of dietary triglyceride absorption and the reduction of inflammation (
suggested that Lactobacillus reuteri 263 ameliorating the levels of lipids and transaminase activities of obese rats' serum and their BW might be caused by increasing the oxygen consumption of white adipose tissue and altering the expression of genes involved in glucose and lipid metabolism. Hence, the energy metabolism of white adipose tissue might be one of the reasons for the ability of L. rhamnosus hsryfm 1301 fermented milk to improve the levels of TC, TG, LDL-C, HDL-C, FFA, ALT, and AST of obese rats' serum and their BW (Figure 1, Figure 2, and Figure 3A). A study has also found that L. rhamnosus HA-114 not only significantly inhibited the BW and body fat of volunteers but also had a positive impact on the self-control ability and appetite of volunteers, which indicates that Lactobacillus's association with decreased BW may be attributed to its ability to restore dietary behavior through the brain-gut axis (
Lacticaseibacillus rhamnosus HA-114 improves eating behaviors and mood-related factors in adults with overweight during weight loss: A randomized controlled trial.
). There were 13 and 10 different metabolites in rat serum that were significantly regulated by feeding a high-fat diet and L. rhamnosus hsryfm 1301 fermented milk, respectively (Table 2, P < 0.05). Thirty-seven metabolic pathways were significantly changed in the M group when compared with the C group (Table 3, P < 0.05), and 20 metabolic pathways were significantly changed in the T group when compared with the M group (Table 3, P < 0.05).
Glycerophosphocholine is a supportive nutrient for the brain and other organs and plays an important role in lipid metabolism (
). The content of GPC in the glycerophospholipid metabolism map of the lipid metabolism metabolic pathway in rat serum was significantly increased by feeding a high-fat diet for 8 wk (Table 2, Table 3; P < 0.05), and a high content of GPC could result in damage to the body by the accumulation of fat and triacylglycerols and disruption of the hepatocyte membrane (
), which led to severe steatosis in the liver and a significant increase in the level of lipids in serum (Figure 2, Figure 4; P < 0.05). However, the content of GPC in the choline metabolism in cancer map of the cancers: overview metabolic pathway and in the glycerophospholipid metabolism map of the lipid metabolism metabolic pathway in serum was significantly decreased after administration of fermented milk for 8 wk (Table 2, Table 3; P < 0.05), and the steatosis of the liver and the level of lipids were improved. In fact, GPC is also related to inflammation, which may lead to significantly decreased levels of TNF-α, IL-6, and IL-8 in serum (
In the lipid metabolism metabolic pathway, the content of OA in the M group serum was also significantly increased (Table 2, Table 3; P < 0.05), and it has been revealed that OA could stimulate ROS production and TG accumulation and further induce peroxidation of lipids (
). Hence, the levels of ROS and TG in the M group serum were significantly higher than those in the C group (Figure 2, Figure 3; P < 0.05). In addition, the high level of FFA in the M group serum could also contribute to TG accumulation and storage by activating signaling pathways (
). After 8 wk of fermented milk intervention, the content of OA in the lipid metabolism metabolic pathway was significantly decreased, suggesting that the decrease in OA content is conducive to improving oxidative stress and inflammation (
), and the levels of ROS, TG, SOD, IL-6, and IL-8 in serum were significantly lower than those in the M group (Table 2, P < 0.05). It was also found that excess fatty acids circulate and accumulate in the liver and may result in insulin resistance, which is a major feature of NAFLD (
). Therefore, OA in the lipid metabolism metabolic pathway was significantly increased and may be one of the important factors for accelerated insulin resistance in the endocrine and metabolic diseases pathway in the M group, and insulin resistance according to this pathway was improved by feeding fermented milk. Because targeting insulin resistance is an effective method for the treatment of NAFLD and has been widely used (
), fermented milk can be used as an adjuvant treatment of NAFLD. Overall, it was inferred that fermented milk could attenuate the symptoms of NAFLD induced by a high-fat diet, at least partially, by regulating GPC and OA metabolism in the lipid metabolism metabolic pathway.
The content of d-aspartic acid in the alanine, aspartate, and glutamate metabolism map of the amino acid metabolism metabolic pathway in serum was significantly increased by high-fat diet intervention for 8 wk but was significantly decreased by the administration of fermented milk (Table 2, P < 0.05). d-Aspartic acid is involved in several steps of steroidogenesis and contributes to increasing the level of serum TC (
), and the level of TC in the T group serum was significantly lower than that in the M group (Figure 2, P < 0.05).
The l-glutamate in serum was enriched in 13 metabolic pathways, such as central carbon metabolism in cancer, protein digestion and absorption, aminoacyl-tRNA biosynthesis, ATP-binding cassette transporters, retrograde endocannabinoid signaling, and long-term depression, and was significantly increased by high-fat diet intervention for 8 wk (Table 2, P < 0.05), whereas it was significantly decreased in the retrograde endocannabinoid signaling, long-term depression, ATP-binding cassette transporters, arginine biosynthesis, proximal tubule bicarbonate reclamation, Huntington disease, and alanine, aspartate, and glutamate metabolism metabolic pathways after administration of fermented milk (Table 3, P < 0.05). Because the decrease in l-glutamate content is beneficial for scavenging ROS and improving the oxidation capacity (
), the level of ROS in serum was significantly decreased, and ALT and AST were significantly increased (Figure 2, Figure 3; P < 0.05).
As an important substance in the nucleotide metabolism metabolic pathway, thymine is closely related to biological processes such as cell growth, metabolism, and genetics; participates in a variety of metabolic and regulatory activities; and is oxidation induced by ROS (
). Consequently, the content of thymine was significantly decreased along with the level of ROS, which was significantly increased in the nucleotide metabolism metabolic pathway in the M group serum, and the content of thymine was increased significantly after administration of fermented milk according to the improved level of ROS (Table 2, Figure 3; P < 0.05).
15-Deoxy-delta-12,14-PGJ2 and arachidonic acid (peroxide free) were enriched in the arachidonic acid metabolic map. 15-Deoxy-delta-12,14-PGJ2 is the most widely studied cyclopentenone prostaglandin with a good anti-inflammatory effect and is an endogenous ligand of peroxisome proliferator activated receptor-γ (
). 15-Deoxy-delta-12,14-PGJ2 has the ability to inhibit the activation of nitrogen oxides and ROS in a peroxisome proliferator activated receptor-γ–dependent manner to lessen lipid peroxidation (
), and it may be that the significantly decreased ROS level resulting from the content of 15-deoxy-delta-12,14-PGJ2 was increased by fermented milk to reverse the damage of lipid peroxidation. Arachidonic acid, as an important inflammatory lipid mediator, could activate hepatocyte surface receptors, enhance the activity of NADPH oxidase, and lead to NAFLD (
), and it was enriched into 14 significant metabolic pathways, such as biosynthesis of UFA, linoleic acid metabolism, arachidonic acid metabolism, retrograde endocannabinoid signaling, long-term depression, regulation of lipolysis in adipocytes, and vascular smooth muscle contraction, in this study. Arachidonic acid is the metabolite that is most involved in the significant metabolic pathways and was significantly increased by feeding a high-fat diet (Table 2, Table 3; P < 0.05); therefore, it can be used as an important biomarker metabolite in NAFLD serum.
With the increasing attention to gut microbiota, growing evidence has emphasized the role of gut microbiota in NAFLD. Ruminococcus abundance was significantly increased, whereas Prevotella abundance was significantly decreased in NAFLD compared with normal individuals, and the ratio of Bacteroidetes (Prevotella)/Firmicutes was decreased (
). However, it was found that Ruminococcus and Prevotella abundance was significantly decreased and increased, respectively, and the ratio of Bacteroidetes (Prevotella)/Firmicutes was also improved by fermented milk according to our previous research (
); therefore, regulating gut microbiota may be another important mechanism by which fermented milk improves NAFLD.
CONCLUSIONS
We showed that the metabolic disorder of rat serum was caused by a high-fat diet, which led to lipid peroxidation, insulin resistance, and inflammation of serum and caused fatty degeneration of the liver, resulting in NAFLD. However, GPC, l-glutamate, OA, ROS, d-aspartic acid, and other metabolites related to lipid peroxidation, insulin resistance, and inflammation in the lipid metabolism metabolic pathway, amino acid metabolic pathway, insulin resistance metabolic pathway, and other metabolic pathways of NAFLD in rat serum were improved with the administration of fermented milk for 8 wk. These improvements may arise from the regulation of gut microorganisms related to the metabolism of lipids and other substances by fermented milk. Nevertheless, how gut microorganisms affect serum metabolites needs to be further explored in future studies.
ACKNOWLEDGMENTS
This study was supported by National Natural Science Foundation of China (no. 32272362, No. 31972094), Natural Science Foundation of Jiangsu Province (no. BK20211325; China), Natural Science Foundation of the Jiangsu Higher Education Institutions (no. 19KJA140004; China), Jiangsu Science and Technology Projects (no. XZ-SZ202042; China), and Key Laboratory of Probiotics and Dairy Deep Processing of Yangzhou (no. YZ2020265; China). We thank Shanghai Applied Protein Technology Co. Ltd. (Shanghai, China) for their technical assistance. The authors have not stated any conflicts of interest.
REFERENCES
Abd El-Gawad I.A.
El-Sayed E.M.
Hafez S.A.
El-Zeini H.M.
Saleh F.A.
The hypocholesterolaemic effect of milk yoghurt and soy-yoghurt containing bifidobacteria in rats fed on a cholesterol-enriched diet.
Evaluating the nutritional and immune potentiating characteristics of unfermented and fermented turmeric camel milk in cyclophosphamide-induced immunosuppression in rats.
Short-chain fatty acids produced by synbiotic mixtures in skim milk differentially regulate proliferation and cytokine production in peripheral blood mononuclear cells.
Int. J. Food Sci. Nutr.2015; 66 (26398897): 755-765
Effect of yogurt fermented by Lactobacillus Fermentum TSI and L. Fermentum S2 derived from a Mongolian traditional dairy product on rats with high-fat-diet-induced obesity.
Lacticaseibacillus rhamnosus HA-114 improves eating behaviors and mood-related factors in adults with overweight during weight loss: A randomized controlled trial.
Oral administration of compound probiotics ameliorates HFD-induced gut microbe dysbiosis and chronic metabolic inflammation via the G protein-coupled receptor 43 in non-alcoholic fatty liver disease rats.
Serum levels of TSP-1, NF-κB and TGF-β1 in polycystic ovarian syndrome (PCOS) patients in northern China suggest PCOS is associated with chronic inflammation.
A search for synbiotics: Effects of enzymatically modified arabinoxylan and Butyrivibrio fibrisolvens on short-chain fatty acids in the cecum content and plasma of rats.
Live yeast supplementation during late gestation and lactation affects reproductive performance, colostrum and milk composition, blood biochemical and immunological parameters of sows.
Consumption of probiotic Lactobacillus fermentum MTCC: 5898-Fermented milk attenuates dyslipidemia, oxidative stress, and inflammation in male rats fed on cholesterol-enriched diet.
00021-8&id=gr1.jpg){kind=link}
00021-8&id=gr2.jpg){kind=link}
00021-8&id=gr3.jpg){kind=link}
00021-8&id=gr4.jpg){kind=link}