All of the F. prausnitzii was isolated from feces collected from healthy Koreans after obtaining informed consent from each subject. The genetic diversity of isolated F. prausnitzii were analyzed, and the detailed results are presented in Supplementary Section ( Figure S1 ). This research was approved by the Institutional Review Board of Dongguk University, Ilsan Hospital (IRB# 2018-06-001-012). The reference strain A2-165 (Deutsche Sammlung von Mikroorganismen [DSM] 17677) was obtained from the Leibniz-Institute DSMZ-German Collection of Microorganism and Cell Cultures). The extremely oxygen-sensitive bacteria were grown in brain-heart infusion medium supplemented with 0.5% (w/v) yeast extract, 0.1% (w/v) cellobiose, 0.1% (w/v) maltose, and 0.05% (w/v) L-cysteine, at 37°C in an anaerobic chamber (atmosphere of 5% CO2, 5% H2, and 90% N2). 16S rRNA gene sequencing for the identification of F. prausnitzii was performed after polymerase chain reaction (PCR) amplification of a region of the 16S rRNA gene. For the animal experiments, all of the F. prausnitzii strains were cultured anaerobically in a soy-peptone based medium with some supplements and centrifuged at 12,000 × g for 5 min. They were then adjusted to an end concentration of 1 × 10⁸ CFU/150 µl using anaerobic PBS with 20% glycerol and stored at −80°C until use.

Seven-week-old male C57BL/6 mice were obtained from Orient Bio (Seongnam, South Korea). The mice were housed under controlled conditions (a 12:12-hr light-dark cycle, temperature of 20 ± 3°C, and humidity 55 ± 5%) with ad libitum access to water and a standard chow diet (FeedLab, Guri, South Korea) for 10 days of acclimatization. All experiments were approved by the institutional animal care and use committee (IACUC) of the Dongguk University (2020-11208) and conducted according to the guidelines of the National Rearch Council (Guide for the Care and Use of Laboratory Animals, 2011). Diets were purchased from Reseach Diets, Inc. (Nwe Brunswick, NJ, USA): normal diet (Nor) containing 10% calories from the fat with 3.85 kcal/g (19.2% protein, 67.3% carbohydrate, and 4.8% fat) and HFD containing 60% calories from the fat with 5.24 kcal/g (26.2% protein, 26.3% carbohydrate, and 34.9% fat).

After acclimatization, 72 mice were weighted and randomly divided into eight groups (n = 9) as follows: (1) Nor group; (2) HFD group; (3) Orilistat-treated HFD group (XEN); (4) F. prausnitzii type strain A2-165-treated HFD group (A2-165); (5) F. prausnitzii EB-FPDK3-treated HFD group (DK3); (6) F. prausnitzii EB-FPDK6-treated HFD group (DK9); (7) F. prausnitzii EB-FPDK11-treated HFD group (DK11); (8) F. prausnitzii EB-FPYYK1-treated HFD group (YK1). Mice had ad libitum access to diets and sterile water. Orlistat (Xenical®; Roche, Basel, Switzerland) was dissolved in sterile PBS at pH 7.4 and administrated orally to the Xen group (10 mg/kg/day). The live bacterial samples were resuspended in sterile PBS and then freshly administrated to the A2-165, DK3, DK9, DK11, and YK1 groups via oral gavage in sterile PBS with 20% glycerol at a dose of 1 × 10⁸ CFU/150 µl per animal. The mice in the NOR and HFD groups were given only sterile PBS with 20% glycerol as a vehicle. The treatments were carried out 6 days per week for 12 weeks. The body weight of mice was assessed weekly, and food intake was recorded three times a week. Fecal samples were collected at weeks 0 and 12 post-treatment and kept at -80°C for further microbiome analysis

At the end of the experiment, mice were fasted for 12 h and blood was collected by cardiac puncture under anesthesia with a mixture of tiletamine-zolazepam (Zoletil 50, Virbac, Carros, France). Serum was separated by centrifuging at 2,000 × g for 15 min at 4°C and kept at −80°C until further biochemical analyses. The spleen, liver, large intestine, small intestine, and adipose tissues was removed, washed with ice-cold PBS, dried, and then weighed. Portions of the liver, large intestine, small intestine, adipose, and hypothalamus were removed and flash-frozen in the liquid nitrogen and stored at −80°C until further use. The other portions of the tissues were fixed with 4% paraformaldehyde (PFA) (Junsei, Tokyo, Japan) overnight at 4°C and then embedded in paraffin.

Serum samples were analyzed for total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) using commercial colorimetric assay kits (Asan Pham. Co., Seoul, South Korea). Insulin levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Morinaga, Yokohama, Japan). Insulin resistance was estimated by homeostasis model assessment of insulin resistance (HOMA-IR) index calculated as follows: HOMA-IR = [(fasting glucose (mg/dL) x fasting insulin (μU/mL))/405. Lower HOMA-IR values indicated better insulin sensitivity and vice versa.

Mice were fasted for 14h with free access to drinking water at week 11 of study period and administrated with glucose solution (2 g/kg body weight) by oral gavage. Blood glucose level was measured from the tail vein using an Accu-Chek Active blood glucose meter (Roche Diagnostics Corp, Rotkreuz, Switzerland) at 0, 30, 60, 90, and 120 min after injection. The glucose area under the curves (AUC) during the OGTT were calculated using GraphPad Prism 5.0 software (San Diego, CA, USA).

PFA-fixed paraffin-embedded sections (4 μm) of the liver, intestine and mesenteric adipose tissues were placed on positively charged glass slides, de-waxed in xylene (Sigma Chemicals, St Louis, MO, USA), dehydrated in a gradual ethanol series, and stained with either hematoxylin and eosin (Sigma-Aldrich, St. Louis, MO, USA), Alcian blue (AB) solution (Abcam, Cambridge, UK) for goblet cells, or Oil Red O (Sigma-Aldrich) for fatty liver tissues according to the manufacturer’s protocol. Tissues were examined under an inverted light microscope (Olympus, Tokyo, Japan). Representative images of the liver and mesenteric adipose tissue were taken from three individual liver and mesenteric adipose samples in each group at 4 x and 20 x magnifications, while for intestine AB stain, representative images were taken from six to seven individual samples in each group. The liver steatosis area and the Oil Red O-stained area were assessed on the images taken with Image-Pro Plus 6.0 software. The AB-positive area, villus length, and the number of the goblet cells were determined from image J software via images. The AB-positive goblet cells were quantified by counting the positive cells per crypt in all crypts per colon section, three sections per mouse. The villus length was measured from the base of the villus to the top in Five to six villi in section, three sections per mouse.

DNA from fecal samples was extracted by the QIAamp® DNA Stool Mini Kit (QIAGEN, Hilden, Germany) as previously described (30). The V1–V2 16S rRNA hypervariable regions were amplified using the Bio-Rad C1000 Touch thermal cycler (Hercules, CA, USA) with primers containing a unique 10-base barcode to tag each PCR product. The PCR amplicons were purified using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and then sequenced using the Ion Torrent PGM system (Thermo Scientific, DE, USA). Sequences below 300 bp in length and low-quality score below 20 were removed. All effective sequences were clustered into operational taxonomic units (OTUs) using SILVA rRNA gene database (http://www.arb-silva.de) with a threshold of 97% sequence identity. The Quantitative Insights into Microbial Ecology (QIIME) software was used to select the representative reads of each OTU and to calculate beta diversity. The clustering pattern of the microbial composition was presented by principal coordinated analysis (PCA) using UniFrac distance matrices. The differences between groups in the taxa with varying abundances were sassed by using the linear discriminant analysis (LDA) effect size (LEfSe). In LEfSe, a size-effect threshold of 2.0 on the logarithmic linear discriminant analysis (LDA) score and an alpha value of 0.05 for the factorial Kruskal-Wallis test among the classes were used. The microbiota sequencing data have been deposited to the NCBI Sequence Read Archive (SRA) database under PRJNA885263. SPSS software (version 19.0) was used to assess the relationship strength between relative abundance and obesity-related parameters by the two-tailed Pearson’s correlation test.

Total RNA form liver, adipose, colon, jejunum, and hypothalamus tissues were isolated with TRIsureTM reagent (Meridian Life Science Inc, TN, USA) following the manufacturer’s protocol. The A260/280 ratio confirmed RNA purity. One μg of total RNA was reverse-transcribed with an AccuPower RT PreMix with oligo-(dT)18 primer (Bioneer, Daejeon, South Korea). Quantitative real-time PCR was performed using the LightCycler® 480 Real-Time PCR. System (Roche, Mannheim, Germany) and SYBR® Green real-time PCR kit (Toyobo, Tokyo, Japan) according to the manufacturer’s instruction. The GAPDH gene was used as a reference. The sequences of primers were shown in the Supplementary Table ( Table S1 ) in the Online Repository.

All data are presented as means ± standard error of the means (SEM). GraphPad Prism 8.0 software (San Diego, CA, USA) was used to determine the statistical significance. Data were analyzed by an unpaired two-tailed Student’s t-test for a two-group comparison or one-way ANOVA with Bonferroni-corrected post hoc tests for multiple comparisons unless stated otherwise. P-values less than 0.05 was considered statistically significant.

Mice fed a 12-week high-fat-diet showed a 2.9 times increase in body weight gain (p < 0.01), 17% increase in calorie intake (p < 0.05), and 2.35 times increase in energy efficiency (p < 0.01) compared to the normal diet group ( Figures 1A–C ). However, the body weight gain was markedly reduced by the microbial strains compared to HFD, with reduced rates of A2-165: 40%; DK3: 37%; DK9: 31%; DK11: 36%; YK1: 44%; XEN: 44% (p < 0.05 or p < 0.01, Figure 1B ). Furthermore, all these treated groups showed a significant decrease in calories intake compared to the HFD group ( Figure 1D ). The increased liver weights by HFD feeding also resulted in significant reductions in the other treatment groups ( Figure 1E ). The total visceral fat mass weight of the HFD mice ( Figure 1F ) was 4.79 times greater than the normal group (p < 0.01). This effect was explained mainly by a reduction in mesenteric and subcutaneous fat upon treatment with all four selected pasteurized F. prausnitzii strains: DK3, DK9, DK11, and YK1. By contrast, only DK3, DK11 and YK1 reduced the epididymal fat significantly ( Figures 1G–I ). In addition, the relative liver and epididymal weights did not show a significant difference among all groups, while other the tissue mass as relative to body weight also showed a similar pattern with absolute organ mass ( Figure S2 ).

The impact of the F. prausnitzii strains treatment on glucose homeostasis and insulin sensitivity in mice was determined by oral glucose tolerance tests (OGTT) ( Figure 2A ). HFD-fed mice showed the 54% higher in fasting glucose level (p < 0.01), the 69% higher in 30 minutes’ glucose level (p < 0.01), and the 64% higher in the OGTT area under the curve (AUC) (p < 0.01) compared with normal low-fat diet food mice ( Figures 2B–D ). The fasting glucose level was changed significantly by the strains A2-165, DK3, and YK1 treatment with reduction rate 20%, 19%, 19% respectively (p < 0.01), while other treatment groups showed a slight reduction compared to the HFD group ( Figure 2D ). The oral gavage glucose solution at 30 minutes recorded the highest glucose level. The HFD group had a significantly higher level than the other groups, which was decreased significantly by all treatments, with the decrease rates of XEN:18%; A2-165%; DK3:18%; DK9:18%; DK11:23%; YK1:31% (p < 0.05 or p < 0.01) ( Figure 2C ). In the OGTT, the AUC values also showed upregulation by HFD feeding compared to the normal group, while it was reduced significantly by treatment with XEN, A2-165, DK11, and YK1, with decrease rates of 13%, 16%, 19%, 23% respectively (p < 0.05 or p < 0.01); the other treatment groups showed a slight decrease compared to HFD group ( Figure 2B ). The serum insulin concentration was elevated significantly in response to HFD feeding, while it was reduced markedly by the microbial strains and XEN treatment, with reduction rate as XEN: 33%; A2-165: 46%; DK3: 41%; DK9: 42%; DK11: 38%; YK1: 42% (p < 0.05 or p < 0.01) ( Figure 2E ). Homeostatic model assessment for insulin resistance index (HOMA-IR) showed a significant increase in the HFD group and a noticeable decrease in the treatment groups compared to the HFD group, with decrease rates as XEN:44%; A2-165:57%; DK3:49%; DK9:47%; DK11:39%; YK1:47% (p<0.01) ( Figure 2F ).

The obesity-associated serum biomarkers were further analyzed to investigate lipid metabolism in response to diet in HFD mice ( Table 1 ). The total cholesterol (TC), triglyceride (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were increased significantly in the HFD group than in the normal group (p < 0.05 or p < 0.01). Moreover, the levels of TC all showed downward trends after treatment. Among them, the XEN and YK1 treatments caused a 22% and 28% reduction in TC levels compared to the HFD group, respectively (p < 0.05 or p < 0.01). In addition, the TG level showed similar results to the TC. The serum TG could be improved by all treatment ways and compared with the HFD group. The YK1 strain reduced the TG level by 42% compared to the HFD group (p < 0.01). All treatment groups could significantly improve the serum AST and ALT levels (p < 0.05 or p < 0.01), which are biomarkers of the hepatic function. These results showed that F. prausnitzii administration improved the lipid metabolism in serum.

For requirements for a specific article type please refer to the Article Types on any Frontiers journal page. Because the hepatic function is associated with HFD- induced obesity (31), the effects of F. prausnitzii supplement on HFD-induced hepatic function were next determined. Histological analysis of H&E staining and lipid staining on the liver sections ( Figures 3A–D ) confirmed the normal histology in normal low-fat diet-fed mice. In contrast, mice after 12 weeks of HFD developed a fatty liver phenotype, featuring a pale liver appearance caused by extensive fat accumulation, including cases of both macrovesicular and microvesicular steatosis. As expected, treatment with DK3, DK9, DK11, and YK1 resulted in markedly reduced vacuolation. The XEN and A2-165 groups also showed a slight improvement in fat accumulation and hepatic steatosis ( Figures 3A, C ). Moreover, the Oil Red O examination showed that the HFD-fed mice had 16.77 times more lipid droplets in the liver tissues compared to the normal group (p < 0.01). As shown in Figures 3B, D , the treatment groups showed obvious down-regulation of the lipid droplets compared to the HFD group, with reduction rates as XEN: 71%; A2-165: 72%; DK3: 64%; DK9: 66%; DK11: 70%; YK1: 73% (p < 0.01). In addition, the hepatic gene expression of ACC1, FAS, and SREBP1c in the normal group was significantly lower than in the HFD group (p < 0.01). In contrast, the expression of ACC1 mRNA was decreased significantly in the liver of the HFD-fed animals treated with all four selected pasteurized F. prausnitzii strains (p < 0.01). From the treatment, except DK9, other selected strains all significantly decreased the FAS and SREBP1c (p < 0.01). Among them, the hepatic expression of SREBP1c was significantly lower in the DK11 and YK1 groups than in the XEN group (p < 0.05) ( Figure 3E ).

On the other hand, the gene expression of (GLUT2), Glucose 6-phosphatase(G6Pase), and peroxisome proliferator-activated receptor-gamma (PPARγ) are associated with gluconeogenesis, but only G6Pase and PPARγ showed significant changes compared to the normal and HFD groups ( Figure 3E ). By treatment, in addition to DK3 and XEN, the other treatment methods upregulated the GLUT2 level markedly, but all treatments could inhibit G6Pase and PPARγ expression significantly. Consistent with the decrease in liver weight, the improvement of hepatic steatosis in histochemical and qPCR analysis showed that the F. prausnitzii treatment could ameliorate the hepatic steatosis and damage caused by the HFD and improve the hepatic function in mice.

For requirements for a specific article type please refer to the Article Types on any Frontiers journal page. Adipocyte hypertrophy is the major mechanism for the expansion of adipose tissue during the obesity development (32). To investigate the effects of F. prausnitzii administration on lipid accumulation in the adipose tissue of HFD-fed mice, three different parts of adipose tissue samples were collected from the mice and the amount of fat was assessed. The fat mass weight and adipocytes size of mesenteric fat was examined by H&E staining.

As shown in the H&E staining pictures ( Figure 4A ), the results revealed up-regulation in the HFD group than the normal group of lipid accumulation in the mesenteric fat and a significant increase in the average fat cell size (p < 0.01). After treatment with different supplements, however, the fat cell size decreased significantly compared to the HFD group, with the reduction rates as XEN: 32%; A2-165: 27%; DK3: 36%; DK9: 33%; DK11: 35%; YK1: 39% (p < 0.05 or p < 0.01) ( Figure 4B ).

Obesity leads to adipose tissue dysfunction and vice versa (33). The expression levels of genes associated with lipogenesis or adipogenesis in mesenteric fat tissue were measured by RT-PCR to clarify the mechanism of the F. prausnitzii strains on HFD-induced obesity ( Figure 4C ). The expression levels of CD36 (p < 0.01), FAS (p < 0.05), and LDL (p < 0.01) mRNAs increased significantly after HFD feeding compared to the low-fat feeding group (normal group), but ACC1 showed an increasing trend in the HFD group. However, FAS and ACC1 levels were markedly reversed after all ways of treatments (p < 0.01). The selected strains, DK3 (p < 0.01), DK9 (p < 0.01), and YK1 (p < 0.01), resulted in markedly reduced CD36 mRNA expression, and DK3 (p < 0.01) and YK1 (p < 0.01) produced a significant decrease in the LDL levels. In addition, after the different treatments, the levels of the glucose and lipid metabolism regulation marker (aP2 and AKT), insulin-related markers (Adiponectin and IRS-1), and leptin, a hormone that facilitates the regulation of feeding and energy homeostasis ( Figure 4C ). A definite, insignificantly lower AKT, IRS-1, and Adiponectin mRNA levels were observed in the HFD groups compared to the normal groups. In contrast, the aP2 (p < 0.01) and leptin (p < 0.01) genes showed significantly higher expressions in the HFD group than in the normal group. After treatment, the mRNA levels of aP2 decreased (p<0.05 or p<0.01)., and except for the DK9 and DK11 groups, the leptin (p < 0.01) level also decreased significantly. Treatment with all four selected strains in the HFD groups, but not the other treatments, upregulated the expression of the Adiponectin gene significantly (p < 0.05 or p < 0.01). All the treatments except DK11 could increase the levels of AKT (p < 0.05 or p < 0.01), and the type strain A2-165 (p < 0.01), DK11 (p < 0.01), and YK1 (p < 0.01) could upregulate the IRS-1 mRNA expression level. Therefore, F. prausnitzii affects the adipose tissue metabolism.

The protective activity of pasteurized F. prausnitzii against HFD-induced inflammation in the colon was evaluated. This study examined the effects of F. prausnitzii in the HFD group on the gene expression of the following, which play vital roles in the inflammation and inflammatory signaling pathways: pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; pro-inflammatory chemokine MCP-1; toll-like receptors TLR2 and TLR4, ( Figures 5A, B ). The colonic mRNA levels of IL-1β (p < 0.01), IL-6 (p < 0.01), TNF-α (p < 0.05), MCP-1 (p < 0.01), TLR2 (p < 0.05), and TLR4 (p < 0.05) were significantly higher in the HFD group than in the normal group. In particular, exposure of the HFD mice to all F. prausnitzii strains depleted the expression of IL-1β, IL-6, MCP-1, and TLR4 and suppressed the expression of the TNF-α gene in the colonic tissue, even though the latter change was insignificant. Treatment of the HFD-fed animals with XEN (p < 0.01), DK3 (p < 0.01), DK11 (p < 0.01), and YK1 (p < 0.05), but not the other treatments, decreased the expression of the TLR2 gene significantly.

Previously studies suggested that HFD feeding induced various dysbiosis of the intestines, which is associated with physiopathological changes, such as damaged mucus production and secretion, and injured the gut integrity and permeability of the intestinal epithelium (34). The intestine tissue was observed by AB staining to determine the effects of F. prausnitzii on the intestinal structure. As expected, HFD - induced obesity mice resulted in marked changes in the intestinal architecture ( Figure 6A ). In particular, a 55% decrease in the AB-stained area was observed in the HFD group compared with the normal group (p < 0.01), which is consistent with these results. The number of goblet cells (p < 0.01) and villus length (p < 0.01) were both lower than the normal group ( Figures 6B–D ). Nevertheless, the AB-positive area that represents the acidic mucins was improved markedly in four selected F. prausnitzii strain groups (p < 0.05 or p < 0.01) ( Figure 6B ). In support of the above results, a significant increase in the villus length was observed after treatment with A2-165 and all four selected strains compared to HFD control, with the increase rate as A2-165: 70%; DK3: 46%; DK9: 46%; DK11: 56%; YK1: 60% (p < 0.05 p < 0.01) ( Figure 6D ).

The F. prausnitzii supplement improved the mRNA expression of the intestine tissue and gut integrity ( Figure 6E ). Tight junctions connect the epithelial cells and play a key role in regulating the intestinal-barrier functionality (35). This study examined the expression levels of colonic mucin 2 (Muc2), ZO-1, Occludin, and JAM-A mRNAs in the intestine tissue by qRT-PCR. Compared to the untreated group (normal group), the mRNA expression of ZO-1 was inhibited significantly by HFD-feeding (p < 0.05). On the other hand, treatment of the HFD group with the XEN (p < 0.05), DK3 (p < 0.05), DK9 (p < 0.05), and DK11 (p < 0.05), but not A2-165 and YK1, increased the mRNA level of JAM-A significantly. The gene expression of occludin was upregulated significantly in the HFD group after treatment with DK9 (p < 0.05), DK11 (p < 0.05), and YK1 (p < 0.05) strains, but not the other regimens. Moreover, the gene expression of Muc2 was upregulated significantly by the oral DK9 (p < 0.01) and DK11 (p < 0.01) strains in the HFD-fed mice.

For requirements for a specific article type please refer to the Article Types on any Frontiers journal page. The gut–brain axis reflects the interactions between the gastrointestinal system and the brain in general. The brain receives both neural and endocrine inputs from the gut in response to food intake, which is integrated with signals from other organs to orchestrate physiological responses (26). Major integrating centers within the brain are the hypothalamic nuclei. Compared with the HFD-fed mice, food intake was decreased significantly by the F. prausnitzii strains, with the reduction rates as A2-165: 24%; DK3: 18%; DK9: 16%; DK11: 16%; YK1: 22%, (p < 0.05 or p < 0.01) ( Figure S3 ). In this study, the hypothalamus, colonic, and small intestine tissue were used to provide evidence of gut-brain cross-talk involved in regulating food intake. Compared to the normal low-fat diet group, the gene expression of PYY (p<0.05), GPR120 (p<0.05), and GPR41 (p<0.05) of colonic tissues was markedly lower in the HFD group in response to feeding HFD. On the other hand, treatment of the HFD-fed animals with all the F. prausnitzii strains and XEN elevated the mRNA level of PYY significantly (p < 0.05 or p < 0.01). Similar to PYY, except for DK3, the other treatments could increase the GPR120 mRNA levels significantly (p < 0.05 or p < 0.01). Exposing the HFD group to type strain A2-165 and DK11, but not other treatments, upregulated the expression of the GLP-1 (p < 0.05) and GPR43 (p < 0.05) genes significantly. After treatment with DK9 (p < 0.05), the mRNA levels of GPR41 were also upregulated markedly compared to the HFD group ( Figure 7A ). In addition, the small intestine mRNA levels of CCK (p < 0.01) and PYY (p < 0.05) were significantly lower in the HFD group than in the normal group. In contrast, the gene expression of GIP (p < 0.01) and ghrelin (p < 0.01) was significantly higher in the HFD group than in the normal group. Exposure of the HFD group to all F. prausnitzii strains upregulated the mRNA level of CCK markedly (p < 0.05 or p < 0.01). In contrast, the treatment of the HFD-fed mice with the F. prausnitzii strains except YK1 depleted the expression of Ghrelin genes significantly (p < 0.05 or p < 0.01). The small intestine expression of the PYY gene in the HFD group was upregulated significantly by XEN, A2-165, DK3, and DK11 (p < 0.05 or p < 0.01). Treatment of HFD-fed mice with A2-165 and YK1, but not other treatments, increased the CCK1R (p < 0.05) gene expression, DK3 and YK1 increased the GIP (p<0.01) gene expression compared with HFD group ( Figure 7B ). Furthermore, the F. prausnitzii supplement modulated the appetite-related hormone mRNA expression in the hypothalamus ( Figures 7C, D ).

The gene expression levels of GHSR, NPY, Leptin R, 5-HT1A, and AgRP in the hypothalamus were significantly higher in the HFD group than in the normal group (p < 0.05 or p < 0.01). On the other hand, treatment of the HFD group with XEN and the F. prausnitzii strains decreased the mRNA levels of GHSR, NPY, Leptin R, and 5-HT1A (p < 0.05 or p < 0.01). The gene expression of AgRP was down regulated significantly in the HFD group upon treatment with type train A2-165 (p < 0.05) and YK1 (p < 0.01), but not the other regimens. Significantly lower expression of the GIPR (p < 0.05) and POMC (p < 0.01) genes was observed in the hypothalamus of the HFD group vs. the normal group. A definite but insignificantly higher mRNA levels of Cart, 5-HT1B, and CCK2R were observed in the HFD group than in the normal group. The mRNA levels of GIPR (p < 0.05 or p < 0.01) and 5-HT1B (p<0.05) were increased significantly in the hypothalamus of the HFD-fed mice by all treatment ways. Cart gene expression in the HFD-fed mice was also upregulated significantly upon treatment with XEN and F. prausnitzii strains, except for the YK1 strain (p < 0.05 or p < 0.01). The POMC mRNA level in the HFD group was increased markedly by A2-165 (p<0.05), DK3 (p<0.01), and DK11 (p < 0.05). In addition, the expression of the CCK2R gene was increased significantly by the DK3 (p < 0.05) strain, but not the other treatments.

Several recent studies have provided significant evidence to reveal a strong association between obesity and gut microbiota (36). 16S rRNA gene analysis of fecal samples from the mice was performed to explore changes to the gut microbiota after F. prausnitzii administration on HFD-fed obesity mice. The bacterial composition was examined by taxonomy-based analysis. The results showed that compared to normal low-fat diet food mice, HFD induced a significant change in the populations of the intestinal microbiota ( Figure 8 ).

First, principal component analysis (PCA) was performed. Figures 8A–D shows the PCA results of the different treated groups. The results showed the different clustering positions of the mice within and across different treatment ways. The fecal samples of the mice with HFD clustered together, whereas most of the samples from the normal chow-diet-fed mice comprised another group. This result indicates that HFD feeding could induce differences in the gut microbiota compared to the normal group. In particular, distinct segregation of the gut microbial communities was observed in the DK3, DK9, DK11, and YK1 groups compared to the normal and HFD groups ( Figures 8A–D ). Unlike with four F. prausnitzii groups, the gut microbiota communities of A2-165 were not significantly different with normal and HFD groups ( Figure S4 ).

Further analysis of the 16S rRNA sequencing data showed that the relative abundance of the gut microbial taxa differed among the experimental groups. The relative abundance of DK3, DK9, DK11, and YK1 was assessed at the genus level to identify the specific taxa related to selected strains supplementation ( Figure S5 ). At the genus level, the relative abundances of Ruminococcus and Lactococcus were suppressed significantly in the HFD group compared to the normal chow diet group (p < 0.01). The F. prausnitzii strains could increase the abundance of Ruminococcus, rc4-4, Parabacteroides, Lactococcus, and Bacteroides significantly (p < 0.05 or p < 0.01). Furthermore, the increased proportions of sequences assigned to Enterococcus were significantly observed in the HFD group (p<0.05). In addition to Dehalobacterium, all the treatment groups significantly suppressed the increase in Enterococcus and Coprococcus (p<0.01). DK3, DK9, and DK11 markedly decreased the Dehalobacterium abundance (p<0.05). Overall, these results showed that administration of the selected strains F. prausnitzii could modulate the changes in these relative abundances of the gut microbiota induced by HFD-induced obesity.

A correlation matrix was established using Pearson’s correlation coefficient to completely investigate the relationships between levels of obesity biomarkers and abundance of gut microbiota ( Figure 9 ). Intriguingly Oscillospira was positively correlated with body weight, fat weight, glucose metabolism related parameters, whereas ruminococcus showed negative correlation. On the other hand, Lactobacillus and Dehalobacterium were positively related with steatosis area (%) and Oil Red O-stained area (%) in liver, adipocyte cell size (μm) in adipose. Furthermore, it was indicated that rc4-4 shows a significant positive association with PYY levels in both colon and small intestine tissues, as well as a negative correlation with serum TC levels. Additionally, rc4-4 showed significant negative correlations with FAS and ACC1 levels in adipose tissues, and TLR2 levels in the colon.