Long-term exposure to polystyrene microplastics promotes HFD-induced obesity in mice through exacerbating microbiota dysbiosis

doi:10.21203/rs.3.rs-3148828/v1

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Long-term exposure to polystyrene microplastics promotes HFD-induced obesity in mice through exacerbating microbiota dysbiosis

https://doi.org/10.21203/rs.3.rs-3148828/v1

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Background:Microplastics (MPs) have become a global environmental problem, emerging as contaminants with potentially alarming consequences. However, long-term exposure to MPs and its effects on the development of obesity are not yet fully understood. This study aimed to investigate the effect of polystyrene (PS)-MPs exposure on high fat diets (HFD)-induced obesity and underlying mechanisms.

Methods:In the present study, C57BL/6J mice were fed a normal diet (ND) or a HFD in the absence or presence PS-MPs via oral administration for 8 weeks. Antibiotic depletion of the microbiota and fecal microbiota transplantation (FMT) were performed to assess the influence of PS-MPs on intestinal microbial ecology. We performed 16S rRNA sequencing to dissect microbial discrepancies, and investigated the dysbiosis-associated mucous layer damage and systemic inflammation.

Results:We found that PS-MPs supplementation led to an increased body weight, increases of liver weight, development of hepatic steatosis, elevated tissues mass of white adipose, and induced glucose intolerance and hyperlipemia. At the molecular level, PS-MPs administration was associated with enhanced protein levels of C/EBPα and PPARγ two critical transcription factors that regulate lipid metabolism in the liver, while reducing the protein level of PGC-1α in HFD-fed mice. Furthermore, 16S rRNA sequencing of the fecal microbiota indicated that PS increased the diversity and changed composition of the gut microbiota in HFD-fed mice. Potential relations analysis revealed that PS induced microbiota dysbiosis was associated with obesity.Interestingly, microbiota-depleted mice were resistance to PS-induced obesity, suggesting that intestinal microbiota played a critical role in PS-induced obesity pathogenesis. Importantly, transplantation of PS-altered microbiota to microbiota-depleted HFD-fed mice promoted colon mucus layer damage, systematic inflammation and obesity.

Conclusions: Our findings provide a new gut microbiota-driven mechanism for PS-induced obesity in HFD-fed mice, suggesting the need to reevaluate the adverse health effects of MPs commonly existed in daily life, particularly in susceptible population.

Microplastics

polystyrene

obesity

mucus layer

gut microbiota

Over the last few decades, the global prevalence of obesity has progressively risen, coinciding with changes in dietary and lifestyle patterns [1]. According to data from the World Health Organization, in 1975, only 2.8% of the global population were considered obese, while by 2016, this figure had risen to 13% of adults, or approximately 650 million people globally. Obesity negatively affects physiological functions and poses a significant threat to public health. It heightens the risk of developing metabolic syndromes such as type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease [2, 3]. Obesity is influenced by a combination of genetic, epigenetics and environmental factors, including lifestyle and eating habits [4]. Previous studies highlighted that mice fed a western diet that includes a lot of fat, that is, a high-fat diet (HFD), developed obesity by transforming gut microbiota [5, 6]. The intestinal microbiome plays a crucial role in regulating several aspects vital to host metabolic function, such as fat accumulation, hepatic lipogenesis, and energy absorption [7, 8]. Despite dietary factors, other environmental factors, such as polymarate-80 and carboxymethylcellulose, two commonly used food additives, have also been proven to promote obesity through intestinal microbiota alteration [9]. Thus, understanding the relationship between diet and other environmental factors is crucial in formulating comprehensive strategies to prevent obesity.

Microplastics (MPs), an emerging class of persistent environmental pollutants, comprise chemically fragmented plastic particles, fibers, and fragments less than 5 mm in diameter [10]. By 2050, nearly 12 billion metric tons of unrecyclable plastic is expected to accumulate in natural environments [11], which results in gradual physicochemical degradation of plastics into smaller-size particles ("secondary" MPs) [12]. Moreover, there is widespread deposition of "primary" MPs which constitute small plastic beads primarily intended for manufacturing or commercial purposes. Recent studies have shown that microplastics are present ubiquitously in various media, environments, and even in food and drinking water [13, 14]. Humans are exposed to MPs through various pathways such as oral, respiratory, or dermal exposure, with the oral route being the most significant [15]. Daily intakes of MPs ranging between 0.5–10 µm for adults have been estimated at 40.1 µg/kg body weight per day [16]. Shockingly, MPs have been found in urine [17], stool [18], blood [19], colectomy specimens [20], placenta, and meconium of human [21]. As a result, serious concerns regarding their potential toxicity to human have been raised, and their general toxicological effects on organisms need to be further evaluated.PS-MPs are a commonly used polymer in the production of styrofoam, which is widely used in food containers and packaging [22, 23]. Research has shown that PS-MPs with a diameter of 5 µm can accumulate in the intestine, resulting in dysfunction of the intestinal barrier and alteration of metabolites in serum in otherwise healthy mice [24–26]. Furthermore, PS-MPs of diameter within 0.5-5 µm have been extensively employed in bioassays to investigate biological interactions and toxicity in living organisms [27–29]. It should also be noted that prolonged exposure to high doses of MPs can lead to apparent adverse effects, such as histological injury and inflammation of the colon [30].

Besides these advances, the potential impacts of PS-MPs on gut microbiota have been a focus of toxicity studies, as 99.54% of PS-MPs were found to be excreted in feces after oral administration of 5 µm PS-MPs [31]. Exposure to PS-MPs have been reported to cause shifts in the microbiota, as indicated by increased Firmicutes and reduced β-diversity in mice feces [32]. Previous studies have also linked dysregulation of the intestinal flora with disorder of hepatic lipid metabolism caused by PS-MPs in mice [33]. According to a study, alterations in the composition of gut microbiota have been associated with the development of obesity and its associated metabolic disorders [34]. Additionally, other studies have demonstrated that gut dysbiosis, which can result from environmental or genetic factors, can impede intestinal integrity in obese animals [35]. Furthermore, ingestion of PS-MPs beads promoted adiposity in mice fed normal diets (ND) [32]. The toxic effects of PS-MPs is related to dietary changes and disease status [36, 37],which indicates that the toxicity of PS may be amplified in obese populations. Nonetheless, it is still not clear whether PS-MPs could disrupt gut barrier function by inducing gut dysbiosis, and how PS-MPs disrupts the gut microbiota and contributes to HFD induced-obesity remains unresolved. Therefore, it is highly plausible that exposure to PS-MPs will impose pressure to determine the species of gut flora and affect the intestinal integrity to further promote HFD-induced obesity.

2.1 Characterization of PS

Aqueous suspension of concentration 25 mg/mL PS was purchased from Shanghai McLean biochemical Technology Co., Ltd., having particle size “5 µm” beads (range = 4.0–4.9 µm, # L815942; 5.0% w/v dispersed in Deionized (DI) water. The morphology was analyzed by TEM spectroscopy (JEOL Ltd., Tokyo, Japan) (Fig. S1 in the Electronic Supplementary Material (ESM)). All suspensions were thoroughly dispersed by sonication, and diluted with water before use.

2.2 Animals and diets

Male C57BL/6J mice at the age of 7 weeks, obtained from Vital River Company (Vital River Laboratory Animal Center, Beijing, China), were selected for the experiment. The mice were housed in climate-controlled cages maintained at a temperature of 24 ± 1°C with 55% ± 5% relative humidity and a 12-hour light-dark cycle. Groups of mice were housed together (n = 6 per group) and provided free access to feed and drinking water. The experimental procedures were approved by the Laboratory Animal Welfare and Animal Experimental Ethical Committee of the China Agricultural University (No. AW72403202-1-31). All groups of mice were co-housed for 1 week to normalize their intestinal microbiota before the experiment. The mice were then given either ND containing 10% D12450B Research Diet (New Brunswick, NJ, USA), or a HFD consisting of 60% D12492 Research Diet (New Brunswick, NJ, USA). Suspensions of 10 mg/L PS was prepared in ultrapure sterile water, which was then sonicated for 10 minutes every day. The mice were given either filtered and autoclaved water (ND and HFD groups) or normal water containing PS beads (ND + PS and HFD + PS groups) at a dose of 10 mg/L per day for 8 weeks. At the end of the experiment, all mice were fasted overnight and then sacrificed. Blood samples were collected and stored at − 80°C for the determination of blood biochemical indicators. Tissues were removed, rinsed with a physiologic saline solution, weighed, and immediately stored at − 80°C.

2.3 Histology

The tissues were fixed in 4% paraformaldehyde at room temperature for 24 hours and embedded in paraffin. The liver and adipose tissue sections, with a thickness of 5 µm, were stained with hematoxylin and eosin (H&E) to allow for general morphological observations. To examine lipid deposition in the liver, Oil Red O staining was performed. Alcian blue/periodic acid-Schiff (AB/PAS) were used to quantify the number of goblet cells. All images were acquired using a light microscope (BX63, OLYMPUS, Japan) and quantified with Image-Pro Plus (Media Cybernetics, USA).

2.4 Quantitative real-time polymerase chain reaction

The total RNA was extracted and then reverse-transcribed into cDNA according to the manufacturer’s instructions, followed by real-time PCR using the SYBR Premix Ex Taq II (Beijing Aidlab Biotechnologies Co., Ltd.) and the ABI-Prism 7500 Sequence Detection System (Applied Biosystems). The fold change in mRNA expression was determined using the 2^−ΔΔCT method. To normalize the gene expression level, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used. The primer sequences used in this study are listed in Table S1 (ESM).

2.5 Western blot analysis

The liver samples were retrieved and kept frozen at -80°C to preserve their natural state. Two mouse liver samples were randomly selected from each group for western blot analysis. To extract tissue proteins, the samples were fully ground in liquid nitrogen. The protein concentration was measured using the bicinchoninic acid method. Equal amounts of protein (40 µg) were separated on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%). Next, they were transferred onto a polyvinylidene fluoride (PVDF) membrane (Beyotime Biotechnology, Shanghai, China). The PVDF membrane was blocked with 5% bovine albumin for an hour. Subsequently, rabbit anti-mouse primary antibodies against C/EBPα (CST, USA), PPARγ (CST, USA) and PGC-1α (Abcam, UK) as well as GAPDH (1:5000) (CST, USA) were incubated overnight at 4°C. After washing the PVDF membrane the next day, it was then incubated with HRP-labelled secondary antibodies for two hours under room temperature. Finally, using the Image Quant LAS 4000 mini system (GE Healthcare, Piscataway, NJ, USA), the PVDF membrane was photographed after washing. All primary and secondary antibodies used were diluted according to the manufacturer’s instructions. By comparing the gray value of each target protein with that of GAPDH, the relative expression of each protein was established.

2.6 Enzyme-linked immunosorbent assay (ELISA)

In this study, the distal colon tissues were weighed and homogenized in PBS to extract the supernatant. The MUC2 level in the collected supernatant was quantified by using a MUC2 ELISA kit (# MM-44508M1) in accordance with the prescribed instructions. Additionally, serum inflammatory factors like interleukin-1β (IL-1β, # MM-0040M1), interleukin-6 (IL-6, # MM-0163M1), interleukin-10 (IL-10, # MM-0176M1), tumor necrosis factor-α (TNF-α, # MM-0132M1), and interleukin-17A (IL-17A, # MM-0170M1) were measured using ELISA kits likewise following the recommended instructions. All ELISA kits used were procured from Jiangsu Meimian Industrial Corporation, China.

2.7 Oral glucose tolerance test (OGTT)

At week 8, OGTT was carried out to evaluate the mice's ability to tolerate glucose according to the methods described by previous study [38]. Mice were deprived of food for 6 h, blood samples were collected from the caudal vein at baseline (0 min) and at various time points (15, 30, 60, 90, 120 min) after administering 2 g/kg body weight through oral gavage. The glucose levels were measured using a glucose analyzer.

2.8 Determination of plasma parameters

Plasma indicators, including total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels, were quantified using kits provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.9 16S rRNA sequencing

Stools were collected every 4 weeks and snap-frozen immediately at − 80°C until further processing. Fecal microbial DNA was extracted using the Omega Bio-tek Stool DNA Kit (Omega, Norcross, GA, USA) following the manufacturer's protocol. Bacterial DNA concentrations were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The V3-4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACNNGGGTATCTAAT-3’), with 8-digit barcode sequences added to the 5’ end of each primer for sample identification (provided by Allwegene Company, Beijing). PCR products were purified using a Beckman Coulter Agencourt AMPure XP Kit (Beckman Coulter, Inc., USA). Sequencing libraries were generated using the NEB Next Ultra II DNA Library Prep Kit (New England Biolabs, Inc., USA) according to the manufacturer's recommendations. Library quality was assessed by Nanodrop 2000 (Thermo Fisher Scientific, Inc., USA), Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA), and ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Inc., USA). Deep sequencing was performed on an Illumina Miseq PE300 (Illumina, Inc., USA) platform at Beijing Allwegene Technology Co., Ltd.. After sequencing, image analysis, base calling, and error estimation were performed using Illumina Analysis Pipeline Version 2.6 (Illumina, Inc., USA). Raw data were filtered and spliced using Pear (v0.9.6) software, and chimeras were removed using Vsearch (v2.7.1) software. Qualified sequences were clustered into operational taxonomic units (OTUs) using the Uparse algorithm of Vsearch (v2.7.1) software with a similarity threshold of 97%. OTU unique representative sequences were obtained, and all tags were mapped to each OTU representative sequence using the BLAST tool. Rarefaction curves were generated, and richness and α-diversity indices were calculated using QIIME (v1.8.0) based on the OTU abundance information. To assess the dissimilarity between multiple samples, PCA was performed using R (v3.6.0) based on the OTU information from each sample. The relative abundances of taxa at multiple levels, including phylum, class, order, family, genus, and species, were computed based on the results of taxonomic annotation and relative abundance. To provide a comprehensive analysis of the microbial diversity differences between samples, statistical analyses, such as Metastats and linear discriminant analysis effect size (LEfSe), were conducted. These methods are effective for mining deeper data on microbial diversity.

2.10 Antibiotic treatment

Antibiotic treatment was administered following an established protocol [39]. In brief, mice were treated daily via oral intragastric gavage with a broad-spectrum antibiotic cocktail (ABX) (Beijing Suo Lai Bao Technology Co., Ltd.) consisting of ampicillin (100 mg/kg body weight (BW)), neomycin (100 mg/kg BW), metronidazole (100 mg/kg BW), and vancomycin (50 mg/kg BW) throughout the 8-week ND/HFD-feeding period.

2.11 Fecal microbiota transplantation (FMT)

Mice were administered a mixture of antibiotics (vancomycin [0.5 g/L], neomycin sulfate [0.5 g/L], metronidazole [1 g/L], and ampicillin [1 g/L]; Sigma-Aldrich) in their drinking water for 5 consecutive days prior to FMT. Fecal transplant was conducted according to established protocols [40–42]. Briefly, 8-week-old male donor mice were fed either HFD or HFD + PS, while recipient mice were fed microbiota derived from either HFD or HFD + PS. Stool samples were collected from the donors and pooled over the course of the 8-week experiment. Donor stools were diluted with PBS (1 g of sample in 10 mL of PBS) and homogenized for one minute using a vortex to create a liquid slurry. The mixture was centrifuged at 1000 rpm for 2 minutes to remove particulate matter. About 200 µl of the supernatant was immediately administered to each mouse by oral gavage three times a week for 8 weeks. Fresh transplant material was prepared within 10 minutes before oral gavage to prevent changes in bacterial composition.

2.12 Statistical analysis

Data were analyzed with GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA). For comparisons between two groups, Student's t-test was used for parametrically distributed data and the Mann-Whitney test was used for nonparametrically distributed data where applicable. Significance was accepted at p < 0.05.

3.1 HFD-induced obesity is aggravated by PS in drinking water

After 8-week ingestion of PS at a dose of 10 mg/L via drinking water, mice treated with PS gained a similar amount of weight under ND but 17% more weight under HFD (Fig. 1B–C), despite consuming less food under both diets (Fig. 1D). These findings suggested that PS-induced weight gain did not result from increased food intake. Additionally, the PS + ND group showed no significant increase in liver weight, while the PS + HFD group exhibited a 16.9% increase (Fig. 1E). Further analysis of liver tissue using H&E and Oil red O staining indicated an increase in liver lipid area in the PS-treated group compared to the non-PS treated group (Fig. 1F–H). Moreover, the PS + HFD group showed increased levels of C/EBPα and PPARγ proteins, while PGC-1α protein levels were reduced (Fig. 1I). Although PS only significantly increased perirenal adipose tissue (pWAT) weight in ND-fed mice, there was a 76% increase in inguinal adipose tissue (iWAT) mass, a 64% increase in pWAT mass, and a 39% increase in epididymal adipose tissue (eWAT) mass in the HFD + PS group (Fig. 1J–L). H&E analysis of iWAT revealed larger adipocyte size in PS-treated mice, particularly those fed with HFD as compared to ND-fed mice (Fig. 1M–N). The increased obesity may further disrupt insulin signaling, reduce insulin sensitivity, and disturb the stability of blood glucose. Hence, glucose tolerance experiments were conducted in mice. According to Fig. 1O, blood glucose concentrations in HFD group were significantly higher after exposure to PS compared to HFD-fed mice who received a glucose gavage. Additionally, blood glucose levels in ND + PS group were only significantly higher than those in the control group at 30 minutes after gavage of glucose. Lipid profiles were analyzed biochemically to determine the effect of PS, which resulted in a significant increase in TC and TG in the liver (Fig. 1P–Q), as well as a significant increase in TC, LDL-C, and LDL-C/HDL-C in serum (Fig. 1S–T and V). There was no difference observed in TG and HDL-C with PS supplementation when compared to the HFD group (Fig. 1R and U). However, in ND-fed group, PS resulted in a significant increase in LDL-C and decrease in HDL-C in serum (Fig. 1T and U). Collectively, these findings demonstrate that oral administration of PS significantly exacerbates HFD-induced obesity.

3.2 PS induced significant alterations in microbiota associated with obesity

The gut microbiota of both obese humans and mice fed with a high-fat diet (HFD) exhibit reduced α diversity, alterations in β diversity, an increased ratio of Firmicutes-to-Bacteroidetes, higher levels of endotoxin-producing bacteria, and diminished immuno-homeostatic bacterial species [43, 44]. In this context, we examined the effects of PS on the composition of gut microbiota by analyzing bacterial 16S rRNA (V3-V4 region) present in faeces using MiSeq sequencing-based analysis. Bacterial diversity analysis revealed that oral intake of PS enhanced the alpha-diversity of gut flora in both ND and HFD-fed mice (Fig. 2A). Additionally, PS administration led to considerable changes in microbiota composition in both groups, as depicted in Fig. 2B–C. The Firmicutes-to-Bacteroidetes (F:B) ratio, a crucial intestinal microbial parameter linked to diet-induced obesity [8], decreased by more than 2-fold in HFD + PS group (Fig. 2D). At the family levels, PS resulted in augmented relative abundances of Lachnospiraceae, Oscillospiraceae, Marinifilaceae, Akkermansiaceae, and Deferribacterales in the HFD + PS group (Fig. 2E-J), and HFD + PS group showed a significant decrease in the relative abundance of Bifidobacteriaceae and Atopobiaceae (Fig. 2H and 2K). These families are associated with obesity [45]. Family Deferribacteres were significantly enriched at higher taxonomic levels such as phylum, class, and order in HFD + PS group. At the genus level, relative abundances of Desulfovibrio, Bifidobacterium and Parabacteroides had pronounced changes in PS + HFD group (Fig. 3L–N). Of note, enriched Desulfovibrio have been reported to promote the development of chronic inflammation and obesity [49]. These results indicated that PS induced obesity-related microbiota changes in HFD + PS group.

3.3 PS-induced microbiota alterations lead to mucus disruption and low-grade systemic inflammation

Intestinal dysbiosis may impair intestinal barrier function, leading to low-grade systemic inflammation [46], To determine if PS disrupted gut integrity, we hypothesized that the PS-induced obesity might be due to intestinal barrier damage. AB-PAS staining was used to verify this hypothesis. The intestinal goblet cell contents and mucin secretion levels were significantly decreased in the PS-exposed mice fed either ND or HFD (Fig. 3A). The epithelial mucin-encoding gene Muc2 and the MUC2 protein ELISA analyses were also significantly decreased by PS (Fig. 3B–C and Fig. 3G–H), suggesting that PS severely damaged the intestinal mucus layer in the ND + PS or HFD + PS group. The tight junction proteins ZO-1, Occludin, and Claudins play a crucial role in maintaining the integrity of the intestinal barrier, and impaired intestinal tight junctions considerably increase paracellular permeability of the intestine [47]. Furthermore, the mRNA expression levels of the barrier-forming tight junction proteins ZO-1, Occludin, and Claudin3 were significantly decreased in the colon compared to those of control mice (Fig. 3D–F and Fig. 3I–K). All of the data indicated that PS resulted in increased gut permeability through the disruption of the intestinal mucus layer. It has been reported that mucus layer damage could induce low-grade inflammation promoting obesity [11]. In this study, we investigated the effects of PS on gut microbiota and its potential role in promoting obesity and inflammation. 16S rRNA sequencing results showed an increase in the relative abundance of Desulfovibrio species (Fig. 2L) with strong endotoxin activity upon PS administration [48]. Based on the above results, we hypothesized that PS-induced gut barrier damage and microbiota changes may lead to inflammation, thereby promoting HFD-induced obesity. To further explore this hypothesis, we evaluated colon length as a measure of intestinal inflammation. We found that ND and HFD-fed mice exhibited shortened colons after PS administration (Fig. 3M). The shortened colons and H&E staining images of colons indicated a significant increase in intestinal inflammation in PS-treated groups (Fig. 3N and Fig. 3L), which was confirmed by elevated IL-17A and TNF-α mRNA expression levels in colon (Fig. S3A–B in the ESM). Increased intestinal permeability allows lipopolysaccharide (LPS) and PS to enter circulation, culminating in low-grade systemic inflammation [35, 49]. Therefore, we tested the levels of inflammatory factors in the serum. The results showed that serum levels of IL-1β, IL-6, TNF-α, and IL-17A were significantly higher in the HFD + PS group than in the HFD group (Fig. 3O–R), which was also confirmed by elevated IL-17A mRNA expression levels in iWAT (Fig. S3C in the ESM). Meanwhile, levels of IL-10 were significantly decreased by PS in HFD-fed mice but no difference was seen in the ND-fed mice (Fig. 3S). These findings suggested that PS causes low-grade systemic inflammation by altering gut microbiota and disrupting the mucus layer in HFD-fed mice.

3.4 Potential relations between obesity parameters and gut microbiota

To comprehensively analyze the relationship between obesity and gut microbiota, we generated a correlation matrix by calculating Pearson's correlation coefficient. Our heatmap correlation analysis showed that the top 15 families affected by PS intervention were significantly associated with at least one parameter of obesity (Fig. 4). Specifically, f__Streptococcaceae, f__Lachnospiraceae, f__Oscillospiraceae, f__Desulfovibrionaceae, and f__Marinifilaceae showed significant positive associations with body weight gain, epididymal fat, perirenal fat, inguinal adipose accumulation, serum TC, fasting glucose levels, serum TC, liver TC and liver TG. Conversely, f__Bifidobacteriaceae and f__Atopobiaceae were positively correlated with colon MUC2 protein levels. Notably, f__Bacteroidaceae showed significant positive correlations with serum IL-1β and IL-6 levels. Furthermore, f__Marinifilaceae were significantly positively correlated with serum TNF-α and IL-17A levels. In contrast, f__Prevotellaceae, f__Muribaculaceae, f__Erysipelotrichaceae, f__Bifidobacteriaceae, and f__Atopobiaceae were negatively correlated with weight gain, liver weight, inguinal adipose, perirenal adipose, epididymal adipose, LDL-C, LDL-C/HDL-C, fasting glucose and liver TG, IL-1β, TNF-α, and IL-17A levels. Additionally, f__Lactobacillaceae were negatively correlated with serum IL-6 levels, while f__Lachnospiraceae, f__Oscillospiraceae, f__Desulfovibrionaceae, f__Marinifilaceae were negatively associated with MUC2 protein levels. Lastly, f__Akkermansiaceae showed significant negative associations with body weight gain, liver weight, epididymal adipose, fasting glucose, and serum TC. These findings suggest that gut microbiota can influence host phenotypes, as well as serum lipid profiles, liver lipid profiles, gut barrier function, and inflammation markers. Overall, our findings illustrate that PS intervention exacerbate gut microbiota dysbiosis induced by HFD, leading to a more unhealthy gut microbiota composition.

3.5 PS-driven inflammation and obesity are microbiota-dependent

Germ-free or microbiota-depleted mice have shown decreased susceptibility to diet-induced obesity and metabolic syndrome, suggesting that the gut microbiota was involved [50]. To further elucidate the role of gut microbiota in diet-induced obesity driven by PS, we employed an antibiotic-treated mouse model (a broad-spectrum antibiotic cocktail (ABX) consisting of ampicillin, neomycin, metronidazole, and vancomycin) [39] to investigate the effects of PS on mice fed ND and HFD. After eight weeks of treatment with ABX by gavage, more than 97% of intestinal bacteria were eliminated (Fig. S4 in the ESM). Importantly, the obesity-related phenomenon induced by PS was not observed in either ND or HFD-fed mice with ABX treatment (Fig. 5), indicating that the fat-accumulation effects driven by PS were dependent on gut microbiota. These findings underscored the vital role of gut microbiota in PS-induced obesity in mice. Interestingly, mucus layer disruption was also not found in the microbiota-depleted mice (Fig. 6A–G), suggesting that PS-induced mucus disruption was also dependent on gastrointestinal flora. It is worth mentioning that the low-grade inflammation caused by PS disappeared with the removal of microorganisms (Fig. 6H–M). These data indicated that gut microbiota was necessary for PS-induced disruption of mucus layers, low-grade inflammation, and obesity.

3.6 The gut microbiota from the HFD + PS group induced obesity in HFD-fed mice

In order to further elucidate the effects of PS-induced obesity mediated by the gut microbiota and its impact on obesity, we transferred microbiota from PS + HFD group and HFD group to ABX (a mixture of antibiotics (vancomycin, neomycin sulfate, metronidazole, and ampicillin) [51] in their drinking water for one week prior to FMT)-pretreated recipient mice fed with HFD. We then examined obesity-related traits (Fig. 7A). After 8 weeks of colonization, the receivers (R-HFD + PS group) that received microbiota from the PS + HFD group exhibited a noteworthy rise in body weight when compared to the receivers (R-HFD group) that received microbiota from the HFD group (Fig. 7B). These recipients exhibited impaired metabolic features, such as liver weight, hepatic steatosis, iWAT, pWAT, eWAT, serum TC, LDH-C, and LDH-C/HDH-C (Fig. 7C–K and 7M–O); however, blood glucose concentrations in the R-HFD + PS group were not significantly higher than those in the R-HFD group after glucose gavage, except at 0 min (Fig. 7L). These results demonstrate that recipient mice recapitulated microbial and obesity phenotypes and suggest that the gut microbiota mediates PS-induced obesity.

3.7 Gut microbiota from the HFD + PS group impaired intestinal barrier function and induced low-grade inflammation in HFD-fed mice

Through the FMT experiment, we found that the gut microbiota from the HFD + PS group impaired the intestinal barrier function and induced low-grade inflammation. Notably, the fecal bacterial solution from the HFD + PS group significantly reduced the protein expression of MUC2 and the relative mRNA levels of Muc2 and ZO-1 in the colon tissues than did the fecal bacterial solution from the HFD group (Fig. 8A–D). H&E staining also suggested that there was obvious pathological lesion in the distal colon (Fig. 8E). Furthermore, a subsequent increase in the expressions of the inflammatory factors IL-17A, TNF-α, and IL-6 (p = 0.086) was observed in the serum of R-HFD + PS mice (Fig. 8F–H). Together, these results demonstrate the crucial role of gut microbiota in PS-mediated influence on intestinal health of HFD-fed mice.

Microplastics (MPs) are ubiquitous in our environment due to human activities. Recent studies have increasingly confirmed the presence of MPs in the human body, raising concerns about their potential hazards. However, the impact of PS-MPs on obesity of human remains unclear. Considering that HFD effectively induces obesity, and HFD combined with other factors (e.g., Azithromycin) may aggravate obesity [52], in this study, we set out to examine the detrimental impact of PS-MPs when administered through drinking water in a HFD-induced obesity model. Our study presented compelling evidence that PS exacerbated HFD-induced obesity and promoted weight gain, which was associated with impaired glucose and lipid metabolism. As a considerable proportion of ingested MPs traversed the gastrointestinal tract following oral intake [31], it was hypothesized that the gut microbiota and epithelial barrier may represent novel targets for PS-MPs toxicity, particularly in obese populations. By utilizing a mouse model of obesity induced by a HFD, and employing ABX treatment and FMT, this study has furnished compelling evidence that PS treatment induced inflammation and obesity by altering the composition of the gut microbiota and compromising the integrity of the intestinal barrier. To our best knowledge, this is the first study to explore the obesity-enhancing effects of the combination of a HFD and PS, as well as to evaluate the role of PS-induced alterations of gut microbiota in HFD-induced obesity. Our findings suggested that PS might exacerbate Western-diet induced obesity and related metabolic disorders in humans, and the gut microbiota is a crucial target for the obesity-promoting effects of PS-MPs.

The daily intake of MPs is still uncertain, despite organisms being constantly exposed to them. This is particularly true for humans, as the pathways of MPs contact are diverse and complex, leading to varying assessments of body intake. The dose selection for PS-MPs took into account both their toxicological and environmental significance. PS-MPs with a yield of 5 µm and a toxic effect were chosen as exposure materials, based on previous studies on male intestinal toxicity, with a range of 5 µg/d to 1 mg/d [30, 53–55]. In terms of environmental significance, on average, humans could potentially ingest 0.1-5 g of MPs per week globally [56]. Based on the weight of the mice, which was approximately 20 g, their daily intake of PS was calculated to range between 37.14 µg/d and 1.86 mg/d. It was estimated that each control mouse consumed 3.6 mL of water per day, resulting in a PS intake of 36 µg/d per mouse [32]. This amount is comparable to the minimum intake for humans. The exposed doses of mice were exposed daily to PS through their drinking water, simulating the major route of PS exposure in mammals [57, 58]. Therefore, the selected doses were of environmental and toxicological significance.

Recently, it has been discovered that MPs have the ability to disrupt glucose and lipid metabolism in rodents [59], however, the role of PS in obesity populations is not yet well understood. Our study first confirmed that PS exacerbated HFD-induced obesity. It is noteworthy that the lipid accumulation in the liver and white adipose tissue caused by PS was more pronounced in HFD-fed mice than in those fed a ND, which suggested that susceptible populations might be more affected by the impact of PS. This suggested that PS-induced obesity was related to diets. This could be attributed to the fact that, in addition to PS consumption, HFD also caused mucosal damage and low-grade inflammation [60]. At the molecular level, the process of adipocyte differentiation is controlled by multiple transcription factors, which facilitate the expression of adipogenic genes and support cell differentiation. Notably, recent research has highlighted the significant role of PPAR-γ and C/EBP-α in regulating this process. [61]. PGC-1α plays a vital role in maintaining energy balance by regulating the transcription of genes responsible for fatty acid oxidation [62]. In our study, PS administration up-regulated adipogenic genes such as PPAR-γ and C/EBP-α, and down-regulated lipolysis genes PGC-1α in the liver of mice fed a HFD. It was suggested that the pro-obesity activity of PS might be mediated by up regulating the expression of transcription regulator genes of lipogenesis and down regulating the expression of transcription regulator genes of lipolysis, which was further corroborated by fat accumulation and the elevation of TG and TC levels in the liver. It is well known that overweight and obesity are associated with significant changes in metabolism, especially glucose and lipid metabolism [63]. We consistently found that glucose tolerance was severely impaired in HFD + PS group, and serum levels of TC, LDL-C and LDL/HDL-C ratio were significantly elevated. Together, our results indicated that alterations in glucose and lipid metabolism played a significant role in the development of PS-induced obesity. Recent studies have revealed that the development of obesity in mice exposed to PS can be effectively dose-controlled under ND conditions [64]. Specifically, relatively low doses of PS have been found to trigger obesity while higher doses do not. In line with these observations, we report that administering 10 mg/L PS in drinking water did not induce obesity in mice on a ND. Intriguingly, and in contrast to the above findings, we observed that 10 mg/L PS exposure promoted fat accumulation in mice fed a HFD, suggesting that PS-induced obesity was highly influenced by diets as well as dosage of PS. Previous studies have reported that oral intake of 0.45–0.53 µm PS-COOH beads or a combination of PS particles with varying particle sizes (5, 50, 100, and 200 µm) led to a reduction weight in HFD-fed mice [49, 65]. This implies that PS with distinct particle sizes and properties may have dissimilar effects on body weight. In the following phase, we will investigate the impact of PS and other microplastic particles on obesity in mice under varying dietary conditions and dosage levels. Our findings demonstrated that exposure to PS at levels below those typically encountered by humans worsened HFD-induced obesity, potentially elucidating the link between increased plastic production and the growing global prevalence of obesity.

Recent studies have shown that PS-induced obesity is related to gut microbiota [32, 64]. Previous research has revealed that obese mice have different intestinal compartments compared to their normal counterparts, with a higher proportion of Firmicutes phylum [66, 67]. Additionally, it has been suggested that Firmicutes phylum may be implicated in obesity, as a larger population of these microorganisms can effectively convert food to energy at accelerated rates [68]. The study found that PS increased the relative abundance of the class Clostridia (phylum Firmicutes), including family Lachnospiraceae, and Oscillospiraceae in the HFD + PS group. Interestingly, PS was also able to reduce the F:B ratio, which is considered a key marker of diet-induced obesity [9, 10]. These findings suggest that PS can modify the population of certain bacteria within the Firmicutes phylum, leading to obesity independent of the F:B ratio.

Therefore, the relative abundance alterations of certain family levels within the Firmicutes bacteria was a significant factor in PS-induced obesity. Previous studies have reported an relative abundance increase of family Lachnospiraceae in both mice and humans with obesity and diabetes [69, 70]. Mechanistic research has shown that Fusimonas intestini, a commensal species of Lachnospiraceae, contributes to diet-induced obesity by producing excessive long-chain fatty acids [71]. In our study, Lachnospiraceae was the predominant family of Firmicutes, comprising over 40% of the total population. The overproduction of long-chain fatty acids could potentially be linked to PS-induced obesity. Family Oscillospiraceae was reported to be increased in HFD-fed mice. This family exhibited a positive correlation with obesity indexes, blood parameters, bacterial translocation parameters, proinflammatory cytokine, TLR4/NF-κB pathways, energy metabolism, and colonic permeability [45, 72]. Consistent with these observations, PS treatment significantly enhanced the percentage of family Oscillospiraceae in the gut microbiota of the HFD + PS group. In addition to alterations in bacterial abundance within the Firmicutes phylum, there is a remarkable relative abundance increase in family Marinifilaceae in HFD + PS group, which were significantly associated with visceral fat and body fat percentage [73]. Correlation analysis also proved that the relative abundance of family Lachnospiraceae, Oscillospiraceae and Marinifilaceae were significantly positively correlated with obesity related indicators.

In particular, the relative abundance of genus Akkermansia significantly increased in the HFD + PS treatment group. Recent research has highlighted the significance of genus Akkermansia as an essential functional bacterium that regulates human immunity and metabolism in the intestine, significantly linked with human health and diseases [74, 75]. Normally, genus Akkermansia colonized the outer mucous layer of the gastrointestinal tract to maintain the dynamic balance of mucin production, thereby upholding the stability of the mucus layer [76]. Furthermore, genus Akkermansia could significantly improve metabolic endotoxemia, insulin resistance, fat accumulation and fasting hyperglycemia in obese mice induced by HFD [77]. However, genus Akkermansia's role in intestinal diseases remains controversial. Some studies have found that genus Akkermansia could improve obesity and obesity-associated metabolic syndrome in mice [77, 78]. In contrast, clinical studies have revealed a high frequency of genus Akkermansia in the fecal matter of infants with eczema. Moreover, it has been established that genus Akkermansia impairs gut barrier integrity and elevates the risk for eczema [79]. Recently, a study demonstrated that a high-sugar diet could augment the relative abundance of genus Akkermansia in mice. This increased abundance resulted in the destruction of intestinal barrier function, leading to colitis via stimulating the activity of mucolytic enzymes within the microbiota [80]. In this study, PS was observed to significantly enhance the relative abundance of genus Akkermansia, while decreasing the thickness of the mucus layer in the colon tissue of HFD-fed mice, which potentially contributed to worsening obesity.

Numerous reports have indicated that the onset of obesity induced by dietary factors is linked to changes in gut microbiota, characterized by a reduction in beneficial microbial genera, which ultimately contributes to obesity and metabolic syndrome [81–83]. Consistently, our study also revealed a reduction in the abundance of beneficial bacteria, including family Atopobiaceae and genus Bifidobacterium, as well as genus Parabacteroides. The function of family Atopobiaceae is not yet fully understood; however, previous studies have reported a significant decrease in the abundance of family Atopobiaceae in HFD-induced diabetic mice. [84]. Consistent with the findings of this study, the relative abundance of family Atopobiaceae was observed to be negatively correlated with the obesity index induced by PS, indicating a potential role of family Atopobiaceae in anti-obesity. Previously, the Bifidobacterium spp. had been shown to reduce obesity [85], which was negatively related to intestinal permeability, metabolic endotoxemia, and low-grade inflammation [86, 87]. Similarly, this study found that PS promoted a significant decrease in the proportion of genus Bifidobacterium in HFD-fed mice, which was a negative correlation with obesity-related indicators. Except for genus Bifidobacterium, a study revealed that Parabacteroides goldsteinii also plays a significant role in anti-obesity effects [88]. We found that PS supplementation caused a notable decrease in the abundance of genus Parabacteroides in mice fed a HFD, indicating that the supplementation of Parabacteroides spp. may be another effective approach to alleviate the adverse health effects induced by PS. Further research is necessary to gain a better understanding of the potential therapeutic applications of Bifidobacterium spp. and Parabacteroides spp. in managing PS-induced obesity and related metabolic disorders. Taken together, microbial dysbiosis is associated with PS-induced obesity.

HFD could significantly reduces the thickness of the intestinal mucus layer through microbial dysbiosis [77]. The mucus layer, which is formed by mucin proteins and hydrated gel with glycosylation, plays an essential role in separating the intestinal epithelium from its contents [89]. It prevents microorganisms from directly contacting epithelial cells and makes the intestinal mucosal barrier essential for human health. Dysfunction in this barrier has been linked to numerous diseases such as inflammatory bowel disease, microbial infections, and metabolic syndrome [90]. Therefore, we hypothesized that PS-induced microbial dysbiosis exacerbated mucosal layer injury. This study discovered that exposure to PS resulted in a reduction of mucus contents in the colon since it down-regulated MUC2 expression along with the tight junction-related genes such as ZO-1, Occludin, and Claudin-3 in HFD-fed mice. As a consequence, the destruction of the mucus layer selected and enriched certain bacteria like Lachnospiraceae, residing in the inter-fold regions of the colon, come in direct contact with the intestinal epithelium, thus initiating inflammation [91]. Previous study have shown that PS exposure induced systemic inflammation and thus induced insulin resistance [49]. Based on these results, we speculated that PS impaired the integrity of intestinal barrier to result in intestinal inflammation, which allows toxic molecules entry into the systemic circulation, thereby triggering systemic inflammation. This hypothesis was supported by the observation of reduced colon length, and up-regulated mRNA expression of inflammatory cytokines in the colon (TNF-α, IL-17A) and inguinal fat tissues (IL-17A), which was further bolstered by elevated serum levels of pro-inflammatory cytokines (TNF-α, IL-17A, IL-1β, and IL-6) and decreased serum levels of anti-inflammatory cytokines (IL-10). Furthermore, correlation analysis also revealed significant associations between the dysregulated gut microbiota induced by PS and indicators related to obesity, MUC2 expression levels, as well as inflammatory factors. Our findings suggest that PS-induced dysbiosis disrupts the gut microbiota, leading to increased penetration of toxic gut molecules into the body via impaired intestinal permeability in HFD-fed mice. This process drived a state of chronic low-grade inflammation that ultimately contributes to the development of obesity. These findings provide further evidence for the role of PS-induced microbial imbalance in perpetuating metabolic dysfunction, and highlight the potential utility of targeting the gut microbiota as a therapeutic strategy for treating obesity and associated disorders.

Alongside germ-free animal models, antibiotic-treated animal models are frequently employed in the investigation of intestinal bacteria [92]. To verify whether the adverse effect of PS on obesity is mediated by the intestinal flora, In this study, a combination of four broad-spectrum antibiotics - namely aminoglycoside, penicillin, neomycin, metronidazole and vancomycin were simultaneously administered for 8 weeks, resulting in a 97% reduction of commensal bacteria within murine intestinal microbiota. This investigation exhibitd certain limitations, most notably our inability to fully eradicated the intestinal microbial population. The findings indicated that antibiotic cocktail effectively resolved PS-induced obesity, thus supporting the hypothesis that gut microbiota play a crucial role in the development of this disorder. A more concerning result was that PS reduced the thickness of the mucus layer depended on the presence of gut flora. Therefore, our results indicated that PS harmed the intestinal barrier by affecting intestinal flora metabolites, leading to a low-grade inflammation that promotes HFD-induced obesity.

Moreover, we discovered that the PS induced low-grade inflammation and obesity were also dependent on intestinal flora. Notably, we found that the genus Desulfovibrio, a sulfate-reducing bacteria (SRB), exhibited an increased abundance in the PS + HFD group. This bacteria has been proven to produce LPS, which possesses 1000-fold endotoxin activity in comparison to other gut bacteria [48]. We also found that family Deferribacteres enriched at higher taxonomic levels such as phylum, class, and order in HFD + PS group. Mounting evidence suggests that family Deferribacteres were strongly associated with upregulated pro-inflammatory cytokines and exacerbation of inflammation and obesity-induced metabolic disease T2D [5, 93]. Furthermore, we observed a decrease in the number of family Bifidobacteriaceae, which is known to have a negative correlation with intestinal permeability, metabolic endotoxemia, and low-grade inflammation in the PS + HFD group. These shifted in gut microbiota induced by PS contributed significantly to the reduced expression levels of Muc2 and tight junction related genes, resulting in damage to the intestinal barrier function and induction of low-grade inflammation in mice colon tissue. We also found variations in the PS-induced microbiota alterations between the HFD-fed and ND-fed groups. However, we attribute this to the influence of the HFD on gut flora as it differs from ND. Nevertheless, ABX treatment did not completely eliminate lipid accumulation induced by an HFD compared to the ND diet, which indicated that gut microbes participated in HFD-induced obesity but were not the sole determining factor. Additionally, FMT exacerbated HFD-induced obesity, further supporting our conclusion that PS induced mucosal layer injury, low-grade inflammation and obesity depended on intestinal flora. Thus, microbiota dysbiosis and the damage of intestinal barrier collaborate to promote low-grade inflammation, which has been known to boost obesity development [94].

Obesity and related chronic inflammatory diseases have become a serious health problem both in developed and developing countries [95]. Recent studies indicate that the relationship between obesity, gut barrier, intestinal microbes, and their interactions is interlinked and chiefly dependent on dietary habits [96]. Our study demonstrated that frequently ingested PS exacerbated HFD-driven destruction of the mucus layer, inflammation, and obesity by altering the gut microbiome. We observed that PS amplified HFD-induced microbiota dysbiosis, demonstrating a superimposed effect between PS and HFD on intestinal integrity and flora. This highlights the importance of considering the impact of intestinal health on individuals with different dietary habits, particularly those consuming western diets, as PS may enhance the progression of obesity via alterations in the microflora.

In summary, our study demonstrated that exposure to PS particles of 5 µm in size reduced the thickness of the mucus layer to promote more toxic molecules entry into the body, thus upregulating systemic inflammation and eventually exacerbating HFD-induced obesity. Through antibiotic treatment and fecal microbiota transplantation experiments, we have demonstrated that PS-altered microbiota play a critical role in obesity process. Together, our results suggest that PS-MPs may promote metabolic syndrome by altering gut microbiota. While many other types of MPs exist widely in daily life, their effects on mucus-bacterial interactions remain largely unknown. Nevertheless, considering the inevitable human exposure to MPs and their potential toxicity link with obesity, it is important to monitor our exposure to these pollutants and their role in promoting inflammation, which may in turn lead to chronic metabolic diseases.

PS, Polystyrene; MPs, Microplastics; HFD, high-fat diet; ND, normal diet; H&E, hematoxylin and eosin; BW, body weight; TC, total cholesterol; TG, total triglyceride; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; eWAT, epididymal white adipose tissues; pWAT, perirenal white adipose tissues; iWAT, inguinal white adipose tissues; DI, Deionized; MUC2, Mucin2; C/EBPα, CAAT/enhancer binding protein; PPARγ, Peroxisome proliferator-activated receptor γ; PGC-1α, Peroxisome proliferator-activated receptor-gamma coactivator-1α; IL-1β, Interleukin-1β; IL-6, Interleukin-6; IL-17A, Interleukin-17A; IL-10, Interleukin-10; TNF-α, Tumor necrosis factor-α; ZO-1, Zona occludin-1; PBS, phosphate buffered saline; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; BCA, Bicinchonininc acid; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF, Polyvinylidene fluoride; BSA, Bovine albumin; PCA, principal component analysis; OTUs, operational taxonomic units; LEfSe, linear discriminant analysis effect size; LDA, linear discriminant analysis.

GRAPHICAL ABSTRACT

Ethics approval and consent to participate

The animal experiments were approved by the Laboratory Animal Welfare and Animal Experimental Ethical Committee of the China Agricultural University (No. AW72403202-1-31).

Consent for publication

Not applicable.

Availability of data and materials

All data generated of this study are included in this article (and its supplementary information). The 16S rRNA raw reads are available at NCBI’s SRA repository (BioProject PRJNA990701).

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31625025 and 32000082), and the 2115 Talent Development Program of China Agricultural University.

Authors’ contributions

Zhian Zhai mainly completed the experimental design, analysis, and manuscript writing. Zhenlong Wu mainly directed the work and completed the revision of the final paper. Ying Yang was involved in writing, review & editing. All the authors have read and approved the final version of the manuscript.

Acknowledgements

We thank the National Natural Science Foundation of China and the 2115 Talent Development Program of China Agricultural University for supporting our research.

Authors’ details

¹State Key Laboratory of Animal Nutrition, Department of Companion Animal Science, China Agricultural University, Beijing 100193, China; ²Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing 100193, China.

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No competing interests reported.

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Long-term exposure to polystyrene microplastics promotes HFD-induced obesity in mice through exacerbating microbiota dysbiosis

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Characterization of PS

2.2 Animals and diets

2.3 Histology

2.4 Quantitative real-time polymerase chain reaction

2.5 Western blot analysis

2.6 Enzyme-linked immunosorbent assay (ELISA)

2.7 Oral glucose tolerance test (OGTT)

2.8 Determination of plasma parameters

2.9 16S rRNA sequencing

2.10 Antibiotic treatment

2.11 Fecal microbiota transplantation (FMT)

2.12 Statistical analysis

3. Results

3.1 HFD-induced obesity is aggravated by PS in drinking water

3.2 PS induced significant alterations in microbiota associated with obesity

3.3 PS-induced microbiota alterations lead to mucus disruption and low-grade systemic inflammation

3.4 Potential relations between obesity parameters and gut microbiota

3.5 PS-driven inflammation and obesity are microbiota-dependent

3.6 The gut microbiota from the HFD + PS group induced obesity in HFD-fed mice

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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