The Effect Of Oxidative Stress Induced By High Fat Diet On Changes Of Gut Microbiota And Inflammation In Mice

It is widely accepted the fact that metabolic syndrome is associated with gut microbiota.But the mechanisms by which high-fat diet affects the gut microbiota and low-gradeinflammation is unknown. Research has shown that a high-fat diet can cause oxidative stress,especially in the intestine. And the intestine is bacteria inhabiting environment. Differentstrains of intestinal bacteria had different tolerance to oxidative stress. Therefore, wehypothesized that long-term high-fat diet (HFD) may influence gut microbiota by changingthe redox state. Then, the indigenous opportunistic bacteria on the interior of Peyer’s patcheswere changed, inducing the secretion of inflammatory cytokines, eventually cause intestinaland systemic inflammation.We have used an animal model of a high-fat diet-induced oxidative stress to prove thehypothesis that long-term HFD may influence gut microbiota directly and/or indirectly bychanging the redox state. Lipoic acid (LA), a universal antioxidant, was used to modulateredox balance. Reactive oxygen species (ROS), total antioxidant capacity (T-AOC), andmalondialdehyde (MDA) were analyzed to profile oxidative stress states. PCR denaturinggradient gel electrophoresis (DGGE) was used to describe gut flora structures, while platecount was employed for the quantitative analysis of gut flora. ROS and MDA, whichsignificantly decreased in LA mice compared with HFD mice. DGGE profiles clearly showedthat there were gut microbiota dysbiosis and special bands in HFD mice. Escherichia coli andenterococcus were significiantly increased in HFD mice, whereas lactobacilli was reduced(P<0.05). ROS and MDA were positively and significantly correlated with Escherichia coliand enterococcus (P<0.05), whereas negatively correlated with lactobacilli. A positivecorrelation was found between T-AOC and lactobacilli (P<0.05), but a negative correlationwas found between T-AOC and Escherichia coli and enterococcus (P<0.05).We conducted an experiment using an animal model of obesity-resistant (OR) andobesity-prone (OP) groups and adding resveratrol (RES) to further demonstrate therelationship between oxidative stress and gut microbiota. Colon were removed to determineoxidative stress and gut inflammation by real-time PCR and to analyze the gut microbiotausing fluorescence in situ hybridization (FISH). The level of oxidative stress: OP>OR>CT.RES treatment decreased the level of oxidative stress. Principal components analysis (PCA)illustrated that significant differences were observed in the composition of bacteria on the OP,OR and Control mice. Lactobacilli and Bifidobacterium were significantly increased in OPmice, whereas Enterobacteriaceae were reduced. The Bacteroidetes-to-Firmicutes ratios: OP<OR<CT. After RES treatment, Lactobacillus and Bifidobacterium were significantlyincreased, whereas Enterobacteriaceae was significantly decreased to that of control levels.Multiple-correlation analyses indicated that Bacteroidetes-to-Firmicutes ratios significantlypositively correlated with ROS and MDA (P<0.05), whereas negatively correlated with totalantioxidant capacity (T-AOC). OP mice increased the mRNA expression of NOD2andCARD9, and selectively activated transcription factors (i.e., NF-κB and AP-1), together withenhancing the production of IL-8, IL-10, TNF-α, and IL-23, modulating adaptive immuneresponses.The differences of indigenous opportunistic bacteria on the interior of Peyer’s patchesbetween OP and OR mice were assessed. OP group had a significantly lower bacteriadiversity than that in the OR group (P<0.05). Significant differences were observed in thecomposition of bacteria on the interior of PPs at the genera level among three groups. Theproportion of Lactococcus and Rhizobium were significantly increased in the OP groupcompared with the CT and OR group, whereas the proportion of Allobaculum andLactobacillus were significantly reduced. Several genera of the microbial community weresignificantly increased in the OP and OR group compared with the CT mice, includingStreptococcus, Pseudomonas, Comamonas and Flavobacterium. Pro-inflammatory cytokines(IL-6and TNF-α) were significantly increased in the OP mice. The mRNA expression of IL-6and TNF-α was positively and significantly correlated with Lactococcus, whereas a positivecorrelation was found between IL-10mRNA and Allobaculum and Lactobacillus (P<0.05).We assessed the composition of Lactobacillus on the interior of Peyer’s patches.Antioxidant properties and the ability to induce PPs secrete different cytokines were analysisin vitro. There were13ERIC-PCR banding patterns of the92Lactobacillus isolates, whichmainly belong to Lactobacillus reuteri and the other four groups. And there were eightdifferent Lactobacillus reuteri strains. Our findings indicated that the Lactobacillus reuteriwith stronger tolerance to oxidative stress were significantly increased in OP mice, whereasLactobacillus reuteri with poorer tolerance to oxidative stress were significantly decreased.Among them, the Lactobacillus reuteri L3with the poorest tolerance to oxidative stress wasdetected only in normal group, and while Lactobacillus reuteri L8with strongest tolerance tooxidative stress was detected only in high-fat group. Furthermore, Lactobacillus reuteriinduced the production of anti-inflammatory cytokine (IL-10), inhibited the production ofIL-6, IL-12, and TNF-α, and protected PPs against LPS injury.Lactobacillus reuteri L3and Lactobacillus reuteri L8was gavaged to CT group andHFD mice, respectively. The effect of the two Lactobacillus reuteri strains to the body’shealth was assessed. HFD mice had higher levels of body weight, fasting glucose, insulin, which were normalized after Lactobacillus reuteri L3treatment. But this effect has nothing todo with the inhibition of food intake. Furthermore, The level of LPS and inflammatorycytokines was significantly decreased after Lactobacillus reuteri L3treatment, whereas therewas no significant effects after Lactobacillus reuteri L8treatment. The mRNA expression ofLPL, Scd1in liver and PPAR-γ, ACC1, Fas in adipose tissue were significant decreased afterLactobacillus reuteri L3treatment.Gut microbiota dysbiosis in high-fat diet mice was due to the increased levels ofoxidative stress. Then, they changed the indigenous opportunistic bacteria on the interior ofPeyer’s patches, inducing the secretion of inflammatory cytokines, eventually cause intestinaland systemic inflammation.