Feces Of Mice Research Articles

Electroacupuncture alleviates functional constipation by upregulating host-derived miR-205-5p to modulate gut microbiota and tryptophan metabolism

Electroacupuncture (EA) has shown promise as a treatment for Functional constipation (FC), with growing evidence suggesting it may enhance gut motility. MicroRNAs (miRNAs) serve as key regulatory molecules mediating host-microbiota interactions. However, the specific fecal miRNAs regulating microbiota composition and metabolism in EA-treated constipated mice, along with their key targets, remain unidentified. We examined fecal microbiome composition, metabolism, and colonic miRNA expression in loperamide-induced constipated mice and EA-treated mice to identify differentially expressed miRNAs and assess their relationships with microbial abundance, metabolism, and gut motility. An antibiotic cocktail and adeno-associated virus were employed to interfere with the gut microbiota and target miRNA in vivo, thereby validating the proposed mechanism. Our results indicate that miR-205-5p, significantly upregulated in fecal and colonic tissues of EA-treated constipated mice, promotes intestinal motility in a microbiome-dependent manner. Specifically, EA promoted the growth of Lactobacillus reuteri, enriched in the feces of constipation-recovered mice, through host-derived miR-205-5p regulation. Furthermore, Lactobacillus reuteri and its tryptophan metabolites (indole-3-acetamide, indole-3-acetic acid, and indole-3-carboxaldehyde) alleviated loperamide-induced constipation. These findings underscore the pivotal role of host-derived miR-205-5p in modulating microbial composition and tryptophan metabolites to enhance intestinal motility through EA.

Hepatoprotective Effect of Nano Selenium Derived from Neem Leaf Extracts against Infection with Eimeria papillata in C57BL/6 Mice

Background Coccidiosis is considered a protozoan disease affecting wild and domestic animal species which leads to economic losses. Regarding drug resistance, scientists are focused on alternative sources rather than coccidiostats with fewer side effects. Recently, nanotechnology has been a new trend against parasitic infections by manipulating atoms and molecules at the nanoscale. Purpose This study assessed the hepatoprotective effects of biosynthesized nano selenium (BSN) derived from Azadirachta indica leaf extracts on biological, biochemical, and oxidative stress markers in mice infected with Eimeria papillata. Methods The formation of nano selenium was achieved using leaf extract from A. indica, and their morphology and size were characterized using scanning electron microscopy (SEM). Twenty-five male C57BL/6 mice were divided into five groups, with five mice in each group. The first group received distilled water, while the second group was administered 0.5 mg/kg of BSN. The third, fourth, and fifth groups were infected with the parasite’s oocysts. For 5 days, the fourth group received BSN (0.5 mg/kg), and the fifth group was treated with amprolium (120 mg/kg). Oocyst shedding was assessed on day 5 post-infection (p.i.). After sacrificing the mice, the liver and blood from each mouse were collected and processed for histological, biochemical, and oxidative damage analyses. Results Oocyst shedding in the feces of mice decreased following oral treatment with BSN. Eimeria infection reduced feed consumption to 31.44%, but this increased to 45.57% after BSN treatment. The infection caused significant liver damage, as evidenced by pathological changes and a notable increase in liver function parameters, including glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), alkaline phosphatase (ALP), and direct bilirubin. These parameters improved after treatment with BSN. Additionally, Eimeria infection disrupted nutrient homeostasis. Levels of soluble proteins and carbohydrates in the liver tissue decreased, while blood glucose and total protein levels increased. Treatment with BSN in mice infected with E. papillata significantly improved nutrient homeostasis. Our research also demonstrated that BSN significantly reduced levels of nitric oxide (NO) and hydrogen peroxide (H2O2), while increasing levels of catalase (CAT) and superoxide dismutase (SOD) to help mitigate oxidative damage during Eimeria infection. Conclusion Our findings demonstrate that BSN has a significant in vivo hepatic response against murine coccidiosis and could be valuable as a coccidiostat in treating the disease.

P1318 Temporal Changes of Gut Microbiota and Micro-RNAs according to Disease Activity in Chronic Colitis

Abstract Background Both gut microbiota and micro-RNAs (miRNAs) have been implicated in the pathogenesis of inflammatory bowel disease. We aimed to investigate the temporal changes and their interactions between gut microbiota and miRNAs according to the disease activity using multi-omics analysis in a murine chronic colitis model. Methods Chronic colitis was induced in the C57BL/6 mice by three rounds of 2.5% DSS exposure. Mice were divided into three groups (n= 8 per group): control group, active group (sacrified at 3 days after the third DSS), and recovery group (sacrificed at 80 days after the third DSS). Clinical activities including weight change and histologic findings of colonic segments were examined. DNA was extracted from mouse feces, and 16S rRNA gene sequencing was performed to evaluate changes in gut microbiota. miRNA expression levels were measured by microarray analysis from mouse colonic tissues. Correlation and network analyses were performed to evaluate the interactions between gut microbiota and miRNAs. Results Alpha diversity indices were increased in the active group compared to control and recovery group. PCoA of metagenomic sequencing showed that the gut microbial profile of the active group was distinctly different from the control group, while the gut microbial profile of the recovery group was similar to the control group. On the other hand, PCoA of miRNA microarray showed that the active group had a significantly different miRNA expression pattern compared to the control group, while the recovery group had a similar miRNA expression pattern to the active group. In taxonomic and miRNA analyses, 147 differential abundant bacterial ASVs and 30 differential expressed miRNAs were identified in the active group compared to the recovery group. Spearman correlation and network analysis between gut microbiota and miRNAs, which showed significant differences between the active and the recovery groups, revealed significant associations between the genera Akkermansia and Phocaeicola and miR-34c-3p, miR-483-5p, miR-200b-3P, and miR-378a-3p. Conclusion In the active phase of chronic colitis, the gut microbial composition and miRNA expression were significantly altered, while in the recovery phase of chronic colitis, the gut microbial profile was similar to that of control, but a significantly different miRNA expression pattern was observed. Specific miRNAs were associated with specific bacterial ASVs, suggesting the interactions between miRNAs and gut microbiota in the development of chronic colitis. Our data suggested significant correlations between the genera Akkermansia and Phocaeicola and miR-34c-3p, miR-483-5p, miR-200b-3P, and miR-378a-3p in the active phase of chronic colitis.

P1335 Dietary Carnosic Acid Eases Colitis by Activating NRF2 Signal via Regulating Gut Microbiota and Metabolites

Abstract Background Ulcerative colitis (UC) is a chronic intestinal inflammatory disease, which is closely related to the imbalance of intestinal microbiota and metabolic alterations. We have studied the role of dietary carnosic acid (CA) in alleviating intestinal inflammation through regulating the intestinal microbiota and its metabolites. Methods Mice were fed with a diet supplemented with CA or a normal diet, and the enteritis model was established using dextran sulfate sodium (DSS). The feces of mice fed with different diets were transplanted into germ-free mice to determine the direct role of the CA-regulated microbiota in alleviating colitis. The intestinal microbiota was analyzed by 16S rRNA sequencing, serum metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS) metabolomics, and downstream targets were analyzed by RNA sequencing. Results Through the analysis of the microbiota based on the 16S rRNA gene, we found for the first time that the addition of CA to the diet improved the imbalance of the intestinal microbiota induced by DSS. The protective mechanism of CA against DSS-induced colitis is to improve the function of the intestinal mucosal barrier by activating the Nrf2 signaling pathway. It is worth noting that the addition of CA to the diet can enrich the microbe-derived 3-indolepropionic acid, thereby activating the Nrf2 signaling in human colonic epithelial cells. In addition, exogenous 3-indolepropionic acid treatment has a similar protective effect on DSS-induced colitis by improving the Nrf2 signaling-dependent mucosal barrier function. Conclusion In conclusion, our research results indicate that the intestinal microbiota - 3-indolepropionic acid - Nrf2 axis plays an important role in maintaining intestinal homeostasis, which may represent a new method for preventing colitis by manipulating the metabolites of the intestinal microbiota.

Role of the gut microbiota in cefoperazone/sulbactam-induced epilepsy in mice with chronic renal failure

Objectives The mechanism of cefoperazone/sulbactam-induced epilepsy in chronic kidney disease (CKD) patients is not yet clear. We hypothesized that cefoperazone/sulbactam-induced epilepsy could be based on two main factors: neurotoxicity caused by drug accumulation after renal failure and an abnormal gut microbiota (GM). Methods A chronic renal failure (CRF) model in mice was established, and then different doses of cefoperazone/sulbactam were injected to induce epilepsy in mice. Normal mouse feces for fecal microbiota transplantation (FMT) were collected. We observed the changes in feces, mental state, and activity of each group of mice. After killing, we collected kidneys and colon for H&E staining. We collected mouse feces for the 16S RNA sequencing of bacteria. Results All CRF mice injected with different concentrations of cefoperazone/sulbactam experienced grade-V seizures and eventually died, whereas normal control mice did not. However, after FMT intervention, the time of epilepsy onset and death in mice was delayed. Early FMT intervention resulted in more mice surviving (p = .0359). Moreover, the villi in the mucosal of group-CS layer fell off, goblet cells missed, and crypts disappeared. The mucosal layer and submucosa were clearly separated. The morphology of intestinal tissue of the CFS and FS group was improved. After FMT, the changes of the GM were observed. Conclusions The GM may be involved in the epilepsy induced by cefoperazone/sulbactam in CRF mice. FMT can delay the onset of epilepsy in CRF mice induced by cefoperazone/sulbactam, and the earlier the intervention, the better the effect.

Effects of feces storage conditions for host-microbiota screenings in C. elegans

Background and aimsCurrent research on host-gut microbiota interactions is hindered by almost infinite bacterial combinations depending on intrinsic characteristics, environment, and health status, which prevents large-scale screenings in mammals. For these reasons, the bacterivore model organism C. elegans has been developed to test the effects of gut microbiota extracts from mammals. This study tested whether storage conditions of mouse feces and fecal extracts modify normal C. elegans healthspan.MethodsFeces from mice were processed for microbiota extraction after collection or after one or twelve months at -80 °C and compared to microbiota extracted six months before and left at room temperature. Extracts were probed for bacterial composition, viability, and nutritional content and tested in synchronized wild-type (strain N2) worms for food preferences and intake, development, fat accumulation, brood size, and maximal lifespan.ResultsLong-term freezing of feces before microbiota extraction modified composition but did not negatively impact subsequent worm development, fat accumulation, reproduction, and maximal lifespan, whereas using samples extracted and left at room temperature after a long period of time resulted in robust avoidance and was detrimental for normal growth.ConclusionsUsing frozen feces to test for impacts of microbiota in C. elegans appears an appropriate method since it did not affect normal biology and healthspan, which supports protocols with already existing feces stored in biobanks for high-throughput phenotype screenings.

Lactobacillus reuteri-Enriched Eicosatrienoic Acid Regulates Glucose Homeostasis by Promoting GLP-1 Secretion to Protect Intestinal Barrier Integrity.

Lactobacillus reuteri is a well-known probiotic with beneficial effects, such as anti-insulin resistance, anti-inflammatory, and improvement of the intestinal barrier. However, the underlying mechanisms remain unclear. Here, we found that gavage of L. reuteri improved the intestinal barrier and glucose homeostasis in HFD-fed mice. Analysis of lipid metabolomics reveals a significant increase in eicosatrienoic acid (ETA) levels in mouse feces after L. reuteri gavage. We found that ETA maintain intestinal barrier integrity and improve glucose homeostasis by promoting GLP-1 secretion. Mechanistically, by using CD36 inhibitor in vivo and CD36 knockdown STC-1 cells in vitro, we elucidate that ETA activates intestinal CD36-activated PLC/IP3R/Ca2+ signaling to promote GLP-1 secretion. In vivo administration of GLP-1R inhibitor and in vitro intestinal organoid experiments demonstrate that GLP-1 upregulates the PI3K/AKT/HIF-1α pathway by GLP-1R and increases intestinal tight junction protein expressions, which in turn enhance the intestinal barrier integrity, reduce serum LPS level, attenuate inflammation in white adipose tissue (WAT), and ultimately improve glucose homeostasis in HFD and db/db mice. Our study elucidates for the first time the mechanism by which L. reuteri and its enriched metabolite ETA inhibit WAT inflammation by ameliorating the intestinal barrier, ultimately improving glucose homeostasis, and provides a new treatment strategy for T2D.

Polysaccharides from fermented garlic attenuate high-fat diet-induced obesity in mice through gut microbes.

The weight loss and lipid-lowering effects of fermented garlic polysaccharides (BGP) in obese mice were analyzed by detecting the intestinal flora and short-chain fatty acids. An obesity model was established by feeding mice a high-fat diet (HFD) for 8 weeks. After euthanasia, biochemical index testing and hematoxylin and eosin staining were performed. Spearman analysis was used to assess the relationship between the 16S rRNA sequencing results and the fatty acid content in mouse feces. Compared with the obese model mice, the BGP group had significantly reduced body weight and serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, malondialdehyde, and free fatty acids in the serum. Moreover, BGP reversed HFD-induced gut microbiota dysbiosis, as indicated by the elevated populations of Paraclostridium, Lachnospiraceae_UCG_006, Enterorhabdus, and Lachnospiraceae-NK4A136. BGP also significantly increased the contents of acetic, propionic, butyric, and valeric acids. These results indicate that BGP may serve as a potential prebiotic agent that modulates particular bacteria in the gut and their byproducts that play a crucial role in preventing diseases associated with obesity.

Water-insoluble dietary fiber from walnut relieves constipation through Limosilactobacillus reuteri-mediated serotonergic synapse and neuroactive ligand-receptor pathways.

Dietary fiber can alleviate functional constipation (FC) by modulating the gut microbiota. To clarify the prebiotic properties of walnut insoluble dietary fiber (WIDF), we explored its structural characteristics and laxative mechanism. A galacturonic acid and glucose-rich WIDF was isolated from walnuts by using a complex enzymatic method. Animal experiments results showed that WIDF could effectively alleviate the symptoms of loperamide-induced FC in mice, including shortening the defecation time, increasing the wet weight and water content of feces, and promoting intestinal motility. WIDF might alleviate FC through activating serotonergic synapse and inhibiting the delta-opioid receptor/inducible nitric oxide synthase (Oprd/iNOS) pathways. Importantly, WIDF treatment altered the structure and composition of the gut microbiota. Correlation analysis revealed that Bacillus and its dominant ASV17, which is considered to be the key microbe for constipation alleviation, were strongly associated with constipation phenotypes. Based on pure culture and 16S rRNA gene phylogenetic analysis, Limosilactobacillus reuteri (L. reuteri), which is 100% similar to ASV17, was isolated and identified from the feces of WIDF-treated mice. L. reuteri relieved FC by modulating serotonergic synapse and the Oprd/iNOS pathways. These results suggested that WIDF and L. reuteri treatment is a prospective strategy for the prevention of constipation.

Prevotellaceae Modulates Colorectal Cancer Immune Microenvironment to Assist Anti-PD-L1 Immunotherapy.

Colorectal cancer (CRC) stands as the third most prevalent cancer on a global scale. In recent years, immunotherapy, such as anti-PD-L1 treatment, has demonstrated promising therapeutic outcomes in CRC. However, studies have suggested that intestinal microbiota may influence the efficacy of anti-PD-L1 immunotherapy. This study aimed to investigate the linkage between intestinal bacteria and anti-PD-L1 therapy. Bioinformatics analysis was employed to study the correlation between the intestinal microbiota of CRC patients and immune infiltration. The study delved into the relationship between Prevotellaceae and immune-related genes in CRC. Mouse experiments were conducted to validate the association between Prevotellaceae abundance and the efficacy of anti-PD-L1 tumor treatment. Prevotellaceae abundance in mouse feces was assayed by 16S sequencing. Flow cytometry was utilized to assay immune cell infiltration in patient tumor tissues, while western blot and quantitative polymerase chain reaction (qPCR) assays measured IFN-γ, IL-2, and PD-L1 levels in tumor tissues. The high immune cell infiltration group demonstrated reduced tumor purity when compared with the group displaying low immune cell infiltration. Substantial variances were discerned in the Stromal Score, Immune Score, ESTIMATE Score, and Tumor Purity among the 3 distinct subtypes. The community evenness in the gut microbiota of CRC patients from cluster 2 and cluster 3 subtypes displayed significant differences. Members of the Prevotellaceae family were significantly enriched in the gut microbiota of cluster 3 subtype patients. In vivo experiments ascertained the supportive role of Prevotellaceae in anti-PD-L1 immunotherapy. The facilitating effect of Prevotellaceae on anti-PD-L1 treatment was demonstrated in CRC. The findings suggest that elevating Prevotellaceae abundance may offer a new direction for assisting in CRC immunotherapy and provide a foundation for devising more effective CRC immunotherapeutic strategies.

Rifaximin alleviates irinotecan-induced diarrhea in mice model

Background Irinotecan is a chemotherapeutic drug widely used to treat solid tumors. However, its effectiveness is limited by the severely delayed onset of diarrhea. This study aimed to confirm the protective effects of the non-systemic oral antibiotic rifaximin on irinotecan-induced mucositis in mice model. Materials and methods Six to eight week-old BALB/c mice were treated with saline, irinotecan (50 mg/kg, i.p. once daily), rifaximin (50 mg/kg, p.o. twice daily), or irinotecan + rifaximin for 9 consecutive days. Signs of diarrhea, bloody diarrhea, and body weight were monitored daily. Intestinal tissues were harvested for histopathological analysis and quantitative PCR. SN38 and SN38G concentration in intestine were detected using LC-MS analysis. Intestinal bacteria β-glucuronidase (BGUS) activity was detected using mouse feces. We performed 16S rRNA sequencing to investigate the gut microbiota composition. Gut permeability was tested in vivo by measuring the fluorescein isothiocyanate-dextran intensity in the serum. Results Rifaximin reduced the frequency of delayed diarrhea and attenuated the severity of diarrhea caused by irinotecan in mice. Rifaximin significantly inhibited SN38 exposure in intestine and irinotecan-induced increase in BGUS activity. Rifaximin alleviated intestinal mucosal inflammation, prevented intestinal epithelial damage caused by irinotecan, and maintained gut barrier function. Moreover, the consecutive use of rifaximin did not cause a disorder in gut microbiota and reduced irinotecan-induced Firmicutes expansion. More importantly, rifaximin inhibited the expansion of some microbiota (such as Blautia, Eggerthella, and f_Enterobacteriaceae) and promoted an increase in beneficial microbiota (such as Lactobacillus intestinalis, Lachnospiraceae NK4A136 group, and f_Oscillospiraceae). Conclusions Preventive use of rifaximin is a feasible method to protect against irinotecan-induced diarrhea.

Cholesterol-Lowering Activity of Lactiplantibacillus pentosus KS6I1 in High-Cholesterol Diet-Induced Hypercholesterolemic Mice.

Hypercholesterolemia is a risk factor of coronary heart disease and cholesterol-lowering probiotics are seen as alternative to drugs for the management of this condition. In the present study, we evaluated the cholesterol-lowering activity of Lactiplantibacillus pentosus KS6I1 in high-cholesterol diet-induced hypercholesterolemic mice. The mice were fed with high-cholesterol diet (HCD) and were divided into three groups: HCD group, KS6I1 group (fed with HCD + 200 μl of 1010 CFU/ml L. pentosus KS6I1), and L.ac group (fed with HCD + 200 μl of 1010 CFU/ml L. acidophilus ATCC 43121 as the positive control). Simultaneously, a normal control diet (NCD) group was maintained. After 6 weeks, the low-density lipoprotein (LDL)-cholesterol and total cholesterol levels were significantly reduced in the blood plasma of KS6I1 group mice. Analysis of liver tissue showed a decrease in total cholesterol and LDL-cholesterol and increase in triglyceride levels in KS6I1 compared to those in HCD group. Fecal total cholesterol and total bile acid content was significantly increased in the KS6I1 group than in other groups. Additionally, gene expression analysis showed that LDLR, SREBF2, CYP7A1 genes were significantly upregulated in KS6I1 group compared to the HCD group, while the expression of NPC1L1 gene was significantly reduced in KS6I1 group compared to HCD group. These observations show that the cholesterol-lowering effect of L. pentosus KS6I1 could be attributed to increased excretion of bile acids and cholesterol in the feces of mice. These results indicate that L. pentosus KS6I1 could be developed into a potential probiotic for hypercholesterolemia.

Bacillus velezensis HBXN2020 alleviates Salmonella Typhimurium infection in mice by improving intestinal barrier integrity and reducing inflammation.

Bacillus velezensis is a species of Bacillus that has been widely investigated because of its broad-spectrum antimicrobial activity. However, most studies on B. velezensis have focused on the biocontrol of plant diseases, with few reports on antagonizing Salmonella Typhimurium infections. In this investigation, it was discovered that B. velezensis HBXN2020, which was isolated from healthy black pigs, possessed strong anti-stress and broad-spectrum antibacterial activity. Importantly, B. velezensis HBXN2020 did not cause any adverse side effects in mice when administered at various doses (1×107, 1×108, and 1×109 CFU) for 14 days. Supplementing B. velezensis HBXN2020 spores, either as a curative or preventive measure, dramatically reduced the levels of S. Typhimurium ATCC14028 in the mice's feces, ileum, cecum, and colon, as well as the disease activity index (DAI), in a model of infection caused by this pathogen in mice. Additionally, supplementing B. velezensis HBXN2020 spores significantly regulated cytokine levels (Tnfa, Il1b, Il6, and Il10) and maintained the expression of tight junction proteins and mucin protein. Most importantly, adding B. velezensis HBXN2020 spores to the colonic microbiota improved its stability and increased the amount of beneficial bacteria (Lactobacillus and Akkermansia). All together, B. velezensis HBXN2020 can improve intestinal microbiota stability and barrier integrity and reduce inflammation to help treat infection by S. Typhimurium.

Preparation and characterization of Tobacco polysaccharides and its modulation on hyperlipidemia in high-fat-diet-induced mice.

This study aimed to investigate the structural properties of tobacco polysaccharide (TP) and its mechanism of modulating hyperlipidemia in high-fat diet-induced mice. The structural properties of TP were characterized by FT-IR, 1HNMR, SEM, AFM and thermogravimetric analysis. And the regulatory mechanism of TP on lipid metabolism was investigated in hyperlipidemia mice. These results showed that TP had a high composition of reducing monosaccharide and the glycosidic bond type was α-glycosidic bond. The intervention by TP resulted in a significant reduction of body weight and improvement in lipid accumulation. And the modulation mechanism by which TP ameliorated the abnormalities of lipid metabolism was associated with the expression levels of lipid metabolism-related genes and serum exosomes miRNA-128-3p, as well as the modulation of structure and abundance of the gut microbiota in mice. In addition, TP treatment significantly increased the content of short-chain fatty acids (SCFAs) in mice feces. The results of molecular docking and dual-luciferase assay exhibited a good interaction between propionic acid and PPAR-α, and it was hypothesized that the interaction might further ameliorate the hyperlipidemia. Therefore, TP can regulate the expression levels of lipid metabolism-related genes through miRNAs from serum exosomes and SCFAs from gut microbiota.

Cross-Kingdom Pathogenesis of Pantoea alfalfae CQ10: Insights from Transcriptome and Proteome Analyses.

In grassland agroecosystems, some plant pathogenic bacteria can cause disease in animals. These strains are known as plant and animal cross-kingdom pathogenic bacteria. In this study, we established an alfalfa root infection model and a mouse model via the gavage administration of the Pantoea alfalfae CQ10 (CQ10) bacterial suspension. It was confirmed that the CQ10 strain caused bacterial leaf blight of alfalfa. Mice inoculated with 0.4 mL of 109 cfu/mL bacterial suspension developed clinical symptoms 48 h later, such as diminished vitality, tendencies to huddle, and lack of appetite, including severe lesions in stomach, liver, kidney, and spleen tissues. CQ10 strains were isolated from mouse feces at different time points of inoculation. Thus, CQ10 is a plant and animal cross-kingdom pathogenic bacterium. Transcriptome and proteome analyses showed that biofilm and iron uptake are important virulence factors of the pathogen CQ10, among which Bap and Lpp regulating biofilm are the key cross-kingdom virulence genes of CQ10. From an evolutionary perspective, insights gained from this dual animal-plant pathogen system may help to elucidate the molecular basis underlying the host specificity of bacterial pathogens. The result provides a theoretical basis for the risk assessment, prevention, and control strategies of new pathogenic bacteria entering a new region.

Milk-derived extracellular vehicles alleviate heart failure with preserved ejection fraction by modulating gut microbiota

Abstract Background Accounting for around 50% of human heart failure, heart failure with preserved ejection fraction (HFpEF) is driven by complex and heterogeneous pathological factors, including gut microbiome dysbiosis. We have previously reported that milk-derived extracellular vesicles (mEVs) modulate the gut microbiota in inflammatory bowel disease. However, whether mEVs can affect gut microbiota and thereby alleviate HFpEF remains unexplored. Aims We aim to uncover potential therapeutic effects of mEVs on HFpEF and if so, the underlying mechanisms. Methods The HFpEF mouse model was generated by feeding C57/B6 mice on a high-fat diet and L-NAME for 15 weeks. Mouse feces samples were analyzed to detect the presence of gut dysbiosis in HFpEF. Mice received bovine mEVs through oral gavage. Heart function was assessed with echocardiography. Mouse feces and plasma were collected to analyze gut microbiota and metabolomics, respectively. Results Gut microbiota was disturbed in HFpEF mice. Oral administration of mEVs effectively attenuated heart failure which was evidenced by less bodyweight gain, reduction in cardiac hypertrophy and lung weight, and improvement in cardiac function parameters such as ratios of E/A (1.8 vs. 1.5, p<0.05) and E/E’ (40 vs. 27, p<0.05). Moreover, the increase in mouse blood pressure in HFpEF was also inhibited upon treatment (SBP 130mmHg vs. 100mmHg, DBP 90mmHg vs. 75mmHg, p<0.05). Mechanistically, we ruled out the possibility of direct action of mEVs on the heart as orally administered mEVs were only distributed in the intestine. On the other hand, mEVs restored gut microbiota by increasing the abundance of beneficial bacteria such as Lachnospiraceae. As a result, oral administration of mEVs elevated blood levels of Lachnospiraceae metabolite, butyric acid, a type of short-chain fatty acid. To confirm the butyric acid-mediated heart-protective effects of mEVs, we administered butyric acid to HFpEF mice. Strikingly, butyric acid largely reproduced the therapeutic effects of mEVs on HFpEF. Conclusions Oral administration of mEVs restores gut microbiota dysbiosis, thereby attenuating heart failure in HFpEF. mEVs may represent a new biological therapeutic for the treatment of HFpEF.

Aspirin inhibits atherosclerosis through macrophage pyroptosis by interacting with Lactobacillus johnsonii and its metabolite butyrate

Abstract Introduction Atherosclerotic cardiovascular disease (ASCVD) is a chronic progressive disease closely related to metabolism and inflammation. Aspirin inhibits cyclooxygenase (COX) and has antiplatelet aggregation effects, considered as the cornerstone of ASCVD treatment. Numerous studies have shown that gut microbiota (GM) and its metabolites affects host metabolism. Studies have also shown that people taking aspirin exhibit specific changes in the microbiota profile. However, whether the interaction of aspirin with GM and its metabolites affects the therapeutic efficacy of aspirin in treating atherosclerosis (AS) have not been reported. Purpose This study aimed to explore the interaction of aspirin with the GM and its metabolites in the treatment of AS, to reveal the specific mechanism of the GM-metabolite axis and provide potential new targets for the combined treatment of AS. Methods To establish the mouse model of atherosclerosis by high-fat diet, and the atherosclerotic plaque burden and composition were analyzed. The role of GM in aspirin anti-atherosclerosis was explored by antibiotics and fecal microbiota transplantation (FMT). The third generation 16S RNA sequencing was used to explore the effects of aspirin on species diversity and abundance of GM, and to screen differential microorganisms for intervention of AS mice. UHPLC-MS and GC-MS were used to explore the changes of intestinal metabolites induced by aspirin. The correlation between gut microbiota and metabolites was also investigated. Finally, in vitro and in vivo experiments were performed to confirm the effects and mechanisms of metabolites on macrophage pyroptosis and atherosclerosis. Results Aspirin has anti-atherosclerosis effect. After removing gut microbiota, the therapeutic effect of aspirin on atherosclerosis was decreased. Three generations of 16S rRNA gene sequencing results showed that aspirin supplementation alters the diversity of gut microbiota, and increased the relative abundance of Lactobacillus johnsonii(L.johnsonii) and Ligilactobacillus murinus(L.murinus). After gavage of L.johnsonii and L.murinus, the plaque burden and macrophage pyroptosis were significantly reduced in AS mice. Colonization with L.johnsonii and L.murinus significantly increased the abundance of probiotics and butyrate level in the feces of AS mice and L.johnsonii and L.murinus were positively correlated with butyrate. Supplementation of butyrate inhibited macrophage pyroptosis and atherosclerosis in vivo. In vitro, butyrate could inhibit the expression of NLRP3/Caspase-1/IL-1β and pyroptosome production, and this process mainly by activating butyrate receptors GPR41, GPR43 and GPR109A. Conclusions The gut microbiota may mediate the anti-AS effect of aspirin by promoting an increase in the abundance of L.johnsonii and L.murinus. Butyrate produced by Lactobacillus may mediate the anti-AS effect of aspirin, and inhibiting macrophage pyroptosis and the progression of AS.

Preventive effects of matrine on LPS-induced inflammation in RAW 264.7 cells and intestinal damage in mice through the TLR4/NF-κB/MAPK pathway

BackgroundMatrine is a tetracyclic quinolizidine alkaloid with diverse bioactive properties, including anti-inflammatory and neuroprotective properties. However, the underlying anti-inflammatory mechanisms remain unclear. PurposeThis study aimed to explore how matrine reduces Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and to assess its protective effects against LPS-induced intestinal damage. MethodsThe effect of matrine on cell viability was assessed using the cell counting kit-8 (CCK-8) assay. Additionally, its impact on inflammatory cytokines and macrophage polarization was assessed using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The effects on intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), nitric oxide (NO) production, and oxidative stress were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate staining and JC-1 and Griess assays. Immunofluorescence staining was used to observe the translocation of the NF-κB p65 subunit. Western blotting (WB) and qRT-PCR were employed to analyze the expression levels of proteins related to the toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. An LPS-induced mouse model was established to study the intestinal inflammation and barrier injury. Mouse feces characteristics, colon length, and disease activity index (DAI) were recorded. Hematoxylin-eosin (H&E) and alcian blue/periodic acid schiff (AB/PAS) staining were used to observe morphological changes and barrier damage in the duodenum, jejunum, ileum, and colon and to measure villus length, crypt depth, goblet cell count, and positive areas in the duodenum, jejunum, and ileum. The content of short-chain fatty acids (SCFAs) in the colon was determined using gas chromatography (GC). ResultsMatrine inhibited LPS-induced inflammatory cytokine levels, suppressed macrophage M1 polarization, and promoted M2 macrophage polarization. Matrine reduced LPS-induced increases in ROS and NO levels and regulates oxidative stress. Additionally, matrine inhibited the nuclear translocation of the NF-κB p65 subunit and exerted anti-inflammatory effects by suppressing the activation of the TLR4/NF-κB/MAPK pathway. In vivo experiments indicated that matrine significantly alleviated LPS-induced diarrhea, increased DAI, and shortened the colon. Matrine reduced the production of the pro-inflammatory cytokine interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α and the pro-inflammatory mediator NO in mouse intestinal tissues while promoting the content of the anti-inflammatory cytokine IL-10. Furthermore, it improved intestinal tissue structure and alleviated LPS-induced intestinal barrier damage. Finally, matrine increased the SCFA levels in the intestine. ConclusionMatrine exerted its anti-inflammatory effects and protects against intestinal injury through the TLR4/NF-κB/MAPK signaling pathway.

EphA2 blockage ALW-II-41-27 alleviates atherosclerosis by remodeling gut microbiota to regulate bile acid metabolism.

Coronary artery disease (CAD), a critical condition resulting from systemic inflammation, metabolic dysfunction, and gut microbiota dysbiosis, poses a global public health challenge. ALW-II-41-27, a specific inhibitor of the EphA2 receptor, has shown anti-inflammatory prosperities. However, the impact of ALW-II-41-27 on atherosclerosis has not been elucidated. This study aimed to examine the roles of pharmacologically inhibiting EphA2 and the underlying mechanism in ameliorating atherosclerosis. ALW-II-41-27 was administered to apoE-/- mice fed a high-fat diet via intraperitoneal injection. We first discovered that ALW-II-41-27 led to a significant reduction in atherosclerotic plaques, evidenced by reduced lipid and macrophage accumulation, alongside an increase in collagen and smooth muscle cell content. ALW-II-41-27 also significantly lowered plasma and hepatic cholesterol levels, as well as the colonic inflammation. Furthermore, gut microbiota was analyzed by metagenomics and plasma metabolites by untargeted metabolomics. ALW-II-41-27-treated mice enriched Enterococcus, Akkermansia, Eggerthella and Lactobaccilus, accompanied by enhanced secondary bile acids production. To explore the causal link between ALW-II-41-27-associated gut microbiota and atherosclerosis, fecal microbiota transplantation was employed. Mice that received ALW-II-41-27-treated mouse feces exhibited the attenuated atherosclerotic plaque. In clinical, lower plasma DCA and HDCA levels were determined in CAD patients using quantitative metabolomics and exhibited a negative correlation with higher monocytes EphA2 expression. Our findings underscore the potential of ALW-II-41-27 as a novel therapeutic agent for atherosclerosis, highlighting its capacity to modulate gut microbiota composition and bile acid metabolism, thereby offering a promising avenue for CAD.

Asiaticoside improves depressive-like behavior in mice with chronic unpredictable mild stress through modulation of the gut microbiota.

Asiaticoside, the main active ingredient of Centella asiatica, is a pentacyclic triterpenoid compound. Previous studies have suggested that asiaticoside possesses neuroprotective and anti-depressive properties, however, the mechanism of its anti-depressant action not fully understood. In recent years, a growing body of research on anti-depressants has focused on the microbiota-gut-brain axis, we noted that disruption of the gut microbial community structure and diversity can induce or exacerbate depression, which plays a key role in the regulation of depression. Behavioral experiments were conducted to detect depression-like behavior in mice through sucrose preference, forced swimming, and open field tests. Additionally, gut microbial composition and short-chain fatty acid (SCFA) levels in mouse feces were analyzed 16S rRNA sequencing and gas chromatography-mass spectrometry (GC-MS). Hippocampal brain-derived neurotrophic factor (BDNF) and 5-hydroxytryptamine receptor 1A (5-HT1A) expression in mice was assessed by western blotting. Changes in serum levels of inflammatory factors, neurotransmitters, and hormones were measured in mice using ELISA. This study revealed that oral administration of asiaticoside significantly improved depression-like behavior in chronic unpredictable mild stress (CUMS) mice. It partially restored the gut microbial community structure in CUMS mice, altered SCFA metabolism, regulated the hypothalamic-pituitary-adrenal axis (HPA axis) and inflammatory factor levels, upregulated BDNF and 5-HT1A receptor protein expression, and increased serum serotonin (5-hydroxytryptamine, 5-HT) concentration. These findings reveal that asiaticoside exerts antidepressant effects via the microbiota-gut-brain axis. These results suggested that asiaticoside exerts antidepressant effects through the microbiota-gut-brain axis in a CUMS mouse model.