Fluoride exposure-induced gut microbiota alteration mediates colonic ferroptosis through N6-methyladenosine (m6A) mediated silencing of SLC7A11.

Fluoride exposure is widespread worldwide and poses a significant threat to organisms, particularly to their gastrointestinal tracts. However, due to limited knowledge of the mechanism of fluoride induced intestinal injury, it has been challenging to develop an effective treatment. To address this issue, we used a series of molecular biology in vitro and in vivo experiments. NaF triggered m6A mediated ferroptosis to cause intestinal damage. Mechanistically, NaF exposure increased the m6A level of SLC7A11 mRNA, promoted YTHDF2 binding to m6A-modified SLC7A11 mRNA, drove the degradation of SLC7A11 mRNA, and led to a decrease in its protein expression, which eventually triggers ferroptosis. Moreover, NaF aggravated ferroptosis of the colon after antibiotics destroyed the composition of gut microbiota. 16 S rRNA sequencing and SPEC-OCCU plots, Zi-Pi relationships, and Spearman correlation coefficients verified that Lactobacillus murinus (ASV54, ASV58, and ASV82) plays a key role in the response to NaF-induced ferroptosis. Collectively, NaF-induced gut microbiota alteration mediates severe intestinal cell injury by inducing m6A modification-mediated ferroptosis. Our results highlight a key mechanism of the gut in response to NaF exposure and suggest a valuable theoretical basis for its prevention and treatment.

Disrupted gut microbiota aggravates spatial memory dysfunction induced by high altitude exposure: A link between plateau environment and microbiome-gut-brain axis.

Approximately 400 million people work and live in high-altitude areas and suffer from memory dysfunction worldwide. Until now, the role of the intestinal flora in plateau-induced brain damage has rarely been reported. To address this, we investigated the effect of intestinal flora on spatial memory impairment induced by high altitudes based on the microbiome-gut-brain axis theory. C57BL/6 mice were divided into three groups: control, high-altitude (HA), and high-altitude antibiotic treatment (HAA) group. The HA and HAA groups were exposed to a low-pressure oxygen chamber that simulated an altitude of 4000 m above sea level (m. a. s.l.) for 14 days, with the air pressure in the chamber set at 60-65 kPa. The results showed that spatial memory dysfunction induced by the high-altitude environment was aggravated by antibiotic treatment, manifesting as lowered escape latency and hippocampal memory-related proteins (BDNF and PSD-95). 16 S rRNA sequencing showed a remarkable separation of the ileal microbiota among the three groups. Antibiotic treatment exacerbated the reduced richness and diversity of the ileal microbiota in mice in the HA group. Lactobacillaceae were the main target bacteria and were significantly reduced in the HA group, which was exacerbated by antibiotic treatment. Meanwhile, reduced intestinal permeability and ileal immune function in mice exposed high-altitude environment was also aggravated by antibiotic treatment, as indicated by the lowered tight junction proteins and IL-1ß and IFN-γ levels. Furthermore, indicator species analysis and Netshift co-analysis revealed that Lactobacillaceae (ASV11) and Corynebacteriaceae (ASV78, ASV25, and ASV47) play important roles in high-altitude exposure-induced memory dysfunction. Interestingly, ASV78 was negatively correlated with IL-1ß and IFN-γ levels, indicating that ASV78 may be induced by reduced ileal immune function, which mediates high-altitude environment exposure-induced memory dysfunction. This study provides evidence that the intestinal flora is effective in preventing brain dysfunction caused by exposure to high-altitude environments, suggesting a relationship between the microbiome-gut-brain axis and altitude exposure.

Fluoride disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated RhoA/ROCK signaling and myosin light chain kinase.

Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca2+]i) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca2+]i, whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca2+-dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury.

Probiotic alleviate fluoride-induced memory impairment by reconstructing gut microbiota in mice.

Fluoride which is widespread in our environment and food due to its geological origin and industrial pollution has been identified as a developmental neurotoxicant. Gut-brain axis provides new insight into brain-derived injury. We previously found the psychoactive effects of a probiotic strain, Lactobacillus johnsonii BS15 against fluoride-induced memory dysfunction in mice by modulating the gut-brain axis. In this study, we aimed to detect the link between the reconstruction of gut microbiota and gut-brain axis through which probiotic alleviate fluoride-induced memory impairment. We also added an hour of water avoidance stress (WAS) before behavioral tests and sampling, aiming to demonstrate the preventive effects of the probiotic on fluoride-induced memory impairment after psychological stress. Mice were given fluoridated drinking water (sodium fluoride 100 ppm, corresponding to 37.8 ± 2.4 ppm F¯) for 70 days and administered with PBS or a probiotic strain, Lactobacillus johnsonii BS15 for 28 days prior to and throughout a 70 day exposure to sodium fluoride. Results showed that fluoride increases the hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis and reduces the exploration ratio in novel object recognition (NOR) test and the spontaneous exploration during the T-maze test in mice following WAS, which were significantly improved by the probiotic. 16S rRNA sequencing showed a significant separation in ileal microbiota between the fluoride-treated mice and control mice. Lactobacillus was the main targeting bacteria and significantly reduced in fluoride-treated mice. BS15 reconstructed the fluoride-post microbiota and increased the relative abundance of Lactobacillus. D-lactate content and diamine oxidase (DAO) activity, two biomarkers of gut permeability were reduced in the serum of probiotic-inoculated mice. ZO-1, an intestinal tight junction protein was reduced by fluoride in mRNA, and its protein levels were increased by the probiotic treatment. Moreover, the hippocampus which is essential to learning and memory, down-regulated mRNA level of both the myelin-associated glycoprotein (MAG), and protein levels of brain-derived neurotrophic factor (BDNF), including the improvement of cAMP response element-binding protein (CREB) by BS15 in fluoride-exposed mice after WAS. Via spearman correlation analysis, Lactobacillus displayed significantly positive associations with the behavioral tests, levels of nerve development related factors, and intestinal tight junction proteins ZO-1, and negative association with TNF-α of the hippocampus, highlighting regulatory effects of gut bacteria on memory potential and gut barrier. These results suggested the psychoactive effects of BS15 on fluoride-induced memory dysfunction after psychological stress. In addition, there may be some correlations between fluoride-induced memory dysfunction and reconstruction of gut microbiota. AVAILABILITY OF DATA AND MATERIALS: 16S rRNA sequencing reads have uploaded to NCBI. The accession code of 16S rRNA sequencing reads in the National Center for Biotechnology Information (NCBI) BioProject database: PRJNA660154.

Preventive effects of Lactobacillus johnsonii on the renal injury of mice induced by high fluoride exposure: Insights from colonic microbiota and co-occurrence network analysis.

Fluoride (F) exposure was widely reported to be associated with renal diseases. Since absorbed F enters the organism from drinking water mostly through the gastrointestinal tract, investigating changes of gut microbes may have profound implications for the prevention of chronic F exposure because increasing evidence supported the existence of the gut-kidney axis. In the present study, we aimed to explore the potential positive effects of probiotics on high F exposure-induced renal lesions and dysfunction in mice by the modulation of the colonic microbiota. Mice were fed with normal (Ctrl group) or sodium-fluoride (F and Prob groups; 100 mg/L sodium fluoride (NaF)) drinking water with or without Lactobaillus johnsonii BS15, a probiotic strain proven to be preventive for F exposure. Mice fed with sodium-fluoride drinking water alone exhibited renal tissue damages, decreased the renal antioxidant capability and dysfunction. In contrast, L. johnsonii BS15 reversed these F-induced renal changes. 16S rRNA gene sequencing shows that L. johnsonii BS15 alleviated the increased community diversity (Shannon diversity) and richness index (number of observed features) as well as the distured structure of colon microbiota in F-exposed mice. A total of 13 OTUs with increased relative abundance were identified as the keystone OTUs in F-exposed mice based on the analysis of degree of co-occurrence and abundance of OTUs. Moreover, Spearman's rank correlation shows that the 13 keystone OTUs had negative effect on renal health and intestinal integrity. L. johnsonii BS15 reversed four of keystone OTUs (OTU 5, OTU 794, OTU 1035, and OTU 868) changes which might be related to the underlying protected mechanism of L. johnsonii BS15 against F-induced renal damages.

Transcriptome analysis revealed ameliorative effect of probiotic Lactobacillus johnsonii BS15 against subclinical necrotic enteritis induced hepatic inflammation in broilers.

Subclinical necrotic enteritis (SNE) broadly occurs in boilers, which reduces the growth performance by causing serious economic and social problems. The following study was conducted to better understand the molecular mechanism of the SNE on liver inflammation and to examine the innovative prevention of Lactobacillus johnsonii BS15 upon SNE. The research was based on the regulatory molecular mechanism of Lactobacillus johnsonii BS15, and its effect on liver inflammatory pathways in the broiler with SNE infection. Day old one hundred and eighty (Cobb 500) broiler chickens were distributed into 3 groups (control, SNE and BS15 group) and reared for 28 days. RNA sequencing was used for the analysis of gene expression extracted from liver samples. Gene expression was detected with the help of quantitative real-time PCR (qRT-PCR). RNA-Seq analysis revealed altered expressions of genes involved in liver inflammatory pathway. A total number of 385 genes were found as differentially expressed (DEGs) in the liver samples that belonged to SNE group as compared with the control liver samples (p < 0.05). Out of those 385 genes, 117 were down-regulated and 268 were up-regulated. The DEGs related to liver inflammation between control group and SNE group or SNE and BS15 groups, included cluster of differentiation 80 (CD80), Interleukin 1 beta (IL1B), Phosphoinositide 3- Kinase regulatory subunit 5 (PIK3R5), Toll-like receptor 4 (TLR4), Toll-like receptor 2 A (TLR2A), and proto-oncogene protein (FOS). The RNA-Seq analysis provided DEGs expression and this result was validated by qRT-PCR. Results confirmed that these genes are essential in the regulation of liver inflammation in the SNE infected chickens. Findings of current research indicated that the hepatic inflammation could be induced by SNE in broilers. Simultaneously, effects of SNE infection on liver could be subsided by improved TLRs signaling pathway with the naturally present prophylactic strategy as BS15.

Preventing non-alcoholic fatty liver disease through Lactobacillus johnsonii BS15 by attenuating inflammation and mitochondrial injury and improving gut environment in obese mice.

The increasing prevalence of obesity worldwide is associated with a parallel increase in non-alcoholic fatty liver disease (NAFLD). To investigate the effect of Lactobacillus johnsonii BS15 on NAFLD, 120 male ICR mice were randomly divided into four groups and administrated with BS15 (2 × 10(7) cfu/0.2 mL or 2 × 10(8) cfu/0.2 mL) or phosphate buffered saline (PBS) throughout a 17-week experimental period. The mice were fed with normal chow diet (NCD) 5 weeks before the experimental period. Afterward, with the exception of the PBS group, NCD was changed into high-fat diet (HFD) for the remaining experimental period. Results showed that BS15-treated HFD mice were protected from hepatic steatosis and hepatocyte apoptosis. BS15 exhibited a positive effect on liver lipid peroxidation through an anti-oxidative stress activity by enhancing the liver antioxidant defense system. In addition, BS15 inhibited the insulin resistance; decreased the mRNA levels of acetyl-CoA carboxylase 1, fatty acid synthase, and peroxisome proliferator-activated receptor γ; and increased the expression of the fasting-induced adipose factor in livers. Meanwhile, BS15 attenuated mitochondria abnormalities when the content of uncoupling protein-2 decreased and cytochrome c increased in NAFLD mice. BS15 also reduced the level of serum lipopolysaccharide in NAFLD mice by lowering the intestinal permeability and adjusting gut flora, followed by the downregulation of the TNFα mRNA level in liver and the serum level of C-reactive protein. These findings suggest that BS15 may be effective in preventing NAFLD induced by HFD.

Effects of Lactobacillus plantarum on production performance, immune characteristics, antioxidant status, and intestinal microflora of bursin-immunized broilers.

Examples of probiotics that can promote host health by improving its intestinal microbial balance and intestinal immunity belong to the genus Lactobacillus. Bursin (BS) is a peptide isolated from the bursa of Fabricius for use as an adjuvant for a variety of immunogens. To investigate the synergistic effects of Lactobacillus plantarum (LP) dietary supplementation and BS immunization on production performance, immune characteristics, antioxidant status, and intestinal microflora in broilers, we randomly allocated 200 1-day-old broilers of mixed sex into 4 treatments in a 2 × 2 factorial arrangement (LP-/BS-, LP-/BS+, LP+/BS-, LP+/BS+) for 42 days. BS immunization enhanced immune response by increasing serum total immunoglobulin G concentration and interleukin-6 concentration, promoted antioxidant capacity by increasing catalase activities in serum and liver and by decreasing serum malondialdehyde (MDA) content at 42 days of age (DOA), and enriched intestinal microflora diversity. LP supplementation enhanced immune response by increasing interleukin-2 concentration at 42 DOA; promoted antioxidant capacity by increasing liver catalase activities, increasing glutathione peroxidase activities in serum and liver at 21 DOA, and decreasing serum MDA content at 42 DOA; promoted intestinal microflora composition by decreasing total aerobes and Escherichia coli counts at 21 DOA, by increasing total anaerobes count at 21 DOA, and by increasing Lactobacillus spp. and Bifidobacterium spp. counts at both 21 and 42 DOA. The interactions between BS and LP had a significant effect on daily body mass gain and feed conversion ratio in the starter period (1-21 DOA); on interleukin-2 concentration and liver MDA content at 21 DOA; and on thymus index, peripheral lymphocyte proliferation, and E. coli counts at 42 DOA. Overall, these data suggest that the combination of LP dietary supplementation and BS immunization promoted the production performance, immune characteristics, antioxidant status, and intestinal microflora of broilers.

Role of disulfidptosis in colorectal adenocarcinoma: implications for prognosis and immunity.

Background: Recent research has found a new way of cell death: disulfidptosis. Under glucose starvation, abnormal accumulation of disulfide molecules such as Cystine in Solute Carrier Family 7 Member 11 (SLC7A11) overexpression cells induced disulfide stress to trigger cell death. The research on disulfidptosis is still in its early stages, and its role in the occurrence and development of colorectal malignancies is still unclear. Method: In this study, we employed bioinformatics methods to analyze the expression and mutation characteristics of disulfidptosis-related genes (DRGs) in colorectal cancer. Consensus clustering analysis was used to identify molecular subtypes of Colorectal Adenocarcinoma (COAD) associated with disulfidptosis. The biological behaviors between subtypes were analyzed to explore the impact of disulfidptosis on the tumor microenvironment. Constructing and validating a prognostic risk model for COAD using diverse data. The influence of key genes on prognosis was evaluated through SHapley Additive exPlanations (SHAP) analysis, and the predictive capability of the model was assessed using Overall Survival analysis, Area Under Curve and risk curves. The immunological status of different patients and the prediction of drug treatment response were determined through immune cell infiltration, TMB, MSI status, and drug sensitivity analysis. Single-cell analysis was employed to explore the expression of genes at the cellular level, and finally validated the expression of key genes in clinical samples. Result: By integrating the public data from two platforms, we identified 2 colorectal cancer subtypes related to DRGs. Ultimately, we established a prognosis risk model for COAD using 7 genes (FABA4+GIPC2+EGR3+HOXC6+CCL11+CXCL10+ITLN1). SHAP analysis can further explained the positive or negative impact of gene expression on prognosis. By dividing patients into high-risk and low-risk groups, we found that patients in the high-risk group had poorer prognosis, higher TMB, and a higher proportion of MSI-H and MSI-L statuses. We also predicted that drugs such as 5-Fluorouracil, Oxaliplatin, Gefitinib, and Sorafenib would be more effective in low-risk patients, while drugs like Luminesib and Staurosporine would be more effective in high-risk patients. Single-cell analysis revealed that these 7 genes not only differ at the level of immune cells but also in epithelial cells, fibroblasts, and myofibroblasts, among other cell types. Finally, the expression of these key genes was verified in clinical samples, with consistent results. Conclusions: Our research findings provide evidence for the role of disulfidptosis in COAD and offer new insights for personalized and precise treatment of COAD.

Preventive effect of Lactobacillus johnsonii YH1136 against uric acid accumulation and renal damages.

Background: Hyperuricemia (HUA) is a prevalent metabolic disorder whose development is associated with intestinal microbiota. Therefore, probiotics have emerged as a potential and safe approach for lowering uric acid (UA) levels. However, the underlying mechanisms of many effective probiotic strains remain unknown. Methods and results: C57BL/6 mice were randomly divided into two groups: control and model groups. The model group received 12 weeks of potassium oxonate. Through 16s sequencing we found that HUA resulted in a significant decrease in the total diversity of all intestinal segments. When each intestinal segment was analyzed individually, the reduction in diversity was only significant in the cecum and colon sections. RDA analysis showed that lactobacilli in the rat colon exhibited a strong correlation with model group, suggesting that Lactobacillus may play an important role in HUA. Consequently, the preventive effects of Lactobacillus johnsonii YH1136 against HUA were investigated. C57BL/6 mice were randomly divided into three groups: control, model and YH1136 groups. The results showed that administering Lactobacillus johnsonii YH1136 effectively reduced serum UA levels in vivo by inhibiting hepatic xanthine oxidase (XOD) activity and promoting renal ABCG2 transporter expression. Moreover, supplementation with Lactobacillus johnsonii YH1136 significantly ameliorated pathological damage in the kidney and liver, thereby reducing UA accumulation. Conclusion: Hyperuricemia is accompanied by an altered composition of multiple gut bacteria, of which Lactobacillus is a key genus. Lactobacillus johnsonii YH1136 may ameliorate renal involvement in HUA via the gut-kidney axis.

Effects of prolonged fluoride exposure on innate immunity, intestinal mechanical, and immune barriers in mice.

The aim of this study is to explore the effects of fluoride on the innate immunity, intestinal mechanical barrier, and immune barrier of C57BL/6 mice, as well as to analyze the degree of structural and tissue damage, providing reference data for related research. Mice were randomly divided into four groups and then treated with 0 mg/L (control), 50 mg/L, 100 mg/L, 125 mg/L sodium fluoride solution, respectively, for 120 days. Histological technique, ELISA, MTT colorimetry methods were used to detect and analyze the effects of different concentrations of fluoride on the intestinal morphology, mechanical barrier and the immune functions and innate immunity of mice. The results showed that compared with the control group, the villi were injured in different degrees of the three fluoride groups, the number of goblet cells, the protein expression levels of connexin ZO-1, Claudin-1 and Occludin, the content of Diamine Oxidase (DAO), endotoxin (ET) and D-lactic acid (D-LA), the activity of natural killer cell (NK cells), the number and percentage of neutrophils and erythrocytes, the phagocytic rate of neutrophils, and the rate of C3bR rosette (which is formed by the adhesion of C3b receptors on the red blood cell membrane to complement sensitized yeast) and IC rosette (which is formed by the adhesion of C3b molecules in the immunecomplex adhered to the red blood cell membrane to non sensitized yeast) of red blood cells, the content of interlenkin 1 beta (IL-1ß) and interlenkin 8 (IL-8), the number and percentage of lymphocytes decreased with the increasing of fluoride concentration. In addition, the content of the Immunoglobulin A (sIgA) showed a trend of increase at first and then decrease in salivary gland and jejunum. It is concluded that excessive intake of fluoride for a long time has a certain damage effect on the intestinal tract, leading to an increase in the permeability of the intestinal tract, thereby destroying the mechanical and immune barrier function of the intestinal tract.

Prolonged fluoride exposure induces spatial-memory deficit and hippocampal dysfunction by inhibiting small heat shock protein 22 in mice.

Millions of residents in areas with high-fluoride drinking water supply ingest excessive levels of fluoride for long periods. This study investigated the mechanisms and impacts of lifelong exposure to naturally occurring moderate-high-fluoride drinking water on spatial-memory function by studying mice in controlled experiments. Spatial-memory deficits and disorders of hippocampal neuronal electrical activity were observed in mice exposed to 25-ppm or 50-ppm-fluoride drinking water for 56 weeks, but not in adult or old mice exposed to 50 ppm fluoride for 12 weeks. Ultrastructural analysis showed severely damaged hippocampal mitochondria, evidenced by reduced mitochondrial membrane potential and ATP content. Mitochondrial biogenesis was impaired in fluoride-exposed mice, manifesting as a significantly reduced mtDNA content, mtDNA-encoded subunits mtND6 and mtCO1, and respiratory complex activities. Fluoride reduced expression of Hsp22, a beneficial mediator of mitochondrial homeostasis, and decreased levels of signaling for the PGC-1α/TFAM pathway-which regulates mitochondrial biogenesis-and the NF-κß/STAT3 pathway-which regulates mitochondrial respiratory chain enzyme activity. Hippocampus-specific Hsp22-overexpression improved fluoride-induced spatial-memory deficits by activating the PGC-1α/TFAM and STAT3 signaling pathways, while Hsp22-silencing aggravated the deficits by inhibiting both pathways. Downregulation of Hsp22 plays a vital role in fluoride-induced spatial-memory deficits by impacting mtDNA-encoding subsets and mitochondrial respiratory chain enzyme activity.

Dynamic Monitoring of Changes in Fecal Flora of Giant Pandas in Mice: Co-Occurrence Network Reconstruction.

Giant pandas are uniquely vulnerable mammals in western China. It is important to develop an animal model to explore the intestinal flora of giant pandas to understand the relationship between digestive diseases and flora. Existing animal models of intestinal flora focus on human flora-associated animals, such as mice, and there is a very limited amount of knowledge regarding giant panda flora-associated animals. To fill this gap, fecal microorganisms from giant pandas were transplanted into pseudosterile and germfree mice using single and multiple gavages. Fecal samples were collected from mice at four time points after transplantation for microbial community analysis. We determined that compared to pseudosterile mice, the characteristics of intestinal flora in pandas were better reproduced in germfree mice. There was no significant difference in microbial diversity between germfree mice and giant panda gut microbes from day 3 to day 21. Germfree mice at the phylum level possessed large amounts of Firmicutes and Proteobacteria, and at the genus level, Escherichia-Shigella, Clostridium sensu stricto 1, and Streptococcus dominated the intestinal flora structure. The microbial community co-occurrence network based on indicator species indicated that germfree mice transplanted with fecal bacteria tended to form a microbial community co-occurrence network similar to that of giant pandas, while pseudosterile mice tended to restore the microbial community co-occurrence network originally present in these mice. Our data are helpful for the study of giant panda flora-associated animals and provide new insights for the in vitro study of giant panda intestinal flora. IMPORTANCE The giant panda is a unique vulnerable mammal in western China, and its main cause of death is digestive system diseases regardless of whether these animals are in the wild or in captivity. The relationship between the intestinal flora and the host exerts a significant impact on the nutrition and health of the giant pandas. However, the protected status of the giant panda has made in vivo, repeatable, and large-sample sampling studies of their intestinal flora difficult. This greatly hinders the research depth of the giant panda intestinal flora from the source. The development and utilization of specific animal models to simulate the structure and characteristics of the intestinal flora provide another means to deal with these research limitations. However, current research examining giant panda flora-associated animals is limited. This study is the first to reveal dynamic changes in the fecal flora of giant pandas in mice after transplantation.

Psychoactive Effects of Lactobacillus johnsonii BS15 on Preventing Memory Dysfunction Induced by Acute Ethanol Exposure Through Modulating Intestinal Microenvironment and Improving Alcohol Metabolic Level.

The negative effects of ethanol (EtOH) abuse on the body have been widely reported in recent years. Building on the microbiota-gut-brain axis hypothesis, our study aimed to demonstrate the potential psychobiotic role of Lactobacillus johnsonii BS15 in the preventive effects of acute EtOH intake on memory impairment. We also determined whether L. johnsonii BS15 intake could effectively improve resistance to acute drinking and alleviate the adverse effects of EtOH. Male mice were fed L. johnsonii BS15 orally with (Probiotic group) or without (Control and Alcohol groups) daily dose of 0.2 × 109 CFU/ml per mouse for 28 days. Gavage with L. johnsonii BS15 significantly modified the ileal microbial ecosystem (assessed by 16S rRNA gene sequencing) in favor of Firmicutes and Lactobacillus, indicating the ability of BS15 to restore the gut microbiota. The acute EtOH exposure model (7 g/kg EtOH per mice) was established by gavage, which was administered to the alcohol and probiotic groups on day 28 of the experiment. The L. johnsonii BS15 intake effectively reduced alcohol unconsciousness time, blood alcohol concentration, and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Meanwhile, the improvement of ethanol resistance time and the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver were shown by BS15 in acute alcohol-induced mice. We found that acute EtOH exposure reduced the exploration ratio (assessed by the novel object recognition test), escape latency, number of errors (assessed by passive avoidance test), and spontaneous exploration (assessed by T-maze test) in mice, which were obviously improved by L. johnsonii BS15. In the hippocampus, L. johnsonii BS15 significantly reversed the decrease in antioxidant capacity of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) and mRNA expression of memory-related functional proteins of brain-derived neurotrophic factor (BDNF) and cyclic ampresponse element binding protein (CREB) in the hippocampal tissue after acute EtOH exposure. In conclusion, L. johnsonii BS15 intake appears as a promising psychoactive therapy to ameliorate alcohol-mediated memory impairment by increasing EtOH metabolic levels.

Lactobacillus johnsonii YH1136 plays a protective role against endogenous pathogenic bacteria induced intestinal dysfunction by reconstructing gut microbiota in mice exposed at high altitude.

Background: Intestinal microbiota plays an important role in maintaining the microecological balance of the gastrointestinal tract in various animals. Disturbances in the intestinal microbiota may lead to the proliferation of potentially pathogenic bacteria that become the dominant species, leading to intestinal immune disorders, intestinal inflammation, and other intestinal diseases. Numerous studies have been confirmed that high-altitude exposure affects the normal function of the intestine and the composition of the intestinal microbiota. However, it is still necessary to reveal the changes in intestinal microbiota in high-altitude exposure environments, and clarify the relationship between the proliferation of potentially pathogenic bacteria and intestinal injury in this environment. In addition, explored probiotics that may have preventive effects against intestinal diseases. Methods and results: C57BL/6 mice were randomly divided into three groups, a high-altitude group (HA), control group (C), and high-altitude probiotic group (HAP). The HA and HAP groups were subjected to hypoxia modeling for 14 days in a low-pressure oxygen chamber with daily gavage of 0.2 mL of normal saline (HA) and Lactobacillus johnsonii YH1136 bacterial fluid (HAP), while the control group was fed normally. L. johnsonii YH1136 was isolated from feces of a healthy Tibetan girl in Baingoin county, the Nagqu region of the Tibet Autonomous Region, at an altitude of 5000 meters. Our observations revealed that gavage of YH1136 was effective in improving the damage to the intestinal barrier caused by high-altitude exposure to hypoxic environments and helped to reduce the likelihood of pathogenic bacteria infection through the intestinal barrier. It also positively regulates the intestinal microbiota to the extent of Lactobacillus being the dominant microbiome and reducing the number of pathogenic bacteria. By analyzing the expression profile of ileal microRNAs and correlation analysis with intestinal microbiota, we found that Staphylococcus and Corynebacterium1 cooperated with miR-196a-1-3p and miR-3060-3p, respectively, to play a regulatory role in the process of high-altitude hypoxia-induced intestinal injury. Conclusion: These findings revealed the beneficial effect of L. johnsonii YH1136 in preventing potential endogenous pathogenic bacteria-induced intestinal dysfunction in high-altitude environments. The mechanism may be related to the regulation of intestinal injury from the perspective of the gut microbiota as well as miRNAs.

Dietary Probiotic Supplementation Suppresses Subclinical Necrotic Enteritis in Broiler Chickens in a Microbiota-Dependent Manner.

Background: Chicken meat is one of the most consumed meats worldwide and poultry production is increasing at an exponential rate. Reducing antibiotic usage has resulted in the recurrence of subclinical necrotic enteritis again and influenced global poultry production. Probiotics are potential antibiotic substitutes that can be used to prevent subclinical necrotic enteriti. However, the precise mechanism of action of probiotics and information on which gut microbes confer this efficacy remain elusive. Methods and results: The subclinical necrotic enteritis animal model was used to reveal the mechanism underlying the effect of probiotics on intestinal health through RNA sequencing and 16S rDNA amplicon sequencing. Bacillus licheniformis H2 feeding significantly reduced the relative abundance of Clostridium perfringens in the ileum and markedly ameliorated the pathological damage in the ileum and liver. In addition, oral administration of B. licheniformis H2 contributed to the enhancement of the intestinal barrier function and epithelial renewal, reducing energy consumption, and improving enteral nutrition absorption. Probiotic B. licheniformis H2 also ameliorated the inflammatory response and increased the immunity of subclinical necrotic enteritis infected broilers. Finally, B. licheniformis H2 feeding regulated liver gene expression to suppress immune response and promoted growth and metabolism depending on the gut microbiota. Conclusions: These results indicated the mechanism of probiotic action of B. licheniformis H2 in maintaining intestinal health and thus promoting growth and B. licheniformis H2 may serve as an antibiotic substitute to prevent subclinical necrotic enteritis in poultry farming.

Psychoactive Effects of Lactobacillus johnsonii Against Restraint Stress-Induced Memory Dysfunction in Mice Through Modulating Intestinal Inflammation and permeability-a Study Based on the Gut-Brain Axis Hypothesis.

Though the underlying mechanism remains elusive, a close relationship between psychological stress and intestinal inflammation has been widely accepted. Such a link is very important to set the basis for our understanding of the critical role of gut-brain axis (GBA) in homeostatic processes in health and disease. Probiotics that could confer benefits to mental health through GBA are referred to as "psychobiotics". This study aimed to further determine whether a potential psychobiotic strain, Lactobacillus johnsonii BS15 could prevent memory dysfunction in mice induced by psychological stress through modulating the gut environment, including intestinal inflammation and permeability. Memory dysfunction in mice was induced by restraint stress (RS), one of the most commonly utilized models to mimic psychological stress. The mice were randomly categorized into three groups including no stress (NS), restraint stress (RS), and probiotic (RS-P) and administered with either phosphate buffered saline (NS and RS groups) or L. johnsonii BS15 (RS-P group) every day from day 1-28. From days 22-28, the mice in RS and RS-P groups were subjected to RS each day. Results revealed that BS15-pretreatment enhanced the performance of RS-induced mice during three different behavioral tests for memory ability and positively modulated the hypothalamic-pituitary-adrenal axis by attenuating the serum corticosterone level. In the hippocampus, L. johnsonii BS15 positively modulated the memory-related functional proteins related to synaptic plasticity, increased neurotransmitter levels, and prevented RS-induced oxidative stress and mitochondria-mediated apoptosis. In the intestines, L. johnsonii BS15 protected the RS-induced mice from damaged gut barrier by enhancing the mRNA levels of tight junction proteins and exerted beneficial effects on the anti-inflammatory cytokine levels reduced by RS. These findings provided more evidence to reveal the psychoactive effect of L. johnsonii BS15 against memory dysfunction in RS-induced mice by modulating intestinal inflammation and permeability.

Analysis of miRNA Expression in the Ileum of Broiler Chickens During Bacillus licheniformis H2 Supplementation Against Subclinical Necrotic Enteritis.

Subclinical necrotic enteritis (SNE) is one of the serious threats to the poultry industry. Probiotics have been proven to exert beneficial effects in controlling SNE. However, their exact mechanisms have not been fully elucidated. Moreover, few studies have focused on their impact on microRNAs (miRNAs). Therefore, the present study aimed to explore the miRNA expression profiles in the ileum of broiler chickens during probiotic supplementation for controlling SNE. A total of 180 newly hatched male broilers were randomly allocated into three groups, including a negative control group, an SNE infection group, and a Bacillus licheniformis H2 pretreatment group. Illumina high-throughput sequencing was conducted to identify the miRNA expression of the three groups. Results showed that 628 miRNAs, including 582 known miRNAs and 46 novel miRNAs, were detected in the miRNA libraries. The target genes of 57 significantly differentially expressed miRNAs were predicted and annotated. Moreover, they were found to be partly enriched in pathways related to immunity and inflammation such as tumor necrosis factor receptor binding, immune response-regulating signaling pathway, Toll-like receptor 2 signaling pathway, interleukin-15 production, activation of NF-κB-inducing kinase activity, and MAP kinase tyrosine/serine/threonine phosphatase activity. Some of the target genes of 57 miRNAs were related to the MAPK signaling pathway. Furthermore, the expression of several miRNAs, which may be involved in the MAPK signaling pathway, was significantly affected by SNE induction and showed no significant difference in the presence of H2. All these findings provide comprehensive miRNA expression profiles of three different treatment groups. They further suggest that H2 could exert beneficial effects in controlling SNE through immune and inflammatory response associated with altered miRNA expression, such as the MAPK signaling pathway.

Probiotic Lactobacillus johnsonii BS15 Promotes Growth Performance, Intestinal Immunity, and Gut Microbiota in Piglets.

Numerous studies have investigated the beneficial effects of Lactobacillus johnsonii strain BS15 on mice and broilers. This study aimed to understand the growth-promoting effects of BS15 on piglets. We determined the effects of L. johnsonii BS15 and a commercial probiotic strain, Bacillus subtilis JS01. Seventy-two suckling piglets (1 ± 2-day-old) were divided into three groups and fed with diets supplemented with 1 × 106 colony-forming units (cfu) BS15 per gram of feed (BS15 group); 1 × 106 cfu JS01 per gram of feed (JS01 group); or de Man, Rogosa, and Sharpe liquid medium (control group) 35 days. Compared with JS01, BS15 significantly improved the daily weight gain and diarrhea index of the piglets. The BS15 group had higher fecal sIgA levels, whereas the JS01 group had high fecal sIgA levels only after 35 days of treatment. Additionally, BS15 altered T cell subsets in peripheral blood by significantly increasing the CD3+CD4+ T cell percentage and CD3+CD4+/CD3+CD8+ ratio and decreasing the CD3+CD8+ T cell percentage. Moreover, BS15 exerted better beneficial effects on fecal microbiota than JS01. Specifically, the BS15 group had markedly increased Clostridium, Peptococcus, and Lactobacillus populations on days 7 and 21 of treatment and reduced Escherichia coli populations on day 35 of treatment. These findings indicated that BS15 can be applied as a probiotic that promotes growth performance and controls diarrhea in piglets.

Lactobacillus johnsonii BS15 improves intestinal environment against fluoride-induced memory impairment in mice-a study based on the gut-brain axis hypothesis.

BACKGROUND: Excessive fluoride can lead to chronic neurodegeneration characterized by neuron and myelin loss and memory dysfunction. The gut-brain axis hypothesis suggests that gut microbiota plays a crucial role in regulating brain function. Thus, using probiotics to adjust the gut microenvironment may be a potential therapy for mental diseases. METHODS: Mice in the prob group were administrated with Lactobacillus johnsonii BS15 for 28 days prior to and throughout a 70-day exposure to sodium fluoride. The drinking water of all groups (F and prob groups) except the control group were replaced by high-fluoride water (100 mg NaF/L) on day 28. Animals in each group were divided into two subsets: one underwent behavioral test, and the other was sacrificed for sampling. The mRNA expression level and protein content related to inflammatory reaction in the ileum and hippocampus were respectively detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of proteins related to myelin structure, apoptosis, and memory in the hippocampus and tight junction proteins in the ileum were determined by RT-qPCR and/or immunohistochemistry. Gut permeability markers (D-lactate and diamine oxidase (DAO)) in the serum were also examined by ELISA. RESULTS: The results showed that fluoride exposure induced a lower spontaneous exploration (P < 0.05) in T-maze test, which indicated an impairment of memory. Spontaneous exploration of BS15-treated mice was significantly higher (P < 0.05) than that in F group. Fluoride reduced (P < 0.05) levels of myelin structural protein (proteolipid protein) and neurogenesis-associated proteins (brain-derived neurotrophic factor and cAMP/Ca2+ responsive element-binding protein), induced disordered inflammatory cytokines (TNF-α, IFN-γ, and IL-6; P < 0.05), increased pro-apoptotic genes (caspase-3; P < 0.05), and decreased anti-apoptotic genes (Bcl-2; P < 0.05) in the hippocampus, of which the influences were reversed by BS15. BS15 treatment exerted significant preventive effects on reversing the gut inflammation induced by excessive fluoride intake by reducing (P < 0.05) the levels of pro-inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)) and remarkably increasing (P < 0.05) the level of anti-inflammatory cytokines (IL-10). Moreover, the serum DAO activity and D-lactate concentration significantly increased by fluoride were also reduced (P < 0.05) by BS15. This result indicated the profitable effect of BS15 on gut permeability. CONCLUSION: L. johnsonii BS15 intake could benefit the neuroinflammation and demyelination in the hippocampus by improving the gut environment and ameliorating fluorine-induced memory dysfunction.