Lactobacillus reuteri inhibits Staphylococcus aureus-induced mastitis by regulating oxytocin releasing and gut microbiota in mice.

Mastitis is the most frequent disease of cows and has well-recognized detrimental effects on animal wellbeing and dairy farm profitability. With the advent of the postantibiotic era, alternative antibiotic agents, especially probiotics, have received increasing attention in the treatment of mastitis. Based on research showing that Lactobacillus reuteri (L. reuteri) has anti-inflammatory effects, this study explored the protective effects and mechanisms of L. reuteri against mastitis induced by Staphylococcus aureus (S. aureus) in mice. First, mice with S. aureus-induced mastitis were orally administered L. reuteri, and the inflammatory response in the mammary gland was observed. The results showed that L. reuteri significantly inhibited S. aureus-induced mastitis. Moreover, the concentration of oxytocin (OT) and protein expression of oxytocin receptor (OTR) were measured, and inhibition of OTR or vagotomy reversed the protective effect of L. reuteri or its culture supernatant (LCS) on S. aureus-induced mastitis. In addition, in mouse mammary epithelial cells (MMECs), OT inhibited the inflammation induced by S. aureus by inhibiting the protein expression of OTR. It was suggested that L. reuteri protected against S. aureus-induced mastitis by releasing OT. Furthermore, microbiological analysis showed that the composition of the microbiota was altered, and the relative abundance of Lactobacillus was significantly increased in gut and mammary gland after treatment with L. reuteri or LCS. In conclusion, our study found the L. reuteri inhibited the mastitis-induced by S. aureus via promoting the release of OT, and treatment with L. reuteri increased the abundance of Lactobacillus in both gut and mammary gland.

Lactobacillus paracasei Jlus66 relieves DSS-induced ulcerative colitis in a murine model by maintaining intestinal barrier integrity, inhibiting inflammation, and improving intestinal microbiota structure.

PURPOSE: Ulcerative colitis (UC) is a serious health problem with increasing morbidity and prevalence worldwide. The pathogenesis of UC is complex, currently believed to be influenced by genetic factors, dysregulation of the host immune system, imbalance in the intestinal microbiota, and environmental factors. Currently, UC is typically managed using aminosalicylates, immunosuppressants, and biologics as adjunctive therapies, with the risk of relapse and development of drug resistance upon discontinuation. Therefore, further research into the pathogenesis of UC and exploration of potential treatment strategies are necessary to improve the quality of life for affected patients. According to previous studies, Lactobacillus paracasei Jlus66 (Jlus66) reduced inflammation and may help prevent or treat UC. METHODS: We used dextran sulfate sodium (DSS) to induce a mouse model of UC to assess the effect of Jlus66 on the progression of colitis. During the experiment, we monitored mouse body weight, food and water consumption, as well as rectal bleeding. Hematoxylin-eosin staining was performed to assess intestinal pathological damage. Protein imprinting and immunohistochemical methods were used to evaluate the protein levels of nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and tight junction (TJ) proteins in intestinal tissues. Fecal microbiota was analyzed based on partial 16S rRNA gene sequencing. RESULTS: Jlus66 supplementation reduced the degree of colon tissue damage, such as colon shortening, fecal occult blood, colon epithelial damage, and weight loss. Supplementation with Jlus66 reduced DSS-induced upregulation of cytokine levels such as TNF-α, IL-1ß, and IL-6 (p < 0.05). The NF-κB pathway and MAPK pathway were inhibited, and the expression of TJ proteins (ZO-1, Occludin, and Claudin-3) was upregulated. 16S rRNA sequencing of mouse cecal contents showed that Jlus66 effectively regulated the structure of the intestinal biota. CONCLUSION: In conclusion, these data indicate that Jlus66 can alter the intestinal biota and slow the progression of UC, providing new insights into potential therapeutic strategies for UC.

Tyrosol inhibits NF-κB pathway in the treatment of enterotoxigenic Escherichia coli-induced diarrhea in mice.

Tyrosol is one of the main polyphenol compounds in white wine and extra virgin olive oil (EVOO), which plays an antioxidant and anti-inflammatory role in vitro. In the present study, we investigated the possible anti-inflammatory mechanism of tyrosol in Escherichia coli (ETEC)-induced diarrhea in mice. ICR mice were randomly divided into control group, ETEC group, and ETEC + Tyrosol group with 10 mice in each group. In addition to the control group, a bacterial diarrhea model was induced in mice by continuous administration of 0.2 ml × 109 CFU/ml ETEC. After 7 days, the ETEC + Tyrosol group was given tyrosol (20 mg/kg) once a day by gavage, during which the body weight of mice and the degree of diarrhea were measured daily. On the 15th day, all animals in this experiment were sacrificed, colon tissue was collected, and colon length was recorded. Our results indicate that tyrosol significantly attenuated the extent of ETEC-induced diarrhea, including inhibition of pro-inflammatory cytokine, repair of the intestinal epithelial mechanical barrier, and significant inhibition of NF-κB activation. This finding is helpful for the development and further application of tyrosol in the treatment of diarrhea.

Cadmium induces liver dysfunction and ferroptosis through the endoplasmic stress-ferritinophagy axis.

Cadmium (Cd) is a type of high-risk heavy metal that can damage organs such as the liver, but its mechanism is not yet clear. Ferroptosis is a newly discovered mode of regulatory cell death. We explored whether ferroptosis is involved in Cd-induced liver damage and the underlying mechanism. Our research showed that Cd induced liver damage by inducing ferroptosis, and the use of ferroptosis inhibitors reduced the degree of liver damage. Moreover, the occurrence of ferroptosis was accompanied by the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway, and inhibiting endoplasmic reticulum (ER) stress reduced ferroptosis demonstrating that ferroptosis induced by Cd is dependent on ER stress. In addition, chloroquine, a common autophagy inhibitor, mitigated ferroptosis caused by Cd exposure. Then, the iron chelator deferoxamine reduced Cd-induced lipid peroxidation and cell death, demonstrating that the iron regulation disorder caused by ferritin phagocytosis contributes to the Cd-induced ferroptosis. In conclusion, our results show that Cd-induced liver toxicity is accompanied by ferroptosis, which contributes to Cd inducing oxidative stress to trigger autophagy and ER stress to promote the process of ferroptosis.

Protective effects of polydatin on LPS-induced endometritis in mice.

Endometritis, a common inflammation of the uterus, often causes severe damage to human and animal reproductive health. Polydatin is a polyphenol extracted from the rhizome of Polygonum cuspidatum that has anti-inflammatory and anti-oxidative effects. The purpose of this study was to investigate the underlying protective effects and mechanisms of polydatin against lipopolysaccharide (LPS)-induced endometritis in mice. The mouse model of endometritis was established by injection of LPS through the vagina. The uterine tissues of each group were gathered to analyze histopathological changes, inflammatory cytokine production, and the degree of activation of the NF-κB and Nrf2 signaling pathways. The myeloperoxidase (MPO) activity assay indicated that polydatin treatment significantly alleviated inflammatory cell infiltration in LPS-induced endometritis mice. Furthermore, polydatin treatment remarkably impeded the expression of TNF-α, IL-1ß, and IL-6 by ELISA assay. Hematoxylin-eosin staining (H&E) showed that polydatin significantly decreased impairment of the uterus. In addition, polydatin was also found to suppress LPS-induced NF-κB activation in a dose-dependent manner. The expression of Nrf2 and HO-1 was enhanced by polydatin treatment. All the results suggest that polydatin helpfully alleviates LPS-induced endometritis by suppressing the NF-ĸB signaling pathway and activating the Nrf2 signaling pathway.

Dimethyl itaconate protects against lippolysacchride-induced mastitis in mice by activating MAPKs and Nrf2 and inhibiting NF-κB signaling pathways.

Mastitis, as the main disease to affect the dry dairy cow with the characterized by increasing number of somatic cells in milk and reducing milk production, has been known as one of the most serious expensive disease for the dairy industry. Escherichia coli (E.coli), a gram negative bacterial, have normally been considered to be an opportunistic pathogen that can invade the mammary gland sometimes to cause inflammatory diseases. Lippolysacchride (LPS), as the co-cell wall component of the Escherichia coli (E.coli), is the main virulence factors to induce acute inflammation. Itaconate is an endogenous metabolite which has recently been reported to regulate the macrophage function and has the ability to reduce the secretion of pro-inflammatory cytokines, such as IL-6 and IL-12. Here, the aim of this study is to investigate the protective role of dimethyl itaconate (DI)-the membranepermeable derivative of itaconate, on LPS-induced mastitis in mice. To establish the model of mastitis, mice 5-7 day after delivery were utilized by nipple duct injection of LPS, while DI was treated 24h intraperitoneally before LPS injection. Further, the hematoxylin-eosin (H&E) staining was used to evaluate the pathological changes of the mammary gland, the inflammatory cytokines of TNF-α and IL-1ß and the myeloperoxidase (MPO) activity were also measured respectively by enzyme-linked immunosorbent assay (ELISA) and MPO assay kit. To clarify the underling mechanisms of the protective role of DI on mastitis, the MAPKs, NF-κB and Nrf2 signaling pathways were detected via western blotting. The results demonstrated that DI markedly decreased the pathological injury of mammary, and considerably reduced the production of TNF-α and IL-1ß, as well as up-regulated the Nrf2, HO-1, phosphorylation of p38 and ERK, but down-regulated TLR4 and phosphorylation of p65 NF-κB. Our research recommended that DI ameliorated LPS-induced mastitis which highlights itaconate may as a potential candidate to protect against mastitis.

Gut/rumen-mammary gland axis in mastitis: Gut/rumen microbiota-mediated "gastroenterogenic mastitis".

BACKGROUND: Mastitis is an inflammatory response in the mammary gland that results in huge economic losses in the breeding industry. The aetiology of mastitis is complex, and the pathogenesis has not been fully elucidated. It is commonly believed that mastitis is induced by pathogen infection of the mammary gland and induces a local inflammatory response. However, in the clinic, mastitis is often comorbid or secondary to gastric disease, and local control effects targeting the mammary gland are limited. In addition, recent studies have found that the gut/rumen microbiota contributes to the development of mastitis and proposed the gut/rumen-mammary gland axis. Combined with studies indicating that gut/rumen microbiota disturbance can damage the gut mucosa barrier, gut/rumen bacteria and their metabolites can migrate to distal extraintestinal organs. It is believed that the occurrence of mastitis is related not only to the infection of the mammary gland by external pathogenic microorganisms but also to a gastroenterogennic pathogenic pathway. AIM OF REVIEW: We propose the pathological concept of "gastroenterogennic mastitis" and believe that the gut/rumen-mammary gland axis-mediated pathway is the pathological mechanism of "gastroenterogennic mastitis". KEY SCIENTIFIC CONCEPTS OF REVIEW: To clarify the concept of "gastroenterogennic mastitis" by summarizing reports on the effect of the gut/rumen microbiota on mastitis and the gut/rumen-mammary gland axis-mediated pathway to provide a research basis and direction for further understanding and solving the pathogenesis and difficulties encountered in the prevention of mastitis.

Gut microbiota-mediated secondary bile acid alleviates Staphylococcus aureus-induced mastitis through the TGR5-cAMP-PKA-NF-κB/NLRP3 pathways in mice.

Although emerging evidence shows that gut microbiota-mediated metabolic changes regulate intestinal pathogen invasions, little is known about whether and how gut microbiota-mediated metabolites affect pathogen infection in the distal organs. In this study, untargeted metabolomics was performed to identify the metabolic changes in a subacute ruminal acidosis (SARA)-associated mastitis model, a mastitis model with increased susceptibility to Staphylococcus aureus (S. aureus). The results showed that cows with SARA had reduced cholic acid (CA) and deoxycholic acid (DCA) levels compared to healthy cows. Treatment of mice with DCA, but not CA, alleviated S. aureus-induced mastitis by improving inflammation and the blood-milk barrier integrity in mice. DCA inhibited the activation of NF-κB and NLRP3 signatures caused by S. aureus in the mouse mammary epithelial cells, which was involved in the activation of TGR5. DCA-mediated TGR5 activation inhibited the NF-κB and NLRP3 pathways and mastitis caused by S. aureus via activating cAMP and PKA. Moreover, gut-dysbiotic mice had impaired TGR5 activation and aggravated S. aureus-induced mastitis, while restoring TGR5 activation by spore-forming bacteria reversed these changes. Furthermore, supplementation of mice with secondary bile acids producer Clostridium scindens also activated TGR5 and alleviated S. aureus-induced mastitis in mice. These results suggest that impaired secondary bile acid production by gut dysbiosis facilitates the development of S. aureus-induced mastitis and highlight a potential strategy for the intervention of distal infection by regulating gut microbial metabolism.

Enterogenic Stenotrophomonas maltophilia migrates to the mammary gland to induce mastitis by activating the calcium-ROS-AMPK-mTOR-autophagy pathway.

BACKGROUND: Mastitis is an inflammatory disease of the mammary gland that has serious economic impacts on the dairy industry and endangers food safety. Our previous study found that the body has a gut/rumen-mammary gland axis and that disturbance of the gut/rumen microbiota could result in 'gastroenterogenic mastitis'. However, the mechanism has not been fully clarified. Recently, we found that long-term feeding of a high-concentrate diet induced mastitis in dairy cows, and the abundance of Stenotrophomonas maltophilia (S. maltophilia) was significantly increased in both the rumen and milk microbiota. Accordingly, we hypothesized that 'gastroenterogenic mastitis' can be induced by the migration of endogenous gut bacteria to the mammary gland. Therefore, this study investigated the mechanism by which enterogenic S. maltophilia induces mastitis. RESULTS: First, S. maltophilia was labelled with superfolder GFP and administered to mice via gavage. The results showed that treatment with S. maltophilia promoted the occurrence of mastitis and increased the permeability of the blood-milk barrier, leading to intestinal inflammation and intestinal leakage. Furthermore, tracking of ingested S. maltophilia revealed that S. maltophilia could migrate from the gut to the mammary gland and induce mastitis. Subsequently, mammary gland transcriptome analysis showed that the calcium and AMPK signalling pathways were significantly upregulated in mice treated with S. maltophilia. Then, using mouse mammary epithelial cells (MMECs), we verified that S. maltophilia induces mastitis through activation of the calcium-ROS-AMPK-mTOR-autophagy pathway. CONCLUSIONS: In conclusion, the results showed that enterogenic S. maltophilia could migrate from the gut to the mammary gland via the gut-mammary axis and activate the calcium-ROS-AMPK-mTOR-autophagy pathway to induce mastitis. Targeting the gut-mammary gland axis may also be an effective method to treat mastitis.

The Role of Rumen Microbiota and Its Metabolites in Subacute Ruminal Acidosis (SARA)-Induced Inflammatory Diseases of Ruminants.

Subacute ruminal acidosis (SARA) is a common metabolic disease in ruminants. In the early stage of SARA, ruminants do not exhibit obvious clinical symptoms. However, SARA often leads to local inflammatory diseases such as laminitis, mastitis, endometritis and hepatitis. The mechanism by which SARA leads to inflammatory diseases is largely unknown. The gut microbiota is the totality of bacteria, viruses and fungi inhabiting the gastrointestinal tract. Studies have found that the gut microbiota is not only crucial to gastrointestinal health but also involved in a variety of disease processes, including metabolic diseases, autoimmune diseases, tumors and inflammatory diseases. Studies have shown that intestinal bacteria and their metabolites can migrate to extraintestinal distal organs, such as the lung, liver and brain, through endogenous pathways, leading to related diseases. Combined with the literature, we believe that the dysbiosis of the rumen microbiota, the destruction of the rumen barrier and the dysbiosis of liver function in the pathogenesis of SARA lead to the entry of rumen bacteria and/or metabolites into the body through blood or lymphatic circulation and place the body in the "chronic low-grade" inflammatory state. Meanwhile, rumen bacteria and/or their metabolites can also migrate to the mammary gland, uterus and other organs, leading to the occurrence of related inflammatory diseases. The aim of this review is to describe the mechanism by which SARA causes inflammatory diseases to obtain a more comprehensive and profound understanding of SARA and its related inflammatory diseases. Meanwhile, it is also of great significance for the joint prevention and control of diseases.

Endoplasmic reticulum stress-mediated autophagy activation is involved in cadmium-induced ferroptosis of renal tubular epithelial cells.

Acute cadmium (Cd) exposure is a significant risk factor for renal injury and lacks effective treatment strategies. Ferroptosis is a recently identified iron-dependent form of nonapoptotic cell death mediated by membrane damage resulting from lipid peroxidation, and it is implicated in many diseases. However, whether ferroptosis is involved in Cd-induced renal injury and, if so, how it operates. Here, we show that Cd can induce ferroptosis in kidney and renal tubular epithelial cells, as demonstrated by elevation of intracellular iron levels and lipid peroxidation, as well as impaired antioxidant production. Treatment with a ferroptosis inhibitor alleviated Cd-induced cell death. Intriguingly, we established that Cd-induced ferroptosis depended on endoplasmic reticulum (ER) stress, by demonstrating that Cd activated the PERK-eIF2α-ATF4-CHOP pathway and that inhibition of ER stress reduced ferroptosis caused by Cd. We further found that autophagy was required for Cd-induced ferroptosis because the inhibition of autophagy by chloroquine mitigated Cd-induced ferroptosis. Furthermore, we showed that iron dysregulation by ferritinophagy contributed to Cd-induced ferroptosis, by showing that the iron chelator desferrioxamine alleviated Cd-induced cell death and lipid peroxidation. In addition, ER stress is likely activated by MitoROS which trigger autophagy and ferroptosis. Collectively, our results indicate that ferroptosis is involved in Cd-induced renal toxicity and regulated by the MitoROS-ER stress-ferritinophagy axis.

Neutralization of Interleukin-17A Attenuates Lipopolysaccharide-Induced Mastitis by Inhibiting Neutrophil Infiltration and the Inflammatory Response.

Mastitis has been recognized as a common and major disease of cows with a strong impact on dairy farming. Interleukin-17A (IL-17A) has been shown to mediate crucial crosstalk between the immune system and various epithelial tissues, initiating a series of defensive mechanisms against bacterial and fungal infections. This crosstalk is especially involved in neutrophil infiltration. To evaluate the role of IL-17A in immune defense in the mammary gland in mice, we tested the effects of depleting IL-17A on changes in pathology, neutrophil infiltration, and pro-inflammatory cytokine levels in the mammary gland stimulated by lipopolysaccharide (LPS). Further, the effects of IL-17A on the activation of the nuclear factor-κB (NF-κB) signaling pathway during mastitis induced by LPS were also studied. The results showed that the production of IL-17A was significantly elevated during mastitis induced by LPS. IL-17A blockade via an intraperitoneal antibody injection protected against LPS-induced mastitis, as indicated by decreased neutrophil infiltration, myeloperoxidase activity, pro-inflammatory cytokines levels, and NF-κB signaling pathway molecule phosphorylation in response to LPS. In conclusion, an elevated IL-17 level plays a crucial role during mastitis, and anti-IL-17A antibody blockade protects against LPS-induced mammary gland inflammation induced through the NF-κB signaling pathway, which provides a new potential treatment target for mastitis.

Taraxasterol Inhibits LPS-Induced Inflammatory Response in BV2 Microglia Cells by Activating LXRα.

Neuroinflammation plays a critical role in the development of neurodegenerative diseases. Taraxasterol, a pentacyclic-triterpene isolated from Taraxacum officinale, has been reported to have anti-inflammatory effect. The aim of this study was to investigate the anti-inflammatory effects and mechanism of taraxasterol in LPS-stimulated BV2 microglia cells. BV2 microglia cells were treated with taraxasterol 12 h before LPS stimulation. The effects of taraxasterol on LPS-induced TNF-α and IL-1ß production were detected by ELISA. The effects of taraxasterol on LXRα, ABCA1, TLR4, and NF-κB expression were detected by western blot analysis. The results showed that taraxasterol dose-dependently inhibited LPS-induced TNF-α and IL-1ß production and NF-κB activation. Taraxasterol also disrupted the formation of lipid rafts and inhibited translocation of TLR4 into lipid rafts. Furthermore, taraxasterol was found to activate LXRα-ABCA1 signaling pathway which induces cholesterol efflux from cells. In addition, our results showed that the anti-inflammatory effect of taraxasterol was attenuated by transfection with LXRα siRNA. In conclusion, these results suggested that taraxasterol inhibits LPS-induced inflammatory response in BV2 microglia cells by activating LXRα-ABCA1 signaling pathway.