Consumption of supplementary inulin modulates milk microbiota and metabolites in dairy cows with subclinical mastitis

Milk microbiota and mediated metabolites directly affect the health of the udder in dairy cows. Inulin, a dietary prebiotic, can modulate the profile of gastrointestinal microbiota. However, whether the inulin intake affects the milk microbial population and metabolites remains unknown. In this study, forty subclinical mastitis (SCM) cows were randomly divided into 5 groups. Five inulin addition doses, 0, 100, 200, 300 and 400 g/d per cow, based on the same basal diet were supplemented, respectively. The experiments lasted for 8 weeks. The results showed lower relative abundance of mastitic-causing and pro-inflammation microbes in milk (i.e., Escherichia -Shigella , Pseudomonas , Rhodococcus and Burkholderia-Caballeronia-Paraburkholderia , etc.), and higher probiotics and commensal bacteria, such as, Lactobacillus , Bifidobacterium , etc. in the cows fed 300 g/d inulin, compared with the control group. Meanwhile, the levels of arachidonic acid pro-inflammatory mediators (leukotriene E3, 20-carboxy-leukotriene B4 and 12-Oxo-c-LTB3) and phospholipid metabolites were reduced, and the levels of compounds with antibacterial and anti-inflammatory potential (prostaglandin A1 and 8-iso-15-Keto-PGE 2 , etc.), and participating energy metabolism (citric acid and L-Carnitine, etc.) were elevated. These data suggested that inulin intake might modulate the microflora and metabolites level in extra-intestinal tissue, such as mammary gland, which provided an alternative for the regulation and mitigation for SCM. IMPORTANCE The profile of microbial community and metabolic activity in milk are main determinant of udder health status and milk quality. Recent studies have demonstrated that diet could directly modulate mammary gland microbiome. Inulin is a probiotic dietary fiber, which can improve the microbiota population in gastrointestinal tract. However, whether inulin intake can further regulate the profile of microbiota and metabolic activities in milk remains unclear. In subclinical mastitic cows, we found that inulin supplementation could reduce the abundance of Escherichia -Shigella , Pseudomonas , Rhodococcus , Burkholderia-Caballeronia-Paraburkholderia and the levels of (±)12, 13 - DiHOME, leukotriene E3 and 20-Carboxy-Leukotriene B4 etc., while, elevated the abundance of Lactobacillus , Bifidobacterium , and Muribaculaceae as well as the levels of prostaglandin A1 (PGA1), 8-iso-15-keto-PGE2 and benzoic acid etc., in milk. These data suggest that inulin intake affects the profile of microorganisms and metabolites in milk, which provides an alternative for the regulation of mastitis.

Prostaglandin A1 inhibits the phosphorylation of tau via activating protein phosphotase 2A in a michael addition mechanism at the site of cysteine 377

Background: Prostaglandin (PG) A1 is a metabolic product of cyclooxygenase 2 (COX-2), which potentially involved in regulating the development and progression of Alzheimer’s disease (AD). As a cyclopentenone (cy) PG, PGA1 is characterized by the presence of a chemically reactive α, β-unsaturated carbonyl. Although PGA1 is potentially involved in regulating multiple biological processes via michael addition, its specific roles in AD remained unclear.Methods: The tauP301S transgenic (Tg) mice were employed as in vivo AD models and neuroblastoma (N) 2a cells as in vitro neuronal models. By intracerebroventricular injected (i.c.v) with PGA1, the binding proteins to PGA1 are analyzed by HPLC-MS-MS. In addition, western blots are used to determine the phosphorylation of tau in PGA1 treated Tg mice in the absence or presence of okadaic acid (OA), an inhibitor of protein phosphotase (PP) 2A. Combining a synthesis of pull down assay, immunoprecipitation, western blots and HPLC-MS-MS, PP2A scaffold subunit A alpha (PPP2R1A) was identified to be activated by directly binding on PGA1 in cysteine 377-dependent manner. Via inhibiting the hyperphosphorylation of tau, morris maze test was employed to determine the inhibitory effects of PGA1 on cognitive decline of tauP301S Tg mice.Results: By incubation with neuroblastoma (n)2a cells and pull down assay, mass spectra (MS) analysis revealed that PGA1 binds with more than 1000 proteins, among which contains the proteins of AD, especially tau protein. Moreover, short-term administration of PGA1 to tauP301S Tg mice significantly decreased the phosphorylation of tau at the sites of Thr181, Ser202 and Ser404 in a dose-dependent manner. To the reason, it’s caused by activating PPP2R1A in tauP301S Tg mice. More importantly, PGA1 has the ability to form michael adduct with PPP2R1A via its cysteine 377 motif, which is critical for the enzymatic activity of PP2A. By activating PP2A, long-term application of PGA1 to tauP301S Tg mice significantly reduced the phosphorylation of tau, which results in improving the cognitive decline of tauP301S Tg mice.Conclusion: Our data provided the first insights needed to decipher the mechanisms underlying the ameliorating effects of PGA1 on cognitive decline of tauP301S Tg mice via activating PP2A in a PPP2R1AC377-dependent Michael adducting mechanisms.