Licochalcone A Protects the Blood–Milk Barrier Integrity and Relieves the Inflammatory Response in LPS-Induced Mastitis

Background/Aims: Mastitis is an acute clinical inflammatory response. The occurrence and development of mastitis seriously disturb women's physical and mental health. Licochalcone A, a phenolic compound in Glycyrrhiza uralensis, has anti-inflammatory properties. Here, we examined the effect of licochalcone A on blood-milk barrier and inflammatory response in LPS-induced mice mastitis.Methods:In vivo, we firstly established mice models of mastitis by canal injection of LPS to mammary gland, and then detected the effect of licochalcone A on pathological indexes, inflammatory responses and blood-milk barrier in this model. In vivo, Mouse mammary epithelial cells (mMECs) were treated with licochalcone A prior to the incubation of LPS, and then the inflammatory responses, tight junction which is the basic structure of blood-milk barrier were analyzed. Last, we elucidated the anti-inflammatory mechanism by examining the activation of mitogen-activated protein kinase (MAPK) and AKT/NF-κB signaling pathways in vivo and in vitro.Result: The in vivo results showed that licochalcone A significantly decreased the histopathological impairment and the inflammatory responses, and improved integrity of blood-milk barrier. The in vitro results demonstrated that licochalcone A inhibited LPS-induced inflammatory responses and increase the protein levels of ZO-1, occludin, and claudin3 in mMECs. The in vivo and in vitro mechanistic study found that the anti-inflammatory effect of licochalcone A in LPS-induced mice mastitis was mediated by MAPK and AKT/NF-κB signaling pathways.Conclusions and Implications: Our experiments collectively indicate that licochalcone A protected against LPS-induced mice mastitis via improving the blood–milk barrier integrity and inhibits the inflammatory response by MAPK and AKT/NF-κB signaling pathways

GLP-2 Suppresses LPS-Induced Inflammation in Macrophages by Inhibiting ERK Phosphorylation and NF-κB Activation

Background/Aims: GLP-2 has been shown to exert anti-inflammatory effects, but the underlying molecular mechanisms remained undefined. As macrophages are important in the development and maintenance of inflammation, we investigated whether exogenous GLP-2 modulates the expression of pro-inflammatory proteins in LPS stimulated murine peritoneal macrophages. Methods: Macrophages were pretreated with various concentrations of GLP-2 for 1 h and then stimulated with LPS. The effects on pro-inflammatory enzymes (iNOS and COX-2), and pro-inflammatory cytokines (TNF-a, IL-1ß and IL-6) were analysed by Western blotting, ELISA and qRT-PCR. We also examined whether NF-κB or MAPK signaling was involved in the effects of GLP-2. Results: In macrophages, GLP-2 blunted the effect of LPS on protein and mRNA expression levels of iNOS, COX-2, TNF-a, IL-1ß and IL-6. Pre-incubation of macrophages with GLP-2 also blunted LPS-induced IκB-a degradation, IκB-a phosphorylation and NF-κB translocation. In the presence of GLP-2, the effect of LPS treatment on ERK phosphorylation was also profoundly blunted. GLP-2 did, however, not significantly modify the effects of LPS on p38 and JNK activities. Conclusions: These findings demonstrate that in LPS primed macrophages, GLP-2 reduced pro-inflammatory enzymes and cytokine production via mechanisms involving the suppression of NF-κB activity and ERK phosphorylation

Quercitrin, an Inhibitor of Sortase A, Interferes with the Adhesion of Staphylococcal aureus

Sortase A (SrtA) is a cysteine transpeptidase of most Gram-positive bacteria that is responsible for the anchorage of many surface protein virulence factors to the cell wall layer. SrtA mutants are unable to display surface proteins and are defective in the establishment of infections without affecting microbial viability. In this study, we report that quercitrin (QEN), a natural compound that does not affect Staphylococcus aureus growth, can inhibit the catalytic activity of SrtA in fibrinogen (Fg) cell-clumping and immobilized fibronectin (Fn) adhesion assays. Molecular dynamics simulations and mutagenesis assays suggest that QEN binds to the binding sites of the SrtA G167A and V193A mutants. These findings indicate that QEN is a potential lead compound for the development of new anti-virulence agents against S. aureus infections

The therapeutic effect of Chlorogenic acid against Staphylococcus aureus infection through Sortase A inhibition

The emergence and wide spread of multi-drug resistant Staphylococcus aureus (S. aureus) requires the development of new therapeutic agents with alternative modes of action. Anti-virulence strategies are hoped to meet that need. Sortase A (SrtA) has attracted great interest as a potential drug target to treat infections caused by S. aureus, as many of the surface proteins displayed by SrtA function as virulence factors by mediating bacterial adhesion to specific organ tissues, invasion of host cells, and evasion of the host-immune responses. It has been suggested that inhibitors of SrtA might be promising candidates for the treatment and/or prevention of S. aureus infections. In this study, we report that Chlorogenic acid (CHA), a natural compound that lacks significant anti–S. aureus activity, inhibit the activity of SrtA in vitro (IC50=33.86±5.55μg/ml) and the binding of S. aureus to fibrinogen (Fg). Using molecular dynamics simulations and mutagenesis assays, we further demonstrate that CHA binds to the binding sites of C184 and G192 in the SrtA. In vivo studies demonstrated that CHA prevent mice from S. aureus-induced renal abscess, resulting in a significant survival advantage. These findings indicate that CHA is a promising therapeutic compound against SrtA during S. aureus infections

Flow cytometry analysis of immuno-deficient HUMAMICE.

<p>Comparative cell analysis by flow cytometry between immuno-deficient HUMAMICE (HLA-A2^+/+/DR1^+/+/H-2-β₂m^-/-/IAβ^-/-/Rag2^-/-/IL2rγ^-/-/Perf^-/-) and immuno-competent C57BL/6 mice. The left panel shows the analysis of immuno-deficient HUMAMICE and the right panel was the immuno-competent C57BL/6 mice. (A) Samples in Fig 1A were double-labelled with mCD3-PE and mCD19-FITC. (B) Samples in Fig 1B were labelled with mCD4-APC and mCD8-PE-Cy5.</p

The humoral immune response of hPBMC-HUMAMICE after immunization with HBsAg vaccine.

<p>(A) The HBsAg specific IgG and (B) HBsAg specific IgM responses of immunized hPBMC-HUMAMICE before (0 w) and every two weeks after immunization (before each immunization boost).</p

The experimental procedure of hPBMC transplantation and immunization with HBsAg vaccine in HUMAMICE.

<p>The experimental procedure of hPBMC transplantation and immunization with HBsAg vaccine in HUMAMICE.</p

The tumor growth kinetics in HUMAMICE after transfer of RAMOS specific splenocytes from parental Sure-L1 mice.

<p>(A) Tumor growth kinetics following transfer of tumor specific splenocytes from Sure-L1 mice in three immuno-deficient HUMAMICE bearing a solid RAMOS tumor (mouse No.926; 948; 952) and the control mouse No.953 without the transfer of tumor-bearing cells. (B) Tumor growth kinetics following transfer of purified tumor specific CD8⁺ T lymphocytes from Sure-L1 mice in two immuno-deficient HUMAMICE bearing a solid RAMOS tumor (mouse No.208 ; 326) and the control mouse No.323 without transfer of tumor-bearing cells. (C) Tumor growth kinetics following transfer of tumor specific splenocytes depleted of CD8⁺ T cells (purified CD8^- T lymphocytes) from Sure-L1 mice in three immuno-deficient HUMAMICE bearing a solid RAMOS tumor (mouse No.139; 212; 337) and the control mouse No.323 without transfer of tumor-bearing cells.</p

Summary of comparative cell analysis was implemented by flow cytometry between immuno-deficient HUMAMICE and immuno-competent C57BL/6 mice.

<p>Summary of comparative cell analysis was implemented by flow cytometry between immuno-deficient HUMAMICE and immuno-competent C57BL/6 mice.</p