Anti-inflammatory Effect And Mechanism Of Chlorogenic Acid On Mastitis

Gram-negative bacteria such as E.coli is the major pathogenic bacteria thatcauses bovine mastitis. LPS, the main component of the outer membranes ofGram-negative bacteria, plays a key role in producing an inflammatory response. LPScould activate TLR4signaling pathway and induce inflammatory response. Studiesshowed that PPAR-γ played an important role in inflammatory response. In this study,we first confirmed the anti-inflammatory effects of chlorogenic acid in LPS-inducedmastitis in mice. Secondly, we investigated the anti-inflammatory mechanism ofchlorogenic acid on LPS-stimulated bovine and mouse mammary epithelial cells. Inthe last, the molecular targets of the anti-inflammatory actions of chlorogenic acidwere identified. All the results will provide the reference and experimental basis forprevention and treatment of bovine mastitis by anti-inflammatory medicine inveterinary clinical practice.Firstly, the mouse mastitis model induced by LPS was established. The lactatingBalb/C mice of5-7d were random divided into two groups: control group and LPSgroup. The fourth pair mammary glands of the mice were infusion with LPS(0.2mg/ml,50μL) through the mammary duct.24h after LPS induction, the mice werekilled. The pathological and histopathological changes of mammary gland wereobserved. Meanwhile, the activity of MPO and the level of TNF-α was detected. Theresults showed that the mammary gland showed redness, swelling, and congestion inLPS group. Meanwhile, the mammary histological examination also indicated thatmammary tissues infused with LPS showed thickening and edema of the alveoluswalls and inflammatory cells infiltration. In addition, the level of TNF-α wassignificantly increased in LPS group. These results demonstrated that the mousemastitis model induced by LPS was successfully established.Secondly, the mouse model of mastitis was induced by injection of LPS throughthe duct of mammary gland. Chlorogenic acid was administered intraperitoneally withthe dose of12.5,25,50mg/kg respectively1h before and12h after induction of LPS. In this study, the effect of chlorogenic acid on LPS-induced mice mastitis wasassessed through histopathological examination, myeloperoxidase (MPO) activityanalysis, ELISA assay, and western blot analysis. The results showed that chlorogenicacid significantly attenuated the activity of MPO and reduced TNF-α, IL-1β, and IL-6production compared with LPS group. Besides, western blot analysis showed thatchlorogenic acid could inhibit the expression of TLR4and the phosphorylation ofNF-κB and IκB induced by LPS. These results suggested that anti-inflammatoryeffects of chlorogenic acid against LPS-induced mastitis may be due to its ability toinhibit TLR4-mediated NF-κB signaling pathway. Therefore, chlorogenic acid may bea potent therapeutic reagent for the prevention of mastitis.Thirdly, to further detect the protective effect of chlorogenic acid on mastitis, themouse mammary epithelial cells were used. The cells were stimulated with LPS in thepresence or absence of chlorogenic acid. The expression of pro-inflammartorycytokines TNF-α, IL-6and IL-1β were determined by ELISA. NF-κB and IκBα weredetermined by Western blotting. The results showed that chlorogenic aciddose-dependently inhibited the expression of TNF-α, IL-6and IL-1β inLPS-stimulated mouse mammary epithelial cells. Western blot analysis showed thatchlorogenic acid suppressed LPS-induced NF-κB activation, IκBa degradation.We also detected the protective effect of chlorogenic acid on LPS-stimulatedbovine mammary epithelial cells. The cells were stimulated with LPS in the presenceor absence of chlorogenic acid. The expression of pro-inflammartory cytokines TNF-α,IL-6and IL-1β were determined by qRT-PCR. TLR4, NF-κB and IκBα weredetermined by Western blotting. The results showed that chlorogenic aciddose-dependently inhibited the expression of TNF-α, IL-6and IL-1β inLPS-stimulated bovine mammary epithelial cells. Western blot analysis showed thatchlorogenic acid suppressed LPS-induced TLR4signaling pathway.In the last, molecular targets of chlorogenic acid was identified. The bindingaffinity and activating of PPAR-γ were detected by PPAR-γ ligand binding assays andWestern blotting. Meanwhile, to detect whether the effects of chlorogenic acid onTLR4expression is dependent on PPAR-γ activation, GW9662, the specific PPAR-γantagonist was used. Then the cells were stimulated by LPS and TLR4expression was detected. The results showed that chlorogenic acid could bind and activate PPAR-γ.Furthermore, the inhibition effects of TLR4by chlorogenic acid can be reversed byGW9662, a specific antagonist for PPAR-γ. These results suggest that PPAR-γ may bethe anti-inflammatory targets of chlorogenic acid.In conclusion, these results suggest that chlorogenic acid activates PPAR-γ,thereby attenuating TLR4expression and TLR4mediated NF-κB activation and therelease of pro-inflammatory cytokines TNF-α, IL-1β and IL-6. These findings suggestthat chlorogenic acid may be a therapeutic agent against inflammatory diseases. Allthe results revealed that emodin may be used as a potential candidate in treatment ofmastitis.