The Anti-inflammatory Effects And Mechanisms Oof Astragalin N LPS-induced Mouse Mastitis

Mastitis is a prevalent and economic disease that affects dairy ruminants and cattle industryworldwide. Lipopolysaccharide (LPS), which is possessed by the outer membrane of E.coli, is thepredominant factor causing clinical bovine mastitis. Nowdays, more and more scientists arefocused on natural compounds to prevent mastitis. In this study, we first investigate the molecularmechanism of astragalin in modifying LPS-induced signaling pathways in RAW264.7cells andprimary cultured mouse epithelial cells, then an LPS-induced mice mastitis model was eatablishedto envaluate its anti-inflammatory and therapeutic effect against mastitis.In the first place, the molecular mechanism of astragalin on LPS-stimulated RAW264.7cellswas studied in vitro. Specifically, a CCK-8assay was used to analyse the effect of astragalin oncell viability; the release of pro-inflammatory cytokinesTNF-, IL-1β, and IL-6in the culturesupernatants were determined by ELISA; proteins iNOS, COX-2, and P65, IκB, P38, ERK, JNK,were analyzed by Western blot. The results showed that this molecule (25,50, and100μg/mL) didnot display any cellular toxicity against RAW264.7cells. Astragalin attenuated the release ofpro-inflammatiory cytokines TNF-, IL-1β, and IL-6; inhibited the production of inflammatorymediator iNOS and COX-2; suppressed the phosphorylation of P65, IκB, and P38, ERK.In the second place, to investigate the potential therapeutic effect of astragalin in mastitis, amurine model of mastitis was induced by administration of LPS in mammary gland. Astragalinwas applied1h before and12h after LPS treatment. The tissue samples were collected, and theMPO activity in the mammary gland was measured; the release of pro-inflammatory cytokinesTNF-, IL-1β, and IL-6in the mammary gland was determined by ELISA. The results showedthat astragalin substancially ameliorated LPS-induced pathlogical changes; reduced the MPOactivity; decreased the expression of TNF-, IL-1β, and IL-6; in a dose-dependent manner inLPS-challenged mice mammary gland.In the third place, the anti-inflammatory and protective mechanism of astragalin was furtherinvestigated in LPS-stimulated mouse epithelial cells, which were primary cultured by collagenaseI/II/trypsin mixture digestion. More precisey, a CCK-8assay was used to analyse the effect ofastragalin on cell viability; the release of pro-inflammatory cytokines TNF-and IL-6in theculture supernatants were determined by ELISA; proteins iNOS, COX-2, and P65, IκB, P38, ERK,JNK, were analyzed by Western blot. The results showed that this molecule (25,50, and100μg/mL) did not display any cellular toxicity against primary cultured mouse mammary epithelialcells. Astragalin attenuated the release of pro-inflammatiory cytokines TNF-and IL-6; inhibitedthe production of inflammatory mediator iNOS and COX-2; suppressed the phosphorylation of In conclusion, our results demenstrated that astragalin attenuated inflammatory response viathe suppression of NF-κB and MAPKs signaling pathways in vitro and in vivo, which indicate thatastragalin may be used as a potential anti-mastitis therapy in the future.