The Anti-inflammatory Effect Of Nuclear Receptor LXRα On Bovine Mastitis And LXRα Agonists Screening

Bovine mastitis is one of the most important diseases that always causes hugeeconomic loss in dairy industry. According to the clinical symptoms, mastitis can bedivided into clinical and subclinical mastitis. The Gram-negative bacteria, such asEscherichia coli often causes clinical mastitis. LPS, the main component of outermembrane of Gram-negative bacteria, often causes severe inflammatory responses inmammary gland by activating TLR4signaling pathway. It is crucial to controlinflammatory responses of mammary gland to treat clinical mastitis. However, limitedby the drug residues in milk and other food safety factors, we lack the safe andeffective anti-inflammatory drugs in the productive practice. Therefore, it is veryimportant to seek new anti-inflammatory target and drugs to control mastitis.Traditional Chinese medicine is expected to treat bovine mastitis due to the drugresidue is relatively low and relatively safe. LXRα, one of the main members of thenuclear receptor superfamily, has been reported to play important roles in metabolism,infection and immunity. Furthermore, LXRα has been reported to have the ability toenhance milk fat production and have anti-inflammatory activity. LXRα may be usedas a new anti-inflammatory target in the future. In this study, we investigated the anti-inflammatory effects of LXRα through LPS-induced mice mastitis model and LPS-stimulated bovine and mouse mammary epithelial cells both in vivo and in vitro.T0901317was used as the agonist of LXRα. Secondly, we screened LXRα agonistfrom Schisandrin A, thymol, Cyanidin-3-O-β-glucoside, glycyrrhizin, Saikosaponin a,alpinetin, emodin, berberine, baicalin, baicalein, forsythiaside, curcumin, chlorogenicacid, astragalin, and taraxasterol in traditional Chinese medicine by LXRα receptorgene assay. Also, we assess the anti-inflammatory effects of the screened LXRαagonist. Finally, we investigated the anti-inflammatory effects and mechanism of thebetter screened LXRα agonist in the treatment of mastitis. First, we estiblished the LPS-induced mice mastitis model and LPS-stimulatedbovine and mouse mammary epithelial cells model to investigate the anti-inflammatory effects and mechanism of LXRα agonist T0901317by detecting MPOactivity; TNF-α expression; TLR4, NF-κB, and IRF3activation; and cholesterol levelin lipid rafts. The results showed that activation LXRα attenuated LPS-inducedmammary histopathologic changes, MPO activity, and inflammatory cytokines TNF-α,IL-1β and IL-6production. Activation LXRα also inhibited LPS-induced TNF-α, IL-1β and IL-6mRNA expression in bovine mammary epithelial cells. Furthermore,Activation LXRα attenuated cholesterol level in lipid rafts, and inhibited LPS-inducedTLR4recruitment into lipid rafts, as well as NF-κB and IRF3activation in LPS-stimulated mouse mammary epithelial cells. In conclusion, activation LXRα has aprotective effect against LPS-induced mastitis. The promising anti-inflammatorymechanisms of LXRα is associated with disrupting lipid rafts by depleting cholesteroland reducing translocation of TLR4to lipid rafts, thereby suppressing TLR4mediatedinflammatory response.Secondly, we screened LXRα agonist from15natural compounds mentionedabove by LXRα receptor gene assay. We assess the anti-inflammatory effects of thescreened LXRα agonist in LPS-stimulated bovine and mouse mammary epithelialcells by detecting TNF-α to choose the strongest LXRα agonist. Our results showedthat Cyanidin-3-O-β-glucoside, glycyrrhizin, Saikosaponin a, and taraxasterol couldactivate LXRα. Cyanidin-3-O-β-glucoside, glycyrrhizin, Saikosaponin a, andtaraxasterol could inhibit LPS-induced TNF-α production in bovine and mousemammary epithelial cells. The anti-inflammatory effects of Cyanidin-3-O-β-glucosideis stronger that taraxasterol, Saikosaponin a, and glycyrrhizin. These results suggestedthat Cyanidin-3-O-β-glucoside, glycyrrhizin, Saikosaponin a, and taraxasterol can beused as LXRα agonists and the anti-inflammatory effects of Cyanidin-3-O-β-glucoside is better than others.Finally, we investigated the anti-inflammatory effects and mechanism ofCyanidin-3-O-β-glucoside (C3G) in LPS-induced mice mastitis model and LPS-stimulated mouse mammary epithelial cells by detecting MPO activity, TNF-α expression, NF-κB, and IRF3activation, and cholesterol level in lipid rafts. Theresults showed that C3G could attenuate mammary histopathologic changes, andmyeloperoxidase (MPO) activity; inhibit TNF-α, IL-1β and IL-6production; causedby LPS. Meanwhile, C3G dose-dependently inhibited TNF-α and IL-6in LPS-stimulated mouse mammary epithelial cells. C3G suppressed LPS-induced NF-κB andIRF3activation. Furthermore, C3G disrupted the formation of lipid rafts by depletingcholesterol. Moreover, C3G activated LXR-ABCG1-dependent cholesterol efflux.Knockdown of LXRα abrogated the anti-inflammatory effects of C3G. In conclusion,C3G has a protective effect on LPS-induced mastitis. The promising anti-inflammatory mechanisms of C3G is associated with up-regulation of the LXRα-ABCG1pathway which result in disrupting lipid rafts by depleting cholesterol,thereby suppressing TLR4mediated NF-κB and IRF3signaling pathways induced byLPS.In conclusion, this study confirmed that LXRα could be used as a potential targetin the treatment of mastitis. We screened four LXRα agonists Cyanidin-3-O-β-glucoside, glycyrrhizin, Saikosaponin a and taraxasterol from15natural compoundsand confirmed that the anti-inflammatory effects of Cyanidin-3-O-β-glucoside is thestrongest one. Furthermore, our results showed that C3G exhibited anti-inflammatoryeffects by activating LXRα-ABCG1to inhibited TLR4signaling pathway. Theseresults provided preliminary experimental data and reference in researching drugs forthe treatment of mastitis based on the target of LXRα.