Complement activation in SCID and nude mice is related to severity of tissue inflammation in the Candida mastitis model

A small animal model of localised candidiasis is needed for the evaluation of new antifungal compounds. Mammary glands of immunocompetent (BALB/cJ) and immunodeficient (SCID and athymic nude) mice were infected with a wild-type of Candida albicans. Some of the animals were treated with amphotericin B (AmB) while others were treated with saline and acted as controls. The histologic changes of infected mammary gland tissues and a number of other organs were evaluated. Complement (C) activation was analysed by immunoelectrophoretic quantification of molecules with C3c epitopes (C3, C3b, iC3b, and C3c) in serum. In all animals the organisms were confined to the mammary glands. Serum C3c levels were significantly higher (P<0.05) in infected untreated BALB/cJ and SCID mice, which also had severe mammary gland tissue inflammation, compared with control mice treated with AmB (4 mg kg(-1) i.p. once daily for 4 days). Both treated and control nude mice showed less tissue inflammation compared to BALB/cJ and SCID mice, and revealed insignificant activation of the complement system. It is concluded that innate immune response is important in the control of candidiasis and that the murine mastitis model is useful for immunopathological studies as well as evaluation of potential antifungal compounds.     

Mitogen-activated protein kinase-defective Candida albicans is avirulent in a novel model of localized murine candidiasis

Candida albicans strains with a deletion of the mitogen-activated protein kinase CEK1 gene are defective in the yeast to hyphal transition on solid surfaces in vitro. The virulence of a cek1Δ/cek1Δ null mutant strain was compared with its wild-type parent strain (WT) in a novel model of localized candidiasis. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 50 μl suspension containing 1×10⁵, 1×10⁶ and 1×10⁷ blastospores before death. Infected and non-infected control glands were evaluated pathologically. All animals infected with cek1Δ/cek1Δ null mutant strains showed no lesions while 65% of animals infected with the WT strain had severe lesions characterized by widespread heterophilic infiltration, necrosis, and abscess formation. As an additional control, animals infected with the disrupted strain complemented with the WT CEK1, on a replicating plasmid, also showed severe pathological changes similar to the WT strain. These results clearly demonstrate that the CEK1 gene codes for a virulence determinant of C. albicans and that the mouse mastitis model is well suited for the discriminative study of the pathogenicity of different C. albicans strains.     

The effects of colchicine on milk secretion, mammary metabolism and blood flow in the goat

Mammary function in the conscious goat was studied during colchicine-induced depression of milk secretion in one mammary gland. Milk yield of the treated gland was reduced to approximately a quarter of previous, while there were significant increases in afternoon milk yield from the untreated glands on the 2nd and 3rd days after treatment in goats in late lactation. Milk composition in the untreated glands was not significantly affected. In the treated gland, milk [Na+], [Cl-], [citrate] and [protein] increased while [K+] and [lactose] decreased, although the time course of these changes differed; milk [fat] was unaffected. Mammary extractions ((A-V)/A) of glucose, acetate and most amino acids were significantly decreased during the period of maximal inhibition of secretion. There were no significant changes in arterial plasma concentrations of glucose, acetate or any essential amino acids. In another series of experiments, mammary blood flow increased and then returned to normal after colchicine treatment even though milk yield and mammary glucose uptake decreased markedly; oxygen uptake was not significantly affected. The results are discussed in relation to the actions of colchicine on the mammary secretory cell, to the normal control of mammary blood flow and to the mechanism of compensation by the untreated gland.     

Effects of BRCA1 transgene expression on murine mammary gland development and mutagen-induced mammary neoplasia

To characterize the role of BRCA1 in mammary gland development and tumor suppression, a transgenic mouse model of BRCA1 overexpression was developed. Using the mouse mammary tumor virus (MMTV) promoter/enhancer, transgenic mice expressing human BRCA1 or select mutant controls were generated. Transgenic animals examined during adolescence were shown to express the human transgene in their mammary glands. The mammary glands of 13-week-old virgin homozygous MMTV-BRCA1 mice presented the morphology of moderately increased lobulo-alveolar development. The mammary ductal trees of both hemizygous and homozygous MMTV-BRCA1t340 were similar to those of control non-transgenic littermates. Interestingly, both hemi- and homozygous mice expressing a splice variant of BRCA1 lacking the N-terminal RING finger domain (MMTV-BRCA1sv) exhibited marked mammary lobulo-alveolar development, particularly terminal end bud proliferation. Morphometric analyses of mammary gland whole mount preparations were used to measure epithelial staining indices of ~35% for homozygous MMTV-BRCA1 mice and ~60% for both hemizygous and homozygous MMTV-BRCA1sv mice versus ~25% for non-transgenic mice. Homozygous MMTV-BRCA1 mice showed delayed development of tumors when challenged with 7,12 dimethylbenzanthracene (DMBA), relative to non-transgenic and homozygous BRCA1t340 expressing mice. In contrast, homozygous MMTV-BRCA1sv transgenic animals were sensitized to DMBA treatment and exhibited a very rapid onset of mammary tumor development and accelerated mortality. MMTV-BRCA1 effects on mortality were restricted to DMBA-induced tumors of the mammary gland. These results demonstrate in vivo roles for BRCA1 in both mammary gland development and in tumor suppression against mutagen-induced mammary gland neoplasia.     

The protein-synthesizing activity of ribosomes isolated from the mammary gland of lactating and pregnant guinea pigs

1. Polyribosome preparations were made from the deoxycholate-treated post-nuclear fractions obtained by the disruption of mammary glands from lactating and pregnant guinea pigs. 2. A high proportion of large polyribosomes was obtained from the glands of lactating animals whereas mainly small polyribosomes were obtained from the glands of pregnant animals. The isolated preparations incorporated [(14)C]phenylalanine into protein. The polyribosomes from the glands of pregnant animals were less active than those from the glands of lactating animals but the activity of the former was stimulated more by poly(U) than was the latter. 3. The ribosomes from mammary gland could be dissociated into subunits after incubation, under conditions necessary for protein synthesis, in the presence of puromycin. The subunits could be recombined to give a preparation that actively polymerized [(14)C]phenylalanine in the presence of poly(U). The subunits from guinea-pig mammary gland could be combined with subunits from liver of either guinea pig or rat. Hybrid ribosomes were also formed from subunits derived from glands of pregnant and lactating animals. The hybrids were as active as were the ribosomes formed by reassociation of subunits from the same tissue, suggesting that in this respect the ribosomes from pregnant animals were not defective. 4. Polyribosomes from mammary glands of lactating animals when incubated with cell sap from the same source were tested for their ability to synthesize alpha-lactalbumin. The polyribosomes were incubated in the presence of [(3)H]leucine and alpha-lactalbumin was isolated from the supernatant. The protein was finally treated with cyanogen bromide and the C-terminal and N-terminal fragments were separated and their radioactivity was determined. Both fragments were radioactive consistent with the synthesis of alpha-lactalbumin. 5. The results are discussed in relation to protein synthesis in the mammary gland after parturition.     

Treatment with Gentamicin on a Murine Model of Protothecal Mastitis

The aim of this study was to establish a murine protothecal mastitis model and to evaluate the treatment efficiency of gentamicin. Challenge routes were determined with a pathogenic Prototheca zopfii genotype 2 (P. zopfii) strain. 25 BALB/c mice were inoculated in mammary glands with graded dosages (10³, 10⁴, 10⁵, 10⁶, 10⁷ CFU of P. zopfii) and killed on the 7th day. Another 25 animals were also killed at 1, 3, 5, 7, and 9 days after inoculation of 1 × 10⁶ CFU of P. zopfii, the milk somatic cell counts, pathological section of mammary glands, and P. zopfii burden were observed. The antimicrobial activity was tested using disc diffusion test and minimum inhibitory concentrations. Gentamicin was given intramuscularly to analyze the therapeutic effect. The results showed that the best infection route was intra-mammary gland, and the mastitis model was established with 1 × 10⁶ CFU of P. zopfii. After infection, the somatic cell counts increased significantly. The pathological reaction mainly consisted of infiltration of inflammatory cells, destruction of acini, accumulation of lymphocyte cells and the severity of the changes was dosage and time-dependent. The P. zopfii burden revealed that P. zopfii continuously replicated. In vitro susceptibility tests indicated that the Prototheca strains were antimicrobial susceptible to gentamicin at concentrations between 0.03 and 4 μg/ml. In vivo therapeutic assay demonstrated that high concentrations of gentamicin (≥20 mg/kg) could inhibit the growth of P. zopfii. We conclude that the murine model of protothecal mastitis was established successfully and gentamicin may be an effective choice for treatment of P. zopfii.     

Influence of melatonin on mammary gland growth: in vivo and in vitro studies

Our objective was to determine whether melatonin influenced mammary growth in response to mammogenic hormones. Prepubertal female BALB/c mice were injected for 9 days with 1 microgram of 17 beta-estradiol and 1 mg of progesterone or 17 beta-estradiol/progesterone plus 50, 100, or 200 micrograms of melatonin. Area of the parenchyma and total DNA content of the second thoracic gland were similar between controls and melatonin-injected mice. However, micrograms of DNA/100 mg of mammary tissue were lower in animals treated with 17 beta-estradiol/progesterone plus 200 micrograms of melatonin than in controls. Triglyceride content of mammary glands from animals treated with 100 or 200 micrograms of melatonin/day increased relative to controls. In an in vitro experiment, thoracic mammary glands of 21-day-old mice were cultured for 6 days in a mammogenic milieu of hormones (17 beta-estradiol/progesterone, aldosterone, bovine prolactin, growth hormone, and insulin) with 0 (control), 10(-6), 10(-9), or 10(-12) M melatonin. Relative to controls, 10(-12) M melatonin increased and 10(-6) M melatonin decreased mammary DNA and uptake of [methyl-3H]thymidine. We conclude that high doses of melatonin reduce mammary development in normal mice and that some of this effect may be mediated directly at the mammary tissue.     

Overexpression of a kinase-deficient transforming growth factor-beta type II receptor in mouse mammary stroma results in increased epithelial branching

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through heteromeric type I and type II serine/threonine kinase receptors. Transgenic mice that overexpress a dominant-negative mutation of the TGF-beta type II receptor (DNIIR) under the control of a metallothionein-derived promoter (MT-DNIIR) were used to determine the role of endogenous TGF-betas in the developing mammary gland. The expression of the dominant-negative receptor was induced with zinc and was primarily localized to the stroma underlying the ductal epithelium in the mammary glands of virgin transgenic mice from two separate mouse lines. In MT-DNIIR virgin females treated with zinc, there was an increase in lateral branching of the ductal epithelium. We tested the hypothesis that expression of the dominant-negative receptor may alter expression of genes that are expressed in the stroma and regulated by TGF-betas, potentially resulting in the increased lateral branching seen in the MT-DNIIR mammary glands. The expression of hepatocyte growth factor mRNA was increased in mammary glands from transgenic animals relative to the wild-type controls, suggesting that this factor may play a role in TGF-beta-mediated regulation of lateral branching. Loss of responsiveness to TGF-betas in the mammary stroma resulted in increased branching in mammary epithelium, suggesting that TGF-betas play an important role in the stromal-epithelial interactions required for branching morphogenesis.     

Quantitative and qualitative analysis of the antifungal activity of allicin alone and in combination with antifungal drugs

The antifungal activity of allicin and its synergistic effects with the antifungal agents flucytosine and amphotericin B (AmB) were investigated in Candida albicans (C. albicans). C. albicans was treated with different conditions of compounds alone and in combination (allicin, AmB, flucytosine, allicin + AmB, allicin + flucytosine, allicin + AmB + flucytosine). After a 24-hour treatment, cells were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) to measure morphological and biophysical properties associated with cell death. The clearing assay was conducted to confirm the effects of allicin. The viability of C. albicans treated by allicin alone or with one antifungal drug (AmB, flucytosine) in addition was more than 40% after a 24-hr treatment, but the viability of groups treated with combinations of more than two drugs was less than 32%. When the cells were treated with allicin alone or one type of drug, the morphology of the cells did not change noticeably, but when cells were treated with combinations of drugs, there were noticeable morphological changes. In particular, cells treated with allicin + AmB had significant membrane damage (burst or collapsed membranes). Classification of cells according to their cell death phase (CDP) allowed us to determine the relationship between cell viability and treatment conditions in detail. The adhesive force was decreased by the treatment in all groups compare to the control. Cells treated with AmB + allicin had a greater adhesive force than cells treated with AmB alone because of the secretion of molecules due to collapsed membranes. All cells treated with allicin or drugs were softer than the control cells. These results suggest that allicin can reduce MIC of AmB while keeping the same efficacy.     

Expression of Lysostaphin in Milk of Transgenic Mice Affects the Growth of Neonates

As an initial step towards enhancing mastitis resistance in dairy animals, we generated BLG-Lys transgenic mice that secrete lysostaphin, a potent antistaphylococcal protein, in their milk. In the current study, we continue our assessment of lysostaphin as a suitable antimicrobial protein for mastitis resistance and have investigated mammary gland development and function in three lines of transgenic mice. As the lines were propagated, there was a tendency for fewer BLG-Lys litters to survive to weaning (51% as compared to 90% for nontransgenic lines, p = 0.080). Nontransgenic pups fostered on dams from these three lines exhibited diminished growth rates during the first week of lactation. Rates of gain became comparable to pups on nontransgenic dams at later time points. Initial slow growth also resulted in decreased weaning weights for pups nursed by transgenic dams (15.35±0.27 g) when compared to pups delivered and nursed by nontransgenic dams (18.61 ± 0.61 g; p < 0.001), but the effect was temporary, as similar weights were attained by adulthood. Milk yield at peak lactation was not different between BLG-Lys (0.79 ± 0.33 g) and nontransgenic (0.91 ± 0.38 g; p = 0.166) dams. Histological examination of the transgenic mammary glands during gestation revealed no differences when compared to control glands; however, at early lactational stages, the BLG-Lys glands exhibited less alveolar area than control glands and a delay in lobulo-alveolar maturation. The results clearly demonstrate reduced growth of neonates on BLG-Lys dams; whether the poor pup performance can be attributed to delayed mammary development or the gland development merely reflects reduced suckling stimuli from the pups remains to be determined.Authors Abhijit Mitra and Kathleen S. Hruska contributed equally to this work     

Study of cytokines involved in the prevention of a murine experimental breast cancer by kefir

Previous studies have shown that compounds released during milk fermentation by Lactobacillus helveticus are implicated in the antitumour effect of this product. Here the effects of the consumption, during 2 or 7 days, of kefir or kefir cell-free fraction (KF) on the systemic and local immune responses in mammary glands and tumours using a murine hormone-dependent breast cancer model were studied. In the tumour control group, mice did not receive these products. At the end of the feeding period, mice were injected subcutaneously with tumour cells in the mammary gland. Four days post-injection, they received kefir or KF on a cyclical basis. Rate of tumour development, cytokines in serum; mammary gland tissue, and tumour isolated cells were monitored. Two-day cyclical administration of both products delayed tumour growth. Both kefir and KF increased IL-10 in serum and decreased IL-6(+) cells (cytokine involved in oestrogen synthesis) in mammary glands. Two-day cyclical administration of KF increased IL-10(+) cells in mammary glands and in tumours and decreased IL-6(+) cells in tumour. This study demonstrated the modulatory capacity of KF on the immune response in mammary glands and tumours and the importance of the administration period to obtain this effect.     

Fine structural studies of rat seminal vesicle in castrated and intact animals following estrogen treatment.

The effect of estradiol and/or testosterone upon secretion by seminal vesicle in castrated and intact rats was assessed in young adult Sprague-Dawley rats, using light microscopy (LM), transmission (TEM) and scanning (SEM)electron microscopy. Hormones were injected daily for ten days beginning ten days after castrations were performed. The normal rat seminal vesicle, as revealed by SEM, was characterized by a large saccular lumen with highly folded walls. Cell surfaces were covered with microvilli, or occasionally displayed a protruding, ruffled surface, sparsely covered with short microvilli. Cytology was normal in testosterone-treated animals. Estradiol treatment of castrated animals stimulated secretion by seminal vesicle epithelial cells as evidenced by the presence of normal secretory bodies, the presence of RER, and moderately hypertrophied Golgi complexes. These glands were not heavier than were glands from castrated, untreated animals, although the epithelial cells were significantly taller. Secretion was maintained in intact animals treated with estradiol, although glands were smaller and epithelial height was reduced. Estradiol and testosterone treatment in combination did not appear to have an additive effect on secretion, weight of the gland, or epithelial height. The following results support the hypothesis that estrogen-induced prolactin synthesis and release may be involved in the mechanism by which estradiol effected stimulation of seminal vesicle epithelium. Prolactin-treated, castrated animals exhibited focal areas of stimulated epithelium. In hypophysectomized animals (untreated controls), the seminal vesicle epithelium retained some secretory bodies and secretory fluid in the glandular lumen; epithelial height was taller than that in castrated controls. Estrogen treatment reduced the epithelial height to that of castrated controls; there was no evidence of secretion. This suggests that in the absence of anterior pituitary hormones, including prolactin, the stimulatory effect of estradiol on seminal vesicle epithelium was nullified. In adrenalectomized/castrated animals, estradiol treatment stimulated secretion in seminal vesicle epithelium just as in non-adrenalectomized/castrated animals. This indicates that the adrenal gland plays a non-essential role in the action of estrogen on seminal vesicle epithelium.     

Solexa sequencing and custom microRNA chip reveal repertoire of microRNAs in mammary gland of bovine suffering from natural infectious mastitis

Identification of microRNAs (miRNAs), target genes and regulatory networks associated with innate immune and inflammatory responses and tissue damage is essential to elucidate the molecular and genetic mechanisms for resistance to mastitis. In this study, a combination of Solexa sequencing and custom miRNA chip approaches was used to profile the expression of miRNAs in bovine mammary gland at the late stage of natural infection with Staphylococcus aureus, a widespread mastitis pathogen. We found 383 loci corresponding to 277 known and 49 putative novel miRNAs, two potential mitrons and 266 differentially expressed miRNAs in the healthy and mastitic cows’ mammary glands. Several interaction networks and regulators involved in mastitis susceptibility, such as ALCAM, COL1A1, APOP4, ITIH4, CRP and fibrinogen alpha (FGA), were highlighted. Significant down‐regulation and location of bta‐miR‐26a, which targets FGA in the mastitic mammary glands, were validated using quantitative real‐time PCR, in situ hybridization and dual‐luciferase reporter assays. We propose that the observed miRNA variations in mammary glands of mastitic cows are related to the maintenance of immune and defense responses, cell proliferation and apoptosis, and tissue injury and healing during the late stage of infection. Furthermore, the effect of bta‐miR‐26a in mastitis, mediated at least in part by enhancing FGA expression, involves host defense, inflammation and tissue damage.     

Vaccination with Staphylococcus aureus fibrinogen binding proteins (FgBPs) reduces colonisation of S. aureus in a mouse mastitis model

Abstract A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus . The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibronogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus .     

Intramammary immunization with live-attenuated Staphylococcus aureus: microbiological and immunological studies in a mouse mastitis model

Abstract Mammary infection was induced in lactating mice by intramammary injection of Staphylococcus aureus . Histopathological analysis revealed infiltration and lesions of varying magnitude that were still apparent 21 days after the challenge. Concomitantly, viable S. aureus was recovered from infected mammary glands. Mice were immunized by the intramammary route with 5 × 10⁶ colony forming units of a temperature-sensitive mutant of S. aureus and subsequently received a boosting injection seven days later. On day 14 mice were challenged by the intramammary route with the wild-type strain. Intramammary immunization induced a significant increase in milk IgA ( P < 0.05), serum IgG ( P < 0.05) and serum IgA ( P < 0.05) on the day of the challenge, when compared with non-immunized mice. Immunization decreased significantly ( P < 0.01) the number of S. aureus colony forming units recovered 96 h after intramammary challenge. In conclusion, the feasibility of immunizing locally with temperature-sensitive S. aureus to induce immunity in the mouse mammary gland was demonstrated. The mouse model of mastitis is proposed as a useful system for screening temperature-sensitive S. aureus strains to be utilized in the development of a vaccine.     

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates

The goal of this study was to examine the ability of a commercially available feed additive (OmniGen-AF) to reduce mammary infections caused by a single strain of mastitic pathogens (Streptococcus uberis, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae) and to examine the effects of the additive on markers of mammary immunity. Four experiments were completed using a murine model of bovine mastitis. Infection progression was examined using Sybr-green- and TaqMan-based quantitative PCR assays of 16S ribosomal DNA. Infection of the mammary gland with all pathogens caused rapid (24 to 48 h) appearance of pathogen DNA in mammary tissue. Provision of the feed additive for 2 weeks before infection significantly (P < 0.05) reduced the extent of pathogen DNA accumulation in models of S. uberis, E. coli and S. aureus infection. The additive was ineffective in reducing mammary infections caused by K. pneumoniae. We examined mechanisms of action of the additive through assessment of mammary concentrations of mammary myeloperoxidase (MPO), major histocompatibility complex 2 class II (MHC) and macrophage inflammatory protein-1α (MIP) messenger RNA (mRNA) concentrations and by examining serum complement C3 concentration. Infection of the mammary gland increased concentrations of MPO and MHC mRNAs (P < 0.05). Ability of the pathogen to elicit changes in mammary MPO and MHC gene expression was enhanced by the provision of the additive for 2 weeks before infection. These data imply that the additive increased the mammary inflammatory response and increased antigen presentation during a mammary infection. Value of the additive in preventing mastitis in cattle awaits additional studies using a bovine model and further evaluation of additional strains of the pathogens used in this study.     

Toll-like receptor 4 is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis

Mastitis, an inflammatory response of the mammary tissue to invading pathogenic bacteria, is a common disease in breast-feeding women and dairy animals. Escherichia coli is a leading cause of mastitis in dairy animals. During the course of the disease the host mounts a strong inflammatory response, but specific bacterial virulence factors have not yet been identified. Here we report the use of a murine mastitis model to investigate the innate inflammatory reaction of the mammary gland. We show that lipopolysaccharide (LPS) infusion induces mastitis in wild-type mice (C3H/HeN), but not in mice expressing mutated Toll-like receptor 4 (TLR4) (C3H/HeJ). The wild-type phenotype was restored by adoptive transfer of TLR4-expressing macrophages into the alveolar milk space of C3H/HeJ mice. In contrast to the LPS treatment, infection with E. coli P4 (ECP4) resulted in inflammation even in the absence of LPS/TLR4 signalling, indicating that additional factors play a role in the pathogenesis of the intact bacteria. Furthermore, in the absence of functional TLR4 the infecting ECP4 invade the epithelial cells with high efficiency, forming intracellular microcolonies. However, adoptive transfer with TLR4-expressing macrophages drastically reduced the epithelial invasion. Taken together, these results indicate that ECP4 has an invasive potential, which is restricted by alveolar macrophages in response to the LPS/TLR4 signalling.     

Protocatechuic acid inhibits LPS-induced mastitis in mice through activating the pregnane X receptor

Mastitis refers to the inflammation in the mammary gland caused by various reasons. Protocatechuic acid (PCA) exerts anti-inflammatory effect. However, no studies have shown the protective role of PCA on mastitis. We investigated the protective effect of PCA on LPS-induced mastitis in mice and elucidated its possible mechanism. LPS-induced mastitis model was established by injection of LPS into the mammary gland. The pathology of mammary gland, MPO activity and inflammatory cytokine production were detected to evaluate the effects of PCA on mastitis. In vivo, PCA significantly attenuated LPS-induced mammary pathological changes, MPO activity, TNF-α and IL-1β production. In vitro, the production of inflammatory cytokines TNF-α and IL-1β was significantly reduced by PCA. Furthermore, LPS-induced NF-κB activation was also inhibited by PCA. In addition, PCA was found to activate pregnane X receptor (PXR) transactivation and PCA dose-dependently increased the expression of PXR downstream molecule CYP3A4. In addition, the inhibitory effect of PCA on inflammatory cytokine production was also reversed when PXR was knocked down. In conclusion, the protective effects of PCA on LPS-induced mastitis in mice through regulating PXR.