Inflammatory Signaling via PEIZO1 Engages and Enhances the LPS-Mediated Apoptosis during Clinical Mastitis.

Bovine clinical mastitis is characterized by inflammation and immune responses, with apoptosis of mammary epithelial cells as a cellular reaction to infection. PIEZO1, identified as a mechanotransduction effector channel in nonruminant animals and sensitive to both mechanical stimuli or inflammatory signals like lipopolysaccharide (LPS). However, its role in inflammatory processes in cattle has not been well-documented. The aim of this study was to elucidate the in situ expression of PIEZO1 in bovine mammary gland and its potential involvement in clinical mastitis. We observed widespread distribution and upregulation of PIEZO1 in mammary epithelial cells in clinical mastitis cows and LPS-induced mouse models, indicating a conserved role across species. In vitro studies using mammary epithelial cells (MAC-T) revealed that LPS upregulates PIEZO1. Notably, the effects of PIEZO1 artificial activator Yoda1 increased apoptosis and NLRP3 expression, effects mitigated by PIEZO1 silencing or NLRP3 inhibition. In conclusion, the activation of the PIEZO1-NLRP3 pathway induces abnormal apoptosis in mammary epithelial cells, potentially serving as a regulatory mechanism to combat inflammatory responses to abnormal stimuli.

Hordenine Alleviates Lipopolysaccharide-Induced Mastitis by Suppressing Inflammation and Oxidative Stress, Modulating Intestinal Microbiota, and Preserving the Blood-Milk Barrier.

Mastitis is a common mammalian disease occurring in the mammary tissue and poses a major threat to agriculture and the dairy industry. Hordenine (HOR), a phenylethylamine alkaloid naturally extracted from malt, has various pharmacological effects, but its role in mastitis is unknown. The aim of this study was to investigate the role of HOR and its underlying mechanism in a lipopolysaccharide (LPS)-induced inflammatory response model of mouse mammary epithelial cells (EpH4-Ev) and mouse mastitis model. The experimental results showed that HOR attenuated LPS-induced mammary tissue damage (from 3.75 ± 0.25 to 1.75 ± 0.25) and restored the integrity of the blood-milk barrier. Further mechanistic studies revealed that HOR inhibited LPS-induced overactivation of the TLR4-MAPK/NF-κB signaling pathway and activated the AMPK/Nrf2/HO-1 signaling pathway. Additionally, HOR altered the composition of the intestinal microbiota in mice, ultimately reducing the extent of inflammatory injury (from 3.33 ± 0.33 to 0.67 ± 0.33) and upregulating the expression of tight junction proteins (ZO-1, occludin, and claudin-3). The findings of this study provide a theoretical basis in the rational use of HOR for the prevention and treatment of mastitis and the maintenance of mammalian mammary gland health.

Endogenous retroviruses modulate the susceptibility of mice to Staphylococcus aureus-induced mastitis by activating cGAS-STING signaling.

Recently studies showed that cow mastitis seriously affected the economic benefit of dairy industry and pathogen infection including S. aureus is the main cause of mastitis. However, there is still a lack of safe and effective treatment for S. aureus-induced mastitis due to its complex pathogenesis. Endogenous retroviruses (ERVs) have long been symbiotic with mammals, and most ERVs still have the ability to produces complementary DNA (cDNA) by reverse transcription, whose induction by commensal or pathogens can regulate host immunity and inflammatory responses through the cGAS-STING pathway. However, whether and how ERVs participate in the pathogenesis of S. aureus-induced mastitis still unclear. In this study, we found that S. aureus treatment increased the levels of ERVs and IFN-ß. Inhibition the transcription of ERVs by emtricitabine alleviated S. aureus-induced mammary injury, reduced mammary bacterial burden, and inhibited the production of mammary proinflammatory factors including TNF-α, IL-1ß and MPO activity. Moreover, inhibition of ERVs restored the function of blood-milk barrier caused by S. aureus. Next, we showed that S. aureus infection activated mammary cGAS-STING signaling pathway, which was mediated by ERVs, as evidenced by emtricitabine inhibited S. aureus-induced activation of the cGAS-STING pathway. Interestingly, inhibition of cGAS-STING by Ru.521 and H151 respectively, significantly alleviated S. aureus-induced mammary injury and inflammatory responses, which was associated with the inhibition of NF-κB and NLRP3 signaling pathways. In conclusion, our study revealed that ERVs regulate the development of S. aureus-induced mastitis in mice through NF-κB- and NLRP3-mediated inflammatory responses via the activation of cGAS-STING pathway, suggesting that targeting ERVs-cGAS-STING axis may be a potential approach for the treatment of S. aureus-induced mastitis.

Seasonal assessment of mastitis in crossbred goats: A thermographic approach.

Sub-clinical mastitis exhibits a higher prevalence in dairy goats than clinical mastitis, necessitating the adoption of non-invasive diagnostic techniques such as infrared thermography (IRT) to detect this economically significant production disease in the dairy sector. Accordingly, this study aims to employ IR imaging of the udder and teat quarters of lactating crossbred goats (Alpine × Beetal and Sanen × Beetal) across various seasons, utilising IRT, to discern cases of sub-clinical (SCM) and clinical mastitis (CM). Over a year, 100-110 lactating crossbred dairy goats underwent consistent IRT screenings, followed by a comprehensive evaluation of udder health status using the California mastitis test and somatic cell count (SCC). The receiver operating characteristic (ROC) analysis was conducted to establish the cut-off values for different thermographic parameters in this study. The results revealed that the SCC increased significantly (p < 0.01) in healthy, SCM, and CM milk samples across the seasons. The analysis of ROC revealed a comparatively higher sensitivity, specificity, and accuracy for udder thermograms during SCM than CM and vice versa for teat thermograms. IRT analysis reflected a difference (p < 0.01) in the udder and teat thermograms among quarters of healthy, SCM, and CM in summer, winter, autumn, and rainy seasons. A significant increase (p < 0.01) in udder thermograms was observed for quarters affected with SCM and CM relative to healthy, with an increase of 1.89 and 2.94 °C in winter, 0.85 and 1.63 °C in summer, 0.73 and 1.41 °C in rainy, and 1.33 and 2.38 °C in autumn, respectively. Similarly, for teat thermograms it was 1.79 and 2.81 °C in winter, 0.76 and 1.41 °C in summer, 0.70 and 1.37 °C in rainy, and 1.09 and 1.93 °C in autumn, respectively. Therefore, regardless of the seasons examined in this study, IRT proves to be an effective and supportive tool for early mastitis detection in lactating crossbred dairy goats.

Activation of ARP2/3 and HSP70 Expression by Lipoteichoic Acid: Potential Bidirectional Regulation of Apoptosis in a Mastitis Inflammation Model.

Mastitis typically arises from bacterial invasion, where host cell apoptosis significantly contributes to the inflammatory response. Gram-positive bacteria predominantly utilize the virulence factor lipoteichoic acid (LTA), which frequently leads to chronic breast infections, thereby impacting dairy production and animal husbandry adversely. This study employed LTA to develop models of mastitis in cow mammary gland cells and mice. Transcriptomic analysis identified 120 mRNAs associated with endocytosis and apoptosis pathways that were enriched in the LTA-induced inflammation of the Mammary Alveolar Cells-large T antigen (MAC-T), with numerous differential proteins also concentrated in the endocytosis pathway. Notably, actin-related protein 2/3 complex subunit 3 (ARPC3), actin-related protein 2/3 complex subunit 4 (ARPC4), and the heat shock protein 70 (HSP70) are closely related. STRING analysis revealed interactions among ARPC3, ARPC4, and HSP70 with components of the apoptosis pathway. Histological and molecular biological assessments confirmed that ARPC3, ARPC4, and HSP70 were mainly localized to the cell membrane of mammary epithelial cells. ARPC3 and ARPC4 are implicated in the mechanisms of bacterial invasion and the initiation of inflammation. Compared to the control group, the expression levels of these proteins were markedly increased, alongside the significant upregulation of apoptosis-related factors. While HSP70 appears to inhibit apoptosis and alleviate inflammation, its upregulation presents novel research opportunities. In conclusion, we deduced the development mechanism of ARPC3, ARPC4, and HSP70 in breast inflammation, laying the foundation for further exploring the interaction mechanism between the actin-related protein 2/3 (ARP2/3) complex and HSP70.

Disturbances of the gut microbiota-derived tryptophan metabolites as key actors in vagotomy-induced mastitis in mice.

Previous studies have demonstrated that gut microbiota dysbiosis promotes the development of mastitis. The interaction of the vagus nerve and gut microbiota endows host homeostasis and regulates disease development, but whether the vagus nerve participates in the pathogenesis of mastitis is unclear. Here, vagotomized mice exhibit disruption of the blood-milk barrier and mammary gland inflammation. Notably, mastitis and barrier damage caused by vagotomy are dependent on the gut microbiota, as evidenced by antibiotic treatment and fecal microbiota transplantation. Vagotomy significantly alters the gut microbial composition and tryptophan metabolism and reduces the 5-hydroxyindole acetic acid (5-HIAA) level. Supplementation with 5-HIAA alleviates vagotomy-induced mastitis, which is associated with the activation of the aryl hydrocarbon receptor (AhR) and subsequent inhibition of the NF-κB pathway. Collectively, our findings indicate the important role of the vagus-mediated gut-mammary axis in the pathogenesis of mastitis and imply a potential strategy for the treatment of mastitis by targeting the vagus-gut microbiota interaction.

Bioactive Compounds and Probiotics Mitigate Mastitis by Targeting NF-κB Signaling Pathway.

Mastitis is a significant inflammatory condition of the mammary gland in dairy cows. It is caused by bacterial infections and leads to substantial economic losses worldwide. The disease can be either clinical or sub-clinical and presents challenges such as reduced milk yield, increased treatment costs, and the need to cull affected cows. The pathogenic mechanisms of mastitis involve the activation of Toll-like receptors (TLRs), specifically TLR2 and TLR4. These receptors play crucial roles in recognizing pathogen-associated molecular patterns (PAMPs) and initiating immune responses through the NF-κB signaling pathway. Recent in vitro studies have emphasized the importance of the TLR2/TLR4/NF-κB signaling pathway in the development of mastitis, suggesting its potential as a therapeutic target. This review summarizes recent research on the role of the TLR2/TLR4/NF-κB signaling pathway in mastitis. It focuses on how the activation of TLRs leads to the production of proinflammatory cytokines, which, in turn, exacerbate the inflammatory response by activating the NF-κB signaling pathway in mammary gland tissues. Additionally, the review discusses various bioactive compounds and probiotics that have been identified as potential therapeutic agents for preventing and treating mastitis by targeting TLR2/TLR4/NF-κB signaling pathway. Overall, this review highlights the significance of targeting the TLR2/TLR4/NF-κB signaling pathway to develop effective therapeutic strategies against mastitis, which can enhance dairy cow health and reduce economic losses in the dairy industry.

Somatic cell count as an indicator of subclinical mastitis and increased inflammatory response in asymptomatic lactating women.

Subclinical mastitis is an asymptomatic inflammatory condition that can be difficult to define and diagnose. In the dairy industry, subclinical mastitis is diagnosed by milk somatic cell counts (SCCs) of ≥250,000 cells mL-1. In this pilot study, we assessed the efficacy of this index to identify human subclinical mastitis by comparing SCC levels with the inflammatory response [interleukin-8 (IL-8) levels] in 37 samples from asymptomatic and 10 clinical mastitis (CM) lactating women. The milk microbiota was determined by 16S rRNA gene sequencing. The SCC of CM samples ranged from 310,000 to 6,600,000 cells mL-1. However, 14 of 37 (37.8%) asymptomatic samples had high SCC (250,000-460,000 cells mL-1), indicating subclinical mastitis. SCC levels significantly (P < 0.001) and positively correlated with milk IL-8 levels reflecting the escalating inflammatory response across subclinical and clinical mastitis samples. Samples with an SCC of ≥250,000 cells mL-1 showed significant increases in IL-8 responses when compared with milk samples from healthy women. The milk microbiome of CM samples was dominated by streptococcal and staphylococcal species (89.9% combined median relative abundance). In contrast, the combined median streptococcal/staphylococcal relative levels were 75.4% and 66.3% in milks from asymptomatic (subclinical mastitis) and healthy groups, respectively. The Streptococcus genus was increased in samples with an SCC of ≥250,000, although this should be interpreted with caution. Thus, the index of ≥250,000 somatic cells mL-1 could be a reliable indicator of subclinical mastitis in humans and should aid future studies investigating the impact of subclinical mastitis on maternal health, breastfeeding behaviors, infant health, and development. IMPORTANCE: This pilot study suggests that SCC at a level of (greater than or equal to) 250,000 cells mL-1, as used in the dairy industry, is a suitable index to identify asymptomatic subclinical mastitis in lactating women since it reflects a significant increase in the inflammatory response compared to milk samples from healthy women. Using this index should aid studies into the short- and long-term consequences of subclinical mastitis for mother and infant.

Mastitis: Rapid Evidence Review.

Mastitis represents a spectrum of inflammatory conditions. Lactational mastitis is the most common, with an approximate incidence of 10% in the United States, and it usually occurs in the first 3 months postpartum. Diagnosis is made clinically based on the presence of symptoms such as fever, malaise, focal breast tenderness, and overlying skin erythema or hyperpigmentation without the need for laboratory tests or imaging. However, obtaining milk cultures should be considered to guide antibiotic therapy, and ultrasonography should be performed to identify abscesses in immuno-compromised patients or those with worsening or recurrent symptoms. Because most cases of mastitis are caused by inflammation and not a true infection, a 1- to 2-day trial of conservative measures (i.e., nonsteroidal anti-inflammatory drugs, ice application, feeding the infant directly from the breast, and minimizing pumping) is often sufficient for treatment. If there is no improvement in symptoms, narrow-spectrum antibiotics may be prescribed to cover common skin flora (e.g., Staphylococcus, Streptococcus). Most patients can be treated as outpatients with oral antibiotics; however, if the condition worsens or there is a concern for sepsis, intravenous antibiotics and hospital admission may be required. Use of probiotics for treatment or prevention is not supported by good evidence. Factors that increase the risk of mastitis include overstimulation of milk production and tissue trauma from aggressive breast massage; therefore, frequent overfeeding, excessive pumping to empty the breast, heat application, and breast massage are no longer recommended because they may worsen the condition. The best prevention is a proper lactation technique, including a good infant latch, and encouraging physiologic breastfeeding rather than pumping, if possible.

Novel insights: crosstalk with non-puerperal mastitis and immunity.

The two primary types of non-puerperal mastitis (NPM) are granulomatous lobular mastitis (GLM) and plasma cell mastitis (PCM). Existing research indicates that immune inflammatory response is considered to be the core of the pathogenesis of GLM and PCM, and both innate and adaptive immune responses play an important role in the pathophysiology of PCM and GLM. However, the regulatory balance between various immune cells in these diseases is still unclear. Consequently, we present a comprehensive summary of the immune-related variables and recent advances in GLM and PCM.

Characterization and antioxidant activity of nano-formulated berberine and cyperus rotundus extracts with anti-inflammatory effects in mastitis-induced rats.

Bovine mastitis caused by infectious pathogens, mainly Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), constitutes a major destructive challenge for the dairy industry and public health. Berberine chloride (BER) and Cyperus rotundus possess a broad spectrum of anti-inflammatory, antioxidant, antibacterial, and antiproliferative activities; however, their bioavailability is low. This research aimed first to prepare an ethanolic extract of Cyperus rotundus rhizomes (CRE) followed by screening its phytochemical contents, then synthesis of BER and CRE loaded chitosan nanoparticles (NPs) (BER/CH-NPs and CRE/CH-NPs), afterward, the analysis of their loading efficiency in addition to the morphological and physicochemical characterization of the formulated NPs employing Scanning Electron Microscopy (SEM), Zeta Potential (ZP), Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD) assessments compared to their crude forms to evaluate the enhancement of bioavailability and stability. Isolation of bacterial strains from the milk of mastitic cows, used for induction of mammary gland (MG) inflammation in female albino rats, and a preliminary investigation of the prophylactic oral doses of the prepared NPs against S. aureus-induced mastitis in female rats. The minimal inhibitory concentration (MIC) of BER/CH-NPs and CRE/CH-NPs is 1 mg/kg b.w. BER/CH-NPs and CRE/CH-NPs alone or in combination show significant (P ≤ 0.05) DPPH radical scavenging activity (69.2, 88.5, and 98.2%, respectively) in vitro. Oral administration of BER/CH-NPs and CRE/CH-NPs to mastitis rats significantly (P ≤ 0.05) attenuated TNF-α (22.1, 28.6 pg/ml), IL-6 (33.4, 42.9 pg/ml), IL-18 (21.7, 34.7 pg/ml), IL-4 (432.9, 421.6 pg/ml), and MPO (87.1, 89.3 pg/ml) compared to mastitis group alongside the improvement of MG histopathological findings without any side effect on renal and hepatic functions. Despite promising results with BER and CRE nanoparticles, the study is limited by small-scale trials, a focus on acute administration, and partially explored nanoparticle-biological interactions, with no economic or scalability assessments. Future research should address these limitations by expanding trial scopes, exploring interactions further, extending study durations, and assessing economic and practical scalability. Field trials and regulatory compliance are also necessary to ensure practical application and safety in the dairy industry. In conclusion, the in vitro and in vivo results proved the antioxidant and anti-inflammatory efficacy of BER/CH-NPs and CRE/CH-NPs in low doses with minimal damage to the liver and kidney functions, supposing their promising uses in mastitis treatment.

Perspectives and Possibilities for New Antimicrobial Agents in the Treatment and Control of Mastitis Induced by Algae of the Genus Prototheca spp.: A Review.

Innovative approaches in nanotechnology provide a potentially promising alternative to untreatable cases of mastitis caused by genus Prototheca spp. algae infections. Drying of the teats of the affected animals or culling are typically the outcomes of mastitis in dairy cattle caused by these pathogens. A major issue in both veterinary medicine and animal breeding is the Prototheca species' widespread resistance to the current methods of managing infections and the available drugs, including antibiotics. Commercial antifungal preparations are also ineffective. Nanotechnology, an emerging discipline, has the potential to create an effective alternative treatment for protothecal mastitis. The aim of the paper is to combine the literature data on the use of nanotechnology in the control of mastitis, taking into account data on combating mastitis caused by Prototheca spp. infections. The databases employed were PubMed, Google Scholar, and Scopus, focusing on literature from the last 20 years to ensure relevance and currency. Studies conducted in vitro have demonstrated that nanomaterials have significant biocidal activity against mastitis infections of different etiologies. Analyzed research papers show that (NPs), such as AgNPs, CuNPs, AuNPs, etc., may not negatively impact various cell lines and may be effective agents in reducing the pathogens' viability. However, it is also critical to assess the risks involved in using nanomaterials.

Biological characterization of the phage lysin AVPL and its efficiency against Aerococcus viridans-induced mastitis in a murine model.

Aerococcus viridans (A. viridans) is an important opportunistic zoonotic pathogen that poses a potential threat to the animal husbandry industry, such as cow mastitis, due to the widespread development of multidrug-resistant strains. Phage lysins have emerged as a promising alternative antibiotic treatment strategy. However, no lysins have been reported to treat A. viridans infections. In this study, the critical active domain and key active sites of the first A. viridans phage lysin AVPL were revealed. AVPL consists of an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and a C-terminal binding domain comprising two conserved LysM. H40, N44, E52, W68, H147, T157, F60, F64, I77, N92, Q97, H159, V160, D161, and S42 were identified as key sites for maintaining the activity of the catalytic domain. The LysM motif plays a crucial role in binding AVPL to bacterial cell wall peptidoglycan. AVPL maintains stable activity in the temperature range of 4-45°C and pH range of 4-10, and its activity is independent of the presence of metal ions. In vitro, the bactericidal effect of AVPL showed efficient bactericidal activity in milk samples, with 2 µg/mL of AVPL reducing A. viridans by approximately 2 Log10 in 1 h. Furthermore, a single dose (25 µg) of lysin AVPL significantly reduces bacterial load (approximately 2 Log10) in the mammary gland of mice, improves mastitis pathology, and reduces the concentration of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in mammary tissue. Overall, this work provides a novel alternative therapeutic drug for mastitis induced by multidrug-resistant A. viridans. IMPORTANCE: A. viridans is a zoonotic pathogen known to cause various diseases, including mastitis in dairy cows. In recent years, there has been an increase in antibiotic-resistant or multidrug-resistant strains of this pathogen. Phage lysins are an effective approach to treating infections caused by multidrug-resistant strains. This study revealed the biological properties and key active sites of the first A. viridans phage lysin named AVPL. AVPL can effectively kill multidrug-resistant A. viridans in pasteurized whole milk. Importantly, 25 µg AVPL significantly alleviates the symptoms of mouse mastitis induced by A. viridans. Overall, our results demonstrate the potential of lysin AVPL as an antimicrobial agent for the treatment of mastitis caused by A. viridans.

Ferroptosis is involved in Staphylococcus aureus-induced mastitis through autophagy activation by endoplasmic reticulum stress.

Cell death caused by severe Staphylococcus aureus (S. aureus) infection is a fatal threat to humans and animals. However, whether ferroptosis, an iron-dependent form of cell death, is involved in S. aureus-induced cell death and its role in S. aureus-induced diseases are unclear. Using a mouse mastitis model and mammary epithelial cells (MMECs), we investigated the role of ferroptosis in the pathogenesis of S. aureus infection. The results revealed that S. aureus-induced ferroptosis in vivo and in vitro as demonstrated by dose-dependent increases in cell death; the level of malondialdehyde (MDA), the final product of lipid peroxidation; and dose-dependent decrease the production of the antioxidant glutathione (GSH). Treatment with typical inhibitors of ferroptosis, including ferrostatin-1 (Fer-1) and deferiprone (DFO), significantly inhibited S. aureus-induced death in MMECs. Mechanistically, treatment with S. aureus activated the protein kinase RNA-like ER kinase (PERK)-eukaryotic initiation factor 2, α subunit (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP) pathway, which subsequently upregulated autophagy and promoted S. aureus-induced ferroptosis. The activation of autophagy degraded ferritin, resulting in iron dysregulation and ferroptosis. In addition, we found that excessive reactive oxygen species (ROS) production induced ferroptosis and activated endoplasmic reticulum (ER) stress, manifesting as elevated p-PERK-p-eIF2α-ATF4-CHOP pathway protein levels. Collectively, our findings indicate that ferroptosis is involved in S. aureus-induced mastitis via ER stress-mediated autophagy activation, implying a potential strategy for the prevention of S. aureus-associated diseases by targeting ferroptosis. In conclusion, the ROS-ER stress-autophagy axis is involved in regulating S. aureus-induced ferroptosis in MMECs. These findings not only provide a new potential mechanism for mastitis induced by S. aureus but also provide a basis for the treatment of other ferroptotic-related diseases.

Tuberculous Mastitis.

This case report describes induration of the right breast with multiple painful ulcers, draining nodules, and fistulae.

Ultrasound-based deep learning radiomics nomogram for differentiating mass mastitis from invasive breast cancer.

BACKGROUND: The purpose of this study is to develop and validate the potential value of the deep learning radiomics nomogram (DLRN) based on ultrasound to differentiate mass mastitis (MM) and invasive breast cancer (IBC). METHODS: 50 cases of MM and 180 cases of IBC with ultrasound Breast Imaging Reporting and Data System 4 category were recruited (training cohort, n = 161, validation cohort, n = 69). Based on PyRadiomics and ResNet50 extractors, radiomics and deep learning features were extracted, respectively. Based on supervised machine learning methods such as logistic regression, random forest, and support vector machine, as well as unsupervised machine learning methods using K-means clustering analysis, the differences in features between MM and IBC were analyzed to develop DLRN. The performance of DLRN had been evaluated by receiver operating characteristic curve, calibration, and clinical practicality. RESULTS: Supervised machine learning results showed that compared with radiomics models, especially random forest models, deep learning models were better at recognizing MM and IBC. The area under the curve (AUC) of the validation cohort was 0.84, the accuracy was 0.83, the sensitivity was 0.73, and the specificity was 0.83. Compared to radiomics or deep learning models, DLRN even further improved discrimination ability (AUC of 0.90 and 0.90, accuracy of 0.83 and 0.88 for training and validation cohorts), which had better clinical benefits and good calibratability. In addition, the information heterogeneity of deep learning features in MM and IBC was validated again through unsupervised machine learning clustering analysis, indicating that MM had a unique features phenotype. CONCLUSION: The DLRN developed based on radiomics and deep learning features of ultrasound images has potential clinical value in effectively distinguishing between MM and IBC. DLRN breaks through visual limitations and quantifies more image information related to MM based on computers, further utilizing machine learning to effectively utilize this information for clinical decision-making. As DLRN becomes an autonomous screening system, it will improve the recognition rate of MM in grassroots hospitals and reduce the possibility of incorrect treatment and overtreatment.

In vivo and in vitro anti-inflammation of Rhapontici Radix extract on mastitis via TMEM59 and GPR161.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhapontici Radix ethanol extract (RRE) is derived from the dried root of Rhaponticum uniflorum (L.) DC belonging to the Asteraceae family. RRE exhibits significant anti-inflammatory and antioxidant properties; however, the potential of RRE in mastitis treatment requires further investigation. AIM OF THIS STUDY: This research was performed to examine the protective properties of RRE against mastitis and the mechanisms underlying the effects of RRE. MATERIAL AND METHODS: RRE components were analyzed by HPLC-MS/MS and DPPH methods. Isochlorogenic acid B (ICAB) was obtained commercially. MTT assay was utilized to assess RRE or ICAB cytotoxicity in bovine mammary alveolar (MAC-T) cells. Immunohistochemistry were used to investigate the pathological alterations in mammary tissue. The protein levels of inflammatory cytokines and mediators were analyzed using ELISA, and the expression of MAPK and NF-κB signaling pathways, as well as p65 nuclear translocation, were analyzed through Western blotting and immunofluorescence techniques, respectively. Target proteins of RRE were screened by RNA-seq and tandem mass tag analyses. Protein interaction was revealed and confirmed using co-immunoprecipitation and CRISPR/Cas9-based knockdown and overexpression of target genes. RESULTS: ICAB was revealed as one of the main components in RRE, and it was responsible for 84.33% of RRE radical scavenging activity. Both RRE and ICAB mitigated the infiltration of T lymphocytes in the mammary glands of mice, leading to decreased levels of inflammatory mediators (COX-2 and iNOS) and cytokines (TNF-α, IL-6, and IL-1ß) in lipopolysaccharide (LPS)-induced MAC-T cells. Furthermore, RRE and ICAB suppressed the LPS-induced phosphorylation of NF-κB inhibitor and p65, thereby impeding p65 nuclear translocation in mouse mammary glands and MAC-T cells. In addition, RRE and ICAB attenuated the LPS-triggered activation of c-Jun N-terminal kinase 1/2, p38, and extracellular regulated protein kinase 1/2. Importantly, co-treated with LPS and ICAB in MAC-T cells, an upregulation of G-protein coupled receptor 161 (GPR161) and transmembrane protein 59 (TMEM59) was observed; the interact between TMEM59 and was found, leading to inhibition of NF-κB activity and inflammatory cytokine production. CONCLUSION: ICAB is a prominent antioxidant in RRE. RRE and ICAB reduce mammary inflammation via MAPK and NF-κB pathways and the interaction between TMEM59 and GPR161 mediates the control of ICAB in NF-κB signaling.