A Cas12a-based fluorescent microfluidic system for rapid on-site human papillomavirus diagnostics.

Persistent infection with human papillomavirus (HPV) is the leading cause of cervical cancer, and early diagnosis is crucial for clinical management. However, the easy and rapid on-site diagnostic for HPV genotyping remains challenging. Here, we develop a Cas12a-based fluorescent microfluidic detection system for diagnosing six HPV subtypes (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33). A panel of crRNAs and recombinase polymerase amplification (RPA) primers targeting the HPV L1 gene was screened for sensitive and specific detection. Furthermore, a one-pot RPA reaction was developed to amplify the six HPV subtypes without cross-reactivity. For on-site detection, we integrated the RPA-Cas12a detection into a microfluidic device, enabling the detection of processed clinical samples within 35 minutes. The assay was validated using 112 clinical swab samples and obtained consistent results with the qPCR assay, with a concordance rate of 99.1%. Overall, our diagnostic method offers a rapid, sensitive, and easy-to-use on-site assay for detecting HPV genotypes and holds promise for improving cervical cancer screening and prevention. KEY POINTS: • The Cas12a-based fluorescent microfluidic detection system for the diagnosis of six HPV subtypes. • A one-pot RPA reaction for amplifying the six HPV subtypes without cross-reactivity. • The RPA-Cas12a-microfluidic system provides results within 35 minutes for on-site detection.

Lysine promotes proliferation and ß-casein synthesis through the SLC6A14-ERK1/2-CDK1-mTOR signaling pathway in bovine primary mammary epithelial cells.

Lysine, as the first limiting amino acid in dairy cows, has been shown to play an important role in milk synthesis and cell proliferation. However, the underlying mechanism remains unclear. In this study, we isolated bovine primary mammary epithelial cells (BMECs) and studied the mechanism in which lysine promotes cell proliferation and ß-casein synthesis through overexpression and knockdown of CDK1 and supplements BCH, U0126, and rapamycin in BMECs. Results show that 0.7 mM lysine can significantly promote cell proliferation and the synthesis of ß-casein in BMECs. In addition, lysine activates the ERK signaling pathway to promote the expression of CDK1. Further studies have shown that CDK1 can promote cell proliferation and the synthesis of ß-casein through the mTOR signaling pathway in BMECs. Lastly, lysine can promote cell proliferation and the synthesis of ß-casein through SLC6A14 in BMECs. The above results indicate that lysine promotes cell proliferation and the synthesis of ß-casein through the SLC6A14-ERK-CDK1-mTOR signaling pathway in BMECs.

Mammary Fibrosis Tendency and Mitochondrial Adaptability in Dairy Cows with Mastitis.

Dairy cow mammary gland fibrosis causes huge economic losses to livestock production, however, research on dairy cow mammary gland fibrosis is in its infancy and it lacks effective treatments. Therefore, the purpose of this experiment was to explore the correlation between mastitis and fibrosis and mitochondrial damage, and to further explore its pathogenesis. In vivo, mammary tissue and milk samples were collected from healthy cows (n = 10) and mastitis cows (n = 10). The results of the study showed that compared with the control group, the mastitis tissue showed tissue damage, accumulation of collagen fibers, and the content of TGF-ß1 in mammary tissue and milk was significantly increased; the level of inflammatory mediators was significantly increased; the fibrotic phenotype, collagen 1, α-SMA, vimentin gene, and protein levels were significantly increased, while the E-cadherin gene and protein levels were significantly decreased. In vitro, based on TGF-ß1-induced bMECs, the above experimental results were further confirmed, and TGF-ß1 significantly promoted the fibrotic phenotype of bMECs. On the other hand, in vivo results showed that fibrotic mammary tissue had a significantly stronger mitochondrial damage phenotype and significantly higher ROS than the control group. In vitro, the results also found that TGF-ß1 induced a significant increase in the mitochondrial damage phenotype of bMECs, accompanied by a large amount of ROS production. Furthermore, in a TGF-ß1-induced bMEC model, inhibiting the accumulation of ROS effectively alleviated the elevated fibrotic phenotype of TGF-ß1-induced bMECs. In conclusion, the fibrotic phenotype of mammary gland tissue in dairy cows with mastitis was significantly increased, and mastitis disease was positively correlated with mammary fibrotic lesions. In an in vitro and in vivo model of cow mammary fibrosis, bMECs have impaired mitochondrial structure and dysfunction. Inhibiting the accumulation of ROS effectively alleviates the elevated fibrotic phenotype, which may be a potential therapeutic approach to alleviate mammary fibrosis.

Lensfree on-chip microscopy based on single-plane phase retrieval.

We propose a novel single-plane phase retrieval method to realize high-quality sample reconstruction for lensfree on-chip microscopy. In our method, complex wavefield reconstruction is modeled as a quadratic minimization problem, where total variation and joint denoising regularization are designed to keep a balance of artifact removal and resolution enhancement. In experiment, we built a 3D-printed field-portable platform to validate the imaging performance of our method, where resolution chart, dynamic target, transparent cell, polystyrene beads, and stained tissue sections are employed for the imaging test. Compared to state-of-the-art methods, our method eliminates image degradation and obtains a higher imaging resolution. Different from multi-wavelength or multi-height phase retrieval methods, our method only utilizes a single-frame intensity data record to accomplish high-fidelity reconstruction of different samples, which contributes a simple, robust, and data-efficient solution to design a resource-limited lensfree on-chip microscope. We believe that it will become a useful tool for telemedicine and point-of-care application.

Oxypeucedanin relieves LPS-induced acute lung injury by inhibiting the inflammation and maintaining the integrity of the lung air-blood barrier.

INTRODUCTION: Acute lung injury (ALI) is commonly accompanied by a severe inflammatory reaction process, and effectively managing inflammatory reactions is an important therapeutic approach for alleviating ALI. Macrophages play an important role in the inflammatory response, and this role is proinflammatory in the early stages of inflammation and anti-inflammatory in the late stages. Oxypeucedanin is a natural product with a wide range of pharmacological functions. This study aimed to determine the effect of oxypeucedanin on lipopolysaccharide (LPS)-induced ALI. METHODS AND RESULTS: In this study, the following experiments were performed based on LPS-induced models in vivo and in vitro. Using myeloperoxidase activity measurement, ELISA, qRT-PCR, and Western blotting, we found that oxypeucedanin modulated the activity of myeloperoxidase and decreased the expression levels of inflammatory mediators such as TNF-α, IL-6, IL-1ß, MPO, COX-2 and iNOS in LPS-induced inflammation models. Meanwhile, oxypeucedanin inhibited the activation of PI3K/AKT and its downstream NF-κB and MAPK signaling pathways. In addition, oxypeucedanin significantly decreased the pulmonary vascular permeability, which was induced by LPSs, and the enhanced expression of tight junction proteins (Occludin and Claudin 3). CONCLUSIONS: In conclusion, this study demonstrated that the anti-inflammatory mechanism of oxypeucedanin is associated with the inhibition of the activation of PI3K/AKT/NF-κB and MAPK signaling pathways and the maintenance of the integrity of the lung air-blood barrier.

Sodium butyrate attenuates bovine mammary epithelial cell injury by inhibiting the formation of neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are an important means by which the body fights against exogenous bacteria. However, studies have shown that excessive NETs release can damage other cells. Accumulating evidence has shown that butyric acid can alleviate the inflammatory response of cells. However, the effect of butyric acid on Staphylococcus aureus-induced NETs formation and its underlying mechanism are still unclear. In this study, western blotting, immunofluorescence and CCK-8 assays were used to examine the effect of NETs formation by sodium butyrate (NaB). The results showed that NaB suppressed the release of S. aureus-induced NETs formation, as indicated by decreases in the levels of DNA, histones, myeloperoxidase, and neutrophil elastase. S. aureus can induce autophagy, and autophagy plays a key role in the formation of NETs. Our data showed that NaB activated mammalian target of rapamycin (mTOR) and the kinases protein kinase B (AKT) and unc-51 like kinase 1 (ULK1) at Ser757 and inhibited AMP-activated protein kinase (AMPK). To explore whether NaB inhibited the formation of NETs by inhibiting autophagy, we added 3-methyladenine (autophagy inhibitor) (3-MA, 5 mM) to bovine neutrophils, and the results showed that 3-MA significantly inhibited NETs release. Furthermore, we found that NETs and their component histones exhibited significantly increased the cytotoxic effects on bovine mammary epithelial cells (BMECs), indicating that NETs and their component histones play a key role in BMEC damage. In conclusion, NaB can reduce the excessive formation of NETs by inhibiting autophagy, thus reducing the damaging effect of NETs on BMECs.

MafK accelerates Salmonella mucosal infection through caspase-3 activation.

Gastrointestinal homeostasis is critical for maintaining host health, and is affected by many factors. A recent report showed that Musculoaponeurotic fibrosarcoma K (MafK) expression is increased in patients that have ulcerative colitis (UC). Even so, MafK's significance in sustaining intestinal homeostasis has not been investigated. In this research, MafK overexpressing transgenic (MafK Tg) mice were found to be more susceptible to infection with Salmonella on the mucosa than the wild-type (WT) mice. Following Salmonella oral infection, MafK Tg mice suffered higher mortality and a lot more weight loss, damage to the intestines, and inflammation in the intestines than WT mice. MafK Tg mice were also unable to control Salmonella colonization and dissemination. In vivo data showed that increased MafK expression promoted epithelial cell apoptosis which was further confirmed by in vitro data. The rapid cleavage of caspase-3 in epithelial cells contributed to Salmonella dissemination and inflammation initiation. This study reveals that MafK participates in Salmonella pathogenesis acceleration by increasing caspase-3 activation.

Tartary buckwheat flavonoids relieve the tendency of mammary fibrosis induced by HFD during pregnancy and lactation.

Mammary gland fibrosis is a chronic and irreversible disease. Tartary buckwheat flavonoids (TBF) are a natural product of flavonoid extracts from buckwheat and have a wide range of biological activities. The purpose of this experiment was to explore whether HFD during pregnancy and lactation induces fibrosis of the mammary tissue and whether TBF alleviates the damage caused by HFD, along with its underlying mechanism. The HFD significantly increased the levels of TNF-α, IL-6, IL-1ß, and MPO; significantly damaged the integrity of the blood-milk barrier; significantly increased the levels of collagen 1, vimentin and α-SMA, and reduced the level of E-cadherin. However, these effects were alleviated by TBF. Mechanistic studies showed that TBF inhibited the activation of AKT/NF-κB signaling and predicted the AKT amino acid residues that formed hydrogen bonds with TBF; in addition, these studies not only revealed that TBF promoted the expression of the tight junction proteins (TJs) claudin-3, occludin and ZO-1 and inhibited the activation of TGF-ß/Smad signaling but also predicted the Smad MH2 amino acid residues that formed hydrogen bonds with TBF. Conclusion: HFD consumption during pregnancy and lactation induced the tendency of mammary fibrosis. TBF alleviated the tendency of mammary fibrosis by inhibiting the activation of AKT/NF-κB, repairing the blood-milk barrier and inhibiting the activation of TGF-ß/Smad signaling.

GPR109A alleviate mastitis and enhances the blood milk barrier by activating AMPK/Nrf2 and autophagy.

Mastitis causes great psychological and physical pain among women. Our previous studies found that niacin has anti-inflammatory effect, and the realization of this function depends on GPR109A. However, there are no previous reports about the anti-inflammatory function of GPR109A in mastitis. In our study, we observed the effect of niacin on the WT and GPR109A-/- mice mastitis model. The results showed that administration of niacin to WT mice reduced the damage, proinflammatory mediators and protected the integrity of the blood milk barrier in mammary gland. While in GPR109A-/- mice, there was no effect on the above indexes. In mammary epithelial cells, GPR109A was able to promote autophagy and Nrf2 nuclear import through AMPK. In LPS-induced mammary epithelial cells, niacin inhibited the LPS-induced inflammatory response and downregulation of tight junction proteins, and these effects were eliminated by knocking down GPR109A, blocking autophagy or inhibiting Nrf2 nuclear import. These results indicate that in mastitis, GPR109A promotes autophagy and Nrf2 nuclear import through AMPK, thereby inhibiting inflammatory damage to the mammary gland and repairing the blood milk barrier. Our results suggested that GPR109A may be a potential target for the treatment of mastitis.

Effect of Palrnatine on lipopolysaccharide-induced acute lung injury by inhibiting activation of the Akt/NF|-κB pathway.

Inflammation plays an important role in the development of acute lung injury (ALI). Severe pulmonary inflammation can cause acute respiratory distress syndrome (ARDS) or even death. Expression of proinflammatory interleukin-|1ß (IL-|1ß) and inducible nitric oxide synthase (iNOS) in the process of pulmonary inflammation will further exacerbate the severity of ALI. The purpose of this study was to explore the effect of Palrnatine (Pa) on lipopolysaccharide (LPS)-induced mouse ALI and its underlying mechanism. Pa, a natural product, has a wide range of pharmacological activities with the potential to protect against lung injury. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to detect the expression and translation of inflammatory genes and proteins in vitro and in vivo. Immunoprecipitation was used to detect the degree of P65 translocation into the nucleus. We also used molecular modeling to further clarify the mechanism of action. The results showed that Pa pretreatment could significantly inhibit the expression and secretion of the inflammatory cytokine IL-1ß, and significantly reduce the protein level of the proinflammatory protease iNOS, in both in vivo and in vitro models induced by LPS. Further mechanism studies showed that Pa could significantly inhibit the activation of the protein kinase B (Akt)/nuclear factor-κB (NF-κB) signaling pathway in the LPS-induced ALI mode and in LPS-induced RAW264.7 cells. Through molecular dynamics simulation, we observed that Pa was bound to the catalytic pocket of Akt and effectively inhibited the biological activity of Akt. These results indicated that Pa significantly relieves LPS-induced ALI by activating the Akt/NF-κB signaling pathway.

Pedunculoside protects against LPS-induced mastitis in mice by inhibiting inflammation and maintaining the integrity of blood-milk barrier.

Mastitis is a disease that seriously threatens the health of the mammary gland after delivery. Pedunculoside (PE) is the main bioactive component of Aquifoliaceae. The purpose of this experiment is to explore the effects of PE on mastitis and its underlying mechanisms. Our research results showed that PE could significantly inhibit the increase in the levels of inflammatory mediators such as TNF-α, IL-6, IL-1ß, MPO and iNOS during mastitis. Mechanism studies have found that PE could significantly inhibit the phosphorylation of AKT protein and binds to the ASP-184 site. Further research found that PE also inhibited the activation of AKT's downstream pro-inflammatory signals NF-κB and MAPK. In addition, PE effectively promote the expression of tight junction proteins occludin and claudin-3 during inflammation, maintaining the integrity of the blood-milk barrier. In summary, our research shows that PE inhibits the phosphorylation of AKT/NF-κB and MAPK signals; It also relieves mastitis by repairing the blood-milk barrier.

Protective effect of myricetin on LPS-induced mastitis in mice through ERK1/2 and p38 protein author.

The inflammatory reaction of mammary gland tissue in dairy cattle leads to the occurrence of mastitis disease and causes huge economic loss. Myricetin (Myr), a flavonoid natural product, is extracted from the root, stem, and leaves of Myrica rubra. It has a wide range of biological activities, such as anti-oxidant, anti-inflammatory, and anti-tumor. The purpose of this experiment is to further explore the effect of Myr on mastitis and further explore its potential mechanism in LPS-induced mice mastitis model and LPS-induced mice mammary epithelial cells (mMECs). The results showed that Myr could significantly inhibit the expression of TNF-α, IL-6, and IL-1ß in the mammary gland of mice. Furthermore, the results of mechanism studies show that Myr can significantly inhibit P38 and ERK1/2 protein phosphorylation levels in mice mammary tissue, and this result has been further verified at the cellular level. These results confirm that Myr can significantly inhibit mammary inflammation, and its potential mechanism is to play a protective role by inhibiting the phosphorylation level of P38 and ERK1/2 protein.

Myricetin protects against H2 O2 -induced oxidative damage and apoptosis in bovine mammary epithelial cells.

High-producing dairy cows are prone to oxidative stress due to their high secretion and strong metabolism, and excessive oxidative stress may cause the apoptosis of bovine mammary epithelial cells (bMECs). Myricetin (Myr) has been shown to have a wide range of pharmaceutical activities. The aim of this study was to evaluate the effect of Myr on hydrogen peroxide (H2 O2 )-induced oxidative stress and apoptosis in bMECs and to clarify the underlying mechanism. bMECs were pretreated with or without Myr and then stimulated with H2 O2 . The results showed that Myr significantly increased the total antioxidant capacity and superoxide dismutase levels and decreased the malondialdehyde (MDA) and reactive oxygen species (ROS) levels in a model of oxidative stress induced by H2 O2 in bMECs. Mechanistic studies found that Myr inhibited H2 O2 -induced oxidative stress in bMECs through the adenosine monophosphate-activated protein kinase/nuclear factor erythroid-2 related factor 2 (AMPK/NRF2) signaling pathway. Additional research found that Myr could also inhibit H2 O2 -induced apoptosis in bMECs through NRF2. These data suggest that Myr effectively alleviated oxidative stress and apoptosis in H2 O2 -induced bMECs through the activation of the AMPK/NRF2 signaling pathway.

Rumen-bypassed tributyrin alleviates heat stress by reducing the inflammatory responses of immune cells.

Heat stress (HS) in summer will seriously affect the health and performance of dairy cows. To alleviate the injury to dairy cows caused by HS, we added the rumen-bypassed tributyrin to the feed. We determined whether cows were in a heat-stressed environment by testing the temperature humidity index in the morning, at noon, and in the evening. The detection of anal temperature and respiratory frequency further proved the HS state of the dairy cows. The quantificational real time PCR results showed that tributyrin could significantly reduce the relative expression of tumor necrosis factor α, interleukin 1ß, and Interleukin 6. Western blot results showed that tributyrin could alleviate the lymphocyte inflammatory response by inhibiting the mitogen-activated protein kinase and nuclear factor-кB signaling pathways. To further detect the effect of tributyrin on HS in dairy cows, routine biochemical and blood tests were carried out. The results showed that the contents of aspartate aminotransferase, total bilirubin, creatinine, albumin, and globulin were significantly reduced by tributyrin. The results showed that tributyrin could significantly alleviate the liver and kidney injury induced by heat stress in dairy cows. Moreover, tributyrin could also significantly reduce the numbers of intermediate cells and increase the level of hemoglobin. Tributyrin could also improve the performance of dairy cows. These results suggested that tributylglycerol may have a positive effect on breast health of dairy cows. In conclusion, these results indicated that tributyrin could relieve HS and increase the production performance of dairy cows by reducing the inflammatory responses of lymphocytes.

The inflammatory environment mediated by a high-fat diet inhibited the development of mammary glands and destroyed the tight junction in pregnant mice.

Long-term intake of a high-fat diet seriously affects the health of pregnant women and leads to increased levels of inflammation in the mammary gland. Therefore, to further explore the effect of a high-fat diet on mammary gland development and the tight junction (TJ) during pregnancy, we placed mice into two groups: a high-fat diet group and a control group. We detected the expression of proteins related to fat synthesis in the mammary gland by western blotting. The results showed that a high-fat diet could lead to an increase in fat synthesis in the mammary gland. Then, the inflammatory levels and acinar cell morphology in the mammary gland were detected by ELISA and H&E staining. We also measured the levels of MAPK and NF-κB signal pathway-related proteins by western blotting. The results showed that a high-fat diet activated the MAPK and NF-κB signaling pathways and promoted the expression of inflammatory factors. Finally, the development of the mammary gland and the integrity of the TJ were determined by immunohistochemistry, immunofluorescence and western blotting. The results showed that a high-fat diet inhibited the development of the mammary gland and the expression of tight junction proteins (TJs). Our study showed that a high-fat diet could promote the expression of inflammatory factors by activating the MAPK and NF-κB signaling pathways and could reshape the microenvironment through extramammary inflammation. Finally, a high-fat diet inhibited the development of the mammary gland during pregnancy and destroyed the integrity of the TJ.

Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway.

Mastitis is one of three bovine diseases recognized as a cause of substantial economic losses every year throughout the world. Niacin is an important feed additive that is used extensively for dairy cow nutrition. However, the mechanism by which niacin acts on mastitis is not clear. The aim of this study is to investigate the mechanism of niacin in alleviating the inflammatory response of mammary epithelial cells and in anti-mastitis. Mammary glands, milk, and blood samples were collected from mastitis cows not treated with niacin (n = 3) and treated with niacin (30 g/d, n = 3) and healthy cows (n = 3). The expression of GPR109A, IL-6, IL-1ß, and TNF-α in the mammary glands of the dairy cows with mastitis was significantly higher than it was in the glands of the healthy dairy cows. We also conducted animal experiments in vivo by feeding rumen-bypassed niacin. Compared with those in the untreated mastitis group, the somatic cell counts (SCCs) and the expression of IL-6, IL-1ß, and TNF-α in the blood and milk were lower. In vitro, we isolated the primary bovine mammary epithelial cells (BMECs) from the mammary glands of the healthy cows. The mRNA levels of IL-6, IL-1ß, TNF-α, and autophagy-related genes were detected after adding niacin, shRNA, compound C, trans retinoic acid, 3-methyladenine to BMECs. Then GPR109A, AMPK, NRF-2, and autophagy-related proteins were detected by Western blot. We found that niacin can activate GPR109A and phosphorylate AMPK, and promote NRF-2 nuclear import and autophagy to alleviate LPS-induced inflammatory response in BMECs. In summary, we found that niacin can reduce the inflammatory response of BMECs through GPR109A/AMPK/NRF-2/autophagy. We also preliminarily explored the alleviative effect of niacin on mastitis in dairy cows.

Butyrate alleviates oxidative stress by regulating NRF2 nuclear accumulation and H3K9/14 acetylation via GPR109A in bovine mammary epithelial cells and mammary glands.

Oxidative stress consistently affects lactation length and quality in dairy cows. Oxidative stress in the mammary gland of high-yielding dairy cows is a serious problem. Therefore, we studied the role of butyrate in dairy cow oxidative stress and further elucidated the mechanism of the antioxidative action of mammary epithelial cells in dairy cows. Oxidative stress and activated GPR109A were present in high-yielding dairy cows. Then, bovine mammary epithelial cells (BMECs) were isolated, and oxidative stress-related protein expression was measured, confirming that sodium butyrate (NaB) exerted antioxidant effects through GPR109A, NRF2 and H3K9/14 acetylation. To further study the antioxidative mechanism of butyrate in dairy cows, we also confirmed that butyrate promoted NRF2 nuclear accumulation and H3K9/14 acetylation through the AMPK signaling pathway by western blotting. Additionally, we preliminarily clarified the interaction between NRF2 and H3K9/14 acetylation by Co-IP and ChIP. Butyrate activated the AMPK signaling pathway through GPR109A to promote NRF2 nuclear accumulation and H3K9/14 acetylation, subsequently exerting antioxidant effects through the synergistic functions of these two processes. Then, we studied the effect of butyrate on oxidative stress in dairy cows in vivo, and the results were consistent with those in vitro. Therefore, butyrate played an antioxidant and antiapoptotic role through the GPR109A/AMPK/NRF2 signaling pathway, while H3K9/14 acetylation could promote NRF2 transcription and enhance the antioxidant capacity of BMECs.

Dioscin Improves Pyroptosis in LPS-Induced Mice Mastitis by Activating AMPK/Nrf2 and Inhibiting the NF-κB Signaling Pathway.

Dioscin, a natural steroid saponin, has been shown to have anti-inflammatory effects, but its protective mechanism against mastitis is still unknown. NLRP3 inflammasome and pyroptosis play important roles in the pathogenesis of many inflammatory diseases, including mastitis. The purpose of this study was to explore the effect of dioscin on lipopolysaccharide- (LPS-) induced mastitis in vivo and in vitro and its mechanism of action. In vivo experiments, dioscin can reduce the inflammatory lesions and neutrophil motility in mammary tissue. Moreover, dioscin also can reduce the production of proinflammatory factors such as interleukin-1 beta (IL-1ß) and inhibit the activation of NLRP3 inflammasome in LPS-induced mice mastitis. In vitro experiments, the results showed that dioscin inhibited the inflammatory response and the activation of NLRP3 inflammasome, but the survival rate of mouse mammary epithelial cells (mMECs) induced by LPS+ATP is increased. Subsequently, the experiment convinces that dioscin can reduce LPS+ATP-induced mMEC pyroptosis by adding Ac-DEVD-CHO (a caspase-3 inhibitor). Further mechanistic studies demonstrate that dioscin can activate AMPK/Nrf2 to inhibit NLRP3/GSDMD-induced mMEC pyroptosis. In summary, this paper reveals a novel function of dioscin on mMEC pyroptosis and provides a new potential therapy of dioscin for the treatment and prevention of mastitis.

Kp-10 promotes bovine mammary epithelial cell proliferation by activating GPR54 and its downstream signaling pathways.

It has been reported that the proliferation and apoptosis of mammary epithelial cells affect milk production. Therefore, ensuring adequate mammary epithelial cells is expected to enhance milk production. This study is devoted to studying the effects of kisspeptin-10 (Kp-10), a peptide hormone composed of 10 amino acids, on bovine mammary epithelial cell (bMEC) proliferation and exploring the underlying mechanism of its action. bMECs were treated with various concentrations of Kp-10 (1, 10, 100, and 1,000 nM), and 100 nM Kp-10 promoted the proliferation of the bMECs. Kp-10 promoted the cell cycle transition from G1 to the S and G2 phases, increased the protein levels of Cyclin D1 and Cyclin D3, and reduced the expression levels of the p21 gene. This study also showed that inhibition of G protein-coupled receptor 54 (GPR54), AKT, mTOR, and ERK1/2 reduced the proliferation of the bMECs that had been induced by Kp-10. In addition, Kp-10 decreased the complexes formed by Rb and E2F1 and increased the expression levels of the E2F1 target genes. These results indicate that Kp-10 promotes bMEC proliferation by activating GPR54 and its downstream signaling pathways.

Schisandrin A protects against lipopolysaccharide-induced mastitis through activating Nrf2 signaling pathway and inducing autophagy.

Schisandrin A (Sch A), a dibenzocyclooctadiene lignan extracted from Schisandra chinensis (Turcz.) Baill., has anti-oxidant and anti-inflammatory effects, but the effect on masitits has not been studied. Therefore, we investigated the effect of Sch A in cell and mouse models of lipopolysaccharide (LPS)-induced mastitis. Studies in vivo showed that Sch A reduced LPS-induced mammary injury and the production of pro-inflammatory mediators. Sch A also decreased the levels of pro-inflammatory mediators and activated nuclear factor-E2 associated factor 2 (Nrf2) signaling pathway in mouse mammary epithelial cells (mMECs). The Nrf2 inhibitor partially abrogated the downregulation of Sch A on LPS-induced inflammatory response. In addition, LPS stimulation suppressed autophagy, while both Sch A and the autophagy inducer rapamycin activated autophagy in mMECs, which down-regulated inflammatory response. Sch A also restrained LPS-induced phosphorylation of mammalian target of rapamycin (mTOR) and activated AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1). In summary, these results suggest that Sch A exerts protective effects in LPS-induced mastitis models by activating Nrf2 signaling pathway and inducing autophagy and the autophagy is initiated by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway.