Interferon-tau protects bovine endometrial epithelial cells against inflammatory injury by regulating the PI3K/AKT/ß-catenin/FoxO1 signaling axis.

Endometritis is one of the most common causes of infertility in dairy cows, and is histopathologically characterized by inflammation and damage of endometrial epithelium. Interferon-tau (IFN-τ) is a novel type I interferon secreted by ruminant trophoblast cells with low cytotoxicity even at high doses. Previous studies suggested that IFN-τ plays an important role in inflammation. However, the mechanisms whereby IFN-τ may modulate the inflammatory responses in the bovine endometrium are unknown. In the present study, primary bovine endometrial epithelial cells (BEEC) isolated from fresh and healthy uterine horns were used for in vitro studies. The integrity of BEEC was assessed by immunofluorescence staining for cytokeratin 18 (CK-18, a known epithelial marker). For the experiments, BEEC were stimulated with different concentrations of lipopolysaccharide (LPS; 0-20 µg/mL) for different times (0-24 h). Cell viability and apoptosis were assessed via CCK-8 and flow cytometry. In a preliminary study, we observed that compared with the control group without LPS, 10 µg/mL of LPS stimulation for 24 h induced apoptosis. In a subsequent study, 20 or 40 ng/mL of IFN-τ alleviated LPS-induced apoptosis. Relative to the LPS group, western blotting further revealed that IFN-τ inhibited the protein abundance of TLR4 and phosphorylated (p-) p65 (p-p65) and Bax/Bcl-2 ratio, suggesting that IFN-τ can protect BEEC against inflammatory injury. Furthermore, the protein abundance of p-phosphoinositide 3-kinase (p-PI3K), p-protein kinase B (p-AKT), p-glycogen synthase kinase-3ß (p-GSK3ß), ß-catenin, and p-forkhead box O1 (p-FoxO1) was lower in the LPS group, whereas IFN-τ upregulated their abundance. The use of LY294002, a specific inhibitor of PI3K/AKT, attenuated the upregulation of p-PI3K, p-AKT p-GSK3ß, ß-catenin, and p-FoxO1 induced by IFN-τ, and also blocked the downregulation of TLR4, p-p65, and Bax/Bcl-2 ratio. This suggested that the inhibition of TLR4 signaling by IFN-τ was mediated by the PI3K/AKT pathway. Furthermore, compared with the LPS group, the ß-catenin agonist SB216763 led to greater p-FoxO1 and lower p-p65 and cell apoptosis. In contrast, knockdown of ß-catenin using small interfering RNA had the opposite effects. To explore the role of FoxO1 on the inhibition of TLR4 by IFN-τ, we employed LY294002 to inhibit the PI3K/AKT while FoxO1 was knocked down. Results revealed that the knockdown of FoxO1 blocked the upregulation of TLR4 and p-p65 induced by LY294002, and enhanced the inhibition of IFN-τ on TLR4, p-p65, and cell apoptosis. Overall, these findings confirmed that IFN-τ can protect endometrial epithelial cells against inflammatory injury via suppressing TLR4 activation through the regulation of the PI3K/AKT/ß-catenin/FoxO1 axis. These represent new insights into the molecular mechanisms underlying the anti-inflammatory function of IFN-τ in BEEC, and also provide a theoretical basis for further studies on the in vivo application of IFN-τ to help prevent negative effects of endometritis.

Upregulated-gene expression of pro-inflammatory cytokines, oxidative stress and apoptotic markers through inflammatory, oxidative and apoptosis mediated signaling pathways in Bovine Pneumonia.

Pneumonia is the acute inflammation of lung tissue and is multi-factorial in etiology. Staphylococcus aureus (S. aureus) is a harmful pathogen present as a normal flora of skin and nares of dairy cattle. In bovine pneumonia, S. aureus triggers to activates Toll-Like Receptors (TLRs), that further elicits the activation of the inflammation via NF-κB pathway, oxidative stress and apoptotic pathways. In the current study, pathogen-associated gene expression of the pro-inflammatory cytokines, oxidative stress and apoptotic markers in the lung tissue of cattle was explored in bovine pneumonia. Fifty lung samples collected from abattoir located in Wuhan city, Hubei, China. Histopathologically, thickening of alveolar wall, accumulation of inflammatory cells and neutrophils in perivascular space, hyperemia, hemorrhages and edema were observed in infected lungs as compared to non-infected lung samples. Furthermore, molecular identification and characterization were carried by amplification of S. aureus-specific nuc gene (270 base pairs) from the infected and non-infected lung samples to identify the S. aureus. Moreover, qPCR results displayed that relative mRNA levels of TLR2, TLR4, pro-inflammatory gene (IL-1ß, IL-6 and TNF-α) and apoptosis-associated genes (Bax, caspase-3 and caspase-9) were up-regulated except Bcl-2, which is antiapoptotic in nature, and oxidative stress related genes (Nrf2, NQO1, HO-1 and GCLC) which was down-regulated in infected pulmonary group. The relative protein expression of NF-κB, mitochondria-mediated apoptosis gene was up-regulated while Bcl-2 and Nrf2 pathway genes were downregulated in infected cattle lungs. Our findings revealed that genes expression levels of inflammatory mediators, oxidative stress and apoptosis were associated with host immunogenic regulatory mechanisms in the lung tissue during infection. Conclusively, the present study provides insights of active immune response via TLRs-mediated inflammatory, oxidative damage, and apoptotic paradox.

Ginsenoside Rb1 protects from Staphylococcus aureus-induced oxidative damage and apoptosis through endoplasmic reticulum-stress and death receptor-mediated pathways.

Acute lung injury (ALI) is acute uncontrolled inflammation of lung tissue that leads to high fatality both in human and animals. Staphylococcus aureus (S. aureus) could be an opportunistic, versatile bacterial etiology of ALI. Ginsenoside Rb1 (Rb1) is extracted from the Panax ginseng, which displays a wide range of biological and pharmacological effects. However, protective effects of Rb1 in S. aureus-induced ALI though endoplasmic reticulum (ER) stress and death receptor-mediated pathways have not yet been reported. Therefore, present study was planned with the aims to investigate the antioxidant and anti-apoptotic properties of Rb1 through regulation of ER stress as well as death receptor-mediated pathways in ALI induced by S. aureus in mice. In this study, four groups of healthy Kunming mice (n = 48) were used. The S. aureus (80 µl; 1 ×107 CFU/10 µl) was administered intranasally to establish mice model of ALI. After 24 h of onset of S. aureus-induced ALI, the mice were injected thrice with Rb1 (40 mg/kg) intraperitoneally six hours apart. Histopathology, enzyme linked immunosorbent assay (ELISA), real time quantitative polymerase chain reaction (RT-qPCR), Immunohistochemistry and western blotting assay were employed in the current study. Our results suggested that Rb1 administration save lungs from pulmonary injury by reducing wet to dry (W/D) ratio, protein levels, total cells, neutrophilic count, reactive oxygen species (ROS), myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx)1 depletion. Meanwhile, Rb1 therapy ameliorated histopathology alteration of lung tissue and pro-inflammatory cytokines secretion. The gene expression of ER stress marker (PERK, AFT-6, IRE1 and CHOP) were upregulated markedly (P < .05) in S. aureus-instilled groups, which was reduced by Rb1 administration that is reveled from the result findings of the RT-qPCR and immunoblot assay. The results of immunohistochemistry for CHOP indicated the increased expression in S. aureus groups which in turn ameliorated by Rb1 treatment. The mRNA expression demonstrated that death receptor-associated genes (FasL, Fas, FADD and caspase-8) showed up-regulation in S. aureus group. The similar findings were observed for the protein expression of caspase-8, FADD and Fas. Rb1 treatment markedly (P < .05) reversed protein and mRNA expression levels of these death receptor-associated genes when compared to the S. aureus group. Taken together, Rb1 attenuated S. aureus-induced oxidative damage via the ER stress-mediated pathway and apoptosis through death receptor-mediated pathway. Conclusively, our findings provide an insight into preventive mechanism of Rb1 in ALI caused by S. aureus and hence proven a scientific baseline for the therapeutic application of Rb1.

miR-148a suppresses inflammation in lipopolysaccharide-induced endometritis.

Endometritis is a postnatal reproductive disorder disease, which leads to great economic losses for the modern dairy industry. Emerging evidence indicates that microRNAs (miRNAs) play a pivotal role in a variety of diseases and have been identified as critical regulators of the innate immune response. Recent miRNome profile analysis revealed an altered expression level of miR-148a in cows with endometritis. Therefore, the present study aims to investigate the regulatory role of miR-148a in the innate immune response involved in endometritis and estimate its potential therapeutic value. Here, we found that miR-148a expression in lipopolysaccharide (LPS)-stimulated endometrial epithelial cells was significantly decreased. Our results also showed that overexpression of miR-148a using agomiR markedly reduced the production of pro-inflammatory cytokines, such as IL-1ß and TNF-α. Moreover, overexpression of miR-148a also suppressed NF-κB p65 activation by targeting the TLR4-mediated pathway. Subsequently, we further verified that miR-148a repressed TLR4 expression by binding to the 3'-UTR of TLR4 mRNA. Additionally, an experimental mouse endometritis model was employed to evaluate the therapeutic value of miR-148a. In vivo studies suggested that up-regulation of miR-148a alleviated the inflammatory conditions in the uterus as evidenced by H&E staining, qPCR and Western blot assays, while inhibition of miR-148a had inverse effects. Collectively, pharmacologic stabilization of miR-148a represents a novel therapy for endometritis and other inflammation-related diseases.

MicroRNA-182 supplies negative feedback regulation to ameliorate lipopolysaccharide-induced ALI in mice by targeting TLR4.

Acute lung injury (ALI), characterized by increased excessive pulmonary inflammation, is a pervasive inflammatory disease with clinically high incidence. MicroRNA (miRNAs) have been associated with the progression of multiple diseases and are regarded as novel regulators of inflammation. However, it remains largely unknown whether the miRNAs-mediated regulatory mechanism has an effect on lipopolysaccharide (LPS)-induced inflammation in ALI. We discovered that miR-182 distinctly lessened expression in the lung tissue of mice with ALI and macrophages stimulated by LPS. We also found that overexpression of miR-182 significantly cut down the secretion of inflammatory cytokines, while this change was reversed by inhibition of miR-182. In addition, miR-182 suppressed the activation of NF-κB by targeting TLR4 expression. And it was confirmed that miR-182 directly regulated TLR4 expression at the posttranscriptional level by binding to the 3'-UTR of TLR4. Together, these data suggested that inhibition of TLR4 expression assuaged LPS-stimulated inflammation through negative feedback regulation of miR-182.

MicroRNA-188-5p promotes apoptosis and inhibits cell proliferation of breast cancer cells via the MAPK signaling pathway by targeting Rap2c.

Breast cancer is a common malignancy that is highly lethal with poor survival rates and immature therapeutics that urgently needs more effective and efficient therapies. MicroRNAs are intrinsically involved in different cancer remedies, but their mechanism in breast cancer has not been elucidated for prospective treatment. The function and mechanism of microRNA-188-5p (miR-188) have not been thoroughly investigated in breast cancer. In our study, we found that the expression of miR-188 in breast cancer tissues was obviously reduced. Our findings also revealed the abnormal overexpression of miR-188 in 4T1 and MCF-7 cells significantly suppressed cell proliferation and migration and also enhanced apoptosis. miR-188 induced cell cycle arrest in the G1 phase. To illuminate the molecular mechanism of miR-188, Rap2c was screened as a single target gene by bioinformatics database analysis and was further confirmed by dual-luciferase assay. Moreover, Rap2c was found to be a vital molecular switch for the mitogen-activated protein kinase signaling pathway in tumor progression by decreasing apoptosis and promoting proliferation and migration. In conclusion, our results revealed that miR-188 is a cancer progression suppressor and a promising future target for breast cancer therapy.

miR-488 mediates negative regulation of the AKT/NF-κB pathway by targeting Rac1 in LPS-induced inflammation.

Endometritis is an inflammatory change in the structure of the endometrium due to various causes and is a common cause of infertility. Studies have confirmed that microRNAs (miRNAs) play a key regulatory role in various inflammatory diseases. However, the miRNA-mediated mechanism of endometrial inflammation induced by lipopolysaccharides (LPS) remains unclear. In this study, real-time quantitative polymerase chain reaction, Western blot analysis, immunofluorescence and Rac family small GTPase 1 (Rac1) interference were used to reveal the overexpression of miR-488 in the LPS-induced bovine uterus, and the effect of protein kinase B κ-light chain enhancement of the nuclear factor-activated B cells (AKT/NF-κB) pathway in intimal epithelial cells. The results showed that the expression of inflammatory cytokines such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α in the experimental group was significantly lower than that in the control group when miR-488 was overexpressed. Similar results were observed in the expression levels of p-AKT, p-IKK, and p-p65 proteins. In addition, the dual-luciferase reporter system confirmed that miRNA-488 may directly target the 3'-untranslated region of Rac1. In turn, the expression of Rac1 was inhibited. Moreover, the nuclear translocation of NF-κB was inhibited, and meanwhile, the accumulation of reactive oxygen species (ROS) in the cells was reduced. Thus, we provide basic data for the negative regulation of miR-488 in LPS-induced inflammation by inhibiting ROS production and the AKT/NF-kB pathway in intimal epithelial cells.

Ginsenoside Rb 1: A novel therapeutic agent in Staphylococcusaureus-induced Acute Lung Injury with special reference to Oxidative stress and Apoptosis.

Acute lung injury (ALI) is considered as an uncontrolled inflammatory response that can leads to acute respiratory distress syndrome (ARDS), which limits the therapeutic strategies. Ginsenosides Rb1 (Rb1), an active ingredient obtained from Panax ginseng, possesses a broad range of pharmacological and medicinal properties, comprising the anti-inflammatory, anti-oxidant, and anti-tumor activities. Therefore, the purpose of the present study was to investigate the protective effects of Rb1 against S. aureus-induced (ALI) through regulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) and mitochondrial-mediated apoptotic pathways in mice (in-vivo), and RAW264.7 cells (in-vitro). For that purpose, forty Kunming mice were randomly assigned into four treatment groups; (1) Control group (phosphate buffer saline (PBS); (2) S. aureus group; (3) S. aureus + Rb1 (20 mg/kg) group; and (4) Rb1 (20 mg/kg) group. The 20 µg/mL dose of Rb1 was used in RAW264.7 cells. In the present study, we found that Rb1 treatment reduced ALI-induced oxidative stress via suppressing the accumulation of malondialdehyde (MDA) and myeloperoxidase (MPO) and increase the antioxidant enzyme activities of superoxidase dismutase 1 (SOD1), Catalase (CAT), and glutathione peroxidase 1 (Gpx1). Similarly, Rb1 markedly increased messenger RNA (mRNA) expression of antioxidant genes (SOD1, CAT and Gpx1) in comparison with ALI group. The histopathological results showed that Rb1 treatment ameliorated ALI-induced hemorrhages, hyperemia, perivascular edema and neutrophilic infiltration in the lungs of mice. Furthermore, Rb1 enhanced the antioxidant defense system through activating the Nrf2 signaling pathway. Our findings showed that Rb1 treated group significantly up-regulated mRNA and protein expression of Nrf2 and its downstream associated genes down-regulated by ALI in vivo and in vitro. Moreover, ALI significantly increased the both mRNA and protein expression of mitochondrial-apoptosis-related genes (Bax, caspase-3, caspase-9, cytochrome c and p53), while decreased the Bcl-2. In addition, Rb1 therapy significantly reversed the mRNA and protein expression of these mitochondrial-apoptosis-related genes, as compared to the ALI group in vivo and in vitro. Taken together, Rb1 alleviates ALI-induced oxidative injury and apoptosis by modulating the Nrf2 and mitochondrial signaling pathways in the lungs of mice.

Peripheral Circulating Exosome-Mediated Delivery of miR-155 as a Novel Mechanism for Acute Lung Inflammation.

Emerging evidence has revealed that excessive activation of macrophages may result in an adverse lung inflammation involved in sepsis-related acute lung injury (ALI). However, it has never been clearly identified whether peripheral circulating serum exosomes participate in the pathogenesis of sepsis-related ALI. Therefore, the purposes of our study were to investigate the effect of serum exosomes on macrophage activation and elucidate a novel mechanism underlying sepsis-related ALI. Here we found that exosomes were abundant in the peripheral blood from ALI mice and selectively loaded microRNAs (miRNAs), such as miR-155. In vivo experiments revealed that intravenous injection of serum exosomes harvested from ALI mice, but not control mice, increased the number of M1 macrophages in the lung, and it caused lung inflammation in naive mice. In vitro, we demonstrated that serum exosomes from ALI mice delivered miR-155 to macrophages, stimulated nuclear factor κB (NF-κB) activation, and induced the production of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. Furthermore, we also showed that serum exosome-derived miR-155 promoted macrophage proliferation and inflammation by targeting SHIP1 and SOCS1, respectively. Collectively, our data suggest the important role of circulating exosomes secreted into peripheral blood as a key mediator of septic lung injury via exosome-shuttling miR-155.

Targeting the ROS/PI3K/AKT/HIF-1α/HK2 axis of breast cancer cells: Combined administration of Polydatin and 2-Deoxy-d-glucose.

It is well established that cancer cells depend upon aerobic glycolysis to provide the energy they need to survive and proliferate. However, anti-glycolytic agents have yielded few positive results in human patients, in part due to dose-limiting side effects. Here, we discovered the unexpected anti-cancer efficacy of Polydatin (PD) combined with 2-deoxy-D-glucose (2-DG), which is a compound that inhibits glycolysis. We demonstrated in two breast cell lines (MCF-7 and 4T1) that combination treatment with PD and 2-DG induced cell apoptosis and inhibited cell proliferation, migration and invasion. Furthermore, we determined the mechanism of PD in synergy with 2-DG, which decreased the intracellular reactive oxygen (ROS) levels and suppressed the PI3K/AKT pathway. In addition, the combined treatment inhibited the glycolytic phenotype through reducing the expression of HK2. HK2 deletion in breast cancer cells thus improved the anti-cancer activity of 2-DG. The combination treatment also resulted in significant tumour regression in the absence of significant morphologic changes in the heart, liver or kidney in vivo. In summary, our study demonstrates that PD synergised with 2-DG to enhance its anti-cancer efficacy by inhibiting the ROS/PI3K/AKT/HIF-1α/HK2 signalling axis, providing a potential anti-cancer strategy.

Sodium selenite induces apoptosis via ROS-mediated NF-κB signaling and activation of the Bax-caspase-9-caspase-3 axis in 4T1 cells.

Sodium selenite (SSE), a source of inorganic selenium, has been widely used as a clinical cancer treatment, but the precise molecular mechanisms of SSE remain to be elucidated. Our in vitro experiments have confirmed that SSE treatment causes a transient increase in intracellular reactive oxygen species (ROS) levels, resulting in the inhibition of nuclear transcription factor-κB (NF-κB) signaling and p65 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha phosphorylation levels in 4T1 cells. The inhibition of NF-κB subsequently increased the expression of the apoptosis gene B-cell lymphoma-2-associated X (Bax) and downregulated the transcription of antiapoptosis genes, such as B-cell lymphoma-2, cellular inhibitor of apoptosis 1, and X-linked inhibitor of apoptosis. Additionally, the accumulation of ROS caused mitochondrial dysfunction, leading to the activation of caspase-9 and -3, thereby resulting in apoptosis. However, modulation of the ROS level by the chemical inhibitor N-acetyl-cysteine reversed these events. Similarly, in vitro murine syngeneic breast tumor models showed that SSE inhibits tumor growth by promoting apoptosis. These results indicate that SSE induces apoptosis via ROS-mediated inhibition of NF-κB signaling and activation of the Bax-caspase-9-caspase-3 axis.

miR-497a-5p attenuates lipopolysaccharide-induced inflammatory injury by targeting IRAK2.

Acute lung injury (ALI) is a severe acute inflammatory reaction of the lungs caused by a variety of factors, which can lead to a high mortality rate. MicroRNAs are a novel therapeutic molecule that play a vital role in many diseases. However, its mechanism of action in lipopolysaccharide (LPS)-induced mouse ALI is not clear. The study aimed to investigate the mechanism of action of miR-497 in LPS-induced ALI. As a result, it was found that the expression of miR-497 in the inflammatory reaction showed a decrease in time and dose trends. Importantly, miR-497 reduced LPS-induced expression levels of related inflammatory factors. In addition, we also demonstrated that IRAK2 is a direct target molecule of miR-497. Interestingly, we further found that miR-497 inhibits the expression of IRAK2 by targeting IRAK2-3'UTR. Therefore, miR-497 can partially negatively regulate the activation of IRAK2-NF-κB pathway in LPS-induced inflammatory responses.

Ginsenoside Rb1 ameliorates Staphylococcus aureus-induced Acute Lung Injury through attenuating NF-κB and MAPK activation.

Acute lung injury (ALI) is clinically characterized by excessive inflammation leading to acute respiratory distress syndrome (ARDS), having high morbidity and mortality both in human and animals. Ginsenoside Rb1 (Rb1) is a major primary bioactive component extracted by Panax ginseng, which has numerous pharmacological functions such as anti-cancer, anti-inflammatory, and antioxidant. However, the anti-inflammatory effects of Rb1 in Staphylococcus aureus (S. aureus)-induced ALI in mice have not been investigated. The aim of the current study was to determine the anti-inflammatory influence of Rb1 on S. aureus-induced ALI in mice, and to explore its possible underlying principle mechanisms in RAW 264.7 macrophage cells. The results of physical morphology, histopathological variation and wet-to-dry weight ratio of lungs revealed that Rb1 significantly attenuated S. aureus-induced lung injury. Furthermore, qPCR results displayed that Rb1 inhibited IL-1ß, IL-6 and TNF-α production both in vivo and in vitro. The activation of Toll-like receptor 2 (TLR2) by S. aureus was inhibited by application of Rb1 as confirmed by results of immunofluorescence assay. The expression of NF-kB and MAPK signaling proteins revealed that Rb1 significantly attenuated the phosphorylation of p65, ERK, as well as JNK. Altogether, the results of this experiment presented that Rb1 has ability to protect S. aureus-induced ALI in mice by attenuating TLR-2-mediated NF-kB and MAPK signaling pathways. Consequently, Rb-1 might be a potential medicine in the treatment of S. aureus-induced lung inflammation.

Shikonin exerts anti-inflammatory effects in LPS-induced mastitis by inhibiting NF-κB signaling pathway.

Previous studies have shown that shikonin(SHI), the bioactive naphthoquinone constituent extracted from Chinese herb Lithospermum Erythrorhizon, possesses the potential to confront inflammation, and has little concerns towards drug residues comparing with antibiotics. While mastitis in dairy industry always trigger great harm to milk yields, effects of SHI on lipopolysaccharides (LPS)-induced mastitis should be measured. Here, we demonstrate anti-inflammatory effects of SHI on LPS-challenged mastitis and elucidate the potential signaling pathway both in vivo and in vitro. As a result, SHI administration mice significantly suffered less impairment of mammary gland and less recruitment of neutrophils than LPS administration mice. SHI significantly suppressed the expression of p-IκBα and p-p65, which are the critical proteins functioning in NF-kB signaling pathway. qPCR results indicate decreasing level of upstream pro-inflammatory cytokines in tissues, such as TNF-α, IL-1ß, and IL-6. The results are corresponding with the results in vitro, suggesting the potential usage of SHI as a therapeutic medicine in mastitis.

Nuciferine alleviates LPS-induced mastitis in mice via suppressing the TLR4-NF-κB signaling pathway.

BACKGROUND: Nuciferine, a major bioactive component from the lotus leaf, has been reported to have notable anti-inflammatory activities such as renal inflammation and acute lung injury in previous studies. Mastitis is one of the most prevalent diseases in the dairy cattle, which causes large economic losses for the dairy industry. However, the effects of nuciferine on lipopolysaccharide (LPS)-induced mastitis have not been reported. METHODS AND RESULTS: Here, we investigated the anti-inflammatory effects of nuciferine on LPS-induced mastitis in mice and illuminated its potential mechanism on the TLR4-mediated signaling pathway in mouse mammary epithelial cells (mMECs). Histopathological changes and myeloperoxidase (MPO) activity assay showed that nuciferine treatment significantly alleviated the LPS-induced injury of mammary gland flocculus, inflammatory cells infiltration. qPCR and ELISA assays indicated that nuciferine dose-dependently reduced the levels of TNF-α and IL-1ß, which indicated that nuciferine might have therapeutic effects on mastitis. Furthermore, nuciferine treatment significantly decreased the expression of TLR4 in a dose-dependent manner. Besides, nuciferine was also found to suppress LPS-induced NF-κB activation. CONCLUSION: These findings indicate that nuciferine potently ameliorates LPS-induced mastitis by inhibition of the TLR4-NF-κB signaling pathway.

Polydatin reduces Staphylococcus aureus lipoteichoic acid-induced injury by attenuating reactive oxygen species generation and TLR2-NFκB signalling.

Staphylococcus aureus (S. aureus) causes severe inflammation in various infectious diseases, leading to high mortality. The clinical application of antibiotics has gained a significant curative effect. However, it has led to the emergence of various resistant bacteria. Therefore, in this study, we investigated the protective effect of polydatin (PD), a traditional Chinese medicine extract, on S. aureus lipoteichoic acid (LTA)-induced injury in vitro and in vivo. First, a significant improvement in the pathological conditions of PD in vivo was observed, suggesting that PD had a certain protective effect on LTA-induced injury in a mouse model. To further explore the underlying mechanisms of this protective effect of PD, LTA-induced murine macrophages were used in this study. The results have shown that PD could reduce the NF-κB p65, and IκBα phosphorylation levels increased by LTA, resulting in a decrease in the transcription of pro-inflammatory factors, such as TNF-α, IL-1ß and IL-6. However, LTA can not only activate NF-κB through the recognition of TLR2 but also increase the level of intracellular reactive oxygen species (ROS), thereby activating NF-κB signalling. We also detected high levels of ROS that activate caspases 9 and 3 to induce apoptosis. In addition, using a specific NF-κB inhibitor that could attenuate apoptosis, namely NF-κB p65, acted as a pro-apoptotic transcription factor in LTA-induced murine macrophages. However, PD could inhibit the generation of ROS and NF-κB p65 activation, suggesting that PD suppressed LTA-induced injury by attenuating ROS generation and TLR2-NFκB signalling.

Polydatin ameliorates Staphylococcus aureus-induced mastitis in mice via inhibiting TLR2-mediated activation of the p38 MAPK/NF-κB pathway.

Recent studies show that Polydatin (PD) extracted from the roots of Polygonum cuspidatum Sieb, a widely used traditional Chinese remedies, possesses anti-inflammatory activity in several experimental models. In this study, we investigated the anti-inflammatory effects of PD on Staphylococcus aureus-induced mastitis in mice and elucidated the potential mechanisms. In mice with S aureus-induced mastitis, administration of PD (15, 30, 45 mg/kg, ip) or dexamethasone (Dex, 5 mg/kg, ip) significantly suppressed the infiltration of inflammatory cells, ameliorated the mammary structural damage, and inhibited the activity of myeloperoxidase, a biomarker of neutrophils accumulation. Furthermore, PD treatment dose-dependently decreased the levels of TNF-α, IL-1ß, IL-6 and IL-8 in the mammary gland tissues. PD treatment also dose-dependently decreased the expression of TLR2, MyD88, IRAK1, IRAK4 and TRAF6 as well as the phosphorylation of TAK1, MKK3/6, p38 MAPK, IκB-α and NF-κB in the mammary gland tissues. In mouse mammary epithelial cells (mMECs) infected by S aureus in vitro, pretreatment with PD dose-dependently suppressed the upregulated pro-inflammatory cytokines and signaling proteins, and the nuclear translocation of NF-κB p65 and AP-1. A TLR2-neutralizing antibody mimicked PD in its suppression on S aureus-induced upregulation of MyD88, p-p38 and p-p65 levels in mMECs. PD (50, 100 µg/mL) affected neither the growth of S aureus in vitro, nor the viability of mMECs. In conclusion, PD does not exhibit antibacterial activity against S aureus, its therapeutic effects in mouse S aureus-induced mastitis depend on its ability to down-regulate pro-inflammatory cytokine levels via inhibiting TLR2-mediated activation of the p38 MAPK/NF-κB signaling pathway.

Puerarin Exerts an Antiinflammatory Effect by Inhibiting NF-kB and MAPK Activation in Staphylococcus aureus-Induced Mastitis.

Mastitis is defined as the inflammation of the mammary gland. There is generally no effective treatment for mastitis in animals. Puerarin, extracted from Radix puerariae, has been proven to possess many biological activities. The present study aims to reveal the potential mechanism that is responsible for the antiinflammatory action of puerarin in Staphylococcus aureus (S. aureus)-induced mastitis in mice. Histopathological changes showed that puerarin ameliorated the inflammatory injury induced by S. aureus. Quantitative real-time polymerase chain reaction and ELISA analysis indicated that puerarin not only suppressed the production of pro-inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 but also promoted the secretion of IL-10. Toll-like receptor 2 (TLR2) is important in the immune defense against S. aureus infection. Research in molecular biology has shown that the expression of TLR2 was inhibited with administration of puerarin. Further studies were performed on NF-kB and mitogen-activated protein kinase signaling pathways using western blot. The results demonstrated that puerarin suppressed phosphorylated IkBα, p65, p38, extracellular signal-regulated kinase 1and 2 (ERK), and c-Jun N-terminal kinase (JNK) in a dose-dependent manner. All of the results suggested that puerarin may be a potential therapy for treating mastitis. Copyright © 2016 John Wiley & Sons, Ltd.

Serological and molecular survey of Toxoplasma gondii infection and associated risk factors in urban cats in Kunming, Southwest China.

Toxoplasma gondii (T. gondii) is a worldwide zoonotic parasite that can infect almost warm-blood animals, including humans, which seriously affect the health of host. Cats are known to be the only definitive host of T. gondii and continuously excrete highly infectious oocysts. This parasite carried by the companion animals leads to a great public health risk. However, there is little information on epidemiology of T. gondii in urban cats in Kunming, Southwest China. In the present study, a total of 231 serum and fecal samples were collected in Kunming aera, and then seroprevalence of T. gondii IgG antibodies in serum and molecular investigation in feces were analyzed to elucidate T. gondii infection in urban cats. The results revealed that 168 of 231 cats (72.7%) were positive for T. gondii antibodies, and 1 of 74 cat feces (1.4%) also showed a positive PCR for T. gondii DNA. The positive fecal sample was sequenced and then phylogenetically analyzed, and the isolate of T. gondii in the present study was closely related to T. gondii strain CN. In addition, the food, water and age of cats were identified as the risk factor for seropositivity. Overall, our findings indicate the widespread occurrence of T. gondii infection in urban cats in Kunming, Southwest China and identify food, water and age are the risk factors associated with T. gondii infection, which can provide effective information for developing strategies to prevent and control this zoonosis.

Effect of high NEFA concentration on lipid metabolism disorders in hepatocytes based on lipidomics.

Introduction: High concentrations of nonesterified fatty acids (NEFA) is the key of characteristic of fatty liver in dairy cows. Therefore, the aim of this study was to investigate the effect of high concentration of NEFA on lipid metabolism in hepatocytes through the lipidomic approach and molecular biology techniques. Methods: Stimulate AML-12 cells with different concentrations of NEFA, observe the cellular lipid accumulation, and select 0.6 mM NEFA stimulation concentration for subsequent experiments. Collect cells for lipidomics analysis. Results: High concentration of NEFA (0.6-2.4 mM) significantly reduced the cell viability in a concentration-dependent manner, indicating that high concentrations of NEFA have lipotoxicity on hepatocytes. In addition, NEFA promoted triglycerides (TAG) accumulation, increased the mRNA expression of the lipogenic molecules SREBP1c and FASN, and decreased the mRNA expression of lipolytic molecules CPT1A and HSL in hepatocytes. Mechanistically, high concentration of NEFA induced lipid metabolism disorders in hepatocytes by regulating metabolic pathways such as glycerol phospholipid metabolism, glycosyl phosphatidylinositol anchored biosynthesis, triglyceride metabolism, sphingolipid metabolism, and inositol phosphate metabolism. Discussion: High concentration of NEFA is lipotoxic to cells, promoting lipid accumulation. LPE (18:2), LPE (18:3), LPE (18:1) via glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, glycerolipid metabolism, sphingolipid metabolism, and inositol phosphate metabolism, indicating their potential regulation role in the pathogenesis of fatty liver.