Search

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.

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.

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.

Mastitis, an inflammation of mammary gland, is a serious disease that affects the health of dairy cows around the world. Myricetin, a flavonoid from Bayberry, has been reported to suppress various inflammatory response. The aim of this study was to evaluate the effect of myricetin on lipopolysaccharide (LPS)-induced in vivo and in vitro mastitis model and clarify the underlying mechanism. In vivo experiments, myricetin attenuated the severity of inflammatory lesion and neutrophil infiltration. Moreover, myricetin pretreatment induced a significant decrease in the activity of myeloperoxidase (MPO) and the production of TNF-α, IL-6, and IL-1β triggered by LPS. Myricetin pretreatment could also increase the integrity of the blood-milk barrier and upregulate the tight junction proteins in LPS-induced mice mastitis. In vitro, myricetin inhibited LPS-induced inflammatory response in mice mammary epithelial cells (mMECs). In the further mechanism studies, we found that the anti-inflammatory effect of myricetin was mediated by inhibiting LPS-induced phosphorylation of AKT, IKK-α, IκB-α, and P65 in vivo and in vitro. Collectively, these data suggested that myricetin effectively ameliorated the inflammatory response by inhibiting the AKT/IKK/NF-κB signaling pathway and repairing the integrity of blood-milk barrier in LPS-induced mice mastitis.

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.

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-1β 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.

Accumulating evidence has shown that activated microglia cause inflammatory immune response, which could lead to neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. α-Cyperone, one of the main ingredients of Cyperus rotundus oil, has been reported to possess anti-inflammatory activity in activated macrophages. In this study, we found that α-cyperone markedly decreased the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in LPS-induced BV-2 cells. Moreover, α-cyperone inhibited NF-κB activation and enhanced heme oxygenase-1 (HO-1), nuclear factor-E2-related factor 2 (Nrf2) and Akt expression. Furthermore, we found that α-cyperone could upregulate HO-1 expression and enhance nuclear translocation of Nrf2 via activating the Akt signaling pathway, and inhibition of Akt, Nrf2 or HO-1 attenuated LPS-induced expression of proinflammatory cytokines in BV-2 cells. Moreover, the toxicities of conditioned medium from activated microglia toward dopaminergic neuronal SH-SY5Y cells and hippocampal neuronal HT22 cells were significantly inhibited by pretreatment with α-cyperone. Taken together, our results indicate that α-cyperone exerts neuroprotective effects by inhibiting the production of inflammatory cytokines in BV-2 cells through activating Akt/Nrf2/HO-1 and suppressing the NF-κB pathway.

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.

Background/Aims: Mastitis is an acute clinical inflammatory response. The occurrence and development of mastitis seriously disturb women's physical and mental health. Licochalcone A, a phenolic compound in Glycyrrhiza uralensis, has anti-inflammatory properties. Here, we examined the effect of licochalcone A on blood-milk barrier and inflammatory response in LPS-induced mice mastitis. Methods: In vivo, we firstly established mice models of mastitis by canal injection of LPS to mammary gland, and then detected the effect of licochalcone A on pathological indexes, inflammatory responses and blood-milk barrier in this model. In vivo, Mouse mammary epithelial cells (mMECs) were treated with licochalcone A prior to the incubation of LPS, and then the inflammatory responses, tight junction which is the basic structure of blood-milk barrier were analyzed. Last, we elucidated the anti-inflammatory mechanism by examining the activation of mitogen-activated protein kinase (MAPK) and AKT/NF-κB signaling pathways in vivo and in vitro. Result: The in vivo results showed that licochalcone A significantly decreased the histopathological impairment and the inflammatory responses, and improved integrity of blood-milk barrier. The in vitro results demonstrated that licochalcone A inhibited LPS-induced inflammatory responses and increase the protein levels of ZO-1, occludin, and claudin3 in mMECs. The in vivo and in vitro mechanistic study found that the anti-inflammatory effect of licochalcone A in LPS-induced mice mastitis was mediated by MAPK and AKT/NF-κB signaling pathways. Conclusions and Implications: Our experiments collectively indicate that licochalcone A protected against LPS-induced mice mastitis via improving the blood–milk barrier integrity and inhibits the inflammatory response by MAPK and AKT/NF-κB signaling pathways.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by selective loss of dopaminergic neurons in the substantia nigra (SN). Neuroinflammation induced by over-activation of microglia leads to the death of dopaminergic neurons in the pathogenesis of PD. Therefore, downregulation of microglial activation may aid in the treatment of PD. Polydatin (PLD) has been reported to pass through the blood-brain barrier and protect against motor degeneration in the SN. However, the molecular mechanisms underlying the effects of PLD in the treatment of PD remain unclear. The present study aimed to determine whether PLD protects against dopaminergic neurodegeneration by inhibiting the activation of microglia in a rat model of lipopolysaccharide (LPS)induced PD. Our findings indicated that PLD treatment protected dopaminergic neurons and ameliorated motor dysfunction by inhibiting microglial activation and the release of pro-inflammatory mediators. Furthermore, PLD treatment significantly increased levels of p-AKT, pGSK-3βSer9, and Nrf2, and suppressed the activation of NF-κB in the SN of rats with LPS-induced PD. To further explore the neuroprotective mechanism of PLD, we investigated the effect of PLD on activated microglial BV-2 cells. Our findings indicated that PLD inhibited the production of pro-inflammatory mediators and the activation of NF-κB pathways in LPS-induced BV-2 cells. Moreover, our results indicated that PLD enhanced levels of p-AKT, p-GSK-3βSer9, and Nrf2 in BV-2 cells. After BV-2 cells were pretreated with MK2206 (an inhibitor of AKT), NP-12 (an inhibitor of GSK-3β), or Brusatol (BT; an inhibitor of Nrf2), treatment with PLD suppressed the activation of NF-κB signaling pathways and the release of pro-inflammatory mediators in activated BV-2 cells via activation of the AKT/GSK3β-Nrf2 signaling axis. Taken together, our results are the first to demonstrate that PLD prevents dopaminergic neurodegeneration due to microglial activation via regulation of the AKT/GSK3β-Nrf2/NF-κB signaling axis. Funding Sources: This work was funded by National Nature Science Foundation of China (project No. 31772547, 31672509), Jilin Scientific and Technological Development Program (project No. 20170623029TC, 20170623083-04TC). Conflicts of Interest: The authors report no conflicts of interest. The study design was approved by the appropriate ethics review board.

Vanillin is used in a variety of food, chemical, and pharmaceutical applications, and exhibits anti-inflammatory properties. However, there are no reports about the effects of vanillin on lipopolysaccharide (LPS)-induced mastitis. In this study, we explored the effects of vanillin on the subsequent inflammatory response and blood-milk barrier in LPS-induced mastitis. Results showed that vanillin suppressed the inflammatory response by a) inhibiting myeloperoxidase activity; b) decreasing the production of pro-inflammatory mediators which include tumor necrosis factor alpha (Tnf-α; from 128.5 ± 14.59 to 67.51 ± 10.88,pg/mL, P 0.01), interleukin-6 (Il-6; from 531.5 ± 196.4 to 109.3 ± 24.14, pg/mL, P 0.05), interleukin-1β (Il-1β; from 2569 ± 1648 to 731.8 ± 171.7, pg/mL, P 0.05), inducible nitric oxide synthase (Inos), and cyclooxygenase-2 (Cox-2); and c) repairing the blood-milk barrier by increasing the protein levels of the tight junction proteins, including zona occludens 1 (Zo-1), claudin-3, and occludin. In vitro experiment, Vanillin can inhibit LPS-induced inflammation and enhance the protein levels of tight junction proteins. Further studies have shown that vanillin inhibits inflammation by inhibiting mitogen-activated protein kinases (MAPKs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. Our findings showed that vanillin protects mammary gland from LPS-induced mastitis by enhancing the blood-milk barrier and inhibiting the inflammatory response.

Peiminine, an alkaloid extracted from Fritillaria plants, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effect of peiminine on a mouse lipopolysaccharide (LPS)-induced mastitis model remains to be elucidated. The purpose of this experiment was to investigate the effect of peiminine on LPS-induced mastitis in mice. LPS was injected through the canals of the mammary gland to generate the mouse LPS-induced mastitis model. Peiminine was administered intraperitoneally 1 h before and 12 h after the LPS injection. In vitro, mouse mammary epithelial cells (mMECs) were pretreated with different concentrations of peiminine for 1 h and were then stimulated with LPS. The mechanism of peiminine on mastitis was studied by hematoxylin-eosin staining (H&amp, E) staining, western blotting, and enzyme-linked immunosorbent assay (ELISA). The results showed that peiminine significantly decreased the histopathological impairment of the mammary gland in vivo and reduced the production of pro-inflammatory mediators in vivo and in vitro. Furthermore, peiminine inhibited the phosphorylation of the protein kinase B (AKT)/ nuclear factor-&kappa, B (NF-&kappa, B), extracellular regulated protein kinase (ERK1/2), and p38 signaling pathways both in vivo and in vitro. All the results suggested that peiminine exerted potent anti-inflammatory effects on LPS-induced mastitis in mice. Therefore, peiminine might be a potential therapeutic agent for mastitis.

Kisspeptins (Kps) play a key role in the regulation of GnRH axis and as an anti-metastasis agent by binding with GPR54. Recently, we observed that the expression of GPR54 was higher in the lactating mammary tissues of dairy cows with high-quality milk (0.81 ± 0.13 kg/day of milk protein yield; 1.07 ± 0.18 kg/day of milk fat yield) than in those with low-quality milk (0.51 ± 0.14 kg/day of milk protein yield; 0.67 ± 0.22 kg/day of milk fat yield). We hypothesized that Kp-10 might regulate the milk protein, β-casein (CSN2) synthesis via GPR54 and its downstream signaling. First, we isolated the bovine mammary epithelial cells (bMECs) from lactating Holstein dairy cows, and treated them with different concentrations of Kp-10. Compared with the control cells, the synthesis of CSN2 is significantly increased at a concentration of 100 nM of Kp-10. In addition, the increased effect of CSN2 synthesis was blocked when the cells were pre-treated with the selective inhibitor of GPR54 Peptide-234 (P-234). Mechanistic study revealed that Kp-10 activated ERK1/2, AKT, mTOR and STAT5 in bMECs. Moreover, inhibiting ERK1/2, AKT, mTOR and STAT5 with U0126, MK2206, Rapamycin and AG490 could block the effects of Kp-10. Together, these results demonstrate that Kp-10 facilitates the synthesis of CSN2 via GPR54 and its downstream signaling pathways mTOR, ERK1/2, STAT5 and AKT.