Apoptosis In Bovine Mammary Epithelial Cells Research Articles

Resveratrol inhibits LPS-induced apoptosis in bovine mammary epithelial cells: the role of PGC1α-SIRT3 axis

Resveratrol (Res) is a bioactive dietary component and alleviates apoptosis in multiple cell types. However, its effect and mechanism on lipopolysaccharide (LPS)-induced bovine mammary epithelial cells (BMEC) apoptosis, which commonly happens in dairy cows with mastitis, is unknown. We hypothesized that Res would inhibit LPS-induced apoptosis in BMEC through SIRT3, a NAD + -dependent deacetylase activated by Res. To test the dose-response effect on apoptosis, 0-50μM Res were incubated with BMEC for 12h, followed by 250μg/mL LPS treatment for 12h. To investigate the role of SIRT3 in Res-mediated alleviation of apoptosis, BMEC were pretreated with 50μM Res for 12h, then incubated with si-SIRT3 for 12h and were finally treated with 250μg/mL LPS for 12h. Res dose-dependently promoted the cell viability and protein levels of Bcl-2 (Linear P < 0.001) but decreased protein levels of Bax, Caspase-3 and Bax/Bcl-2 (Linear P < 0.001). TUNEL assays indicated that cellular fluorescence intensity declined with the rising doses of Res. Res also dose-dependently upregulated SIRT3 expression, but LPS had the opposite effect. SIRT3 silencing abolished these results with Res incubation. Mechanically, Res enhanced the nuclear translocation of PGC1α, the transcriptional cofactor for SIRT3. Further molecular docking analysis revealed that Res could directly bind to PGC1α by forming a hydrogen bond with Tyr-722. Overall, our data suggested that Res relieved LPS-induced BMEC apoptosis through the PGC1α-SIRT3 axis, providing a basis for further in vivo investigations of applying Res to relieve mastitis in dairy cows.

Lentinan inhibits oxidative stress and alleviates LPS-induced inflammation and apoptosis of BMECs by activating the Nrf2 signaling pathway

Lentinan (LNT) has been reported to have a wide range of functions, including anti-inflammatory, antioxidant and anticancer properties. LNT may provide a protective effect in dairy cow mastitis. In this study, we investigated the effect of LNT on lipopolysaccharide (LPS)-induced injury of bovine mammary epithelial cells (BMECs) and the possible mechanism. First, we treated BMECs with different concentrations of LPS to study the effects of LPS on oxidative stress and inflammation in BMECs. Then, we examined the effects of LNT by dividing the cells into seven groups: the control group (CON), LPS treatment group (LPS), Acetyl-l-cysteine (NAC) pretreatment group (NAC + LPS), LNT pretreatment group (LNT + LPS), ML385 and LNT pretreatment group (ML385 + LNT + LPS), LNT treatment group (LNT) and NAC treatment group (NAC). The results showed that LPS-triggered intracellular ROS production and the downregulation of Nrf-2 and HO-1 in BMECs were blocked by LNT pretreatment. LNT inhibited the expression of inflammatory genes and proteins by inhibiting of NF-κB and MAPK. In addition, LNT attenuated LPS induced-apoptosis in BMECs. However, ML385 reversed the protective effect of LNT. Taken together, LNT can be used as a natural protective agent against LPS-triggered BMECs damage through its anti-inflammatory, antioxidant and antiapoptotic effects through modulation of the Nrf2 pathway.

Matrine attenuates bovine mammary epithelial cells inflammatory responses induced by Streptococcus agalactiae through inhibiting NF-κB and MAPK signaling pathways

Streptococcus agalactiae is one of the main pathogens associated with bovine mastitis. The invasion of S. agalactiae in bovine mammary epithelial cells (BMECs) has been implicated as a key event in the pathogenesis of mastitis. Matrine is known for its various pharmacological activities, such as immune response regulation and anti-inflammation. The primary aim of the research was to investigate the preventive effect of matrine on S. agalactiae-induced inflammation in BMECs along with underlying molecular mechanisms. Our data showed matrine at the concentrations of 50–100 μg/mL promoted BMECs proliferation without infection, and decreased cytotoxicity induced by S. agalactiae. Subsequently, BMECs were pre-treated with matrine (50, 75, or 100 μg/mL) for 24 h, followed by the infection with S. agalactiae for an additional 6 h. Pretreatment with matrine followed by S. agalactiae treatment decreased cell apoptosis of BMECs. Also, pretreatment of matrine to BMECs prevented the invasion of S. agalactiae. The mRNA abundances of IL-1β, IL-6, IL-8, and TNF-α were down-regulated in S. agalactiae-infected cells pretreated with matrine. In addition, the greater ratios of protein NF-κB p-p65/p65, p-IκBα/IκBα, p-38/38, and p-ERK/ERK induced by S. agalactiae were attenuated due to matrine treatment. Furthermore, pretreatment of BMECs with matrine impeded the degradation of TAK1 induced by S. agalactiae infection. These results suggest matrine could be a potential modulator in immune response of the mammary gland. In conclusion, matrine prevents cellular damage due to S. agalactiae infection by the modulation of NF-κB and MAPK signaling pathways and pro-inflammatory cytokine production.

Non-Esterified Fatty Acid Induces ER Stress-Mediated Apoptosis via ROS/MAPK Signaling Pathway in Bovine Mammary Epithelial Cells.

Elevated concentrations of non-esterified fatty acid (NEFA) induced by negative energy balance (NEB) during the transition period of dairy cows is known to be toxic for multiple bovine cell types. However, the effect of NEFA in bovine mammary epithelial cells (BMECs) remains unclear. The present study aimed to explore the role and molecular mechanism of NEFA in endoplasmic reticulum (ER) stress and the subsequent apoptosis in BMECs. The results showed that NEFA increased ER stress and activated the three unfolded protein response (UPR) signaling sub-pathways by upregulating the expression of GRP78, HSP70, XBP1, ATF6, phosphor-PERK, and phosphor-IRE1α. We also found that NEFA dose-dependently induced apoptosis in BMECs, as indicated by flow cytometry analysis and increased apoptotic gene expression. RNA-seq analysis revealed that NEFA induced apoptosis in BMECs, probably via the ATF4-CHOP axis. Mechanistically, our data showed that NEFA increased reactive oxygen species (ROS) levels, resulting in the activation of the MAPK signaling pathway. Moreover, quercetin, a well-known antioxidant, was found to alleviate ER stress-mediated apoptosis in NEFA-treated BMECs. Collectively, our results suggest that NEFA induces ER stress-mediated apoptosis, probably via the ROS/MAPK signaling pathway, as quercetin has been shown to alleviate ER stress-mediated apoptosis in NEFA-treated BMECs.

Nicotinamide mononucleotide alleviates heat stress-induced oxidative stress and apoptosis in BMECs through reducing mitochondrial damage and endoplasmic reticulum stress

Heat stress is directly correlated to mammary gland dysfunction in dairy cows, especially in summer. Abnormally high environmental temperature induces oxidative stress and apoptosis in bovine mammary epithelial cells (BMECs). Nicotinamide mononucleotide (NMN) has beneficial effects in maintaining the cellular physiological functions. In this study, we evaluate the protective effect of NMN on heat stress-induced apoptosis of BMECs and explore the potential underlying mechanisms. Our results showed that heat stress considerably decreased cell viability in BMECs, whereas pretreatment of BMECs with NMN (150 μM) for 24 h significantly alleviated the negative effects of heat stress on cells. NMN protected BMECs from heat stress-induced oxidative stress by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing the activity of antioxidant enzymes. It also inhibited apoptosis by reducing the ratio of Bax/Bcl2 and blocking proteolytic the cleavage of Caspase-3 in heat stressed-BMECs. Importantly, NMN treatment could reduce mitochondrial damage through mediating the expression of mitochondrial fission and fusion-related genes, including Dynamin related protein 1 (Drp1), Mitochondrial fission 1 protein (Fis1), and Mitofusin1, 2 (MFN1, 2); and suppress endoplasmic reticulum stress through unfolded protein response regulator Glucose regulated protein 78 (GRP78), and downstream elements Recombinant activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). Above all, our results demonstrate that NMN supplemention attenuates heat stress-induced oxidative stress and apoptosis in BMECs by maintaining mitochondrial fission and fusion, and regulating endoplasmic reticulum stress, which provides the convincing evidence that NMN has valuable potential in alleviating mammary gland injury of dairy cows caused by environmental heat stress.

SIRT3 protects bovine mammary epithelial cells from heat stress damage by activating the AMPK signaling pathway

With global warming, heat stress has become an important challenge for the global dairy industry. Sirtuin 3 (SIRT3), an important mitochondrial NAD+dependent decarboxylase and a major regulator of cellular energy metabolism and antioxidant defense, is integral to maintaining normal mitochondrial function. The aim of this study was to assess the protective effect of SIRT3 on damage to bovine mammary epithelial cells (BMECs) induced by heat stress and to explore its potential mechanism. Our results indicate that SIRT3 is significantly downregulated in heat-stressed mammary tissue and high-temperature-treated BMECs. SIRT3 knockdown significantly increased the expression of HSP70, Bax, and cleaved-caspase 3 and inhibited the production of antioxidases, thus promoting ROS production and cell apoptosis in BMECs. In addition, SIRT3 knockdown can aggravate mitochondrial damage by mediating the expression of genes related to mitochondrial fission and fusion, including dynamin-related protein 1, mitochondrial fission 1 protein, and mitochondrial fusion proteins 1and 2. In addition, SIRT3 knockdown substantially decreased AMPK phosphorylation in BMECs. In contrast, SIRT3 overexpression in high-temperature treatment had the opposite effect to SIRT3 knockdown in BMECs. SIRT3 overexpression reduced mitochondrial damage and weakened the oxidative stress response of BMECs induced by heat stress and promoted the phosphorylation of AMPK. Taken together, our results indicate that SIRT3 can protect BMECs from heat stress damage through the AMPK signaling pathway. Therefore, the reduction of oxidative stress by SIRT3 may be the primary molecular mechanism underlying resistance to heat stress in summer cows.

S-allyl cysteine ameliorates heat stress-induced oxidative stress by activating Nrf2/HO-1 signaling pathway in BMECs

Heat stress-induced oxidative stress in bovine mammary epithelial cells (BMECs) threatens the normal growth and development of bovine mammary tissue, resulting in lower milk production of dairy cows. The aim of the present study is to investigate the protective effects of S-allyl cysteine (SAC), an organosulfur component extracted from aged garlic, on heat stress-induced oxidative stress and apoptosis in BMECs and to explore its underlying mechanisms. Our results showed that heat stress treatment considerably decreased cell viability, whereas SAC treatment dose-dependently restored cell viability of BMECs under heat-stress conditions. In addition, SAC protected BMECs from heat stress-induced oxidative damage by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing the activity of antioxidant enzymes. It also inhibited heat stress-induced apoptosis by reducing the ratio of Bax/Bcl-2 and blocking proteolytic the cleavage of caspase-3 in BMECs. Interestingly, we found that the protective effect of SAC on heat stress-induced oxidative stress and apoptosis was dependent on the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. SAC promoted the Nrf2 nuclear translocation in heat stress-induced BMECs. The results were also validated by Nrf2 and Keap1 knockdown experiments further demonstrating that Nrf-2 was indeed involved in the protective effect of SAC on heat stress-induced oxidative damage and apoptosis. In summary, our results showed that SAC could protect BMECs from heat stress-induced injury by mediating the Nrf2/HO-1 signaling pathway, suggesting that SAC could be considered as a therapeutic drug for attenuating heat stress-induced mammary gland diseases.

Sodium valproate attenuates the iE-DAP induced inflammatory response by inhibiting the NOD1-NF-κB pathway and histone modifications in bovine mammary epithelial cells

The anti-inflammatory effects of sodium valproate (VPA) in vivo and in vitro have been demonstrated in recent studies. The aim of this study was to evaluate whether VPA can suppress inflammation in bovine mammary epithelial cells (BMECs) stimulated by γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP). First, the concentration and treatment points of iE-DAP and VPA were optimized. Then, BMECs were cultured in complete media and separated into four groups: untreated control cells (CON group), cells stimulated by 10 μg/mL iE-DAP for 6 h (DAP group), cells stimulated by 0.5 mmol/L VPA for 6 h (VPA group), and cells pretreated with VPA (0.5 mmol/L) for 6 h followed by 10 μg/mL of iE-DAP for 6 h (VD group). The results showed that the level of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the culture medium increased in the iE-DAP-treated cells and that pretreatment with VPA reversed this increase. iE-DAP increased both mRNA and protein expression levels of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and receptor-interacting protein kinas (RIPK2) and activated inhibitor of NF-κB (IκB) and nuclear factor-kappa B p65 (NF-κB p65) through phosphorylation. Upon activation of the NF-κB pathway, the expression of the pro-inflammatory cytokines IL-6, interleukin-8 (IL-8) and interleukin-1β (IL-1β), the acute phase protein serum amyloid A 3 (SAA3) and the lingual antimicrobial peptide (LAP) but not haptoglobi (HP) or bovine neutrophil beta defensing 5 (BNBD5) were increased in the DAP group. The VPA pretreatment induced the acetylation of signal transducers and activators of transcription(STAT1) and histone 3 (H3) by inhibiting histone deacetylase (HDAC) and then suppressed the NF-κB pathway. Moreover, VPA induced autophagy and reduced apoptosis in BMECs in the VD group. These results suggested that VPA treatment can attenuate the inflammatory response induced by iE-DAP.

Matrine and baicalin inhibit apoptosis induced by Panton-Valentine leukocidin of Staphylococcus aureus in bovine mammary epithelial cells

It was previously thought that the Panton-Valentine leukocidin (PVL) toxin of Staphylococcus aureus (S. aureus) was not the main cause of cow mastitis. However, in recent years, detection of the gene encoding PVL has been increasing in dairy cow mastitis, which implies that PVL may be related to bovine mastitis. Therefore, we wanted to search for drugs inhibiting PVL or PVL-induced apoptosis. In this report, we investigated the apoptosis mechanism of PVL in bovine mammary epithelial cells (BMEC) and the inhibition mechanism of matrine and baicalin on PVL-induced apoptosis of BMEC. The results demonstrated that BMEC were damaged and underwent apoptosis by a standard PVL-producing strain of S. aureus (ATCC 49775), a PVL knockout mutant Δpvl 49775, complemented mutant C-Δpvl 49775, or recombinant (r)PVL in vitro. The rates of apoptosis and necrosis induced by S. aureus ATCC 49775 and C-Δpvl 49775 were significantly higher than those induced by Δpvl 49775, demonstrating that BMEC apoptosis and necrosis were associated with PVL. In addition, this research found matrine and baicalin could inhibit the apoptosis of BMEC induced by PVL-producing S. aureus and by rPVL. Matrine downregulated protein expression levels of endogenous and exogenous cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9, and the effect was pronounced at a concentration of 50 μg/mL. Baicalin downregulated the expression of cleaved caspase-9. These results suggested that matrine and baicalin may have potential value against cow mastitis caused by the toxin PVL.

Induction of Chemerin on Autophagy and Apoptosis in Dairy Cow Mammary Epithelial Cells.

Simple SummaryThe process of mammary gland involution during the early is accomplished by both apoptosis and autophagy. Chemerin, a novel adipocytokine, plays a pivotal role in immune response and lipid metabolism, which was involved in the regulation of programmed cell death. This study focused on the relationship between autophagy and apoptosis in the presence of Chemerin in bovine mammary epithelial cells (BMECs). The results indicated that Chemerin could activate the complete autophagy process and induce apoptotic cascade in BMECs. The addition of Chloroquine (CQ), an inhibitor of autophagy, prompted Chemerin to have more obvious effects on apoptosis-related factors, which suggests that Chemerin-induced autophagy involves the intrinsic apoptotic pathway of BMECs. We found that the pro-apoptotic potential of Chemerin is further enhanced under conditions in which autophagy is effectively inhibited. Thus, exploring the role of autophagy and apoptosis on the involution of mammary epithelial cells will undoubtedly be of great importance in productivity improvement of dairy animals. Involution of the mammary gland is a complex process controlled by various endocrine hormones and cytokine. As a novel adipocytokine, Chemerin not only plays a pivotal role in physiological and pathological processes such as immune response and lipid metabolism, but is also involved in the regulation of programmed cell death, including autophagy and apoptosis. The purpose of the present study was to elucidate whether autophagy and apoptosis of bovine mammary epithelial cells (BMECs) was triggered by Chemerin. BMECs were cultured and treated with Chemerin in vitro. The expression of autophagosome-forming marker, microtubule-associated protein 1 light chain 3 II (LC3-II) and sequestosome-1 (SQSTM 1, best known as p62), a substrate of autophagosome degradation were detected. The result showed that Chemerin significantly decreased the expression of p62 and markedly induced the conversion of LC3-I to LC3-II. The ratio of Bcl2-associated X and B-cell lymphoma-2 (Bax/Bcl-2) and the activity of caspase-3 were up-regulated after being treated by Chemerin, and the apoptotic rate was also significantly increased. These results suggested that Chemerin promoted the occurrence of autophagy and apoptosis in BMECs. Chloroquine (CQ), which is an inhibitor of autophagy. To explore effects of Chemerin on apoptosis, we prevented Chemerin-induced autophagy by pre-adding CQ in BMECs. Interestingly, this part of the experiment helped us find that all effects of Chemerin on apoptosis of BMECs could be enhanced with the inhibition of autophagy. Our study demonstrates that Chemerin-induced autophagy and apoptosis are mutually regulated in BMECs, but the specific mechanism remains to be further researched.

Comparison of the effect of recombinant bovine wild and mutant lipopolysaccharide-binding protein in lipopolysaccharide-challenged bovine mammary epithelial cells.

Lipopolysaccharide (LPS)-binding protein (LBP) plays a crucial role in the recognition of bacterial components, such as LPS that causes an immune response. The aim of this study was to compare the different effects of recombinant bovine wild LBP and mutant LBP (67 Ala → Thr) on the LPS-induced inflammatory response of bovine mammary epithelial cells (BMECs). When BMECs were treated with various concentrations of recombinant bovine lipopolysaccharide-binding protein (RBLBP) (1, 5, 10, and 15 μg/mL) for 12 h, RBLBP of 5 μg/mL increased the apoptosis of BMECs induced by LPS without cytotoxicity, and mutant LBP resulted in a higher cell apoptosis than wild LBP did. By gene-chip microarray and bioinformatics, the data identified 2306 differentially expressed genes that were changed significantly between the LPS-induced inflamed BMECs treated with 5 μg/mL of mutant LBP and the BMECs only treated with 10 μg/mL of LPS (fold change ≥2). Meanwhile, 1585 genes were differently expressed between the inflamed BMECs treated with 5 μg/mL of wild LBP and 10 μg/mL of LPS-treated BMECs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these differentially expressed genes were involved in different pathways that regulate the inflammation response. It predicted that carriers of this mutation increase the risk for a more severe inflammatory response. Our study provides an overview of the gene expression profile between wild LBP and mutant LBP on the LPS-induced inflammatory response of BMECs, which will lead to further understanding of the potential effects of LBP mutations on bovine mammary glands.

The role of native bovine α-lactalbumin in bovine mammary epithelial cell apoptosis and casein expression

Folding variants of alpha-lactalbumin (alpha-la) are known to induce cell death in a number of cell types, including mammary epithelial cells (MEC). The native conformation of alpha-la however has not been observed to exhibit this biological activity. Here we report that native bovine alpha-la reduced the viability of primary bovine mammary epithelial cells (BMEC) and induced caspase activity in mammospheres, which are alveolar-like structures formed by culturing primary BMEC on extracellular matrix in the presence of lactogenic hormones. These observations suggest a possible role for bovine alpha-la in involution and/or maintaining the luminal space in mammary alveoli during lactation. In addition, co-incubation of bovine alpha-la in an in-vitro mammosphere model resulted in decreased beta-casein mRNA expression and increased alphas1- and kappa-casein mRNA expression. This differential effect on casein expression levels is unusual and raises the possibility of manipulating expression levels of individual caseins to alter dairy processing properties. Manipulation of alpha-la levels could be further investigated for its potential to enhance milk protein expression and/or improve lactational persistency by influencing the balance between proliferation and apoptosis of BMEC, which has a major influence on the milk-producing capacity of the mammary gland.