Blue LED light induces cytotoxicity via ROS production and mitochondrial damage in bovine subcutaneous preadipocytes.

The purpose of this study was to investigate the effect and mechanism of blue light irradiation on bovine subcutaneous preadipocytes. In this study, preadipocytes were divided into dark group (control) and blue light group. Results show that blue light exposure time-dependently reduced the viability of preadipocytes and induced mitochondrial damage, in accompaniment with the accumulation of intracellular reactive oxygen species (ROS). Meanwhile, blue light caused oxidative stress, as evidenced by the increased MDA level, the reduced T-AOC contents, as well as the decreased activities of antioxidant enzymes. Additionally, blue light treatment induced apoptosis and G2/M phase arrest via Bcl-2/Bax/cleaved caspase-3 pathway and P53/GADD45 pathway, respectively. Protein expressions of LC3-II/LC3-I and P62 were up-regulated under blue light exposure, indicating blue light initiated autophagy but impeded autophagic degradation. Moreover, blue light caused an increase in the secretion of pro-inflammatory factors (TNF-α, IL-1ß, and IL-6). Pretreatment with N-acetylcysteine (NAC), a potent ROS scavenger, restored the loss of mitochondrial membrane potential (Δψ) and reduced excess ROS. Additionally, the above negative effects of blue light on cells were alleviated after NAC administration. In conclusion, this study demonstrates blue light induces cellular ROS overproduction and Δψ depolarization, resulting in the decrease of cell viability and the activation of apoptosis, autophagy, and inflammation, providing a reference for the application of blue light in the regulation of fat cells in the future.

Low temperature exposure inhibits proliferation and induces apoptosis of bovine subcutaneous preadipocytes via p38 MAPK/JNK activation.

Cold stress can cause changes in cell growth states. Cell proliferation and apoptosis are important physiological processes to maintain normal growth and development of cells and tissues. We sought to identify the impact of low temperature exposure on bovine subcutaneous preadipocyte proliferation and apoptosis. We first cultured preadipocytes at different low temperatures (35°C, 33°C, and 31°C) for 12 h, and then at 31°C for 24 and 48 h. The results showed that compared with the 37°C group, exposure to 31°C significantly reduced cell viability and number as well as inhibited cell cycle progression in preadipocytes. Moreover, low temperature also significantly upregulated the apoptosis rate of preadipocytes. After low temperature treatment, the mRNA levels of Cyclin E, CDK2 and B-cell lymphoma 2 (Bcl-2) were decreased in preadipocytes, whereas that of p53, p21 and Bcl-2 associated x protein (Bax) were increased in preadipocytes. Concurrently, low temperature increased the proteins levels of p53, Bax and Cleaved Caspase3, and reduced the protein level of Bcl-2 in preadipocytes. Furthermore, the elevated phosphorylation levels of p38 mitogen-activated protein kinases (p38 MAPK) and c-jun NH2-terminal kinase (JNK) were detected in cold-treated preadipocytes. The influence of low temperature exposure on preadipocyte proliferation and apoptosis were obviously weakened after blocking the p38 MAPK and JNK signaling pathways. In conclusion, low temperature exposure could inhibit proliferation and cell cycle progression and induce apoptosis through activation of p38 MAPK and JNK signaling pathways in bovine subcutaneous preadipocytes.

Procyanidin B2 Alleviates Heat-Induced Oxidative Stress through the Nrf2 Pathway in Bovine Mammary Epithelial Cells.

The objective of this study was to investigate the protective effects and potential molecular mechanisms of procyanidin B2 (PB2) in MAC-T (mammary alveolar cells-large T antigen) cells during heat stress (HS). The MAC-T cells were divided into three treatment groups: control (37 °C), HS (42 °C), and PB2 + HS (42 °C). Compared with MAC-T cells that were consistently cultured at 37 °C, acute HS treatment remarkably decreased cell viability, reduced activities of catalase (CAT), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC), and elevated intracellular levels of malondialdehyde (MDA) and reactive oxygen species (ROS). Additionally, nuclear factor erythroid 2-related factor 2 (Nrf2) was activated and translocated to the nucleus, in accompaniment with upregulation of Nrf2, heme oxygenase 1 (HO-1), thioredoxin reductase 1 (Txnrd1), and heat shock protein 70 (HSP70). In parallel, both mRNA transcript and actual protein secretion of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), were increased by heat stress. Pretreatment of MAC-T cells with 0~25 µM PB2 alleviated the decline of cell viability by HS in a dose-dependent fashion and protected cells against HS-induced oxidative stress, as evidenced by significantly improved CAT, SOD, and T-AOC activity, as well as with decreased MDA and ROS generation. Furthermore, PB2 further activated the Nrf2 signaling pathway and reversed the inflammatory response induced by HS. Silencing of Nrf2 by si-Nrf2 transfection not only exacerbated HS-induced cell death and provoked oxidative stress and the inflammatory response, but also greatly abolished the cytoprotective effects under HS of PB2. In summary, PB2 protected MAC-T cells against HS-induced cell death, oxidative stress, and inflammatory response, partially by operating at the Nrf2 signal pathway.

Preconditioning with procyanidin B2 protects MAC-T cells against heat exposure-induced mitochondrial dysfunction and inflammation.

Heat stress (HS) induced by high environmental temperature is a main factor causing mastitis and reduced milk production in dairy cows. Procyanidin B2 (PB2) is a phenolic compound with strong anti-inflammatory and antioxidant properties. By using the MAC-T (mammary alveolar cells-large T antigen) cells as the in vitro cell model, this study determines PB2 effects on HS-induced MAC-T mitochondrial dysfunction, cell apoptosis, and inflammation. Cells were divided into three groups: Con (37 °C), HS (42 °C), and PB2 +HS. Results show that, under HS-exposure, MAC-T cells exhibited an increased accumulation of reactive oxygen species (ROS) and Ca2+, a decreased mitochondrial membrane potential (Δψ) and ATP content. Besides, HS markedly induced cell apoptosis, as evidenced by flow cytometry and significantly increased mRNA and protein expressions of apoptosis-related genes, including cytochrome C (Cyto-c) and cleaved caspase-3, etc. HS also led to mitochondrial fission and fusion dynamic disorder. Meanwhile, HS induced a significant inflammatory response by activating the Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κß) signaling pathway and the NOD-like receptor with pyrin domain containing-3 (NLRP3) inflammasome. Notably, preconditioning of PB2 alleviated the accumulation of ROS and Ca2+ concentration induced by HS, increased Δψ and ATP content, and maintained the dynamic balance of mitochondrial fission and fusion, thus improving mitochondrial function. PB2 also blocked the HS-induced mitochondrial caspase apoptosis pathway. Furthermore, PB2 preconditioning inhibited HS-induced activation of the TLR4/NF-κß signaling pathway and the NLRP3 inflammasome, as well as IL-1ß release, reversing HS-induced inflammation. In conclusion, PB2 has an important protective effect against the mitochondrial dysfunction, inflammatory response, and apoptosis of MAC-T cells induced by HS.

BMP2 increases hyperplasia and hypertrophy of bovine subcutaneous preadipocytes via BMP/SMAD signaling.

The study aims to characterize functions of bone morphogenetic protein 2 (BMP2) gene in the process of subcutaneous (SQ) fat deposition of bovine, thereby providing insights into mechanisms for the use of BMP2 in fat management. Our results show that BMP2 was extensively expressed in bovine and relatively rich in adipose tissue. Exogenous BMP2 significantly enhanced proliferation of bovine preadipocytes. Consistently, si-BMP2 apparently induced cell cycle arrest at G0/G1 phase and decreased proliferation of preadipocytes. Meanwhile, exogenous BMP2 mildly enhanced preadipocyte differentiation at day 3 of differentiation, as evidenced by accelerated lipid accumulation, as well as increased mRNA and protein expressions of adipogenic key transcription factor PPARγ; contrary results about lipids were found by BMP2 interference treatment. No difference was observed concerning BMP2 or si-BMP2 treatment at day - 2 and day 0 of differentiation. Additionally, LDN-193189 (inhibitor of BMP type I receptor) pretreatment diminished the enhancement of preadipocyte proliferation and differentiation induced by BMP2, as evidenced by constant proliferation rate and PPARγ expressions. Furthermore, BMP2 markedly enhanced phosphorylation level of SMAD1/5/9, and LDN-193189 could diminish the difference caused by BMP2. Thus, our results suggest that BMP2 triggers BMP/SMAD signaling pathway, promoting both hyperplasia and hypertrophy of bovine preadipocytes.