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 Ca, 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-κβ) signaling pathway and the NOD-like receptor with pyrin domain containing-3 (NLRP3) inflammasome. Notably, preconditioning of PB2 alleviated the accumulation of ROS and Ca 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-κβ signaling pathway and the NLRP3 inflammasome, as well as IL-1β 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.