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-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 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-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.