This study investigated if in vitro supplementation of
vitexin could mitigate the adverse effects of
hyperthermia on buffalo mammary epithelial cells (BuMECs). Immortalized BuMECs were divided into seven groups (n = 3): (1) a negative control group at 37 °C; (2) BuMECs exposed to heat stress as a positive control at 42 °C for 1 h; (3-7) heat stressed BuMECs pre-treated or co-treated with different concentrations of
vitexin (5 μM, 10 μM, 20 μM, 50 μM, and 100 μM), respectively.
Hyperthermia was induced by exposing the cells to 42 ºC for 1 h. For the pre-treatment experiment, BuMECs were treated with
vitexin for 2 h before
hyperthermia exposure. For co-treatment,
vitexin was added simultaneously with
hyperthermia for 1 h. Subsequently, the cells were allowed to recover for 12 h at 37 °C. Results showed that pre-treatment with
vitexin was more effective than co-treatment in protecting BuMECs from
hyperthermia in a dose-dependent manner, with higher concentrations (50 μM and 100 μM) being the most effective. Pre-treatment with
vitexin maintained cellular viability and prevented
inflammation by inducing the expression of the anti-apoptotic gene (BCL-2) and reducing the expression of the pro-apoptotic gene (Bax) and pro-inflammatory mediators (IL-1β, IL-6) in heat-stressed BuMECs. Pre-treatment with
vitexin reduced oxidative stress and induced thermotolerance by increasing the expression of
antioxidants mediators such as SOD, GPx and CAT at
mRNA and
protein levels, and modulating the expression of
heat shock proteins. The findings suggest that
vitexin has the potential as a therapeutic agent to protect the mammary gland from the negative impact of
hyperthermia in dairy cows.