Pre-treatment but not co-treatment with vitexin alleviates hyperthermia induced oxidative stress and inflammation in buffalo mammary epithelial cells.

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-1ß, 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.

Curcumin induces thermotolerance by reducing oxidative stress, apoptosis, and inflammation in buffalo mammary epithelial cells under heat shock conditions.

The epithelial cell is the main basic unit of the udder in which milk synthesis takes place. Curcumin is well known for its antioxidant, anti-apoptotic, and anti- inflammatory properties. The present study was performed to test whether in vitro curcumin supplementation can alleviate the unfavorable impact of hyperthermia on buffalo mammary epithelial cells (BuMECs). The spontaneously immortalized BuMECs were divided into 7 groups (n = 9); 1) unstressed BuMECs (negative control, 37 °C); 2) BuMECs exposed to hyperthermia without curcumin treatment (positive control); 3-7) BuMECs cultured with different concentrations of curcumin (5, 10, 20, 40 and 60 µM), respectively, followed by hyperthermic exposure (42ºC) for 1 h and then returned to 37ºC. Changes in viability (MTT assay), proliferation (BrdU colorimetric immunoassay) and concentrations of antioxidant enzymes, CAT, and SOD (ELISA) of BuMECs were recorded. The gene expression study was performed using qRT-PCR. Lower concentrations of curcumin (5, 10 µM) maintained viability, enhanced proliferation, and content of antioxidant enzymes of heat stressed BuMECs. Curcumin induced thermotolerance and antioxidant status by upregulating the expression of antioxidants genes, anti-apoptotic genes and heat shock proteins in heat stressed BuMECs compared to the positive control group. Besides, curcumin reduced apoptosis and inflammation in BuMECs exposed to hyperthermia by downregulating the expression of genes and transcriptional factors associated with apoptosis and inflammatory immune response. The results reveal the potential roles of curcumin in eliminating the negative impact of hyperthermia on BuMECs by regulating the pathways of apoptosis, inflammation, and oxidative stress.