Caveolin 1 ameliorates nonesterified fatty acid-induced oxidative stress via the autophagy regulator beclin 1 in bovine mammary gland epithelial cells.

High blood concentrations of nonesterified fatty acids (NEFA) during ketosis enhance uptake by the mammary gland and impair autophagy while causing oxidative stress. Caveolin 1 (CAV1) is closely related to autophagy and plays a role in regulating oxidative stress. The aim of this study was to explore the potential role of CAV1 on oxidative stress and autophagy during a high-NEFA challenge in the immortalized bovine mammary epithelial cell line (MAC-T). Mammary gland tissue biopsies and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) Holstein cows at 3 to 10 (average = 6) days in milk. Compared with healthy cows, ketotic cows had lower DMI, daily milk yield, and serum glucose, and greater serum NEFA and BHB, accompanied by greater milk fat and lower milk protein. Malondialdehyde (MDA) was greater, but activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase were lower in cows with clinical ketosis. A lower protein abundance of CAV1, beclin 1, autophagy relative gene 5 (ATG5), and microtubule-associated protein 1 light chain 3 (LC3) as well as greater protein abundance of sequestosome-1 (SQSTM1, also called p62) were detected in mammary tissue of cows with clinical ketosis. In vitro, the MAC-T cells were treated with 0, 0.6, and 1.2 mM NEFA for 12 h or treated with 1.2 mM NEFA for various lengths of time (0, 0.5, 1, 2, 4, 8, 12, and 24 h). Compared with 0 mM NEFA, protein abundances of CAV1, beclin 1, ATG5, and LC3 were greater in the MAC-T challenged with 0.6 mM NEFA but lower in the 1.2 mM NEFA group. Protein abundance of p62 was lower with 0.6 mM NEFA but higher with 1.2 mM NEFA. In response to increasing doses of NEFA, mRNA abundance of CAV1, total antioxidant capacity and SOD activity decreased, whereas the levels of reactive oxygen species and MDA content increased. The protein abundances of CAV1 and beclin 1 peaked at 0.5 h, the protein abundances of ATG5 and LC3 peaked at 1 h, and these proteins start to fade away at later time points under NEFA treatment, resulting in both linear and quadratic effects. The protein abundance of p62 decreased, reaching a nadir at 4 h in both a linear and quadratic manner. The silencing of CAV1 in MAC-T cells aggravated the 1.2 mM NEFA-induced decrease in beclin 1 expression, impaired autophagy, and increased severe oxidative stress, whereas the overexpression of CAV1 alleviated these effects. Pretreatment of MAC-T cells with beclin 1 small interfering RNA (si-beclin 1) and the overexpression of CAV1, followed by challenge with 1.2 mM NEFA, resulted in reversed CAV1-induced autophagy, thereby aggravating oxidative stress. Overall, these data suggest that CAV1 protects bovine mammary epithelial cells from NEFA-induced oxidative stress through enhancing the expression of beclin 1 and activating autophagy.