Puerarin attenuates S. aureus-induced mastitis through inhibiting inflammation and ferroptosis by activating the SIRT1/p53/SLC7A11 signaling pathway

Abstract

Mastitis, one of the most prevalent diseases in humans and animals, is commonly caused by local infection of the mammary gland. Puerarin inhibits mitochondrial oxidative stress and acute liver injury in mice. However, the therapeutic effect of puerarin on Staphylococcus aureus-induced mastitis is still not well understood. This study was designed to investigate the therapeutic role and mechanism of puerarin on S. aureus-induced mastitis. S. aureus was injected through the mammary gland to induce mouse mastitis. Puerarin was administered to mastitis mice via intraperitoneal injection. The iron concentration was measured using an iron assay. TNF-α and IL-1β levels in the mammary gland tissues were assessed using the ELISA kits. The signaling pathways were detected by western blot analysis. Hematoxylin and eosin staining revealed that puerarin reversed pathological injury to mammary gland tissue induced by S. aureus. Puerarin also inhibited TNF-α and IL-1β production induced by S. aureus. The significant increases in iron concentration, myeloperoxidase activity, and the malondialdehyde (MDA) level in the mammary gland tissue were attenuated by puerarin. Moreover, puerarin treatment significantly reversed the decreases in the relative glutathione (GSH) and GPX4 expression. In addition, puerarin increased the expression of SIRT1 and solute carrier family 7 member 11 (SLC7A11) and decreased p53 expression. In vitro, the inhibitory effects of puerarin on S. aureus-induced inflammation and ferroptosis were prevented by the SIRT1 inhibitor EX-527. In conclusion, the therapeutic mechanism of puerarin involves the activation of the SIRT1/p53/SLC7A11 pathway to inhibit ferroptosis and the inflammatory response.IMPORTANCEAntibiotics are the main drugs used to treat mastitis, but they can easily cause bacterial resistance and food safety issues. Therefore, finding safe and effective drugs is crucial to prevent and treat mastitis. Puerarin is the main isoflavonoid active ingredient in Pueraria lobata and has a wide range of pharmacological effects. We found that it could attenuate mastitis induced by Staphylococcus aureus.

Keywords: SIRT1; ferroptosis; inflammation; mastitis; puerarin.

Fig 1

Effects of puerarin on S. aureus-induced mammary histopathological changes. Histopathologic sections of mammary tissues (H&E, ×100). Twenty-four hours after puerarin treatment, the mammary gland tissues were collected for the histological analysis by H&E staining (A) control, (B) puerarin (100 mg/kg) group, (C) S. aureus group, and (D–F) Puerarin (25, 50, and 100 mg/kg) + S. aureus groups.

Fig 2

Effect of puerarin on MPO activity in the mammary gland. Twenty-four hours after puerarin treatment, the mammary gland tissues were collected, and the MPO activity in mammary gland tissues was detected by the MPO detection kit. The values presented are the mean ± SD and were analyzed using one-way ANOVA. ^#P < 0.01 indicates a significant difference from the control group; **P < 0.01 indicates a significant difference from the S. aureus group.

Fig 3

Effect of puerarin on inflammatory cytokine production in the mammary gland. Twenty-four hours after puerarin treatment, the mammary gland tissues were collected, and the levels of inflammatory cytokines were measured by ELISA. The values presented are the mean ± SD and were analyzed using one-way ANOVA. ^#P < 0.01 indicates a significant difference from the control group; **P < 0.01 indicates a significant difference from the S. aureus group.

Fig 4

Effect of puerarin on GPX4 and ferritin expression, MDA, iron, and GSH production in the mammary gland. Twenty-four hours after puerarin treatment, the mammary gland tissues were collected, and the MDA, iron, and GSH production in mammary gland tissues were detected by the detection kits. The expression of GPX4 and ferritin was detected by western blot analysis. The values presented are the mean ± SD and were analyzed using one-way ANOVA. ^#P < 0.01 indicates a significant difference from the control group; **P < 0.01 indicates a significant difference from the S. aureus group.

Fig 5

Effect of puerarin on NF-κB activation in the mammary gland. Twenty-four hours after puerarin treatment, the mammary gland tissues were collected, and the expression of NF-κB signaling pathway proteins was measured by western blot analysis. The values presented are the mean ± SD and were analyzed using one-way ANOVA. ^#P < 0.01 indicates a significant difference from the control group; **P < 0.01 indicates a significant difference from the S. aureus group.

Fig 6

Effect of puerarin on SIRT1, p53, and SLC7A11 expression in the mammary gland. Twenty-four hours after puerarin treatment, the mammary gland tissues were collected, and the expression of SIRT1, p53, and SLC7A11 was measured by western blot analysis. The values presented are the mean ± SD and were analyzed using one-way ANOVA. ^#P < 0.01 indicates a significant difference from the control group; **P < 0.01 indicates a significant difference from the S. aureus group.

Fig 7

SIRT1 inhibitor reverses the inhibition of puerarin on S. aureus-induced inflammation and ferroptosis. The effect of puerarin on the viability of mMECs was detected by the CCK8 method. mMECs were treated with EX-527 (10 µm) and puerarin (60 µm) and stimulated with S. aureus. Samples were collected after 24 hours for follow-up testing. The expression of IL-1β and TNF-α was detected by qRT-PCR. The production of ferroptosis indicators (MDA, GSH, and Fe²⁺) was detected by the detection kits. The values presented are the mean ± SD and were analyzed using one-way ANOVA. ^#P < 0.01 indicates a significant difference from the control group; **P < 0.01 indicates a significant difference from the S. aureus group.