Evaluation the protective role of baicalin against H2O2-driven oxidation, inflammation and apoptosis in bovine mammary epithelial cells

Abstract

Mastitis is one of the most common diseases in dairy farms. During the perinatal period, the bovine mammary epithelial cells (BMECs) of High-yielding dairy cows accelerate metabolism and produce large amounts of reactive oxygen species (ROS). It is one of the primary causes of mastitis and will lead to the breakdown of redox balance, which will induce oxidative stress, inflammation, and apoptosis. Baicalin is a flavonoid substance extracted from the root of natural plant Scutellaria baicalensis, which has anti-inflammatory, anti-oxidant, anti-viral and other biological functions. In this research, hydrogen peroxide (H₂O₂) was used to construct a mastitis oxidative stress model, and relevant mechanisms were analyzed by immunofluorescence techniques, qRT-PCR and Western Blot to explore how baicalin affects BMECs' oxidative stress and inflammation caused by H₂O₂, as well as to provide new perspectives on the combined application of baicalin in the prevention and treatment of mastitis. The results demonstrated that baicalin treatment could reduce the accumulation of H₂O₂-induced intracellular ROS and decrease the expression of inflammatory cytokines Tumor Necrosis Factor-α (TNF-α), interleukin 6 (IL-6), interleukin-1β (IL-1β) and the apoptosis rate. The inhibitory effect of baicalin on H₂O₂-induced intracellular ROS accumulation and the expression of inflammatory cytokines and apoptotic factors in BMECs was blocked by pretreatment with the Nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor retinoic acid (RA) prior to H₂O₂ and/or baicalin treatment. In summary, baicalin could served as a natural antioxidant agent to regulate cell apoptosis through its anti-inflammatory, antioxidant and anti-apoptotic effects to combat BMECs damage caused by H₂O₂.

Keywords: apoptosis; baicalin; bovine mammary epithelial cells (BMECs); inflammation; nuclear factor erythroid 2-related factor 2 (Nrf2); oxidative stress.

Figure 1

Exploration of the optimal concentration and time of H₂O₂ and baicalin. (A) Effect of different concentrations of H₂O₂ (0, 100, 200, 400, 600, 800, and 1,000 μM) on the cell viability of BMECs upon stimulation for 4, 6, and 8 h. *P < 0.05 compared with the CON, **P < 0.05 compared with the 200 μM H₂O₂ group, ^#P < 0.05 compared with the 400 μM H₂O₂ group. (B) Effect of different concentrations of H₂O₂ on ROS levels in BMECs upon stimulation for 6 h. *P < 0.05. (C) Effect of different concentrations of baicalin (0, 5, 10, 25, 50, 100 μM) on the cell viability of BMECs upon stimulation for 12, 24 and 48 h. *P < 0.05 compared with the CON, **P < 0.05 compared with the 10 μM baicalin group, ^#P < 0.05 compared with the 50 μM baicalin group. (D) BMECs were treated with baicalin at different concentrations, and 200 μM H₂O₂ was added 6 h before the end of treatment to determine the cell viability at 24 h. *P < 0.05. (E) Effect of different concentrations of baicalin on H₂O₂-induced ROS levels in BMECs. *P < 0.05. The data from the CON were used to normalize the data of each treatment group. Each treatment was repeated three times, and the results are expressed as the means ± SEM.

Figure 2

Effect of baicalin on H₂O₂-induced changes in ROS, MDA, SOD and T-AOC in BMECs. BMECs were treated with 5 μM RA for 1 h, and baicalin (10 μM) was then added for 18 h before cotreatment with 200 μM H₂O₂ for 6 h. (A) The ROS fluorescence intensity was imaged using a fluorescence microscope (scale bar is 200 μm). (B) Changes in ROS levels in BMECs. (C) Changes in MDA content in BMECs. (D) Changes in SOD activity in BMECs. (E) Changes in T-AOC levels in BMECs. *P < 0.05. RA, Nrf2 inhibitor.

Figure 3

Activation of the Keap1/Nrf2 signaling pathway by baicalin. BMECs were treated with 5 μM RA for 1 h, and baicalin (10 μM) was then added for 18 h before cotreatment with 200 μM H₂O₂ for 6 h. (A) Western blot analysis of Keap1 and Nrf2 protein levels in BMECs. Immunoreactive bands are shown on the left, and quantitative results are shown on the right. (B) Nrf2 mRNA levels in BMECs. (C) Keap1 mRNA levels in BMECs. (D) HO-1 mRNA levels in BMECs. (E) NQO1 mRNA levels in BMECs. *P < 0.05.

Figure 4

Baicalin inhibits the H₂O₂-induced NF-κB signaling pathway. BMECs were treated with 5 μM RA for 1 h, and baicalin (10 μM) was then added for 18 h before cotreatment with 200 μM H₂O₂ for 6 h. (A) Western blot analysis of IκBα, p65 and p-p65 protein levels in BMECs. Immunoreactive bands are shown on the left, and quantitative results are shown on the right. (B) IL-6 mRNA levels in BMECs. (C) IL-1β mRNA levels in BMECs. (D) TNF-α mRNA levels in BMECs. *P < 0.05.

Figure 5

Baicalin inhibits the H₂O₂-induced caspase3/Bcl-2 signaling pathway. BMECs were treated with 5 μM RA for 1 h, and baicalin (10 μM) was then added for 18 h before cotreatment with 200 μM H₂O₂ for 6 h. (A) Western blot analysis of caspase3 and Bax protein levels in BMECs. Immunoreactive bands are shown on the left, and quantitative results are shown on the right. (B) Caspase3 mRNA levels in BMECs. (C) Bax mRNA levels in BMECs. (D) Bcl-2 mRNA levels in BMECs. (E) Ratio of Bcl-2 to Bax mRNA levels in BMECs. *P < 0.05.

Figure 6

Schematic summary of the effect and mechanism of baicalin on H₂O₂-induced oxidative stress, inflammation and apoptosis in BMECs. Baicalin resists H₂O₂-induced oxidative damage in BMECs by activating the Keap1/Nrf2 signaling pathway and reducing ROS production, which in turn inhibits NF-κB inflammation and the caspase3/Bcl-2 apoptotic pathway to maintain intracellular redox homeostasis.