Effect of the flavonoid baicalin on the proliferative capacity of bovine mammary cells and their ability to regulate oxidative stress

Mammary tissue sampling

Lactating Holstein multiparous (third lactation) cows (n = 3) raised at the INRA experimental barn (UMR PEGASE, Le Rheu, France) were used for mammary tissue collection. The cows were milked twice daily before mammary tissue collection at the peak of lactation (after 60 DMI). Biopsies from the left and the right halves of the udder (one sample per quarter) were taken approximately halfway between the base of the teat and the dorsal body wall in a region containing a large amount of secretory tissue, according to a method adapted from Farr et al. (1996). Mammary biopsies were sliced into small explants (five mm³) for tissue digestion and cell isolation.

BMEC preparation and primary culture

Mammary explants were digested and dissociated as described by Perruchot et al. (2016). To ensure that the isolated BMECs corresponded to mature secretory cells, the cells were sorted by flow cytometry using fluorescein isothiocyanate (FITC) anti-rat IgG1 CD49f (α6 integrin) (ref. 13-097-246; Miltenyi Biotec, Bergisch Gladbach, Germany). Isotype controls were used for each antibody to eliminate nonspecific background fluorescence. Flow cytometric analysis was performed on a data set of 30,000 events (single cells) using a MACSQuant® Analyzer10 (Miltenyi Biotec, Bergisch Gladbach, Germany), and the data were analyzed using MACSQuantify analysis software (Miltenyi Biotec, Bergisch Gladbach, Germany). The results are expressed as percentages (dot plot analysis) (Fig. 1A).

The BMECs were cultured in 96-well plates at a density of 5,000 cells/100 μL/well in MGE-P epithelial cells growth medium containing 10% FBS, 1× penicillin/streptomycin, 0.25× insulin-transferrin-selenium (ITS) without lactogenic hormones according to methods described in Perruchot et al. (2016). All cell culture products were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). All BMEC cultures were grown at 37 °C under 95% air and 5% CO₂.

Cell viability, cell death and proliferation assays

Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich, St. Louis, MO, USA) to assess the metabolic activity of cells. Briefly, confluent monolayers of BMECs were treated with increasing concentrations of baicalin (0, 1, 10, 100 and 200 µg/mL) for 24 h. The media were removed at the end of the treatment period, and BMEC monolayers were then exposed to 200 μL of MTT solution (0.5 mg/mL in PBS) for 2 h. After washing the cells with PBS, the formazan crystals were solubilized with DMSO (200 μL per well; Sigma-Aldrich Chimie) before the plates were measured at 540 nm using a Multiskan Spectrum microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a spectrophotometer. The results are presented as the mean ± standard error of the mean of triplicate assays from three independent experiments.

Cell death was assessed using annexin-V/PI (propidium iodide)-double labeling to visualize apoptotic and necrotic cells. An Alexa Fluor 488 Annexin-V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer’s recommendations. Briefly, BMECs cultured with or without increasing doses of baicalin (0–200 µg/mL) during 24 h were incubated for 15 min with annexin-V-FITC (5 μg/mL) and PI (1 μg/mL) and then diluted in 400 μL of annexin-binding buffer. Cells were then analyzed by flow cytometry. The following staining controls were used: unstained cells, cells labeled with annexin-V-FITC alone (without PI) and cells labeled with PI alone (without annexin-V-FITC). For each sample, 2 × 10⁴ events were analyzed.

To assess proliferative capacity, BMECs were cultured for 24 h with increasing doses of baicalin (from 0 to 200 µg/mL), then labeled using 10 μM BrdU per well and re-incubated overnight following the manufacturer’s instructions. The amount of labeling was quantified by measuring the absorbance at 370 nm using a scanning multiwell spectrophotometer (Mithras LB 940, Berthold Technologies, Oak Ridge, TN, USA) with a reference wavelength of 492 nm. Increased optical density (OD) values indicate a higher proliferation rate.

Oxidative stress assays

ROS production in BMECs cultured with different doses of baicalin (1–100 µg/mL) was determined by fluorimetric assay using a five μM carboxy derivative of fluorescein, carboxy-H (DCFDA), added to the medium. After 90 min or 24 h of incubation, respectively, the hydroxyl radicals produced by the cells were estimated by quantifying dichlorofluorescein (DCF) using a multidetection microplate reader (Mithras, LB 940) with excitation and emission wavelengths at 485 and 535 nm, respectively. Samples incubated with 50 U of PEG-SOD were used as negative controls to assess the specificity of the assay.

Hydrogen peroxide was also applied to primary BMECs as an in vitro oxidative stress model (Gille & Joenje, 1992). The cytotoxicity of H₂O₂ to BMECs was first established by incubating BMECs with increasing concentrations of H₂O₂ (0–1,000 µM) diluted in cell culture media. Cytotoxicity was estimated after 24 h of treatment as described above. Because a large decrease in cell viability was observed at the highest doses of H₂O₂, the following tests were performed with H₂O₂ concentrations below 200 µM. ROS production was monitored in BMECs exposed to increasing doses of H₂O₂ (0, 25, 50, 100 and 200 µM) as described above. Finally, the potential of baicalin treatment to protect BMECs against H₂O₂-induced oxidative stress was estimated by pretreating BMECs with increasing concentrations of baicalin (24 h) and then exposing the cells to H₂O₂ (0, 25, 50 or 100 µM) in culture media for an additional 24 h. Cell viability and ROS production were examined at the end of this period as described above.

Protein extraction and western blot analysis

Total proteins were extracted with Radioimmunoprecipitation buffer (Fisher Scientific Illkirch, Illkirch-Graffenstaden, France) from BMECs cultured for 24 h with or without increasing concentrations of baicalin (0, 5, 10 and 100 µM/mL). Protein concentrations were determined using a BCA Protein Assay Kit (Fisher Scientific Illkirch, Illkirch-Graffenstaden, France) according to the manufacturer’s instructions. The proteins were separated by SDS-PAGE on 4–12% SDS-polyacrylamide gels (NuPage 4–12% Bis-Tris, NP0323BOX, Invitrogen Life Technology, Berlin, Germany), transferred to polyvinylidene difluoride membranes (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and incubated overnight with primary antibodies against catalase (SC-50508, Santa Cruz) or nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NFR-2; SC-722; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Beta-actin (A5441; Sigma Aldricy, St. Louis, MO, USA) was used as a control. Horseradish peroxidase-conjugated secondary antibody was used at 1:2,500, and chemiluminescence was visualized using an ECL Kit and an ImageQuant LAS4000 Biomolecular Imager digital imaging system (GE Healthcare, Velizy-Villacoublay, France).

Statistical analysis

The data were first tested for normality. Experiments were repeated three times. Data were analyzed by one-way analysis of variance (ANOVA) using the following model: y_ij = µ + time_i + ε_ij (y = viability, proliferation, ROS production, western blot data; µ = mean; i = baicalin dose and ε = residuals). Tukey’s post hoc pairwise analysis was used. Differences were considered significant at p < 0.05. All statistical analyses were performed using RStudio (RStudio Team, 2018).

Effects of baicalin exposure on the viability and apoptotic and proliferative capacities of BMECs in primary culture

To study the effects of baicalin on BMECs, we chose to use primary BMECs isolated from bovine mammary glands. We digested mammary secretory tissue from lactating dairy cows and isolated adherent BMECs. Prior to use, we verified that the BMECs expressed the classical epithelial cell marker CD49f. Indeed, it is obvious from Fig. 1A that the primary BMECs used for this study were epithelial since 91% expressed CD49f.

Next, we evaluated baicalin cytotoxicity in culture by increasing baicalin doses and evaluating the number of viable cells. Viability was significantly enhanced with 10 µg/mL baicalin but was similar under control conditions and medium supplemented with one µg/mL baicalin. Higher doses of baicalin (100 and 200 µg/mL) dramatically lowered cell viability (Table 1). Specifically, cell viability was two-fold lower after exposure to 200 µg/mL baicalin for 24 h than under control conditions (p < 0.001). Apoptosis was indirectly evaluated through the expression of annexin-V in cultured BMECs. The proportion of apoptotic cells was lower (5% vs. 8%; p < 0.05) with 10 µg/mL baicalin but was similar under control conditions and medium supplemented with one µg/mL baicalin. Higher doses of baicalin resulted in a dose-dependent increase in the proportion of apoptotic cells (Table 1), with 1.6-fold more apoptotic cells with 200 µg/mL baicalin compared with control conditions. The effects of increasing doses of baicalin were also characterized in terms of BMEC proliferation after 24 h of culture (Fig. 1B). Although the lowest concentration of baicalin (1 µg/mL) slightly increased BMEC proliferation (+10%, p < 0.05) when compared with the control, large doses of baicalin (100 µg/mL and 200 µg/mL) in medium decreased cell proliferation (p < 0.05), with more than 50% fewer proliferative cells at the highest dose of baicalin (200 µg/mL) compared with the control. Phenotypic differences between BMECs cultured in the absence of baicalin (control) or with a high dose of baicalin (100 µg/mL) are shown in Fig. 1C, with lower cell density and more disrupted BMEC colonies in the cells treated with 100 µg/mL baicalin. Taken together, these results show that low concentrations (1 and 10 µg/mL) of baicalin have no or slightly positive effects on BMEC viability, apoptosis and proliferation, whereas large doses (100 and 200 µg/mL) of baicalin clearly lowered proliferation, impaired viability and increased apoptosis of BMECs.

Baicalin lowered ROS production in confluent BMECs

Acute exposure (90 min) of baicalin to confluent BMECs, independent of dose, was associated with a large decrease in the amount of ROS released by BMECs into the culture media (−80%, p < 0.001, Fig. 2A). Chronic exposure (24 h) to baicalin was similarly associated with reduced ROS production by BMECs (Fig. 2B) when compared to BMECs cultured in a medium without baicalin. However, the inhibitory effect of baicalin on ROS production was lower at high concentrations (100 and 200 µg/mL) than at low doses (1–50 µg/mL).

To investigate the mechanisms that might be involved in the reduction of ROS production by BMECs when exposed to baicalin, we analyzed the intracellular amounts of catalase, a powerful antioxidant enzyme and of Nrf-2, a transcription factor that controls the expression of antioxidant genes. A subset of baicalin doses (5, 10 and 100 µg/mL) was tested on BMECs cultured for 24 h and compared to the control. Treatment of BMECs with baicalin caused a marked increase in catalase and Nrf-2 protein (Figs. 3A and 3B). The greatest effect was observed at 10 µg/mL baicalin, with a 3-fold increase in catalase and Nrf-2 in treated cells compared with control cells. Taken together, low doses of baicalin may improve oxidative stress in BMECs by lowering ROS production and activating antioxidant intracellular defenses.

Baicalin pretreatment limits H₂O₂-induced ROS production in BMECs

To study the possible benefits of baicalin pretreatment of BMECs during oxidative stress, we used an in vitro model of H₂O₂-induced oxidative stress. We first evaluated the effect of H₂O₂ on BMEC viability to determine the optimal working concentration. The data presented in Fig. 4A show that BMEC viability was significantly affected after treatment with 200 µM H₂O₂. Next, we investigated ROS production by BMECs when increasing doses of H₂O₂ were added to the cell medium. An exponential relationship between ROS production and increasing doses of H₂O₂ was observed (Fig. 4B), with high doses of H₂O₂ (200 µM) resulting in a 6-fold increase in ROS production compared to zero µM H₂O₂. Finally, increasing doses of H₂O₂ were added to BMECs pretreated for 24 h with increasing doses of baicalin. Although baicalin was not able to inhibit H₂O₂-induced ROS production in BMECs, ROS production was significantly lower in pretreated BMECs than in untreated cells (Fig. 4C). The largest reduction was observed after treatment with the highest doses of baicalin (100 and 200 µg/mL), independent of H₂O₂ dose. Finally, improved viability after the H₂O₂ was observed in cells pretreated with baicalin (Fig. 4D) compared to untreated cells, with the highest improvement observed at five µg/mL baicalin pretreatment. Altogether, pretreatment of BMECs with baicalin reduced ROS production under normal and oxidative stress conditions and improved cell viability when exposed to an acute stressor.