Introduction

Wound may be the result of an accident; however, it can also occur intentionally for reasons such as surgery. Wound healing following trauma is described as the normalization of cellular, biochemical and systemic processes with the formation of new tissue. It is also an important physiological process that preserves the integrity of the tissue (1). Wound healing consists of three phases; (i) the hemostasis/inflammation phase, (ii) the proliferation phase, and (iii) the remodeling phase (2). Chronic and difficult to heal wounds causes a significant economic burden because of reducing the patient’s quality of life (3).

There are many factors that affect wound healing such as infection, oxygenation defects, stress, aging, and smoking. At low concentrations, reactive oxygen species (ROS) are involved in the wound healing process, defense against invading microorganisms, and signals necessary for cell survival. Production of high concentrations of ROS or impaired ROS detoxification is the main cause of chronic non-healing wounds (4). One of the causes of cellular oxidative stress (SOD) is the overproduction of hydrogen peroxide, a kind of ROS. Reducing oxidative balance is very important for the functioning of fibroblasts (5). On the other hand, the inflammatory response that begins after tissue injury accompany with the growth factor, cytokine, and chemokine expressions and those are necessary for wound healing. In the early phase of wound healing, cytokines and chemokines such as interleukine-1-alpha (IL-1α), IL-1-β, transforming growth factor-β (TGF-β), vascular endothelial growth factor, tumor necrosis factor alpha (TNF-α) and IL-8 are substantial and play important roles (6).

Scutellaria baicalensis Georgi. (Lamiaceae), contains baicalein as a major component, is a flowering plant species and its dried roots are used in traditional Chinese medicine for the treatment of cold, diarrhea, dysentery, hypertension, bleeding, insomnia, inflammation and respiratory tract infections (7). Baicalein has lots of beneficial effects including anti-oxidant, anti-inflammatory, anti-cancer, cardioprotective, neuroprotective, and hepatoprotective properties (8-10).

Fibroblasts are one of the key factors in wound healing and remain at the wound site until epithelialization occurs. First, they migrate to the wound site, then proliferate there, after that promote normal wound healing to form new extracellular matrix, and finally induces collagen structures to support other cells associated with effective wound healing (11).

In this study, it was aimed to investigate the possible protective effects of baicalein in H₂O₂-induced L929 fibroblast cell model by determining the parameters of SOD and inflammation process (TNF-alpha, TGF-beta, IL-10).

Materials and Methods

Cell Culture and Viability Test

The mouse fibroblast cell line L929 was purchased from American Type Culture Collection (ATCC, USA). It was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific), 1% antibiotic (Penicillin, Streptomycin, Amphotericin, Gibco, Thermo Fisher Scientific), under 95% humidity and 5% CO₂, and 37 °C temperature. Baicalein (MedChemExpress, Cat.No: HY-N0196), dissolved in 10% DMSO.

Baicalein Administration

For determine the baicalein doses, L929 cells incubated with varying doses (800 µM, 400 µM, 200 µM, 100 µM, 80 µM and 40 µM, 20 µM) for 24 hours. It was observed that the 800 µM dose reduced the viability by 26% and the 400 µM dose by 17% and 20 µM dose by 13%. The 40 µM dose increased the viability by only 5%. Therefore, these doses were not included in the experiment. 200 µM (B200), 100 µM (B100), 80 µM (B80) doses were used in the experiment.

H₂O₂ Administration

L929 cells were incubated for 24 hours with varying doses of H₂O₂ (1 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, 0.1 mM) while determining H₂O₂ damage. It was observed that cell viability decreased by 77% at 1 mM, 78% at 0.5 mM, 78% at 0.4 mM, 83% at 0.2 mM and 88% at 0.1 mM. So, 1 mM dose was chosen for the experiment. The groups with H₂O₂ were named as H+B200, H+B100 and H+B80.

CVDK-8 Cell Viability Test

Cell viability test was performed to see the effect of baicalein on cell proliferation. Cells were first seeded into 96-well plates, 2 µL of baicalein was applied at the specified doses (200 µM, 100 µM and 80 µM) after 70% confluence was detected. Then, 1 mM hydrogen peroxide (H₂O₂) was applied to related wells 23 hours after baicalein application. After 1 hour, 10 µL of CVDK-8 was added to each well. It was incubated at 37 ^oC for 4 hours for the formation of formazan crystals and the optical density (OD) value was detected with a spectrophotometer (Thermo, MultiSkan GO) at a wavelength of 450 nm.

Semi-Quantitative ELISA Technic

Cells (2x10⁴) were seeded 48-well plates in 200 µL medium for each well plate. The medium was collected 24 hours after the H₂O₂ damage and the addition of the specified doses of baicalein. In this study, we aimed to measure the levels of following parameters; TGF-beta ELISA Kit (BT Laboratory, Shanghai, China), TNF-alpha ELISA Kit (BT Laboratory, Shanghai, China), IL-10 ELISA Kit (BT Laboratory, Shanghai, China), SOD ELISA Kit (BT Laboratory, Shanghai, China). The ELISA technic was applied according to the manufacturer’s protocol. At the end of the experiments, the microplate was read at a wavelength of 450 nm with ELISA reader (Thermo, MultiSkan GO). The OD values were noted and the calculations were performed.

Immunofluorescence Assay

Cells were seeded 24-well plates in 400 µL medium for each well plate. After the H₂O₂ and baicalein co-incubation, the medium was removed and washed twice with PBS. For fixation, 400 mL of 4% formaldehyde was added to per well and after 4 minutes formaldehyde was removed and cells were washed with PBS. For permeabilization of cells, they were incubated with 99.9% methanol for 20 minutes at room temperature and then washed with PBS. Five ml each of fluorescent diacetate (FDA, ThermoFisher, Cat.No: F1303) and propidium iodide (PI, ThermoFisher, Cat.No: P1304MP) were added to per well, visualized with an inverted microscope (Invitrogen Evos FL) with fluorescence attachment after 10 minutes of incubation.

Statistical Analysis

The results were expressed as means ± SEM. Statistical significance was evaluated by One-Way ANOVA followed by Tukey post-test. All data were analyzed using GraphPad Prism, version 5.0 for Windows (Graph Pad Software, San Diego, California, USA). The probability level of p<0.05 was considered statistically significant.

Results

Baicalein Increased Cell Viability with/without H₂O₂-Induced

To test whether baicalein has a cytotoxic effect on healthy L929 fibroblast cells, a viability test was performed. It was observed that high doses of baicalein (200 µM, 100 µM, 80 µM) did not decrease cell viability, on the contrary, it induced cell proliferation (18%, 21%, 13%, respectively). The highest cell proliferation was observed at the dose of 100 µM.

After 1 h incubation with 1 mM H₂O₂, the viability decreased by approximately 77%. The cell viability increased after 24 hours of baicalein H₂O₂ co-incubation in all doses especially 100 µM. The 100 µM baicalein application reduced the damage of H₂O₂ up to 63% (Figure 1).

Baicalein Had Anti-Apoptotic Effect on H₂O₂ Damaged Cells

One hundred and ninety-six immunofluorescence assay (IFA) findings to show the anti-proliferative and anti-apoptotic effects of baicalein on L929 cells were similar with the cell viability assay findings. Baicalein given to healthy cells did not seem to decrease cell viability (Figure 2). It was observed that the number of apoptotic cells increased in H₂O₂ damaged group. The number of apoptotic cells decreased due to the increasing dose of baicalein, and the groups with the highest number of viable cells were 200 µM and 100 µM doses (Figure 3). Baicalein has been shown to have an anti-apoptotic effect with/without H₂O₂ in a dose-dependent manner.

Baicalein Effects the Levels of Inflammatory Cytokine

When the levels of TNF-alpha, a pro-inflammatory cytokine, were examined, a significant increase was observed in the H₂O₂ damage group compared to control group. Application of baicalein reduced H₂O₂ damage (Figure 4). Additionally, the levels of IL-10 and TGF-beta increased in H₂O₂ damage group compared to control group; however, the levels of those anti-inflammatory cytokines were decreased in the baicalein application groups (Figure 5). The activity of SOD enzyme showed a significant decrease in H₂O₂ damage group compared to control. An increase in the activity of SOD enzyme was observed in H₂O₂+baicalein application groups. In B200, B100, and B80 groups, the activity of SOD enzyme increased when compared to H₂O₂ group (Figure 6).

Discussion

To our knowledge, it was shown for the first time that baicalein has a protective effect on H₂O₂-induced L929 cell damage by effecting two important cellular process: SOD and inflammation. In current literature, it is well-known that the anti-inflammatory, antioxidant, anti-angiogenesis, and immunomodulatory effects of baicalein have been proven (12). A study reported that baicalein decreased cell viability in human lung fibroblast MRC-5 cell line (13). In this study, baicalein increased cell viability in L929 fibroblast cells, while it was shown that cell viability decreased in H₂O₂ damage group (5,14,15).

IFA findings were compatible with the cell viability assay findings. The ratios of FDA, which gives green fluorescent color to living cells, and PI to DNA helix of dead cells, have shown that baicalein has no toxic effects on healthy cells. A study reported similar findings with our study that baicalein did not cause death in Chinese hamster lung fibroblast cells (V79-4) (16). After H₂O₂ penetrates the cells, it produces a highly reactive hydroxyl radical that attacks cellular components including lipids, proteins and DNA (17,18). Membrane lipid peroxidation induced by H₂O₂ is one of the most important damages responsible for the loss of cell viability. It was detected that there were less death cells in H₂O₂ + baicalein treatment groups when compared to control. Consistent with the literature, it can be said that baicalein has a protective effect on H₂O₂ damaged cells (17,19).

SOD is one of the important factors inducing organ damage and associated with over-released of reactive oxygen species such as hydrogen peroxide (20). On the other hand, SOD is an important reactive oxygen species (ROS) scavenger, which can transform superoxide radicals into H₂O₂ and prevent ROS attack to important organs (21). It is known that SOD has therapeutic effects in physiological and pathological cellular conditions including inflammatory diseases, cancer, and neurodegenerative diseases (22). In our study, it was observed that high-dose baicalein application increased the SOD level, and low-dose baicalein slightly decreased the activity of SOD enzyme in related experiment groups. Tan et al. (23) performed a study and reported that pretreatment of baicalein increased the activity of SOD. Additionally, Ye et al. (24) also reported similar findings that baicalein activated apoptosis through induced intracellular ROS generation, and SOD apparently inhibited intracellular ROS production. These information shows us that baicalein has a potential to be effective on cellular SOD parameters.

On the other hand, all kinds of cellular damage including H₂O₂ can cause an inflammation in tissue and cytokine release. In our study, H₂O₂ caused an increase in the levels of TNF-alpha, TGF-beta, and IL-10; while different doses of baicalein application decreased the levels of mentioned cytokines. In current literature, it can be seen that baicalein has anti-inflammatory effects of some experimental models. Lin et al. (25) reported that TNF-alpha and IL-6 were suppressed by baicalein pretreatment; however, IL-10 level was significantly elevated by baicalein in contrast to our study. Generally, studies in literature support the current data about the inflammatory cytokine levels (26-28).

Study Limitations

The study have some limitations. First, I should have used more than one cell culture to compare the exact effects of baicalein on hydrogen-peroxidase damage. Second, I should have tried to determine more inflammatory and oxidative stress parameters. For example, to better explain the anti-inflammatory and anti-oxidant effects, additional parameters including catalase (CAT), lipid peroxidase (LPO), Malondialdehyde (MDA), Interleukin-1-beta (IL-1-B), Interleukin-6 (IL-6), Interleukin-17 (IL-17), and Interleukin-22 (IL-22) should have been determined. Third, seconder cellular signaling pathways related with the inflammation and oxidative stress processes should have been checked.

Conclusion

As a conclusion, pretreatment with baicalein results in significant findings including the suppression of inflammatory cytokines, TNF-alpha, TGF-beta, IL-10 and increase of SOD parameter, SOD, in L929 cells. Taking everything into account, baicalein which is a natural product may be an alternative therapeutic option for prevention of cellular SOD and inflammatory processes. To better understand the potential beneficial effects of baicalein, molecular mechanisms and cellular targets should be investigated.

Ethics

Ethics Committee Approval: This study is a cell culture study and therefore ethics committee approval was not obtained.

Informed Consent: Cell culture study.

Peer-reviewed: Externally peer-reviewed.

Financial Disclosure: The author declared that she did not receive any financial support during this article.