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Effects of Minocycline on H2O2-induced Cell Death and Interleukin-8 Production in Human Small Airway Epithelial Cells

Takahiro Sato, Ayano Hirohara, Yuki Izumi, Takayuki Nakatomi, Kuninori Iwayama, Ko-Ichi Ohtaki and Nobumasa Hayase

Background and Objective: The antibiotic minocycline (MINO) is known to have anti-inflammatory activity. This study aimed to examine the effects of MINO on hydrogen peroxide (H2O2)-induced cell death and interleukin (IL)-8 production, which have been shown to increase during inflammation, using small airway epithelial cells (SAECs). Materials and Methods: The SAECs were pretreated with MINO (1, 5 and 10 μM) for 24, 48 or 72 h and subsequently exposed to H2O2 (100 μM) for 6 h. Cell viability was assessed using the WST-8 assay and levels of IL-8 protein were measured using an enzyme-linked immunosorbent assay (ELISA). Tukey’s multiple comparison test was used for analysis of statistical significance. Results: Treatment of SAECs with 10 μM MINO or greater than 100 μM H2O2 significantly decreased cell viability. However, pretreatment with 1 μM MINO for 24 h significantly suppressed the H2O2-induced decrease in cell viability. In addition, IL-8 production increased in the H2O2 treated group as compared to the control group (not treated with H2O2), whereas IL-8 production decreased significantly following pretreatment with MINO (1, 5 or 10 μM) for 48 or 72 h. Conclusion: It was suggested that pretreatment of SAECs with low concentrations of MINO for 24 or 48 h has a cytoprotective effect and pretreatment for 48 or 72 h suppresses IL-8 production indicating a potential anti-inflammatory effect.

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Takahiro Sato, Ayano Hirohara, Yuki Izumi, Takayuki Nakatomi, Kuninori Iwayama, Ko-Ichi Ohtaki and Nobumasa Hayase, 2019. Effects of Minocycline on H2O2-induced Cell Death and Interleukin-8 Production in Human Small Airway Epithelial Cells. International Journal of Pharmacology, 15: 509-514.

DOI: 10.3923/ijp.2019.509.514

Received: November 05, 2018; Accepted: January 12, 2019; Published: April 15, 2019

Copyright: © 2019. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.


Macrolides and tetracyclines possess anti-bacterial action and also exhibit distinct anti-inflammatory effects1-4. Chronic respiratory tract infections, in which oxidative stress is an important pathogenic factor5, are characterized by a vicious cycle of infection and inflammation, with inflammatory cytokines, mainly interleukin (IL)-8, observed to increase in bronchial epithelial cell sat the infection site1,6. This precedes the development of cytotoxicity, including the death of bronchial epithelial cells1,2.

Recently, the effect of clarithromycin (CAM), a macrolide antibiotic effective for diffuse panbronchiolitis (DPB), on H2O2-induced cytotoxicity in human small airway epithelial cells (SAECs) was investigated7,8. The results revealed that long-term treatment with low-concentration CAM inhibits the production of IL-8 and enhances anti-oxidant enzyme activity, e.g., glutathione peroxidase, which protects cells from H2O2-induced oxidative toxicity. On the other hand, minocycline (MINO) is used for the treatment of mycoplasma and chlamydia pneumonia, which are classified as diffuse lung diseases9 like DPB. This therapeutic effect may involve anti-inflammatory actions other than the antibacterial activity possessed by MINO. That is, MINO has been found to have multiple non-antibacterial biological activities that are beneficial in experimental models of various diseases, including autoimmune diseases such as dermatitis, rheumatoid arthritis and inflammatory bowel disease3,4. The MINO also produces a neuroprotective effect10,11 that may ameliorate conditions such as ischemia, traumatic brain injury and neuropathic pain as well as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis, spinal cord and its effects have been confirmed in experimental models of several neurodegenerative diseases3,4.

Based on these findings, it is possible that MINO may have the same effect as CAM against chronic inflammatory airway diseases. However, there are no reports describing the direct effects of MINO on H2O2-induced cytotoxicity in bronchial epithelial cells7, 8. The purpose of this study was to examine the inhibitory effects of MINO on H2O2-induced cell death and IL-8 production in SAECs under the same conditions as long-term treatment of CAM at low concentrations previously demonstrated to show the cytoprotective effects on SAECs. Although it is not possible to directly translate a pharmacological effect in cells to treatment efficacy in clinical practice, the present study presented possible evidence that MINO therapy is effective against chronic inflammatory airway diseases through the inhibitions of cell death and IL-8 release in bronchial epithelial cells.


The cultured cell study was conducted at the Faculty of Pharmaceutical Sciences, Hokkaido University of Science, Sapporo, Japan. The total period of this research was 7 months, from September, 2017 to March, 2018.

Materials: The MINO hydrochloride, H2O2 (30%) and the WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium) assay system were purchased from Wako Pure Chemical Industries, Ltd., (Osaka, Japan). A human IL-8 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Bender Med Systems (Vienna, Austria). All other chemicals used in this experiment were of research grade.

Cell culture and drug treatments: Cell culture and treatments were according to methods described previously7,8. Briefly, a normal human small airway epithelial cell line (SAECs) and culture media were purchased from Lonza (Walkersville, MD, USA). Cells were seeded in 75 cm2 filter vent flasks (Corning, NY, USA) and were grown to 80% confluence (3×106 cells/well) for each experimental condition at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cells were cultured in the presence or absence of MINO (1, 5 or 10 μM) for 24-72 h and were then stimulated with H2O2 (100 μM) for an additional 6 h. Pre-treatments with MINO were carried out with a different set of cells for each concentration and treatment duration. For stimulation of the cells with H2O2, the medium was changed to small airway basal medium (SABM) containing no supplements12.

Cell viability: Cell viability was assessed using the cell counting kit-8 assay13, WST-8. After treatment with MINO and H2O2, the SAEC medium was changed to SABM containing 10% WST-8 solution and the cells were incubated at 37°C for 2 h. Viable cells convert the WST-8 solution to an orange-colored formazan product with an absorbance at 450 nm. The optical density (OD) of the culture medium was measured at 450 nm with a spectrophotometric microliter plate reader (Bio-Rad, Hercules, CA, USA). The cell proliferation and viability were expressed as the ratio (%) of surviving cells to H2O2 untreated cells.

IL-8 measurement: After the treatments with MINO and H2O2, the culture media supernatants were collected and centrifuged to remove cell debris (12,000×g for 10 min). The IL-8 protein levels in the culture supernatants were analyzed using ELISA according to the manufacturer’s instructions. Briefly, experimental samples were added into individual wells coated with human monoclonal anti-body specific for IL-8 and incubated for 3 h at room temperature. After 6 washes with phosphate-buffered saline (PBS) containing 0.1% Tween 20 to remove unbound protein, tetramethyl-benzidine was added to each well and incubated for 10 min at room temperature. The reaction was terminated by the addition of 1 M phosphoric acid. The color generated in each sample was determined by OD measurement at 450 nm using a spectrophotometric microliter plate reader. The IL-8 protein levels of unknown samples were calculated using a standard curve.

Statistical analysis: All data were expressed as Means±SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) and differences, which were estimated by Tukey’s multiple comparison test after the Goodness of fit test and Bartlett’s test were considered to be statistically significant at p<0.05.


Effects of MINO and H2O2 on SAEC cell growth: Treatment of SAECs with 1 or 5 μM MINO for up to 72 h produced no significant change in cell viability. However, at a high concentration (10 μM), cell viability decreased significantly (p<0.01) compared to the control group indicating the cytotoxicity of MINO (Table 1). On the other hand, following H2O2 treatment (100, 300 and 500 μM) for 6 h, cell viability decreased in a concentration-dependent manner to 77.6±10.4, 65.9±6.4 and 26.4±1.5%, respectively. Furthermore, treatment with H2O2 for 24 h resulted in a stronger reduction in cell viability at 300 and 500 μM (Table 2).

Effect of MINO pre-treatment on H2O2-induced cell death in SAECs: Pre-treatment of SAECs with 1 μM MINO for 24 or 48 h prior to H2O2 (100 μM) treatment significantly (p<0.01 or p<0.05) suppressed the H2O2-induced decrease in cell viability compared to100 μM H2O2 treatment alone. Additionally, pre-treatment with 5 μM MINO for 24 h significantly suppressed the H2O2-induced decrease in cell viability (p<0.01). However, at a high concentration (10 μM) of MINO, the H2O2-induced decrease in cell viability was not suppressed at any time point assayed (Table 3).

Effect of MINO pre-treatment on H2O2-induced IL-8 protein release in SAECs: The exposure of SAECs to H2O2 (100 μM) caused an increase in supernatant IL-8 levels (significantly increased at 48 h) compared to the control group. Pre-treatments with 1 or 5 μM MINO for 48 or 72 h and with 10 μM MINO for up to 72 h prior to H2O2 treatment significantly (p<0.01 or p<0.05) decreased H2O2-induced IL-8 protein release compared to H2O2 treatment alone (Fig. 1).

Table 1: Effect of MINO on SAEC cell growth
Cell viabilities of the MINO untreated group (control cells) at each treatment time are expressed as 100%, data are expressed as Means±SD of six independent experiments, **p<0.01 vs. control cells

Fig. 1: Effect of MINO pretreatment on IL-8 production
  Data are expressed as Means±SD of three independent experiments, *p<0.05, **p<0.01 vs. control cells, #p<0.05, ##p<0.01 vs. cells stimulated with H2O2 alone

Table 2: Effect of H2O2 on SAEC cell growth
Cell viabilities of the H2O2 untreated group (control cells) at each treatment time are expressed as 100%, data are expressed as Means±SD of six independent experiments, **p<0.01 vs. control cells

Table 3: Effect of MINO pre-treatment on H2O2-induced cell death in SAECs
Cell viabilities of the MINO and H2O2 untreated group (control cells) at each pre-treatment time are expressed as 100%, data are expressed as Means±SD of six independent experiments, *p<0.05, **p<0.01 vs. control cells, #p<0.05, ##p<0.01 vs. cells stimulated with H2O2 alone


The results of this work showed that pre-treatment of SAECs with low concentrations (1 or 5 μM) of MINO for 24 or 48 h significantly suppressed the H2O2-induced decrease in cell viability and pre-treatment for 48 or 72 h significantly decreased IL-8 protein release.

The MINO concentrations of 1, 5 and 10 μM used in this study were deemed reasonable because an adult taking 200 mg of MINO per day would exhibit blood levels of approximately14 1-5 μM. In addition, a 2-4 fold higher concentration (2-20 μM) was detected in human sputum and bronchial secretion fluid15. However, in a study examining the anti-tumor effect of MINO on mouse epithelial ovarian cancer cells16 and human leukemia cells17, MINO decreased cell viability, with IC50 values of 10-30 μg mL–1 (about 22-66 μM). Therefore, it was suggested that the cytotoxic concentration of MINO obtained in this study was lower than those reported values due to differences in experimental conditions, such as the cell type used and the evaluation method of cell viability. On the other hand, during inflammatory conditions in the lower respiratory tract, neutrophil-derived H2O2 in the respiratory tract viscous fluid is reported to reach a concentration of 50 μM18. Since H2O2 is also produced directly from bronchial epithelial cells following stimulation with pollutants such as bacteria and lipopolysaccharide19, it was proposed that the H2O2 concentration (100 μM) used in this study reflects conditions observed during chronic inflammatory airway diseases.

Inflammation is the immune system’s response to harmful stimuli such as pathogens, damaged cells, toxic compounds, reactive oxygen species or radiation20 and uncontrolled inflammation finally leads to a loss of tissue function or cell viability at the tissue or cell level21. It has been shown that MINO causes a concentration-dependent protective effect on cell viability after oxidative stress22,23. Also in SAECs of this study, MINO suppressed cell death due to H2O2 (100 μM)-induced oxidative stress at low concentrations (1 and 5 μM). However, since cytotoxicity was observed at10 μM MINO, the decrease in cell viability induced by H2O2 may be further decreased depending on the MINO concentration in excess of the toxic threshold. Accordingly, when the MINO pre-treatment time is prolonged, evidence of cytotoxicity appears at low MINO concentrations, notably at 5 μM and the inhibitory effect of MINO on H2O2-induced cell death may be attenuated.

In regards to cell viability, the greatest effect was obtained by pretreatment with MINO for 24 h; however, the effect on IL-8 production released during the inflammatory process as an inflammatory cytokine1,6,24 was inconsistent with this result. It was thought that inflammatory cytokines other than IL-8 might be involved in the mechanism of MINO’s cytoprotective effect. Because MINO suppresses the activation of nuclear transcription25 factor (NF)-κB, it is possible that the production of IL-1, IL-6 or tumor necrosis factor (TNF)-α induced by NF-κB is also suppressed. In this study, IL-8, which is considered the principal inflammatory mediator1, 6 was used as an index; however, the expression pattern of each cytokine varies depending on the MINO and H2O2 treatment conditions25,26. Therefore, it is possible that there will be a difference in the relationship between the production of respective cytokines and cell viability. Nevertheless, pretreatment with 10 μM MINO significantly reduced the production of IL-8 at all time points assayed. The reason for this observation may be that the cytotoxicity from 10 μM MINO reduced the number of IL-8-producing SAECs. It is known that reactive oxygen species such as H2O2 regulate the DNA binding activity of nuclear transcription factors such as NF-κB, altering gene expression and activating a number of major signaling pathways leading to cell death27.

Therefore, compounds with antioxidant activity are considered to be effective for the inhibition of cytotoxicity and cell death caused by oxidative toxicity. Similar to α-tocopherol, MINO contains a multi-substituted phenol ring in its chemical structure and shows reactive oxygen species scavenging ability23. It was suggested that the direct anti-oxidant activity of MINO contributes, at least in part, to the suppression of H2O2-induced cell death and IL-8 production in SAECs.


This study showed that pre-treatment of SAECs with low concentrations (1 or 5 μM) of MINO for 24 or 48 h suppresses cell death after H2O2 treatment (100 μM) and pre-treatment for 48 or 72 h inhibits IL-8 release. Therefore, it was expected that MINO may also exhibit cytoprotective and anti-inflammatory effects in bronchial epithelial cells.


This study revealed possible cytoprotective effects on H2O2-induced cell death and IL-8 protein release in SAECs after pretreatment with MINO, which can be beneficial for the treatment of chronic airway inflammatory diseases. This study will assist researchers in uncovering a critical area of inflammatory-mediated bronchial epithelial cell death through oxidative stress that has previously been difficult to assess. Previous studies have not explored anti-inflammatory activities after pretreatment with MINO on chronic airway inflammatory diseases but instead focused on dermatitis, rheumatoid arthritis, central nerve system (CNS) diseases, neuropathic pain, ischemia, inflammatory bowel disease and cancer. Thus, new therapeutic regimens that effectively combat chronic airway inflammatory diseases may be developed.

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