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, 2018, 5059469
eCollection

Differential Regulation of NF- κ B and Nrf2 by Bojungikki-Tang Is Associated With Suppressing Lung Inflammation

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Differential Regulation of NF- κ B and Nrf2 by Bojungikki-Tang Is Associated With Suppressing Lung Inflammation

Soo Ryun Park et al. Evid Based Complement Alternat Med.

Abstract

Bojungikki-tang (BT), an Asian herbal remedy, has been prescribed to increase the vitality of debilitated patients. Since a compromised, weakened vitality often leads to illness, BT has been widely used to treat various diseases. However, little is known about the mechanism by which BT exerts its effect. Given that BT ameliorates inflammatory pulmonary diseases including acute lung injury (ALI), we investigated whether BT regulates the function of key inflammatory factors such as NF-κB and Nrf2, contributing to suppressing inflammation. Results show that BT interrupted the nuclear localization of NF-κB and suppressed the expression of the NF-κB-dependent genes in RAW 264.7 cells. In similar experiments, BT induced the nuclear localization of Nrf2 and the expression of the Nrf2-dependent genes. In a lipopolysaccharide-induced ALI mouse model, a single intratracheal administration of BT to mouse lungs ameliorated alveolar structure and suppressed the expression of proinflammatory cytokine genes and neutrophil infiltration to mouse lungs. Therefore, our findings suggest that suppression of NF-κB and activation of Nrf2, by which BT suppresses inflammation, are ways for BT to exert its effect.

Figures

Figure 1
Figure 1
Cytotoxic effect of BT. (a) RAW 264.7 cells were treated with varying amounts of BT for 16 h. The cytotoxicity induced by BT was measured by MTT assay. Data are shown as the mean ± SEM of three independent measurements. There was no statistical difference among different groups. (b) RAW 264.7 cells were treated with LPS (100 ng/ml) or BT (50 μg/ml) for 16 h. Cells producing intracellular ROS were determined by a flow cytometer. The percentile of ROS-positive cells is shown in the upper right side of each panel. All experiments were repeated three times independently and representative results are shown.
Figure 2
Figure 2
BT suppresses the transcriptional activity of NF-κB. (a) RAW 264.7 cells, pretreated with PBS (lanes 1 and 2) or the indicated amounts of BT for 16 h (lanes 2 and 4 to 6), were stimulated with TLR4-specific LPS (100 ng/ml) for 30 min (lanes 3 to 6). Nuclear proteins were isolated from the cells and analyzed by immunoblotting of p65 RelA. For ensuring equal loading, the membrane was stripped and reprobed for lamin B. (b) The intensity of each band was measured by ImageJ and calculated over lamin B. Data represent the mean ± SEM of three independent measurements. P was less than 0.05, compared to the control treated with LPS only. (c) RAW 264.7 cells were treated with BT for 16 h and subsequently with TLR4-specific LPS (100 ng/ml) for 4 h. The levels of COX-2, TNF-α, IL-1β, and IL-6 were analyzed by a semiquantitative RT-PCR. (d) The intensity of each PCR band was measured by ImageJ and calculated over that of a housekeeping gene GAPDH. Data represent the mean ± SEM of three independent measurements. P was less than 0.05, compared to the control treated with LPS only.
Figure 3
Figure 3
BT activates the transcriptional activity of Nrf2. (a) RAW 264.7 cells were treated with varying amounts of BT for 16 h. As for Nrf2 control, cells were similarly treated with 5 μM of sulforaphane (SFN), a potent Nrf2 activator. Nuclear proteins were isolated from these cells and analyzed by immunoblotting of Nrf2. The membrane was stripped and blotted with α-lamin A/C antibody for nuclear protein controls. A similar experiment was repeated at least three times. (b) RAW 264.7 cells were treated similarly to (a), and total RNA was extracted and analyzed by a semiquantitative RT-PCR for the expression of HO-1, GCLC, and NQO-1. (c) PCR bands in (b) were analyzed by ImageJ, and relative expressions of individual genes were shown in graphs after being normalized to GAPDH. (d) RAW 264.7 cells were treated with BT for 16 h and subsequently with TLR4-specific LPS (100 ng/ml) for 4 h. A semiquantitative RT-PCR was performed for the expression of HO-1, GCLC, and NQO-1. Resultant PCR bands were analyzed by ImageJ, and relative expressions of individual genes were shown in graphs after being normalized to GAPDH (e). Data represent the mean ± SEM of three independent measurements. P was less than 0.05, compared to untreated controls.
Figure 4
Figure 4
BT ameliorates alveoli structure of mouse lungs. C57BL/6 mice (n = 5 per group) received a single i.t. PBS (a) or 2 mg/kg of i.t. LPS ((b), (c), and (d)). Two hours later, mice received 1 mg/kg (c) or 10 mg/kg body weight of i.t. BT (d). Mouse lungs were harvested at 24 h after LPS treatment and stained with HE for histological examination (magnification, ×100, and bar = 100 μm). Representatives of at least five different areas of a lung are shown.
Figure 5
Figure 5
BT suppresses neutrophil infiltration to mouse lungs. C57BL/6 mice (n = 5 per group) received 2 mg/kg of i.t. LPS and then 2 h later two different amounts of i.t. BT were administered (1 mg/kg or 10 mg/kg body weight). Bronchoalveolar lavage (BAL) was conducted 24 h after LPS treatment. Total cells (a) and macrophages and neutrophils (b) in the BAL fluid were counted. Data are shown in the mean ± SEM of three independent measurements. P was less than 0.05, compared to the LPS-treated mice (post-ANOVA comparison with Tukey's post hoc test). (c) The lysate was prepared from the lung and MPO assay was performed. Values are expressed as the mean ± SEM of 5 mice. P was less than 0.05, compared to the LPS-treated mice (post-ANOVA comparison with Tukey's post hoc test). Total RNA was extracted from the lung tissue (n = 5 per group) and analyzed by a semiquantitative RT-PCR. PCR products were analyzed by ImageJ and normalized over GAPDH for the expressions of TNF-α, IL-1β, and IL-6 (d) and of Nrf2-dependent genes, such as NQO-1, HO-1, and GCLC (e). Representatives of three mice in each group are shown. P was less than 0.05, compared to the LPS-treated mice (post-ANOVA comparison with Tukey's post hoc test).

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