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. 2020 Feb 7;20(1):40.
doi: 10.1186/s12906-020-2828-6.

The methanol extract of Guettarda speciosa Linn. Ameliorates acute lung injury in mice

Affiliations

The methanol extract of Guettarda speciosa Linn. Ameliorates acute lung injury in mice

Kyun Ha Kim et al. BMC Complement Med Ther. .

Abstract

Background: Guettarda speciosa is mainly found in tropical areas in Asia. Although G. speciosa is traditionally used to treat some of the inflammatory disorders, the experimental evidence supporting the anti-inflammatory effect of G. speciosa is limited. Here, we sought to obtain evidence that G. speciosa has anti-inflammatory activity using an acute lung injury (ALI) mouse model and to explore possible underlying mechanisms for the activity.

Methods: The methanol extract of G. speciosa Linn. (MGS) was fingerprinted by HPLC. Cytotoxicity was determined by MTT and flow cytometer. As for an ALI mouse model, C57BL/6 mice received an intratracheal (i.t.) injection of lipopolysaccharide (LPS). The effects of MGS on lung inflammation in the ALI mice were assessed by differential cell counting and FACS of inflammatory cells and hematoxylin and eosin staining of lung tissue. Proteins were analyzed by immunoprecipitation and immunoblotting, and gene expression was by real-time qPCR. Neutrophil elastase activity was measured by ELISA.

Results: MGS did not cause metabolic disarray or produce reactive oxygen species that could induce cytotoxicity. Similar to ALI patients, C57BL/6 mice that received an i.t. LPS developed a high level of neutrophils, increased pro-inflammatory cytokines, and inflicted tissue damage in the lung, which was suppressed by i.t. MGS administered at 2 h after LPS. Mechanistically, MGS activated Nrf2, which was related to MGS interrupting the ubiquitin-dependent degradation of Nrf2. MGS suppressed the nuclear localization of NF-κB induced by LPS, suggesting the inhibition of NF-κB activity. Furthermore, MGS inhibited the enzymatic activity of neutrophil elastase.

Conclusion: MGS could suppress lung inflammation in an ALI mouse model, the effect of which could be attributed to multiple mechanisms, including the activation of Nrf2 and the suppression of NF-κB and neutrophil elastase enzymatic activity by MGS.

Keywords: Acute lung injury; Anti-inflammation; Guettarda speciosa Linn.; NF-κB; Neutrophil elastase; Nrf2.

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Conflict of interest statement

The authors do not have a commercial or other association that might have a conflict of interest.

Figures

Fig. 1
Fig. 1
Fingerprinting of Guettarda speciosa Linn. The chemical constituents of the methanol extract of G. speciosa Linn. (MGS) was analyzed by HPLC. The compounds used as index chemicals are indicated as follow: 1, 3-O-caffeoylquinic acid (chlorogenic acid); 2, quercetin rutinosied (rutin); 3, quercetin 3-O-galactoside (hyperin); 4, quercetin 3-O-glucoside, (isoquercitrin); 5: 3,4-di-O-caffeoylquinic acid (isochlorogenic acid A); and 6, apigenin 7-O-glucuronide
Fig. 2
Fig. 2
ROS production and cytotoxicity by MGS. (a) A flow cytometric analysis of intracellular ROS. After treated with LPS (100 ng/mL) or MGS (50 μg/mL and 100 μg/mL in PBS) for 16 h, RAW 264.7 cells were stained with carboxy-H2DCFDA, a ROS indicator. One thousand cells were counted, and cells producing intracellular ROS are shown in percentile, compared with untreated control. (b) MTT assay for cell viability. Increasing amounts of MGS were added to RAW 264.7 cells. After 16 h, metabolically active cells were determined after measuring the formazan product by a spectrophotometer. Data represent the mean ± SEM of triplicate samples. No statistical differences between groups were found (ANOVA)
Fig. 3
Fig. 3
MGS decreases neutrophils and tissue damage in mouse lungs. C57BL/6 mice (n = 5 per group) were administered with a single i.t. PBS (control) or 2 mg/kg of i.t. LPS, along with MGS (1 mg/kg body weight or 10 mg/kg body weight). BAL was performed with PBS to obtain cells infiltrated to the lung. Total cells (a) and macrophages and neutrophils (b) in the BAL fluid were counted. From the cell-free BAL fluid, albumin was measured by using ELISA (c). Data are shown in the mean ± SEM of three independent measurements. *P and **P were less than 0.001, compared to the LPS treated (post-ANOVA comparison with Tukey’s post hoc test). (d) Lung sections prepared were histologically analyzed after HE staining. At least three sections per mouse were analyzed under a microscope. Representative areas of each section are shown (100 magnifications, scale bar = 50 μm)
Fig. 4
Fig. 4
MGS decreases the number of cells expressing inflammatory cytokines. From C57BL/6 mice (n = 3/group) treated with i.t. PBS (control), i.t. LPS (2 mg/kg body weight), or LPS with i.t. MGS (1 mg/kg body weight), single-cell suspension of the lung was prepared and incubated with anti-Ly-6G, −TNF-α, −IL-1β, or -IL-6 antibodies. Representative histograms after flow cytometric analyses are shown. The percentile of positive cells is denoted in each panel
Fig. 5
Fig. 5
MGS activates Nrf2. (a) RAW 264.7 cells incubated with the indicated amounts of MGS or sulforaphane (5 μM) for 16 h were harvested. Nuclear fraction was analyzed by immunoblotting for Nrf2. The same blot was stripped and incubated with anti-lamin B antibody. A similar experiment was performed three times. (b) Total RNA extracted from RAW 264.7 cells treated with MGS was analyzed for the expression of GCLC, HO-1, and NQO-1 by a quantitative real-time PCR. Data represent the mean ± SEM of three independent measurements. *P, **P, and **P were less than 0.001, compared to untreated controls
Fig. 6
Fig. 6
MGS suppresses the ubiquitination of Nrf2. HEK 293 cells transfected with plasmids encoding V5-Nrf2, HA-Ub, and Keap1 were treated with MGS for 16 h. After V5-Nrf2 was immunoprecipitated by an anti-V5 antibody, the ubiquitinated V5-Nrf2 was analyzed by an anti-HA antibody. The cell lysate was analyzed by immunoblotting for V5-Nrf2 and GAPDH as for inputs
Fig. 7
Fig. 7
MGS suppresses NF-κB and neutrophil elastase activity. (a) RAW 264.7 cells were treated with MGS for 16 h and subsequently with 100 ng/mL of LPS for 15 min. Nuclear proteins were analyzed by immunoblotting for p65 RelA. The same blot was stripped and reblotted for lamin B. Similar experiments were performed three times. (b) MGS diluted in PBS was added to neutrophil elastase at final concentrations of 0.01, 0.1, 1, 10 μg/mL. Elastase activity was measured every minute for 10 min by ELISA assay per the instruction of the manufacturer. Data represent the mean ± SEM of three independent measurements. *P and **P were less than 0.01 and 0.001, respectively, compared to the control elastase activity

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