Astragalus mongholicus Bunge Water Extract Exhibits Anti-inflammatory Effects in Human Neutrophils and Alleviates Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice


doi: 10.3389/fphar.2021.762829.


eCollection 2021.

Affiliations

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Wei-Jen Cheng et al.


Front Pharmacol.


.

Abstract

Neutrophils are the primary immune cells in innate immunity, which are related to various inflammatory diseases. Astragalus mongholicus Bunge is a Chinese medicinal herb used to treat various oxidative stress-related inflammatory diseases. However, there are limited studies that elucidate the effects of Astragalus mongholicus Bunge in human neutrophils. In this study, we used isolated human neutrophils activated by various stimulants to investigate the anti-inflammatory effects of Astragalus mongholicus Bunge water extract (AWE). Cell-free assays were used to examine free radicals scavenging capabilities on superoxide anion, reactive oxygen species (ROS), and nitrogen-centered radicals. Imiquimod (IMQ) induced psoriasis-like skin inflammation mouse model was used for investigating anti-psoriatic effects. We found that AWE inhibited superoxide anion production, ROS generation, and elastase release in human neutrophils, which exhibiting a direct anti-neutrophil effect. Moreover, AWE exerted a ROS scavenging ability in the 2,2′-Azobis (2-amidinopropane) dihydrochloride assay, but not superoxide anion in the xanthine/xanthine oxidase assay, suggesting that AWE exhibited anti-oxidation and anti-inflammatory capabilities by both scavenging ROS and by directly inhibiting neutrophil activation. AWE also reduced CD11b expression and adhesion to endothelial cells in activated human neutrophils. Meanwhile, in mice with psoriasis-like skin inflammation, administration of topical AWE reduced both the affected area and the severity index score. It inhibited neutrophil infiltration, myeloperoxidase release, ROS-induced damage, and skin proliferation. In summary, AWE exhibited direct anti-inflammatory effects by inhibiting neutrophil activation and anti-psoriatic effects in mice with IMQ-induced psoriasis-like skin inflammation. Therefore, AWE could potentially be a pharmaceutical Chinese herbal medicine to inhibit neutrophilic inflammation for anti-psoriasis.


Keywords:

astragalus mongholicus bunge; inflammation; neutrophil; psoriasis; traditional Chinese medicine.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures


FIGURE 1



FIGURE 1

The chemical fingerprint of Astragalus mongholicus Bunge water extract (AWE). Chromatograms were obtained from high-performance liquid chromatography (HPLC) at 260 nm. (A) HPLC fingerprint of AWE and (B) of three standard references including 1: Calycosin-7-O-β-D-glucoside; 2: Ononin; 3: Calycosin; (C) Identification of astragaloside IV in AWE using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). (D) Structures of the parent ion and product ions of astragaloside IV.


FIGURE 2



FIGURE 2

AWE inhibited superoxide anion generation in activated human neutrophils. Human neutrophils (3 × 105 or 6 × 105 cells/mL) were incubated with ddH2O (as the control) or AWE (10, 30, or 100 μg/ml) for 5 min, and then were activated by (A) fMLF, (B) MMK-1 for 10 min, or (C) PMA for 5 min. Superoxide anion production was monitored using ferricytochrome c reduction at 550 nm. (D) Human neutrophils were incubated with ddH2O (as the control) or different concentrations of AWE for 15 min. Cytotoxicity was represented by LDH release as a percentage of the total. The Total LDH release was determined by lysing cells with 0.1% of Triton X-100 at 37°C for 30 min. The cell viability was calculated as one minus percent of LDH release. Data are expressed as mean ± S.E.M. (n = 3 or 4). Statistical analysis was conducted using the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test if there were statistical differences in the results of ANOVA. Adjusted p-values of Tukey’s multiple comparison tests were defined as ***p < 0.001 and **p < 0.01 compared with the control.


FIGURE 3



FIGURE 3

AWE inhibited elastase release in activated human neutrophils by different stimulants. Human neutrophils (6 × 105 cells/mL) were incubated with ddH2O (as the control) or AWE (100 μg/ml) for 5 min, and then activated by (A) fMLF, (B) MMK-1, (C) LTB4, or (D) IL-8 and CB (0.5 μg/ml for fMLF and MMK-1 and 2 μg/ml for IL-8 and LTB4) for another 10 min. Elastase release was measured by spectrophotometrically at 405 nm. Data are expressed as mean ± S.E.M. (n = 3 or 4). Statistical analysis was conducted using the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test if there were statistical differences in the results of ANOVA. Adjusted p-values of Tukey’s multiple comparison tests were defined as ***p < 0.001, **p < 0.01, and *p < 0.05 compared with the control.


FIGURE 4



FIGURE 4

AWE has a significant ROS scavenging effect but neither superoxide anion nor nitrogen-centered radicals. (A) 1 mM CaCl2 in HBSS and xanthine oxidase were incubated with ddH2O (as the control). AWE (10, 30, and 100 μg/ml) was added for 3 min and then xanthine (0.1 mM) for another 10 min. Reduction of WST-1 by superoxide anion was measured spectrophotometrically at 450 nm. Superoxide dismutase (SOD) 20 U/ml was used as the positive control. Fluorescein decay curve induced by AAPH and cleared by (B) Trolox and (C) AWE in different concentrations. (D) The area under the curve of AWE and Trolox. (E) DPPH (100 μM in 99% EtOH) was incubated with ddH2O (as the control), AWE (10, 30, and 100 μg/ml) or α-tocopherol (3, 15, and 30 μM). The reduction of DPPH was measured spectrophotometrically at 517 nm. (F) ABTS (7 mM in ddH2O) was incubated with ddH2O (as the control), AWE (10, 30, and 100 μg/ml) or L-ascorbic acid (10, 15, and 30 μM). The reduction of ABTS was measured spectrophotometrically at 734 nm. Data are expressed as the mean ± S.E.M. (n = 3 or 4). Statistical analysis was conducted using the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test if there were statistical differences in the results of ANOVA. Adjusted p-values of Tukey’s multiple comparison tests were defined as ***p < 0.001 compared with ddH2O alone.


FIGURE 5



FIGURE 5

AWE both inhibited neutrophil activation and scavenged ROS generation in fMLF-activated human neutrophils in luminol-enhanced chemiluminescence assay. Human neutrophil (7 × 105 cells/mL) were incubated with ddH2O (as the control) or AWE (10, 30, and 100 μg/ml) for 5 min and stimulated with (A) fMLF (0.1 μM) for another 6 min or (B) PMA (10 nM) for 30 min. The area under curve (AUC) of chemiluminescence in (C) fMLF or (D) PMA are shown as the mean ± S.E.M. (n = 5 or 6). Statistical analysis was conducted using the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test if there were statistical differences in the results of ANOVA. Adjusted p-values of Tukey’s multiple comparison tests were defined as ***p < 0.001 and *p < 0.05 compared with the control.


FIGURE 6



FIGURE 6

AWE inhibits intracellular superoxide anion generation in fMLF-activated human neutrophils. Human neutrophils labeled with HE were incubated with ddH2O (as the control) or AWE (10, 30, and 100 μg/ml) for 5 min and then stimulated with (A) fMLF (0.1 μM) or (B) PMA (10 nM) for an additional 5 min. The representative histograms demonstrating typical fluorescence-activated cell sorting profiles are shown. Mean fluorescence intensities are shown as (C) and (D). Data are expressed as the mean ±S.E.M. (n = 3–6). Statistical analysis was conducted using the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test if there were statistical differences in the results of ANOVA. Adjusted p-values of Tukey’s multiple comparison tests were defined as ***p < 0.001 and **p < 0.01 compared with the control.


FIGURE 7



FIGURE 7

AWE suppressed the adhesion of fMLF-activated human neutrophils to bEnd.3 cells by inhibiting CD11b expression. (A) Human neutrophils were incubated with ddH2O (as the control) or AWE (100 μg/ml) for 5 min and then activated by fMLF (0.1 μM)/CB (1 μg/ml) for an additional 5 min. Representative histograms demonstrating typical fluorescence in the absence or presence of FITC-labeled anti-CD11b. (B) Mean fluorescence intensities are shown. (C) Hoechst 33342-labeled human neutrophils (4 × 106 cells/mL) were incubated with ddH2O or AWE (100 μg/ml) for 5 min, then fMLF (0.1 μM) was used as the stimulant. The activated human neutrophils were added into bEnd.3 cells for 15 min. Adherent neutrophils on bEnd.3 cells were detected using a fluorescence microscope. Scale bar represented 200 μm. Representative histograms for fluorescent microscopy are shown. (D) Adherent human neutrophils were counted and quantified. Data are expressed as the mean ± S.E.M. (n = 5). Statistical analysis was conducted using the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test if there were statistical differences in the results of ANOVA. Adjusted p-values of Tukey’s multiple comparison tests were defined as ***p < 0.001 and **p < 0.01 compared with the control.


FIGURE 8



FIGURE 8

AWE ameliorated the severity of IMQ-induced psoriasis-like skin inflammation. (A) The skin change of IMQ-induced psoriasis-like skin inflammation was observed using a digital camera and a hand-held digital microscope. The scale bar of the picture in a hand-held digital microscope was 1 mm. (B) PASI scores, including erythema, thickness, scaling, and cumulative scores in study groups, are shown. Data are expressed as mean S.E.M. (n = 5). Statistical analysis was conducted using the Kruskal–Wallis test, followed by multiple comparison test with the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli if there were statistical differences in the results of the Kruskal–Wallis test. Adjusted q-values of multiple comparison tests using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli were defined as ***q < 0.001 and *q < 0.05 compared with the IMQ plus vehicle group.


FIGURE 9



FIGURE 9

AWE reduced the epidermal thickness and neutrophil infiltration in IMQ-induced psoriasis-like skin inflammation in mice. The mice were sacrificed by deep anesthesia on day 6 after topical IMQ and AWE or vehicle treatment for 5 days. Their back skins were collected and stored in 4% formaldehyde. Hematoxylin-eosin (H&E) staining and immunohistochemical (IHC) staining for Ly6G, MPO (the neutrophil infiltration and activation marker), Ki67 (the epidermal proliferation marker), and 4-HNE (the ROS marker) were performed for all samples (marked as orange triangles). The representative images (A) were observed under a microscope. The scale bar was 100 μm. The IMQ plus vehicle group exhibited psoriasis-like skin inflammation. The typical histological manifestations are marked as yellow arrows (hyperkeratosis), double-sided red arrows (acanthosis), and green triangles (elongation of rete-like ridges). AWE significantly decreased neutrophil infiltration and epidermal thickness in IMQ-induced psoriasis-like skin inflammation in mice. (B) The quantified image data using ImageJ. Epidermal area and average epidermal thickness were calculated in H&E staining. Ly6G-, MPO-, Ki67-, and 4-HNE-positive area and integrated density were calculated in IHC staining. Data are expressed as mean S.E.M. (n = 5). Statistical analysis was conducted using the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test if there were statistical differences in the results of ANOVA. Adjusted p-values of Tukey’s multiple comparison tests were defined as ***p < 0.001, **p < 0.01, and *p < 0.05 compared with the IMQ plus vehicle group.

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