Galloyl - Hexahydroxydiphenoyl (HHDP)-Glucose Isolated From Punica granatum L. Leaves Protects Against Lipopolysaccharide (LPS)-Induced Acute Lung Injury in BALB/c Mice

Abstract

The hydroalcoholic extract and ethyl acetate fraction of Punica granatum leaves have been known to exhibit anti-inflammatory activities. In this study, we investigated the therapeutic effects of galloyl-hexahydroxydiphenoyl (HHDP)-glucose isolated from pomegranate leaves on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice were treated with different doses of galloyl-HHDP-glucose (5, 50, and 100 mg/Kg) or dexamethasone at 5 mg/Kg (per os) 6 h after intra-tracheal instillation of LPS. Vehicle-treated mice were used as controls. Twenty-four hours after LPS challenge, bronchoalveolar lavage fluid (BALF), and lung samples were collected for analyses. They were evaluated by monitoring the expression of NF-κB, JNK, and cytokine genes and proteins, as well as cell migration and lung function. All doses of galloyl-HHDP-glucose inhibited LPS-induced JNK and NF-κB activation. Likewise, the galloyl-HHDP-glucose-treated animals presented reduced expression of the TNF-α, IL-6, and IL-1β genes in the lungs and reduced TNF-α, IL-6, IL-1β, and IL-8 protein levels when compared with the vehicle-treated LPS-challenged mice. In addition, the ALI mice treated with galloyl-HHDP-glucose also presented reduced lung inflammatory cell accumulation, especially that of neutrophils, in their BALF and lungs. In addition, galloyl-HHDP-glucose treatment markedly ameliorated the LPS-induced pulmonary mechanism complications and attenuated weight loss. Overall, we showed for the first time that galloyl-HHDP-glucose protects against ALI, and may be useful for treating ALI and other inflammatory disorders.

Keywords: acute lung injury; anti-inflammatory effects; cytokines; galloyl-HHDP-glucose; leukocytes; pomegranate.

Figure 1

Structure of galloyl-HHDP-glucose isolated from the hydroalcoholic extract of Punica granatum L.

Figure 2

Effects of galloyl-HHDP-glucose treatments on the activation of NF-κB (A) and JNK (B) in the lungs of BALB/c mice. BALB/c mice were randomly allocated to 6 groups (n = 4/group). Mice received galloyl-HHDP-glucose (5, 50, and 100 mg/kg; p.o), dexamethasone (DEXA; 5 mg/kg; p.o), or vehicle (PBS, 10 mL/kg; p.o). The results were calculated as % increase relative to that in the control. β-actin was used as a constitutive protein. The values are presented as median and interquartile. Significances were calculated by Kruskal–Wallis followed by Dunn multiple comparison test analysis of the groups vs. saline-injected non-treated mice (^*p < 0.05) and vs. lipopolysaccharide (LPS)-installed non-treated mice (^#p < 0.05). The gel is representative of results that were obtained in an experiment repeated four times.

Figure 3

Effects of galloyl-HHDP-glucose treatment on TNF-α (A), IL-1β (B), IL-6 (C), and IL-10 (D) gene expression in isolated lung tissues of BALB/c mice. BALB/c mice were randomly allocated to 6 groups (n = 6/group). Mice received galloyl-HHDP-glucose (5, 50, and 100 mg/kg; p.o), dexamethasone (DEXA; 5 mg/kg; p.o), or vehicle (PBS; p.o). The values are presented as median and interquartile range. Significances were calculated by Kruskal–Wallis followed by Dunn multiple comparison test analysis of the groups vs. saline-injected non-treated mice (^*p < 0.05) and vs. lipopolysaccharide (LPS)-installed non-treated mice (^#p < 0.05).

Figure 4

Effects of galloyl-HHDP-glucose treatments on TNF-α (A), IL-1β (B), IL-8 (C), IL-6 (D), and IL-10 (E) levels in homogenized isolated lung tissues of BALB/c mice. BALB/c mice were randomly allocated to 6 groups (n = 6/group). Mice received galloyl-HHDP-glucose (5, 50, and 100 mg/kg; p.o), dexamethasone (DEXA; 5 mg/kg; p.o), or vehicle (PBS; p.o). The values are presented as median and interquartile. Significances were calculated by Kruskal–Wallis followed by Dunn multiple comparison test analysis of the groups vs. saline-injected non-treated mice (^*p < 0.05) and vs. lipopolysaccharide (LPS)-installed non-treated mice (^#p < 0.05).

Figure 5

Analyses of body weights of mice. (A) Effects of galloyl-HHDP-glucose treatment on the total number of leukocytes (B), neutrophils (C), or macrophages (D) in bronchoalveolar lavage fluid (BALF), and total proteins in lung (E). BALB/c mice were randomly allocated to 6 groups (n = 6/group). Mice received galloyl-HHDP-glucose (5, 50, and 100 mg/kg; p.o), dexamethasone (DEXA; 5 mg/kg; p.o), or vehicle (PBS; p.o). The values are presented as mean and SD. Significances were calculated by One-way ANOVA followed by Tukey test analysis of the groups vs. saline-injected non-treated mice (^*p < 0.05) and vs. lipopolysaccharide (LPS)-installed non-treated mice (^#p < 0.05).

Figure 6

Effect of galloyl-HHDP-glucose treatment on leukocyte accumulation in the lungs of mice treated with saline-injected non-treated mice (A), lipopolysaccharide (LPS)-installed non-treated mice (B), 5 mg/kg galloyl-HHDP-glucose and LPS (C), 50 mg/kg galloyl-HHDP-glucose and LPS (D), 100 mg/kg galloyl-HHDP-glucose and LPS (E), 5 mg/kg dexamethasone and LPS (F), and total cells in lung (G). Black arrows: bronchiolar inflammatory cell infiltrates. Sections (4 μm) were stained with hematoxylin and eosin, and viewed with a microscope at 100x and 400x magnification respectively. Significances were calculated by One-way ANOVA followed by Tukey test analysis of the groups vs. saline-injected non-treated mice (^*p < 0.05) and vs. lipopolysaccharide (LPS)-installed non-treated mice (^#p < 0.05).

Figure 7

Efficacy of galloyl-HHDP-glucose treatments in reducing respiratory system and lung tissue elastance in lipopolysaccharide (LPS)-induced ALI mice. The respiratory system resistance (Rrs) and elastance (Ers), tissue damping (Gtis) and elastance (Htis), and airway resistance (Raw) (A–E). The mice received different doses of galloyl-HHDP-glucose (5, 50, and 100 mg/kg) 6 h after intra-tracheal instillation of LPS. The values are presented as mean and SD. Significances were calculated by One-way ANOVA followed by Tukey test. Significances were calculated by One-way ANOVA followed by Tukey test analysis of the groups vs. saline-injected non-treated mice (^*p < 0.05) and vs. lipopolysaccharide (LPS)-installed non-treated mice (^#p < 0.05).

Figure 8

Effect of Galloyl-HHDP-glucose for cell viability of RAW 264.7 cells. Results are expressed as means followed by standard deviation of three independent experiments performed in triplicate. (^*) indicates statistically significant different compared to control group by Kruskal–Wallis followed by Dunn multiple comparison test analysis.