A novel chalcone derivative exerts anti-inflammatory and anti-oxidant effects after acute lung injury

Introduction

Acute lung injury (ALI) manifests clinically as serious and acute respiratory dysfunction. Despite improvements in treatment, ALI has high morbidity and mortality rates, particularly in the elderly [1]. The pathogenesis of ALI involves disruption of the alveolar capillary-epithelial barrier due to exaggerated pulmonary inflammation, increased permeability, and exudation of protein-rich serous fluid [2]. As a consequence, lung edema develops, and pulmonary gas exchange is suppressed [3]. Oxidative damage and the resulting activation of multiple signaling pathways is associated with the pathogenesis of ALI [4]. Intratracheal administration of lipopolysaccharide (LPS), a pathogenic endotoxin found in the outer membrane of Gram-negative bacteria [5], induces pulmonary inflammation by enhancing the production of reactive oxygen species (ROS) and activating inflammatory responses. LPS is therefore frequently used to induce ALI in animal models [6].

Chalcones, a group of naturally occurring flavonoid compounds, exert antibacterial, antioxidant, anti-inflammatory, and anticancer effects [7, 8]. Previously, we synthesized several novel (E)-3,4-diphydroxychalcone derivatives and screened them for antioxidant activity. One of them, compound 33, exerted a particularly strong cytoprotective effect on hydrogen peroxide (H₂O₂)-induced oxidative damage in vitro and a neuroprotective effect against ischemia/reperfusion brain injury in vivo [9], its chemical structure is shown in Figure 1. However, whether compound 33 ameliorates LPS-induced inflammation and ALI is unknown.

Figure 1. Chemical structure of compound 33.

LPS induces various intracellular signaling events, including activation of the mitogen-activated protein kinase (MAPK) pathway and nuclear factor κB (NF-κB) [10]. NF-κB, a rapid-acting pleiotropic transcription factor, induces overproduction of inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), cyclooxygenase (COX2), and inducible nitric oxide synthase (iNOS) [11]. Activation of NF-κB is also implicated in the progression of LPS-induced ALI [12]. In addition, synthesis of the antioxidant enzyme heme oxygenase-1 (HO-1), which exerts a beneficial effect in rats with LPS-induced ALI, is regulated by nuclear factor erythroid-2-related factor 2 (Nrf2) [13].

In this study, we investigated the anti-inflammatory and antioxidant activity of compound 33 in LPS-challenged RAW 264.7 macrophages and in an animal model of LPS-induced ALI.

Results

Compound 33 prevented LPS-induced ALI in vivo

No mice died in compound 33 group and vehicle group in the toxicity experiment. Mean body weights were also similar between the two groups (Figure 2A), indicating that compound 33 is realtively safe for use in mouse models. Only one mouse died after 24h of LPS challenge, and survival rates did not differ significantly between the groups (Figure 2B). Compared to the control group, the lungs of mice challenged intratracheally with LPS exhibited thickening of the alveolar walls and interstitial spaces, disruption of endothelial and epithelial integrity, and neutrophil infiltration around the pulmonary blood vessels and airways (Figure 2C). However, pretreatment with compound 33 or dex prevented these LPS- induced pathological changes in the lungs. Moreover, pretreatment with compound 33 (20 mg/kg) or dex significantly reversed LPS-induced increases in lung wet weight (WW)/dry weight (DW) ratios and total protein concentration in the bronchoalveolar lavage fluid (BALF). More specifically, compound 33 reduced WW/DW ratio by 34% and protein levels by 42%. These effects were slightly stronger than those observed for dex, which reduced WW/DW ratio by 27% and protein amounts by 40% (Figure 2D, 2E). These results indicate that compound 33 ameliorated LPS-induced pathological changes in the lungs of ALI model mice.

Figure 2. Effects of compound 33 on mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI). (A) Mean body weights for the two groups over 15 days. (B) Survival rates of mice with LPS-induced ALI. (C) Lung tissues were stained with hematoxylin and eosin to show morphological changes. (D) Degree of tissue edema was evaluated using lung wet-to-dry weight ratios. (E) Total protein amounts in cell-free supernatant of bronchoalveolar lavage fluid (BALF). Data are presented as means ± standard error of the mean (SEM; n = 3–6). ^# P <0.05, ^## P <0.01, compared to the control group. * P <0.05, ** P <0.01, compared to the LPS group.

Compound 33 ameliorated LPS-induced inflammation and oxidative damage

LPS stimulation markedly increased TNF-α, IL-6, and IL-1β levels in cell-free bronchoalveolar lavage fluid (BALF) supernatant compared to the control group. Pretreatment with compound 33 (10 or 20 mg/kg) or dex reversed the LPS-induced increases in the levels of these three proinflammatory cytokines (Figure 3A). More specifically, 20 mg/kg compound 33 reduced TNF-α levels by 56%, IL-6 levels by 32%, and IL-1β levels by 63%. Pretreatment with compound 33 prior to LPS administration also increased SOD activity in the BALF, which was significantly lower in the LPS group than in the control group (Figure 3D). Moreover, LPS markedly increased iNOS and COX-2 levels (Figure 3E, 3F), and compound 33 significantly reversed these increases. Thus, compound 33 exerts anti-inflammatory and antioxidant effects in vivo.

Figure 3. Effects of compound 33 on LPS-induced inflammation and oxidative damage in mice. (A–C) Levels of proinflammatory cytokines in cell-free supernatant of the BALF as determined using enzyme-linked immunosorbent assays. (D) Superoxide dismutase (SOD) activity in the BALF. Protein levels of the proinflammatory mediators (E) iNOS and (F) COX2 in the lungs. Data are presented as means ± SEM (n = 3–6). ^# P <0.05, ^## P <0.01, compared to the control group. * P <0.05, ** P <0.01, compared to the LPS group.

Compound 33 inhibited the MAPK/NF-κB pathway and activated the Nrf2/HO-1 pathway in vivo

Pretreatment with 20 mg/kg, but not 10 mg/kg, compound 33 reduced LPS-induced phosphorylation of P38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) (Figure 4A–4C). Furthermore, phosphorylated-IκBα (p-IκBα), P65, and p-P65 levels in lung tissues decreased in the presence of compound 33, especially for the 20 mg/kg dose (Figure 4D–4F). Immunofluorescence staining showed that nuclear NF-κB p65 levels in lung tissue increased upon exposure to LPS, and pretreatment with compound 33 blocked this nuclear translocation of NF-κB p65 (Figure 4G). Immunohistochemical analysis showed that Nrf2 levels were slightly higher in the LPS group than in the control group. Compound 33 also increased Nrf2 levels, and 20 mg/kg compound 33 increased Nrf2 levels more than LPS did (Figure 4H). HO-1 levels also increased after administration of compound 33 (Figure 4I). The MAPK/NF-κB and Nrf2/HO-1 pathways are therefore implicated in the effects of compound 33 on LPS-induced ALI in vivo.

Figure 4. Effects of compound 33 on the MAPK/NF-κB and Nrf2/HO-1 signaling pathways in vivo. (A) p-P38, (B) p-ERK, (C) p-JNK, (D) p-IκBα, (E) P65, and (F) p-P65 protein levels relative to total P38, total ERK, total JNK, and GAPDH (loading control) were assayed using their respective antibodies. (G) Translocation of NF-κB P65 visualized by immunofluorescence staining. Immunohistochemical analysis of (H) Nrf2 and (I) HO-1 expression in lung tissue. Data are presented as means ± SEM (n = 3–6). ^# P <0.05, ^## P <0.01, compared to the control group. * P <0.05, ** P <0.01, compared to the LPS group.

Compound 33 inhibited overproduction of proinflammatory markers and ROS

TNF-α, IL-6, and IL-1β mRNA levels increased markedly in RAW 264.7 cells challenged with LPS for 12h compared to vehicle-treated cells. Compound 33 reversed these LPS-induced cytokine level increases in a concentration-dependent manner, with 10 μM compound 33 exerting the most robust protective effect (Figure 5A–5C). Furthermore, the LPS-induced increase in iNOS and COX2 levels was also significantly reversed by compound 33 in a dose-dependent manner (Figure 5E, 5F). Finally, flow cytometry showed that pretreatment with compound 33 reversed the LPS-induced increase in intracellular ROS levels in a concentration-dependent manner (Figure 5D).

Figure 5. Effects of compound 33 on LPS-induced increases in proinflammatory marker and reactive oxygen species (ROS) levels in RAW 264.7 cells. RAW264.7 cells were stimulated with LPS or phosphate-buffered saline (PBS) for 12 h after pretreatment with 2.5, 5, or 10 μM compound 33. Total RNA was extracted and (A–C) proinflammatory cytokine mRNA levels were assayed. (D) Intracellular ROS level was assessed using loro-dihydro-fluorescein diacetate. (E) iNOS and (F) COX2 protein levels in RAW264.7 cells as determined using Western blotting. Data are presented as means ± SEM (n = 3–6). ^# P <0.05, ^## P <0.01, compared to the control group. * P <0.05, ** P <0.01, compared to the LPS group.

Compound 33 inhibited the MAPK/NF-κB pathway and activated the Nrf2/HO-1 pathway in vitro

Western blotting showed that LPS significantly increased phosphorylation of P38, ERK, JNK, IκBα, and P65. Compound 33 reversed this effect, with 10 μM compound 33 exhibiting the highest potency (Figure 6A–6E). Immunofluorescence analysis revealed that LPS stimulation promoted nuclear translocation of NF-κB p65, and compound 33 markedly reversed this effect as well (Figure 6F). In addition, compound 33, but not LPS, increased Nrf2 and HO-1 protein levels (Figure 4G, 4H). These results suggest that the the MAPK/NF-κB and Nrf2/HO-1 pathways may mediate the protective effects of compound 33.

Figure 6. Effects of compound 33 on the NF-κB and HO-1/Nrf2 signaling pathways in vitro. RAW264.7 cells were treated with LPS for 1 h after pretreatment with 2.5, 5, or 10 μM compound 33. Protein levels were determined using Western blotting, and (A) p-P38/P38, (B) P-ERK/ERK, (C) p-JNK/JNK, (D) p-IκBα/IκBα, and (E) p-P65/P65 expression was quantified. (F) Nuclear translocation of NF-κB P65 visualized using immunofluorescence staining. Cells were pretreated with 2.5, 5, or 10 μM compound 33 or dimethyl sulfoxide for 1 h, and then exposed to LPS for 12 h. (G) Nrf2 and (H) HO-1 protein levels in cell lysates were analyzed; GAPDH was used as the control. Data are presented as means ± SEM (n = 3–6). ^# P <0.05, ^## P <0.01, compared to the control group. * P <0.05, ** P <0.01, for comparisons with the LPS group.

Nrf2-siRNA transfection and Tin protoporphyrin IX (SnPP) reversed the anti-inflammatory effect of compound 33

Western blot analysis showed that Nrf2 siRNA reduced Nrf2 levels compared to the negative control (Figure 7A). Nrf2 siRNA, but not control siRNA, also significantly reversed the compound-33-induced decrease in TNF-α, IL-6, and IL-1β levels (Figure 7B–7D). In addition, pretreatment with SnPP, an inhibitor of HO-1, blocked about 60–79% of the compound-33-induced reduction in IL-6, IL-1β, and TNF-α levels (Figure 7E–7G). Similarly, flow cytometry showed that SnPP increased ROS generation (Figure 7H). The Nrf2/HO-1 pathway therefore mediates the anti-inflammatory and antioxidant activities of compound 33.

Figure 7. Effects of Nrf2-siRNA transfection and tin protoporphyrin IX (SnPP) on compound 33-induced suppression of proinflammatory cytokine and ROS synthesis. (A) Nrf2 protein levels were assayed using Western blotting after transfection with Nrf2-siRNA or Nrf2-negative control siRNA. Effects of Nrf2 silencing on compound 33-induced suppression of (B) TNF-α, (C) IL-6, and (D) IL-1β. To explore the function of the Nrf2/HO-1 signaling pathway in inflammatory responses and oxidative stress, SnPP, an inhibitor of HO-1, was administered to RAW264.7 cells; compound 33 was administered 1 h later. (E) TNF-α, (F) IL-6, and (G) IL-1β mRNA and (H) ROS levels were analyzed. * P <0.05, ** P <0.01, compared to the 33+ LPS group.

Discussion

Inflammation facilitates ROS generation, which in turn promotes inflammatory reactions [14]. Inflammation and oxidative stress are linked biological processes that contribute to the pathogenesis of ALI [15, 16]. Intratracheal administration of LPS reportedly triggers a severe inflammatory response and oxidative damage by increasing the production of proinflammatory cytokines and ROS. We therefore used LPS to induce ALI in mice with histopathological characteristics similar to those of human ALI [6, 17]. ALI was induced in mice by intratracheal instillation of LPS (10 mg/kg) and was characterized by tissue edema, inflammatory cell infiltration, and disruption of the alveolar structure.

The low toxicity and diverse biological properties of natural and synthetic chalcones make them suitable candidates for drug discovery [18, 19]. We previously synthesized multiple chalcone derivatives and screened them for antioxidant potential. One of them, compound 33, exerted a significant protective effect in an animal model of middle cerebral artery occlusion and against H₂O₂-induced cellular injury in vitro. Compound 33 is therefore a candidate agent for the treatment of ischemic disorders [9]. However, relatively little was known about its anti-inflammatory activity and efficacy against LPS-induced ALI. Here, we report that intragastric administration of compound 33 ameliorated LPS-induced ALI, as indicated by histopathological features and decreased lung WW/DW ratios and total protein concentrations in BALF.

Inflammatory cells are activated in the early phase of ALI and lead to excessive production of proinflammatory cytokines (e.g., TNF-α, IL-6, and IL-1β). This results in the disruption of the alveolar epithelium, abnormal gas exchange, and a reduction in lung compliance [20]. IL-1β enhances the production of iNOS and COX2, which regulate the synthesis of nitric oxide and prostaglandin E2 [21]. Thus, inhibition of proinflammatory cytokine production is vital for the prevention of inflammatory reactions [22]. In this study, compound 33 significantly reduced TNF-α, IL-6, and IL-1β mRNA levels and inhibited the LPS-induced overproduction of these three proinflammatory cytokines in mice with ALI. Compound 33 also decreased iNOS and COX2 levels in vivo and in vitro. Notably, the effects exerted by compound 33 were equivalent to or greater than those of dex. Excessive activation and accumulation of inflammatory cells during ALI promotes the synthesis of proinflammatory factors and ROS. In this study, compound 33 suppressed the LPS-induced generation of ROS in RAW 264.7 cells. Compound 33 also upregulated SOD activity in vivo. Thus, compound 33 prevents LPS-induced oxidative damage by scavenging ROS. Taken together, these results indicate that compound 33 exhibits potent anti-inflammatory and antioxidant activity and attenuates LPS-induced tissue and cell damage.

Among the intracellular signaling pathways involved in inflammatory and immune responses, MAPK/NF-κB pathway might be particuarly important in mediating the effects of compound 33. Other chalcone analogues reportedly inhibit activation of the MAPK/NF-κB pathway in vivo and in vitro [7, 23]. The MAPK pathway plays a vital role in inflammation, and its activation is implicated in LPS-induced tissue injury, such as ALI [10]. Three subfamilies of MAPKs—P38, ERK, and JNK—are activated and phosphorylated in response to inflammatory stimuli such as LPS, as well as during LPS-induced ALI [4, 24]. Indeed, LPS stimulation markedly increased phosphorylation of P38, ERK, and JNK, and this effect was reversed by pretreatment with compound 33 in vivo and in vitro. NF-κB is a master transcription factor that plays a vital role in regulating the synthesis of proinflammatory markers during ALI [25]. Stimulants such as LPS activate the NF-κB pathway by promoting the phosphorylation and degradation of IκBα. This results in nuclear translocation of activated NF-κB, which then induces the transcription of genes encoding the proinflammatory cytokines iNOS and COX2 [26]. Our results suggest that compound 33 suppressed LPS-induced phosphorylation of NF-κB p65 and IκBα in RAW 264.7 cells. Moreover, compound 33 reversed the LPS-induced increase in nuclear translocation of NF-κB p65. These findings confirm that inhibition of the MAPK/NF-κB pathway is involved in the anti-inflammatory effects of compound 33.

The Nrf2 transcription factor regulates the expression of genes that encode antioxidant factors, including SOD and HO-1, by binding to antioxidant-response elements [27]. Activation of Nrf2/HO-1 results in antioxidant and antiapoptotic effects in many models of cell and tissue injury [28, 29]. In addition, the Nrf2/HO-1 signaling pathway was associated with the antioxidant effects of compound 33 after ischemia/reperfusion-related brain injury [9]. In this study, LPS significantly upregulated Nrf2 and HO-1 levels invivo and invitro, indicating that the Nrf2/HO-1 pathway was activated in response to LPS-induced injury. In addition, Nrf2 siRNA largely reveresed compound-33-induced decreases in TNF-α, IL-6, and IL-1β levels. Moreover, SnPP, an inhibitor of HO-1, significantly reversed the compound-33-induced suppression of proinflammatory cytokine and ROS synthesis, suggesting that the Nrf2/HO-1 pathway is functionally linked to the anti-inflammatory and anti-oxidative effects of compound 33.

In conclusion, compound 33 protected against LPS-induced injury, inflammation, and oxidative damage invivo and invitro. The protective effects of compound 33 may be mediated by the inhibition of the MAPK/NF-κB pathway and activation of the Nrf2/HO-1 pathway (Figure 8).

Figure 8. Illustration of protection provided by compound 33 against LPS-induced lung injury.

Abbreviations

ALI: Acute lung injury; LPS: Lipopolysaccharide; ROS: Reactive oxygen species; H₂O₂: Hydrogen peroxide; MAPK: Mitogen-activated protein kinase; NF-κB: Nuclear factor κB; TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6; COX2: Cyclooxygenase; iNOS: Inducible nitric oxide synthase; HO-1: Heme oxygenase-1; Nrf2: Nuclear factor erythroid-2-related factor 2; BALF: bronchoalveolar lavage fluid; WW: Wet weights; DW: Dry weights; p- IκBα: phosphorylated-IκBα; ERK: Extracellular signal-regulated kinase; JNK: Jun N-terminal kinase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; SnPP: Tin protoporphyrin IX; PBS: Phosphate buffered saline; DMEM: Dulbecco’s modified Eagle’s medium; qRT-PCR: Quantitative reverse transcriptase-polymerase chain reaction.

Author Contributions

Yuting Lin and Man Zhang performed the animal experiments; Qingdi Lu and Jingwen Xie performed the cell experiments. Jianzhang Wu and Chengshui Chen designed the experiments. Yuting Lin and Man Zhang wrote the manuscript. Jianzhang Wu and Chengshui Chen revised the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

The study was supported by the National Natural Science Foundation of China (81570075, 81770074). The National Key Research and Development Program of China (2016YFC1304000).

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