Anti-inflammatory and protective effects of D-carvone on lipopolysaccharide (LPS)-induced acute lung injury in mice

1 Introduction

Lung inflammation or acute lung injury is characterized with pulmonary edema, severe hypoxemia, overwhelming accumulation of neutrophils that leads to mortality caused by multiple organ failure (San et al., 2014). Overall incidence of mortality due to acute lung inflammation is increasing every year throughout the world (Li et al., 2019). The causes for lung inflammation and injury include bacterial infections, surgical defects, and uncontrolled infiltration of inflammatory cells (Lee et al., 2016). Lipopolysaccharide (LPS) is well-known to cause severe lung injury through activation IL-6, TNF-α, IL-1β, etc (Li et al., 2019; Ding et al., 2019). Inhibition and prevention of pro-inflammatory cytokines and their regulatory pathway has given tremendous results in modulating LPS-initiated lung inflammation (Ding et al., 2019; Huang et al., 2015). Antioxidant compounds have the ability to modulate inflammatory cytokine productions and control the overall progression of parenchymal inflammatory process in lung injury (Lee et al., 2018).

Carvone is an unsaturated monoterpenoid ketone, found as major phytochemical constituent in caraway seeds (Carumcarvi L.) as well in essential oil extracts of aromatic medicinal plants such as spearmint (Mentha spicata), dill (Anethum graveolens), and many more (Gopalakrishnan et al., 2019). Among carvone isomers, D-carvone has shown pharmaceutical values like anti-hyperlipidemic, anti-microbial, anti-carcinogenic, chemopreventive, anti-hypertensive, and anti-tumorigenic (Vinothkumar et al., 2013; Moro et al., 2018). Carvone and a few other monoterpenes have been reported to possess immunomodulatory and anti-inflammatory values (Rita De Cássia Da Silveira et al., 2013). However, there are no previous studies reported with specification to D-carvone on its modulating effects in lung inflammation. Hence, it was aimed to inspect the anti-inflammatory and protective role of D-carvone on lipopolysaccharide (LPS)-instigated lung damage in mice.

2 Materials and methods

3 Results

3.1

3.1 Effects of D-carvone on lung wet-to-dry (W/D) ratio of LPS-induced mice

Relative lung weight W/D ratio of mice treated with LPS was measured to determine lung pulmonary edema. Sharp increase (p < 0.05) was noticed in the W/D ratio of lung of LPS-instigated mice (Fig. 1). Pre-treatment with D-carvone and dexamethasone brought down (p < 0.05) the W/D ratio of LPS-challenged mice.

Close

Fig. 1

Effect of D-carvone on lung W/D ratio in LPS-induced mice. Values expressed as mean ± SEM (n = 8) of final body weight of mice. # p < 0.05 as compared to normal control group. * p < 0.05 as compared to LPS-induced acute lung injury model group.

Export to PPT

3.2

3.2 Effects of D-carvone on accumulation of BALF inflammatory cells on LPS-instigated mice

Total inflammatory cells were counted in BALF of mice instigated with LPS and were found that the amount of lymphocytes, macrophages, neutrophils, and eosinophils were increased (p < 0.05) in contrast to the BALF of normal mice (Fig. 2). The amount of cells in the BALF of D-carvone treated mice were efficiently reduced (p < 0.05) compared with LPS-instigated model animals. Positive control dexamethasone treated mice also demonstrated decrease (p < 0.05) in the concentration of inflammatory cells in the BALF as compared to LPS-challenged model mice.

Close

Fig. 2

Effect of D-carvone on of inflammatory cell counts in BALF of LPS-induced mice. Values expressed as mean ± SEM (n = 8) of final body weight of mice. # p < 0.05 as compared to normal control group. * p < 0.05 as compared to LPS-induced acute lung injury model group.

Export to PPT

3.5

3.5 Histopathological modifications of D-carvone in LPS-instigated mice

Observation of lung histopathology of normal control mice showed no changes and healthy arrangements of alveolar spaces (Fig. 3). In contrast, the LPS-challenged model mice demonstrated lung edema, infiltration of inflammatory cells, distorted alveolar spaces and hemorrhage. Pre-treatment with D-carvone significantly attenuated the histopathological changes caused by LPS in a dose-dependent manner. Positive control drug dexamethasone also prevented the histopathological modifications induced by LPS.

Close

Fig. 3

Histopathological alterations due to D-carvone administration in LPS-induced mice. H&E staining at 400 × magnification. (A) Normal control lung showing no pathological changes with healthy arrangements of alveolar spaces; (B) LPS-challenged model lung demonstrating thickening of alveolar wall, infiltration of inflammatory cells, lung edema, distorted alveolar spaces and hemorrhage; (C) & (D) Lung pre-treated with D-carvone at 25 and 50 mg/kg b.w. shows signs of recovery and reduced inflammation; (E) Lung pre-treated with dexamethasone at 5 mg/kg b.w. shows significant prevention of inflammation against LPS.

Export to PPT

4 Discussion

Acute lung injury or lung inflammation is a fatal disease, which is practically incurable, hence prevention is the best therapeutic option to overcome the problem (Jung et al., 2017). Antioxidant compounds are well-known to possess anti-inflammatory effects in many pathological conditions (Wang et al., 2014; Zhang et al., 2017). LPS is found in gram-negative bacterial outer membrane that induces severe inflammatory response once entered the blood stream, which may eventually cause death (Jing et al., 2015). The lung inflammation response induced by LPS in a mice model closely resembles the pathological changes during acute lung injury in humans (Jing et al., 2015; Jeong et al., 2014). Therefore, D-carvone was chosen to be studied against lung damage instigated by LPS in mice to learn its ability in preventing the progression of lung inflammation. From the results, it was evident that LPS had induced severe lung inflammation in mice, characterized with inflammatory cells accumulation, pulmonary edema, and elevation of pro-inflammatory cytokines similar to past results (San et al., 2014; Ding et al., 2019; Huang et al., 2015).

Lung W/D ratio was increased in LPS-instigated model animals exhibiting edema caused by inflammation. Pre-treatment with D-carvone decreased the W/D ratio by reducing the pulmonary edema caused by inflammation. Increased accumulation of neutrophils, eosinophils, macrophages, and lymphocytes were detected in BALF of LPS-instigated model animals, which relates to the pulmonary edema formation during inflammation. These results were similarly explained by Ran et al. (2014) and Huang et al. (2019). D-carvone treatment efficiently decreased the cell counts in BALF, which explains that D-carvone attenuated the inflammatory reaction induced by LPS. Histopathological findings of this study further evidence the lung inflammation and injury caused by LPS in the model group with edema formation, alveolar walls thickening, and infiltration of inflammatory cells. The pathological alterations caused by LPS were prevented by D-carvone by preserving the alveolar spaces, reduced inflammatory cells infiltration, and preventing the development of edema, suggesting the protective effect of D-carvone against acute lung injury. The histopathological results were similar to other studies using antioxidant compound against LPS-initiated lung damage in mice (Huang et al., 2015; Niu et al., 2017).

LPS triggers inflammatory reaction vigorously in the initial stage of lung damage (Lee et al., 2016). The accumulation of inflammatory cells and edema development is preceded by inflammatory response by pro-inflammatory cytokines and related proteins (Ding et al., 2019). It is learned that inflammatory cytokines are accountable in the pathogenesis of acute lung damage triggered by LPS (Li et al., 2019; Ran et al., 2014). In the results, cytokines were increased in the LPS-instigated model animals, which elucidate the rigorousness of lung damage and inflammation. In contrast, D-carvone treatment significantly downregulated the activities of TNF-α, IL-1β, and IL-6 from LPS-initiated inflammation. The results are similarly reported by Jung et al. (2017) and Fu et al. (2019). Oxidative stress is an important progenitor in inflammatory response (Jing et al., 2015). The levels of GSH, SOD, and CAT were suppressed whereas MDA was elevated in LPS-induced model mice, exhibiting oxidative stress condition due to depletion of defense mechanism. D-carvone pre-treatment prevented lipid peroxidation by means of reduced MDA formations. Amelioration of oxidative stress by D-carvone in LPS-initiated lung inflammation is similar to Chu et al. (2016) report. Overall, the study shows that pre-treatment of D-carvone has successfully prevented LPS from causing lung damage and inflammation in mice as supported by the biochemical and histopathological results.

5 Conclusion

In conclusion, this study proved that D-carvone has anti-inflammatory and preventive effects on LPS-initiated lung damage in mice. D-carvone significantly ameliorated the lung W/D ratio, oxidative stress markers, pro-inflammatory cytokines, and histopathological changes in LPS-challenged mice, compatible with the results of positive control dexamethasone. Together, these results support the use of D-carvone as preventive agent against acute lung injury and inflammation. Further research on the pharmacokinetics, pathway and mechanism of action of D-carvone are necessary prior to conducting clinical trials to promote D-carvone as a commercial anti-inflammatory drug.

Contribution of authors

Dr. Jianhui Du designed the experiment. Dr. Meng Zhao collected data from the experiments. Dr. Jianhui Du and Dr. Meng Zhao analyzed the data and compiled the acquired outcome. Both authors equally prepared and edited the manuscript.