The upregulation of GPR84 expression induced by IL-33
We firstly evaluated the expression of GPR84 through detecting its transcription and translation in lung tissue in LPS-induced mice and BEAS-2B cells (Fig. 1A-B). The results showed that GPR84 was significantly upregulated through LPS treatment. Furthermore, the upregulation of GPR84 was further induced by the co-treatment of LPS and IL-33 both lung tissue (Fig. 1C-D) and pulmonary epithelial cells (Fig. 1E-F).
GPR84 inhibitor ameliorated lung injury
In this experiment, after LPS-induced ALI in mice for 12h, the lung lobules of mice in the control group were intact, with no abnormal alveolar walls, no infiltration of inflammatory cells and no alveolar exudation (Fig. 2A). The lung tissues of mice in LPS group were infiltrated by a large number of inflammatory cells and the alveolar septa were thickened. In addition, the alveolar interstitium is marked with hyperplasia and edema. Compared with the model group, the pathological morphological damage of lung in the GPR84 inhibitor group of low dose was improved to varying degrees. In the high dose group, a small amount of exudates such as neutrophils and red blood cells were observed, edema and hyperplasia were significantly improved (Fig. 2A). The wet/dry weight ratio of lung tissue reflects the degree of noncardiogenic pulmonary edema[12]. Compared with the control group, there was significant difference in the wet/dry weight ratio of lung tissue in LPS group. The wet/dry weight ratio of lung tissue in the GPR84 treatment group was markedly decreased when compared with control group (B).
GPR84 inhibitor changed the levels of inflammatory markers
ELISA results of bronchoalveolar lavage fluid showed that the expressions of TNF-a,IL-1β and IL-6 in the LPS group were significantly higher than those in the control group 12 hours after LPS administration (Fig. 3A). But the expression of TNF-a༌IL-1β and IL-6 in BALF showed a significant decrease after GPR84 treatment. MPO is mainly synthesized and released by neutrophils, the activity of which in lung tissue is an important indicator to reflect neutrophil infiltration during the inflammatory process[13]. After LPS-induced ALI in mice for 12h, the MPO activity in lung tissues of mice in LPS group was increased compared with the control group (Fig. 3B). The MPO activity in the lung tissues of mice in LPS + GPR84 inhibitor group was lower than that in the model group. With the increase of GPR84 dose, MPO activity was significantly reduced. Cox-2 is an important downstream inflammatory mediator of NF- κB pathway and plays an important role in the pathogenesis of ALI and is associated with disease severity[14]. After 12 hours of LPS-induced ALI in mice, the protein expressions of COX-2 and the ratio of p-IKKβ/ IKKβand p-p65/p65 in lung tissues of mice in LPS group were significantly increased compared with those in the control group (Fig. 3C). After the intervention of GPR84, these two proteins were significantly lower than those in the LPS group, indicating the suppression of GPR84 inhibitor for NF-κB pathway.
GPR inhibitor reduced apoptosis levels
In this study, TUNEL assay was used to detect the effect of GPR inhibitor on apoptosis of lung tissue in mice with acute lung injury induced by LPS. Compared with the control group, the number of apoptotic cells in the lung tissues of the LPS group was significantly increased, indicating that LPS stimulation could promote cell apoptosis (Fig. 4A). The number of apoptotic cells in lung tissue of GPR inhibitor group was significantly lower than that of LPS group, indicating that GPR inhibitor could reduce the number of apoptotic cells in lung tissue of acute lung injury. Bcl-2 can form heterodimers with Bax, resulting in decreased cell permeability. When Bax is overexpressed, apoptosis can increase, while when Bcl-2 is overexpressed, its products can combine with Bax, thus inhibiting apoptosis. The results showed that the proteins promoting apoptosis were significantly reduced while the protein inhibiting apoptosis was increased (Fig. 4B).
GPR inhibitor suppressed the activation of MAPK
The levels of P38, ERK and JNK phosphorylated proteins in the lung tissues of mice in the model group was significantly increased compared to control group (Fig. 5A). The levels of above proteins in GPR inhibitor group were significantly reduced and the effect was dose-dependent when comparing with LPS group. Therefore, we predicted that the suppression of GPR inhibitor for inflammation and apoptosis could derived from the suppression for AMPK signalling pathway. Next, the hypothesis was further validated in BEAS-2B cells treated by LPS. As shown in Fig. 5B, the treatment of BEAS-2B with GPR84 inhibitor resulted in increased cell viability. Moreover, similar effects of GPR84 inhibitor on AMPK signalling pathway were also be observed in BEAS-2B cells with LPS stimulation (Fig. 5C).
The inhibition of MAPK signalling rescued the effects of GPR84 inhibitor on NF-κB signalling
In addition to the treatment of LPS or GPR84 inhibitor, anisomycin (25 µg/mL) as a activator of p38, or TPA (200 nM) as a activator of ERK was also used to treat BEAS-2B cells. The expression of TNF-α, IL-1β and IL-6 was significantly increased in LPS group compared to control group. In addition, we found that anisomycin or TPA treatment could significantly counteract the effects of GPR84 inhibitor (Fig. 6A). NF- κB P65 is a nuclear transcription factor present in the cytoplasm that participates in and regulates inflammatory responses. The ratio of phosphorylated protein to total protein was used to reveal the change of phosphorylated protein[15]. Compared with the normal group, the expression of NF- κB p65 and Iκκβ phosphorylated protein LPS group was significantly up-regulated while GPR84 inhibitor could effectively reduce their expression (Fig. 6B). Intriguing, anisomycin and TPA markedly reversed these effects of GPR84 inhibitor.
The inhibition of MAPK signalling reversed the effects of GPR84 inhibitor on apoptosis
As we aimed at investigating the action mechanism of GPR84 in apoptosis, we next set out experiments to analyze whether anisomycin or TPA affected the effects of GPA84 on apoptosis. We found that the effects of GPR84 inhibitor on apoptosis could also reversed by anisomycin and TPA, suggesting that the suppression of apoptosis by GPR84 was mediated by MAPK signally (Fig. 7A-B).