Introduction

Acute lung injury (ALI) is a life-threatening medical condition associated with high morbidity and mortality rates [1], which always characterized by widespread lung inflammation and loss of endothelial integrity [2, 3]. Unfortunately, there is currently no effective pharmacological treatment for ALI. Lipopolysaccharide (LPS) is commonly used to induce an immune response in endothelial cells and is widely employed in mouse models to induce ALI [4, 5]. The development of acute pulmonary inflammation is closely tied to the activation of the mitogen-activated protein kinase (MAPK) and nuclear transcription factor-kappa B (NF-κB) signaling pathways [6, 7]. Abnormal NF-κB activation is a defining feature of ALI [4A, B). RT-PCR further verified the success of FGF18 knockout. (Supplementary Fig. 4B). WT and FGF18+/− mice were subject to LPS administration for 12 h and then euthanized to get lung tissues. In addition, we found that the deletion of FGF18 exacerbated lung injury and inflammatory cell recruitment (Fig. 4C). Moreover, FGF18 deficiency significantly aggravated the elevated levels of VCAM-1, ICAM-1, IL-6, and TNF-α, compared to levels in WT LPS-treated mice (Fig. 4D). Meanwhile, more cell adhesion molecules with less VE-cadherin were also observed in FGF18+/− mice (Fig. 4E, Supplementary Fig. 3B). Taken together, these results indicate that FGF18 ablation aggravates LPS-induced lung inflammation and injury.

Fig. 4
figure 4

FGF18 deletion exacerbates LPS-induced lung injury. (A) FGF18+/+ and FGF18+/− mice were subjected to western blotting analysis. The expression of FGF18 was detected. (n = 5 per group). (B) Immunofluorescent staining of FGF18 (red) and DAPI (blue) in FGF18+/+ and FGF18+/− mice were detected. (n = 5 per group, Scale bar = 150 μm). (C) HE staining in lung tissues of FGF18+/+ and FGF18+/− mice after PBS or LPS injection for 12 h (n = 5 per group). (D) Western blotting was performed and quantitatively analyzed to determine the protein levels of VCAM-1, ICAM-1, IL-6, and TNF-α in the lungs from FGF18+/+ and FGF18+/− mice in the presence of LPS (n = 5 per group). (E) Immunofluorescent staining of ICAM-1/VCAM-1 (red) and DAPI (blue) in FGF18+/+ and FGF18+/− mice were detected. (n = 5 per group, Scale bar = 150 μm)

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The knockdown of FGF18 exacerbates LPS-induced HUVEC injury

To further verify the function of FGF18 in LPS treated HUVEC, western blotting analysis showed that FGF18 was significantly decreased after treated with si-FGF18 (Fig. 5A, Supplementary Fig. 4A). To explore the function of FGF18 in vitro, we found that the protein level of VCAM-1, ICAM-1, IL-6, and TNF-α was further increased in HUVECs transfected with si-FGF18 (Fig. 5B), and the mRNA level of ICAM-1 and IL-6 was also increased (Supplementary Fig. 1G, H), suggesting that deletion of FGF18 could exacerbate LPS-induced HUVEC damage. Moreover, we found that the elevated level of ICAM-1 and VCAM-1 (Fig. 5C, D) and decreased level of VE-cadherin (Supplementary Fig. 2B) under LPS conditions were further increased or decreased by si-FGF18 treatment as measured by immunofluorescent staining, respectively. Collectively, these data provide further evidence that knocking down FGF18 could aggravate LPS-induced HUVEC injury.

Fig. 5
figure 5

The knockdown of FGF18 exacerbates LPS-induced HUVEC injury. (A) Western blotting of FGF18 in HUVECs was detected. (n = 5 per group). (B) HUVECs were subjected to western blotting analysis. The expression of VCAM-1, ICAM-1, IL-6, and TNF-α were detected. (n = 5 per group). (C) Immunofluorescent staining of ICAM-1 (red) and DAPI (blue) in HUVECs were detected. (n = 3 per group, Scale bar = 50 μm). (D) Immunofluorescent staining of VCAM-1 (red) and DAPI (blue) in HUVECs were detected. (n = 3 per group, Scale bar = 50 μm)

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FGF18 promotes lung repair and attenuates HUVEC injury by inhibiting the NF-κB pathway

LPS treatment caused significant activation of the NF-κB pathway by enhancing the phosphorylation of IκBα and NF-κB p65 in HUVECs and reversed by FGF18 treatment (Fig. 6A). Meanwhile, western blotting analyses revealed that IκBα and NF-κB p65 phosphorylation was significantly decreased in the lungs of AAV-FGF18-treated mice than in the lungs of control mice (Fig. 6B), and higher in FGF18+/− mice than in lungs of WT mice (Supplementary Fig. 3A). Additionally, results from in vitro experiments showed that si-FGF18 treatment increased the phosphorylation of IκBα and NF-κB p65 (Fig. 6C), indicating that FGF18 knockdown enhanced the activation of the NF-κB signaling pathway. Moreover, a nuclear-cytoplasmic separation experiment demonstrated that FGF18 treatment reduced the entry of NF-κB p65 into the nucleus after LPS stimulation, while si-FGF18 treatment increased the nuclear translocation of NF-κB p65 (Fig. 6D, E). These findings suggest that FGF18 plays a direct protective role against LPS-induced endothelial impairment by inhibiting the activation and nuclear translocation of NF-κB p65. To clarify whether FGF18 protects endothelial cell by inhibiting the NF-κB pathway, we used NF-κB p65 inhibitors JSH-23 and Bay11-7082, which inhibits the expression of p-p65 [19, 20]. The results showed that TNF-α and IL-6 were enriched after si-FGF18 treatment, and this phenomenon was alleviated by the treatment of JSH-23 and Bay11-7082 (Fig. 7A, B), suggesting that FGF18 could suppress the TNF-α and IL-6 expression via p65. In addition, JSH-23 and Bay11-7082 treatment eliminated the ICAM-1 and VCAM-1 exacerbations induced by si-FGF18 (Fig. 7C, D).

Fig. 6
figure 6

FGF18 promotes lung repair and attenuates HUVEC injury by inhibiting the NF-κB pathway. (A) HUVECs were subjected to western blotting analysis. The expression of p-p65, p65, p-IκBα, and IκBα were detected. (n = 5 per group). (B) C57BL/6J mice were treated with AAV-FGF18 and AAV-LacZ in the presence of LPS and then subjected to western blotting analysis. (n = 5 per group). (C) HUVECs were subjected to western blotting analysis. The expression of p-p65, p65, p-IκBα, and IκBα were detected. (n = 5 per group). (D) Immunofluorescent staining of p65 (red) and DAPI (blue) in HUVECs were detected. (n = 5 per group, Scale bar = 50 μm). (E) Immunofluorescent staining of p65 (red) and DAPI (blue) in HUVECs were detected. (n = 5 per group, Scale bar = 50 μm)

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Fig. 7
figure 7

FGF18 promotes HUVECs repair by inhibiting NF-κB p65 activation. (A) HUVECs transfected with si-FGF18 were then treated with JSH-23 (20 µM) for 3 h, HUVECs were subjected to western blotting analysis. The expression of TNF-α and IL-6 were detected. (n = 5 per group). (B) HUVECs transfected with si-FGF18 were then treated with Bay11-7082 (10 µM) for 3 h, HUVECs were subjected to western blotting analysis. The expression of TNF-α and IL-6 were detected. (n = 5 per group). (C) Immunofluorescent staining of ICAM-1/VCAM-1 (red) and DAPI (blue) in HUVECs were detected. (n = 3 per group, Scale bar = 50 μm). (D) Immunofluorescent staining of ICAM-1/VCAM-1 (red) and DAPI (blue) in HUVECs were detected. (n = 3 per group, Scale bar = 50 μm)

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Overall, the data indicate that FGF18 ameliorates lung injury in LPS-induced ALI by inhibiting the NF-κB signaling pathway and protecting against endothelial impairment.

FGF18 is consistent with MAPK kinase inhibitors in reducing the activation of NF-κB pathway

MAPK kinases were widely reported to regulate the inflammation-related signaling pathway. Because of the tight association between the MAPK and NF-κB signaling pathways, a large number of studies have shown that MAPK kinase inhibitors could attenuate phosphorylation of NF-κB p65 [19,20,21]. In the inhibitors group, mice received SB203580 by intraperitoneal injection (Fig. 8A). Moreover, the other two groups of mice underwent gavage of SP600125 and U0126 (Fig. 8B). HE staining revealed that the AAV-FGF18 group and MAPK kinase inhibitors could alleviate lung injury and inflammatory cell aggregation (Fig. 8C). In addition, LPS stimulation enhanced the phosphorylation of IκBα and p65, but this activation was significantly ameliorated by FGF18 and MAPK kinase inhibitors treatment, indicating that FGF18 inhibited LPS-induced activation consistent with MAPK kinase inhibitors in ALI mice (Fig. 8D). Overall, these findings confirm that FGF18 exhibits a comparable protective effect against LPS-induced lung injury as MAPK kinase inhibitors.

Fig. 8
figure 8

FGF18 inhibits NF-κB pathway activation in ALI mice in line with MAPK kinase inhibitors. (A, B) A schematic diagram demonstrates the animal experiment design. (C) HE staining in lung tissues of MAPK kinase inhibitors-treated mice in the presence of AAV-FGF18 after LPS injection for 12 h. (n = 5 per group, Scale bar = 50 μm). (D) C57BL/6J mice were treated with AAV-FGF18 and MAPK kinase inhibitors in the presence of LPS and then subjected to western blotting analysis. (n = 5 per group)

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The in vitro experiments conducted in HUVECs demonstrated that FGF18, as well as MAPK kinase inhibitors (SP600125 and SB203580), and si-Erk could alleviate the phosphorylation of IκBα and p65 (Supplementary Fig. 7A). This indicates that FGF18 and MAPK kinase inhibitors have a similar effect in inhibiting the activation of the NF-κB signaling pathway. Furthermore, western blotting analysis confirmed that pretreatment with FGF18 and the inhibition of MAPK kinases reduced the nuclear localization of IκBα and NF-κB p65 induced by LPS (Supplementary Fig. 7B). This suggests that FGF18 and MAPK kinase inhibitors can prevent the translocation of NF-κB p65 into the nucleus. Immunofluorescent staining further supported these findings by demonstrating that FGF18 and MAPK kinase inhibitors could alleviate the entry of NF-κB p65 into the nucleus (Supplementary Fig. 7C). Intriguingly, the combination of FGF18 and MAPK kinase inhibitors could further inhibit the NF-κB pathway (Supplementary Fig. 8A). Overall, these results provide strong evidence that the therapeutic effect of FGF18 is consistent with that of MAPK kinase inhibitors in inhibiting the NF-κB signaling pathway. This suggests that FGF18 may serve as a novel approach for the treatment of acute lung injury.

Discussion

ALI and ARDS are the two main causes of acute lung failure, which is characterized by high morbidity and mortality and for which effective therapeutic strategies are lacking. Thus, identifying novel treatments for ALI is urgently needed. Several lines of evidence show that FGFs play a central role in pulmonary inflammation. Dhlamini, Q et al. found that FGF1 alleviates LPS-induced ALI via suppression of inflammation and oxidative stress [22]. Tichelaar JW et al. confirmed that FGF7 improved survival during ALI in adult mouse lungs after short-term expression [23]. Wang Q et al. demonstrated that the anti-inflammatory effect of FGF10 on NF-κB signaling was mediated through the regulation of oxidative stress [24].

It is well-realized that FGF18 is released by interstitial cells and possibly endothelial cells in the lung and is known to drive cell migration [25], especially for endothelial cells [26]. FGF18 transgene induction also enhanced the expression of other genes that may be involved in angiogenesis, including endothelial cell growth and differentiation factor Wnt2 [27]. Interestingly, previous findings identified FGF18 as a likely important player in the control of alveolar angiogenesis [18], an event that is an absolute requirement for alveolarization and is compromised in bronchopulmonary dysplasia. However, the role of FGF18 in the pathological development of ALI has not been reported. In this study, we revealed that FGF18 protects against pulmonary injury by inhibiting the NF-κB pathway both in vivo and in vitro (Fig. 6). FGF18 inhibits nuclear accumulation of NF-κB p65 and thereby, alleviates cellular inflammation and pulmonary repair. These findings provide new clues and ideas for develo** potential methods to treat ALI and promote pulmonary repair.

Our previous study demonstrated that FGF18 plays a protective role in the liver, especially in liver fibrosis and hepatic ischemia-reperfusion [28, 29]. In the present study, we found that FGF18 was increased upon LPS stimulation, and FGF18 treatment could decrease the phosphorylation of IκBα, and this effect was also correlated to a parallel decrease in the nuclear translocation of the NF-κB p65 as confirmed by immunofluorescence and western blotting analysis. On the contrary, reducing the expression of FGF18 in vivo and in vitro exacerbates lung injury and endothelial cell damage, respectively (Fig. 4). Our work showed that the elevated p-IκBα and p-p65 expression in LPS-treated HUVECs was largely reversed by FGF18 treatment along with alleviated HUVEC injury (Fig. 6). This is the first time for us to explore the relationship between FGF18 and the NF-κB pathway in the context of acute lung injury.

Double immunofluorescent staining indicated that FGF18 was mainly co-localized with CD34 expression, suggesting that endothelial cells are the main source of FGF18 in mice lungs. Consistently, FGF18 was upregulated in LPS-treated HUVECs (Fig. 1). Lung vascular leakage in response to an unchecked cytokine storm generated by the activation of innate immune cells is a hallmark of sepsis-induced inflammatory injury [30,31,32]. The vascular leakage in the lungs is the result of endothelial barrier breakdown and the change level of cell adhesion molecules [33, 34]. Endothelial permeability is normally tuned by the interaction of VE-cadherin in endothelial monolayers [35, 36]. Multiple studies have demonstrated that modifications of FGFs are important for the structure of endothelial cells [37]. Furthermore, aberrant NF-κB activation contributes to the development of vascular leakage [38], among other inflammatory disorders, by mediating the transcription of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β, which in turn enhance the inflammatory response. Here, FGF18 pretreatment significantly suppressed LPS-induced phosphorylation of IκBα and NF-κB p65 in mice, consistent with MAPK kinase inhibitors (Fig. 7).

Hyper-phosphorylation of MAPK molecules can lead to the activation of NF-κB and the subsequent production of inflammatory molecules [19,20,21]. The MAPK and NF-κB pathways have been identified as important targets in LPS-induced ALI. In our study, it was observed that FGF18 significantly attenuated the phosphorylation of NF-κB p65 in a dose-dependent manner, although the specific data was not shown. This finding is consistent with previous studies that have shown MAPK kinase inhibitors can alleviate p65 phosphorylation [39, 40]. Based on these observations, it is reasonable to speculate that FGF18 may affect the NF-κB pathway in a similar manner to MAPK kinase inhibitors. The potential mechanism of FGF18 inhibits p-IκBα and p-p65 may involve several aspects. Firstly, FGF18 may interfere with the activity of kinases involved in the NF-κB signaling pathway, such as the IKK complex (IκB kinase), reduces the phosphorylation of IκBα, thereby inhibiting the activation and intranuclear transfer of NF-κB. Secondly, FGF18 may indirectly affect the NF-κB pathway by affecting the upstream signals of NF-κB activation, such as those of inflammatory factors (TNF-α or IL-1β). Further investigation is still required to fully understand the underlying mechanisms and confirm the specific interactions between FGF18 and the NF-κB pathway.

It appears that our study demonstrated the beneficial effects of FGF18 in alleviating LPS-induced inflammation and preventing endothelial cell leakage in the lung. The overexpression of FGF18 suppressed NF-κB activation, as evidenced by decreased levels of ICAM-1/VCAM-1 and increased levels of VE-cadherin. On the other hand, FGF18 knockdown in mouse models and HUVECs exacerbated endothelial cell leakage. These findings highlight a previously unknown role of FGF18 in regulating the activation of vascular endothelial cells and shed light on the role and mechanism of FGF18 in the pathophysiology of ALI. Consequently, targeting FGF18 may represent a promising therapeutic strategy for the treatment of ALI. It is important to note that FGF18 as a therapeutic agent for ALI would need to be further investigated in preclinical and clinical studies before it can be considered for clinical use.

Conclusion

In conclusion, these data suggest that FGF18 is associated with ALI and that FGF18 effectively protects against ALI by inhibiting NF-κB mediated by p65 activation. This study enriched the regulatory mechanism of NF-κB in sepsis-associated ALI, suggesting that FGF18 may be a therapeutic pathway for ALI. Considering that inflammation is involved in the pathological processes of many diseases, and that FGF18 effectively inhibits the NF-κB pathway in LPS-induced ALI model, we can further validate its therapeutic potential in many inflammation-related diseases.