Hypoxia-inducible factor prolyl-hydroxylase inhibitor roxadustat (FG-4592) alleviates sepsis-induced acute lung injury

https://doi.org/10.1016/j.resp.2020.103506Get rights and content

Highlights

  • HIF-1α has a protective effect on acute lung injury in LPS induced septic mice.

  • FG-4592 treatment remarkably ameliorated the LPS-induced lung injury through the stabilization of HIF-1α.

  • The clinical use of FG-4592 might be extended to ALI.

Abstract

Acute lung injury (ALI) is one of the most severe outcomes of sepsis which still waiting for effective treatment method. Roxadustat (FG-4592) which is often used for treatment of anemia in patients with chronic kidney disease (CKD), its affection on LPS-induced ALI haven’t been evaluated. MH-S and MLE-12 cell injury and ALI mouse model was induced LPS. Several assays were used to explore the role of FG-4592 in reducing the damage caused by LPS. FG-4592 treatment significantly upregulated HIF-1α and HO-1 and strikingly attenuated inflammation in vivo and in vitro. Furthermore, septic mice overexpressing HIF-1α had high level of survival rate and lower expression of inflammatory factors while down-regulation can enhance the damage of LPS. HIF-1α has a protective effect on acute lung injury in LPS induced septic mice. FG-4592 treatment remarkably ameliorated the LPS-induced lung injury through the stabilization of HIF-1α. Besides the role in treating CKD anemia, the clinical use of FG-4592 also might be extended to ALI.

Introduction

Sepsis is a life-threatening organ dysfunction circumstance resulting from dysregulated host responses to infection, that can lead to multiple organ failure, acidosis, and death (Cecconi et al., 2018). Although the surgical and pharmacological treatment methods of sepsis are continually improving with the help of increasing studies on its pathogenesis, the mortality of sepsis patients is still very high (Duran-Bedolla et al., 2014). Therefore, it is urgent to develop effective treatments for sepsis.

Sepsis or sepsis-associated multiple organ dysfunction syndrome is the worldwide foremost reason of death in intensive care unit (ICU) by mainly attacking the lung, kidney, and cardiovascular system (Ferreira and Sakr, 2011). Lung is the organ that collapse first when respond to sepsis (Schultz and van der Poll, 2002). Meanwhile, ALI as an inflammation syndrome of the respiratory system, is one of the most severe outcomes of sepsis and responsible for sudden deaths of patients in the ICU (Rubenfeld et al., 2005; Sevransky et al., 2004). The nosogenesis of ALI have been studied previously (Kissoon et al., 2016; Liu et al., 2018). However, there is still a lack of effective drugs for ALI therapy.

Endotoxin is the main component of the outer membrane of gram-negative bacteria and known as a stable complex containing lipopolysaccharide (LPS). The initial pathogenesis of ALI induced by LPS is the production of reactive oxygen species, which makes leukocytes infiltrate into the lung. Plasma proteins leakage with leukocytes infiltration results in hypoventilation and hypoxia. Metabolism pathways related to oxygen homeostasis activate and genes involved up-regulate such as HIFs. HIFs are heterodimeric basic helix-loop-helix transcription factors which were composed of two dimeric subunits (Keith et al., 2011). Among them, HIF-1α is a transcription factor that causes cytoprotection and metabolic changes in response to hypoxia and inflammation (Kaelin and Ratcliffe, 2008). Previous studies have shown that HIF-1α is very important for innate and adaptive immunity (Knowles et al., 2006). In addition, overexpression of HIF-1α have anti-inflammatory effects (Westra et al., 2010). However, HIF prolyl hydroxylase domain proteins (HIF PHDs) can rapidly reduce the HIF activity under normoxic conditions. Thus, the suppression of HIF PHDs activity appears to have considerable clinical perspectives. Researchers investigated the effect of dimethyloxalylglycine (DMOG), a HIF PHDs inhibitor, on ALI while it wasn’t ‘clinical’ PHD inhibitor (Nagamine et al., 2016; Tojo et al., 2018). Therefore, and it is well worthy to give a shot on effect evaluation of the ‘clinical’ HIF PHDs inhibitors for ALI.

Roxadustat (FG-4592) is a transient small molecule HIF PHDs inhibitor (Del Vecchio and Locatelli, 2018). The potency of FG-4592 has been proved by lots of studies. For instance, it can increase HIF activity which is sufficient to stimulate effective erythropoiesis, enhance the endogenous EPO levels near the physiological range, protect the severe premature infant against retinal oxygen toxicity (Hoppe et al., 2016) and protect kidney from the cisplatin induced nephropathy (Yang et al., 2018). Moreover, FG-4592 have the higher potency than DMOG (Singh et al., 2020). However, whether FG-4592 treatment could against acute lung injury is still unknown.

Thus, the role of FG-4592 in LPS induced ALI and its potential mechanisms were examined in this study. We investigated the effects of FG-4592 on mouse alveolar macrophage cell line (MH-S) and mouse lung epithelia type II cell line (MLE-12) in vitro, and an LPS induced sepsis mouse model in vivo. Moreover, the possible mechanisms involved in the protective effects of FG-4592 against LPS-induced inflammation were also been explored.

Section snippets

Cell culture

MH-S and MLE-12 were purchased from BeNa Culture Collection. MH-S cells were grown in 1640 medium and MLE-12 cells were cultured in DMEM F12 medium with 10 % FBS supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin at 37 ℃ in a humidified atmosphere of 5 % CO2 (26.4 % O2).

Cell counting Kit-8 (CCK-8) assay

Cell viability was determined by CCK-8 assay kit. MH-S and MLE-12 cells were treated with FG-4592 (5 μM–100 μM) for 24 h, then 10 μL CCK-8 reagent was added to medium and incubated for 1 h. OD450 nm was read with a

FG-4592 alleviates LPS-induced cell inflammation

Firstly, we evaluated the effect of FG-4592 on LPS-induced cell injury in vitro. Cell viability of MH-S and MLE-12 was not affected by FG-4592 treatment at the concentrations of 5 μM, 10 μM, 15 μM, and 25 μM when compared to vehicle control group, while it was depressed around 10 % and 50 % under 50 μM and 100 μM FG-4592, respectively (Fig. 1A). These results indicated that the safe dose of FG-4592 with no noticeable cellular toxicity on MH-S and MLE-12 was within 25 μM. In addition, the effect

Discussion

Increasing evidence suggest that lung is the first organ to fail in response to sepsis, and the major cause of death for sepsis patients is respiratory failure. LPS-induced leukocytes infiltration results in generation ROS, hypoventilation and hypoxia, and thus plays important roles in ALI (Rojas et al., 2005). HIF has been identified as an important mechanism of cellular adaptation to low oxygen and ROS condition (Kenneth and Rocha, 2008). However, HIF PHDs can rapidly reduce the HIF activity

Conclusions

HIF-1α has a protective effect on acute lung injury in LPS induced septic mice. It’s important to enhance the expression of HIF-1α under ALI condition. FG-4592 had anti-inflammatory effects on the LPS-induced MH-S and MLE-12 cell injury model. FG-4592 has therapeutic effects on LPS-induced ALI mouse model. We firstly found that FG-4592 could reduce cell injury and ALI. We also demonstrated the potent effect of HIF stabilizer on protecting against ALI. Our findings reveal a clinical potential of

Funding

This work was supported by the national natural science foundation of China (81530064 and 81801913)

Acknowledgments

We thank for prof. Hongtao Wang which providing language help and writing assistance.

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    These authors contributed equally to this work.

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