Protectin conjugates in tissue regeneration 1 alleviates sepsis-induced acute lung injury by inhibiting ferroptosis

doi:10.21203/rs.3.rs-2374878/v1

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Research Article

Protectin conjugates in tissue regeneration 1 alleviates sepsis-induced acute lung injury by inhibiting ferroptosis

https://doi.org/10.21203/rs.3.rs-2374878/v1

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Journal Publication

published 30 Apr, 2023

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Background

Acute lung injury (ALI) is a common and serious complication of sepsis with high mortality. Ferroptosis, categorized as programmed cell death, contributed to the development of lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is an endogenous lipid mediator, exerting protective effects in multi-organ injury. However, the role of PCTR1 in the ferroptosis of sepsis-related ALI remains unknown.

Methods

Pulmonary epithelial cell line and the mouse model of ALI with lipopolysaccharides (LPS) stimulation were established in vitro and in vivo studies. Ferroptosis biomarkers including Fe²⁺, GSH, MDA and 4-HNE were detected by relevant assay kits. GPX4 and PTGS2 protein were determined by western blotting. Lipid peroxides were examined by fluorescence microscope and flow cytometry. Cell viability was detected by CCK-8 assay kit. Ultrastructure of mitochondria was observed with transmission electron microscopy. Morphology and inflammatory cytokine level predicted the severity of lung injury. Afterwards, related inhibitors were used to explore the potential mechanism by which PCTR1 regulated ferroptosis.

Results

PCTR1 treatment protected mice from LPS-induced lung injury, which was consisted with the effect of ferroptosis inhibitor ferrostatin-1. PCTR1 treatment decreased Fe²⁺, PTGS2 and lipid ROS contents, increased GSH and GPX4 levels and ameliorated mitochondrial ultrastructural injury. Administration of LPS or ferroptosis agonist RSL3 resulted in reduced cell viability, which was rescued by PCTR1. Mechanically, inhibition of PCTR1 receptor ALX, protein kinase A (PKA) and transcription factor cAMP-response element binding protein (CREB) partly decreased PCTR1 up-regulated GPX4 expression and CREB inhibitor blocked the effects of PCTR1 on ferroptosis inhibition and lung protection.

Conclusion

This study suggests that PCTR1 suppresses LPS-induced ferroptosis via ALX/PKA/CREB signalling pathway, which may offer a promising therapeutic prospect in the sepsis-related ALI.

PCTR1

ferroptosis

sepsis

acute lung injury

CREB

Sepsis, a life-threatening systemic illness, is primarily responsible for hospital mortality, and is also the dominant cause of acute respiratory distress syndrome or acute lung injury (ARDS/ALI) [1, 2]. Previous studies have demonstrated that inflammation, coagulation dysfunction and oxidative stress play significant roles in the development of sepsis-associated ALI, which manifested as massive inflammatory cell infiltration, progressive pulmonary filling, intractable arterial hypoxemia and dysfunction of lung tissue [3, 4]. However, the pathological mechanism of sepsis-induced ALI is still not fully understood. Noteworthy, recent studies indicated that ferroptosis plays an important role in the pathogenesis of sepsis-induced ALI.

Ferroptosis, which is different from apoptosis, autophagy and other forms of cell death, is driven by iron-dependent lipid peroxidation. Biochemically, the mechanisms underlying ferroptosis mainly contain intracellular iron storage, glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4) inactivation and lipid peroxidation [5–8]. In the occurrence of ferroptosis in ALI, it is found that the production of lipid reactive oxygen species (lipid ROS) induced by iron overload and dysfunction of antioxidant system lead to the destruction of pulmonary epithelial cells and pulmonary vascular endothelial cells [9, 10]. In addition, ferroptosis has attracted tremendous interest for its unique correlation among a number of organ injuries during sepsis, covering cardiovascular dysfunction, liver injury and kidney injury [11–14]. Hence, targeting ferroptosis is a possible therapeutic option for sepsis-induced multiple-organ dysfunction.

Protectin conjugates in tissue regeneration 1 (PCTR1), a new member of specialized pro-resolving mediators (SPMs), is derived from docosahexaenoic acid (DHA) in leukocytes and highly aplenty in lymphatic tissues [15]. PCTR1 not only promotes macrophage phagocytosis and efferocytosis to facilitates inflammation resolution but also accelerates tissue reparation [16, 17]. Our previous study found that PCTR1 increased serum superoxide dismutase (SOD) and GPX4 levels as well as reduced reactive oxygen species (ROS) production in mice, thus ameliorating sepsis-related multiple organ damage [18]. Nevertheless, the connection between PCTR1 and ferroptosis has not been discoverd.

The cAMP-response element binding protein (CREB), which is localized in the nucleus, binds to the cAMP response element (CRE) on the promoters of the target genes, once phosphorylated at Ser133 by different protein kinases, including mitogen-activated protein kinases (MAPK), calmodulin-dependent protein kinase (CaMK), protein kinase A (PKA) and other kinases [19]. Phosphorylation of CREB has been shown to be protective in lung injury models, such as mediating transcription of VE-cadherin to promote lung vascular integrity and enhancing ENaC expression to remove alveolar fluid [20, 21]. Recently, transcription factor CREB was identified to bound to the ‑250 to ‑1 region of the GPX4 promoter in H1299 and A549 lung cancer cells so as to inhibit ferroptosis [22].

In this study, we aimed to investigate the role of PCTR1 on the regulation of ferroptosis in sepsis-induced ALI and the potential mechanism, which mainly focuses on the CREB-correlative regulation of ferroptosis.

Reagents

PCTR1 (17-hydroxydocosa-4,7,10,12,14,19-hexaenoic acid) and H89 (PKA antagonist) were purchased from Cayman Chemical (Ann Arbor, MI). LPS (L2637, Escherichia coli 055: B5) and Ferrostatin-1(SML0583) were purchased from Sigma (St. Louis, MO, USA). 1S,3R-RSL3(HY-100218A) and 666 − 15 (CREB antagonist) were from Med Chem Express (New Jersey, USA). BOC-2 (ALX receptor inhibitor) was purchased from Biomol-Enzo Life Sciences (Farmingdale, NY).

Animal Preparation

Male C57BL/6 mice (8–12 weeks old, 22–25 g), were purchased from Shanghai Experimental Animal Center of China. Mice were housed in specific pathogen-free rooms, which maintained a light/dark cycle for 12 hours with controlled air temperature (22–26℃) and relative humidity (60–65%). Mice were free to contact water and food. This study was authorized by the Animal Studies Ethics Committee of Wenzhou Medical University and was implemented in accordance with the Guide for the Care and Use of Laboratory Animals. The sepsis-associated acute lung injury model was established by intraperitoneal injection of LPS (15 mg/kg) (or an equal volume of saline as the Control group) according to our previous experiments [23].

Cell Culture

H1299 cells (human NSCLC cell line) were purchased from ATCC. H1299 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Cells were cultured in an incubator with 37°C constant temperature and 5% carbon dioxide. Cells were passaged when growth and fusion achieved 70–80%. The third to fifth generation H1299 cells were used for the experiments.

Cell Viability Determination

Cell viability was checked by using Cell Counting Kit-8 (CCK-8, CK04, Dojindo, Tokyo, Japan) according to the manufacturer’s protocol. H1299 cells were seeded in 96-well plates at a density of 5000 cells/well and plated for 24 hours prior to various treatment. At the indicated time, 10 µL CCK-8 solution was added to each well, and the cells were incubated for another 3 h at 37°C. The optical density (OD) values at 450nm were measured using a microplate spectrophotometer.

Western Blotting

Lung tissues and H1299 cell proteins were extracted with lysis buffer. The total protein concentrations were then quantified with a BCA protein assay kit (Rockford, IL, USA). Equal amounts of protein (30 mg) from each group were loaded onto 10–12% SDS-PAGE gels and then transferred to PVDF membranes. After being blocked with quick blocking buffer (NCM Biotech) for 30 minutes, membranes were incubated with the primary antibodies: GPX4 (1:1000, BOSTER, Wuhan, China), PTGS2 (1:1000, Abcam, Cambridge, MA, USA), PKA (1:1000, Abcam, Cambridge, MA, USA), p-PKA(1:1000, Abcam, Cambridge, MA, USA), CREB(1:1000, Abcam, Cambridge, MA, USA), p-CREB (1:1000, Abcam, Cambridge, MA, USA), and β-actin (1:1000, BOYUN, Shanghai, China) overnight at 4 ℃. After being rinsed with PBS three times, the membranes were incubated with secondary antibodies (1:1000, Santa Cruz Company) at room temperature for 1 h. The protein bands were detected by Image Quant LAS 4000 mini (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and the band intensities were evaluated with Image J.

Quantitative Real-time Pcr

Total RNA in lung tissues were extracted using TRIzol reagent purchased from Invitrogen (Carlsbad, CA, United States). cDNAs were reverse transcribed from mRNA by reverse transcription kit in accordance with the manufacturer’s instructions (Thermo, Rockford, IL, USA). Gene expression was evaluated using TB Green System (TaKara, Japan). Gene expression levels were normalized to the housekeeping gene β-actin. Data were analyzed by using the 2-ΔΔCt method. The gene-specific primers were summarized below.

TNF-α-F 5′-CCCTCACACTCACAAACCAC-3′ and TNF-α-R 5′- ACAAGGTACAACCCATCGGC-3′

IL-1β-F 5′-ACAGCAGCATCTCGACAAGAGC-3 ′ and IL-1β-R 5′-CCACGGGCAAGACATAGGTAGC-3′;

LI-6-F 5′-TGCCACCTTTTGACAGTGATG-3′ and LI-6-R 5′- TGATGTGCTGCTGCGAGATT-3′;

β-actin-F 5′-ACCCTAAGGCCAACCGTGAA-3′ and β-actin-R 5′- ATGGCGTGAGGGAGAGCATAG-3′.

Mda, Gsh ,4-hne And Fe Measurement

Glutathione (GSH), malondialdehyde (MDA), 4-hydroxynonenal (4HNE) and Fe²⁺ in lung tissues or cells were measured using reduced GSH assay kit (A006-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), MDA Test Kit (A003-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), 4HNE ELISA kit (H268, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and Iron Assay Kit (A039-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) respectively, in accordance with the manufacturer’s instructions.

Lipid Ros Detection

Cellular lipid ROS was determined using C11 BODIPY 581/591 molecular probe (Invitrogen, Carlsbad, CA, USA). Differently treated H1299 cells were washed with PBS for three times, then incubated with 1 ml medium containing 10 µM C11 BODIPY 581/591 reagent for an additional 1 hour. At the end time point, H1299 cells were washed with PBS for three times, then added with 1 ml medium, finally observed using an inverted fluorescence microscope (Nikon Eclipse Ts2). For quantitative analyses of lipid ROS in cells, flow cytometry was applied. The cells were collected, washed three times with PBS, then suspended in 1ml PBS. Oxidized C11-BODIPY 581/591 probe was determined by Cytoflex (Beckman Coulter) and the data were analyzed by CytExpert 2.4 software.

Pulmonary Histopathological Pathologic Analysis

Left lungs were fixed with 4% paraformaldehyde at room temperature for 24 h, then were dehydrated, embedded in paraffin and stained using hematoxylin and eosin (H&E), and finally observed with a light microscope. The lung injury scores were quantified by two observers blinded to the treatment of each group according to the applicable histopathological scoring system.

Immunofluorescence

The H1299 cells were fixed in 4% paraformaldehyde for 15 min, permeabilized for 20 min with 0.1% Triton X-100, and blocked for 30 min with 10% donkey serum. Cells were incubated in anti-GPX4 (1:200) overnight at 4°C and then incubated with secondary antibody for 1 hour at 37°C. The cell nucleus was counterstained with DAPI. Eventually, a fluorescence microscopy (Leica) was used to capture the cell images.

Transmission Electron Microscopy

Lung tissue samples were pre-fixed in 2.5% glutaraldehyde for 24 hours and post-fixed in 1% osmium tetroxide for 1 h. After three washes with PBS, the specimens were sequentially dehydrated in gradient acetone followed by being embedded in epoxy resin. The samples were dyed with uranyl acetate and lead citrate, and then cut into 50–70 nm ultrathin sections. Subsequently, the images were obtained by Zeiss EM 10C transmission electron microscope (Hitachi, H-7500, Tokyo, Japan).

Statistical analysis

Data are presented as means ± SD. Among multiple groups, data were analyzed using one-way ANOVA, followed by the Tukey’s post hoc test. Between two groups, unpaired Student’s t test was performed. Statistical analyses were performed using GraphPad Prism software (version 8.0.1). Statistical significance was set at p < 0.05.

1.Ferroptosis is induced in LPS-challenged ALI and associated with lung injury

The mouse model of sepsis-induced ALI was established by intraperitoneal (i.p.) injection of 15 mg/kg LPS. To explore the dynamic changes of ferroptosis in ALI, mice were respectively exposed to LPS for 0, 12, 24 and 48 h. Glutathione peroxidase 4 (GPX4) and prostaglandin-endoperoxide synthase 2 (PTGS2) are prominent ferroptotic biomarkers. As shown in Fig. 1A-C, the downregulation of GPX4 and upregulation of PTGS2 were persistly induced by LPS, which were more obvious at 24 h compared with 12 h or 48 h. Fe²⁺ and lipid peroxidation malondialdehyde (MDA) also peaked at 24 h, accompanied by a remarkable decrease in glutathione (GSH) (Fig. 1D-F). Therefore, we chose 24 hours as the stimulation duration of LPS in the subsequent experiments. Next, ferroptosis inhibitor ferrostatin-1 was used to verify the effect of ferroptosis resistance on LPS-exposed lung injury. As illustrated in Fig. 1G-H, compared with the control group, LPS stimulation dramatically led to pathological structure damage of lungs indicated by increased acute lung injury scores which decided on hemorrhage, alveolar edema, and inflammatory cell infiltration. However, treatment with ferrostatin-1 effectively ameliorated these changes as evidenced by declined lung injury scores. Furthermore, relative mRNA levels of the proinflammatory cytokines including IL-1β, IL-6 and TNF-α were markedly increased in septic mice relative to normal mice, whereas these levels were distinctly declined by treatment of ferrostatin-1 (Fig. 1I-K). These results suggest that ferroptosis is implicated in the process of ALI development and suppressing ferroptosis could reduce lung damage to some extent.

2.pctr1 Alleviates Lung Injury By Inhibiting Ferroptosis

We then aimed to investigate the actions of PCTR1 on ferroptosis in LPS-induced ALI in vivo. First, after administration of LPS, PCTR1 (100 ng or 200 ng per mice) was intravenously injected into mice to evaluate the effect of PCTR1 on ferroptosis. Although 100 ng PCTR1 treatment decreased the LPS-stimulated Fe2 + levels, there were no significantly statistical differences on the levels of GPX4, PTGS2, GSH and MDA (Fig. 2A-F). Compare with the LPS group, PCTR1 with 200 ng remarkably promoted GPX4 expression and inhibited PTGS2 expression (Fig. 2A-C). Additionally, LPS-increased MDA and Fe2 + along with decreased GSH were also reversed by 200 ng PCTR1. Consequently, PCTR1 with a concentration of 200 ng was selected in our follow-up experiments. Mitochondrial morphology is important evidence to characterize ferroptosis. In Fig. 2G, a transmission electron microscope was used to observe the ultrastructure of mitochondria. LPS challenge made mitochondria shrinkaged, mitochondrial bilayer membrane density increased and mitochondria cristae disappeared, which was reversed by RCTR1 treatment. We further tested the effect of PCTR1 on LPS-induced ALI. As shown in the HE staining, with the LPS injection, the lung tissues displayed significant interstitial edema, pulmonary architecture destruction, hemorrhaging, and inflammatory cells infiltration. However, these pathological changes in the lungs were evidently reversed by PCTR1 intervention. The quantification of acute lung injury was agreed with these pathological changes (Fig. 2H-I). Additionally, the PCTR1 treatment markedly diminished the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, mRNA expressions in lung tissues, which were obviously increased with LPS stimulation (Fig. 2J-L). Thus, these data imply that PCTR1 suppresses ferroptosis and exerts protective effects on LPS-induced ALI.

3.pctr1 Dampens Ferroptosis By Activating Alx/pka/creb In Lps-induced Ali

Subsequently, we explored the potential mechanism involved in the regulatory action of PCTR1 on ferroptosis in vivo. Our previous work reported that PCTR1 bounded to extracellular domains of its receptor ALX (a G-protein-coupled receptor) to exert its biological functions [18 ], which is critical for activation of intracellular signaling cascades. Protein kinase A (PKA) that was found to be a downstream protein of ALX in our previous research phosphorylates CREB at Ser-133 [19, 24]. Phosphorylated CREB (p-CREB) can improve GPX4 expression at transcriptional level [22]. Therefore, we explored whether PCTR1 activates CREB by ALX/PKA pathway to upregulate GPX4 expression via using specific inhibitors. After LPS stimulation, mice were pretreated with BOC-2 (ALX receptor inhibitor), H89 (PKA inhibitor), or 666 − 15 (CREB antagonist) before PCTR1 treatment. As shown in Fig. 3A, B, phosphorylated PKA (p-PKA) level in the LPS group was obviously reduced compared with the control group, and PCTR1 treatment significantly enhanced the expression of p-PKA. However, the use of BOC-2 restrained the upregulation effect of PCTR1 on p-PKA, indicating that PCTR1 activates the ALX/PKA pathway. Next, we found that LPS-induced inhibition of p-CREB expression was dramatically released in LPS + PCTR1 group, whereas BOC-2 or H89 treatment markedly blocked PCTR1-induced phosphorylation of CREB (Fig. 3C, 3D). Finally, it was observed that BOC-2, H89, or 666 − 15 abolished PCTR1-mediated upregulation of GPX4 (Fig. 3E, F). Collectively, these findings suggest that PCTR1 activates CREB by ALX/PKA pathway to promote GPX4 expression. Since CREB could mediate PCTR1 to increase the expression of GPX4, we further verified whether CREB mediated the effect of PCTR1 on the elimination of lipid peroxides caused by GPX4 depletion in ferroptosis. Compared to the LPS + PCTR1 group, using CREB inhibitor 666 − 15 significantly increased MDA and 4-HNE levels (Fig. 4A-B). Typical ferroptotic mitochondria with reduced or absent crista, shrinked volume and increased membrane density were more in LPS + PCTR1 + 666 − 15 group (Fig. 4C). In addition, the lung tissues were more severely damaged and the inflammatory reaction was more intense due to the inhibition of CREB (Fig. 4D-F). These results suggest that PCTR1 probably dampens ferroptosis by activating ALX/PKA/CREB in LPS-induced ALI.

4.PCTR1 inhibits LPS-induced ferroptosis in vitro

Alveolar epithelial cells, a critical component of the alveolar epithelial-endothelial barrier, play central roles in the pathogenesis of ALI. Ferroptosis was reported to participate in the injury of alveolar epithelial cells, which is induced by LPS [9]. Cell viability was examined by the CCK-8 assay kit after H1299 cells were treated with different doses of LPS (0-1000 µg/ml) for 24 h. LPS did not give rise to the mortality of H12e99 cells until its dose was up to 50 µg/ml (Fig. S1A). However, when the administration time of LPS was extended to 48 hours, 5 or 10 µg/ml LPS was enough to reduce the cell viability statistically. And there was a statistical difference between 5 and 10 µg/ml (Fig. S1B). Accordingly, we used 10 µg/ml LPS in cells for 48 hours to establish a cell damage model in the subsequent experiments. Moreover, ferrostatin-1, a specific ferroptosis inhibitor, was used to confirm that ferroptosis occurs in the decline of cell viability following LPS stimulation. As shown in Fig. S1C, LPS-induced cell mortality was significantly abolished by ferrostatin-1 in a dose-dependent manner. We then investigated the role of PCTR1 in LPS- induced ferroptosis in vitro. As illustrated in Fig. 5A, B, PCTR1 distinctly caused dose-depend increases in the cell viability, which was markedly inhibited by LPS or a ferroptosis agonist RSL3. Moreover, LPS challenge resulted in an obvious decrease of GPX4 and an increase of PTGS2, which were reversed by PCTR1 treatment (Fig. 5C-E). There were also a significant reduction of Fe2 + and MDA and an increase of GSH in LPS + PCTR1 group compared with LPS group (Fig. 5F-H). In addition, the production of oxidized C11 BODIPY 581/591, representing the level of lipid ROS, was observed by fluorescence microscope and flow cytometry. It was found that lipid ROS was remarkably accumulated in H1299 cells, which was captured by the increase of green fluorescence, while PCTR1 treatment significantly diminished the fluorescence intensity (Fig. 5I). Consistently, the inhibitory effect of PCTR1 on lipid ROS was also confirmed by flow cytometry (Fig. 5J). Taken together, these data demonstrate that PCTR1 protectes alveolar epithelial cells against LPS-induced ferroptosis.

5.PCTR1 suppresses ferroptosis by ALX/PKA/CREB activation in vitro

Similarly, we further explored whether PCTR1 phosphorylates CREB to elevate the expression of GPX4 via ALX/PKA in vitro. The protein level of p-PKA was obviously promoted in PCTR1-treated cells, but that was diminished by co-treatment with BOC-2 (Fig. 6A, 6B). PCTR1 elevated the LPS-reduced expression of p-CREB, while the effect was partly abolished by BOC-2 or H89 administration (Fig. 6C, 6D). Moreover, PCTR1 enhanced the LPS-declined GPX4 expression, whereas BOC-2, H89, or 666 − 15 reversed that (Fig. 6E, F). These results indicate that PCTR1 activates CREB through ALX/PKA pathway, thereby increasing the expression of GPX4. Next, we also investigated whether PCTR1 upregulated the expression of GPX4 and decreased accumulation of lipid peroxide via CREB in vitro. Immunofluorometric analysis demonstrated that GPX4 expression in the LPS + PCTR1 + 666 − 15 group was decreased compared with LPS + PCTR1 group, which accorded with the western blotting result (Fig. 7A). The end products of lipid peroxidation of ferroptosis such as MDA and 4-HNE were enhanced when CREB inhibitor 666 − 15 was pretreated before PCTR1 administration (Fig. 7B, C). In parallel, the results of fluorescence microscope and flow cytometry also indicated that inhibition of CREB with 666 − 15 attenuated the inhibitory effect of PCTR1 on LPS-induced lipid ROS (Fig. 7D, E). These results suggest that ALX/PKA/CREB signal is responsible for the inhibitory effect of PCTR1 on LPS-induced ferroptosis.

ALI/ARDS is a serious respiratory disease correlated with dysfunction of alveolar-capillary barrier caused by multiple factors [25, 26]. As the disease is related to the high morbidity and mortality rates, it constitutes a primary public health burden [27, 28]. To date, its clinical treatments mainly contain protective mechanical ventilation, without effective drug for its management [29, 30]. Therefore, it is important to elucidate its pathogenesis and develop effective drugs for its therapy. In the present study, we confirmed that PCTR1 could effectively ameliorate acute inflammation and morphological damage in LPS-stimulated lung injury mechanically via inhibiting ferroptosis. Using antagonists BOC-2, H89, and 666 − 15 blocked the promoting effect of PCTR1 on GPX4 expression demonstrating that PCTR1 elevated the expression level of GPX4 via activating ALX/PKA/CREB signalling pathway. CREB inhibitor 666 − 15 partly abolished PCTR1-induced ferroptosis inhibition, implying that CREB was the key point to the regulation of PCTR1 on ferroptosis. Moreover, our in vitro experiments were also consistent with the findings in vivo studies. Taken together, PCTR1 alleviates LPS-induced lung injury via suppressing ferroptosis, which depends on activating the ALX/PKA/CREB pathway (summarized in Fig. 8).

Ferroptosis is tightly controlled by redox equilibrium, iron homeostasis and lipid metabolism. Fe²⁺ could directly generate excessive ROS through the Fenton reaction and increase the activity of some enzymes responsible for oxygen homeostasis and lipid peroxidation [31]. In both in vivo and in vitro experiments, it was observed that the level of Fe²⁺ was increased upon LPS stimulation, while experiencing a drop with PCTR1 treatment. System xc−/GSH/GPX4 axis is critical antioxidant system. The amino acid antiporter system xc − sustains the production of GSH [32]. Two electrons offered mainly by GSH and the catalytic selenocysteine residue of GPX4 are required for GPX4 to reduce lipid peroxides (PL-OOH) to their corresponding alcohols (PL-OH) [33]. In the present study, GSH and GPX4 remarkedly declined after LPS attack, but that was reversed by PCTR1. Lipid peroxidation is a free radical-propelled reaction leading to the formation of various active compounds including MDA and 4HNE, resulting in cellular damage [32]. We discovered that PCTR1 administration downregulated lipid peroxide caused by LPS. Besides, treatment of RSL3, an inhibitor of GPX4 [34], induced a decline in cell viability, which was rescued by PCTR1 in vitro, suggesting the specific regulation of PCTR1 on ferroptosis. In the matter of morphological features of ferroptosis, changes in mitochondrial ultrastructure manifested as reduced or absent crista, shrinked volume and increased membrane density are considered as major events [32]. Mitochondria like that were identified in our LPS-induced ALI model, while the mitochondrial structure was ameliorated after PCTR1 treatment. In addition, we found that LPS-induced inflammatory reaction and tissue damage were distinctly reduced by PCTR1, which is similar to the effect of ferroptosis inhibitor ferrostatin-1. These findings provided evidence that PCTR1 might restores sepsis-related ALI by inhibiting ferroptosis.

PCTR1, a new member of the protectin family of specialized pro-resolving mediators, was reported to decrease classical proinflammatory, exert characteristic tissue regenerative role and enhance macrophage phagocytosis and efferocytosis [16, 17]. Our previous findings revealed that PCTR1 could protect against septic acute lung injury at least partly by promoting alveolar fluid clearance and restoring lung vascular glycocalyx, ultimately improving survival rates [35, 36]. Moreover, we also found that PCTR1 improved serum SOD and GPX4 levels in septic mice with multiple-organ dysfunction [18], so we speculated that PCTR1 might effectively inhibit ferroptosis by regulating GPX4. Conrad group has furnished early evidence that loss of GPX4 induced lipid-peroxidation-dependent cell death in the embryonic fibroblasts of conditional GPX4 knockout mice [37]. A recent study further proposed that GPX4 plays an essential role in preventing lipid-peroxidation-induced ferroptosis and acute renal failure [38]. Despite the prevailing importance of GPX4 for ferroptosis, little is known about how GPX4 is governed in ferroptosis in ALI. The majority of previous research have focused on nuclear factor-erythroid-2-related factor 2 (Nrf2), which shifts into the nucleus to elevate the expression level of GPX4, resulting in ferroptosis inhibition [39]. Knockout of Yes-associated protein 1 (YAP1), a key regulator of the Hippo signaling pathway, accelerated ferroptosis and also exacerbate CLP-induced ALI by decreasing expression of GPX4 [40]. Currently, several studies have proposed that CREB is involved in regulation of GPX4 transcription, making CREB an important focus of ferroptosis. Wang Z et al. have affirmed that CREB directly binds the promoter of GPX4 in H1299 cells via ChIP experiments [22]. Qi Xiong et al. demonstrated that PM2.5 exposure induces ferroptosis in neuronal cells via inhibiting CREB [41]. Ke Chen et al. indicated that bioflavonoid galangin delivered its anti-ferroptosis effects via activating CREB in a hepatic ischemia-reperfusion model [42]. Indeed, a link between CREB and GPX4 has previously been found during enterocytic cell differentiation [43]. However, another recent study found the opposite effect of CREB on ferroptosis. It was found that CREB regulates Acyl-CoA synthetase long-chain family member 4 (ACSL4) expression in Hepatoma to promote ferroptosis and induce hepatotoxicity [44]. Therefore, the effect of CREB on ferroptosis might depend on the binding gene or the disease condition. In this study we discovered the positive effect of CREB on ferroptosis inhibition in the sepsis-induced ALI model. In our experiments, we detected that PCTR1 activates CREB by ALX/PKA pathway to promote GPX4 expression by western blotting and immunofluorescence assays. Inhibition of CREB with 666 − 15 weakened the scavenging effect of PCTR1 on MDA and 4-HNE and also hindered the ability to reduce lung damage of PCTR1. Additionally, CREB also negatively regulates other cell death modes such as apoptosis and pyroptosis. Mice lacking CREB in the central nervous system show massive apoptosis of postmitotic neurons during development [45]. Transcriptional cooperation between CREB and Nrf2 enhances resistance to not only apoptosis but also pyroptosis, exerting an anti-inflammatory effect [46, 47].

Apart from regulating redox balance, PCTR1, as a lipid metabolite, seems to have a complex network with lipid metabolism. As well known, Lipid metabolism is another key mechanism of ferroptosis. The peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position is a critical step in promoting ferroptosis [48]. Arachidonic acid and adrenic acid (AA/AdA) are the core substrates of lipid peroxidation reactions in ferroptosis [49]. PUFAs were cleaved into free PUFAs and lysophospholipids by phospholipase A2 (PLA2). Only free AA/ADA could combinate with CoA to form AA/AdA-CoA, which subsequently reacts with membrane phosphatidylethanolamine (PE) to synthesize AA/AdA–PE. Interestingly, the previous study from our group found that PCTR1 inhibits the translation of bound AA to free AA by inhibiting the expression of PLA2 [18]. Next, the mammalian ALOX family, including six members, mediate AA/AdA-PE to generate AA/AdA-PE-OOHs in tissue- or cell-dependent manner, thus causing ferroptosis. For example, Arachidonate-15-Lipoxygenase (ALOX15) participates in TP53-induced ferroptosis in H1299 Cells [50].On the other hand, ALOX15 is also the key enzyme in the first step of PCTR1 production .Firstly, ALOX15 catalyzes the production of 17S-HpDHA from DHA, then 17S-HpDHA produces 16S,17S-epoxy-Protectin through epoxidation reaction, and finally PCTR1 is generated by human recombinant leukotriene C4 synthase (LTC4S) and glutathione S-transferases (GSTs) from its epoxy compound precursor (16S,17S-epoxy-PD) [51, 52]. Therefore, it is possible that the production of endogenous PCTR1 competes with lipid peroxidation reaction because ALOX15 is required in common. We also hypothesized that exogenous administration of PCTR1 maybe negatively feedback ALOX15 enzymatic activity, thereby inhibiting the catalytic conversion of AA/AdA-PE to AA/ ADA-PE-OOHs. The complex cross-talk between PCTR1 and ferroptosis in terms of lipid metabolism awaits further investigation in our follow-up study.

In summary, these data demonstrated that ferroptosis implicated in the development of ALI and PCTR1 potently protected against lung injury by inhibiting ferroptosis, which is mediated by the ALX/PKA/CREB activation. This study enriches the regulatory mechanism of ferroptosis in sepsis-associated ALI and indicates that PCTR1 may become a therapeutic approach for ALI.

ALI, acute lung injury; ARDS, acute respiratory distress syndrome; AA, Arachidonic acid; AdA, adrenic acid; ALOX15, Arachidonate-15-Lipoxygenase; CREB, cAMP-response element binding protein; CRE, cAMP response element; CaMK, calmodulin-dependent protein kinase; DHA, docosahexaenoic acid; Fe²⁺, Ferrous; GSH, glutathione; GPX4, glutathione peroxidase 4; GSTs , glutathione S-transferases; LPS, lipopolysaccharides; Lipid ROS, lipid reactive oxygen species; LTC4S, leukotriene C4 synthase; MAPK, mitogen-activated protein kinases; PCTR1, Protectin conjugates in tissue regeneration 1; PTGS2, prostaglandin-endoperoxide synthase 2; PUFAs, polyunsaturated fatty acids; PLA2, phospholipase A2; PE, phosphatidylethanolamine; PKA, protein kinase A; SPMs, specialized pro-resolving mediators; SOD, serum superoxide dismutase.

Competing interests

The authors declare that there is no conflict of interests.

Authors' contributions

YL, DM-C and XY-T carried out most of the in vitro and in vivo experiments. YL and YG drafted the manuscript. YG，SW-J and FG-S conceived, supervised the work and revised the manuscript. CC-X and LN-D performed the in vitro studies. JC-L, SY-Z and YX-C performed the in vivo studies. ZR, YQ-G and BY-Y discussed experimental designs and revised the manuscript. All authors discussed the data, commented on the manuscript, and approved the final manuscript.

Availability of data and material

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82070082, 82002093 and 82270098), Primary Research and Development Plan of Zhejiang Province (2019C0301), Natural Science Foundation of Zhejiang Province (LQ20H150002), Wenzhou Municipal Science and Technology Bureau (Y20220086) and Clinical Research Foundation of the 2nd Affiliated Hospital of Wenzhou Medical University (SAHoWMU-CR2018-11-134).

Acknowledgements

Not applicable.

Ethics approval and consent to participate

All procedures involving animals were approved by the Animal Studies Ethics Committee of Wenzhou Medical University.

Consent for publication

Not applicable.

Vincent J-L, Marshall JC, Namendys-Silva SA, et al; ICON Investigators. Assessment of the worldwide burden of critical illness: the Intensive Care Over Nations (ICON) audit. Lancet Respir Med.2014;2(5):380-386.
Hudson L D, Milberg J A, Anardi D et al. Clinical risks for development of the acute respiratory distress syndrome. Am J Respir Crit Care Med, 1995, 151: 293-301.
Matthay MA, Zemans RL, Zimmerman GA, Arabi YM, Beitler JR, Mercat A, et al. Acute respiratory distress syndrome. Nat Rev Dis Prim. 2019; 5: 18.
Beitler JR, Thompson BT, Baron RM, Bastarache JA, Denlinger LC, Esserman L, et al. Advancing precision medicine for acute respiratory distress syndrome. Lancet Respir Med. 2021;10:P107–120.
Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev. 2014;2014:360438. doi:10.1155/2014/360438.
Dixon S, Stockwell B. The role of iron and reactive oxygen species in cell death. Nat Chem Biol. 2014;10:9–17. doi:10.1038/nchembio. 1416.
Stockwell B, Friedmann Angeli J, Bayir H, et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell. 2017; 171: 273–285. doi: 10.1016/j.cell.2017.09.021.
Shah R, Margison K, Pratt D. The potency of diarylamine radical-trapping antioxidants as inhibitors of ferroptosis underscores the role of autoxidation in the mechanism of cell death. ACS Chem Biol. 2017;12:2538–2545. doi:10.1021/acschembio.7b00730
Zhang Jing, Zheng Yongping, Wang Yun et al. viaYAP1 alleviates sepsis-induced acute lung injury inhibiting ferritinophagy-mediated ferroptosis. Front Immunol, 2022, 13: 884362.
Wang Wei, Xu Rongli, Zhao Haomiao et al. CircEXOC5 promotes ferroptosis by enhancing ACSL4 mRNA stability via binding to PTBP1 in sepsis-induced acute lung injury. Immunobiology, 2022, 227: 152219.
Van Coillie Samya, Van San Emily, Goetschalckx Ines et al. Targeting ferroptosis protects against experimental (multi)organ dysfunction and death. Nat Commun, 2022, 13: 1046.
Fang Xuexian, Ardehali Hossein, Min Junxia et al. The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease. Nat Rev Cardiol, 2022, undefined: 1-17.
Wei Shasha, Bi Jianbin, Yang Lifei et al. Serum irisin levels are decreased in patients with sepsis, and exogenous irisin suppresses ferroptosis in the liver of septic mice. Clin Transl Med, 2020, 10: e173.
Maremonti Francesca, Meyer Claudia, Linkermann Andreas, Mechanisms and Models of Kidney Tubular Necrosis and Nephron Loss. J Am Soc Nephrol, 2022, 33: 472-486.
Dalli J, Colas RA, Arnardottir H, Serhan CN. Vagal regulation of group 3 innate lymphoid cells and the immunoresolvent PCTR1 controls infection resolution. Immunity. 2017, 46: 92–105.
Serhan CN, de la Rosa X, Jouvene C. Novel mediators and mechanisms in the resolution of infectious infammation: evidence for vagus regulation. J Intern Med. 2019, 286: 240–58.16.
Dalli J, Colas RA, Arnardottir H, Serhan CN. Vagal regulation of group 3 innate lymphoid cells and the immunoresolvent PCTR1 controls infection resolution. Immunity. 2017, 46: 92–105
Liu YJ, Li H, Tian Y, Han J, Wang XY, Li XY, Tian C et al; PCTR1 ameliorates lipopolysaccharide-induced acute inflammation and multiple organ damage via regulation of linoleic acid metabolism by promoting FADS1/FASDS2/ELOV2 expression and reducing PLA2 expression. Lab Invest 2020 Jul;100(7).
Alberini, C. M. (2009). Transcription factors in long-term memory and synaptic plasticity. Physiol. Rev. 89, 121–145. doi: 10.1152/physrev.00017.2008.
Xiong S, Hong Z, Huang LS, Tsukasaki Y, Nepal S, Di A, Zhong M, Wu W, Ye Z et al; IL-1β suppression of VE-cadherin transcription underlies sepsis-induced inflammatory lung injury. J Clin Invest 2020 Jul 01;130(7).
Lu K, Chen X, Zhu W, Mao X, Yang Y, Qiu J, Zhang M, Cheng R et al. Terbutaline alleviates the lung injury in the neonatal rats exposed to endotoxin: Potential roles of epithelial sodium channels. Pediatr Pulmonol 2019 Mar;54(3).
Wang Z, Zhang X, Tian X, Yang Y, Ma L, Wang J, Yu Y et al; CREB stimulates GPX4 transcription to inhibit ferroptosis in lung adenocarcinoma. Oncol Rep 2021 Jun;45(6)
Cao F, Tian X, Li Z, Lv Y, Han J, Zhuang R, Cheng B, Gong Y, Ying B, Jin S, Gao Y; Suppression of NLRP3 Inflammasome by Erythropoietin via the EPOR/JAK2/STAT3 Pathway Contributes to Attenuation of Acute Lung Injury in Mice. Front Pharmacol 2020;11
Zheng Sisi, Ma Minqi, Li Zhongwang et al. Posttreatment of Maresin1 Inhibits NLRP3 inflammasome activation via promotion of NLRP3 ubiquitination. FASEB J, 2020, 34: 11944-11956.
Y. Butt, A. Kurdowska, T.C. Allen, Acute lung injury: a clinical and molecular review, Arch. Pathol. Lab. Med. 140 (4) (2016) 345–350
K.T. Hughes, M.B. Beasley, Pulmonary manifestations of acute lung injury: more than just diffuse alveolar damage, Arch. Pathol. Lab. Med. 141 (7) (2017) 916–922
Fowler AA, Truwit JD, Hite RD, Morris PE, DeWilde C, Priday A, et al. Effect of vitamin C infusion on organ failure and biomarkers of inflammation and vascular injury in patients with sepsis and severe acute respiratory failure: the CITRIS-ALI Randomized Clinical Trial. JAMA. 2019, 322: 1261–70
Ware LB. Autopsy in ARDS: insights into natural history. Lancet Respir Med. 2013, 1: 352–4
Beitler JR, Thompson BT, Baron RM, Bastarache JA, Denlinger LC, Esserman L, et al. Advancing precision medicine for acute respiratory distress syndrome. Lancet Respir Med. 2021, 21: S2213-2600.
Yaqub N, Wayne G, Birchall M, Song W. Recent advances in human respiratory epithelium models for drug discovery. Biotechnol Adv. 2021, 54: 107832
Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012)
Tang Daolin, Chen Xin,Kang Rui et al. Ferroptosis: molecular mechanisms and health implications.[J] .Cell Res, 2021, 31: 107-125.
Maiorino M, Conrad M & Ursini F GPx4, Lipid Peroxidation, and Cell Death: Discoveries, Rediscoveries, and Open Issues. Antioxid Redox Signal, doi:10.1089/ars.2017.7115 (2017)
Yang WS et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331, doi: 10. 1016/j. cell.2013.12.010 (2014)
Zhang PH, Han J, Cao F, Liu YJ, Tian C, Wu CH, Smith FG, Hao Y, Jin SW. PCTR1 improves pulmonary edema fluid clearance through activating the sodium channel and lymphatic drainage in lipopolysaccharide-induced ARDS. J Cell Physiol 2020 Dec; 235(12)
Wang XY, Li XY, Wu CH, Hao Y, Fu PH, Mei HX, Chen F. Protectin conjugates in tissue regeneration 1 restores lipopolysaccharide-induced pulmonary endothelial glycocalyx loss via ALX/SIRT1/NF-kappa B axis. Respir Res 2021 Jul 03; 22(1)
Seiler A et al. Glutathione Peroxidase 4 Senses and Translates Oxidative Stress into 12/15-Lipoxygenase Dependent- and AIF-Mediated Cell Death. Cell Metab 8, 237–248 (2008)
Friedmann Angeli Jose Pedro,Schneider Manuela,Proneth Bettina et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.[J] .Nat Cell Biol, 2014, 16: 1180-91.
Dalli J, Colas RA, Arnardottir H, Serhan CN. Vagal regulation of group 3 innate lymphoid cells and the immunoresolvent PCTR1 controls infection resolution. Immunity. 2017, 46: 92–105
Serhan CN, de la Rosa X, Jouvene C. Novel mediators and mechanisms in the resolution of infectious infammation: evidence for vagus regulation. J Intern Med. 2019, 286: 240–58.16.
Qi Xiong, Xiang Tian, Congyue Xu, Baomiao Ma, Wei Liu, Binlian Sun, Qin Ru, Xiji Shu; PM2.5 exposure-induced ferroptosis in neuronal cells via inhibiting ERK/CREB pathway. Environ Toxicol. 2022 Sep, 37(9): 2201-2213.
Ke Chen, Ran Xue, Yaping Geng, Shenshen Zhang; Galangin inhibited ferroptosis through activation of the PI3K/AKT pathway in vitro and in vivo. FASEB J. 2022 Nov, 36(11): e22569
Speckmann B, Bidmon HJ, Pinto A, Anlauf M, Sies H, Steinbrenner H; Induction of glutathione peroxidase 4 expression during enterocytic cell differentiation. J Biol Chem 2011 Mar 25;286(12).
Hu M, Zhong Y, Liu J, Zheng S, Lin L, Lin X, Liang B, Huang Y et al; An adverse outcome pathway-based approach to assess aurantio-obtusin-induced hepatotoxicity. Toxicology 2022 Aug;478.
Mantamadiotis T, Lemberger T, Bleckmann SC, Kern H, Kretz O, Martin Villalba A, Tronche F; Disruption of CREB function in brain leads to neurodegeneration. Nat Genet 2002 May;31(1).
Mylroie H, Dumont O, Bauer A, Thornton CC, Mackey J, Calay D, Hamdulay SS et al. PKCε-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis. Cardiovasc Res 2015 Jun 01;106(3).
Zhang T, Wang Y, Yao W, Chen Y, Zhang D et al. Metformin antagonizes nickel-refining fumes-induced cell pyroptosis via Nrf2/GOLPH3 pathway in vitro and in vivo
Yang, W. S. et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc. Natl. Acad. Sci. USA 113, E4966–E4975 (2016).
Kagan, V. E. et al. Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat. Chem. Biol. 13, 81–90 (2017)
Ou, Y., Wang, S. J., Li, D., Chu, B. & Gu, W. Activation of SAT1 engages polyamine metabolism with p53-mediated ferroptotic responses. Proc. Natl. Acad. Sci. USA 113, E6806–E6812 (2016).
Tsai WC, Kalyanaraman C, Yamaguchi A, Holinstat M, Jacobson MP, Holman TR. In Vitro Biosynthetic Pathway Investigations of Neuroprotectin D1 (NPD1) and Protectin DX (PDX) by Human 12-Lipoxygenase, 15-Lipoxygenase-1, and 15-Lipoxygenase-2. Biochemistry 2021 Jun 08;60(22).
Jouvene Charlotte C,Shay Ashley E,Soens Mieke A et al. Biosynthetic metabolomes of cysteinyl-containing immunoresolvents. FASEB J, 2019, 33: 13794-13807.

supplementaryfigure.1.docx
Additional file 1. Fig. S1 (A) Cell viability of H1299 cells stimulated by LPS with different concentrations for 24 hours. (B) Cell viability of H1299 cells stimulated by LPS with different concentrations for 48 hours. (C) H1299 cells were pretreated with or without different concentrations of ferrostatin-1 for 30 min, followed by LPS (10 ug/ml) for 48 h. Fold change of cell viability. Data are presented as mean ± SD, n = 5-6. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns: p> 0.05.

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published 30 Apr, 2023

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Protectin conjugates in tissue regeneration 1 alleviates sepsis-induced acute lung injury by inhibiting ferroptosis

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Reagents

Animal Preparation

Cell Culture

Cell Viability Determination

Western Blotting

Quantitative Real-time Pcr

Mda, Gsh ,4-hne And Fe Measurement

Lipid Ros Detection

Pulmonary Histopathological Pathologic Analysis

Immunofluorescence

Transmission Electron Microscopy

Statistical analysis

Results

1.Ferroptosis is induced in LPS-challenged ALI and associated with lung injury

2.pctr1 Alleviates Lung Injury By Inhibiting Ferroptosis

3.pctr1 Dampens Ferroptosis By Activating Alx/pka/creb In Lps-induced Ali

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1