β-aminoisobutyric Acid, a Metabolite of BCAA, Activates the AMPK/Nrf-2 Pathway to Prevent Ferroptosis and Ameliorates Lung Ischemia-Reperfusion Injury

doi:10.21203/rs.3.rs-2595170/v1

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Research Article

β-aminoisobutyric Acid, a Metabolite of BCAA, Activates the AMPK/Nrf-2 Pathway to Prevent Ferroptosis and Ameliorates Lung Ischemia-Reperfusion Injury

https://doi.org/10.21203/rs.3.rs-2595170/v1

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published 04 Dec, 2023

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Background: Lung ischemia-reperfusion injury is a serious clinical problem and there is no effective treatment. Ischemia-reperfusion (I/R) injury is always accompanied with changed branched chain amino acid (BCAA) metabolism. Enhancing BCAA metabolism can protect against ischemia-reperfusion injury. We believe that this phenomenon is related to bioactive molecules produced by BCAA metabolism. And, L-β-aminoisobutyric acid (L-BAIBA) is a metabolite of valine, a member of BCAA.

Methods: Adult C57BL/6 mouse were treated with L-BAIBA (150mg/kg/day) in the drinking water for 10 consecutive days before lung L/R injury. Then, lung function indexes including pathology and respiratory function were detected. Potential mechanisms were delineated by molecular biology experiment analysis in A549 cells, including western blot or immunofluorescence staining or biochemical detection and so on.

Results:We find that L-BAIBA can protects lung during I/R injury. Further studies show that L-BAIBA can up-regulate the expression of GPX4 and SLC7A11, thereby inhibit ferroptosis. The regulation of L-BAIBA on the expression of GPX4 and SLC7A11 depends on the Nrf-2 signaling pathway. Interfering Nrf-2 eliminates the protective effect of L-BAIBA. We further find that L-BAIBA regulates Nrf-2 by activating AMPK signaling pathway. Meanwhile, in the presence of compound c, the protective effects of L-BAIBA on lung I/R injury are blocked.

Conclusion:Our study reveals that L-BAIBA can alleviate lung I/R injury by inhibiting ferroptosis, which is an promising therapeutic target candidate.

L-BAIBA

Lung Ischemia-Reperfusion Injury

Ferroptosis

Branched chain amino acid

Nrf-2

AMPK

Medical conditions such as transplantation, shock, cardiopulmonary bypass surgery, ARDS and pulmonary embolism can provoke ischemia/reperfusion (I/R) injury in lung, which have been associated with a high mortality rate and seriously affects the success rate of lung transplantation (de Perrot et al. 2003^; den Hengst et al. 2010^; Kuratani et al. 1992). Lung ischemia-reperfusion injury is a aseptic inflammation characterized by pulmonary edema and hypoxemia (Chen et al. 2017). Prevention of lung I/R injury is of great significance in improving the survival rate of patients with severe cardiopulmonary disease or in need of lung transplantation (Capuzzimati, Hough & Liu 2022^; de Perrot et al. 2003^; Hartwig & Davis 2004). Therefore, more strategies and research are needed to prevent and treat lung I/R injury (Chen & Date 2015^; de Perrot et al. 2003).

Pathologically, alveolar epithelial cells and vascular endothelial cells of lung tissues are damaged, which is closely related to the accumulation of inflammatory cells and the production of reactive oxygen species (ROS) (Ferrari & Andrade 2015). Ferroptosis due to the imbalance between the generation and degradation of intracellular lipid reactive oxygen species is thought to play an important pathological role in I/R injury (Chen et al. 2021). Inhibition of ferroptosis is considered to be an effective method to prevent lung I/R injury.

Branched amino acids (BCAA) include leucine, isoleucine and valine, all of them are essential amino acids (Dimou, Tsimihodimos & Bairaktari 2022). Changes in BCAA metabolism are thought to play a important role in ischemia-reperfusion injury. Ischemia-reperfusion injury is often accompanied with changed BCAA catabolism, and strengthening BCAA metabolism is regard to improve ischemia-reperfusion injury (Li et al. 2017^; Lian et al. 2020). The explanation for this phenomenon, firstly, the weakened BCAA metabolism may cause tissue energy supply disorder (Li et al. 2017). Secondly, it may also be connected with the enhanced BCAA metabolism can reduce its accumulation in organs, excessive accumulation of BCAA can produce toxic effects (Li et al. 2017^; Li et al. 2020). Finally, it may also relate with the increase of BCAA metabolites. BCAA metabolism can generate a variety of bioactive molecules, and some of bioactive molecules have been reported to protect against organ ischemia-reperfusion injury (Dong et al. 2016). Thereby, We believe that enhanced BCAA metabolism can play a protective role against I/R injury through the production of protective BCAA metabolites.

L-β-Aminoisobutyric acid (L-BAIBA) is a natural metabolite of valine, and exercise can significantly improve the level of L-BAIBA in plasma (Kammoun & Febbraio 2014). Recent studies have shown that L-BAIBA can inhibit inflammation and induce fatty acid oxidation (Jung et al. 2015^; Roberts et al. 2014). Moreover, L-BAIBA can also protect cells from ROS-induced damage, all of which indicate that L-BAIBA, as a humeral factor, might regulate the functions of various organs (Kitase et al. 2018^; Sawada et al. 2019). However, whether L-BAIBA can protect organs from I/R injury remains unclear. The aim of our study is to verify the protective effect of L-BAIBA preconditioning on lung I/R injury and to study its potential protective mechanism.

2.1 Animals and reagents. Eight-week-old C57BL/6J mouse (20–30g, SPF Biotechnology, Beijing, China), obtained from the animal center of Army Medical University, were fed with normal mouse diet. All animal experiments are conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85–23, revised 1996) and approved by the animal care and use committee of Army Medical University (AMUWEC20201184). We confirm that the study was carried out in compliance with the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Testing, and Education by the New York Academy of Sciences, Ad Hoc Animal Research Committee.

2.2 Mouse lung ischemia-reperfusion injury model. The animal model of lung I/R injury is constructed according to the previous article as follows (Chen et al. 2017). Mouse were anesthetized by sodium pentobarbital (50 mg/kg i.p.), and placed on a heating pad to maintain their body temperature. The right femoral arteries and veins of mouse were cannulated with polyethylene tubing for arterial blood sampling. After intubation through the tracheostomy, mouse were connected to a mouse ventilator (Hugo Sachs Elektronik) with an inspiratory pressure set to 7 ml/kg and the breathing rate was set at 100 breaths per minute. The left hilum was clamped with a vascular clamp for 1 hour and then released for 1 hour to reperfusion. After I/R treatment, blood samples were obtained immediately for arterial blood gas analysis (Gem primer 3000, Instrumentation Laboratory) or enzyme-linked immunosorbent assay (ELISA). The survival analysis method also refers to the previous article of our team (Smith et al. 1997).

2.3 Histological study. Lung tissue samples were stored in 4% paraformaldehyde for 24 hours, then embedded in paraffin, sectioned, and placed on glass slides. After dewaxing and rehydrating with xylene and ethanol of different concentrations, the sections were processed by the following steps: staining with hematoxylin solution for 7 min, rinsing with running water for 5 min, soaking in 1% acid alcohol for 1 min, and counterstaining with eosin Y solution for 2 min. Finally, the plates were dehydrated and sealed with neutral resin. Smith pathological score system was used for pathological injury of lung tissue (Smith et al. 1997).

2.4 Wet/dry lung weight ratio. After the mouse were sacrificed, the lungs were taken and weighed to determine the wet weight, then placed in an oven at 80°C for 24 hours, and reweighed to determine the dry weight for calculation of the wet/dry weight ratio (Luo et al. 2022).

2.5 ELISA. Blood samples were collected from the right femoral artery by catheterization and allowed to coagulate at room temperature, then the collected whole blood samples were quickly centrifuged at 1000g for 5 minutes and the supernatant was collected. Care should be taken to avoid hemolysis during the whole process. The IL-1β, IL-6 and TNF-α concentrations were detected by ELISA (Elabscience). For details of ELISA, referred to the the manufacturer’s instruction.

2.6 Measuring the activities of MDA, MPO, SOD and the ratio of GSH and GSSG. Myeloperoxidase (MDA), GSH and GSSG was measured by using kits produced by Beyotime Biotechnology. Myeloid peroxidase (MPO) assay kit and superoxide dismutase (SOD) assay kit were purchased from Jiancheng biological engineering institute. Briefly, previously obtained lung tissue was homogenized in the sample preparation solution of each kit and then follow the steps on the manufacturer's instructions.

2.7 Tunel staining. Paraffin specimens of lung tissue previously preserved in sections are obtained. Firstly, dewaxing is carried out. Next, the sections were placed in the prepared antigen repair solution and then boiled together in a water bath for 30min. Thirdly, immunostaining sealer will be used for 1 hour after natural cooling. Then, the Tunel staining solution was prepared according to the instructions, added to the specimens, and incubated at 37 ℃ for 30min. Finally, after the sections were dried, the tablets were sealed with DAPI-containing immunostaining tablets. Images were observed and taken by using laser confocal microscope.

2.8 DHE staining. Superoxide production in the mouse lung tissue was evaluated with the fluorescent dye DHE (Beyotime). Frozen section of the lung tissue was stained with DHE (10⁻⁵ M) for 30 min in 37°C. After the tissues were washed by PBS for three times, images were taken by a confocal microscope at an excitation wavelength of 490 nm and an emission wavelength of 590 nm. All sections were processed under the same conditions. The DHE fluorescence intensity was quantified by ImageJ (NIH, Bethesda).

2.9 Western blotting. Total proteins were isolated from the lung tissues or cells. 20 mg of each lung specimen was lysed with RIPA buffer containing protease and phosphatase inhibitors cocktails (Solarbio) by using a Benchmark Beadblast tissue homogenizer (Benchmark scientific). Protein concentration was measured with BSA as a reference standard according to BCA Protein Assay Kit (Beyotime Biotechnology). Nuclear and Cytoplasmic Protein Extraction Kit was purchased from Beyotime Biotechnology, and the specific use method was carried out according to the instruction manual.

SDS-PAGE gels at 8%, 10%, and 15% concentrations were used. According to the different experimental conditions of different target proteins, the same group of protein samples were added into the different groups of swimming lanes to obtain the bands of target protein and loading controls respectively. After separating by SDS-PAGE, proteins were transferred to nitrocellulose filter membrane (Millipore Sigma). Then, NC membrane were cropped according to molecular weight and incubated with antibodies. Image J (NIH, Bethesda, MD) was used to analyze the average gray value of each band.

The antibodies used in this study were as follows: anti-β-actin (1:500, SantaCruz, SC-130657), anti-H3 (1:3000, Cell Signaling Technologies, #4658), anti-GPX4 (1:500, Proteintech, 67763-1-Ig), anti-SLC7A11 (1:1000, Invitrogen, PA1-16893), anti-Nrf-2 (1:1000, Cell Signaling Technologies, #12721; 1:500, Protrintech, 80593-1-RR), anti-P53 (1:1000, Protrintech, 60283-2-Ig), anti-BAP1 (1:500, Affinity, AF7925), anti-PARP1 (1:1000, Protrintech, 22999-1-AP), anti-ATF3 (1:500, SantaCruz, sc-81189), anti-AMPK (1:1000, Cell Signaling Technologies, #2532), anti-Phospho-AMPK (1:1000, Cell Signaling Technologies, #2535).

2.10 A/R (anoxia/reoxygenation)-induced injury in A549 cells. A549 cells, were obtained from Procell Life Science & Technology in Wuhan and cultured in DMEM high glucose medium (Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were cultured at 37℃ in 95% air and 5% CO₂ atmosphere. After changing to serum-free medium, cells were exposed to an anoxic chamber with 5% CO₂ and 95% N₂ at 37℃ for 2 hours followed by reoxygenation in 95% air and 5% CO₂ atmosphere for another 2 hours. Exogenous L-BAIBA was given to cells 48 hours before it becomes anoxic.

2.11 Plasmid transfection and ARE Luciferase Activity Assays. DH5-competent E. coli was used for plasmid screening and amplification. A549 cells were seeded in 6-well plates and transfected with Lipofectamine 3000 at a cell confluency of approximately 50% according to the standard experimental procedures provided by the manufacturer's instructions. For the luciferase activity assay, the double-stranded oligonucleotide tandem repeat sequence spanning the Nrf-2 binding site 5-tgactcagca-3 was inserted into the restriction site of the pGL2 promoter plasmid to construct the antioxidant response element (ARE) luciferase vector. Then, the cell lysate was first mixed with the luciferase substrate solution, and the luciferase activity was measured using a luminometer (Xiao et al. 2018).

2.12 CCK8 and LDH release assay. A549 cells were seeded into 96-well culture plates (Corning, Lowell, MA). The L-BAIBA preconditioning group was treated with different concentrations of L-BAIBA (sigma) for 48h. In addition, cells were preconditioned with Nrf-2, AMPK, ERK, JNK, PI3K inhibitor (Brusatol, ZnPP, compound c, PD98059, SP600125 and LY294002) 30 min before adding L-BAIBA. ZnPP, compound c, PD98059, SP600125, and LY294002 were purchased from Sigma-Aldrich and Brusatol was purchased from MCE. A549 cells were allowed to grow to subconfluence (70-80%). After the cells were subjected to A/R, CCK8 reagent (Bimake) and complete medium were added to the 96-well plate at a ratio of 1:9, then incubate at 37°C for 1 hour, thereafter, using a microplate reader to read the absorbance reading at 450nm. LDH release was detected by LDH cytotoxicity assay kit (Beyotime Biotechnology). Collect the cell culture supernatant in a 96-well plate, add LDH detection working solution separately, incubate in the dark at 37°C for 30 minutes, and then measure the absorbance at 490nm.

2.13 Fe2+ staining. The cells were seeded in glass-bottomed petri dishes (Thermo Scientific). After cells were treated, 1uM Fe2+indicator (Maokang Biotechnology) configured in fresh medium was added to the cells for probe loading for 30min in a cell incubator at 37℃. After loading, cells were washed with PBS. Finally, confocal microscope was used to observe and take images.

2.14 Immunofluorescence staining. Cells were cultured on the cell slide firstly. Next, cells were fixed with 4% paraformaldehyde for 15 minutes after the treatment is completed. Then, the cell slide were mixed with immunostaining blocking solution for 1h at room temperature to prevent nonspecific antibody binding. After blocking, the sections were incubated with anti-Nrf-2 antibody (1:100) at 4℃ overnight. After washing with PBS for 5 min 3 times, the sections were incubated with secondary antibody, Cy3-labeled goat anti-rabbit IgG (1:200), at 37℃ for 30 min. Finally, after washing with PBS (5 min, 3 times), the sections were stained with DAPI before being imaged under confocal microscope.

2.15 Statistical analysis. All data were represented as mean±standard deviation. Firstly, we carefully checked whether the experimental data of each group conformed to the normal distribution by Kolmogorov-Smirnov test. For data that did not conformed to normal distribution, we used the Kruskal-Wallis test for analysis. For data with normal distribution and homogeneity of variance, one-way analysis of variance (ANOVA) was used to analyze the differences between multiple groups, and LSD was used for pair comparison. Tamhane test was performed when assumptions of homogeneity of variance were questionable. SPSS 26 (IBM Corporation, Armonk, NY) was used for the statistical analyses. A value of P<0.05 was considered significant. For survival analysis, the Log-rank (Mantel-Cox) test was used to evaluate the significance of differences in cumulative survival. Graphpad prism 8.0.2 software was used for the survival analysis. A value of P<0.05 was considered significant.

3.1. L-BAIBA relieved lung injury caused by ischemia-reperfusion.

To determine whether L-BAIBA preconditioning has a protective effect on I/R injury, we first preconditioned the mouse with L-BAIBA (150mg/kg/day) in the drinking water for 10 consecutive days. Then, pulmonary I/R injury models were established. We found that L-BAIBA preconditioning improved the survival of mouse subjected to the I/R treatment (Figure 1A). Meanwhile, mouse treated with L-BAIBA showed significantly reduced pathological scores and alleviated pulmonary edema compared with mouse subjected to I/R injury only (Figure 1B and 1C). Moreover, hypoxemia is another mark associated with lung function, which is indicated by partial pressure of oxygen (PaO₂), partial pressure of carbon dioxide (PaCO₂). Our results showed that L-BAIBA treatment improved the lowered blood oxygen partial pressure (Figure 1D), and alleviated the increased retention of CO₂ in the I/R injury group (Figure 1E). Apoptosis is another characteristic of ILRI, and results of TUNEL staining shows that L-BAIBA treatment reduced the number of TUNEL stained positive cells in lung tissue after I/R treatment, which demonstrate the protective effect of L-BAIBA on apoptosis in lung I/R injury (Figure 2A). In addition, L-BAIBA reduced the concentration of pro-inflammatory cytokines, including IL-1β and IL-6 and TNF-α, in plasma isolated from mouse subjected to the I/R treatment (Figure 2B-2D). L-BAIBA preconditioning also reduced the activity of MPO in lung tissue, another indicator of inflammation (Figure 2E). These results indicated that L-BAIBA had a certain beneficial effects on lung function after I/R injury, and has anti-inflammatory and anti-apoptotic effects.

3.2 L-BAIBA treatment ameliorated ferroptosis induced by ischemia-reperfusion.

Ferroptosis is a novel form of programmed cell death caused by iron-dependent accumulation of lipid peroxides. Lipid oxidation is characteristic of ferroptosis, while the impairment of antioxidant system is the pathological basis of ferroptosis, and the role of ferroptosis in ischemia-reperfusion injury has been widely reported (Chen et al. 2021). L-BAIBA is an effective antioxidant, which can regulate the expression of various antioxidant molecules (Sawada et al. 2019). However, whether the antioxidant effect of L-BAIBA can inhibit ferroptosis is still unclear. In this study, we found that L-BAIBA preconditioning significantly reduced the level of ROS and oxidative stress in lung tissues, determined by the measurement of the level of DHE, MDA, SOD and the ratio of GSH and GSSG, which confirmed that the antioxidant effect of L-BAIBA still existed in lung I/R injury models (Figure 3A-3D). Further studies revealed that GPX4 and SLC7A11, as two important proteins against ferroptosis (Chen et al. 2021), were up-regulated in lung tissues of I/R models pretreated with L-BAIBA (Figure 3E). This result was further verified by in vitro experiment. We performed anoxia/reoxygenation on A549 cells, and results showed that L-BAIBA also up-regulated the impaired expression of GPX4 and SLC7A11 in A549 cells after hypoxia-reoxygenation exposure (Figure 4A). Meanwhile, L-BAIBA also effectively protected the cell damage, determined by CCK8, LDH and MDA (Figure 4B-4D). It should be noted that different concentrations of L-BAIBA in the physiological state were not toxic to A549 cells (Figure S1A). We further tested the role of L-BAIBA in the regulation of ferroptosis by blocking experiments. Results showed that two inducers of ferroptosis, RSL-3 and erastin, could eliminate the protective effect of L-BAIBA on cell damage induced by A/R, demonstrated by levels of CCK8, LDH and MDA (Figure 4E-4G). The above results further confirmed that the protective effect of L-BAIBA was achieved by inhibiting ferroptosis. Furthermore, the image of Fe²⁺ probe staining showed that the level of Fe²⁺ in A549 cells was significantly increased after A/R exposure, while L-BAIBA could reduce the intracellular Fe²⁺ enrichment, which further demonstrated the inhibitory effect of L-BAIBA on ferroptosis (Figure 4H). Results presented in this section shows that L-BAIBA can protect against lung I/R injury by up-regulating the expression of GPX4 and SLC7A11 and inhibiting ferroptosis.

3.3. L-BAIBA prevented ferroptosis through Nrf-2 signaling pathway.

We further explored the possible molecular mechanism by which L-BAIBA regulated the expression of GPX4 and SLC7A11 to inhibit ferroptosis. According to previous literature reports, several signaling molecules which may regulate GPX4 and SLC7A11 were screened for detection (Fu et al. 2022^;Hong et al. 2021^;Wang et al. 2021^;Ye et al. 2022^;Zeng et al. 2022). Results detected by western bolt showed that L-BAIBA had the most significant regulatory effect on Nrf-2, which suggested that Nrf-2 might be the potential signaling pathway of L-BAIBA in regulating GPX4 and SLC7A11 (Figure 5A). Therefore, further experiments to verify the regulatory effect of L-BAIBA on Nrf-2 was administrated. Firstly, the regulatory effect of BAIBA on Nrf-2 signaling pathway was also confirmed in lung tissues, results showed that L-BAIBA promoted the nuclear translocation of Nrf-2 in lung tissues regardless of exposure to I/R injury (Figure 5B). Then, results from cells experiment also showed that L-BAIBA could increase the level of Nrf-2 in nucleus and enhance its transcriptional activity in antioxidant response element in a concentration-dependent manner (Figure S2A and S2B). Then, CCK8 and LDH experiments were administrated and results showed that in the presence of brusatol, an inhibitor of Nrf-2, the protective effect of L-BAIBA against cells damage induced by A/R was blocked (Figure 5C and 5D). Finally, blocking experiments were performed by transfecting Nrf-2 SiRNA, and the results showed that interfering the expression of Nrf-2 could eliminate the regulatory effect of L-BAIBA on GPX4 and SLC7A11 expression (Figure 5E). These evidence strongly supports that L-BAIBA plays an inhibitory role in ferroptosis by regulating Nrf-2 signaling pathway.

3.4. L-BAIBA, by activating AMPK, promoted Nrf-2 nuclear translocation.

We further studied the mechanisms underlying the regulation effect of L-BAIBA on the Nrf-2 signaling pathway. It is reported that some signaling pathways, such as AMPK, ERK, JNK, PI3K/Akt signaling pathways, could regulate the nuclear translocation of Nrf-2 (Huang et al. 2020^;Prieto et al. 2010^;Xingyue et al. 2021). We evaluated the effects of these signaling pathway by the administration of individual inhibitors, including compound c (10μm), PD 98059 (10μm), SP 600125 (20μm) and LY 294002 (25μm). Results showed that only AMPK inhibitor compound c blocked the promotion of L-BAIBA on the nuclear translocation of Nrf-2 (Figure 6A), and further experiments showed that L-BAIBA could promote the phosphorylation of AMPK at Thr172 in A549 cells (Figure 6B and Figure S3A). Meanwhile, in the presence of compound c, an AMPK inhibitor, the protective effect of L-BAIBA on A549 cell was blocked, which was confirmed by CCK8 and LDH experiments (Figure 6C and 6D). And the inhibitory effect of compound c on Nrf-2 nuclear translocation induced by L-BAIBA was also confirmed by immunofluorescence staining (Figure 6E). Compound c also abolished the protective effect of L-BAIBA on A/ R-induced oxidative stress in vitro (Figure 6F). The results presented in this section confirm that L-BAIBA promotes Nrf-2 signaling by activating AMPK.

3.5. The protective effect of L-BAIBA on lung I/R injury was abolished by blocking AMPK signaling pathway.

Our results showed that in the presence of compound c (10mg/kg/day i.v.), the protective effects of L-BAIBA on lung I/R injury was eliminated. After treatment with compund c, the pathological damage of lung tissue was significantly aggravated compared with L-BAIBA group (Figure 7A). In addition, the ratio of wet weight to dry weight in lung showed that the edema of lung was significantly aggravated after compound c treatment compared with L-BAIBA treatment group (Figure 7B). The arterial blood gas analysis also showed PaCO2 was increased while PaO2 was decreased in mouse treated by compound c during IR injury compared with L-BAIBA treatment group, which suggested that blocking AMPK signaling pathway eliminated the protective effect of L-BAIBA on lung function after I/R injury (Figure 7C and 7D). Meanwhile, inflammatory factors showed the same results (Figure 7E). Finally, the administration of compound c also eliminated the protective effect of L-BAIBA on oxidative damage of lung I/R injury, demonstrated by measurement of the level of DHE, MDA, SOD and the ratio of GSH and GSSG (Figure 7F-7I). These results further confirmed that AMPK signaling is essential for the protective effect of L-BAIBA on lung I/R injury.

Evidences presented in this study support the hypothesis that L-BAIBA preconditioning can inhibit ferroptosis by activating AMPK/Nrf-2 signaling pathway, thereby alleviate lung I/R injury. L-BAIBA preconditioning can reduce the excessive production of ROS and inhibit ferroptosis in lung tissue after I/R, thereby improve the lung function. The anti-ferroptosis effect of L-BAIBA is achieved by prompting the AMPK/Nrf-2 signaling pathway. Biochemical experimental studies have shown that L-BAIBA prompts the nuclear translocation of Nrf-2 by activating the AMPK signaling pathway, thereby the expression of GPX4 and SLC7A11 is up-regulated and ferroptosis is inhibited. Based on these data, we conclude that L-BAIBA helps protect lung from ferroptosis and improve lung function during I/R injury via AMPK/Nrf-2 signaling.

Organ ischemia-reperfusion injury is thought to be accompanied by changed metabolism of BCAA, and enhancing BCAA metabolism can protect against ischemia-reperfusion injury (Lian et al. 2020). We hypothesized that it might be related to the increase of BCCA active metabolites. L-BAIBA is a non-protein β amino acid produced in skeletal muscle after the metabolization of L-valine, a member of BCAA, which is also regarded as an important sports muscle-derived metabolic factor (Kammoun & Febbraio 2014). Previous studies have found that L-BAIBA increases energy consumption by activating the β-oxidation pathway of liver fatty acids and triggers the browning of white adipose tissue (Roberts et al. 2014). L-BAIBA also improves insulin resistance and regulates glucose and lipid metabolism disorders through AMPK signaling (Jung et al. 2015). Because of these characteristics, L-BAIBA has become an attractive drug in the therapy of metabolic diseases. Meanwhile, L-BAIBA has also been found to protect against multiple organ damage, which rely on the effects of anti-oxidative stress, anti-inflammatory, and alleviating endoplasmic reticulum stress. Meanwhile, the concentration of L-BAIBA in circulation is negatively related to cardio-metabolic risk factors (Kitase et al. 2018^; Roberts et al. 2014^; Shi et al. 2016). In addition, it is worthwhile noting that L-BAIBA has excellent pharmacological properties, such as stable property and convenient to store. These evidences suggest that L-BAIBA may be an excellent potential therapeutic agent, and due to its potential medicinal value, there have been toxicological and pharmacokinetic studies on L-BAIBA (Krieger et al. 2022^; Shanmugasundaram et al. 2022).

In our study, compared with the lung I/R injury group, preconditioning mouse with L-BAIBA can alleviate pathological injury and improve lung function, manifested by alleviated tissue septal edema, reduction of inflammatory cell infiltration and alveolar exudation, as well as the improvement of gas exchange function. In addition, L-BAIBA also reduces the level of cell apoptosis in lung tissue and the level of serum inflammatory factors such as MPO, IL-1β, IL-6 and TNF-α. These results further confirm the protective effect of L-BAIBA on ischemia/reperfusion injury.

Excessive oxidative stress can directly lead to lung cell cytotoxic damage by impairing mitochondrial function or aggravating the inflammatory response (Orrenius, Gogvadze & Zhivotovsky 2007). Ferroptosis is a type of cell death caused by oxidative stress, and acts as a hub linking oxidative stress, inflammation, and cell death (Yu et al. 2021). Intracellular iron ions interact with fatty acids to produce lipid reactive oxygen species. When the intracellular glutathione (GSH) antioxidant system is not detoxicated in time, lipid peroxidation and ferroptosis will occur. Therefore, dysfunction of lipid peroxide clearance, the presence of redox-active Fe²⁺, and the oxidation of phospholipids containing polyunsaturated fatty acids are considered to be the three essential features of ferroptosis (Yu et al. 2021). Ferroptosis is thought to play an important role in lung ischemia-reperfusion injury (Ferrari & Andrade 2015). Recent studies have found that ferroptosis is also closely related to the occurrence and development of many lung diseases (Li, Yang & Yang 2022^; Yu et al. 2021). There is no doubt that inhibition of oxidative stress and ferroptosis is an effective strategy to prevent lung I/R injury.

In this study, increased MDA level and decreased of SOD activities, accompanied with the unbalanced ratio of GSH and GSSG are found in the lung injured by I/R. However, pretreating with L-BAIBA reduces oxidative stress in lung tissue, inhibits the increase of intracellular Fe²⁺ levels and restores impaired expression of GPX4 and SLC7A11, which confirm the anti-oxidant and anti-ferroptosis effects of L-BAIBA in lung I/R injury.

Further experiments show transcription factor Nrf-2 is involved in the protective effects of L-BAIBA. Nrf-2 regulates the expression of many genes involved in organ and cell defense, including antioxidant proteins, detoxification enzymes, drug transporters and so on. Transcription of these protective genes enables cells to maintain redox equilibrium and eliminate proteins damaged by oxidative stress. Nrf-2 signaling pathway has also been shown to have a strong anti-ferroptosis effect (Wang et al. 2022^; Zhang et al. 2022). Mounting evidence have shown that the up-regulation of Nrf-2 can protect various organs from ischemia-reperfusion injury (Ucar et al. 2021). In this study, we find that L-BAIBA prompts the nuclear translocation of Nrf-2. Inhibition of Nrf-2 can eliminate the protective effects of L-BAIBA on cell activity and oxidative stress. The up-regulation of L-BAIBA on the expression of GPX4 and SLC7A11 is also eliminated by interfering Nrf-2. These evidences further confirm the contribution of Nrf-2 in the L-BAIBA-mediated protection of lung injury.

AMPK signaling pathway is an important regulator in controlling energy metabolism, and has been shown to play a protective role against I/R injury (Ding et al. 2020). AMPK signaling also acts as a regulator of intracellular redox equilibrium, and increasing evidence have also shown that the activation of AMPK can prompt the transposition of Nrf-2 into the nucleus (Wang et al. 2019^; Wang et al. 2022^; Zhang et al. 2022). In our study, by screening a variety of signaling pathways which have been reported to regulate Nrf-2 signaling pathway, we find that only AMPK inhibitor can eliminate the regulatory role of L-BAIBA on the nuclear translocation of Nrf-2. In order to further clarify its clinical significance, we treat the I/R injured mouse with both compound c and L-BAIBA, and found that compound c eliminated the protective effect of L-BAIBA. Therefore, these evidences indicate that the preventive effect of L-BAIBA on lung I/R injuryI depends on the AMPK signaling pathway.

Results presented in this study show that L-BAIBA exerts a protective effect on lung I/R injury by inhibiting ferroptosis, which depended on the up-regulation of the expression of GPX4 and SLC7A11 by activation of AMPK/Nrf-2 signaling pathway. L-BAIBA treatment may be an promising strategy for the prevention and treatment of lung I/R injury.

L-BAIBA: L-β-aminoisobutyric Acid

BCAA: Branched Chain Amino Acid

GPX4: Glutathione Peroxidase 4

SLC7A11: Solute Carrier Family 7, Member 11

Nrf-2: Nuclear factor E2 related factor 2

AMPK: AMP-activated Protein Kinase

I/R: Ischemia-reperfusion

ARDS: Acute Respiratory Distress Syndrome

ROS: Reactive Oxygen Species

ELISA: Enzyme-Linked Immunosorbent Assay

IL-1β: Interleukin 1β

IL-6: Interleukin 6

TNF-α: Tumor Necrosis Factor α

MDA: Myeloperoxidase

GSH: Reduced Glutathione

GSSG: Oxidized Glutathione Disulfide

MPO: Myeloid Peroxidase

SOD: Superoxide Dismutase

DHE: Dihydroethidium

PBS: Phosphate Buffered Saline

BSA : Bovine Serum Albumin

RIPA: Radio Immunoprecipitation Assay

SDS-PAGE: Dodecyl Sulfate,Sodium Salt - Polyacrylamide Gel Electrophoresis

NC: Nitrocellulose Filter

BAP1: BRCA1 Associated Protein 1

PARP1: Poly (ADP-ribose) polymerase 1

ATF3: Activating Transcription Factor 3

A/R: Anoxia/Reoxygenation

FBS: Fetal Bovine Serum

ARE: Antioxidant Response Element

CCK8: Cell Counting Kit-8

LDH: Lactate Dehydrogenase

ERK: Extracellular Signal-regulated Kinase

JNK: c-Jun N-terminal kinase

PI3K/AKT: Phosphatidylinositol 3 Kinase /Protein KinaseB

SiRNA: SmallinterferingRNA

Ethics approval and consent to participate

All animal experiments are conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85–23, revised 1996) and approved by the animal care and use committee of Army Medical University (AMUWEC20201184).

Consent for publication

Not applicable

Availability of data and materials

The research article data used to support the findings of this study are included within the article. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that there is no conflict of interest regarding the publication of this paper.

Funding

This work was supported by the National Natural Science Foundation of China (81930008, 31730043, 82000476), Program of Innovative Research Team by National Natural Science Foundation (81721001), and the National Key R&D Program of China (2018YFC1312700); Clinical Technology Innovation and Cultivation Program of AMU (CX2019JS220).

Authors’ Contributions

ZZ, XBL contribute to this article equally. ZZ and XBL contributed to the conception or design of the work, the acquisition, analysis, or interpretation of data for this work, meanwhile, drafted this work and revised it; JG, BH, LW, RY and XYL contributed to the acquisition, analysis, or interpretation of data for this work. DF, XY and DY contributed to the conception or design of the work. MT contributed to revise the manuscript. YH contributed to the conception or design of the work, revised the manuscript, supervised the implementation of experiments and final approval of the version to be published. HW and CZ agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgments

Not applicable

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Graphical Abstract: L-BAIBA up-regulate the expression of GPX4/SLC7A11 by promoting Nrf-2 nuclear translocation via AMPK signaling pathway, thereby protecting against I/R-induced ferroptosis in lung tissues.
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β-aminoisobutyric Acid, a Metabolite of BCAA, Activates the AMPK/Nrf-2 Pathway to Prevent Ferroptosis and Ameliorates Lung Ischemia-Reperfusion Injury

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2. Material And Methods

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Abbreviations

BAP1: BRCA1 Associated Protein 1

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