The effect of chronic intermittent hypobaric hypoxia improving liver damage in metabolic syndrome rats through ferritinophagy

doi:10.21203/rs.3.rs-2952446/v1

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Research Article

The effect of chronic intermittent hypobaric hypoxia improving liver damage in metabolic syndrome rats through ferritinophagy

https://doi.org/10.21203/rs.3.rs-2952446/v1

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Journal Publication

published 25 Sep, 2023

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Background and aim

Studies have confirmed that hepatic iron overload is one of the important factors causing liver damage in metabolic syndrome (MS). As a special form of autophagy, ferritinophagy is involved in the regulation of iron metabolism. Our previous studies have shown that chronic intermittent hypobaric hypoxia (CIHH) can improve the iron metabolism disorder. The aim of this study was to investigate how CIHH improves liver damage through ferritinophagy in rats with MS.

Methods

Male Sprague-Dawley rats aged 8–10 weeks were randomly divided into four groups: control (CON), CIHH (exposed to hypoxia at simulated altitude of 5000 meters for 28 days, 6 hours daily), MS model (induced by 16-week high fat diet and 10% fructose water feeding) and MS + CIHH (exposed to CIHH after 16-week MS inducement) groups. Liver index, liver function, iron content, tissue morphology, ferritinophagy, ferroptosis and iron metabolism related protein expression were measured, and the ferritinophagy flux in liver was further analyzed.

Results

Compared with CON rats, MS rats had increased liver index, damaged liver tissue morphology and function, increased total iron and free iron content, disrupted iron metabolism, significantly increased oxidative stress indicators in the liver, significantly increased expression of ferroptosis-related proteins, reduced expression of nuclear receptor coactivator 4 (NCOA4) and ferritinophagy flux. After CIHH treatment, the degree of liver damage and various abnormal indicators in MS rats were significantly improved.

Conclusions

CIHH may improve liver damage by promoting NCOA4-mediated ferritinophagy, reducing iron overload, and alleviating ferroptosis in MS rats.

hypobaric hypoxia

metabolic syndrome

NCOA4

ferritinophagy

ferroptosis

Metabolic syndrome (MS) is a pathological condition, characterized by metabolic disorders of protein, fat, carbohydrate and other substances, often accompanied by a variety of complications such as non-alcoholic fatty liver disease (NAFLD), hypertension, atherosclerosis, coronary heart disease and stroke^{[1, 2]}. In recent years, with the rapid increase in the proportion of obese people worldwide, the incidence of MS has been increasing year by year, In China, the incidence of MS is approximately 25%^[3], and it has become one of the leading factors that harm human health^[4].

NAFLD is an abnormal liver manifestation in MS. A large number of epidemiological surveys and studies have found that excessive accumulation of iron is an important factor leading to NAFLD and even cirrhosis. Iron overload leads to hypertriglyceridemia, insulin resistance, and oxidative stress, thus damages the liver function and accelerates the development of MS, which is extremely adverse to the prognosis and outcome of the disease^[1].

Ferritinophagy, as a special form of autophagy^[5], mediates the degradation and release of iron ion from ferritin in autophagic lysosomes through nuclear receptor coactivator4(NCOA4), and participates in the regulation of iron metabolism homeostasis^[6]. Studies have found that the down-regulation of NCOA4 expression and inhibition of ferritinophagy lead to the accumulation of ferritin in tissues, iron overload^{[7, 8]} and oxidative stress, which resulting in the liver damage^[9]. The mechanism research of ferritinophagy on liver injury has attracted increasing attention.

The chronic intermittent hypobaric hypoxia (CIHH) treatment refers to a method in which animals or humans are intermittently exposed to a simulated plateau hypobaric hypoxia environment, while the rest of the time they are in a normal oxygen state. Previous studies have found that CIHH has beneficial effects on various tissues of the body^[10] and can prevent or treat certain diseases^{[11, 12]}. For example, it can improve the abnormal iron metabolism parameters by inhibiting the expression of hepcidin in the liverof obese rats^[13]; can also protect the liver of MS rats by activating autophagy^[14–16]. However, it is unclear whether CIHH participates in the improvement of liver injury by regulating ferritinophagy in MS rats.

This study was to investigate the effects and the mechanisms of CIHH improving the liver damage in high-fat-high-fructose inducing MS rats, and to provide a new theoretical basis for the prevention and treatment of liver injury in related diseases, which observe iron metabolism, ferritinophagy and ferroptosis through biochemical, morphological, immunological, molecular biological and other methods.

Animal treatments

All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals adopted and issued by the National Institutes of Health, and were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Hebei Medical University.Male Sprague Dawley rats aged 8–10 weeks (weight 80-120g, provided by Animal Center of Hebei Medical University) were randomly divided into four groups: control group (CON), metabolic syndrome model group (MS), chronic intermittent hypobaric hypoxia treatment group (CIHH), and MS with CIHH treatment group (MS + CIHH). According to our previous study^[14], CON and CIHH rats were fed with chow diet (containing 22% protein, 4% fat, and 50% carbohydrate; specific nutrient composition per 1000 g: 99.50 g water, 216.97 g protein, 50.38 g fat, 56.87 g coarse ash, 7724.00 g fiber, 13.29 g calcium, 9.17 g phosphorus) and drinking water, while MS and MS + CIHH rats were fed with high-fat diet and fructose water (containing 24% protein, 12% fat, and 42% carbohydrate; specific nutrient composition: 8% lard, 2% soy flour and 90% chow diet, and 10% (wt/vol) fructose in water), respectively. After feeding for 16 weeks with different diets, CIHH and MS + CIHH ratswere treated 6 h daily (from 9 a.m. to 3 p.m.) under hypobaric hypoxic conditions (PB = 404 mmHg, P_O2=84 mmHg) simulating an altitude of 5000 m, for 28 days in a hypobaric chamber, and the remainder of time were in a normoxic environment. CON and MS rats were always exposed to normoxic conditions. All animals were housed in a temperature-controlled room (22 ± 1°C) with a 12 h/12 h light/dark cycle, had ad libitum access to food and water, and diet was as usual. During the experiment, the weight and blood pressure of rats were measured regularly every week.

Serum biochemical assay

At the end of the experiments, rats fasted overnight and were euthanized with an intraperitoneally injected over-dose of pentobarbital sodium (50 mg/kg). Blood was taken from the right atrium and centrifuged (3500rpm, 10min) to obtain serum. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by automatic biochemical analyzer (Hitachi labospect 008, Japan).

Liver index

After intraperitoneal anesthesia and euthanasia by bleeding, liver tissue was quickly taken out, and liver index was calculated as liver weight /body weight×100%.

Haematoxylin-eosin (HE), Masson’s and Prussian blue staining

After perfusion with 4℃ saline in vivo through peristaltic pump, removing the blood, a small segment of tissue block (1 cm³) was fixed in 4% formaldehyde solution, dehydrated in an ethanol gradient, embedded in paraffin and cut down in slices of 4 µm. Subsequently, the tissue sections were deparaffinized and stained with HE, Masson, and Prussian blue stains, respectively. The slices were observed using a light microscope and captured with an attached camera (Leica DM6000, Germany).

Iron content in liver

20mg liver tissue was collected and got total iron content by using a high-resolution continuous source atomic absorption spectrometer (Analytik Jena AG, Germany).

200mg fresh liver tissue was taken to make cell suspension for detection of intracellular free iron level. Cell suspensions were stained with Calcein-AM and the Calcein-AM green fluorescence value was read by flow cytometry (BD FACSLyric clinical, America) according to the manufacturer's instructions.

Oxidative stress parameters

Liver tissue was lysed with Radio Immunoprecipitation Assay Lysis (RIPA ) buffer (Biyuntian Biotechnology, Shanghai, China). The supernatant (12000g, 10min) was centrifuged at 4˚C, and the protein concentration was determined using the BCA Protein assay kit. The levels of lipid peroxide (LPO), malonaldehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPX) in the liver were measured with respective kits (Jiancheng Bioengineering Institute, Nanjing, China).

Western blot analysis

The liver tissues were lysed with RIPA buffer (Biyuntian Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors for 30min, centrifuged at 12000g for 15min at 4℃, and protein concentration was determined by BCA protein detection kit. The sample (100µg) were separated by 10% SDS-PAGE electrophoresis and transferred to PVDF membranes and treated with 5% skim milk for at least 1h to block non-specific protein binding. The membranes were incubated with the primary antibody (including ferritin heavy chain1 (FTH1, 1: 1000), transferrin receptor (TfR, 1:1000), ferroportin1 (FPN1, 1:1000), NCOA4 (1:1000), xCT (1:1000), GPX4 (1:2000), transforming growth factor β1 (TGF-β1, 1:1000), α-smooth muscle actin (α-SMA, 1:1000), GAPDH (1: 10000), and β-actin (1:10000)) overnight at 4 ℃, then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The bands were visualized by chemiluminescence and protein levels were quantified by using the Image J software. Protein content and GAPDH or β-actin protein content was normalized on the same blot.

Immunofluorescence Co-localization Staining

Cell suspension was prepared from fresh liver tissue, infiltrated with Triton-X at room temperature for 30min, smeared on the slide, sealed with goat serum for 30min, further incubated with rabbit-derived NCOA4 antibody (1: 1000) and mouse-derived FTH1 antibody (1:1000) overnight at 4 ℃. Then the goat anti rabbit IgG coupled with IgG-488 (goat anti rabbit IgG Alexa Fluor 488, 1:1000) and goat anti mouse IgG coupled with IgG-Cy3 (Cy3-labeled goat anti mouse IgG, 1:1000) were incubated at room temperature for 1 hour and counterstained with DAPI (Solarbio, Beijing, China). Capturing and analyzing stained images with a fluorescence microscope (Leica DM6000, Munich, Germany).

Co-Immunoprecipitation

According to the instructions (Absin Bioscience Inc, Shanghai, China), the cells were lysed with the buffer solution containing protease inhibitors. Lysates were centrifuged at 14000g for 10 min at 4°C, and the supernatant was taken and incubated with protein A/G agarose beads to clarify the soluble fraction. The protein in the clarified supernatant was immunoprecipitated with the indicated primary antibody (rabbit-derived NCOA4 antibody or rabbit-derived FTH1 antibody) at 4°C, and then incubated with protein A/G beads at 4°C for 12 hours. The immunoprecipitated complex was washed and subjected to Western blotting. The supernatant without primary antibody was used as the control group (INPUT group).

Reagents and Antibodies

FTH1 antibody was purchased from Cell Signaling Technology (Danvers, MA, America); TfR, FPN1, and α-SMA antibodies were purchased from Novus (Saint Charles, MO, America); NCOA4 antibody was purchased from Biorbyt (Cambridge, England); xCT, GPX4 antibodies were purchased from Abcam (Cambridge, England); GAPDH, β-actin antibody was purchased from Abways Company (Shanghai, China); TGF-β1 antibody and Goat anti-Rabbit IgG-Alexa Fluor 488 were purchased from Aibixin Biotechnology (Shanghai, China); Cy3-labeled Goat anti-Mouse IgG was purchased from Shanghai Biyuntian Biotechnology (Shanghai, China).

Statistical Analysis

SPSS and GraphPad Prism 8 statistical software were used for data analysis and chart drawing in this experiment, and all data are expressed as mean ± standard error (mean ± SEM). Two-way analysis of variance (two-way ANOVA) was used to compare the data among the four groups. A value of P < 0.05 was considered statistically significant.

Effect of CIHH on liver index

Two-way analysis of variance showed that there was no interaction between MS and CIHH on liver index (F = 4.272, P > 0.05), so the main effect of liver index was analyzed. The results showed that compared with CON rats, the liver index of MS rats was significantly higher (P < 0.01); after CIHH treatment, the liver index of MS + CIHH rats was significantly lower compared with MS rats (P < 0.01, Fig. 1). The results indicated that CIHH treatment can improve liver hypertrophy in MS rats.

Effect of CIHH on liver histomorphology

The results of HE staining showed that the liver tissue of MS rats showed obvious vacuolar degeneration and cellular edema compared with CON rats; after CIHH treatment, the vacuolar degeneration and cellular edema in the liver tissue of MS + CIHH rats was reduced compared with MS rats (Fig. 2).

Masson staining showed that there were obvious blue collagen fibers in the liver tissue of MS rats compared with CON rats, and the blue collagen fibers in the liver tissue of MS + CIHH rats were reduced compared with MS rats (Fig. 2).

Prussian blue staining showed that compared with CON rats, deep blue particles were deposited in the liver tissue sections of MS rats; after CIHH treatment, dark blue particles in liver tissue sections of MS + CIHH rats decreased compared with MS rats (Fig. 2).

The results indicated that CIHH treatment could significantly improve the morphological damage of liver tissue, and reduce tissue fibrosis and iron deposition in MS rats.

Effects of CIHH on serum ALT and AST levels

Two-way ANOVA showed that there was an interaction between MS and CIHH on ALT and AST (F = 18.829, 84.153, P < 0.05), and separate effect analysis was performed. The results showed that the serum ALT and AST levels of MS rats were significantly higher than those of CON rats (P < 0.01); ALT and AST levels were significantly lower in MS + CIHH rats than those of MS rats (P < 0.01, Fig. 3). The results indicated that CIHH could significantly improve the damaged liver function in MS rats.

Effects of CIHH on liver iron content levels

Two-way ANOVA showed that there was an interaction between MS and CIHH on the total and intracellular free iron level in rat liver (F = 8.445, 10.206, P < 0.05), and separate effect analysis was performed. The intracellular free iron concentration was inversely proportional to the fluorescence value detected by flow cytometry^[17]. The results showed that total and intracellular free iron content in the liver of MS rats were significantly higher than those of CON rats (P < 0.01, Fig. 4); the total and intracellular free iron content in the liver of MS + CIHH rats was significantly lower than those of MS rats (P < 0.01, Fig. 4). The results indicated that CIHH could significantly reduce iron overload in the liver of MS rats

Effects of CIHH on hepatic oxidative stress

Two-way ANOVA showed that there was an interaction between MS and CIHH on LPO, MDA, SOD and total GPX in rat liver (F = 9.856, 39.776, 110.357, 189.496, P < 0.05), and separate effect analysis were performed. The results showed that compared with CON rats, the expressions levels of LPO and MDA in the liver of MS rats were significantly increased (P < 0.01, Fig. 5), while the expressions levels of total GPX and SOD were significantly decreased (P < 0.01, Fig. 5); after CIHH treatment, the expressions levels of LPO and MDA were significantly decreased (P < 0.01, Fig. 5), and the expressions levels of total GPX and SOD were significantly increased in MS + CIHH rats compared with those of MS rats (P < 0.01, Fig. 5). The results indicated that CIHH could significantly reduce oxidative stress in the liver of MS rats.

Effect of CIHH on ferroptosis-related protein expressions in the liver

Two-way analysis of variance showed that there was no interaction between MS and CIHH on xCT (F = 2.620, P > 0.05), so the main effects of xCT were analyzed. Two-way analysis of variance showed that there was an interaction between MS and CIHH on GPX4 (F = 5.477, P < 0.05), and separate effect analysis was performed. The results showed that compared with CON rats, the protein expressions of GPX4 and xCT in the liver of MS rats were significantly decreased (P < 0.05, Fig. 6); after CIHH treatment, the protein expressions of GPX4 and xCT in MS + CIHH rats were significantly increased compared with MS rats (P < 0.01, P < 0.05, Fig. 6). These results indicated that CIHH can effectively inhibit ferroptosis in the liver of MS rats.

Effect of CIHH on rat liver TGF-β1 and α-SMA protein expressions

Two-way ANOVA showed that there was an interaction between MS and CIHH on rat liver TGF-β1 (F = 6.873, P < 0.05), and separate effect analysis was performed. There was no interaction between MS and CIHH on α-SMA in rat liver (F = 0.831, P > 0.05), so the main effect of α-SMA was analyzed. Compared with CON rats, the protein expressions of TGF-β1 and α-SMA in the liver tissue of MS rats were significantly increased (P < 0.05); after CIHH treatment, the protein expression of TGF-β1 was significantly decreased in MS + CIHH rats compared with MS rats (P < 0.01, Fig. 7), and the expression of α-SMA protein tended to decrease although without significant statistical difference (P = 0.083, Fig. 7). The results indicated that CIHH could improve liver fibrosis in MS rats.

Effects of CIHH on protein expressions of liver iron metabolism and ferritinophagy flux

Two-way ANOVA showed that there was an interaction between MS and CIHH on protein expressions of TfR, FPN1 and FTH1 in rat liver (F = 7.71, 7.288, 12.611, P < 0.05). There was no interaction between MS and CIHH on NCOA4 in rat liver (F = 1.977, P > 0.05), so the main effect of NCOA4 was analyzed. The results showed that compared with CON rats, the protein expressions of FTH1 and TfR in the liver tissue of MS rats were significantly increased (P < 0.01, Fig. 8), and the expressions of FPN1 and NCOA4 were significantly decreased (P < 0.01, P < 0.05 Fig. 8); after CIHH treatment, all the indexes of MS + CIHH rats were significantly improved compared with MS rats (P < 0.05, Fig. 8).

Co-immunoprecipitation results and immunofluorescence showed that NCOA4 and FTH1 were co-localized and directly interacted with each other (Fig. 9, Figure S1). Further analysis showed that there was an interaction between MS and CIHH on ferritinophagy flux (NCOA4/FTH1)^[18] in rat liver tissue (F = 10.619, P < 0.05), and separate effect analysis was performed. The results showed that compared with CON rats, ferritinophagy flux in the liver tissue of MS rats was significantly decreased (P < 0.01), and after CIHH treatment, ferritinophagy flux in MS + CIHH rats was significantly increased compared with MS rats (P < 0.01, Fig. 9). These results indicated that CIHH can increase ferritinophagy flux in the liver of MS rats.

In this study, the MS rat exhibited increased liver index, impaired liver tissue morphology and function, liver fibrosis, iron overload, oxidative stress, increased ferroptosis levels, and lower level of NCOA4 and ferritinophagy flux. After CIHH treatment, the aforementioned deviations of MS rats were significantly reverted or improved. These results suggest that CIHH play a protective role in the impaired liver of MS rats, which may have occurred by up-regulating NCOA4, promoting ferritinophagy, thereby reducing iron overload, oxidative stress and ferroptosis.

Iron, as an important cofactor for metabolic enzymes, is an essential element for cell proliferation and growth, which is closely related to many biological processes such as neurotransmitter transmission, oxygen transport, cell division and energy generation^[19]. Iron is redox-active and involved in reactions with oxygen radicals. Iron deficiency is a common cause of anemia, while iron overload can lead to oxidative stress and tissue damage, associated with multiple diseases such as MS, NAFLD, heart failure, neurodegenerative diseases, and cancer^[20–22]. Therefore, maintaining iron homeostasis is crucial. NAFLD which is the liver manifestation of MS, is characterized by mild hepatic iron accumulation accompanied by moderate hyperferritinemia^{[2, 23]}. It is called dysmetabolic iron overload syndrome (DIOS), which occurs in 15% of patients with MS and half of patients with NAFLD^{[2, 23]}. It is known that excessive iron can stimulate the secretion of inflammatory factor TGF-β1, which promote the activation of hepatic stellate cells (HSC, α-SMA is the activation marker) as a pro-fibrotic cytokine^[24], cause ECM accumulation and collagen deposition, aggravate liver damage, and finally lead to liver fibrosis^{[25, 26]}, cirrhosis and hepatocellular carcinoma^{[27, 28]}.

A large amount of research has confirmed that CIHH has a protective effect on the body. For example, it can improve the abnormal iron metabolism parameters by inhibiting the expression of hepcidin in the liver of obese rats^[13]; can also protect the liver of MS rats by activating autophagy^[14–16]. In this study, we found that CIHH can reduce the total liver iron and labile iron content, significantly reduce the levels of ALT, AST and protein expressions of TGF-β1 and α-SMA in the liver of MS rats. This indicates that CIHH improves liver injury and liver fibrosis by reducing liver iron overload in MS rats.

Ferroptosis is a form of cell death caused by excessive iron ions, resulting in oxidative damage to cellular components and mitochondrial function, ultimately leading to apoptosis and cell death^{[29, 30]}. Its hallmark is an increase in lipid peroxides and a decrease in GPX4^[31]. It is a form of cell death that is iron-dependent and occurs through a non-apoptotic and non-autophagy-dependent mechanism. Glutathione (GSH) is a tripeptide synthesized from cysteine, glutamate, and glycine, whose uptake is regulated by the cysteine/glutamate antiporter system (system Xc-) containing a light chain subunit xCT (SLC7A11) and a heavy chain subunit CD98hc (SLC3A2), specifically by the light chain subunit xCT^[32]. A reduction in xCT activity inhibits the absorption of cysteine, leading to a decrease in GSH synthesis, which in turn reduces GPX4 activity and cellular antioxidant capacity. GPX4 is the core regulatory protein of ferroptosis, whose activity is maintained by oxidizing GSH to L-oxidized glutathione^[33]. Lipid peroxides and iron ions generate ROS through the Fenton reaction, leading to lipid peroxidation and tissue damage, triggering ferroptosis^[33–35]. Therefore, xCT plays a critical role in inhibiting oxidation reactions and maintaining cell survival under oxidative stress conditions. The levels of MDA and LPO, lipid metabolism products, reflect the extent of oxidative damage during oxidative stress^[31]. Superoxide dismutase (SOD) is an antioxidant metal enzyme that plays a key role in balancing oxidation and antioxidant processes by catalyzing the decomposition of superoxide anions into oxygen and hydrogen peroxide. Our research shows that CIHH treatment significantly reduces the levels of MDA and LPO in the livers of MS rats, increases the levels of total GPX, GPX4, SOD, and xCT, indicating that it may alleviate liver damage by reducing oxidative stress and inhibiting ferroptosis.

Liver is the main iron storage organ. Iron is imported into cells by transferrin combined with TfR. The unused iron is stored in the form of ferritin in hepatocytes and liver macrophages. Ferritin is made up of ferritin light chain (FTL) and FTH. FTH has iron oxidase activity, which converts Fe²⁺ into Fe³⁺ when iron enters the ferritin shell, while FTL promotes the formation of iron core storing Fe³⁺, and reduces the content of free iron^[27]. When iron is deficient, Fe³⁺ is released from ferritin and reduced to Fe²⁺ again, and then Fe²⁺ is exported to the extracellular space by FPN1^[36] for metabolism of other cells in the body. Therefore, ferritin plays an important role in iron homeostasis^[37] by storing and releasing iron, and its overload can aggravate oxidative stress and subsequent ferroptosis^{[38, 39]}, causing liver tissue injury.

It is known that there are two ironrelease pathways from ferritin: the ferritinophagy pathway and the ubiquitin-proteasome system pathway^[40]. Ferritinophagy is a kind of selective autophagy, which mediates the transfer of ferritin to lysosomes to release free iron for use through the interaction between autophagic substrate NCOA4 and FTH1^[6]. When the NCOA4 gene is knocked out or its content is lower, FTH1 is distributed more diffusely in the cell. Under basal and iron-depleted conditions, the lack of NCOA4 also prevents the co-localization of ferritin and lysosomes, resulting in a decrease in ferritin degradation and iron bioavailability, and the occurrence of ferroptosis ^{[7, 8]}. Our results showed that the protein expressions of TfR and FTH1 were up-regulated, while the protein expressions of FPN1 and NCOA4, and the ferritinophagy flux were significantly down-regulated in the liver of MS rats; CIHH treatment increased the binding of NCOA4 to FTH1 and ferritinophagy flux, promoted FTH1 degradation and iron release to other tissues. Therefore, it is reasonable to assume that CIHH improve liver damage by promoting ferritinophagy and reducing iron overload in MS rats.

In summary, this study investigated the molecular mechanism by which CIHH ameliorates liver injury in an animal model of MS induced by a high-fat and high-fructose diet. CIHH may have a protective effect on the injured liver by promoting NCOA4-mediated ferritinophagy to alleviate iron overload, oxidative stress, and ferroptosis (Fig. 10). These results further elucidate the mechanism of CIHH in liver injury and provide a new therapeutic approach for preventing and treating MS-induced liver injury.

Ethics approval

The study was approved by the Ethics committee of Hebei Medical University. No human subjects were involved in the study. All institutional and national guidelines for the care and use of laboratory animals were followed.

Consent to Participate

not applicable

Consent for Publication

All authors agree with the content of the manuscript, reviewed the manuscript, approved the final version and gave consent to submit this work.

Competing interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author’s Contributions

All authors contributed to the article and approved the submitted version; F.C and H.C.M performed the experiments and drafted the manuscript; R.T.W, A.J L and Y.T.D analyzed the data; F.L and S.Y.C prepared the images; Y.C.S took care of animals; M.S was responsible for conception and design of the research, revised and approved the final manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and /or publication of this article: Project H2020206396 supported by Hebei Natural Science Foundation and USIP2022103 supported by University Students' Innovative Experiment Program.

Availability of data and materials

All data that support the finding of the current study are available from corresponding author upon reasonable request.

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No competing interests reported.

fig.S1.tif
Supplementary Figure S1 The cellular distribution of endogenous NCOA4and FTH1.The red fluorescence represents NCOA4, and the green fluorescence represents FTH1, which are displayed in the same position after the merge.

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published 25 Sep, 2023

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Editorial decision: Major revision
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The effect of chronic intermittent hypobaric hypoxia improving liver damage in metabolic syndrome rats through ferritinophagy

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Journal Publication

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Animal treatments

Serum biochemical assay

Liver index

Haematoxylin-eosin (HE), Masson’s and Prussian blue staining

Iron content in liver

Oxidative stress parameters

Western blot analysis

Immunofluorescence Co-localization Staining

Co-Immunoprecipitation

Reagents and Antibodies

Statistical Analysis

RESULTS

Effect of CIHH on liver index

Effect of CIHH on liver histomorphology

Effects of CIHH on serum ALT and AST levels

Effects of CIHH on liver iron content levels

Effects of CIHH on hepatic oxidative stress

Effect of CIHH on ferroptosis-related protein expressions in the liver

Effect of CIHH on rat liver TGF-β1 and α-SMA protein expressions

Effects of CIHH on protein expressions of liver iron metabolism and ferritinophagy flux

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1