LCN2 attenuates sepsis-induced liver injury by alleviating PTGS2-mediated Ferroptosis

doi:10.21203/rs.3.rs-3754334/v1

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LCN2 attenuates sepsis-induced liver injury by alleviating PTGS2-mediated Ferroptosis

https://doi.org/10.21203/rs.3.rs-3754334/v1

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Sepsis, which is strongly linked to sepsis-induced liver injury, is a significant contributor to mortality in critical care units. In this study, we explore the role of lipocalin 2 (LCN2) in sepsis-induced liver injury. We observed increased expression of LCN2 in the livers of septic mice induced by caecal ligation and puncture (CLP), as well as in hepatocytes treated with lipopolysaccharide (LPS). To elucidate the function of LCN2 in sepsis-induced liver injury, we established septic mice with LCN2 knockdown. To evaluate liver injury in mice, levels of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were measured in both serum and liver samples. Oxidative stress was evaluated by measuring the levels of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione (GSH) in serum and liver samples. Additionally, ferroptosis was assessed by examining the expression of Prostaglandin-Endoperoxide Synthase 2 (Ptgs2), solute carrier family 7 member 11 (Slc7a11) and glutathione peroxidase 4 (Gpx4) in the liver tissue of the mice. Our findings demonstrated that LCN2 knockdown significantly exacerbated sepsis-induced liver injury, oxidative stress, and ferroptosis. Moreover, in an in vitro study, LCN2 overexpression notably ameliorated LPS-induced liver injury, oxidative stress, and ferroptosis in hepatocytes by inhibiting PTGS2 expression. In conclusion, our study provides evidence that LCN2 attenuates sepsis-induced liver injury by alleviating PTGS2-mediated ferroptosis.

Health sciences/Biomarkers

Health sciences/Medical research

Health sciences/Pathogenesis

sepsis

liver injury

ferroptosis

LCN2

Sepsis is widely known as the main cause of death in critical care units, and its survivors often face more severe pathology and higher readmission rates compared to those without sepsis^1,2. Multiple organs, including the liver, brain, heart, and lung, can be affected by sepsis^2–4. Notably, liver injury has emerged as an early prognostic indicator of poor outcomes in sepsis patients, and interventions aimed at restoring liver function have shown promise in improving prognosis^5,6. However, the precise mechanism of sepsis-induced liver injury remains unclear.

Ferroptosis is a type of regulated cell death that has recently gained interest in scientific research. It is distinguished by the buildup of reactive oxygen species (ROS) and free iron within cells⁷. It has been established that iron metabolism and oxidative stress-related processes contribute significantly to the development of ferroptosis^7,8. Key processes involved in ferroptosis include the accumulation of free iron, depletion of GSH, and the buildup of lipid oxidative damage leading to cell membrane degeneration^8,9. Interruption of these processes has been shown to impede the development of ferroptosis. Several genes, such as Slc7a11 and Gpx4, act as negative regulators of ferroptosis by inhibiting ROS production^10,11. Conversely, positive regulators like Ptgs2 and Tfr1 promote ROS production, thereby enhancing ferroptosis¹⁰. Notably, ferroptosis has been associated with several disease-related cell death events, such as liver damage.

In this study, we explored the involvement of LCN2 in sepsis-induced liver injury. Our findings revealed a significant increase in the expression of LCN2 in both sepsis-induced liver tissues and hepatocytes treated with LPS. Interestingly, LCN2 knockdown exacerbated sepsis-induced liver injury, oxidative stress, and ferroptosis. Further analysis revealed that LCN2 overexpression suppressed hepatocyte ferroptosis in sepsis by inhibiting Ptgs2 expression. These findings highlight the protective role of LCN2 in sepsis-induced liver injury by mitigating oxidative stress and hepatocyte ferroptosis. Collectively, our data provides valuable insights into the intricate mechanisms underlying sepsis-related liver damage and suggests LCN2 may be a promising target for the treatment of liver injury caused by sepsis

Animal

We obtained 8-week-old male C57 BL/6 mice from Caygen (Suzhou, China) and kept them in a controlled environment with a temperature of 24°C ± 2°C, 50% relative humidity, and a 12-hour light/dark cycle. The mice had free access to food and water. All procedures involving animals were performed in compliance with the guidelines set forth by the Experimental Animal Welfare and Ethics Committee of Shanghai University of traditional Chinese Medicine in accordance with ARRIVE guidelines and conducted according to the AAALAC and the IACUC guidelines.

Cecal ligation and puncture (CLP) Model

We induced sepsis in mice using the CLP technique, as described in previous studies ¹². In brief, the mice were anesthetized using sevoflurane and a 2-cm incision was made along the midline of the abdomen. The cecum was then ligated halfway along its distal end after moving the stool towards the tip. Bacterial peritonitis was induced by puncturing the cecum with a 22-gauge needle.

LCN2 knockdown

Recombinant adenoviruses expressing shRNA targeting LCN2 under the mouse albumin promoter (shLCN2) were purchased from Genechem Co., LTD (Shanghai, China). During this experiment, a solution containing viruses was prepared by diluting them in PBS. Each mouse then received an injection of the viral solution into the tail vein at a concentration of 2 × 10⁹ plaque-forming units. On the 21st day following viral injection, the CLP procedure was performed. Liver and serum samples were collected 16 hours after CLP-induced sepsis for further analysis.

Primary hepatocyte isolation and culture

Hepatocytes were extracted from 8-week-old mice according to previously established protocols. Briefly, hepatocytes were extracted by digestion with type II collagenase (Gibco™, 17101015), and subsequent centrifugation through a 25–50% Percoll gradient. The isolated primary hepatocytes were then grown in DMEM culture medium enriched with 10% FBS and 0.01 mM dexamethasone.

LCN2 overexpression in hepatocytes

To overexpress LCN2, primary hepatocytes were infected with ad-LCN2 obtained from Shanghai Genechem Co., LTD (Shanghai, China), and transfection was performed according to the manufacturer's instructions.

Cell septic model

The septic model in hepatocytes was induced by LPS treatment as previously described. Hepatocytes were stimulated with LPS (100 ng/mL) for 24 hours.

Immunohistochemistry (IHC) Staining

Mouse liver tissues were collected after perfusion with cold PBS and fixed with 10% formalin. For IHC staining, the liver tissue sections were incubated overnight at 4°C with primary antibodies against LCN2. Then the tissue sections were treated with a secondary antibody conjugated to horseradish peroxidase (HRP) and incubated at room temperature.

Western-blotting analysis

Liver tissues and hepatocytes were lysed in RIPA assay buffer (Invitrogen, USA) supplemented with a protease inhibitor cocktail (Invitrogen, USA). Protein concentration was determined using a BCA kit (Invitrogen, USA). Western-blotting was performed as previously described. PVDF membranes were incubated with primary antibodies against LCN2 (1:1000, ab125075, Abcam), PTGS2 (1:1000, ab179800, Abcam), GPX4 (1:1000, ab125066, Abcam), SLC7A11 (1:1000, ab175186, Abcam), and beta-actin (1:5000, ab8226, Abcam) overnight at 4°C. The membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:5000) at room temperature.

Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from liver tissues and hepatocytes using Trizol reagent (Invitrogen, USA). Reverse transcription PCR was performed using the Super-SMART-PCR-cDNA-Synthesis Kits (TaKaRa, JPN). qPCR was conducted on an ABI-7500 Real-Time PCR System (Applied Biosystems, USA) using PowerUp SYBR Green Mix (Invitrogen, USA). The fold changes of RNA transcripts were determined using the 2^−ΔΔCt method, with 18s as the reference gene.

Liver function analysis

The levels of GSH, SOD, ROS, ALT, ALP, and AST were analyzed using biochemical kits from Wuhan Elabscience Biotechnology Co., Ltd.

Statistical analysis

All statistical analyses were performed using SPSS 21 (IBM, NY, USA). Data are presented as mean ± standard deviation (SD) from three independent experiments. The significance between two groups was assessed using Student's t-test, and the significance between more than two groups was determined using one-way ANOVA followed by the Tukey-Kramer multiple comparisons test. A p-value less than 0.05 was considered statistically significant.

LCN2 was upregulated in the livers of CLP-induced mice and in LPS-induced hepatocytes.

To verify the key genes associated with the sepsis-induced liver injury, four Gene Expression Omnibus (GEO) datasets (GSE6008, GSE92703, GSE26472, and GSE71530) were obtained, and the differentially expressed genes (DEGs) were selected by LogFC > 1 and P ༜0.05, the common genes were obtained by Venn diagram. Figure 1A showed that a total of ten common DEGs (Lcn2, Apcs, Orm2, Saa1, Saa2, Icam1, Cpne8, S100a9, S100a8, and Socs3) were found. Since LCN2 was the most significantly different gene and was reported to be associated with liver injury in alcoholic hepatitis¹³ and non-alcoholic steatohepatitis¹⁴. Therefore, LCN2 was selected to investigate whether it is involved in sepsis-induced liver injury. Firstly, LCN2 expression was assessed in CLP-induced mice by qRT-PCR. Figure 1B showed that the LCN2 mRNA level was markedly increased in the liver of septic mice compared with control mice. Moreover, LCN2 protein level was analyzed by western-blotting and IHC (Figs. 1C-F). Consistent with the qRT-PCR results, LCN2 protein expression was significantly increased in the liver of septic mice compared with control mice. In addition, the expression of LCN2 was also examined in the LPS-induced sepsis cell model. We found that LCN2 was also significantly increased in the LPS-induced sepsis cell model (Figs. 1G-I). Our data suggest that LCN2 may contribute to sepsis-induced liver injury.

LCN2 knockdown promoted liver injury in CLP-induced mice.

To explore the function of LCN2 in CLP-induced liver injury, we established a septic mouse model with or without LCN2 knockdown. LCN2 expression was successfully reduced using siRNA, as shown in Figs. 2A-C. To assess liver injury, we performed hematoxylin and eosin (HE) staining on liver tissues. As depicted in Fig. 2D, the septic mouse liver exhibited disrupted hepatic local inflammation, indicating liver injury. Surprisingly, LCN2 knockdown exacerbated the liver injury induced by CLP, as evidenced by more severe alterations in the hepatic lobules and cords. Furthermore, we evaluated the levels of liver-specific enzymes, such as AST, ALT, and ALP, in the blood and liver to further confirm liver damage. The levels of AST, ALT, and ALP were significantly elevated in the serum and liver of septic mice compared to the control group (Figs. 2E-J). Notably, LCN2 knockdown further increased the levels of AST, ALT, and ALP in the serum and liver of septic mice, indicating worsened liver injury in the absence of LCN2. Taken together, our data suggest that LCN2 plays a protective role in sepsis-induced liver injury.

LCN2 knockdown deteriorated oxidative stress in CLP-induced mice.

It is well-known that oxidative stress and impaired antioxidant system function contribute to liver injury¹⁵, and LCN2 has been suggested to play a role in modulating oxidative stress levels ¹⁶. Based on this understanding, we propose the hypothesis that LCN2 may have a beneficial effect on septic liver injury through its antioxidant mechanisms. To explore this further, we examined the levels of key antioxidant defense markers, including SOD and GSH-Px, as well as the oxidative stress marker MDA, in the serum and liver of septic mice with or without LCN2 knockdown. As depicted in Figs. 3A-F, we observed a significant reduction in the activities of GSH and SOD, coupled with a substantial increase in MDA levels, in the serum and liver of septic mice. Remarkably, LCN2 knockdown exacerbated the CLP-induced oxidative stress in mice. These findings provide additional evidence supporting the protective role of LCN2 in sepsis-induced liver injury.

LCN2 knockdown promoted CLP-induced hepatocyte ferroptosis.

Oxidative stress is a well-known contributor to the development of ferroptosis, and LCN2 has been implicated in the promotion of ferroptosis in various diseases¹⁷. To this end, we investigated the presence of ferroptosis in the liver of septic mice with or without LCN2 knockdown. Figure 4A showed that the iron content in the liver of septic mice was significantly increased, indicating the occurrence of ferroptosis. Interestingly, LCN2 knockdown further elevated the iron content in the liver of septic mice, suggesting an exacerbation of ferroptosis. To gain further insights into the molecular mechanisms involved, we assessed the expression of ferroptosis-related genes, namely PTGS2, GPX4, and SLC7A11, in the liver of septic mice with or without LCN2 knockdown by qRT-PCR and western blotting assay (Figs. 4B-F). The results demonstrated a marked decrease in the expression of GPX4 and SLC7A11, while PTGS2 expression was significantly increased in the liver of septic mice. These findings suggest that sepsis induces hepatocyte ferroptosis. Notably, LCN2 knockdown exacerbated hepatocyte ferroptosis by modulating the expression of PTGS2, but not GPX4 or SLC7A11. Taken together, our data indicate that LCN2 knockdown aggravates sepsis-induced liver injury by enhancing hepatocyte ferroptosis, possibly by regulating PTGS2 expression.

LCN2 overexpression suppressed LPS-induced oxidative stress, and ferroptosis of the hepatocyte by inhibiting PTGS2 expression.

To investigate the protective function of LCN2 in sepsis-induced liver injury, an in vitro sepsis cell model was established by stimulating hepatocytes with LPS, with or without LCN2 overexpression (LCN2-OE). We confirmed the successful transfection of LCN2 through qRT-PCR and western blotting (Figs. 5A-C). To assess the protective role of LCN2 in liver injury, we measured the levels of AST and ALT in the medium of hepatocytes. Figures 5D and 5E demonstrated that LPS stimulation markedly increased the levels of AST and ALT, suggesting liver damage. However, when LCN2 was overexpressed, the effect of LPS on ALT and AST leakage from hepatocytes was restored, suggesting that LCN2-OE inhibited the LPS-induced liver injury. Furthermore, we investigated whether LCN2 plays a protective role in sepsis-induced liver damage by regulating oxidative stress and ferroptosis. Similarly, Figs. 5F-H revealed that LPS stimulation resulted in a significant increase in ROS levels and MDA content, indicating oxidative stress in hepatocytes. However, when LCN2 was overexpressed, these effects were attenuated, with decreased ROS levels and MDA content, and increased GSH activity. These findings suggest that LCN2 plays a role in suppressing oxidative stress in hepatocytes during sepsis-induced liver injury. Moreover, Figs. 5I-K demonstrated that LPS stimulation increased the expression of PTGS2, a marker of ferroptosis, in hepatocytes. However, LCN2 overexpression blocked this increase in PTGS2 expression. These results suggest that LCN2 may also play a role in inhibiting ferroptosis in hepatocytes during sepsis-induced liver injury. In summary, our findings indicate that LCN2 exerts a protective function in sepsis-induced liver injury by suppressing oxidative stress and ferroptosis in hepatocytes.

Liver injury in sepsis is regarded as a major complication that contributes to the severity of the sepsis and limits positive outcomes. However, the underlying mechanism of sepsis-induced liver injury is still unclear. In the current study, we demonstrated that LCN2 attenuates sepsis-induced liver injury by alleviating oxidative stress-induced ferroptosis, as evidenced by: 1) LCN2 was upregulated in the livers of CLP-induced mice and in LPS-induced hepatocytes. 2) LCN2 knockdown promoted liver injury in CLP-induced mice. 3) LCN2 knockdown deteriorated oxidative stress in CLP-induced mice. 4) LCN2 knockdown promoted CLP-induced hepatocyte ferroptosis. 5) LCN2 overexpression suppressed LPS-induced oxidative stress and ferroptosis of hepatocytes by inhibiting PTGS2 expression.

LCN2 is a member of the lipocalin family, a group of secreted proteins that act as transporters for small lipophilic molecules, including iron and lipopolysaccharides, within the bloodstream ¹⁸. Previous studies have confirmed that LCN2 is strongly activated in inflamed organs such as the liver, spleen, kidney, lungs, and heart¹⁹. In addition, the expression of LCN2 has been confirmed to be upregulated during bacterial infection¹⁹. Yvonne R. Chan et al. reported that LCN2 is a crucial component of mucosal immune defense against pulmonary infection with K. pneumonia ²⁰. Ming-Jiang Xu et al. reported that the hepatocyte-derived LCN2 suppresses bacterial infection and accelerates the regeneration of the liver after partial hepatectomy²¹. According to a study conducted by Hongjie Li et al., hepatocytes secrete extracellular LCN2, which serves as a protective mechanism against systemic bacterial infection. Meanwhile, neutrophils transport LCN2 to the site of infection, where it contributes to local defense against bacterial infection through the formation of neutrophil extracellular traps ²². These studies support the notion that LCN2 plays a crucial role in mucosal immune defense against bacterial infections, liver regeneration after hepatectomy, and limiting systemic bacterial infections. Consistent with previous research, our study revealed a significant upregulation of LCN2 in septic liver injury models, both in vitro and in vivo. Furthermore, recent studies have shown that LCN2 levels in urine and plasma can serve as independent predictors of acute-on-chronic liver failure, which is associated with liver failure and systemic inflammation. Additionally, LCN2-deficient mice exhibited more severe liver damage when exposed to short-term applications of LPS, CCl4, and Concanavalin A, further supporting the hepatoprotective function of LCN2 ²³. Another study indicated that LCN2 may have a protective effect on the liver in the context of a fructose diet, as it coincided with reduced markers of oxidative stress and mitochondrial dysfunction ²⁴. All these studies supported the hypothesis that LCN2 has a hepatoprotective function in liver damage. In line with these studies, we found that LCN2 was upregulated in the liver of sepsis-induced mice and in the LPS-treated hepatocytes, and LCN2 knockdown exacerbated the liver injury in CLP-induced septic mice, suggesting LCN2 has a protective function in sepsis-induced liver injury. The precise mechanisms underlying the protective effects of LCN2 are currently not fully understood and require further investigation. In recent years, numerous studies have implicated LCN2 in various diseases by regulating ferroptosis, a kind of iron-dependent non-apoptotic cell death characterized by the accumulation of ROS. Indeed, we found that LCN2 knockdown significantly increased oxidative stress, a core inducer of ferroptosis, in the liver of CLP-induced septic mice, suggesting that LCN2 may improve liver injury by inhibiting ferroptosis.

Ferroptosis is a kind of iron-dependent form of non-apoptotic cell death that is characterized by the accumulation of toxic lipid ROS. Recently, ferroptosis has gained more and more attention in the field of liver dysfunction, as the accumulation of ROS and excessive iron contribute to many severe liver illnesses. Ferroptosis appears to be a two-edged sword in the occurrence and development of liver injury. Aimin Wu et al. found that FGF 21 attenuates iron overload-induced liver injury and fibrosis by inhibiting ferroptosis ²⁵. Zili Zhang et al. reported that ELAVL1 regulates hepatic stellate cell ferroptosis involved in the development of liver fibrosis ²⁶. Therefore, ferroptosis could promote the development of liver fibrosis. On the contrary, some studies have reported that ferroptosis can be considered as a new target to inhibit the activation of HSCs and alleviate the fibrosis of the liver. Miao Sui et al. found that magnesium isoglycyrrhizinate treatment obviously induced HSC ferroptosis by accelerating the accumulation of lipid peroxides and iron, whereas inhibition of ferroptosis by the specific inhibitor ferrostatin-1 (Fer-1) completely abolished the anti-fibrosis function of MgIG ²⁷. In conclusion, our study provides evidence that LCN2 attenuates sepsis-induced liver injury by alleviating oxidative stress-induced ferroptosis. The upregulation of LCN2 in septic liver injury, its protective role demonstrated through knockdown and overexpression experiments, and the modulation of oxidative stress and ferroptosis support the hypothesis that LCN2 plays a crucial hepatoprotective function in sepsis-induced liver injury.

Funding statement

This work was supported by the Medical Innovation Research Special Project of Shanghai Science and Technology Commission (grant number 21Y11920500);the Three-year Action Plan of Shanghai to Further Accelerate the Inheritance, Innovation and Development of Traditional Chinese Medicine (ZY [grant number 2021-2023)-0101-02].

Data Availability statement

All relevant data supporting the conclusions of this article is included within the manuscript.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Ethics approval and consent to participate

Animal experimental procedures were approved by Experimental Animal Welfare and Ethics Committee of Shanghai University of traditional Chinese Medicine. The animal protocols were carried out in strict accordance with the National Guidelines for Animal Usage in Research (China) in accordance with ARRIVE guidelines.

Acknowledgements

Not Applicable.

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No competing interests reported.

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LCN2 attenuates sepsis-induced liver injury by alleviating PTGS2-mediated Ferroptosis

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Abstract

Figures

Introduction

Materials and Methods

Animal

Cecal ligation and puncture (CLP) Model

LCN2 knockdown

Primary hepatocyte isolation and culture

LCN2 overexpression in hepatocytes

Cell septic model

Immunohistochemistry (IHC) Staining

Western-blotting analysis

Quantitative real-time PCR (qRT-PCR)

Liver function analysis

Statistical analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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