Curcumin Alleviates Hepatic Ischemia-Reperfusion Injury by Inhibiting Neutrophil Extracellular Traps Formation

Abstract

Background

Hepatic ischemia-reperfusion injury (IRI) is a common innate immune-mediated sterile inflammatory response in liver transplantation and liver tumor resection. Neutrophil extracellular traps (NETs) can aggravate liver injury and activates innate immune response in the process of liver IRI. However, Curcumin (Cur) can reverse this damage and reduce NETs formation. Nevertheless, the specific regulatory mechanism is still unclear in liver IRI. This study aimed to explore the potential mechanisms that how does Cur alleviate hepatic IRI by inhibits NETs production and develop novel treatment regimens.

Methods

We established a hepatic IRI model by subjecting C57BL/6J mice to 60 min of ischemia, followed by reperfusion for 2 h, 6 h, 12 h, and 24 h respectively. Subsequently, we were separated into 5 groups, namely the I/R group, Cur group, DNase-1 group, Cur + DNase1 group and sham operation group. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST), Hematoxylin-eosin staining, immunofluorescence, and TUNEL analysis were applied to assess liver injury degree and NETs levels. Western blot assay was used to detect the protein levels of apoptosis-related proteins and MEK pathway proteins.

Results

Cur could alleviate hepatic IRI by inhibiting the generation of NETs via suppressing the MEK/ERK pathway. In addition, this study also revealed that DNase-1 is vital for alleviating hepatic IRI by reducing the generation of NETs.

Conclusions

Cur combined with DNase-1 was more effective than the two drugs administered alone in alleviating hepatic IRI by inhibiting the generation of NETs. These results also suggested that curcumin combined with DNase-1 was a potential therapeutic strategy to mitigate hepatic IRI.

Keywords:

• Neutrophil extracellular traps (NETs)
• hepatic ischemia reperfusion injury
• Curcumin (Cur)
• DNase-1

Introduction

Liver ischemia-reperfusion injury (IRI) refers to the phenomenon that the cell function and structure of the ischemic liver tissue cannot be recovered in time, and they will aggravate when the blood flow is restored. It frequently happens with partial liver resection, hemorrhagic shock, liver trauma, and liver transplantation [1]. Severe hepatic IRI often leads to liver congestion and thrombosis, endangering the patient’s life [2]. As there is no effective and safe therapeutic regimen yet, treating liver IRI is still quite challenging. Therefore, the in-depth investigation of the mechanism pertaining to ischemia-reperfusion is essential for developing new therapeutic strategies in this regard.

Hepatic IRI is a strong aseptic inflammatory reaction, often accompanied by the activation of immune cells, especially during the whole process of reperfusion, a large number of neutrophils will accumulate in the liver [3, 4]. NETs present a grid-like structure when released by neutrophils after stimulation, and the reticular structure mainly relies on DNA as the skeleton, and is filled with histones, neutrophil elastase (NE), myeloperoxidase (MPO), and cathepsin G [5, 6]. Although researches have revealed that NETs formation depends on reactive oxygen species (ROS) and NADPH oxidase, which is involved in the Raf-MEK-ERK pathway [6], the cellular signaling pathways that regulate NETs formation has not yet been fully elucidated. Marin-Esteban [7] et al. used a protein kinase C (PKC) activator (PMA or H2O2) to stimulate the differentiation and maturation of human myeloid leukemia cell line PLB-985 in vitro to generate NETs. Although NETs are beneficial to the body in terms of immune defense, NETs can induce impairment to normal cells and tissues in the body as well. Researches have revealed that NETs are vital for the aggravation of IRI in small intestine, kidney and liver [8–10]. Therefore, reducing or inhibiting NETs formation during IRI is crucial for ameliorating organ damage.

Curcumin (Cur) is one of the most important bright yellow polyphenols in the rhizome of Chinese herbal medicine turmeric, and has a variety of pharmacologic activities like anti-inflammation, antioxidation and anti-cancer efficacy [11, 12]. Due to its low water solubility, curcumin needs high dose administration to achieve ideal blood concentration and pharmacokinetic stability in the body [13].The study found that sodium carboxymethyl fiber dissolved in water can significantly increase the solubility of drugs and is harmless to the body, and it is one of the most commonly used solvents in animal experiments [13, 14]. Although many recent researches have revealed that curcumin is pivotal for the treatment and prevention of hepatic IRI. However, these researches are limited to the functional level, and the mechanism of action has not been deeply explored. Therefore, establishing an effective liver ischemia-reperfusion model to reveal relevant drug mechanisms can provide a more powerful basis for effective drug screening. In this study, we investigated whether curcumin could mitigate liver IRI via suppressing the MEK/ERK signal path and NETs generation through a mouse liver ischemia-reperfusion model.

Material and Methods

Animals

Eight-week-old male C57BL/6 mice (body weight 18-25 g) were provided by Beijing Speifu Experiment Animal Center. The entire animal assays were completed as per the "3 R" principles of laboratory animal welfare and the guidance of the International Association for the Study of Pain (IASP). The animal protocol was accepted by the Ethical Board of our institution (Approval No: QYFYWZLL26589).

Construction of a Mouse Model of Liver IRI

The animals were subjected to anesthetization by intraperitoneally injecting 1% pentobarbital sodium (80 mg/kg) and then they were put in a supine position. Afterwards, the abdominal surgery areas of the mice were culled with 10% iodophor for disinfection. A 1 cm-long incision was made at the midline of the abdomen, and to reveal the portal vein and hepatic artery that carry blood to the left and center lobes of the liver, the skin, muscle, and peritoneum were gently detached layer by layer. A noninvasive vascular clip was used to block the hepatic artery and portal vein for 60 minutes, after which the vascular clip was promptly released to reestablish blood flow to the ischemic liver lobe for 2, 6, 12, and 24 hours, respectively. The wound was then sutured, and cleaned and disinfected. Mice in the sham-operated group underwent the same surgery without clipping the portal vein and liver artery.

Experimental Protocols

The C57BL/6 mice were stochastically separated into five groups, namely the I/R group (n = 5), curcumin group (n = 5), DNase-1 group (n = 5), curcumin + DNase1 group(n = 5) and sham operation group (n = 5). Curcumin (100 mg/kg) was injected intraperitoneally 3 h before ischemia. DNase1 (10 mg/kg) was administered via tail vein injection 0.5 h prior to the recovery of ischemia. In order to eliminate interference, all experimental groups had corresponding control groups.

Histology and TUNEL Analysis

Fresh liver tissue was fixed in 10% formaldehyde liquor, subjected to paraffin embedment, sectioned, dyed with HE, and the samples were studied via an optical microscope (Lecia, Gremany). The HE outcomes were utilized to assess hepatic injury via Suzuki scoring. The programmed cell death was identified via a TUNEL tool (Beyotime, China) as per the supplier’s instruction.

Immunohistochemistry Assay

Hepatic sections were stained for Ly6G as per the supplier’s guidelines for immunohistochemical analyses. The stained samples were examined and evaluated under a light microscope.

Immunofluorescence Assay

Mouse liver tissue sections were fixed and cultivated with anti-MPO (1:500, A1374, ABclonal), anti-Ly6G (1:150, 65140-1-Ig, proteintech), and anti-citrullinated histone-3 (Cit-H3, 1:200, 17168-1-AP, proteintech) overnight at 4 °C. Secondary antibodies were afterwards supplemented before cultivation for 1 h 28 under ambient temperature. DAPI was utilized for DNA dyeing. Photos were collected via microscopy.

Biochemistry Index Identification

The mouse blood specimens were collected and put under ambient temperature for 2 h, then subjected to centrifugation under 2-8 °C at 3000 rpm for 15 min, and the supernate was collected for immediate observation. The contents of serum ALT and AST were measured using a fully automatic biochemical analyzer (Shenzhen Redu Life Technology, Chemray 240, China). TNF-α, IL-6 and IL-1β contents were identified via ELISA kits (eBioscience, USA).

Mouse Primary Neutrophils and Neutrophil Stimulation

Blood samples (1.5 ml) were taken from mouse eyeballs, and then neutrophils were isolated and obtained by Ficoll Dextran techniques. Next, neutrophils were cultured in DMEM medium containing different concentrations of Curcumin (5 μM, 10 μM, 20 μM, and 40 μM; Ziker, Shenzhen, China). Then all experimental groups except the blank control group were added under 37 °C, 5% carbon dioxide, and stimulated with phorbol 12-myristate 13-acetate (100 nm; PMA; Beyotime, Shanghai, China) for 1 h. Subsequently, we selected the best Cur concentration (40uM) group and observed different stimulation time (30 min, 60 min, 90 min, or 120 min) for PMA.

Quantification of NETs

Mouse serum NETs levels were detected mainly by the mouse MPO ELISA kit (Sigma-Aldrich, RAB0374-1KT) as per the supplier’s protocol.

Quantification of Extracellular DNA

Extracellular DNA/NETs level of neutrophils was measured using Quant-iT Picogreen dsDNA Assay Kit (Thermo Fisher Scientific, California, USA) to evaluation of NETs formation following the product guide. All samples were carried out experiment for three times.

Western Blot Assay

Proteins from hepatic samples were acquired via 15% SDS-PAGE and moved onto PVDF membranes. The PVDF membranes were cultivated for one night under 4 °C with primary Abs, which included Cit-H3 (1:2000, 17168-1-AP, proteintech, China), Bax(1:1000, 2772, Cell Signaling Technology [CST], American), Bcl-2(1:1000, 3498, CST, American), cleaved caspase-3(1:200, ab13585, Abcam, UK), cleaved PARP (1:200, ab13585, Abcam, UK), MEK1/2(1:1000, E-AB-221, Elabscience, China), p-MEK1/2(1:1000, E-AB-70310, Elabscience, China), ERK1/2(1:1000, E-AB-22162, Elabscience, China), p-MRK1/2(1:500, AP0121, ABclonal, China), and β-actin (1:3000, 17168-1-AP, proteintech, China)

Statistical Analysis

Data are displayed as the average ± SD of 3 or more diverse duplicates of independently finished assays. Group contrast was completed via ANOVA and Student’s t-test or Wilcoxon rank-sum tests (non-parametric values). Statistic assay was completed via the GraphPad Prism 8.0 program (America).

Results

Curcumin Can Alleviate Hepatic IRI and Reduce Neutrophil Infiltration

To clarify the role of curcumin in liver IRI, we established a mouse liver ischemia-reperfusion model. Since the severity of hepatic impairment is tightly related to the ischemia-reperfusion duration, we randomly divided the mice into the following five groups according to the reperfusion duration: sham, 2 h, 6 h, 12 h, and 24 h. The outcomes revealed that the contents of ALT and AST were elevated at 2 h posterior to reperfusion and reached peaks at 12 h, afterwards reduced at 24 h. In addition, the entire 4 temporal points, the contents of those hepatic enzymes were significantly lower in curcumin-treated mice versus ischemia-reperfusion group (Figure 1A). To substantiate the aforesaid outcomes, our team assessed the pathologic variations in hepatic samples, and the vector group presented severe hepatic injury, and those characteristics were aggravated over time and peaked at 12 h, which was evidently observed based on the enlarged necrosis regions of their liver tissues. Conversely, the necrosis regions of hepatic samples were remarkably decreased in curcumin-treated mice (Figure 1B and C). Immunohistochemical outcomes revealed that in contrast to the vector group, the content of Ly6G (a neutrophil-specific biomarker) positive cells in the curcumin-treated group was significantly lower (Figure 1D).

Figure 1. Curcumin can alleviate hepatic IRI and reduce neutrophil infiltration. (A) Serum contents of AST/ALT in controls (I/R) and Cur groups, **P < 0.01, ****P < 0.0001 versus Cur groups, respectively. (B and C) Typical histologic H&E dyeing photos showing necrosis regions in livers from controls and Cur mice exposed to sham or ischemia challenge before reperfusion for diverse durations (magnification, ×100; scale bar, 100 μm; n = 5). (D) Immunohistochemical staining showing Ly6G localization in livers from control groups and Cur treatment groups (magnification, ×200; scale bar, 100 μm) after hepatic I/R for diverse durations. (E) Hepatic apoptosis detected by TUNEL within control groups and Cur treatment groups after hepatic I/R for diverse durations (magnification, ×200; scale bar, 100 μm; n = 5). (F) Number of TUNEL + cells per high power field (HPF, n = 3). (G and H) The expressing levels of proteins of cleaved caspase-3, cleaved PARP, Bcl-2, and Bax were identified via WB in different groups. The statistic diversities amongst groups were evaluated via one-way ANOVA. Data are displayed as the average ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

In addition, we also examined the effect of hepatic IRI on hepatocyte apoptosis, and the results showed that the liver programmed cell death was remarkably lower in curcumin-treated mice versus the vector group (Figure 1E and F). In order to further confirm that curcumin could reduce the apoptosis of hepatocytes, we detected the expression of apoptotic proteins in the liver tissue of the reperfusion group for 12 h. The expressing levels of cleaved caspase 3, cleaved PARP, and Bax were remarkably regulated upward, while the expressing levels of Bcl-2 were remarkably regulated downward. However, the curcumin treatment group down-regulated cleaved caspase 3, cleaved PARP, and Bax expression and up-regulated Bcl-2 expression (Figure 1G and H). These results suggest that neutrophils are vital for liver IRI. In addition, we reveal that curcumin can alleviate hepatic IRI and inhibit neutrophil infiltration.

Neutrophil Extracellular Traps Are Formed In Vivo After Liver IRI

The above results have confirmed that neutrophils are vital for liver IRI. In order to detect whether infiltrating neutrophils form NETs posterior to liver IR, we first measured the formation of NETs in the serological samples of mice receiving I/R. By detecting the MPO-DNA complex content, our team discovered that the MPO-DNA content was remarkably elevated versus the sham group, and reached a peak at 12 h (Figure 2A). Subsequently, to further corroborate the forming of NETs in hepatic samples, western blotting was completed on ischemia liver lobes, which showed remarkably greater contents of citrulline histone 3 (Cit-H3, a specific biomarker of NETs generation) posterior to I/R versus the sham group (Figure 2B and C). In addition, the co-localization of Cit-H3 and MPO was considerably elevated when the reperfusion duration was increased, particularly posterior to the 12 h of reperfusion treatment, which confirmed NETs deposition in liver samples (Figure 2D). Therefore, we took the key time point (12 h after reperfusion) for subsequent experiments. These results suggested that NETs formation after hepatic I/R might be involved in persistent liver injury.

Figure 2. Neutrophil extracellular traps are formed in vivo after liver IRI. (A) According to the evaluation of serum MPO-DNA complex contents, the contents of NETs formed posterior to 1 h ischemic treatment followed by 2 h, 6 h, 12 h, and 24 h of reperfusion. Outcomes are displayed as the comparative fold elevation of MPO-DNA complexes versus sham; average ± SD (n = 6). (B and C) Cit-histone H3 protein contents were identified via WB at different reperfusion time points (D) Typical photos of immunofluorescent dyeing of hepatic samples (initial amplification ×400; scale bars, 50 μm). The existence of Cit-H3 (green), MPO (red), and DAPI (blue) is shown. Data are displayed as the average ± SD (n = 3-6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs sham.

Curcumin Inhibits NETs Formation in Vitro and Vivo

We have confirmed that curcumin can not only reduce liver IRI but also reduce neutrophil infiltration. At present, we strongly assume that Cur reduces liver IRI by preventing the creation of NETs since studies have shown that NETs formation is associated to neutrophils aberrant activation. In order to verify our conjecture, neutrophils were isolated from three healthy mice, and then cultured in DMEM medium with different concentrations of curcumin (20 μM, 40 μM, 80 μM, and 160 μM), and then 80 nM PMA was added to each well to activate the neutrophils. At the same time, we determined the optimal concentration (80 μM) of curcumin and then examined the relationship between different stimulation time points (30 min, 60 min, 90 min, and 120 min) and the formation of NETs. The outcomes demonstrated that different concentrations of curcumin pretreatment group may significantly limit the synthesis of NETs when compared to the positive control group, but 80 μM curcumin can greatly inhibit the production of NETs when compared to the other concentrations (Figure 3A and B). Subsequently, we used DMEM medium containing 80 μM curcumin to incubate neutrophils to study whether curcumin inhibited NETs in a time-dependent manner. Analysis revealed that the development of NETs steadily decreased with the extension of the curcumin incubation time (Figure 3D and E). The results also were identical to those above when we measured the amount of extracellular DNA/NETs in the neutrophil supernatant (Figure 3C and F). We also identified the expressing level of Cit-H3 in liver tissue via western blot assay, and discovered that the expressing level of Cit-H3 was remarkably up-regulated in the IR12h group versus the sham group, while the curcumin treatment group could significantly down-regulate the expression of Cit-H3 (Figure 3G and H). To further clarify that curcumin can inhibit the formation of NETs, we also detected the content of MPO-DNA in the serum. The outcomes revealed that the content of MPO-DNA complex in the curcumin treatment group was significantly lower in contrast to the I/R group (Figure 3I).

Figure 3. Curcumin inhibits NETs formation in vitro and vivo. (A) Neutrophils obtained from mice were cultured in DMEM medium containing different concentrations of curcumin (20 μM, 40 μM, 80 μM, 160 μM) for 1 h, and then stimulated with 80 nM PMA for 60 min. An upright fluorescent microscope was used to view the NETs’ structural details (magnification, ×200). (B) Quantitative analysis of the percentage of neutrophils NETosis under different conditions. (C) Quantitative analysis of the extracellular DNA/NETs levels. (D) The separated neutrophils from mice were cultured in DMEM medium with curcumin (80 μM) for different time(30 min, 60min, 90min and 120 min), and then stimulated with 80 nM PMA for 60 min. (E) Quantitative evaluation of the percentage of neutrophils experiencing NETosis under various circumstances. (F) Quantitative evaluation of the percentage of neutrophils NETosis under different conditions. (G and H) Detection of Cit-H3 expression by western blot under different conditions. (I) Serum MPO-DNA was measured by ELISA. The statistic diversities amongst groups were evaluated via one-way ANOVA. Data are displayed as the average ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Curcumin Inhibits NETs Formation and Alleviates Hepatic IRI by Inhibiting MEK/ERK Pathway

To investigate the spontaneous formation of NETs by peripheral venous neutrophils after liver ischemia in mice, we divided them into ischemia reperfusion group, curcumin preconditioning group, sham group and PMA stimulation group as positive control groups. Results analysis showed that the ischemia reperfusion group could spontaneously form NETs without any stimulation, while the curcumin treatment group could reduce the formation of NETs (Figure 4A and B). In addition, the results of liver tissue immunofluorescence also showed that the intrahepatic NETs in Cur-treated mice were remarkably reduced versus I/R mice (Figure 4D-E). It has been reported that the MRK/ERK pathway can affect the formation of NETs [6], but whether curcumin reduces the formation of NETs by inhibiting the MRK/ERK pathway has not yet been studied.

Figure 4. Curcumin inhibits NETs formation and alleviates hepatic IRI by inhibiting MEK/ERK pathway. (A) From mice in various treatment groups, neutrophils were extracted and cultivated for two hours in the absence of stimulation. PMA (80 nM) stimulated group was used as the positive control group. An upright fluorescent microscope was used to view the NETs’ structural details (magnification,×200). (B) Quantitative evaluation of the percentage of neutrophils experiencing NETosis under various circumstances. (C) Dual immunofluorescent assay of MPO (red) and Cit-H3 (green) in liver issues (initial amplification ×400; scale bars, 50 μm). (D and E) Quantitative analysis of MPO and Cit-H3 positive area. (F–H) The expressing levels of proteins of Cit-H3, p-Mek1/2, Mek1/2, p-Erk1/2, and Erk1/2 were identified via WB. Data are displayed as the average ± SD (n = 3-6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Herein, reveal the causal link by which curcumin treats hepatic IRI, WB assay was completed to identify the expressing levels of p-Mek1/2, Mek1/2, p-Erk1/2, Erk1/2, and Cit-H3 at 12 h after reperfusion. Analysis showed that curcumin significantly reduced the expressing levels of Cit-H3, p-Erk1/2, and p-Mek1/2(Figure 4F-H). These findings implied that Cur significantly reversed the formation of NETs induced by ischemia-reperfusion through inhibition MEK/ERK signaling pathway.

Curcumin and DNase-1 Have Synergistic Effects on Reducing Hepatic IRI

Previous studies have confirmed that DNase-1 can alleviate intestinal IRI by reducing the formation of NETs [15], but the role of DNase-1 in hepatic IRI is still unclear, and whether DNase-1 combined with curcumin has a synergistic effect on alleviating hepatic IRI remains elusive. Therefore, herein, our team explored the effect of curcumin and DNase-1 combined therapy on hepatic IRI by HE staining, TUNEL, tissue immunofluorescence, ELISA, and WB experiments. The results showed that the administration of curcumin (100 mg/kg) or DNase-1 alone could reduce neutrophil infiltration, NETs formation and alleviate hepatocyte necrosis and apoptosis to a certain degree. However, the combined therapy of curcumin and DNase-1 exhibited remarkably superior therapeutic efficacy in treating liver IRI versus utilizing them individually as therapeutic regimens (Figure 5A and B). ALT, AST, TNF-α, IL-6, and IL-1β assay results also revealed that combining Cur with DNase-1 was more efficient compared with use of either treatment (Figure 5C-G).

Figure 5. Curcumin and DNase-1 have synergistic effects on reducing hepatic IRI. (A) Effects of Curcumin and DNase-1 on neutrophil infiltration, NETs formation, hepatocyte apoptosis and histopathological changes in liver tissue with liver IRI (amplification, ×200; scale bar, 100 μm) (n = 5). (B) WB assay of the expressing levels of proteins of cleaved caspase-3, cleaved PARP, Bcl-2, and Bax in hepatic IRI with curcumin and DNase-1 administration. (C-G) The expressing levels of AST, ALT, TNF-α, IL-6, and IL-1β were identified via an ELISA tool (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Discussion

Hepatic ischemia-reperfusion (IR) is an unavoidable complicating disease in hepatic surgery, which negatively influences the prognoses of sufferers. Although the mechanism of liver IR-induced hepatic tissue damage, inflammatory responses, and hepatocyte apoptosis has been studied [16], the specific mechanism remains unclear. Previous studies have reported that damage-associated molecular patterns (DAMPs) produced by liver cell apoptosis elicit NETs generation and afterwards aggravate liver IRI [17]. Therefore, NETs have now been considered as new underlying treatment targets for liver IRI [18].

Curcumin (Cur) is a phenolic pigment extracted from the traditional Chinese medicine turmeric tuber with anti-inflammatory, antioxidant and anti-ischemic effects. However, curcumin’s underlying therapeutic mechanism for hepatic IRI is not fully understood. This study found that Cur reduced hepatic apoptosis, and improved liver function. It reduced hepatic neutrophil aggregation, and inhibited NETs formation, alleviating hepatic IRI. In addition, this study is the first to demonstrate that curcumin and DNase-1 have a synergistic effect in relieving hepatic IRI.

Studies also have reported that NETs are associated with IRI-induced inflammation [19, 20], and other researches have revealed that NETs are vital for the aggravation of IRI in the small intestine, kidney and liver [8–10]. Currently, numerous studies have demonstrated that preventing the development of NETs can effectively reduce IRI [21]. In the present study, we further verified this effect through in vivo and in vitro experiments of Cur inhibiting the formation of NETs. We discovered that Cur suppressed NETs production in a time- and concentration-dependent way through in vitro research. In addition, in-vivo tests further demonstrated that Cur pretreatment can drastically lower the expression of NETs in IRI mice’s liver and peripheral blood. These phenomena indicated that curcumin inhibited the formation of NETs in vivo and in vitro. However, there is still no unified conclusion on the specific mechanism about Cur inhibited the formation of NETs. Therefore, our research intended to further explore the molecular mechanism of Cur intervention in the formation of NETs. In recent years, multiple researches have unveiled that Cur can repress NETs formation via Nrf2-associated ROS inhibition [22]. Although it has been pointed out that the formation mechanism of NETs has been evidenced to be associated with the stimulation of NADPH oxidase, ERK and p38 MAPK signal path, it’s still elusive whether Cur participates in the forming of NETs and what effects it exerts. Therefore, in this paper, our results demonstrated that Cur could suppress the forming of NETs by down-regulating the expression of the MEK/ERK signaling pathway, and finally alleviate liver damage.

Previous studies have reported that NETs can be degraded by deoxyribonuclease I (DNase-1) [23]. In addition, studies have also shown that DNase-1 can alleviate acute kidney, intestinal, liver and myocardium I/R injury [15, 24–26]. Although treatment with Cur or DNase-1 in the mouse liver I/R model can reduce the generation of NETs and alleviate hepatic IRI in vivo, the synergies of Cur and DNase-1 haven’t been investigated yet. Hence, our team assessed the synergies of Cur and DNase-1 in liver IRI, hepatic pathologic lesions, hepatocyte apoptosis, and hepatic inflammation mediation factors (TNF-α, IL-6, IL-1β) via a mouse hepatic I/R model. The discoveries herein reveal that, unlike individual therapeutic regimens, which failed to completely improve liver IRI in mice, the combination of curcumin and DNase-1 realized the full symptom mitigation of liver injury.

Conclusion

In conclusion, our results unveil that Cur can alleviate neutrophil infiltration after liver ischemia-reperfusion in mice, and it can reduce the production of NETs by inhibiting the activity of the MEK/ERK signaling pathway, which can further alleviate hepatic IRI. In addition, our findings also substantiate that multi-channel combination therapies are more valid for treating liver IRI.

Disclosure Statement

No potential conflict of interest was reported by the author(s).