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Introduction

Acute kidney injury (AKI) is an acute disorder marked by rapid failure in renal function, associated with an increased incidence and fatality rate (1). AKI is caused by decreased renal perfusion pressure, trauma, mechanical and nephrotoxic agents. Ischemia-reperfusion (IR) damage is a primary reason for acute tubular necrosis in the kidney, resulting IR injury (IRI) of the kidney (2,3). The involvement of inflammatory processes, micro-vascular dysfunctions and adaptive responses of kidney tubules in the pathogenesis of AKI may provide novel treatment and diagnostic approaches.

During IRI, the reactive oxygen species (ROS) generation, elevated calcium levels inside the cell and organelle destruction lead to intrinsic kidney cell damage (4). IRI can trigger an immune cascade of inflammation in stressed kidneys, which induces numerous mechanisms of cell death, such as apoptosis, necrosis and ferroptosis (5). Increased ROS production is a hallmark of IRI. Hydrogen peroxide (H2O2), owing to its self-stability and permeability of the membrane (6), is a principal mediator of renal tubular destruction in diverse disorders and has been used to provoke ROS-mediated oxidative destruction in tubular epithelial cells of the kidney, particularly within the cellular inflammatory immune cascade triggered by kidney IRI (7).

Previous studies have revealed that targeting the apoptosis progression in tubular epithelial cells is a valuable strategy for protecting AKI (8–10). Activating mitochondrial-dependent pathways is essential for apoptosis and kidney injury owing to IR (11). One of the key components of this pathway, Bax, activates and transfers to the mitochondrial membrane, resulting in activation of effector caspase3, an inducer of AKI (12). Studies have demonstrated that inhibition of Bax protects human tubular epithelial cells of the kidney against damage caused by cisplatin and lipopolysaccharide (13,14). Therefore, investigating the role of the aforementioned mitochondrial-dependent apoptosis pathway on the mechanism or management of IRI-AKI is important.

β-elemene (ELE) is a sesquiterpene present in spices, herbs and root vegetables, used for the treatment of different malignancies, such as lung, liver cancer, esophageal cancer, nasopharyngeal cancer, brain cancer, and bone metastasis (15). ELE kills the tumor cells, whereas, to the best of our knowledge, there is no known impact on healthy cells, such as the peripheral blood lymphocytes, at concentrations below 50 µg/ml (16). β-Elemene was found to have effectively suppressed M2 macrophage recruitment and MCP-1 expression through inhibiting the Prx-1/NF-κB/HIF-1α signaling pathway in lung cancer (17). In glioblastoma cells, β-elemene induced ROS production in a dose- and time-dependent manner, mediated oxidative damage and inhibited cancer growth (18). Although the anti-tumor effects of ELE have been extensively reported, the role of ELE in AKI remains unclear. The present study assessed the anti-inflammatory role of ELE in rat proximal tubular epithelial (NRK52E) cells and a mouse model of IRI to support the potential use of ELE in AKI.

Materials and methods

Reagents and antibodies

ELE (cat. no. 63965) was acquired from Merck KGaA. Antibodies targeting toll-like receptor (TLR) 4 (cat. no. AF7017), myeloid differentiation primary response gene 88 (MyD88; cat. no. AF5195), P65 (cat. no. BF8005), ERK1/2 (cat. no. AF0155), F4/80 (cat. no. DF2789), β-actin (cat. no. AF7018), phosphorylated (p)-P65 (cat. no. AF2006), Bax (cat. no. AF0120), JNK (cat. no. AF6318), Bcl-2 (cat. no. AF6139), P38 (cat. no. AF6456), caspase-3 (cat. no. AF6311), cleaved (c-)caspase-3 (cat. no. AF7022), p-JNK (cat. no. AF3318), p-P38 (cat. no. AF4001) and p-ERK1/2 (cat. no. AF1015) were acquired from Affinity Biosciences, Ltd. N-acetylcysteine (NAC; cat. no. S1623) was acquired from Selleck Chemicals. The blood urea nitrogen (BUN) test (cat. no. C013-2-1) and creatinine assay kits (cat. no. C011-2-1) were obtained from the Nanjing Jiancheng Bioengineering Institute.

Animal experiments

The animal experimental ethics committee at Youjiang Medical University for Nationalities (Baise, China) authorized all animal experiments. A total 30 of male C57BL/6 mice (age, 8–10 weeks old and body weight of 18–22 g) were obtained from Changzhou Cavens Experimental Animal Co. Ltd. (cat. no. 202358084) and housed in the specific-pathogen-free facility of Youjiang Medical University for Nationalities (certification no. SYXK 2022–0004). Mice were housed under standard conditions and had unlimited access to sterilized food and distilled water. Room temperature was maintained at 25±2°C and relative humidity at 60±10% and a 12-h light/dark cycle was used. Mice were randomly separated into four groups (n=6/group): Sham, ELE, IRI and IRI + ELE. The IRI model was established as previously described (19). Sham group underwent surgical exposure of the kidney without ischemia induction. ELE group received intraperitoneal injection of ELE (40 mg/kg/day) without ischemia induction for 7 days; In the IRI group, ischemia was induced by clamping both renal arteries for 45 min, followed by reperfusion. Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). A midline abdominal incision was made to expose both renal arteries, which were clamped with non-invasive vascular clips for 45 min. The clamps were removed to restore blood flow. Kidney tissue was collected 24 h post-surgery and 0.5–1.0 ml of blood were collected for analysis using cardiac puncture. All IRI model mice underwent 24 h reperfusion after ischemia to simulate the commonly observed IRI time window in clinical settings (20,21). The IRI + ELE group was pre-treated with ELE (40 mg/kg/day) for 1 week prior to the IRI procedure. ELE injection continued until euthanasia.

All mice were anesthetized with 1% (w/v) pentobarbital sodium solution, administered at a dose of 50 mg/kg via intraperitoneal injection, before surgery. At the end of the experiments, all mice were euthanized by carbon dioxide via a gas anesthesia machine, controlling the CO2 flow rate at 70% of the chamber volume/min, followed by cervical dislocation. Death was confirmed by cessation of respiration for >5 min, (2) absence of pedal reflex.

Cell culture

Rat proximal tubular epithelial NRK52E cells were obtained from American Type Culture Collection. Cells underwent incubation in a 5% CO2 atmosphere with 10% fetal bovine serum and Dulbecco's modified eagle medium (both Gibco; Thermo Fisher Scientific, Inc.) at 37°C. NRK52E cells were treated with ELE (0, 5, 10, 20, 40 and 80 µM) for 1 day at 37°C. Subsequently, NRK52E cells were treated with 150, 300, 450 and 600 µM/ml H2O2 (PeproTech, Inc.) for3, 6 and 12 h at 37°C. In addition, the ROS scavenger NAC was used to intervene in NRK52E cells at 2, 5 and 10 µM for 12, 24, 48 h at 37°C.

Renal function and histology

Mouse blood samples were allowed to coagulate at room temperature for 30–60 min, then centrifuged at 14,000 × g for 10 min at room temperature to obtain a serum sample. Levels of BUN and serum creatinine (Scr) were identified using the aforementioned commercial kits according to the manufacturer's instructions. For histological examination, the mouse renal tissues underwent fixation with 4% formaldehyde at room temperature for 24 h and were immersed in paraffin for staining with H&E to analyze the renal morphology, as previously described (22).

Immunohistochemistry

Kidney tissue from mice of different treatment groups were fixed in 4% paraformaldehyde solution for 24 h and dehydrated in increasing order of alcohol at room temperature. After permeabilization with xylene for 30 min, the heart tissue was embedded in paraffin and cut into 4–5 µm sections. These tissue sections were deparaffinized with xylene. For antigen repair, they were placed in staining jars containing citrate buffer and boiled in a pressure cooker over medium heat for 15 min. To inhibit endogenous peroxidase activity, a 3% hydrogen peroxide solution was added for 10 min at room temperature. Sections were incubate with 10% goat serum (No. SAP-9100; Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) for 30 min at room temperature, followed by incubation with anti-F4/80 primary antibody (1:200; No. A2547, Sigma-Aldrich) for 12 h at 4°C, followed by incubation with horseradish peroxidase (HRP)-IgG secondary antibody (1:50, No. A0208; Beyotime Institute of Biotechnology) for 60 min at 24°C. The expression of F4/80 protein in kidney tissue was observed using DAB staining. A light microscope (Nikon, Japan) was used. Five visual field images of tissue were obtained from each histochemical section. The number of DAB-positive cells was determined by ImageJ (version 1.8.0; National Institutes of Health).

TUNEL staining

TUNEL Apoptosis Detection kit (Item No. KGA1401-100, Jiangsu Kaiji Biotechnology Co., Ltd.) was used to detect apoptosis in mouse kidney tissues (5 µm) and NRK52E cells (1×105). NRK52E cells were treated with 600 µM/ml H2O2 and ELE (10 or 20 µM) at 37°C. Briefly, cells and kidney tissue sections were fixed on coverslips with 4% paraformaldehyde for 30 min at room temperature. Cells were treated with 0.1% Triton X-100 for 10 min at room temperature. The kidney tissue and cells were washed with PBS. Cells and kidney tissue sections were treated with proteinase K working solution for 10 min at 37°C and incubated with 50 µl of TUNEL reaction mixture for 1 h at 37°C. Nuclei were counterstained by mounting with antifade medium containing DAPI (cat. no. P0131, Beyotime Biotechnology). for 10 min at room temperature. Images were captured using an inverted fluorescence microscope (Nikon Corporation) at high magnification (×400). Quantification of TUNEL-positive cells in at least 3 randomly selected areas was performed using ImageJ software (version 1.8.0; National Institutes of Health).

Cell viability

Detection of ELE-induced cytotoxicity in NRK52E cells was performed using Cell Counting Kit-8 (CCK-8) assay (cat. no. M4839; Abmole China Branch). NRK52E cells were seeded into 96-well plates at a density of 3,000 cells/well and incubated with CCK-8 solution for 2 h. Cell absorbance at 450 nm was determined using a microplate reader (Titertek-Berthold).

RNA analysis

Total RNA was extracted from kidney tissue and NRK52E cells (1×106) with TRIzol (Thermo Fisher Scientific, Inc.) and its quality was assessed using a spectrophotometer (Beckman Coulter, Inc.). RNA was reverse-transcribed to cDNA using ReverTra Ace qPCR RT premix (FSQ-201, TOYOBO) according to the manufacturer's instructions. QuantiNova SYBR Green PCR kit (Qiagen GmbH) and Analytik Jena qTOWER 3 G Real-Time PCR System (Jena, Germany) was used for RT-PCR according to the manufacturer's instructions. all PCRs were performed in triplicate with the following cycling conditions: i) initial denaturation at 95°C/10 min; ii) 40 cycles each at 95°C 30 sec, 60°C/1 min; and 72°C/30 sec. The 2-ΔΔCq method was used for all PCRs. Quantification of mRNA levels was performed using the 2-ΔΔCq method and normalised against the internal reference gene GADPH (23). Primers are listed in Table SI.

Small interfering RNA (siRNA) knockdown

To inhibit MyD88 expression, siRNA sequences were used. MyD88 (5′-GCCAGCGAGCTAATTGAGAAA-3′; cat. no. SC-106986; Santa Cruz Biotechnology, Inc.) and negative control siRNA (5′-GCCAGCGAGCTAATTGAGAAA-3′; cat. no. SC-106986; Santa Cruz Biotechnology, Inc.) were used to transfect cells. NRK52E cells (1×105) were inoculated in each well of a 6-well plate. 12 h later, cells were transfected with 80 nM control siRNA and MyD88 siRNA. Lipofectamine® 2000 Reagent (Art. No. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection (5 µl/well). After 18 h of incubation at 37°C, the transfection medium was replaced with fresh Dulbecco's modified eagle medium and the cells were further incubated at 37°C for 24 h.

Western blot analysis

Total proteins were extracted from kidney tissues and NRK52E cells (1×107/per) using RIPA buffer (Beyotime Institute of Biotechnology) and protein concentration was measured using BCA assay kit (ZJ101, Epizyme). Proteins were separated using 10% SDS-PAGE (50 µg/lane) and transferred to PVDF membrane. After being closed with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with F4/80 (1:1,000), TLR4 (1:1,000), MyD88 (1:2,000), P65 (1:1,000), ERK1/2 (1:1,000), β-actin (1:10,000), p-P65 (1:1,000), Bax (1:1,000), JNK (1:1,000), Bcl-2 (1:1,000), P38 (1:1,000), caspase-3 (1:1,000), c-caspase-3 (1:1,000), p-JNK (1:1,000), p-P38 (1:1,000) and p-ERK1/2 (all 1:1,000) primary antibodies overnight at 4°C. The membrane is left at room temperature for 1 h with an HRP coupled secondary antibody (1:2,000). Finally, the bands were visualized using ECL Reagent (SQ201L, Epizyme, Shanghai, China). ImageJ software (version 1.8.0, National Institutes of Health) was used.

Statistical analysis

All data are presented as the mean ± standard deviation (n=3–6 repeats/group). Multiple comparisons were conducted using one-way ANOVA with Tukey's post hoc test using GraphPad Prism (version 8.0, Dotmatics) P<0.05 was considered to indicate a statistically significant difference.

Results

ELE ameliorates IR-induced kidney injury

Levels of the inflammatory cytokine TNF-α were measured at 6, 24 and 48 h post-reperfusion. A pronounced increase in inflammatory cytokines occurred at 24 h, while there was no significant difference at 48 h (Fig. 1A). Therefore, 24 h was selected as the optimal timepoint. The blood concentrations of Scr and BUN in mice subjected to a renal IR intervention were elevated compared with sham animals. ELE + IRI significantly prevented increased Scr and BUN levels (Fig. 1B and C). ELE alone did not cause kidney injury in mice (Fig. 1D and E); renal tissue demonstrated infiltration of inflammatory cells and destruction of the tubules, including a loss of a tubular brush edging and luminal dilatation in the IRI compared with the sham group, which was ameliorated by ELE pretreatment. The levels of the macrophage biomarker F4/80 protein were measured to assess the extent of inflammatory cell interstitial infiltration in the kidney of mice. Levels of F4/80 protein were significantly elevated in IRI mice compared with the sham group, while ELE markedly decreased the F4/80 protein expression levels in IRI mice (Fig. 1F and G).

Figure 1. β-elemene ameliorates kidney damage due to I/R. (A) RT-qPCR was utilized to identify TNF-α mRNA expression. The serum (B) BUN and (C) Scr levels. (D) Representative H&E staining. (E) Tubular damage. (F) Representative western blotting and (G) F4/80 protein expression in renal tissue. (H) Representative immunohistochemistry. Scale bar, 50 µm. (I) F4/80 expression in renal tissue. RT-qPCR was used to identify mRNA expression levels of (J) TNF-α, (K) IL-1β, (L) MCP-1 and (M) ICAM-1. *P<0.05, **P<0.01. RT-q, reverse transcription-quantitative; ns, not significant; ELE, β-elemene; IRI, ischemia-reperfusion injury; MCP-1, monocyte chemoattractant protein-1; ICAM-1, intercellular adhesion molecule 1.

Immunohistochemical experiments also confirmed that ELE + IRI mice exhibited significant inhibition in F4/80 deposition compared with IRI mice (Fig. 1H and I). IRI mice exhibited increased levels of monocyte chemoattractant protein-1 (MCP-1), IL-1β, intercellular adhesion molecule 1 (ICAM-1) and TNF-α expression compared with sham mice (Fig. 1J-M). By contrast, levels of MCP-1, TNF-α, IL-1β and ICAM-1 expression were decreased in ELE + IRI mice compared with IRI mice. These results suggested that ELE partially reduced kidney injury by reducing the inflammatory infiltration in IR-induced AKI.

ELE protects NRK52E cells from H2O2-induced inflammation

Effects of ELE on H2O2-induced inflammation were assessed in NRK52E cells. CCK-8 assay revealed that 600 µM H2O2 for 12 h significantly decreased NRK52E cell viability (Fig. 2A). ELE induced cytotoxicity in NRK52E cells. NRK52E cell viability was not altered by ELE at concentrations of 5–20 µM (Fig. 2B). Decreased NRK52E cell viability caused by H2O2 was recovered by ELE at 5, 10 and 20 µM (Fig. 2C). Therefore, these concentrations were selected for subsequent experiments. MCP-1, IL-1β, TNF-α and ICAM-1 mRNA levels in H2O2-stimulated NRK52E cells increased when compared with control cells. However, MCP-1, TNF-α, IL-1β and IL-6 levels were suppressed by ELE pretreatment in a dose dependent manner (Fig. 2D-G).

Figure 2. β-elemene protects NRK52E cells from H2O2-induced inflammation. (A) Optimal duration of H2O2 treatment in NRK52E cells was identified by employing the CCK-8 assay. (B) CCK-8 analysis was employed to identify the ELE impact on viability of NRK52E cells. (C) NRK52E cells underwent pretreatment with various doses of ELE and were treated for 6 h with or without 600 µM H2O2. CCK-8 analysis was used to determine cell viability. Reverse transcription-quantitative PCR was employed to evaluate the mRNA expression of (D) IL-1β, (E) TNF-α, (F) MCP-1 and (G) ICAM-1. *P<0.05, **P<0.01, ***P<0.001. CCK-8, Cell Counting Kit-8; OD, optical density; ELE, β-elemene; ns, not significant; MCP-1, monocyte chemoattractant protein-1; ICAM-1, intercellular adhesion molecule 1.

ELE inhibits the inflammatory response by suppressing TLR4/MyD88/NF-κB pathway activation in vivo and in vitro

As MCP-1, ICAM-1, and TNF-α are cytokine markers for the NF-κB signal (24), the levels of p65 and p-p65 protein were detected in mice and NRK52E cells. Expression of p-p65 protein increased in mice renal tissues after IRI compared with sham mice (Fig. 3A). ELE prevented the increase in p-p65 expression. Furthermore, TLR4 and MyD88 protein expression upstream of the NF-κB signaling pathway was significantly elevated in IRI compared with the sham mice. ELE decreased the TLR4 and MyD88 expression in IRI mice. In NRK52E cells, ELE inhibited H2O2-induced increases in MyD88, TLR4 and p-P65 protein levels in a dose-dependent manner (Fig. 3B). These findings demonstrated that ELE exerts anti-inflammatory effects, at least partially, via the TLR4/MyD88/NF-κB pathway.

Figure 3. ELE inhibits the inflammatory response by suppressing TLR4/MyD88/NF-κB pathway activation in vivo and in vitro. (A) Renal p-P65, MyD88, TLR4 and P65 expression. (B) Western blotting of the p-P65, MyD88, TLR4 and P65 expression in NRK52E cells treated with H2O2 and various concentrations of ELE. *P<0.05, **P<0.01. ELE, β-elemene; IRI, ischemia-reperfusion injury; TLR4, toll-like receptor 4; MyD88, myeloid differentiation primary response gene 88; p-, phosphorylated-; ns, not significant.

ELE suppresses apoptosis in IRI mice

Apoptosis serves a key function in renal IR as it can cause cell death, and the extent of the apoptosis may be directly associated with the intensity of the damage (25). TUNEL staining in the renal tubule increased in IRI compared with the sham mice (Fig. 4A). ELE pretreatment decreased IRI-induced kidney cell apoptosis. Bax/Bcl-2 ratio and c-caspase3 protein expression were downregulated by ELE pretreatment compared with IRI-alone (Fig. 4B). TUNEL staining revealed that ELE exhibited an anti-apoptotic effect in the IRI model.

Figure 4. ELE suppresses apoptosis in a mouse model of IRI. (A) TUNEL analysis of mouse kidney sections; scale bar, 100 µm. (B) c-caspase3, Bcl-2, Bax and caspase-3 protein levels in the kidney of mice were quantified using western blotting. **P<0.01. ELE, β-elemene; IRI, ischemia-reperfusion injury; ns, not significant; OD, optical density; c, cleaved.

ELE ameliorates H2O2-induced NRK52E cell apoptosis

To detect the ELE-induced anti-apoptotic effect on H2O2-treated NRK52E cells, a TUNEL assay was performed. H2O2 markedly upregulated the proportion of TUNEL-positive cells compared with the control group (Fig. 5A). The proportion of TUNEL-positive cells was downregulated in ELE 10 and 20 µm + H2O2 groups compared with the H2O2 group, with the most pronounced decrease in ELE 20 µm + H2O2 group. ELE pretreatment dose-dependently inhibited the ratio of Bax/Bcl-2 and protein expression of c-caspase3 in H2O2-treated NRK52E cells (Fig. 5B). Additionally, to investigate whether ELE exerts its effects through the oxidative stress pathway, the ROS scavenger NAC was used. NAC at 5 mM inhibited proliferation after 24 h in NRK52E cells compared to the control group (Fig. 5C). In H2O2-treated NRK52E cells, the combination of ELE + NAC significantly suppressed the Bax/Bcl-2 ratio and the protein expression of c-caspase3 compared with ELE alone (Fig. 5D). The data suggested that ELE may exert its anti-apoptotic effects through the oxidative stress pathway.

Figure 5. ELE ameliorates H2O2-induced NRK52E cell apoptosis. (A) TUNEL assay; scale bar, 50 µm. (B) Bax, c-caspase3, Bcl-2 and caspase-3 protein expression were measured using western blotting. (C) CCK-8 analysis was performed to identify the NAC impact on viability of NRK52E cells. (D) Identification and semi-quantification of Bax, c-caspase3, Bcl-2 and caspase-3 protein expression in NRK52E cells. *P<0.05, **P<0.01. ELE, β-elemene; c-, cleaved; ns, not significant; OD, optical density; NAC, N-Acetylcysteine.

ELE decreases inflammatory and apoptosis signaling by inhibiting MAPK signal activation

MAPK pathway signaling is affected by the pro-apoptosis and pro-inflammatory cytokine IL-1β, TNFα (26,27). Expression of MAPK signaling pathway members was assessed in the kidney of IRI mice and H2O2-treated NRK52E cells. The IRI group revealed increased p-ERK, p-JNK and p-p38 protein expression compared with the sham group, while ELE pretreatment decreased ERK, JNK and p38 protein phosphorylation levels in the kidney (Fig. 6A). Similarly, p-JNK, p-ERK and p-p38 protein levels were increased in NRK52E cells stimulated by H2O2; levels of p-JNK, p-ERK and p-p38 diminished with elevated ELE levels (Fig. 6B). These data suggest that ELE may function by inhibiting MAPK signaling pathway activation.

Figure 6. ELE decreases inflammatory and apoptosis signaling by inhibiting MAPK signaling pathway activation. (A) ERK, p-JNK, p-P38, JNK, p-ERK and P38 in the kidney. (B) Identification and semi-quantification of p-JNK, p-ERK1/2, p-P38, JNK, ERK1/2 and P38 expression in NRK52E cells. *P<0.05, **P<0.01. ELE, β-elemene; IRI, ischemia-reperfusion injury; ns, not significant; p-, phosphorylated.

MyD88 knockdown inhibits apoptosis and MAPK signaling pathway activation in H2O2-treated NRK52E cells

Effects of H2O2 in MyD88 knockdown NRK52E cells was examined. MyD88 protein expression was not affected by the negative control siRNA (Fig. 7). Following siRNA-MyD88 transfection, a reduction in MyD88 protein expression was observed. MyD88 knockdown downregulated the ratio of Bax/Bcl-2 and protein expression of c-caspase3 in NRK52E cells following H2O2 treatment. The impact of MyD88 knockdown on MAPK signaling pathway member expression in NRK52E cells was assessed. p-JNK, p-ERK and p-p38 protein expression levels were significantly decreased following H2O2 stimulation, while MyD88 knockdown reversed the effects of H2O2.

Figure 7. MyD88 knockdown suppresses apoptosis and MAPK signaling pathway activation in H2O2-treated NRK52E cells. (A) MyD88 expression in NRK52E cells. (B) MyD88, Bax, c-caspase3, Bcl-2, JNK, caspase-3 p-ERK, p-JNK, ERK, p-P38 and P38 expression in NRK52E cells treated with H2O2. *P<0.05, **P<0.01. c-, cleaved; p-, phosphorylated; ns, not significant; si, small interfering.

Discussion

Previous studies and pathogenic mechanisms support a key role for renal tubules in postischemic AKI (28–30). Apoptosis of renal tubule cells causes lethal damage to renal epithelium cells, aggravating kidney damage (31). Accordingly, the discovery of a therapeutic drug that exhibits anti-inflammatory and anti-apoptosis activity may contribute to IRI-AKI treatment. Studies have demonstrated that ELE has anti-inflammatory and anti-apoptosis activity (32–34). Nevertheless, the protective function of ELE in renal IRI has not been explored. In the present experiments, in vivo and in vitro models of renal IRI revealed that ELE could alleviate renal damage by TLR4/MyD88/NF-κB/MAPK pathway downregulation and suppression of apoptosis.

ELE was initially reported as an adjunctive medicine for anti-tumor agents due to its ability to cause apoptosis in malignant cells (35). Gan et al (36) revealed that ELE improves bladder cancer cell apoptosis by targeting Bcl-2 family protein. Lee et al (37) demonstrated that ELE directly suppresses ovarian malignancy cell proliferation by inducing cell cycle arrest. ELE has been explored in inflammatory disorders: Zhou et al (38) revealed that ELE treats chronic inflammation caused by obesity by enabling the migration of Foxp3CD4T+ cells to the adipose tissue. Our previous study demonstrated that ELE decreases renal fibrosis by inhibiting MyD88/JAK/STAT signaling pathway activation (22). Nevertheless, the mechanism underlying the effects of ELE on renal IRI are unknown. The inflammatory reaction is initiated after IRI and worsening the condition during disease progression. Chemotaxis in inflammatory cells is an important feature of IRI (39). In IRI, damaged renal tubule epithelial cells produce chemokines, which release inflammatory factors that contribute to inflammation, causing renal tissue damage (40). In the present study, ELE decreased the Scr and BUN serum levels in IRI mice, as well as morphological changes such as infiltration of inflammatory cells in the kidney and renal tubule damage. In addition, ELE significantly inhibited the F4/80 infiltration in IRI renal interstitial tissue. Additionally, ELE resulted in the inhibition of inflammatory cytokine (TNF-α and IL-1β), MCP-1 and ICAM-1 expression in vivo and in vitro. ELE may suppress inflammatory responses and restore renal function caused by IRI.

The innate immune system can recognize cells undergoing ischemic injury and stimulate inflammatory responses using pattern recognition receptors. TLR proteins serve a role in inflammatory responses of the kidney (41) and renal IRI. Chen et al (42) revealed that TLR4 regulates leukocytosis infiltration in the kidney during ischemia. Wu et al (43) revealed that knockout of TLR4 in IRI-induced mice decreases infiltration of macrophages and neutrophils. Notably, MyD88 may be a key downstream protein for TLR4 to regulate renal IRI (43). Zhang et al (44) revealed that MyD88 inhibitor TJ-M2010-2 markedly alleviates TGF-β-induced renal fibrosis in mice by inhibiting the TLR-4/MyD88 pathway. In the present study, IRI mice and H2O2-induced NRK52E cell treated with ELE exhibited decreased inflammation and TLR4 and MyD88 protein levels, indicating a feedback loop between ELE and TLR/MyD88 signaling.

The impairment of renal tubule epithelium function caused by apoptosis is a key feature of renal IRI progression. Pro-inflammatory cytokines induce apoptosis, primarily through inducing the caspase family of proteases (45). Caspase-3 activation aggravates renal injury by initiating the final enzymatic apoptosis cascade following ischemia of the kidney (12,46). Here, ELE mechanisms in apoptosis were assessed by measuring the activity of caspase-3 protein and using TUNEL staining in IRI models. The IRI group demonstrated elevated c-caspase3 protein activity compared with the sham group, but ELE significantly decreased c-caspase3 protein expression levels in IRI mice. The increased activity of the c-caspase3 protein was associated with TUNEL-positive staining of renal tubule epithelium cells. The proteins of the Bcl-2 family serve a key role in maintaining renal tubule epithelium cell apoptosis and ameliorating renal dysfunction (25). ELE pretreatment inhibited the ratio of Bax/Bcl-2 protein in IRI mice and H2O2-treated NRK52E cells. Therefore, the cytoprotective function of ELE may be induced by suppressing caspase-3 and Bcl-2 family protein activity.

Dysregulated NF-κB activation during renal ischemia-reperfusion injury promotes tubular cell damage via inflammatory responses (47). Ischemia of the kidney induces translocation of nuclear NF-κB in the renal tubules, promoting ischemia-induced cell death (48). Zou et al (49) revealed that inhibiting the NF-κB signaling pathway decreases kidney inflammatory responses in IRI mice. Our previous study revealed that maslinic acid decreases renal interstitial fibrosis by suppressing NF-κB signal activation (50). The aforementioned study revealed that ELE reverses NF-κB signaling activation in the kidneys of IRI-induced mice and H2O2-treated NRK52E cells. Furthermore, MyD88 inhibition decreases TLR4 signaling and NF-κB protein levels (22). It was hypothesized that the TLR4/MyD88/NF-κB pathway creates a directional signal axis and is involved in the inflammatory reaction of IRI-induced mice. ELE pretreatment decreased the expression of TLR4 protein and MyD88 protein, to inhibit the downstream NF-κB signaling activation. Similar to the present study, a previous study demonstrated that fucoxanthin alleviates lipopolysaccharide-induced acute lung injury by inhibiting the TLR4/MyD88 signaling axis (51). Although the models differ (lung vs. kidney injury), both demonstrated targeting of the TLR4/MyD88 pathway, suggesting that this pathway has a general role in organ ischemia/inflammation (52). ELE may exert multi-organ protective effects via a similar mechanism, which requires further validation.

MAPK signaling members (such as ERK, JNK and p38 protein) mediate proximal renal tubular cell injury mediator (52,53). Activation of MAPK family proteins is associated with NF-κB signal activity and generally considered to mediate apoptosis and inflammatory responses (54,55). In the present study, ELE pretreatment reversed IRI and H2O2-induced NRE52K cell injury by suppressing p-JNK, p-ERK and p-p38 protein expression. Additionally, inhibition of MyD88 markedly reduced the p-ERK, p-p38 and p-JNK protein expression in H2O2-treated NRE52K cells. It was hypothesized that ELE may decrease the inflammatory response and apoptosis caused by IR renal damage by targeting the TLR4/MyD88/NF-κB/MAPK signaling pathway.

The present study had limitations. The present study did not identify a phenotype of ELE in IRI-AKI. Further research is required to explore potential roles or mechanisms of ELE. Secondly, the present study revealed the protective effects of ELE on kidney injury solely in IRI. It is essential to investigate the protective role of ELE on AKI by establishing different models of AKI, including sepsis- and nephrotoxin-induced AKI. IRI-AKI is a dynamic process; different time points following reperfusion may reveal distinct aspects of injury and repair mechanisms. The present study focused on the 24 h time point because it is associated with the acute phase of IRI-AKI and is suitable for evaluating therapeutic interventions (20,21). Other time points should also be studied (for example, 6, 48 and 72 h) to gain a more comprehensive understanding of the injury progression and recovery processes.

In conclusion, ELE suppressed the inflammatory response and apoptosis by downregulating the stimulation of TLR4/MyD88/NF-κB/MAPK signaling, which further prevented kidney dysfunction following IR. ELE may be an anti-inflammatory agent to treat AKI.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by Shanghai Jiao Tong University Affiliated Sixth People's Hospital Basic Research Youth Cultivation Project (grant no. ynqn201313), Scientific Research Project of Shanghai Qingpu District Health and Wellness Committee (grant no. QWJ2022-19), Shanghai Municipal Health Commission Key Clinical Medicine Discipline (grant no. 2024ZDXK0008) and Shanghai Qingpu District High-Level Discipline (grant no. GF2023-7).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

WS, SB and QG conceived and designed the study. QG, YW, FL, YH, LL and DC interpreted data. QG and YW wrote the manuscript. WS revised the manuscript. WS and QG confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The animal experiments were approved by the ethics committee of Youjiang Medical University (approval no. 2023090601). All methodologies are documented in compliance with ARRIVE standards (arriveguidelines.org) for submitting reports of animal research.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References