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.

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.

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.

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).

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 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).

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).

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.

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.

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.

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.

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).

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.

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).

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.

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.

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.

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.

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.