Notably, alpinetin has been shown to activate the Nrf2 signaling pathway26. Furthermore, activation of Nrf2 can upregulate the expression of its downstream target genes such as SLC7A11 and GPX4, which are core molecules inhibiting ferroptosis. Therefore, we propose the following scientific hypothesis: Alpinetin may activate the Nrf2/SLC7A11/GPX4 signaling pathway, enhance cellular antioxidant capacity, thereby inhibiting the ferroptosis process, and ultimately protecting against LPS/D-GalN-induced acute liver injury. This study aims to investigate the protective effect of Alpinetin and its underlying mechanism, to reveal how it intervenes in ferroptosis, and to provide a new candidate drug and a theoretical basis for the treatment of liver injury.
Alpinetin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Lipopolysaccharide (LPS) (Escherichia coli lipopolysaccharide, O55:B5), d-galactosamine hydrochloride (D-GalN) and Ferrostatin-1 (Fer-1) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) and reduced glutathione (GSH) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Iron assay kit was purchased from Solarbio (Beijing, China). RIPA lysis buffer was purchased from Beyotime (Shanghai, China). Protease inhibitor cocktail was purchased from MedChemExpress (NJ, USA). Primary antibodies against transferrin receptor 1 (TFR1) (ab214039), divalent metal transporter 1 (DMT1) (ab55735), SLC7A11 (ab307601), ferritin heavy chain (FTH) (ab183781), GPX4 (ab125066) were purchased from abcam (Cambridge, UK). Primary antibody against Nrf2 (R380773) was purchased from ZEN-BIO (Chengdu, China). Primary antibody against β-actin (66009-1-IG) was purchased from Proteintech (Rosemont, IL, USA). The secondary HRP-conjugated goat anti-rabbit IgG (H + L) (BA1054) and the HRP-conjugated goat anti-mouse IgG (H + L) (BA1050) were purchased from Boster Biological Technology (Wuhan, China). PVDF membranes were purchased from Merck Millipore (MA, USA). TRIzol reagent, a first-strand cDNA synthesis kit and SYBR Green PCR Master Mix were purchased from LABLEAD (Beijing, China).
Male BALB/c mice (6-8 weeks old, weight 20-25 g) were obtained from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All mice were housed in a specific pathogen-free (SPF) facility under controlled conditions (40-60% humidity, 22-25 °C temperature, 12:12 dark-light cycle) and acclimated for one week before experiments. The mice were housed in groups of six per cage with ad libitum access to standard chow and water. All cages were cleaned regularly, with mice remaining in their home cages until the end of the study. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study was approved by the Animal Care and Research Ethics Committee of Binzhou Medical University (Ethics Approval No. 2022 - 420). This study followed the ARRIVE Guidelines 2.0 for reporting animal research.
Thirty-six mice were randomly divided into six groups (n = 6): (1) Control group; (2) LPS/D-GalN group; (3) Fer-1 (5 mg/kg) + LPS/D-GalN group; (4-6) Alpinetin (12.5, 25, or 50 mg/kg) + LPS/D-GalN groups. Groups 3-6 received daily intraperitoneal (i.p.) injections of saline, Fer-1 (5 mg/kg) or Alpinetin (12.5, 25, or 50 mg/kg) for three consecutive days. The ferroptosis-specific inhibitor Fer-1 was employed as a positive control to confirm the model's responsiveness to ferroptosis inhibition and to verify the involvement of ferroptosis in the injury. One hour after the final dose, mice (except the Control group) were challenged via an intraperitoneal (i.p.) injection of LPS (30 µg/kg) and D-GalN (600 mg/kg), which were dissolved in saline, sonicated for homogeneity, and administered to induce acute liver injury.
At 6 h post-challenge, all mice were deeply anesthetized with an i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). The absence of pedal and corneal reflexes confirmed a surgical plane of anesthesia. Euthanasia was then performed by cervical dislocation, with death verified by the absence of a heartbeat and respiration. Blood and hepatic tissue were subsequently collected for analysis (Fig. 2).
The liver index was measured on the following equation:
liver index (%) = liver wet weight/mouse body weight × 100%.
Serum was collected to measure ALT and AST levels. Approximately 100 mg of hepatic tissue was homogenized in 1 mL of ice-cold PBS, and the homogenate supernatant was used to quantify MDA and GSH. The levels of ALT, AST, MDA and GSH were tested by specific determination kits according to the manufacturer's instruction. The absorbance of the resulting reaction products was measured using a microplate reader (Thermo, MA, USA).
Serum Fe content was measured using a commercial iron assay kit according to the manufacturer's instructions. The absorbance value at 593 nm was measured with a microplate reader (Thermo, MA, USA) and the iron content of each sample was obtained based on the standard curve.
The hepatic tissue was collected and fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin and sliced into 4-µm sections. After hematoxylin and eosin (H&E) staining, pathological changes of the hepatic tissue were observed under a light microscope. Liver histopathological changes were scored as described previously. 0-1: no injury, 2-3: mild, 4-6: moderate, 7-9: severe.
Approximately 50 mg of hepatic tissue was homogenized in RIPA lysis buffer supplemented with protease inhibitor cocktail to extract total protein. The protein concentration was determined by BCA method. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder for 2 h at room temperature and then incubated with specific primary antibodies overnight at 4 °C. Subsequently, the membranes were washed three times with TBST and incubated with secondary antibodies for 1 h at room temperature. The membranes were washed again three times with TBST and the target protein blots were visualized by chemiluminescent substrates.
Total RNA was extracted from approximately 30 mg of hepatic tissue using TRIzol reagent according to the manufacturer's instructions and the concentration and quality of RNA were assessed. After mRNA was subsequently reverse transcribed into cDNA using a first-strand cDNA synthesis kit, qRT-PCR was performed using SYBR Green PCR Master Mix. The results were normalized to β-actin. The primers used to amplify specific gene fragments are listed in Table 1.
Statistical analysis and graph generation were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). All data are expressed as the mean ± SEM. The Shapiro-Wilk and Brown-Forsythe tests were employed to verify the normality of the data and the homogeneity of variances, respectively. Comparisons among groups were made with one-way analysis of variance (ANOVA) followed by Dunnett's test. P < 0.05 was considered statistically significant.