Protective effect of rapeseed pollen extract on alcoholic liver injury in mice

Abstract

This study aimed to evaluate the hepatoprotective effects of rapeseed pollen extract (RPE) in mice with alcohol-induced liver injury and to investigate the underlying mechanisms. The chemical composition of RPE was characterized using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). An experimental murine model of alcoholic liver injury was established to assess its biological activity. A total of 64 compounds were identified, predominantly comprising flavonoids, triterpenoids, alkaloids, lipids, and sterols. RPE administration significantly reduced serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). Hepatic activities of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were increased, whereas malondialdehyde (MDA) levels were decreased. Histopathological examination demonstrated attenuation of liver injury, and immunohistochemical analysis revealed increased expression of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and NAD(P)H quinone dehydrogenase 1 (NQO1). These results indicate that RPE exerts protective effects against alcohol-induced liver injury through modulation of oxidative stress and inflammatory responses. This study provides a theoretical basis for the development and application of rapeseed pollen in functional foods for liver protection.

Introduction

Global alcohol consumption has increased substantially with improved living standards, resulting in significant public health concerns. According to the World Health Organization (WHO), approximately 3 million deaths occur annually due to long-term excessive alcohol consumption, accounting for 5.3% of total global mortality¹. Chronic alcohol intake induces damage to multiple organs, including the brain, heart, gastrointestinal system, and liver². Alcoholic liver disease (ALD) is the most prevalent alcohol-related liver disorder and encompasses a spectrum of hepatic abnormalities such as fatty liver and hepatitis induced by prolonged alcohol exposure³. The liver is the principal organ for alcohol metabolism, and ALD pathogenesis is strongly associated with oxidative stress imbalance and inflammatory activation^4,5. Alcohol metabolism generates excessive reactive oxygen species (ROS), leading to impairment of antioxidant enzymes, including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), promoting hepatocyte apoptosis and necrosis⁶. Currently used therapeutic agents for ALD include corticosteroids, silymarin, glycyrrhizic acid preparations, and taurine. However, these agents are associated with adverse effects, limited long-term applicability, and a lack of disease specificity^7,8, necessitating the development of safer therapeutic strategies.

Rapeseed pollen contains abundant flavonoids, polyphenols, and other active components⁹, and exhibits antioxidative¹⁰, anti-inflammatory^11,12, lipid-lowering¹³, and hepatoprotective properties¹⁴. Previous studies have demonstrated that rapeseed pollen extract (RPE) possesses antioxidant and anti-inflammatory activities and may protect against non-alcoholic fatty liver disease^15,16. However, its role in alcohol-induced liver injury has not been fully elucidated. In this study, rapeseed pollen was extracted using 80% ethanol (v/v), and its chemical composition was analyzed via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). An acute alcohol-induced liver injury model was established by a single intragastric administration of 50% ethanol (v/v) at a dose of 12 mL/kg body weight. The pharmacological effects of RPE were evaluated, and its preliminary mechanism of action was investigated. This study provides a theoretical basis for the utilization of rapeseed pollen resources and supports the development of functional foods for the alleviation of alcoholic liver injury.

Materials and methods

Materials

The rapeseed pollen (Brassica napus L.) used in this study was purchased from Henan Aimile Industrial Co., Ltd. (Zhengzhou, China). The company holds all necessary permissions for the collection and processing of this agricultural product. A representative sample of rapeseed pollen (No. 20251021) has been deposited in the Laboratory of Pharmacognosy, School of Medicine, Huanghe Science & Technology University, and is available upon reasonable request from the corresponding author. The plant material was authenticated by Prof. Wang Li from the School of Medicine, Huanghe Science & Technology University, Zhengzhou, China. This study did not involve wild plants or endangered species. Brassica napus L. is a widely cultivated agricultural crop. All procedures complied with relevant institutional, national, and international guidelines and legislation. No specific permits were required for the use of this commercially available agricultural product. Food-grade ethanol (95%, v/v) by volume) was purchased from Hunan Kelun Pharmaceutical Co., Ltd. (Hunan, China). Dihydromyricetin (purity ≥ 98%, HPLC) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Biochemical assay kits for alanine aminotransferase (ALT), aspartate transaminase (AST), triglyceride (TG), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), alcohol dehydrogenase (ADH), and acetaldehyde dehydrogenase (ALDH) were purchased from Nanjing Jiancheng Bioengineering Institute. ELISA kits for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were obtained from Lianke Biotechnology Co., Ltd. Primary antibodies against Keap1, Nrf2, and NQO1 were purchased from Chengdu Zhengneng Biotechnology Co., Ltd. All other reagents used were of analytical grade.

Methods

Preparation of RPE

Following cell wall disruption^17,18, the rapeseed pollen was dried, pulverized, and passed through a 100-mesh sieve. Two hundred grams of the disrupted pollen powder were placed into a 2000 mL round-bottom flask. An 8-fold volume of 80% (v/v) ethanol solution was added and thoroughly mixed. The mixture was subjected to two rounds of heat reflux extraction, each lasting 2 h. After completion of the extraction, the resulting solutions were filtered under vacuum. The filtrates were combined, and the solvent was removed using rotary evaporation under reduced pressure to obtain the RPE. The obtained extract was stored at 4 °C until further use.

UPLC-MS/MS compositional analysis

The prepared RPE was identified and analyzed using UPLC-MS/MS. The chromatographic conditions were as follows: a Waters HSS T3 column (100 × 2.1 mm, 1.8 μm) was used for separation; the column temperature was maintained at 40 °C; the flow rate was 0.3 mL/min; and the injection volume was 2 µL. The mobile phase consisted of (A) Milli-Q water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid, with a gradient elution program: 0 min, A/B (100:0, v/v); 1 min, A/B (100:0, v/v); 12 min, A/B (5:95, v/v); 13 min, A/B (5:95, v/v); 13.1 min, A/B (100:0, v/v); 17 min, A/B (100:0, v/v).

Mass spectrometric analysis was performed using a Q Exactive HFX hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a heated electrospray ionization (HESI) source. Data acquisition was carried out using a Full MS/dd-MS² data acquisition method. The electrospray ionization source parameters were set as follows: sheath gas pressure, 40 arb; auxiliary gas flow rate, 10 arb; spray voltage, + 3000 V/-2800 V; capillary temperature, 350 °C; and ion transfer tube temperature, 320 °C. The primary mass spectrometry scan range (m/z range) was set at 70-1050 Da, with a primary resolution of 70,000 and a secondary resolution of 17,500. Compound identification was performed by comparing accurate mass-to-charge ratios obtained from primary mass spectrometry with fragment ion information from secondary mass spectrometry. Identification was further confirmed through database matching and comparison with relevant literature.

Establishment of an alcoholic liver injury model and drug administration

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Huanghe Science & Technology University (Approval No. 2025-012) and were conducted in accordance with relevant ethical guidelines. Male KM mice (6–8 weeks old, 18–22 g) were obtained from the Laboratory Animal Center of Huanghe Science & Technology University School of Medicine and maintained under specific pathogen‑free conditions at 22 ± 2 °C with a 12 h light/dark cycle and free access to standard diet and water.

Fifty male KM mice were randomly divided into five groups (n = 10 per group): normal control group, model group, dihydromyricetin group (200 mg/kg DHM), low-dose RPE group (200 mg/kg), and high-dose RPE group (400 mg/kg). The doses of RPE (200 and 400 mg/kg) were selected based on preliminary dose-finding experiments in which 200 and 400 mg/kg exhibited optimal hepatoprotective effects, as well as on previous literature¹⁹. Except for the normal and model groups, all treatment groups received intragastric administration of their respective drugs once daily. The normal and model groups received a vehicle only. This treatment continued for 7 consecutive days. Thirty minutes after the final administration, mice, except those in the normal control group, received a single intragastric dose of 50% (v/v) ethanol at a dose of 12 mL/kg body weight (equivalent to 5.5 g/kg) to induce acute alcoholic liver injury, as previously reported with minor modifications^20,21. The normal control group received an equal volume of saline.

Exactly 12 h after the ethanol challenge, mice were euthanized after a 12 h fasting period (with free access to water). Blood samples were collected and centrifuged to obtain serum, which was stored at -80 °C. Mice were euthanized by intraperitoneal injection of sodium pentobarbital (75 mg/kg body weight; Beijing Chemical Reagent Company, Batch No. 081213). Liver tissues were collected after confirmation of death (absence of respiratory movement and heartbeat) and fixed in 4% paraformaldehyde solution for subsequent analysis.

Measurement of serum AST, ALT, and TG levels

Serum samples stored at -80 °C were thawed and homogenized. The levels of AST, ALT, and TG in serum samples from each group were measured according to the instructions provided in the corresponding biochemical assay kits.

Measurement of hepatic ADH, ALDH, MDA, and SOD levels

Liver tissues were accurately weighed and homogenized in ice-cold normal saline (9 mL/gram tissue) under ice conditions. The homogenate was centrifuged at 4 °C and 4000 r/min for 10 min. The supernatant was collected and aliquoted for subsequent analysis. Levels of ADH, ALDH, MDA, and SOD in the liver tissue samples from each group were measured according to the instructions provided in the corresponding biochemical assay kits.

Measurement of serum TNF-α and IL-6 levels

Serum samples stored at -80 °C were thawed and homogenized. The levels TNF-α and IL-6 in serum samples from each group were measured using commercial ELISA kits according to the manufacturer’s instructions.

Histopathological analysis of liver tissue

Liver tissues fixed in 4% paraformaldehyde were processed through dehydration in a graded ethanol series, cleared in xylene, embedded in paraffin, and sectioned into 4 μm-thick slices using a microtome. Sections were then deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). After drying, sections were mounted with coverslips. Histopathological changes were observed and evaluated under an optical microscope.

Immunohistochemical staining

Liver Sect.  (4 μm) were deparaffinized, rehydrated, and subjected to heat‑induced antigen retrieval in citrate buffer (pH 6.0) at 95 °C for 15 min. Endogenous peroxidase activity was blocked using 3% H₂O₂ for 10 min, and non‑specific binding was blocked with 5% normal goat serum for 1 h at room temperature. Sections were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200), Nrf2 (1:200), and NQO1 (1:400) (Chengdu Zhengneng, China). HRP‑conjugated secondary antibody (1:500) was applied for 1 h at 37 °C. Immunoreactivity was visualized using a DAB kit (ZSGB‑BIO, China), and sections were counterstained with hematoxylin. Images (×200) were captured using an Olympus BX53 microscope. Semi‑quantitative analysis was performed with Image‑Pro Plus 6.0 by measuring integrated optical density (IOD) in five randomly selected fields per section.

Statistical analysis

Raw data were analyzed using SPSS 19.0 software and expressed as mean ± standard deviation (x̄ ± SD). One-way analysis of variance (ANOVA) was used for group comparisons, followed by Tukey’s honest significant difference (HSD) post-hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant. Graphs were generated using GraphPad Prism 8.0.2 software.

Results and analysis

Chemical profiling of RPE by UPLC-MS/MS

The chemical constituents of RPE were characterized using UPLC-Orbitrap-MS analysis (Fig. 1A and B). Raw data were processed with Progenesis QI software (Waters Corporation, Milford, USA). A total of 64 compounds were identified through database matching, primarily including flavonoids, triterpenoids, alkaloids, lipids, and sterols, as presented in Table 1. Previous studies have reported that flavonoids^22,23, triterpenoids^24–26, and alkaloids^27,28 display hepatoprotective effects.

Fig. 1.

Total ion chromatograms of RPE in positive (A) and negative (B) ion modes.

Table 1.

Major constituents identified in RPE by UPLC-MS/MS analysis.

NO	Name	Ion Mode	RT(min)	Calc m/z	Formula	Super Class
1	Isorhamnetin	NEG	10.446	315.05096	C16 H12 O7	Flavonoids
2	Naringenin	NEG	9.864	271.06091	C15 H12 O5	Flavonoids
3	Rutin	NEG	6.539	609.14478	C27 H30 O16	Flavonoids
4	Glycitein	NEG	8.323	283.06082	C16 H12 O5	Flavonoids
5	Kaempferol	NEG	10.251	285.04028	C15 H10 O6	Flavonoids
6	Isoquercitrin	NEG	6.95	463.08731	C21 H20 O12	Flavonoids
7	Astragalin	NEG	7.458	447.09247	C21 H20 O11	Flavonoids
8	Hispidulin	NEG	12.221	299.05582	C16 H12 O6	Flavonoids
9	Taxifolin	NEG	6.914	303.05063	C15 H12 O7	Flavonoids
10	3-methoxy-5,7,3’,4’-tetrahydroxy-flavone	NEG	9.452	315.05096	C16 H12 O7	Flavonoids
11	Quercetin	POS	6.268	303.04999	C15 H10 O7	Flavonoids
12	Orientin	POS	6.295	449.1079	C21 H20 O11	Flavonoids
13	Morin	POS	9.115	303.05029	C15 H10 O7	Flavonoids
14	Pinocembrin	POS	12.368	257.08112	C15 H12 O4	Flavonoids
15	Chrysin	POS	12.45	255.06533	C15 H10 O4	Flavonoids
16	Hydroxyecdysone	NEG	14.704	525.30524	C27 H44 O7	Terpenoids
17	Stevioside	NEG	14.674	803.36841	C38 H60 O18	Terpenoids
18	Bilobalide	NEG	4.858	325.09275	C15 H18 O8	Terpenoids
19	Astragaloside iii	NEG	17.691	783.44849	C41 H68 O14	Terpenoids
20	Cuminaldehyde	POS	9.371	149.09636	C10 H12 O	Terpenoids
21	Baliospermin	POS	12.466	563.35541	C32 H50 O8	Terpenoids
22	Artemisinin	POS	14.623	582.32745	C15 H22 O5	Terpenoids
23	Oleandrin	POS	12.226	577.33679	C32 H48 O9	Terpenoids
24	Andrographolide	POS	10.204	351.21707	C20 H30 O5	Terpenoids
25	Xanthosine	NEG	1.559	283.06807	C10 H12 N4 O6	Alkaloids
26	Trigonelline	POS	0.989	138.05525	C7 H7 N O2	Alkaloids
27	Protopine	POS	7.519	354.13364	C20 H19 N O5	Alkaloids
28	Stachydrine	POS	1.258	144.10214	C7 H13 N O2	Alkaloids
29	Harmine	POS	6.446	213.10254	C13 H12 N2 O	Alkaloids
30	4-methyl-5-thiazoleethanol	POS	1.807	144.04816	C6 H9 N O S	Alkaloids
31	Monocrotaline	POS	3.541	326.15991	C16 H23 N O6	Alkaloids
32	Benzoylecgonine	POS	4.292	290.13892	C16 H19 N O4	Alkaloids
33	Talatisamine	POS	15.521	422.28992	C24 H39 N O5	Alkaloids
34	Palmitic acid	NEG	18.357	255.23259	C16 H32 O2	Lipids
35	Linoleic acid	NEG	17.248	279.2326	C18 H32 O2	Lipids
36	Oleic acid	POS	21.397	265.25238	C18 H34 O2	Lipids
37	Arachidonic acid	POS	18.066	305.24749	C20 H32 O2	Lipids
38	A-linolenic acid	POS	18.968	279.23135	C18 H30 O2	Lipids
39	Corticosterone	NEG	16.061	345.20691	C21 H30 O4	Steroids
40	Cortisol	POS	16.28	345.2056	C21 H30 O5	Steroids
41	Dehydroepiandrosterone (dhea)	POS	17.315	271.20575	C19 H28 O2	Steroids
42	Glycocholic acid	POS	15.941	466.31628	C26 H43 N O6	Steroids
43	Testosterone	POS	16.57	289.21606	C19 H28 O2	Steroids
44	5a-dihydrotestosterone	POS	15.486	291.23178	C19 H30 O2	Steroids
45	Epibrassinolide	POS	12.687	481.35297	C28 H48 O6	Steroids
46	Bufotalin	POS	14.611	445.25626	C26 H36 O6	Steroids
47	Cinobufagin	POS	15.754	443.24048	C26 H34 O6	Steroids
48	Gamabufotalin	POS	15.764	403.24783	C24 H34 O5	Steroids
49	Telocinobufagin	POS	15.605	403.24551	C24 H34 O5	Steroids
50	Galangin	NEG	12.616	269.04526	C15 H10 O5	PK Polyketides
51	Kaempferitrin	NEG	6.962	623.16296	C27 H30 O14	PK Polyketides
52	Dodecyl sulfate	NEG	13.648	265.14746	C12 H26 O4 S	PK Polyketides
53	Hematoxylin	NEG	10.036	301.07156	C16 H14 O6	PK Polyketides
54	Cannabidiolic acid	NEG	17.159	357.20688	C22 H30 O4	PK Polyketides
55	Nigericin	NEG	18.749	723.47135	C40 H68 O11	PK Polyketides
56	Afzelin	NEG	8.141	431.09787	C21 H20 O10	PK Polyketides
57	Cannabigerolic acid	NEG	17.514	359.22247	C22 H32 O4	PK Polyketides
58	Kaempferol-3-o-rutinoside	NEG	7.218	593.15094	C27 H30 O15	PK Polyketides
59	Esculetin	NEG	5.139	177.01934	C9 H6 O4	Phenylpropanoids
60	Fraxetin	NEG	5.84	207.03	C10 H8 O5	Phenylpropanoids
61	4-coumaric acid	POS	6.463	147.04425	C9 H8 O3	Phenylpropanoids
62	Coniferin	POS	1.212	360.16528	C16 H22 O8	Phenylpropanoids
63	Scopoletin	POS	7.836	193.04979	C10 H8 O4	Phenylpropanoids
64	Trans-anethole	POS	13.081	131.08563	C10 H12 O	Phenylpropanoids

Key compounds identified in RPE included quercetin, naringenin, kaempferol, astragalin, taxifolin, isorhamnetin, rutin, α-linolenic acid, and linoleic acid. Quercetin is a strong natural antioxidant that mitigates hepatic oxidative stress, inflammation, and steatosis induced by alcohol, carbon tetrachloride, and other agents^29,30. Naringenin modulates hepatic alcohol-metabolizing enzymes, reduces systemic oxidative stress, and improves alcoholic fatty liver disease³¹. Kaempferol and its glycoside astragalin (kaempferol-3-O-glucoside) show hepatoprotective potential due to their antioxidant and anti-inflammatory properties, reducing toxin-induced hepatocyte injury^32,33. Taxifolin, a natural flavonoid, has attracted widespread attention for its strong antioxidant, anti-inflammatory, and hepatoprotective activities^34,35. Isorhamnetin, a methylated derivative of quercetin, shows enhanced bioavailability with similar anti-inflammatory and liver-protective efficacies³⁶. Rutin and its metabolite isoquercetin, which exert their effects following in vivo hydrolysis, can stabilize hepatocyte membranes and attenuate liver injury³⁷. α-Linolenic acid and linoleic acid, as n-3 and n-6 polyunsaturated fatty acid precursors, respectively, contribute to improved lipid metabolism and inflammatory regulation^38,39. Among these, quercetin, kaempferol, and naringenin deserve special attention. They have been repeatedly identified as core flavonoid constituents of rapeseed pollen in independent chemical studies^40–42. Moreover, they possess the strongest published evidence specifically against alcoholic liver injury, including modulation of alcohol-metabolizing enzymes, alleviation of oxidative stress, and suppression of inflammatory responses^29–31,33. Therefore, these three flavonoids are considered the most likely primary active components underlying the hepatoprotective effect of RPE. The other identified flavonoids and fatty acids may contribute synergistically or via complementary pathways.

Effects of RPE on serum AST, ALT, and TG levels in mice

AST and ALT are intracellular enzymes predominantly localized in hepatocyte cytoplasm. Alcohol exposure induces hepatocyte membrane damage, leading to increased permeability and subsequent leakage of these enzymes into the bloodstream. Therefore, increased serum ALT and AST levels directly reflect the extent of hepatocellular injury and serve as important indicators for assessing liver damage severity³⁹.

As shown in Fig. 2A and B, serum AST and ALT levels were significantly elevated in the model group compared with the normal control (^##p < 0.01), indicating successful establishment of alcohol-induced liver injury. In comparison with the model group, serum AST and ALT levels were significantly reduced in both the DHM group and the low- and high-dose RPE groups (^*p < 0.05 and ^**p < 0.01).

Fig. 2.

Effects of RPE on serum levels of AST (A), ALT (B), and TG (C). Data are presented as mean ± standard deviation (x̄ ± SD), with n = 10 per group, ^##p < 0.01 compared with the normal control group; ^*p < 0.05 and ^**p < 0.01 compared with the model group. Statistical significance was determined using one-way ANOVA.

Alcoholic liver injury is closely associated with dysregulated lipid metabolism and elevated serum lipid levels. TG is an important indicator of hepatic injury, as increased TG levels lead to excessive lipid accumulation or impaired lipid metabolism in the liver⁴³. Numerous studies have shown that acute alcohol exposure damages the structure and function of cellular organelles and enzymes in hepatocytes, impairs mitochondrial β-oxidation of fatty acids, and induces lipid peroxidation, ultimately resulting in TG accumulation⁴⁴.

As shown in Fig. 2C, serum TG levels were significantly higher in the model group than in the normal control group (^##p < 0.01). TG levels were significantly decreased in the DHM group and both the low- and high-dose RPE groups (^*p < 0.05 and ^**p < 0.01) relative to the model group.

These results demonstrate that RPE exerts protective effects against alcohol-induced liver injury and lipid metabolic disturbance through modulation of serum AST, ALT, and TG levels.

Effects of RPE on hepatic levels of MDA, SOD, and GSH-Px in mice

Ethanol-induced liver injury is strongly associated with oxidative stress, which is a key pathological mechanism in disease progression⁴⁵. Excessive ethanol intake increases the production of reactive oxygen species (ROS) due to incomplete metabolism, disrupting the body’s antioxidant defense system. This results in oxidative stress, hepatocyte apoptosis, lipid peroxidation, and ultimately liver injury⁴⁶. In the hepatic antioxidant system, SOD and GSH-Px work in concert to maintain redox homeostasis⁴⁷. MDA, a lipid peroxidation product generated under oxidative stress, is positively correlated with the extent of hepatocyte injury⁴⁸.

As shown in Fig. 3A-C, hepatic MDA levels were significantly elevated, whereas SOD and GSH-Px levels were significantly reduced in the model group compared with the normal control group (^##p < 0.01). This indicates the successful induction of oxidative stress and confirms the establishment of the alcoholic liver injury model.

Fig. 3.

Effects of RPE on hepatic levels of MDA (A), SOD (B), and GSH-Px (C) in mice. Data are presented as mean ± standard deviation (x̄ ± SD), with n = 10 per group, ^##p < 0.01 compared with the normal control group; ^*p < 0.05 and ^**p < 0.01 compared with the model group. Statistical significance was determined using one-way ANOVA.

Treatment with DHM and both low- and high-dose RPE significantly reduced hepatic MDA levels (^*p < 0.05 and ^**p < 0.01) and significantly increased SOD and GSH-Px levels (^*p < 0.05 and ^**p < 0.01), except for GSH-Px in the low-dose RPE group, compared with the model group.

These results demonstrate that RPE ameliorates alcohol-induced liver injury by modulating hepatic levels of MDA, SOD, and GSH-Px.

Effects of RPE on hepatic levels of ADH and ALDH in mice

Alcohol dehydrogenase (ADH) catalyzes the conversion of ethanol to acetaldehyde and is a key enzyme regulating the rate of alcohol metabolism, influencing overall alcohol metabolism efficiency⁴⁹. Aldehyde dehydrogenase (ALDH) subsequently converts acetaldehyde, a toxic intermediate, into harmless acetate, which is further metabolized and excreted. ALDH activity is closely associated with acetaldehyde clearance, reducing its accumulation in the body and alleviating symptoms of alcohol toxicity^50,51. Therefore, ADH and ALDH activities are widely used as indicators of hepatic alcohol detoxification capacity.

As shown in Fig. 4A-B), hepatic ADH and ALDH levels were significantly reduced in the model group compared with the normal group (^##p < 0.01), indicating impaired alcohol metabolism following ethanol exposure. Treatment with the DHM group and both low- and high-dose RPE significantly increased ADH and ALDH levels (^*p < 0.05 and ^**p < 0.01), except for ALDH in the low-dose RPE group, compared with the model group.

Fig. 4.

Effects of RPE on hepatic levels of ADH (A) and ALDH (B) in mice. Data are presented as mean ± standard deviation (x̄ ± SD), with n = 10 per group, ^##p < 0.01 compared with the normal control group; ^*p < 0.05 and ^**p < 0.01 compared with the model group. Statistical significance was assessed using one-way ANOVA.

These results indicate that RPE promotes alcohol metabolism by enhancing ADH and ALDH activities, thus ameliorating alcoholic liver injury.

Effects of RPE on serum levels of TNF-α and IL-6 in mice

Excessive alcohol consumption can lead to liver injury and the production of inflammatory cytokines⁵². Studies have indicated a close association between alcoholic liver damage and levels of inflammatory mediators. Interleukin-6 (IL-6) primarily mediates inflammatory responses, while tumor necrosis factor-alpha (TNF-α) promotes neutrophil migration and the generation of neutrophil-derived proteases and reactive oxygen species, resulting in hepatocyte damage⁵³.

As shown in Fig. 5A-B, serum levels of TNF-α and IL-6 were significantly elevated in the model group compared with the normal control group (^##p < 0.01), indicating successful induction of a strong inflammatory response. Compared with the model group, both the DHM group and the low- and high-dose RPE groups showed significant reductions in serum TNF-α and IL-6 levels (^*p < 0.05 and ^**p < 0.01). These results suggest that rapeseed pollen extract exerts hepatoprotective effects by inhibiting the production of inflammatory factors in vivo.

Fig. 5.

Effects of RPE on serum levels of TNF-α (A) and IL-6 (B) in mice. Data are presented as mean ± standard deviation (x̄ ± SD) with n = 10 per group, ^##p < 0.01 compared with the normal control group; ^*p < 0.05 and ^**p < 0.01 compared with the model group. Statistical significance was determined using one-way ANOVA.

Effects of RPE on histopathological changes in mouse liver tissue

As shown in Fig. 6, liver tissue structure in the normal control group was intact. Hepatic lobules and nuclear structures were clearly defined, with hepatocytes arranged radially and orderly around the central vein. Hepatocytes appeared intact, with no evident pathological alterations. In the model group, the architecture of hepatic lobules and cellular nuclei was significantly disrupted. The central veins were severely deformed and disorganized, accompanied by hepatocellular edema, consistent with previously reported pathological features^54,55. Based on histopathological observations, the DHM group and high-dose RPE group displayed a relatively preserved hepatic architecture compared with the model group. Liver sinusoids appeared closer to normal size, with no obvious inflammatory cell infiltration. Hepatocytes were regularly and neatly arranged, and hepatic sinusoids showed no significant dilation or compression. These results indicate that RPE exerts a protective effect against acute alcohol-induced liver injury by alleviating histopathological damage.

Fig. 6.

Effects of RPE on histopathological changes in mouse liver tissue (×200).

Effects of RPE on hepatic Keap1, Nrf2, and NQO1 expression detected by immunohistochemistry

Oxidative stress leads to excessive production of ROS. When the balance between oxidation and antioxidation is disrupted, it results in cellular and tissue damage⁵⁶. The Keap1/Nrf2/NQO1 signaling pathway is a key endogenous defense mechanism against oxidative stress and represents a primary cellular response to oxidative challenges^57–59. Under normal physiological conditions, Nrf2 is sequestered in the cytoplasm through its binding to the key regulatory protein Keap1. However, under oxidative stress, the interaction between Nrf2 and Keap1 is disrupted, allowing Nrf2 to translocate into the nucleus. In the nucleus, Nrf2 functions as a crucial transcriptional activator, inducing the expression of multiple antioxidant genes, including NQO1, HO-1, and SOD^60,61, which play essential roles in mitigating oxidative damage. Numerous studies have reported that the Keap1-Nrf2 signaling pathway is critically involved in the pathogenesis of alcoholic liver injury^62–65.

As shown in Fig. 7A-B, immunohistochemical staining intensities of Keap1, Nrf2, and NQO1 were significantly decreased in the model group compared with the normal group (p < 0.01). All treatment groups showed significantly increased expression levels of these proteins compared with the model group (^*p < 0.05 and ^**p < 0.01), except for Nrf2 in the low-dose RPE group, which showed no statistically significant difference (p > 0.05).

Fig. 7.

Effects of RPE on the immunohistochemical staining of Keap1, Nrf2, and NQO1 in mouse liver tissue (×200). (A) Representative immunohistochemical images (×200). (B) Semi-quantitative analysis of staining intensity expressed as integrated optical density. Data are presented as mean ± standard deviation (x̄ ± SD) with n = 5 per group, ^##p < 0.01 compared with the normal control group; ^*p < 0.05 and ^**p < 0.01 compared with the model group. Statistical significance was determined using one-way ANOVA.

In conclusion, our IHC results demonstrate increased expression of Keap1, Nrf2, and NQO1 in RPE-treated mice. However, the simultaneous increase in Keap1 and Nrf2 warrants further discussion. Our immunohistochemical results showed a simultaneous increase in Keap1 and Nrf2 expression in RPE-treated mice. An apparent paradox in this observation is that it seems to contradict the classical model where Nrf2 activation requires Keap1 downregulation. However, this is not unprecedented. Da et al.⁶⁶ recently reported that in alcoholic liver injury rats, treatment with Tetracera asiatica flavonoids significantly upregulated the transcription of both Keap1 and Nrf2, and still concluded activation of the Keap1/Nrf2/HO-1 antioxidant pathway. Mechanistically, Keap1 function is primarily regulated by oxidative modification of its cysteine residues (C151, C273, C288) upon ROS exposure, which disrupts its ability to bind Nrf2 without necessarily reducing its protein abundance⁶⁷. Therefore, increased Keap1 protein level, as detected by IHC, does not imply maintained repressive activity; it may instead reflect a compensatory transcriptional response to sustained oxidative stress. Importantly, IHC measures total protein and cannot assess Nrf2 nuclear translocation, which is the definitive indicator of pathway activation. Future studies employing Western blot analysis of nuclear and cytoplasmic fractions will be required to directly confirm Nrf2 activation by RPE.

Additionally, Several limitations should be acknowledged. First, although our IHC results showed increased Nrf2 and NQO1 expression, we did not perform Western blot for nuclear Nrf2 translocation. These are important limitations of the present study Future investigations should include orthogonal methods such as Western blot and qPCR to definitively establish the activation of the Keap1/Nrf2/NQO1 pathway by RPE.

Conclusion

This study investigated the protective effects of RPE against alcoholic liver injury using an animal model. The results demonstrated that RPE significantly reduced serum levels of ALT, AST, TG, TNF-α, and IL-6 in mice. It increased the activities of ADH, ALDH, and SOD in liver tissue while decreasing MDA levels. Furthermore, RPE treatment significantly enhanced the immunohistochemical expression of Keap1, Nrf2, and NQO1 in liver tissues. These results suggest that RPE exerts hepatoprotective effects against alcoholic liver injury, potentially through activation of the Keap1/Nrf2/NQO1 signaling pathway. This study clarifies the pharmacological effects of RPE and provides preliminary mechanistic evidence supporting its development as a hepatoprotective agent.

Author contributions

Y.X. and X.H.: writing – original draft, methodology, investigation, formal analysis and conceptualization. C. Y and L.W.: methodology, visualization and funding acquisition; A. J.: writing – review & editing and funding acquisition; C. W.: investigation and resources. All authors have thoroughly reviewed and provided their consent to the final version of the manuscript for publication.

Funding

This work was supported by the Postgraduate Education Reform and Quality Improvement Project of Henan Province (YJS2026YBGZZ62); Henan Province Science and Technology Research Project (242102311266); Zhengzhou Science and Technology Research project (2024TLZDJH017); Henan Provincial Key Discipline Initiative (2023 − 414), sponsored by Department of Education of Henan Province, and Henan Provincial Department of Education Funding Program for Discipline and Specialty Development in Private Regular Institutions of Higher Education(2022 − 219), and Henan Provincial Medical Education Research Project (WJLX2025208).

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.