Ripe fruits of the Chinese olive variety ‘Tanxiang No.1’ were collected in September 2022 from Puning, Guangdong Province, China (116°09′58″E, 23°17′53”N). The fruits were thoroughly washed with water and pitted. Subsequently, the olives were crushed and stored at −20 °C for further analysis.

Dimethyl sulfoxide (DMSO) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) containing 4,500 mg/L glucose, penicillin G (100 units/mL) and streptomycin (100 μg/mL) were purchased from Thermo Fisher Scientific Inc. (Massachusetts, United States). TRNzol Universal Reagent, the miRNAcute miRNA Isolation Kit, FastKing gDNA Dispelling RT SuperMix, the miRNA 1st Strand cDNA Synthesis Kit, TIANScript II RT Kit and RNase-free ddH₂O were purchased from TIANGEN Biotech Co., Ltd. (Beijing, China). SYBR Green Master Mix was procured from Nanjing Vazyme Biotech Co., Ltd. (Nanjing, China). Oil Red O and the BCA protein assay kits were purchased from Beyotime Biotech Inc. (Shanghai, China). Other reagents were of analytical grade. The Triglyceride (TG) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Rabbit anti-phosphorylated AMPK alpha (Thr172; pAMPK) and rabbit anti-AMPK primary antibodies were purchased from Affinity Biosciences (OH, United States). Rabbit anti-β-actin primary antibody was purchased from ABclonal Biotechnology Co., Ltd. (MA, United States). HRP-conjugated secondary antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (PA, United States). Other reagents were of analytical grade.

OP was prepared using an ethanol-based extraction procedure adapted from published approaches (13), with modifications optimized to enhance extraction efficiency and maximize phenolic yield. Briefly, olives were extracted with 70% ethanol at a ratio of 1:25 (w/v) using ultrasound (370 W for 20 min) followed by microwave treatment (500 W for 3 min). The extracts were centrifuged at 6000 rpm for 5 min and the supernatants were collected. Solvent was removed by rotary evaporation and the residue was freeze-dried to yield the extract used in this study. We note that extraction conditions can influence chemical composition; therefore, our conclusions are specific to this preparation.

HepG2 cells, obtained from the Cell Resource Center at the Institute of Basic Medical Sciences (Beijing, China), were cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin. The cells were incubated at 37 °C in a 5% CO₂ incubator (Esco Technologies Pty Ltd., Singapore).

The viability of HepG2 cells was assessed using the methylene blue assay (14). The cells were inoculated into 96-well plates at a concentration of 2.5 × 10⁵ cells per well and cultured at 37 °C with 5% CO₂ for 24 h. The growth medium was then replaced with sodium oleate at concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1,000 μM, and the cells were cultured for an additional 24 h. Following this, 50 μL of methylene blue solution (composed of HBSS, 1.25% (v/v) glutaraldehyde and 0.6% (w/v) methylene blue) was added and incubated at 37 °C for 1 h. Subsequently, 100 μL of elution buffer (50% ethanol, 49% PBS and 1% acetic acid; v/v/v) was added to each well and shaken for 15 min. The cell viability for each treatment was expressed as a percentage of the absorbance value at 595 nm of the treated cells divided by that of the control group.

Sodium oleate solution was prepared according to the method described by Zhang et al. (15) with minor modifications. The experimental treatments were as follows: (1) normal group (control); (2) SO-Treated group treated with 400 μM sodium oleate (SO-Treated); (3) low-concentration OP group treated with 400 μM sodium oleate and 50 μg/mL OP (OP50); (4) medium-concentration OP group treated with 400 μM sodium oleate and 100 μg/mL OP (OP100); (5) high-concentration OP group treated with 400 μM sodium oleate and 250 μg/mL OP (OP250). All groups were co-treated for 24 h at 37 °C in a 5% CO₂ humidified atmosphere, after which the cells were collected for further study.

HepG2 cells were washed three times with PBS and fixed with 4% formaldehyde for 30 min. Following fixation, the cells were washed with 60% isopropanol and stained with Oil Red O solution for 30 min at room temperature. The cells were then washed once with 60% isopropanol and subsequently with PBS. Fat droplets within the cells were observed using an inverted microscope (Nikon Eclipse, Melville, NY, United States). For quantification, 1 mL of isopropanol was added to each well to elute the stain, and absorbance was measured at 510 nm. Intracellular TG content was assessed using a commercial assay kit (Nanjing Jiancheng Technology Co., Ltd., Nanjing, China). Cell lysates were mixed with the enzyme working solution, incubated at 37 °C for 10 min, and absorbance was measured at 550 nm. A standard curve was prepared by serial dilution of the provided calibrator. TG concentration was calculated from the curve based on sample absorbance.

Total proteins were extracted from lysed cells using RIPA lysis buffer, and the protein content of the lysate was quantified using the BCA Kit. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was immersed in a PBS containing 5% skimmed milk powder and incubated on a shaker for 30 min. Primary antibodies were added and incubated overnight at 4 °C, including anti-AMPK (Rabbit mAb, 1:1,000), anti-pAMPK (Thr172; Rabbit mAb, 1:1000), and anti-β-actin (Rabbit mAb, 1:50,000). After three washes with PBS containing Tween 20 (PBST), the membranes were incubated with HRP-conjugated secondary antibodies (1:5,000) for 1 h at room temperature. Finally, enhanced chemiluminescence (ECL) detection reagent was employed to visualize the proteins. Western blot images were analyzed using Image-Pro Plus software.

Total RNA was isolated from HepG2 cells using the TRNzol Universal Reagent. The reverse transcriptase (RT) reaction (20 μM) was performed with 2 μg of total RNA, 4 μL of 5 × FastKing-RT SuperMix and RNase-free ddH₂O. MicroRNA was extracted from HepG2 cells using the miRNAcute miRNA Isolation Kit, and the RT reaction (20 μM) was conducted using the miRNA 1st Strand cDNA Synthesis Kit, following the manufacturer’s instructions and employing a stem-loop method. Quantitative polymerase chain reaction (qPCR) detection was executed using SYBR Green Master Mix. The primers used for real-time PCR analysis were listed in Supplementary Tables S1, S2. The expression levels of genes and miRNAs were normalized to GAPDH and U6, respectively, and the expression of the respective genes or miRNAs was quantified using the 2^−ΔΔCt method.

The phenolic composition was determined using an ExionLC™ UHPLC system coupled to a QTRAP^® 6,500⁺ triple quadrupole mass spectrometer (SCIEX, MA, United States). A Waters Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) was selected due to its moderate pH range (2.0–8.0) and ability to strongly retain highly polar organic compounds and metabolites. The mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was 100% acetonitrile. The elution followed a linear gradient from 2 to 50% B for 0–10 min, 50 to 95% B for 10–11 min, remained at 95% B for 2 min (11–13 min), 95 to 2% B for 13–13.1 min, and finally remained at 2% B for 2 min. The flow rate was set at 400 μL/min, and the sample injection volume was 2 μL. The temperature of the column chamber was maintained at 40 °C. The ESI source conditions were set as follows: the capillary voltage was set at +5,500 V or −4,500 V in positive or negative mode, respectively; the curtain gas pressure was set at 35 psi; the temperature was set at 400 °C; the ion source gas pressure was set at 60 psi; and the DP was set at ±100 V.

All data were expressed as the mean ± standard deviation (SD, n = 3). Statistical analyses were performed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons tests with IBM SPSS Statistics version 26 software. Differences were considered significant at p < 0.05.

Given that HepG2 cells retain most hepatic metabolic functions (16), this study employed a sodium oleate-induced steatosis model to evaluate the lipid-lowering effects of OP in vitro. Prior to model establishment, the cytotoxicity of both OP and sodium oleate was assessed using the methylene blue assay.

As shown in Figure 1a, OP concentrations ranging from 50 to 250 μg/mL did not induce any significant decline in HepG2 cell viability (p > 0.05), indicating negligible cytotoxicity and confirming that these concentrations would not interfere with subsequent molecular experiments. However, concentrations exceeding 250 μg/mL led to a gradual decrease in cell viability. Figure 1b illustrated the cytotoxicity of sodium oleate, showing that cell viability remained comparable to that of untreated controls between 100 and 400 μM. Therefore, 400 μM sodium oleate was selected as the optimal concentration for model induction. Furthermore, to rule out cytotoxicity from the combined treatment, cell viability was assessed under co-treatment conditions (sodium oleate + OP). As shown in Figure 1c, no significant reduction in viability was observed across the tested concentrations. It should be noted that the OP concentrations used in this study (50–250 μg/mL) were optimized for an in vitro mechanistic investigation. While these concentrations may be higher than the typical physiological plasma levels that can be achieved through dietary intake alone, they were selected to produce measurable biochemical responses and to establish clear concentration -response relationships without inducing cytotoxicity.

To evaluate the effects of OP on intracellular lipid deposition, Oil Red O staining and triglyceride (TG) assays were performed. As shown in Figure 2a, minimal lipid droplets were observed in the control group, whereas the SO-Treated group exhibited a pronounced increase in red-stained cytoplasmic lipid droplets. Quantitative TG analysis (Figure 2b) revealed a fivefold increase in intracellular TG levels after 24 h of 400 μM sodium oleate induction compared to the control. However, OP treatment markedly reduced TG accumulation in a concentration-dependent manner.

Consistent results were observed in lipid staining (Figure 2c), where OP supplementation alleviated the sodium oleate–induced increase in intracellular lipid content (p < 0.05). These findings align with previously reported Oil Red O staining patterns in steatotic hepatocytes (9), collectively demonstrating the lipid-inhibitory potential of OP.

To explore the regulatory effects of OP on hepatic lipogenesis, mRNA expression levels of SREBP-1c, ACC1, FASN, and DGAT2 were quantified. SREBP-1c serves as the master transcription factor that regulates the expression of genes involved in fatty acid and TG synthesis. As shown in Figure 3a, there was a 10-fold increase in SREBP-1c expression in the SO-Treated group, while treatment with OP50 significantly reduced expression by 36%. The levels of SREBP-1c were further decreased by 78 and 94% in the OP100 and OP250 groups, respectively, nearly restoring them to normal levels.

ACC1 is essential for catalyzing fatty acid biosynthesis and serves as a significant regulator of lipogenesis (17). Figure 3b illustrated a 4.6-fold increase in ACC1 expression in the SO-Treated group. Treatment with OP50 reversed this increase by 43%. ACC1 levels were further diminished by 84 and 96% in the OP100 and OP250 groups, respectively, bringing them to approximately 25 and 80% lower than those in the control group. FASN is a key enzyme in the final step of fatty acid synthesis, converting free fatty acids into long-chain fatty acids and upregulating TG levels, which contributes to intracellular lipid accumulation (18). As shown in Figure 3c, sodium oleate significantly impacted FASN expression, with OP50 treatment resulting in a 17% reduction. FASN levels were further decreased by 79 and 94% in the OP100 and OP250 groups, respectively.

DGAT2 plays a crucial role in fat digestion and absorption and is vital for converting diacylglycerol to TG (19). Figure 3d indicates a 4.8-fold increase in DGAT2 expression in the SO-Treated group, which was significantly reduced by 34% following OP50 treatment. Similar decreases were observed in the OP100 and OP250 groups, with expressions approximately 79% lower than in the SO-Treated group and 69% lower than in the control group.

The SREBP-1c/ACC1/FASN pathway is a classical lipogenic cascade that tightly regulates cellular lipid synthesis. Under physiological conditions, a delicate balance is maintained between lipid production and clearance. However, when this balance is disrupted, excessive lipid accumulation occurs, promoting hepatic steatosis and metabolic dysfunction (20). Among the key regulators of this pathway, ACC1 is essential for maintaining the balance between lipogenesis and lipolysis in the liver (21). Under conditions of lipid oversynthesis, ACC1 is activated and dissociates from SREBP-1c, subsequently inducing the expression of downstream target genes such as FASN to promote fatty acid synthesis. The mRNA levels of SREBP-1c, ACC1 and FASN —key regulators of fatty acid biosynthesis—are closely associated with lipid accumulation in hepatocytes (16). Furthermore, SREBP-1c acts as a transcription factor for both ACC1 and FASN, facilitating the conversion of excess carbohydrates into fatty acids via acetyl-CoA. Consequently, inhibition of SREBP-1c, ACC1, and FASN expression may alleviate hepatic steatosis and decelerate the progression of fatty liver disease. In addition, DGAT2, which is involved in triglyceride synthesis and lipid absorption, also contributes significantly to hepatic triglyceride accumulation (19). Collectively, OP treatment significantly reduced lipid accumulation. This reduction was accompanied by the coordinated downregulation of SREBP-1c and its downstream targets (ACC1, FASN, and DGAT2), consistent with the suppression of the lipogenic pathway.

To further elucidate the mechanisms underlying OP’s lipid-lowering effects, the mRNA expression of four major β-oxidation regulators—PGC-1α, PPARα, CPT-1A, and ACOX1—was analyzed following 24 h of treatment (Figure 4).

PGC-1α is a master transcription factor that regulates the expression of genes associated with β-oxidation (22). As shown in Figure 4a, PGC-1α expression was reduced by 56% in the SO-Treated group, indicating a significant change; however, treatment with OP50 did not reverse this decline. In contrast, PGC-1α levels increased by 57 and 95% in the OP100 and OP250 groups, respectively, compared to the SO-Treated group, although they remained significantly lower than in the control group.

The nuclear receptor PPARα plays a central role in mediating hepatic fatty acid metabolism. As illustrated in Figure 4b, PPARα expression decreased by 38% in the SO-Treated group, while OP50 treatment nearly restored it to normal levels. PPARα levels were further elevated in the OP100 and OP250 groups, which were approximately 41 and 61% higher than those in the control group, respectively. Figure 4c showed that CPT-1A expression decreased by 44% in the SO-Treated group; however, OP50 treatment reversed this decline, bringing levels back to normal. In the OP100 and OP250 groups, CPT-1A levels increased by approximately 45 and 73%, respectively, compared with the control group. In Figure 4d, sodium oleate significantly affected ACOX1 mRNA expression, with OP50 treatment leading to a 51% increase. However, there were no significant changes in ACOX1 levels in the OP100 and OP250 groups compared to the control group.

The homeostasis of lipid metabolism depends on a coordinated balance among lipogenesis, lipolysis, and fatty acid β-oxidation. The PGC-1α/PPARα signaling axis is a well-characterized pathway that enhances lipolysis and upregulates β-oxidation (23). PPARα, in particular, serves as a key transcriptional regulator of genes involved in fatty acid β-oxidation. Its activation has been shown to alleviate sodium oleate-induced lipid accumulation in HepG2 cells, leading to reduced triglyceride levels and overall lipid content. Under conditions of lipid overload, PPARα is activated and cooperates with PGC-1α to transactivate downstream targets such as CPT-1A and ACOX1, thereby promoting fatty acid β-oxidation (24). Thus, the expression of PGC-1α, PPARα, CPT-1A, and ACOX1—central regulators of β-oxidation—collectively contributes to the suppression of lipid accumulation in hepatocytes.

In the present study, treatment with 400 μM sodium oleate significantly decreased the expression of fatty acid β-oxidation genes, including PGC-1α, PPARα, CPT-1A and ACOX1. OP treatments effectively counteracted this suppression and, at concentrations of 100 and 250 μg/mL, elevated the expression of PGC-1α, PPARα and CPT-1A in a concentration-dependent manner compared with the SO-Treated group. Although ACOX1 expression was also significantly increased by OP, no clear concentration-dependent effect was observed. These results indicate that OP treatment effectively restores the expression of β-oxidation-related genes. The concurrent upregulation of PGC-1α, PPARα, and their downstream targets (CPT-1A and ACOX1) suggests that OP may exert its effects by modulating this transcriptional network, although direct transcriptional activation remains to be verified.

To examine whether OP treatment is associated with changes in AMPK signaling, the protein expression levels of AMPK and p-AMPK were measured. AMPK serves as a key energy sensor and metabolic regulator that is rapidly activated during energy deprivation (25).

As shown in Figure 5, Western blot analysis revealed a significant decrease in p-AMPK protein levels in the SO-Treated group compared to the Control group. In contrast, OP treatment considerably increased p-AMPK expression in a concentration-dependent manner. Consistent with these protein findings, mRNA levels of PGC-1α and PPARα were significantly downregulated in the SO-Treated group, while OP administration markedly restored their expression. Conversely, SREBP-1c mRNA expression was elevated in the SO-Treated group and significantly suppressed by OP treatment. We further assessed key genes involved in lipogenesis and fatty acid oxidation. The SO-Treated group exhibited upregulation of lipogenic genes (ACC1, FASN and DGAT2) and downregulation of fatty acid β-oxidation genes (CPT-1A and ACOX1; Figures 3b–d, 4c,d). OP treatment reversed these alterations, suppressing lipogenic gene expression and enhancing fatty acid oxidation-related transcripts. These changes occurred in parallel with increased AMPK phosphorylation.

These findings are consistent with previous reports that Chinese olive preparations reduce lipid accumulation and are accompanied by AMPK activation in hepatic models (9). AMPK, a heterotrimeric complex composed of a catalytic α subunit and two regulatory β and γ subunits, serves as a master regulator of cellular metabolism (26). Its activation alleviates lipid accumulation through dual mechanisms: suppression of lipogenesis and promotion of lipid catabolism. Phosphorylated AMPK inhibits lipogenesis by inactivating key regulators such as SREBP-1c and ACC1 (27). SREBP-1c is a major transcription factor governing fatty acid desaturation and lipogenic enzyme expression, while ACC1 catalyzes a critical step in de novo lipogenesis and concurrently suppresses fatty acid oxidation (28). In line with this, our study showed that OP enhanced p-AMPK expression while reducing mRNA levels of SREBP-1c, ACC1, and its target gene FASN in sodium oleate-induced HepG2 cells. These results align with reported AMPK-mediated repression of SREBP-1c/FASN signaling in NAFLD models.

Beyond suppressing lipogenesis, AMPK activation also promotes fatty acid β-oxidation via upregulation of PPARα and its target genes CPT-1A and ACOX1. Fatty acid oxidation plays an essential role in maintaining hepatic lipid homeostasis, and its dysregulation leads to excessive triglyceride accumulation and steatosis (27). PPARα is a nuclear receptor that transcriptionally activates fatty acid catabolic genes, thereby reducing hepatic lipid burden (29). CPT-1A, a PPARα target, acts as the rate-limiting enzyme in mitochondrial β-oxidation. In the present study, OP treatment significantly elevated the expression of PPARα and CPT-1A compared to the SO-Treated group.

Overall, OP reduced lipid accumulation in HepG2 cells and was accompanied by increased AMPK phosphorylation, decreased SREBP-1c-related lipogenic transcripts, and increased PPARα-related β-oxidation transcripts. Further mechanistic validation is required to establish pathway causality.

MiRNAs are critical post-transcriptional regulators of lipid metabolism. To assess whether OP treatment is associated with miRNA changes, the expression levels of miR-122 and miR-21 were analyzed following 24 h of treatment (Figure 6).

In the present study, sodium oleate induction led to a 28-fold upregulation of miR-122 in the SO-Treated group (Figure 6a). This observation is consistent with recent findings by Hu et al. (30), who reported that high-fat diet and fatty acid exposure significantly upregulate hepatic miR-122 expression in vivo and in vitro. Furthermore, previous studies have shown that the overexpression of miR-122 facilitates lipid accumulation by targeting genes such as SREBP-1c and DGAT2 (31). OP treatment significantly reversed this increase in a concentration-dependent manner, reducing miR-122 expression by 30, 49, and 67% in the OP50, OP100, and OP250 groups, respectively. Consistent with the reported role of miR-122, the mRNA levels of SREBP-1c and DGAT2 were also elevated upon sodium oleate induction (Figures 3a,d), accompanied by upregulation of downstream lipogenic genes. These results are consistent with prior reports linking miR-122 to lipogenic regulation (6). Because SREBP-1c and DGAT2 have been reported as miR-122–related targets, the parallel changes observed here suggest a possible association between OP-induced miR-122 reduction and decreased lipogenic gene expression, however, causality remains to be experimentally validated.

We further examined the effect of OP on miR-21, another miRNA implicated in lipid homeostasis. As shown in Figure 6b, miR-21 expression increased by 7.5-fold in the SO-Treated group. OP50 treatment significantly reduced its level by 30%, and further decreases of 69 and 72% were observed in the OP100 and OP250 groups, respectively, though the response was not strictly concentration-dependent. PPARα has been identified as a downstream target of miR-21 (4). Its overexpression suppresses PPARα signaling and attenuates β-oxidation in hepatocytes. OP treatment decreased miR-21 levels and increased PPARα and related β-oxidation transcripts in parallel. These findings suggest a potential link between the modulation of miR-122/miR-21 and the lipid-lowering effects of OP. However, whether the downregulation of these miRNAs is the primary driver of the observed phenotypic changes requires further investigation using miRNA mimics or inhibitors.

Hepatic lipid accumulation is a hallmark of metabolic dysfunction and a key contributor to the development of fatty liver and related metabolic disorders. Previous studies have demonstrated that various natural extracts derived from fruits, vegetables, grains, and their by-products, such as pomegranate leaf extract (32), associated with improvements in lipid metabolism. These extracts are often characterized by complex phytochemical compositions, among which polyphenolic compounds represent a major constituent class.

In the present study, UPLC–MS/MS profiling identified 39 phenolic compounds in OP, comprising seven phenolic acids, six tannins, 18 flavonoids, and eight lignans and coumarins. The reported biological activities of representative compounds are summarized in Table 1. Given the compositional complexity of the extract, these constituents may collectively contribute to the lipid-associated effects observed in HepG2 cells; however, individual contributions cannot be distinguished based on the current experimental design.

Among the detected compounds, 17 constituents, including gallic acid, methyl brevifolincarboxylate, and ellagic acid-4-O-xyloside, showed relatively high signal responses in the UPLC–MS/MS analysis and were therefore discussed as representative features of the phenolic profile. Previous studies have reported that some of these compounds are associated with lipid metabolism–related cellular processes, such as AMPK phosphorylation and regulation of lipogenic gene expression (12, 33). In the present study, such findings provide background information rather than direct mechanistic evidence linking individual compounds to the observed lipid-associated effects.

Consistent with the observations of Yeh et al. (9), our results demonstrate that treatment with a Chinese olive extract is associated with reduced lipid accumulation in hepatocytes. Compared with previous studies, the present work provides additional phytochemical characterization of the extract used, allowing discussion of candidate phenolic features that may be relevant to the observed phenotype, while acknowledging that this study does not establish which compounds are necessary or sufficient to elicit the effect.

It should also be recognized that the phytochemical composition of Chinese olive can vary with cultivar, geographic origin, and cultivation conditions. Here, we focused on the commercially dominant cultivar ‘Tanxiang No.1’ to ensure experimental consistency, and the reported phenolic profile should therefore be interpreted as representative of this specific cultivar. Broader sampling across multiple origins, together with quantitative analyses, will be valuable in future studies to further characterize compositional variability and support standardization of polyphenol-rich Chinese olive extracts.