Molecular hybridization of syringaldehyde and fibrate pharmacophores yields a novel derivative with potent, multi-target lipid-lowering activity

Abstract

This study aimed to reduce the hepatotoxicity of traditional fibrate drugs. A molecular hybridization strategy was adopted to synthesize a series of syringaldehyde-based fibrate derivatives. Screening revealed that T2 exhibited the most pronounced reduction in TG and TC levels in a dose-dependent manner in the Triton WR 1339-induced hyperlipidemia model. Moreover, a high-fat dietary regimen (HFD)-induced hyperlipidemia model was utilized to assess the lipid-lowering potential of T2. The findings indicated that T2 exerted a significant lipid-lowering effect and reduced the ALT and AST levels, thereby ameliorating pathological alterations in the liver tissue. Additionally, the activity of SOD was significantly enhanced. It was observed that the content of the lipid peroxidation product MDA was reduced considerably, and the levels of IL-6 and TNF-α were decreased. These changes suggest that T2 is capable of exerting anti-inflammatory and antioxidant effects. Findings from research on the lipid-lowering mechanism indicate that T2 enhances PPAR-α protein expression in the liver and interacts strongly with its active site. These results suggest that T2 is a potential novel multifunctional lipid-lowering fibrate candidate compound.

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1. Introduction

In recent years, with changes in lifestyle and the aging of the population, the incidence and prevalence rates of hyperlipidemia (HLP) have demonstrated a marked upward trend.¹ This condition is always associated with the emergence and progression of cardiovascular diseases and other grave ailments,² positioning it as a worldwide health concern. HLP is characterized by an abnormal rise in blood lipids, such as triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and a reduced level of high-density lipoprotein cholesterol (HDL-C). The primary indication of this condition is an unusual surge in the levels of these lipids. Epidemiological studies conducted locally and globally have found that hypertriglyceridemia (HTG), a common lipid metabolism abnormality, can promote the formation of atherosclerosis and then lead to coronary heart disease, myocardial infarction, stroke, and a series of severe cardiovascular diseases (CVD).^2–6 It is not just an independent risk factor for acute pancreatitis but also a complex interaction with type 2 diabetes.⁷ The elevation of TG and the TG/HDL-C ratio have been identified as independent risk factors for type 2 diabetes. HTG not only elevates the likelihood of developing type 2 diabetes but may also aggravate the progression of diabetes.^8,9 Additionally, HTG can activate the endoplasmic reticulum stress response in renal tubular cells, trigger cell apoptosis programs, and exacerbate kidney damage.^10–12 Therefore, the prevention and treatment of HTG are of profound significance.

Fibrate-based lipid-lowering drugs have significant clinical effects in treating HTG and are the preferred drugs for reducing TG. Commonly used fibrate drugs in clinical practice include chlorothalonil acid and benzyl chlorothalonil (Fig. 1).¹³ The primary mechanism of action of these agents involves activating peroxisome proliferator-activated receptor α (PPAR-α), leading to the positive regulation of lipoprotein lipase (LPL) expression. LPL promotes TG metabolism into fatty acids, reduces the TG content in lipoproteins, and improves blood lipid abnormalities. However, due to the single target, there are significant side effects, such as liver injury, indigestion, abdominal pain, headache, and myositis. Among them, liver injury is the most representative adverse reaction.^14,15 Recent research has shown that liver damage caused by fibrate-lowering drugs is closely related to oxidative stress, inflammation, and other effects.¹⁶ Therefore, it is vital to develop a novel class of lipid-lowering medications that not only lower blood lipids but also exert a hepatoprotective effect to meet the long-term treatment needs of patients with hyperlipidemia.

Fig. 1 Design of syringaldehyde-based fibrate derivatives using the molecular hybridization strategy.

Currently, the potential value of multi-target drug design in HTG therapy is gradually gaining attention.¹⁷ Naturally occurring substances are widely employed as precursor molecules in the quest for novel pharmaceutical agents. At the same time, techniques involving molecular fusion have found extensive applications,¹⁸ which can improve efficacy while reducing side effects, such as liver toxicity. Syringaldehyde (SA) is a phenolic compound found in the Araliaceae plant Acanthopanax senticosus.¹⁹ Contemporary investigations have indicated that SA displays a broad spectrum of biological effects, including antioxidant, anti-inflammatory, lipid-modulating, antibacterial, and antitumor properties.^20–22 The design of the compounds in this study is based on the common structural characteristics of fibrate compounds (the structure in the pink dashed circle). A molecular hybridization strategy was adopted to replace the aromatic oxygen structure in the fibrate compounds with SA (Fig. 1). SA was connected to the alkyl ester structure through ether bonds to design and synthesize syringaldehyde-based fibrate derivatives. The efficacy of the target compound in reducing lipid levels and protecting liver function was evaluated using acute hyperlipidemia models, molecular docking, chronic hyperlipidemia models, etc., in order to obtain candidate compounds with TG-lowering effects and mild liver damage.

2. Results

2.1 Chemistry

Syringaldehyde (SA) is dissolved in N,N-dimethylformamide (DMF), and K₂CO₃ and KI are added; the mixture is then reacted with bromoalkyl ester compounds a–g in an oil bath maintained at 80 °C (Fig. 2). The advancement of the reactions is detected using thin-layer plate chromatography. After the reaction is completed, the crude product is first obtained and subsequently purified by silica gel column chromatography using a solvent mixture of petroleum ether and ethyl acetate at ratios of either 5 : 1 or 8 : 1, resulting in the isolation of the target compounds T1–T7. The target compound was structurally characterized using HR-MS, ¹H-NMR, and ¹³C-NMR.

Fig. 2 Method for synthesizing the target compounds T1–T7.

2.2 Biological evaluation

2.2.1 Acute lipid-lowering activity evaluation. In comparison to the control group, TG and TC levels exhibited a notable increase in the model group (P < 0.01), indicating successful modeling. When contrasted with the model group, the T1–T7 group reduced TG levels by 31% (P < 0.01), 33% (P < 0.01), 30% (P < 0.01), 37% (P < 0.01), 44% (P < 0.01), 36% (P < 0.01), and 35% (P < 0.01), concurrently. The positive drug CF reduced TG levels by 34% (P < 0.01). A comparison of the TC levels in the T1–T7 groups with those of the model group revealed significant differences of 18% (P < 0.01), 33% (P < 0.01), 14%, 13%, 19%, 27% (P < 0.05), and 22%, respectively. A reduction of 22% in TC levels was also observed with the positive CF drug (Fig. 3). The results of the structure–activity relationship (SAR) study provide several key insights. For compounds with analogous carbon chain lengths (T1: linear propanoate vs.T2: branched 2-methylpropanoate), the introduction of a branched side chain (T2) significantly enhanced efficacy in reducing both TG and TC levels compared to its linear counterpart (T1). This suggests that the steric bulk and conformational rigidity at this position are favorable for activity. In contrast, although T3 has the same number of carbon atoms as T2, its linear butyl chain results in weaker activity, reinforcing the importance of branching over simple chain extension. For the longer-chain esters (T4–T7), a clear trend in TG reduction was not observed, indicating an optimal chain length range for activity. However, the efficacy in reducing TC levels generally increased with chain length from T4 to T6, suggesting that longer, more flexible aliphatic chains may interact more effectively with hydrophobic subpockets within the target binding site to influence cholesterol metabolism. The lack of a clear pattern for T7 may be due to excessive chain length, leading to unfavorable entropic penalties or steric clashes. Overall, the comprehensive TG and TC results indicate that T2 achieves the best lipid-lowering effect among the series.

Fig. 3 Impact of compounds T1–T7 on triglyceride (a) and total cholesterol (b) levels in a Triton WR-1339-induced hyperlipidemia mouse model. The administration time is a week, and the dosage for T1–T7 is 100 mg kg⁻¹ and that of clofibrate is 65 mg kg⁻¹. The data are presented as the mean ± SEM, n = 8. ^##P < 0.01 vs. control group. ^*P < 0.05, ^**P < 0.01 vs. model group.

2.2.2 Molecular docking. Fibrate-based lipid-lowering drugs mainly activate the PPAR-α protein to reduce TG content. In this study, syringaldehyde-based fibrate derivatives, which possess skeletal structures identical to those of fibrate compounds, were synthesized using a molecular heterocyclic approach.

Therefore, to preliminarily investigate the lipid-lowering effect of the target compounds, molecular docking technology was employed. This involved examining the interaction between these compounds and the PPAR-α protein, as well as assessing the binding affinity of compound T1–T7 for the protein using a scoring system. This study installed the docking module in MOE software on Win 11 and downloaded the PPAR-α protein file (ID code: 6KXX) from the PDB database. For docking studies, clofibrate was selected as the reference compound to interact with the PPAR-α protein. The binding scores, reflecting the affinity of target compounds T1–T7 for PPAR-α receptors, are presented in Table 1 and are as follows: −5.5054, −5.5480, −5.4305, −5.8633, −5.9863, −5.7964, and −5.6619. The docking score of clofibrate is −5.6776, and the docking score of natural ligand T02 is −5.1242. The molecular docking results are consistent with the TG results of the acute lipid-lowering assessment mentioned above.

Table 1 Rating of the PPAR-α receptor affinity activity for the target compounds, clofibrate, and natural ligands^a

Compounds	Scores (kcal mol^−1)
a PDB code: 6KXX. b CF as the positive control drug. c T02 is formally designated as 1-(4-chlorophenyl)-6-methyl-3-propan-2-yl-prazolo[3,4-b] pyridine-4-carboxalic acid. It functions as a native ligand for the PPAR-α protein, which is identified by the International Protein Index (IPI) code 6KXX.
T1	−5.5054
T2	−5.5480
T3	−5.4305
T4	−5.8633
T5	−5.9863
T6	−5.7964
T7	−5.6619
Clofibrate^b	−5.6776
T02	−5.1242

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Fig. 4 illustrates the outcomes of the 3D and 2D molecular docking analyses. The T2 docking result showed that the docking score between T2 and 6KXX was −5.5480. Within this configuration, the oxygen atom of the ester bond functions as a hydrogen bond acceptor, forming a hydrogen bond with the basic amino acid Lys448. The separation between these two entities is measured at 2.93 Å, accompanied by a binding free energy of −1.1 (kcal mol⁻¹). The docking result of positive drug CF showed that the docking score of CF with 6KXX was −4.6301. The basic amino acid Arg465 interacted with the hydrogen atom (arene-H) on the aromatic ring of the CF structure, forming hydrogen bonds. The distance between the two was measured to be 4.36 Å, with a binding free energy of −0.5 (kcal mol⁻¹).

Fig. 4 Visualization of T2 and CF docking to the PPAR-α protein. (a) 3D structure of the docked T2. (b) 2D interaction scheme for T2. (c) 3D structure of the docked CF. (d) 2D interaction scheme for CF.

2.2.3 Dose-dependent study on the lipid-lowering activity of T2. The acute lipid-lowering effect showed that T2 exhibited the best performance among the target compounds. Therefore, a further evaluation of the dose-dependent lipid-lowering effect of T2 was conducted in a mouse model of Triton WR-1339-induced hyperlipidemia. The model group serves as the reference point, while the T2 (L), T2 (M), and T2 (H) groups reduced TG by 13%, 31% (P < 0.01), and 39% (P < 0.01), respectively, and reduced TC by 7%, 24% (P < 0.01), and 27% (P < 0.01), respectively. The positive drug CF reduced TG levels by 32% (P < 0.01) and TC levels by 15% (Fig. 5). The results indicate that T2 reduces TG and TC in a dose-dependent manner.

Fig. 5 Effects of T2 at low (50 mg kg⁻¹), medium (100 mg kg⁻¹), and high (150 mg kg⁻¹) doses on triglyceride (TG) (a) and total cholesterol (TC) (b) levels in Triton WR-1339-induced hyperlipidemia mice. Clofibrate (65 mg kg⁻¹) served as the positive control. The administration lasted one week. Data are presented as mean ± SEM, n = 8. ^##P < 0.01 vs. control group. ^**P < 0.01 vs. model group.

2.2.4 Study on the chronic hyperlipidemia activity of T2. The target compound T2 demonstrated a favorable lipid-lowering activity, with its effect dose-dependent. Therefore, a model of chronic HLP induced by a high-fat diet was used in this study. This model was used to conduct further evaluation of T2's lipid-lowering efficacy and to appraise its hepatoprotective properties.

The consequences of serum lipid levels are as follows (Fig. 6). In comparison to the control group, the model mice exhibited a marked rise in serum levels of TG, TC, and LDL-C (P < 0.01), and HDL-C noticeably decreased (P < 0.01), suggesting the successful establishment of the model. When compared to the model group, after 28 days of T2 administration, the serum levels of TG, TC, and LDL-C in the mice decreased by 38% (P < 0.01), 33% (P < 0.01), and 57% (P < 0.01), respectively, and HDL-C levels exhibited a marked increase of 49% (P < 0.01). In comparison with the model group, the positive drug CF exerted a substantial influence on the serum levels of TG and LDL-C in murine subjects, reducing them by 42% (P < 0.01) and 22% (P < 0.01), respectively. Concurrently, TC levels decreased by 12%, while HDL-C significantly increased by 79% (P < 0.01), indicating a significant difference in the effects between T2 and CF.

Fig. 6 Impact of the target compound T2 on serum triglyceride (a), total cholesterol (b), HDL-cholesterol (c), and LDL-cholesterol (d) levels in a 28 day high-fat diet-induced chronic hyperlipidemia mouse model. T2 was administered at a dosage of 100 mg kg⁻¹, and clofibrate was used as a positive control at 65 mg kg⁻¹. The results are presented as mean ± SEM, n = 8. ^##P < 0.01 vs. control group. ^**P < 0.01 vs. model group.

The hepatic lipid levels are as follows (Fig. 7). Compared to the control group, the hepatic levels of TG, TC, and LDL-C in the model group mice were significantly elevated (P < 0.01), whereas HDL-C levels were notably decreased (P < 0.01). The findings revealed that following 28 days of administering the target compound T2, compared to the model group, the hepatic levels of TG, TC, and LDL-C in the T2 group mice were reduced by 17% (P < 0.01), 18% (P < 0.05), and 52% (P < 0.01), respectively, while the level of HDL-C was significantly elevated by 73% (P < 0.01). In comparison to the model group, the affirmative drug CF had a significant influence on the hepatic levels of TG and LDL-C in mice, decreasing them by 21% (P < 0.01) and 57% (P < 0.01), respectively. TC levels decreased by 9%, while HDL-C levels increased by 70% (P < 0.01), indicating a significant difference in efficacy. The lipid-lowering effects in the liver and serum are generally harmonious and consistent with the results from acute lipid-lowering evaluations and dose-dependent studies.

Fig. 7 Impact of the target compound T2 on serum triglyceride (a), total cholesterol (b), HDL-cholesterol (c), and LDL-cholesterol (d) levels in 28 day high-fat diet-induced chronic hyperlipidemia mice. T2 was administered at 100 mg kg⁻¹, and clofibrate was used as a positive control at 65 mg kg⁻¹. The results are presented as mean ± SEM, n = 8. ^##P < 0.01 vs. control group. ^*P < 0.05, ^**P < 0.01 vs. model group.

The indicators of liver function in the serum are as follows (Fig. 8). The model group exhibited notably elevated ALT and AST levels in serum compared to the control group (P < 0.01 and P < 0.05), suggesting hepatic tissue damage in hyperlipidemia mice. After 28 days of administering compound T2, the serum ALT and AST levels in the T2 group were markedly decreased by 52% (P < 0.01) and 49% (P < 0.01), respectively, compared to the model group. These findings suggest that T2 possesses a liver-protective effect. The positive control drug CF lowered ALT and AST levels by 9% and 16%, respectively, but did not significantly improve liver injury.

Fig. 8 Impact of the target compound T2 on serum ALT (a) and AST (b) levels in 28 day high-fat diet-induced chronic hyperlipidemia mice. T2 was administered at 100 mg kg⁻¹, and clofibrate was used as a positive control at 65 mg kg⁻¹. The results are expressed as mean ± SEM, n = 8. ^#P < 0.05, ^##P < 0.01 vs. control group. ^**P < 0.01 vs. model group.

The liver function indicators in the liver tissue manifest as follows (Fig. 9). Following a 28 day treatment with the test compound T2, in comparison to the model group, the T2 group exhibited a marked reduction in liver ALT levels by 44% (P < 0.01), and a decrease in AST levels by 79% (P < 0.05). The reference drug CF led to reductions of 8% and 67% in ALT and AST, respectively. These findings suggest that T2 may help alleviate liver damage.

Fig. 9 Impact of the target compound T2 on liver ALT (a) and AST (b) levels in 28 day high-fat diet-induced chronic hyperlipidemia mice. T2 was administered at 100 mg kg⁻¹, and clofibrate was used as a positive control at 65 mg kg⁻¹. The results are presented as mean ± SEM, n = 8. ^##P < 0.01 vs. control group. ^*P < 0.05, ^**P < 0.01 vs. model group.

Macroscopic findings of the livers are shown below (Fig. 10a). The index of liver function indicates that T2 can improve liver tissue injury in HLP. The pathological tissue sections of the liver tissue were further analyzed in this research. The model group of mice exhibited obvious fat spot accumulation in their livers, which were clearly yellow and had caused liver tissue damage. After 28 days of T2 treatment, the accumulation of liver fat observed in chronic high-fat diet mice belonging to the T2 group was significantly reduced, becoming almost invisible, with a reddish-brown color similar to that of control group mice. The fat spot accumulation in the positive drug CF group was markedly less than that observed in the model group mice. Small fat spots remained visible within the livers of the chronic high-fat mice in this CF group, and their liver tissue retained a faint yellowish hue. However, the liver pathology of the positive drug CF group showed enhancement compared to the model group.

Fig. 10 Effect of target compound T2 on the liver tissue of a 28 day high-fat diet-induced chronic HLP mouse model. T2 was administered at 100 mg kg⁻¹, while clofibrate served as a positive control at 65 mg kg⁻¹. (a) Visual examination of liver tissue dissection across all groups. (b) Liver histology: hematoxylin and eosin (HE) staining at 100× magnification. (c) Liver histology: oil red O staining at 200× magnification. Groups are arranged from left to right as follows: control, model, T2, and positive drug (CF).

The HE staining of the livers is shown below (Fig. 10b). The liver cells of the control group exhibit a complete structure and regular morphology, with no significant pathological findings observed. The model group consisted of mice fed a chronic high-fat diet, and their livers were analyzed, revealing an obvious vacuolar morphology, increased liver cell nuclei, partial cell steatosis, significant bleeding, and infiltration of inflammatory cells. The pathological changes within the livers of the mice subjected to the T2 treatment were significantly reduced, and the slices exhibited a reduction in the extent of bleeding, inflammatory response, and cell damage. Further histological analysis confirms that T2 shows significant efficacy in alleviating liver damage caused by HLP. After CF treatment in hyperlipidemic mice, the extent to which inflammatory cells infiltrate the tissue was reduced, and liver tissue pathology was improved in the positive drug CF group. However, a marked increase in the volume of liver cell nuclei was observed in the positive drug CF group, with swelling, blurred edges, and cell lysis, which may be related to the damage caused by the clofibrate drug to the liver cells (Fig. 10b).

Oil red O staining of the livers is shown below (Fig. 10c). In the control group, liver cells exhibit a tight arrangement, and lipid accumulation is not apparent. There was a marked rise in the accumulation of red fat in the liver cells of the model group, with distinct characteristics of steatosis. In the T2 group, liver cell red lipid accumulation and the degree of lipid degeneration were significantly reduced. After clofibrate intervention, the red lipid accumulation in liver cells of the positive drug CF group was also reduced, and the degree of lipid degeneration was improved.

Induced by a chronic high-fat diet, the livers of mice, as shown by HE staining and pathological evaluation of mouse liver tissue, exhibited significant pathological changes, including inflammatory cell infiltration and other characteristics. Based on the above pathological features, further detection of inflammatory indicators in serum, including IL-6 and TNF-α, was conducted. The model group exhibited markedly elevated concentrations of IL-6 and TNF-α in comparison to the control group (Fig. 11) (P < 0.01 and P < 0.05). This suggests the occurrence of inflammatory reactions under HLP. After 28 days of T2 intervention, compared to the model group, the levels of IL-6 and TNF-α in the T2 group reduced by 16% (P < 0.01) and 13% (P < 0.01), respectively. In contrast to the model group, the positive drug CF reduced the levels of IL-6 and TNF-α in the livers of mice by 9% and 8%, respectively. The results suggest that T2 has an improving effect on the inflammatory response under HLP and may have a protective effect on liver injury.

Fig. 11 Effect of target compound T2 on serum IL-6 (a) and TNF-α (b) levels in a 28 day high-fat diet-induced chronic HLP mouse model. T2 was administered at a dosage of 100 mg kg⁻¹, while clofibrate served as a positive control at 65 mg kg⁻¹. The results are expressed as mean ± SEM, n = 8. ^##P < 0.01 vs. control group. ^**P < 0.01 vs. model group.

According to literature reports, oxidative stress reactions may occur in HLP. Therefore, this study further tested antioxidant indicators (Fig. 12). Compared with the control group, the model group demonstrated significantly reduced SOD levels (P < 0.01) and significantly elevated MDA levels (P < 0.01), the latter being a lipid peroxidation product, indicating the occurrence of an oxidative stress response in HLP. The serum SOD level in the T2 group was considerably augmented by 15% (P < 0.01) compared with the model group, and the MDA content considerably decreased by 17% (P < 0.05), indicating that T2 may effectively alleviate oxidative stress damage induced by HLP. In contrast to the model group, the positive CF drug boosted SOD by 3% and decreased MDA by 5%, neither of which reached statistical significance.

Fig. 12 Impact of administering target compound T2 at 100 mg kg⁻¹ and clofibrate at 65 mg kg⁻¹ on serum superoxide dismutase (a) and malondialdehyde (b) concentrations in a chronic HLP mouse model induced by a 28 day high-fat diet. The data are represented as mean ± SEM, n = 8. ^##P < 0.01 vs. control group. ^*P < 0.05, ^**P < 0.01 vs. model group.

Preliminary research found that compound T2 exhibits lipid-lowering effects and has a strong affinity for the PPAR-α protein. This prompted us to employ Western blot analysis to detect PPAR-α protein expression in liver tissue to further elucidate the lipid-lowering mechanism (Fig. 13). As shown in Fig. 13, β-actin was used as a loading control to ensure equal protein loading. The quantification of band optical density was performed using Gel-Pro Analyzer software, and the relative expression levels of PPAR-α were normalized to β-actin. In comparison with the control group, the normalized expression of PPAR-α protein was substantially reduced in the high-fat diet-induced model group. The target compound T2 markedly elevated the normalized expression of PPAR-α protein (P < 0.05), and administration of CF also markedly elevated the normalized expression of PPAR-α protein (P < 0.01) compared to the model group. The results suggest that the mode of action of T2-induced lipid lowering may be linked to PPAR-α activation.

Fig. 13 Impact of administering target compound T2 and clofibrate on PPAR-α protein expression in the liver of a chronic HLP mouse model induced by a 28 day high-fat diet. The upper panel shows the representative immunoblots of PPAR-α and the loading control β-actin. The lower panel presents the quantitative analysis of PPAR-α protein levels, which are normalized to β-actin. The data are represented as mean ± SEM, n = 3 per group. ^##P < 0.01 vs. control group. ^*P < 0.05, ^**P < 0.01 vs. model group.

3. Discussion

This study employed a molecular hybridization strategy to design and synthesize a series of syringaldehyde-based fibrate derivatives, with compound T2 demonstrating the most potent lipid-lowering and hepatoprotective activities. The molecular hybridization of classical drugs with bioactive natural product scaffolds is a promising strategy for enhancing therapeutic profiles. However, a critical challenge in developing new fibrates is not only to improve their efficacy but also to mitigate their well-documented hepatotoxicity. Our study directly addresses this challenge through a more rational design philosophy, treating hepatoprotection as a primary objective from the outset. We analyzed the structure and action of existing related fibrate derivatives. First, such indole-based hybrids and aminothiazole, which focus on anti-obesity or hypoglycemic effects, do not address the inherent hepatotoxicity of the fibrate scaffold.^23,24 Second, dithiophene relied on H₂S release enhances the liver's detoxification function.²⁵ In stark contrast, based on the structure–activity relationships of fibrate lipid-lowering drugs, this study designed target compounds by replacing the aromatic ring in the fibrate drug scaffold with syringaldehyde, a natural monomer with antioxidant and anti-inflammatory effects. It screened out compound T2, which has lipid-lowering, antioxidant, anti-inflammatory, and hepatoprotective effects. Additionally, the lipid-lowering effect and its dose dependence were first evaluated in the Triton WR1339-induced hyperlipidemia mouse model. The lipid-lowering, anti-inflammatory, antioxidant, and hepatoprotective effects of compound T2 were assessed in a high-fat diet-induced hyperlipidemia model. The results showed that T2 had a significant lipid-lowering effect in a dose-dependent manner. In addition, T2 had a protective effect against liver injury under hyperlipidemic conditions, improved liver function indices, and reduced hepatic lipid accumulation. Based on this, T2 has therapeutic potential for hyperlipidemia and hepatoprotective effects. Finally, the study found that T2 can upregulate PPAR-α protein expression in the liver and has a strong affinity for PPAR-α. We speculate that the regulation of PPAR-α protein levels mediates the lipid-lowering effect of T2. In contrast, its hepatoprotective effect may be associated with its significant antioxidant and anti-inflammatory effects.

From the perspective of structure–activity relationship (SAR) analysis, the superior activity of T2 stems from its sophisticated molecular design. By replacing the conventional aryloxy group with a syringaldehyde scaffold, we found that among compounds with similar carbon chain lengths, side-chain branching is the key determinant for enhancing activity over linear structures, like T1. Molecular docking results elucidate that T2 forms a stable interaction with the PPAR-α receptor, anchored by a key hydrogen bond (2.93 Å) between its ester oxygen and Lys448 and reinforced by a network of hydrophobic contacts (Fig. 4). Crucially, this branched conformation restricts the molecule's flexibility, creating a pre-organized effect that optimizes its spatial orientation for receptor activation. Additionally, regarding triglyceride (TG) regulation, both branched T2 and longer linear-chain T5 showed significant activity. For T5, docking revealed that its extended, flexible chain allows for deeper penetration into the hydrophobic pocket of the PPAR-α ligand-binding domain, forming a stable complex that effectively promotes TG clearance. However, in the regulation of total cholesterol (TC), the structure of T2 offers a unique advantage. The branched conformation of T2 confers a distinct three-dimensional shape and a significant steric bulk. This unique architecture forces T2 to adopt a binding mode on the PPAR-α receptor that is fundamentally different from that of linear molecules, like T5. We believe that this steric hindrance effect may induce conformational changes in the receptor or selectively act on the allosteric site, unlike classical PPAR-α agonists. This provides a plausible explanation for T2's significant TC-lowering effect, an activity not observed in flexible linear-chain molecules. This structure–activity relationship not only rationalizes T2's superior profile but also highlights its unique potential to modulate lipid metabolism using a distinct mechanism.

To comprehensively evaluate the lipid-lowering activity and dose–response relationship of the target compound, two HLP models were utilized: the Triton WR-1339-induced acute HLP model and the chronic high-fat diet (HFD)-induced HLP model.^26,27 Using both models, we need to comprehensively evaluate the lipid-lowering potential of T2 from both acute effects and chronic regulation perspectives. The Triton WR-1339-induced acute HLP model, characterized by a short setup time, ease of use, and reliability, serves as a suitable tool for the preliminary screening of lipid-lowering effects and dose–response relationships in compounds. The state of acute HLP is simulated using the Triton WR-1339 acute HLP model, which inhibits lipoprotein lipase (LPL) activity and induces a sharp increase in plasma lipid levels. The advantage of this model is that it can observe the effects of a series of compounds on blood lipid levels within a short period, thereby quickly screening for target compounds with potential lipid-lowering activity. Our research results show that T2 exhibits a significant lipid-lowering effect in this model (Fig. 3) and displays a dose-dependent relationship (Fig. 5), laying a solid foundation for further in-depth research. Meanwhile, given the limitations of the acute HLP model for simulating chronic metabolic disorders induced by a long-term high-fat diet in humans, we further adopted an HFD-induced chronic HLP model that is more closely aligned with human dietary habits. Compared with the Triton WR-1339 model, the HFD model can more accurately simulate the occurrence and development of human HLP. In this model, individual animals exhibit subtle differences in their food and water intake, allowing us to monitor the lipid-lowering efficacy and potential side effects of the target compound T2 over the long term. After 28 days of HFD induction, the levels of blood lipids in the mice exhibited a significant increase. After 28 consecutive days of T2 treatment, the mice demonstrated substantial improvement in blood lipid levels. At the same time, pathological and physiological processes related to HLP, such as oxidative stress, inflammatory response, and liver injury, were effectively alleviated. These results suggest that T2 not only performs well in acute HLP models but also exhibits good lipid-lowering activity and comprehensive regulatory effects in chronic HLP models that are more closely aligned with human physiological states. Overall, this experiment comprehensively evaluated the lipid-lowering activity and dose–response relationship of target compound T2 from both acute effects and chronic regulation perspectives through the integration of acute HLP models induced by Triton WR-1339 and chronic HLP models established using HFD. This provides strong experimental evidence that compound T2 could be a potential multifunctional lipid-lowering compound.

HLP, especially HTG, has received increasing attention. Fibrate compounds have significant lipid-lowering effects in clinical practice, usually with an aryloxyalkanoate structure as the core skeleton. In this study, a molecular hybridization strategy was adopted to synthesize a series of target compounds by combining ligand dehydrogenases of different chain lengths and substituents that conform to the structural characteristics of fibrate lipid-lowering drugs. Fibrates interact with the ligand-binding domain of PPAR-α via the structure of aryloxyalkanoates, which stabilize the binding conformation through hydrogen bonding and hydrophobic interactions, thereby activating PPAR-α.²⁸ Research has demonstrated that activated PPAR-α forms a heterodimer with the retinoic acid X receptor. This complex then binds to the peroxisome proliferator-activated receptor response element (PPRE) of the target gene, upregulating the expression of genes, such as LPL and apolipoprotein A-I, and promoting T-cell-mediated degradation of HDL-C^29,30 while simultaneously downregulating the inhibitory effect of apolipoprotein CIII on TG hydrolysis, thereby ultimately improving hyperlipidemia.^31,32 The target compound T2 retains the core pharmacophore. Therefore, it is speculated that its lipid-lowering mechanism is consistent with that of fibrate drugs. Research data show that T2 significantly reduces triglyceride, total cholesterol, and LDL-C levels in mouse serum and liver, and substantially increases HDL-C (Fig. 6 and 7). A strong binding interaction between T2 and PPAR-α was demonstrated by molecular docking results (Table 1, Fig. 3), further confirming its properties as a PPAR-α agonist. Notably, PPAR-α agonists are closely related to lipid-lowering mechanisms.^28,33,34 Furthermore, Western blot analysis showed that after 28 days of administration, T2 resulted in markedly higher PPAR-α protein expression in the liver (Fig. 13). These results suggest that T2 may also act as a PPAR-α agonist, regulating gene expression related to lipoprotein metabolism and exerting significant lipid-lowering effects.

It has been reported in the literature that HTG can cause oxidative stress, which leads to LDL oxidation to oxLDL, thus aggravating the disease and possibly causing serious cardiovascular diseases, such as atherosclerosis and hypertension. The research results showed that in the chronic HLP model, T2 significantly increased SOD activity and reduced MDA content in hyperlipidemia mice (Fig. 12), indicating its ability to clear free radicals and alleviate oxidative stress. The research mechanism suggests that fibrate blockers can induce hepatic peroxisome proliferation through PPAR-α activation,³⁴ which leads to heightened activity of enzymes crucial for fatty acid metabolism, coupled with excessive reactive oxygen species (ROS) production, worsening oxidative stress-related injury to hepatocytes.³⁵ At the same time, SA may also affect the expression of antioxidant enzymes by activating antioxidant pathways, thereby increasing SOD activity, reducing MDA levels, and exerting antioxidant effects.³⁶ At the same time, SA may also affect the expression of antioxidant enzymes by activating antioxidant pathways, thereby increasing SOD activity, reducing MDA levels, and exerting antioxidant effects.^37,38 Additionally, derivatives containing SA are used as tetraphenolic antioxidants to exert their antioxidant effects.³⁹ In summary, target compound T2 also includes the SA structure. Therefore, we speculate that the antioxidant activity of compound T2 may be related to the presence of the SA structure.

Based on previous studies, we have learned that LDL is oxidized to oxLDL under HLP and that oxidative stress activates the inflammatory pathway, leading to the release of downstream inflammatory factors, including TNF-α and IL-6, which, in turn, drive an inflammatory response.⁴⁰ The findings of this research indicated that compound T2 substantially decreased serum IL-6 and TNF-α concentrations in mice with high lipid levels (Fig. 11), indicating its ability to clear inflammatory factors. As indicated by scholarly articles, SA possesses anti-inflammatory properties that are achieved by inhibiting the inflammatory pathway.⁴¹ Additionally, related derivatives of SA have been shown to inhibit the release of inflammatory factors, thereby achieving anti-inflammatory effects.⁴² Since the target compound T2 in this study also contains the SA structure, we postulate that its anti-inflammatory action may be linked to its SA moiety. This anti-inflammatory effect reduces infiltration of inflammatory cells into the liver (Fig. 10b) and synergistically improves liver steatosis (Fig. 10c).

Under chronic HLP, oxidative stress and inflammatory reactions can occur, which lead to lipid accumulation in the liver, lipid peroxidation, and damage to liver cells, thereby forming a vicious cycle and exacerbating the complications of HTG. According to literature reports, the hepatotoxicity of traditional fibrate drugs may be closely related to mitochondrial dysfunction. The active products generated by CYP450 metabolism can cause metabolic disorders or trigger related apoptotic pathways, leading to cell apoptosis.⁴³ The target compound T2, designed through a molecular hybridization strategy in this study, not only significantly reduced serum and hepatic TG/TC concentrations but also demonstrated hepatoprotective activity. The research data revealed that both ALT and AST levels in the serum and liver were reduced (Fig. 8 and 9). Analyzing the mechanism, compound T2 may achieve hepatoprotective effects through the following pathways. First, the research results indicate that T2 has antioxidant and anti-inflammatory effects, and histopathology shows that the T2 group significantly reduces liver tissue pathological damage, which is manifested by reduced bleeding, reduced infiltration of inflammatory cells, and reduced hepatic fat accumulation (Fig. 10). In view of this, the target compound T2 also contains an SA structure, so we speculate that the hepatoprotective mechanism of compound T2 may be related to its SA structure. Further confirmation suggests that T2 may have a hepatoprotective effect, protecting the liver from damage caused by lipid peroxidation and inflammatory responses.

A key finding of our study is the distinct therapeutic profile of T2 compared to the standard fibrate, clofibrate (CF). Although CF demonstrated a substantial effect on upregulating PPAR-α protein expression, it failed to significantly ameliorate hyperlipidemia-induced liver injury, as evidenced by negligible changes in serum ALT/AST levels and persistent histopathological damage. In stark contrast, T2, while exhibiting comparable lipid-lowering efficacy, conferred robust hepatoprotection. This was demonstrated by a dramatic reduction in serum ALT and AST (by 52% and 49%, respectively) and a significant reduction in hepatic steatosis and inflammation. The superior hepatoprotective effect of T2 is likely not merely a consequence of lipid reduction but is attributable to the intrinsic antioxidant and anti-inflammatory properties conferred by the syringaldehyde moiety, which effectively counteracts the oxidative stress pathways implicated in fibrate-induced liver damage.

Although our study demonstrates the multi-functional effects of T2, we acknowledge that a primary limitation is the correlative nature of our mechanistic evidence linking its effects to PPAR-α activation. The current data, including molecular docking and Western blot analysis, suggest but do not definitively prove direct target engagement or functional pathway activation. To address this critical gap and establish a causal link, our future research will be multifaceted. First, we employ biophysical techniques, such as surface plasmon resonance (SPR), to directly validate the binding affinity and kinetics of the T2–PPAR-α interaction. Second, we perform functional assays by quantifying mRNA expression of key PPAR-α downstream genes (e.g., CPT1A, ACOX1, and LPL) in liver tissues from HFD-treated mice via qRT-PCR, providing direct evidence of pathway activation. Finally, to conclusively demonstrate target dependency, we utilize PPAR-α knockout models to determine whether the lipid-lowering and hepatoprotective effects of T2 are abolished in the absence of the receptor. Integrating these approaches allows us to construct a definitive mechanistic model for T2's action, providing the robust evidence required for its further development.

4. Conclusion

In conclusion, this study successfully synthesized a syringaldehyde-based fibroblast derivative and evaluated its lipid-lowering activity in vitro and in vivo. Among these target compounds, T2 activity is the most prominent and exhibits a dose-dependent pattern. T2 can significantly reduce blood lipid levels in a mouse model of HLP. Molecular docking studies have shown that T2 has a high affinity for PPAR-α. Western blot analysis revealed that T2 significantly upregulates PPAR-α protein expression in the liver, reduces liver lipid accumulation, lowers liver enzyme levels, and improves pathological liver tissue damage. Additionally, T2 exhibits antioxidant and anti-inflammatory properties. Its multifunctional effects make it a potential candidate compound for lowering blood lipids in HTG and its complications.

5. Materials and methods

5.1 Materials

All materials, reagents, and test kits were purchased from commercial suppliers, including Nanjing Jiancheng Bioengineering Research Institute, Tianjin Tianli Chemical Reagent Co., Ltd., Qingdao Marine Chemical Co., Ltd., Chengdu Cologne Chemical Co., Ltd., Shanghai Aladdin Biochemical Technology Co., Ltd., and Shaanxi Zhonghui Hecai Biopharmaceutical Purchase Co., Ltd., for direct use, unless otherwise specified. The reaction process was followed using thin-layer chromatography (GF254, Qingdao Marine Chemical Co., Ltd.). The compound was divorced and purified using silica gel column chromatography (silica gel: 200–300 mesh) (Qingdao Marine Chemical Co., Ltd.). Using DMSO-d₆ as a solvent on a BRUKER AVANCE 400 spectrometer (Bruker, Germany), the analyses of nuclear magnetic resonance hydrogen spectroscopy (1H-NMR) and nuclear magnetic resonance carbon spectroscopy (¹³C-NMR) were conducted, with chemical shifts recorded in ppm (δ) and coupling constants (J) given in hertz. Mass spectrometry (MS) data were obtained using ESI-MS (TSQ Altis, Thermofisher, USA). Furthermore, the experimental animals used in the study were obtained from Chengdu Dashuo Experimental Animal Co., Ltd. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Shaanxi University of Chinese Medicine and approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine (no. SUCMDL20240827004).

5.2 Synthesis of the target compounds T1–T7

Ethyl 2-(4-formyl-2,6-dimethoxyphenoxy)propanoate (T1). 1.20 g (6.59 mmol) SA was dissolved in DMF (30.00 mL), and 1.79 g (9.89 mmol) ethyl 2-bromopropionate, 1.82 g (13.18 mmol) K₂CO₃, and 0.65 g KI were added. The mixture was stirred in an 80 °C oil bath, and the response was monitored using thin-layer chromatography (TLC). The mixture was filtered after the reaction was completed. The filtrate was removed by adding an appropriate amount of pure water and using ethyl acetate (30 mL × 3). Wash the organic phase solution twice with a 5% potassium carbonate solution (120 mL). Be granted a suitable volume of ethyl acetate solution from the aqueous phase solution once. All organic phase solutions were combined, and the organic phase was washed twice with 120 mL of a saturated sodium chloride solution. Then, it was dried using anhydrous magnesium sulfate. Finally, the mixture was filtered using a filter, and the filtrate was collected and evaporated using a rotary evaporator to obtain the crude product. The purification of the substance is achieved through column chromatography, which employs a mixture of petroleum ether and ethyl acetate in a 5 : 1 ratio, yielding a light yellow oily crude product. Light yellow oily compound T1 (1.07 g) was obtained, resulting in a yield of 57.53%. Molecular weight 282.29 g mol⁻¹. Molecular formula C₁₄H₁₈O₆. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.89 (s, 1H), 7.26 (s, 2H), 4.77 (q, J = 6.8 Hz, 1H), 4.12 (q, J = 7.1 Hz, 2H), 3.85 (s, 6H), 1.44 (d, J = 6.8 Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, DMSO) δ 192.17, 171.27, 153.13, 141.04, 131.99, 107.15, 76.85, 60.89, 56.56, 18.68, 14.40.

Ethyl 2-(4-formyl-2,6-dimethoxyphenoxy)-2-methylpropanoate (T2). 1.18 g SA was dissolved in DMF (30.00 mL), and 0.98 g ethyl 2-bromoisobutyrate, 1.38 g K₂CO₃, and 0.60 g KI were added. The identical process utilized for the synthesis of compound T1 was implemented. Following the conclusion of the experiment, the crude product T2 was obtained. Thereafter, the crude product T2 underwent purification through column chromatography using a solvent blend of petroleum ether and ethyl acetate in a ratio of 8 : 1. A brown oily compound T2 (0.67 g) was obtained, resulting in a yield of 44.97%. Molecular weight 296.32 g mol⁻¹. Molecular formula C₁₅H₂₀O₆. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.89 (s, 1H), 7.24 (s, 2H), 4.16 (q, J = 7.1 Hz, 2H), 3.80 (s, 6H), 1.38 (s, 6H), 1.23 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, DMSO) δ 192.28, 172.80, 154.56, 138.60, 132.43, 106.73, 81.12, 60.87, 56.40, 25.09, 14.49. HR-MS C₁₅H₂₀O₆ [M + H]⁺ cal. 297.13381, found 297.13261.

Ethyl 2-(4-formyl-2,6-dimethoxyphenoxy)butanoate (T3). 2.13 g SA was dissolved in DMF (30.00 mL), and 1.76 g ethyl 2-bromobutyrate, 2.49 g K₂CO₃, and 0.75 g KI were added. The duplicate process used for the synthesis of compound T1 was implemented. Following the conclusion of the experiment, the crude product T3 was obtained. Thereafter, the crude product T3 underwent purification through column chromatography using a solvent blend of petroleum ether and ethyl acetate in a ratio of 5 : 1. A yellow oily compound T3 (2.59 g) was obtained, resulting in a yield of 97.00%. Molecular weight 296.32 g mol⁻¹. Molecular formula C₁₅H₂₀O₆. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.88 (s, 1H), 7.26 (s, 2H), 4.59 (t, J = 6.2 Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.85 (s, 6H), 1.91–1.71 (m, 2H), 1.20 (t, J = 7.1 Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C-NMR (101 MHz, DMSO) δ 192.09, 170.87, 152.79, 141.57, 131.76, 107.25, 82.08, 60.72, 56.54, 26.37, 14.49, 9.52.

Ethyl 2-(4-formyl-2,6-dimethoxyphenoxy)acetate (T4). 0.80 g SA was dissolved in DMF (30.00 mL), and 1.10 g ethyl bromoacetate, 1.21 g K₂CO₃, and 0.40 g KI were added. The identical process utilized for the synthesis of compound T1 was implemented. Following the conclusion of the experiment, the crude product T4 was obtained. Thereafter, the crude product T4 underwent purification through column chromatography using a solvent blend of petroleum ether and ethyl acetate in a 5 : 1 ratio. A bright yellow solid compound T4 (1.37 g) was obtained, resulting in a yield of 77.40%. Mp: 34.9–35.5 °C. Molecular weight: 268.27 g mol⁻¹. Molecular formula: C₁₃H₁₆O₆. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.87 (s, 1H), 7.26 (s, 2H), 4.69 (s, 2H), 4.15 (q, J = 7.1 Hz, 2H), 3.85 (s, 6H), 1.21 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, DMSO) δ 192.22, 168.99, 152.83, 141.36, 131.93, 107.31, 69.20, 60.88, 56.62, 14.50.

Ethyl 4-(4-formyl-2,6-dimethoxyphenoxy)butanoate (T5). 1.00 g SA in DMF (30.00 mL) was dissolved, and 0.76 g ethyl 4-bromobutyrate, 1.08 g K₂CO₃, and 0.60 g KI were added. The identical process used for the synthesis of compound T1 was implemented. Following the conclusion of the experiment, the crude product T5 was obtained. Thereafter, the crude product T5 underwent purification through column chromatography using a solvent blend of petroleum ether and ethyl acetate in an 8 : 1 ratio. A yellow brown oily compound T5 (0.95 g) was obtained, resulting in a yield of 82.61%. Molecular weight: 296.32 g mol⁻¹. Molecular formula: C₁₅H₂₀O₆. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.89 (s, 1H), 7.26 (s, 2H), 4.08 (q, J = 7.1 Hz, 2H), 4.00 (t, J = 6.2 Hz, 2H), 3.87 (s, 6H), 2.53 (t, J = 7.3 Hz, 2H), 1.96–1.82 (m, 2H), 1.20 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, DMSO) δ 192.21, 173.12, 153.92, 142.27, 132.13, 107.09, 72.13, 60.20, 56.48, 30.30, 25.54, 14.51.

Ethyl 5-(4-formyl-2,6-dimethoxyphenoxy)pentanoate (T6). 1.70 g SA in DMF (30.00 mL) was dissolved, and 1.50 g ethyl 5-bromovalerate, 1.98 g K₂CO₃, and 0.65 g KI were added. The identical process used for the synthesis of compound T1 was implemented. Following the conclusion of the experiment, the crude product T6 was obtained. Thereafter, the crude product T6 underwent purification through column chromatography using a solvent blend of petroleum ether and ethyl acetate in a 5 : 1 ratio. A light yellow oily compound, T6 (1.91 g), was obtained, resulting in a yield of 85.65%. Molecular weight: 310.35 g mol⁻¹. Molecular formula: C₁₆H₂₂O₆. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.88 (s, 1H), 7.25 (s, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.96 (t, J = 5.9 Hz, 2H), 3.85 (s, 6H), 2.35 (t, J = 7.1 Hz, 2H), 1.68 (ddtd, J = 19.5, 8.8, 6.2, 5.2, 2.7 Hz, 4H), 1.18 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, DMSO) δ 192.31, 173.34, 153.96, 142.45, 132.04, 107.17, 72.60, 60.14, 56.52, 33.53, 29.34, 21.51, 14.57.

Ethyl 6-(4-formyl-2,6-dimethoxyphenoxy)hexanoate (T7). 1.52 g SA in DMF (30.00 mL) was dissolved, and 1.50 g 6-bromohexanoate ethyl ester, 1.78 g K₂CO₃, and 0.65 g KI were added. The identical process used for the synthesis of compound T1 was implemented. Following the conclusion of the experiment, a crude product, T7, was obtained. Thereafter, the crude product T7 underwent purification through column chromatography using a solvent blend of petroleum ether and ethyl acetate in a ratio of 5 : 1. A light yellow oily compound, T7 (0.91 g), was obtained, resulting in a yield of 43.54%. Molecular weight: 324.37 g mol⁻¹. Molecular formula: C₁₇H₂₄O₆. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.87 (s, 1H), 7.24 (s, 2H), 4.05 (q, J = 7.1 Hz, 2H), 3.95 (t, J = 6.4 Hz, 2H), 3.85 (s, 6H), 2.29 (t, J = 7.3 Hz, 2H), 1.59 (dq, J = 26.0, 7.3 Hz, 4H), 1.43 (tdd, J = 9.0, 6.4, 3.5 Hz, 2H), 1.17 (t, J = 7.1 Hz, 3H). ¹³C-NMR (101 MHz, DMSO) δ 192.31, 173.36, 153.95, 142.52, 131.98, 107.15, 72.87, 60.13, 56.48, 33.98, 29.71, 25.32, 24.62, 14.53.

5.3 Biological evaluation

5.3.1 Study on acute lipid-lowering activity. A mouse model characterized by hyperlipidemia was established through the administration of Triton WR-1339, and the acute lipid-lowering potential of the experimental compound was preliminarily examined. 80 SPF grade ICR mice (20.0 ± 2.0 g), male, were assigned randomly to the following experimental groups after 3 days of an adaptive diet:

Control group: equal volume 0.5% CMC Na

Model group: equal volume 0.5% CMC Na

Target compound (T1–T7) group: 100 mg kg⁻¹

Positive drug group (clofibrate): 65 mg kg⁻¹ (equivalent volume conversion based on clinical dose).

Eight animals per group received oral administration for seven consecutive days at a dosage of 10 mL per kg body weight. During this period, the mice had access to food and water ad libitum. Mice in the model group, positive drug CF group, and target compound group (T1–T7) were administered Triton WR-1339 intravenously via the tail vein at a dose of 400 mg kg⁻¹ 18 hours before the final gavage to induce acute hyperlipidemia. The control group received an equivalent volume of saline via tail vein injection. Blood samples were harvested from the eye sockets of each mouse within six hours after the last gavage. Serum was isolated by centrifugation at low temperature (3500 rpm for 15 minutes) and stored at −20 °C. Subsequently, serum total cholesterol and triglyceride levels were evaluated according to the kit instructions.

5.3.2 Molecular docking. In vitro molecular docking experiments were performed using MOE 2022.02 software. The crystal structure of the PPAR alpha protein was sourced from the Protein Database (PDB). The PDB code for PPAR-α is 6KXX. Before docking, the protein structure was prepared using the Quick Prep module in MOE, which involved removing all non-protein heteroatoms and water molecules and adding missing hydrogen atoms. The 3D structures of the target compounds (T1–T7), positive control (clofibrate), and native ligand (T02) were constructed and energy-minimized. After checking the software instructions, the corresponding docking parameters we used are as follows: the placement methodology was set to “triangle matcher” as the sampling algorithm, and the scoring function for initial placement was “London dG”. The final docking score, representing the estimated free energy of binding (ΔG), was reported in kilocalories per mole (kcal mol⁻¹). The results were visualized and analyzed in 2D and 3D to investigate ligand-PPAR-α active site interactions.

5.3.3 Dose-dependent study on compound T2. The experimental methodology mirrors that of the investigation into acute hyperlipidemia activity described earlier. Three concentrations of compound T2 were administered: a low dose (50 mg kg⁻¹), an intermediate dose (100 mg kg⁻¹), and a high dose (150 mg kg⁻¹). According to the reagent guidelines, serum total cholesterol and triglyceride levels were required to be determined for each cohort of mice.

5.3.4 Study on the chronic hyperlipidemia activity of compound T2. A high-fat diet was used to replicate a chronic high-fat diet model (high-fat feed formula: 81.3% basic feed, 10% lard, 5% white sugar, 0.2% propylthiouracil, 0.5% sodium cholate, and 3% cholesterol). 32 male SPF grade ICR mice (20 ± 2 g) were used; eight were chosen as the control group and given regular feed, while the other 24 mice received high-fat feed. After successful replication of the high-fat model, it was randomly separated into the following three groups:

Model group: equal volume 0.5% CMC Na,

Target compound T2 group: 100 mg kg⁻¹,

Positive drug group (clofibrate): 65 mg kg⁻¹ (equivalent volume conversion based on clinical dose).

Each group consisted of 8 animals and was administered orally continuously for 28 days. The control group was orally administered an equal volume of 0.5% CMC Na. During the experiment, the mice had free access to water and food. On day 28, both the control and high-fat diet groups were fasted without water for six hours. Blood samples were drawn from the posterior eye socket and centrifuged at 3500 rpm for 15 minutes to obtain serum, which was stored at −80 °C for later use. After euthanizing the mice, their livers were dissected, cleaned of excess connective tissue and blood vessels, weighed, and photographed, and a portion was preserved in a 4% paraformaldehyde solution. The rest was frozen at −80 °C after liquid nitrogen treatment for future analysis.

The instructions provided in the kit were strictly followed to detect relevant biochemical indicators, including blood lipid levels, liver function indicators (AST and ALT), and antioxidant indicators (SOD and MDA). An ELISA kit was used to detect inflammatory factors, including TNF-α and IL-6.

5.4 Histopathological analysis

Histopathological analysis was performed using hematoxylin and eosin (H&E) and oil red O staining, as described previously.⁴⁴

5.5 Western blot

The Western blot process is described in a previous study.⁴⁴ The quantification of band optical density was performed using Gel-Pro Analyzer software. For standardization, the optical density of each target protein band was normalized to that of the housekeeping protein β-actin.

5.6 Statistical analyses

GraphPad Prism 10.1.2 is used for statistical analysis of experimental data and for drawing bar charts. The data are presented as mean ± SEM, and comparisons between multiple groups are conducted using one-way analysis of variance (ANOVA). When P < 0.05, the difference is statistically significant.

Author contributions

Wenjing Li: conceptualization, methodology, investigation, writing – original draft, visualization. Boling Zhou: investigation, data curation, formal analysis. Kexin Xu: investigation, data curation, formal analysis. Yunbi Zhang: investigation, data curation, formal analysis. Huanxian Shi: data curation, formal analysis. Ling Ding: data curation, formal analysis. Huizi Shangguan: investigation, formal analysis. Yongheng Shi: data curation. Xinya Xu: formal analysis. Jiping Liu: conceptualization, supervision. Yundong Xie: project administration, writing – review & editing, and funding acquisition.

Conflicts of interest

We declared no conflict of interest.

Data availability

The data of this study will be made available from the corresponding author on request (Email: E-mail: eng522@126.com).

Supplementary information (SI): the SI concluded the spectrum of target compounds. See DOI: https://doi.org/10.1039/d5md00765h.

Acknowledgements

We are grateful for the financial support received for this work from the National College Student Innovation Training Program (No. 202410716037) at Shaanxi University of Chinese Medicine, the Science and Technology Innovative Talent Program of Shaanxi University of Chinese Medicine (2024-LJRC-04), and the Xianyang city “scientists + engineers” team construction project (No. L2024-CXNL-KJRCTD-DWJS-0028).

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