Male ApoE knock-out mice (ApoE−/−, 6 weeks) on the C57BL/6 background were purchased from Suzhou Saiye Biotechnology Co., Ltd (Suzhou, China). The mice were housed in a specific pathogen-free facility under controlled conditions (temperature, 22°C ± 2°C; relative humidity, 55% ± 15%; noise, <60 dB; light/dark cycle, 12/12 h). Male ApoE−/− mice were fed with a high-fat diet (HFD) for 12 weeks to induce atherosclerosis. C57BL/6 mice served as the control group, while ApoE−/− mice induced by HFD were randomly divided into 5 groups with 6 mice in each group. The groups were as follows: model group (Model), CACN136 10 mg/kg group (Low), CACN136 20 mg/kg group (Middle), CACN136 40 mg/kg group (High), Sim 10 mg/kg group (Sim). The mice were orally gavaged with the respective treatments at a fixed time every day for 28 days. The model group mice were orally gavaged with an equal volume of saline. The mice were weighed once a week at a fixed time, and their body weights were recorded. All animal experiments were conducted in accordance with the ethical guidelines for animal welfare and approved by the ethical review board of Southwest Medical University, with the approval number: 20221116-022.

After the adaptation period, C57BL/6 mice continued to have free access to regular diet, and the ApoE−/− mice were switched to a high-fat diet (HFD) for 12 weeks (Zhou et al., 2017). The mice were weighed once a week at a fixed time, and their body weights were recorded.

The mice were fixed in a supine position on a metal electrode on an ultrasound table, with their nose and mouth placed in a mask continuously delivering isoflurane anesthesia. The operating table was maintained at a constant temperature of 37°C. The MS-400 probe of Vevo 2,100 (VisualSonics Inc., Canada) was used to obtain images of the aortic arch section and the parasternal short-axis section next to the sternum. Doppler pulse images of the descending aorta were collected in the aortic arch section, and B-mode images of the aortic arch and its branches were obtained. M-mode images were obtained in the parasternal short-axis section, allowing measurement or calculation of left ventricular anterior wall thickness in diastole and systole (LVAWd, LVAWs), left ventricular posterior wall thickness in diastole and systole (LVPWd, LVPWs), left ventricular internal diameter in diastole and systole (LVIDd, LVIDs), and interventricular septal thickness in diastole and systole (IVSd, IVSs).

After the treatment period, all animals were euthanized by decapitation after blood collection from the orbital sinus. The collected blood from the orbital sinus was collected in centrifuge tubes coated with EDTA and centrifuged at 5,000 rpm for 15 min. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the mouse plasma samples were measured using a veterinary biochemical analyzer (BS-240VET).

H&E staining was performed on paraffin sections of various organ tissues and aortic tissue from the mice.

The fixed intact aorta was removed from the paraformaldehyde and placed in PBS. The aorta was finely trimmed under a somatic microscope to remove as much excess connective tissue and peripheral fat around it as possible. Subsequently, the trimmed aorta was stained by immersion in oil red O stain for 30 min and washed three times with 70% alcohol for 5 min each time after completion of staining to remove excess stain. Next, the spread aorta was cut along the midline with microscopic scissors to fully expose the inner wall of the aorta.

The aortic root sample was removed from the −80°C refrigerator and trimmed to a length of approximately 2 mm. A layer of OCT embedding adhesive was applied to the sample tray, the trimmed tissue was placed on it, and then covered with OCT adhesive to completely encapsulate the tissue, which was then placed into a thermostatic frozen slicer for freezing and solidification. After the samples were solidified, they were serially sectioned with a thickness of 5–6 μm using a thermostatic freezer sectioning machine. After sectioning, the following steps were performed sequentially: formaldehyde fixation for 10 min, distilled water washing for 3 times (10 min each time), staining with oil-red O staining solution for 10 min, and then distilled water washing for 3 times (10 min each time), hematoxylin re-staining, hydrochloric acid-ethanol differentiation for 2–3 s, tap water rinsing (bluing) for 1–3 min, distilled water washing for 1–3 min, and then staining with OCT staining solution. −3 min, distilled water washing 3 times (10 min each), ammonia reblueing for 3–4 s, and finally distilled water washing 3 times (10 min each). The slices were dried slightly and then sealed with glycerol. Image acquisition of the sections was performed using a BA210Digital trinocular camera microscope camera system manufactured by McAudi Industrial Group Ltd.

The red areas were considered as atherosclerotic lesions, and the lesion area (%) was calculated using ImageJ software:

Lesion area   ( % ) = (Red area)/(Total area of the aorta) × 100 %

Network pharmacology Disgenet database (https://www.disgenet.org/), Genecards database (https://www.genecards.org/), and OMIM database (https://www.omim.org/) were used to identify target genes related to atherosclerosis disease. Swiss Target Prediction (http://www.swisstargetprediction.ch) was employed to predict the target proteins of CACN136 (Synthesized by the Pharmacy Laboratory of Southwest Medical University with independent intellectual property rights, purity >99%). The intersection of disease-drug targets was determined. The intersection targets were then input into the String database (https://string-db.org/) to construct protein-protein interaction networks. Enrichment analysis of KEGG pathways and GO functions for the intersection targets was performed using the David database (https://david.ncifcrf.gov/).

RNA extraction was performed using TRIzol reagent (Invitrogen, cat. NO 15596026) following the method described by Chomczynski et al. (Chomczynski and Sacchi, 1987). The three groups of samples (blank group, LPS model group, drug group) were subjected to RNA extraction. The KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina^® (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd. China) was used for chain-specific RNA sequencing library preparation according to the manufacturer’s instructions. Finally, PE150 sequencing was performed in the DNBSEQ-T7 sequencer (MGI Tech Co., Ltd. China). Differential gene expression was determined using the DESeq R package. Differential genes with Log2|Fold Change|≥0.58 between the LPS model group and the blank group, as well as between the LPS model group and the drug group, were selected. The intersection of the two sets of differential genes was obtained. The obtained intersection targets were subjected to topological analysis using Cytoscape software to identify core targets. Finally, KEGG pathway enrichment analysis was performed using the David website.

The RAW264.7 macrophage cell line derived from mice was obtained from the Institute of CVD, Southwest Medical University. After resuscitation, RAW264.7 cells were transferred to DMEM high-glucose culture medium (Gbico) containing 10% fetal bovine serum (Zhejiang Tianhang Biological Technology Co., Ltd.) and 2% penicillin-streptomycin solution (Shanghai Biyuntian Biotechnology Co., Ltd.). The cells were cultured in a cell incubator at a temperature of 37°C and 5% CO₂. Polarization model: Well-growing RAW264.7 cells were evenly seeded in various wells of a plate. After 4 h of incubation, the cells adhered to the bottom. The cells were randomly divided into Control group, LPS group (1 μg/mL), CACN136 (0.6 μg/mL, 0.3 μg/mL, 0.15 μg/mL) + LPS (1 μg/mL) group. Foam cell model: Well-growing RAW264.7 cells were evenly seeded in various wells of a plate. After 4 h of incubation, the cells adhered to the bottom. The cells were then starved in serum-free medium for 6 h and randomly divided into Control group, ox-LDL group (60 μg/mL), CACN136 (0.6 μg/mL, 0.3 μg/mL, 0.15 μg/mL) + ox-LDL (60 μg/mL) group.

The impact of the drug on cell proliferation viability was determined using the MTT colorimetric assay, as previously described (Wei et al., 2022). The drug concentrations were set as a gradient of 4.8 μg/mL, 2.4 μg/mL, 1.2 μg/mL, 0.6 μg/mL, 0.3 μg/mL, and 0.15 μg/mL.

Total RNA was extracted from cell samples using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Nanjing NuoWeiZan Biotech Co., Ltd.). HiScript III Qrt SuperMix (Nanjing NuoWeiZan Biotech Co., Ltd.) was used for reverse transcription. ChamQ Universal SYBR qPCR Master Mix (Nanjing NuoWeiZan Biotech Co., Ltd.) was used for mRNA level detection. The primer sequences (Shanghai Bioengineering Co., Ltd.) are shown in Table 1.

After collecting and washing the cell samples, they were dissolved in RIPA buffer. The protein concentration was measured using the BCA assay kit. The proteins were separated by SDS-PAGE electrophoresis using an appropriate concentration and then transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with the blocking buffer, the membranes were incubated overnight at 4°C with antibodies against iNOS(80517-1-RR, Proteintech, China), Arg1 (66129-1-Ig, Proteintech, China), ABCA1(66217, Abcam, United States), ABCG1 (13578-1-AP, Proteintech, China), CD36 (18836-1-AP, Proteintech, China), and SRA1 (24655-1-AP, Proteintech, China). Then, the membranes were incubated with a secondary antibody (goat anti-rabbit IgG H&L (HRP) at room temperature for 30 min with shaking. Tubulin antibody (66031-1-Ig, Proteintech, China) was used as an internal control, and ImageJ software was used for protein band densitometry analysis.

After washing twice with pre-chilled PBS buffer, the cells were fixed in Cell ORO Fixative at room temperature for 15–25 min in the dark, followed by washing with 60% isopropanol. The cells were then stained with Oil Red O staining solution in a dark room at 37°C for 15 min, followed by washing with 60% isopropanol and rinsing with ultrapure water for 10 min. After drying, the cells were observed and photographed under a microscope.

Cholesterol quantification was performed using the total cholesterol assay kit (BC1985, Solarbio, China) and the free cholesterol assay kit (BC1895, Solarbio, China), following the manufacturer’s instructions.

Following the manufacturer’s instructions, 20 μg/mL Dil-ox-LDL (YB-0010, Yiyuan, China) was added to each well containing cells on coverslips in the dark. After 4 h of cell uptake, the coverslips were removed, stained with DAPI dye (KGF0218, Keygen, China), and coverslipped. The cells were then observed and photographed using a fluorescence inverted microscope.

Following the manufacturer’s instructions, while treating the cells with the drug, each group of cells was simultaneously treated with 5 μg/mL of 25-NBD-cholesterol. After 24 h of incubation, the culture medium was discarded, and each group of cells was exchanged with serum-free DMEM medium containing 50 μg/mL HDL. The cells were further incubated for 4 h, and the supernatant from each well was collected into a 96-well plate. Blank medium was added to the original cell wells in the same volume as the supernatant wells. The fluorescence intensity was measured using a microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The cholesterol efflux rate (%) was calculated using the following formula:

Cholesterol efflux rate (%) = Fluorescence   intensity s u p e r n a t a n t Total   fluorescence   intensity s u p e r n a t a n t + c e l l s × 100%

Total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels in mouse plasma samples were measured using a veterinary biochemical analyzer (BS-240VET).

According to the manufacturer’s instructions, corresponding levels of inflammatory factors TNF-α and IL-6 in mouse plasma were detected using Elisa kits (920, 14206, Meimian, China).

First, paraffin-embedded sections of mouse aortic tissue were deparaffinized and rehydrated. The sections were then boiled in 0.01 M sodium citrate buffer (pH 6.0) for 10 min (temperature maintained above 95°C) for antigen retrieval. Next, endogenous peroxidase was blocked using 3% hydrogen peroxide, and after room temperature blocking, the sections were incubated overnight at 4°C with primary antibodies against iNOS, Arg1, ABCG1, and CD36 (1:500). Finally, the sections were incubated with secondary antibody reagents for 30 min at room temperature.

Mouse atherosclerosis modeling results was evaluated using Ultrasound Imaging System. After 12 weeks of modeling, the control group mice exhibited clear boundaries, smooth vessel walls, and uniform thickness in the aortic arch and its three branches (from top to bottom: innominate artery, left carotid artery, left subclavian artery). In contrast, the model group mice showed significant dilation of the aortic arch, with a large area of plaque formation at the junction with the left carotid artery. The innominate artery exhibited wall dilation and the presence of calcified lesions, while the outlines of the left carotid artery and left subclavian artery were blurred and almost completely covered by plaques (Figure 2A). These findings indicate the successful establishment of the atherosclerosis mouse model. Compared to the control group mice, the atherosclerosis model mice exhibited a significant decrease in ejection fraction and fractional shortening of 10.63% (p < 0.05) and 9.43% (p < 0.05), respectively (Table 2). The left ventricular corrected mass significantly increased by 30.92 mg (p < 0.05), and the aortic blood flow velocity decreased significantly by 65.70% (p < 0.01) (Table 2). These results indicate a significant decrease in cardiac function, increased cardiac burden, and gradual transition to myocardial hypertrophy in the model mice. The mouse growth curve (Figure 2C) revealed that during the modeling period, all mice’s body weight gradually increased. However, during the treatment period, the body weight of all dosing groups continued to decrease in a concentration-dependent manner. At the end of the treatment, the weight loss effect of all CACN136 groups was slightly better than that of the positive control group treated with Sim.

Due to the first-time use of CACN136 for treating ApoE−/− mice, the liver function was evaluated by measuring the blood biochemical indicators, and the organ safety was assessed using HE staining. As shown in Figure 2B, compared to the model group, the ALT and AST levels were significantly decreased in the blank group and treatment group, while the positive drug Sim group showed a significant increase compared to the model group, with an ALT value exceeding the normal range (28–132 U/L) reaching 177.2 ± 17.78 U/L. The HE staining results of the mouse organs in each group are shown in Figure 2D. The blank group and CACN136 high-dose group showed no significant pathological changes in any organs. However, the model group exhibited disrupted liver tissue structure, diffuse fatty degeneration of liver cells with abundant lipid droplets in the cytoplasm, and localized plaque formation in the aorta tissue. The CACN136 medium-dose group showed mild vacuolar degeneration in liver cells, and a small amount of plaque was observed in the aorta tissue. The low-dose group showed mild vacuolar degeneration in liver cells, and a small amount of plaque was observed in localized areas of the aorta tissue. The Sim group showed slight fatty degeneration in liver cells and extremely few plaques were observed in the aorta tissue.

The ability of CACN136 to alleviate atherosclerosis lesions was evaluated using Ultrasound Imaging System and Oil Red O Staining. As shown in Figure 3A, the blank group of mice had no atherosclerosis lesions in the aortic arch and its three branches. In contrast, the model group of mice exhibited large plaque areas in the aortic arch, calcification lesions in the arterial walls of the branch arteries, indistinct contours of the innominate and left carotid arteries almost completely covered by plaques, and dilatation of the left subclavian artery. The positive drug Sim group and the CACN136 treatment group showed clearer contours of the aortic arch compared to the model group, with significantly reduced plaque formation. In particular, the high-dose and medium-dose groups exhibited significant improvement in arterial wall calcification, while the positive drug and low-dose groups showed more calcification in the innominate artery wall. As shown in Figure 3B, the blood flow velocity in the three branches of the model group was significantly lower compared to the blank group of mice. However, in all treatment groups, the blood flow velocity in the three branches significantly increased compared to the model group, indicating that CACN136 can reduce plaque formation and improve blood flow velocity, bringing it closer to normal levels. Figure 3C shows the cross-section of the aorta stained with oil-red O. The positive area in the model group reached 54.63% ± 6.91%. The positive areas in the treatment groups, from high to low, were as follows: CACN136 low-dose group (46.13% ± 3.78%), Sim group (36.83% ± 2.95%), CACN136 medium-dose group (33.74% ± 1.68%), and CACN136 high-dose group (25.92% ± 4.09%). Figure 3D shows the entire aorta stained with oil-red O. The positive area in the model group reached 0.059 ± 0.006 cm². The positive areas in the treatment groups, from high to low, were as follows: Sim group (0.041 ± 0.003 cm²), CACN136 low-dose group (0.0034 ± 0.002 cm²), CACN136 medium-dose group (0.028 ± 0.002 cm²), and CACN136 high-dose group (0.018 ± 0.001 cm²).

To predict the mechanism of CACN136 anti-atherosclerosis, a total of 5,249 target genes related to atherosclerosis were obtained from the OMIM, Genecards, and DisGeNET databases. Additionally, 100 potential target genes of CACN136 were predicted using the SwissTargetPrediction database. The intersection of the predicted atherosclerosis target genes and CACN136 target genes resulted in 64 common target genes (Figure 4A). The KEGG enrichment analysis results (Figure 4B) showed that CACN136 had the most significant enrichment effect on lipid metabolism and atherosclerosis pathways. The enriched pathways included lipid metabolism and atherosclerosis pathway, Toll-like receptor signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, vascular endothelial growth factor (VEGF) signaling pathway, T cell signaling pathway, IL-17 signaling pathway, C-type lectin receptor signaling pathway, and AGE-RAGE signaling pathway in diabetic complications. The GO enrichment analysis results (Figure 4C) showed that the mechanisms by which CACN136 exerts its anti-atherosclerosis effects are mainly involved in biological processes such as LPS-mediated signaling pathway, cellular response to tumor necrosis factor, positive regulation of protein phosphorylation, positive regulation of macrophage chemotaxis, positive regulation of gene expression, and molecular functions such as MAP kinase activity, ATP binding, protein serine/threonine kinase activity, nitric oxide synthase regulator activity, protein kinase activity, and tyrosine phosphorylation binding.

Using RNA-seq, the differentially expressed genes between macrophage communities after treatment with CACN136 were investigated. A total of 4,068 differential genes (1784 upregulated and 2,284 downregulated) were observed in the LPS group relative to the model group, as shown by the volcano plot visualization. Similarly, in the CACN136 group relative to the LPS group, 397 differential genes (188 upregulated and 209 downregulated) were identified (Figure 4D). A total of 228 target genes were obtained by plotting the Venn diagram to take the intersection (Figure 4E). A protein-protein interaction (PPI) network was further constructed and topological analysis was performed using Cytoscape, identifying 34 core target genes (Figure 4F). KEGG enrichment analysis using David (Figure 4G) revealed that these enriched pathways mainly included the IL-17 signaling pathway, TNF signaling pathway, lipid and atherosclerosis signaling pathway, and fluid shear stress signaling pathway.

The effect of CACN136 on the viability of RAW264.7 cells was evaluated using the MTT assay (Figure 5A). Based on the results, the administration concentrations of CACN136 for subsequent experiments were chosen to be 0.6, 0.3 and 0.15 μg/mL. Under the microscope, the morphology of RAW264.7 cells after LPS induction was observed (Figure 5B). The cells in the control group were mostly round or elliptical with high refractivity, appearing small and transparent. In the LPS group, most of the cells transformed into spindle shapes and extended elongated pseudopodia. The treatment groups with CACN136 showed a dose-dependent improvement in the irregular shape and number of pseudopodia in RAW264.7 cells. Subsequently, the mRNA expression levels of IL-1β, iNOS, TGF-β, and Arg1 in RAW264.7 macrophages were measured using q-PCR (Figure 5C). After 24 h of CACN136 intervention, the mRNA levels of IL-1β and iNOS showed a significant dose-dependent decrease (P < 0.0001). The high, medium, and low concentrations of CACN136 significantly increased the mRNA expression levels of TGF-β and Arg1, also in a dose-dependent manner. Furthermore, the results of Western blot experiments showed that the relative expression levels of iNOS protein in the CACN136 intervention group were significantly reduced to 0.59 times (0.6 μg/mL) and 0.7 times (0.3 μg/mL) compared to the model group (Figure 5D). At the same time, the relative expression levels of Arg1 protein in the CACN136 intervention group increased to 1.72 times (0.6 μg/mL) and 1.35 times (0.3 μg/mL) compared to the model group (Figure 5D).

The formation of lipid droplets in cells after ox-LDL induction was observed using Oil Red O staining. (Figure 6A). The results showed that compared to the model group, the number of red lipid droplets in the cells of all CACN136 groups decreased, and the color became lighter. This indicates that CACN136 can inhibit the formation of macrophage-derived foam cells. Furthermore, the free cholesterol and total cholesterol levels in RAW264.7 cells treated with CACN136 were measured using assay kits. It was found that compared to the ox-LDL group, the levels of both free cholesterol (Figure 6B) and total cholesterol (Figure 6C) in the cells of all CACN136 groups were significantly reduced dose-dependent. Next, RAW264.7 cells were stained with Dil-ox-LDL fluorescent dye to observe the effect of CACN136 on the macrophage’s ability to engulf lipoproteins. The results (Figure 6D) showed that compared to the model group, the fluorescence intensity in the cells treated with CACN136 gradually weakened. This indicates that CACN136 can inhibit the uptake of ox-LDL by macrophages. In addition, RAW264.7 cells were intervened with NBD cholesterol to verify whether CACN136 could inhibit foam cell formation by promoting cholesterol efflux. The cholesterol efflux rate was calculated, and the results (Figure 6E) showed that the efflux rate in the model group was 12.95% ± 4.11%. After CACN136 intervention, the high, medium, and low dose groups increased the efflux rate by 4.32 times, 3.59 times, and 2.98 times, respectively.

The regulatory effect of CACN136 on lipid metabolism in RAW264.7 macrophages was also verified by measuring mRNA and protein expression levels associated with lipoprotein uptake and efflux. In terms of mRNA levels (Figure 6F) of ABCA1 and SR-B1, which control cholesterol efflux, and CD36 and SRA1, which control cholesterol uptake, compared to the control group, the model group showed a significant decrease in ABCA1 and SR-B1 mRNA levels (P < 0.0001) and a significant upregulation of CD36 and SRA1 mRNA levels (P < 0.01). After CACN136 intervention, the mRNA levels of ABCA1 showed a significant dose-dependent increase, and the mRNA levels of SR-B1 were significantly higher compared to the model group. CD36 and SRA1 are two specific receptors controlling cholesterol uptake. In the model group, the mRNA expression levels of CD36 and SRA1 were 5.44-fold and 3.65-fold higher than those in the control group, respectively. CACN136 significantly downregulated the mRNA levels of CD36 (P < 0.0001) and SRA1 (P < 0.01). At the protein level (Figure 6G), the high dose group showed a 1.88-fold increase in ABCG1 and a 4.28-fold increase in ABCA1 compared to the model group after CACN136 intervention. Meanwhile, CD36 and SRA1 were significantly downregulated.

HFD-induced atherosclerosis promotes inflammation which plays a crucial role in the development of atherosclerosis (Huang et al., 2023). To evaluate the anti-inflammatory effect of CACN136 in the pathological context of atherosclerosis, the expression levels of IL-6, TNF-α were examined (Figure 7A). Compared to the blank group of mice, the levels of inflammatory factors IL-6 and TNF-α were significantly elevated in the ApoE−/− model group of mice with atherosclerosis. In contrast, the levels of inflammatory factors in the plasma of mice treated with CACN136 and the positive drug Sim decreased significantly compared to the model group, with statistical significance. Furthermore, the levels of inflammatory factors in the plasma of mice treated with CACN136 were lower than those treated with the positive drug Sim, suggesting that CACN136 has a stronger anti-inflammatory effect than Sim. To further confirm the mechanism by which CACN136 inhibits inflammation, immunohistochemical staining for iNOS and Arg1 was performed on the aortic tissues of mice in each group. Compared to the blank group, iNOS was significantly upregulated (Figure 7B), while Arg1 was significantly downregulated (Figure 7C) in the model group, indicating inflammatory infiltration in the aortas of model mice. After CACN136 treatment, the expression of iNOS was significantly downregulated in a concentration-dependent manner, and Arg1 was significantly upregulated in a concentration-dependent manner, consistent with the cellular-level results. These findings suggest that CACN136 can inhibit the inflammatory response in atherosclerosis mice induced by HFD by regulating macrophage polarization.

To evaluate the lipid metabolism effect of CACN136 in the pathological context of atherosclerosis, the expression levels of TC, TG, LDL-C, and HDL-C were examined. Due to lipid metabolism disorders, the levels of plasma TC, TG, and LDL-C were significantly higher in the atherosclerosis model group of mice compared to the blank group of mice. After treatment, CACN136 exhibited a significantly better effect in reducing plasma TC, TG, and LDL-C levels compared to the positive drug Sim (Figure 7D). Further, immunohistochemical staining revealed that CACN136 significantly downregulated the expression of CD36 and elevated the expression of ABCG1. (Figures 7E,F), which is consistent with the cellular-level results. These findings indicate that CACN136 corrects lipid metabolism disorders in HFD-induced atherosclerosis mice by simultaneously inhibiting cholesterol intake and increasing cholesterol efflux.