Abstract
Introduction
Curcumin has been found to inhibit atherosclerosis. However, its poor
stability and low activity severely limit its further application. To
overcome the shortcomings of curcumin, our team successfully designed a
novel curcumin analog, CACN136. This study aims to explore the
anti-atherosclerosis effects of CACN136 and its mechanisms.
Method and Result
Oil Red O staining results showed that CACN136 significantly improved
atherosclerosis plaques in the aorta and aortic root of ApoE-/- mice.
RNA sequencing analysis (RNA-seq) indicated that CACN136 inhibits
atherosclerosis by regulating lipid metabolism and inflammation-related
pathways. In vitro, CACN136 significantly upregulates the mRNA and
protein expression of iNOS and Arg1 in LPS-induced RAW264.7 cells. In
ox-LDL-induced RAW264.7 foam cells, CACN136 significantly reduced free
cholesterol and total cholesterol levels, and the levels of ABCA1,
CD36, and SRA1 mRNA and protein were significantly altered. In vivo,
CACN136 significantly reduced lipid and inflammatory levels, with
superior safety and efficacy compared to the same dose of simvastatin.
Discussion
CACN136 improves atherosclerotic plaque by regulating macrophage
polarization and lipid metabolism, suggesting that CACN136 may be a
promising new drug for the treatment of atherosclerosis.
Keywords: atherosclerosis, curcumin analog, macrophage, M1/M2, lipid
metabolism, CACN136
1 Introduction
Cardiovascular disease (CVD) is a common and serious illness that poses
a significant threat to human life and health, accounting for over 31%
of global deaths ([50]Roth et al., 2020). It ranks first in terms of
incidence and mortality rates in countries such as China, Europe, the
United States, and other developed nations ([51]The, 2023; [52]Lv et
al., 2024; [53]Perry et al., 2023). Currently, atherosclerosis has
emerged as the major etiology underlying various CVDs ([54]Nedkoff et
al., 2023), including hypertension, coronary heart disease, and acute
myocardial infarction. Atherosclerosis is responsible for approximately
17.6 million deaths annually, posing a severe threat to human health
([55]Roth et al., 2020).
Atherosclerosis is a complex dynamic pathological process that
primarily affects large and medium-sized arteries ([56]Hou et al.,
2023; [57]Jing et al., 2023). In the early stages of atherosclerosis,
endothelial cells attract monocytes to the arterial wall through
chemokine-receptor interactions and increased expression of
intercellular adhesion molecules ICAM-1 and vascular cell adhesion
molecule VCAM-1 ([58]Pickett et al., 2023; [59]Cybulsky and Gimbrone,
1991). Once monocytes migrate to the vessel wall, they differentiate
into macrophages, which further polarize into M1 and M2 phenotypes
under chronic inflammatory stimulation. At the same time, the increased
activity of cholesterol transport pathways leads to a large uptake of
cholesterol by pro-inflammatory M1 macrophages. In addition, the
expression of receptors (including scavenger receptors) on macrophages
increases. With the promotion of scavenger receptor activity,
cholesterol accumulated in foam cells will lead to inflammation and
aggravate the progress of atherosclerosis ([60]Al-Hawary et al., 2023).
Ultimately, lipid deposition forms heterogeneous AS plaques, and
inflammation renders the plaques vulnerable, with thin fibrous caps,
enlarged necrotic cores, and increased susceptibility to rupture and
expose pro-thrombotic material, leading to thrombosis and arterial
occlusion. Thus, the progression of atherosclerosis is consistently
associated with macrophage lipid transformation and chronic
inflammation. Currently, lipid-lowering drugs (including statins and
PCSK9 inhibitors) are the standard therapeutic agents for
atherosclerosis. Although statin therapy has been shown to reduce the
risk of atherosclerosis events ([61]Ference et al., 2017), long-term
use of statins may lead to liver enzyme abnormalities, muscle toxicity,
and diabetes mellitus, with poor patient compliance ([62]Björnsson,
2017), and PCSK9 inhibitors also have the inherent drawbacks of
monoclonal antibody drugs, such as high dosage requirements and
frequent administration ([63]Maningat et al., 2013). Therefore, given
the shortcomings of current first-line clinical drugs, it is
particularly necessary and urgent to develop safe and effective new
anti-atherosclerosis drugs.
In recent decades, researchers have endeavoured to search for potential
anti-atherosclerotic agents among natural compounds. Among them,
curcumin, a naturally occurring bioactive polyphenol, has been
extensively studied in anti-atherosclerosis due to regulating
cholesterol homeostasis and macrophage polarization ([64]Menon and
Sudheer, 2007; [65]Momtazi-Borojeni et al., 2019; [66]Lin et al.,
2020). Although curcumin has shown certain advantages and a good
safety, it was severely restricted for further development and
application due to its poor stability and low activity. Researchers are
simultaneously searching for analogues of curcumin to improve the
drawbacks of its clinical application ([67]Tagde et al., 2021;
[68]Rafiee et al., 2019).
Therefore, a series of curcumin analogues were screened and synthesized
by our group in previous studies ([69]Zhao et al., 2021-09). Among
them, a novel methyl monocarbonyl curcumin analogue named CACN136
([70]Figure 1) showed significant antidepressant in vitro and in vivo
([71]Zhao et al., 2021-09), which was able to show significant
protection against oxidative stress injured cells at all
concentrations, and compared with ascorbic acid and curcumin, the
CACN136 possesses a stronger
2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free radical
ion scavenging ability ([72]Zhao et al., 2021-09; [73]Chen et al.,
2024). Whereas the high presence of free radicals leading to oxidative
stress imbalance is one of the reasons for accelerating the early onset
of atherosclerosis. It has also been shown that oxygen free radical is
an important mediator in inflammation. A number of scholars have found
that there is a causal relationship between depression and
atherosclerosis co-morbidity ([74]Babic et al., 2023; [75]Bezna et al.,
2022; [76]Neil et al., 2017). Meanwhile, CACN136 had a higher
solubility (150.4 μg/mL) than curcumin, suggesting the possibility of
better in vivo absorption ([77]Wen et al., 2024). Our research team
also found that orally administering CACN136 to rats fed high-fat diets
has a significant lipid-lowering effect. Based on our previous
experimental results, we speculate that CACN136, as a novel curcumin
analogue, has a stronger anti-atherosclerosis effect compared to
curcumin.
FIGURE 1.
[78]Chemical structure transformation showing the conversion of
Curcumin (Cur) to CACN136. Curcumin has two methoxy groups and two
hydroxyl groups connected to aromatic rings, separated by a diketone
chain. CACN136 depicts a structural change with an amide bond replacing
the central diketone group.
[79]Open in a new tab
CACN136 is a structural analog of curcumin.
This study aimed to systematically investigate the atherosclerosis
effects and mechanisms of CACN136 to provide a theoretical basis and
evidence support for developing novel atherosclerosis therapeutic
agents. Firstly, an atherosclerosis model was established in high-fat
diet-induced ApoE−/− mice, and CACN136 was confirmed to inhibit the
formation of atherosclerosis plaques in mice using Oil Red O staining.
Then, network pharmacology and transcriptomics were utilized to predict
the mechanism of action and targets of CACN136. In vitro, the ability
of CACN136 to regulate polarization and lipid metabolism was assessed
by the LPS-induced RAW264.7 cell polarization model and
oxidized-LDL-induced RAW264.7 foam cell model, respectively. In vivo,
Elisa assay and immunohistochemistry were performed to assess the in
vivo relevant mechanisms and targets of CACN136. The experimental
results suggest that CACN136 is expected to be a new drug candidate for
the treatment of atherosclerosis.
2 Methods and materials
2.1 Evaluation of the anti-atherosclerosis effect of CACN136 in vivo
2.1.1 Animals
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.
2.1.2 Atherosclerosis animal model establishment and evaluation
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 ([80]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).
2.1.3 CACN136 in vivo safety evaluation
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.
2.1.4 Oil red O staining in en face and aortic root of apoE−/− 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:
[MATH: Lesion
area (%
)=(Red area)/(Total area of the
aorta)×100% :MATH]
2.2 Prediction of the mechanism of action of CACN136 anti-AS
2.2.1 Network pharmacology
Network pharmacology Disgenet database ([81]https://www.disgenet.org/),
Genecards database ([82]https://www.genecards.org/), and OMIM database
([83]https://www.omim.org/) were used to identify target genes related
to atherosclerosis disease. Swiss Target Prediction
([84]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 ([85]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 ([86]https://david.ncifcrf.gov/).
2.2.2 Transcriptomics
RNA extraction was performed using TRIzol reagent (Invitrogen, cat. NO
15596026) following the method described by Chomczynski et al.
([87]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.
2.3 Investigating the mechanism of CACN136 anti-atherosclerosis action in
vitro
2.3.1 Cell culture and grouping
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% CO[2]. 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.
2.3.2 Cell cytotoxicity assay
The impact of the drug on cell proliferation viability was determined
using the MTT colorimetric assay, as previously described ([88]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.
2.3.3 Q-PCR experiment
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 [89]Table 1.
TABLE 1.
Primer sequences.
Gene Primer sequence 5′-3′,F Primer sequence 5′-3′,R
IL-1β ACCCCAAAAGATGAAGGGCTGCTT TGCCTGCCTGAAGCTCTTGTTGAT
iNOS CTTGGAGCGAGTTGTGGATTG GGTCGTAATGTCCAGGAAGTAGGT
TGF-β GCGGACTACTATGCTAAAGAGG CACTGCTTCCCGAATGTCT
Arg1 GGAGAAGGCGTTTGCTTAGTTC GGAGAAGGCGTTTGCTTAGTTC
SR-B1 TTTCAGCAGGATCCATCTGGTGGA AGTTCATGGGGATCCCAGTGAC
ABCA1 GGAGCTGGGAAGTCAACAAC ACATGCTCTCTTCCCGTCAG
CD36 GGAGCCATCTTTGAGCCTTCA GAACCAAACTGAGGAATGGATCT
SRA1 TTCACTGGATGCAATCTC CTTGGCTTGCTTCGGAACTC
[90]Open in a new tab
2.3.4 Western blot experiment
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.
2.3.5 Oil red O staining experiment
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.
2.3.6 Cholesterol detection experiment
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.
2.3.7 Dil-ox-LDL uptake experiment
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.
2.3.8 Cholesterol efflux function assay
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 (%) =
[MATH:
Fluorescence in
tensitysuper
natantTotal fl
uorescence intensitysuper
natant+<
mi>cells :MATH]
× 100%
2.4 Investigating the mechanism of CACN136 anti-AS action in vivo
2.4.1 Plasma cholesterol level measurement
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).
2.4.2 Elisa assay
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).
2.4.3 Immunohistochemistry
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.
2.5 Statistical analysis
The data are presented as mean ± SEM (standard error of the mean).
Statistical significance was evaluated using one-way analysis of
variance (ANOVA) with SPSS 22.0 and GraphPad Prism 5.0 for multiple
group comparisons. P < 0.05 indicates statistical significance.
3 Results
3.1 The anti-atherosclerosis effect of CACN136
3.1.1 Successful construction of atherosclerosis model mice
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 ([91]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
([92]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) ([93]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 ([94]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.
FIGURE 2.
[95]Panel A shows ultrasound images comparing a control to a model,
highlighting differences in arteries. Panel B features bar graphs
displaying plasma levels of ALT and AST across different treatments and
dosages. Panel C is a line graph depicting weight changes over
seventeen weeks among different groups. Panel D presents histological
images of heart, liver, spleen, lung, kidney, and aorta tissues from
control, model, Sim (10 mg/kg), and CACN136 treatments at various
dosages.
[96]Open in a new tab
CACN136 has good safety. (A) Mice aortic arch ultrasound B-Mode image;
(B) Plasma ALT and AST levels in mice (
[MATH: x¯ :MATH]
± s, n = 3).^##P < 0.01, compared with the Control group.*P < 0.05, **P
< 0.01, ***P < 0.001, compared with the model group. (C) mouse body
weight curve (
[MATH: x¯ :MATH]
± s, n = 6); (D) HE staining of mice organs (100×).
TABLE 2.
Cardiac ultrasound related indexes of mice (
[MATH: x¯ :MATH]
± s, n = 3).
Indexes Units Control Model
EF % 77.62 ± 8.16 66.99 ± 6.67*
SF % 45.51 ± 7.74 36.08 ± 5.16*
LVM mg 113.80 ± 22.06 186.68 ± 92.70
LVCM mg 91.04 ± 17.65 121.96 ± 17.22*
Flow velocity mm/s 892.42 ± 118.95 586.32 ± 106.76***
BP mmHg 3.23 ± 0.82 1.41 ± 0.51**
[97]Open in a new tab
3.1.2 CACN136 safety is good
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 [98]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 [99]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.
3.1.3 CACN136 alleviated atherosclerosis lesions in HFD-induced ApoE−/− mice
The ability of CACN136 to alleviate atherosclerosis lesions was
evaluated using Ultrasound Imaging System and Oil Red O Staining. As
shown in [100]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 [101]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. [102]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%). [103]Figure 3D shows the entire aorta stained with
oil-red O. The positive area in the model group reached 0.059 ±
0.006 cm^2. The positive areas in the treatment groups, from high to
low, were as follows: Sim group (0.041 ± 0.003 cm^2), CACN136 low-dose
group (0.0034 ± 0.002 cm^2), CACN136 medium-dose group (0.028 ±
0.002 cm^2), and CACN136 high-dose group (0.018 ± 0.001 cm^2).
FIGURE 3.
[104]Figure showing multiple panels related to a study on CACN136
effects. Panel A: Ultrasound images showing artery conditions under
different treatments. Panel B: Bar graphs displaying blood flow
velocities in various arteries. Panel C: Histological sections
illustrating lesion areas, with a graph comparing lesion percentages.
Panel D: Images of arteries with a graph showing lesion area
comparisons. Panels depict control, model, Simvastatin, and different
CACN136 dosages.
[105]Open in a new tab
CACN136 alleviated atherosclerosis lesions in HFD-induced ApoE−/− mice.
(A) Mice aortic arch ultrasound B-Mode image; (B) Blood flow velocity
(mm/s) in the innominate artery, left common carotid artery, and left
subclavian artery of mice (
[MATH: x¯ :MATH]
± s, n = 3); (C) Oil red O staining of mice aortic root and
Quantification of oil red O staining of mice aortic root (
[MATH: x¯ :MATH]
± s, n = 3); (D) Oil red O staining of the entire aorta and
Quantification of oil red O staining of the entire aortic (
[MATH: x¯ :MATH]
± s, n = 3). ###P < 0.001, compared with the Control group.*P < 0.05,
**P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the model
group.
3.2 Prediction of the anti-atherosclerosis mechanism of CACN136
3.2.1 Network pharmacology analysis
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
([106]Figure 4A). The KEGG enrichment analysis results ([107]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 ([108]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.
FIGURE 4.
[109]Venn diagrams, charts, and graphs illustrating gene analysis and
pathway enrichment. Image A shows overlapping genes in a Venn diagram.
B displays a dot plot of pathways, with size and color indicating gene
count and significance. C is a bar graph categorizing processes. D has
volcano plots for gene expression changes. E is another Venn diagram
comparing conditions. F is a network diagram connecting genes,
highlighting key proteins. G is a dot plot focusing on significant
pathways.
[110]Open in a new tab
CACN136 inhibits atherosclerosis by regulating lipid metabolism and
inflammation-related pathways. (A) Disease-drug target veen plots; (B)
Network pharmacology KEGG enrichment plots; (C) Network pharmacology GO
enrichment plots; (D). Transcriptome differential gene volcano plots;
(E) LPS vs. Control vs. CACN136 vs. LPS differential gene veen plots;
(F) Differential gene core targets; (G) Transcriptome differential gene
KEGG enrichment plots.
3.2.2 Transcriptomic analysis of differentially expressed genes
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 ([111]Figure 4D). A total of 228
target genes were obtained by plotting the Venn diagram to take the
intersection ([112]Figure 4E). A protein-protein interaction (PPI)
network was further constructed and topological analysis was performed
using Cytoscape, identifying 34 core target genes ([113]Figure 4F).
KEGG enrichment analysis using David ([114]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.
3.3 CACN136 regulates macrophage polarization and lipid metabolism in vitro
3.3.1 Regulation of LPS-induced macrophage RAW264.7 M1/M2 polarization by
CACN136
The effect of CACN136 on the viability of RAW264.7 cells was evaluated
using the MTT assay ([115]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
([116]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 ([117]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 ([118]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 ([119]Figure 5D).
FIGURE 5.
[120]A composite image showing various experimental results. Panel A
presents a bar graph of percentage cell viability at different
concentrations of CACN136 to screen for safe concentrations for
Raw264.7 cells, indicating increased viability compared to control.
Panel B shows microscopic images demonstrating morphologic differences
in cells treated with CACN136, control, and LPS. Panel C contains bar
graphs displaying mRNA expression levels of IL-1β, iNOS, TGF-β, and
Arg1 under different conditions, highlighting significant changes.
Panel D includes Western blot images for iNOS, Arg1, and Tubulin,
accompanied by bar graphs of relative protein expression levels for
iNOS and Arg1, comparing control and treatment groups.
[121]Open in a new tab
CACN136 regulates macrophage polarization through the iNOS/Arg1 axis.
(A) Effect of CACN136 intervention for 24h on RAW264.7 viability (
[MATH: x¯ :MATH]
± s, n = 3); (B) Morphological changes in RAW264.7 cells; (C) Effect of
CACN136 on mRNA levels in RAW264.7 cells (
[MATH: x¯ :MATH]
± s, n = 3); (D) Effect of CACN136 on protein levels in RAW264.7 cells
(
[MATH: x¯ :MATH]
± s, n = 3). ^##P < 0.01,^###P < 0.001, ^####P < 0.0001, compared with
the Control group. *P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001,
compared with the LPS model group.
3.3.2 CACN136 inhibits ox-LDL-induced macrophage RAW264.7 transition to foam
cells
The formation of lipid droplets in cells after ox-LDL induction was
observed using Oil Red O staining. ([122]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 ([123]Figure 6B) and total
cholesterol ([124]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 ([125]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 ([126]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.
FIGURE 6.
[127]A multi-panel image displays experimental data on cholesterol
levels and gene expression. Panel A shows cell morphology under
different conditions. Panels B and C present bar graphs of free and
total cholesterol content, respectively. Panel D shows fluorescence
microscopy images with an accompanying bar graph of fluorescence
intensity. Panel E presents a bar graph of cholesterol efflux rates.
Panel F includes bar graphs of mRNA levels for genes ABCA1, SR-B1,
CD36, and SRA1. Panel G shows Western blots for specific proteins and
corresponding bar graphs of protein expression levels, indicating
CACN136's effect on cholesterol metabolism and related gene expression.
[128]Open in a new tab
CACN136 modulates cholesterol metabolism pathways in foam cells. (A)
Oil red O staining for detection of foam cell formation; (B) Free
cholesterol content in RAW264.7 cells (
[MATH: x¯ :MATH]
± s, n = 3); (C) Total cholesterol content in RAW264.7 cells (
[MATH: x¯ :MATH]
± s, n = 3); (D) Ability of RAW264.7 cells to phagocytose Dil-ox-LDL
(blue fluorescence for Dapi, red fluorescence for Dil) and
Quantification of Dil fluorescence intensity (
[MATH: x¯ :MATH]
± s, n = 3); (E) Cholesterol efflux rate (
[MATH: x¯ :MATH]
± s, n = 3); (F) Effect of CACN136 on mRNA levels in RAW264.7 cells (
[MATH: x¯ :MATH]
± s, n = 3); (G) Effect of CACN136 on protein levels in RAW264.7 cells
(
[MATH: x¯ :MATH]
± s, n = 3).^#P < 0.05,^##P < 0.01,^###P < 0.001, ^####P < 0.0001,
compared with the Control group. *P < 0.05 **P < 0.01 ***P < 0.001,
****P < 0.0001, compared with the ox-LDL model group.
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 ([129]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 ([130]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.
3.4 CACN136 regulates inflammation and lipid metabolism in vivo
3.4.1 CACN136 inhibited inflammation in HFD-induced ApoE−/− mice
HFD-induced atherosclerosis promotes inflammation which plays a crucial
role in the development of atherosclerosis ([131]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 ([132]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 ([133]Figure 7B),
while Arg1 was significantly downregulated ([134]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.
FIGURE 7.
[135]Graphs and histological images showing the effects of different
treatments on various biological markers. Panel A: Bar graphs of plasma
levels of IL-6 and TNF-α. Panel B: Histological images and bar graph of
iNOS. Panel C: Histological images and bar graph of Arg1. Panel D: Bar
graphs of plasma levels of TC, TG, LDL-C, and HDL-C. Panel E:
Histological images and bar graph of CD36. Panel F: Histological images
and bar graph of ABCG1. Statistical significance is indicated with
asterisks.
[136]Open in a new tab
CACN136 improves inflammatory response and lipid metabolism in
atherosclerotic mice. (A) Plasma levels of inflammatory factor TNF-α
and IL-6 in mice (
[MATH: x¯ :MATH]
± s, n = 3); (B) Aortic iNOS staining and Percentage area of positive
aortic iNOS staining (%) (
[MATH: x¯ :MATH]
± s, n = 3); (C) Aortic Arg1 staining and Percentage area of positive
aortic Arg1 staining (%) (
[MATH: x¯ :MATH]
± s, n = 3); (D) Plasma Cholesterol level assay in mice (
[MATH: x¯ :MATH]
± s, n = 3); (E) Aortic CD36 staining and Percentage area of positive
aortic CD36 staining (%) (
[MATH: x¯ :MATH]
± s, n = 3); (F) Aortic ABCG1 staining and Percentage area of positive
aortic ABCG1 staining (%) (
[MATH: x¯ :MATH]
± s, n = 3).^#P < 0.05,^###P < 0.001, ^####P < 0.0001, compared with
the Control group.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001,
compared with the model group.
3.4.2 CACN136 regulated lipid metabolism in HFD-induced ApoE−/− mice
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 ([137]Figure 7D). Further, immunohistochemical staining
revealed that CACN136 significantly downregulated the expression of
CD36 and elevated the expression of ABCG1. ([138]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.
4 Discussion
During the last decades, tremendous efforts have been made to improve
atherosclerosis. Current treatments for atherosclerosis are mainly
chemotherapy and surgical procedures such as recanalization,
reconstruction or bypass grafting of stenotic or occluded arteries
([139]Singh et al., 2013; [140]Lordan et al., 2021). Statins, as the
current first-line drugs for the treatment of atherosclerosis, have
relatively large side effects, and their long-term use can lead to
liver enzyme abnormalities, muscle toxicity, and diabetes mellitus,
with poor patient compliance ([141]Björnsson, 2017). Therefore, the
development of safe and effective new anti-atherosclerosis drugs is
particularly necessary and urgent. In recent years, herbal medicines
have been deeply accepted and widely used as supplements and
alternative therapies for various diseases in many countries
([142]Bergien et al., 2020; [143]Missenda et al., 2023; [144]Zhao et
al., 2021). Modern studies have found that curcumin, which is mainly
derived from the rhizomes of turmeric-like plants, has pharmacological
effects that inhibit atherosclerosis ([145]Si et al., 2015),
specifically in terms of reducing inflammation and oxidative stress,
anticoagulation, improving lipid metabolism, promoting glucose
metabolism, modulating smooth muscle cell proliferation and migration,
lowering blood pressure, reducing the number of pathologic
neovascularization, and protecting endothelial cells ([146]Peled et
al., 2014; [147]Sanson et al., 2013; [148]Moore et al., 2013). Although
curcumin has shown some advantages in anti-atherosclerosis, its
deficiencies such as poor stability and activity have greatly limited
its further developmental applications ([149]Lee et al., 2014;
[150]Chen et al., 2019). Zheng found that curcumin analog L3 reduced
dyslipidemia and hyperglycemia, attenuated oxidative stress, enhanced
the activity of antioxidant enzymes, increased the level of nitric
oxide in plasma and aortic arch, and ameliorated adiposity and
atherosclerotic degeneration, thereby preventing diabetic
atherosclerosis ([151]Kumar et al., 2020). Thus, curcumin analogs may
be an effective way to address their deficiencies.
Our group screened and synthesized a series of curcumin analogs in the
previous study, in which a novel curcumin analog, CACN136, was found to
significantly reduce lipid levels with good safety profile compared
with lovastatin positive and negative control groups in hyperlipidemic
rats by gavage for 90 days. CACN136 has demonstrated significant
antidepressant effects in vitro and in vivo ([152]Zhao et al.,
2021-09), with robust protection against oxidative stress-induced
cellular damage. Compared to ascorbic acid and curcumin, CACN136
exhibits superior ABTS radical scavenging capacity ([153]Zhao et al.,
2021-09; [154]Chen et al., 2024). Excessive free radicals drive
oxidative stress imbalance, a recognized contributor to early
atherosclerosis pathogenesis. Notably, oxygen radicals also act as key
mediators of inflammation. Emerging evidence supports a causal link
between depression and atherosclerosis-related complications
([155]Babic et al., 2023; [156]Bezna et al., 2022; [157]Neil et al.,
2017). Collectively, our findings highlight CACN136s therapeutic
potential, leveraging its lipid-lowering, antioxidant and
anti-inflammatory properties to inhibit AS initiation and progression.
In the early stages of atherosclerosis development, endothelial cells
attract monocytes to the arterial wall through chemokine-receptor
interactions ([158]Cybulsky and Gimbrone, 1991). Once monocytes migrate
to the arterial vascular wall, they differentiate into macrophages,
which further polarize due to chronic inflammation. There are two
polarization phenotypes: the M1 type, which secretes pro-inflammatory
factors such as IL-6 and TNF-α, and the M2 type, which secretes
anti-inflammatory factors such as TGF-β. Concurrently, the activity of
cholesterol transport pathways increases, leading M1-type
macrophages—which mediate pro-inflammatory lesions—to take up large
amounts of cholesterol. Subsequently, macrophages increase the
expression of various receptors, including scavenger receptors
([159]Al-Hawary et al., 2023). The cholesterol accumulated by scavenger
receptors causes macrophages to gradually transform into foam cells,
ultimately leading to plaque formation. Thus, macrophages play a
crucial role in the development of atherosclerosis. Therefore, this
study focuses on macrophages, using a patented compound developed by
the research team ([160]Zhao et al., 2021-09), and systematically
investigates the anti-atherosclerotic effects and mechanisms of action
of the curcumin analog CACN136 through HFD-induced atherosclerosis
mouse model, network pharmacology analysis, transcriptomics analysis,
and experiments using RAW264.7 macrophages.
Since CACN136 was used for the first time in ApoE−/− mice, we first
verified its safety by measuring the blood biochemical indicators and
organ HE staining. Safety evaluation results showed that CACN136 did
not have a significant detrimental effect on liver function in both the
control and ApoE−/− mice, and even exhibited a slight protective
effect. On the other hand, Sim showed strong liver toxicity in ApoE−/−
mice, indicating a lower safety profile. In the model group, liver
tissue structure was disrupted, while the low and medium dose groups
showed mild vacuolar degeneration of liver cells. The high dose group
did not exhibit significant pathological changes in liver cells. This
suggests that the slight liver damage in the low and medium dose groups
was not caused by CACN136 itself but rather by liver injury due to the
atherosclerosis model. CACN136 has a reparative function, but the
reparative effect at low and medium doses is not sufficient. However,
when the dosage of CACN136 reached 40 mg/kg, the damaged liver was
corrected to a normal level.
We next confirmed the anti-atherosclerosis effect of CACN136. Through
Oil Red O staining of aortic root cross-section sections and intact
aortas of mice, we found that CACN136 significantly reduced
atherosclerotic plaques in mice, and the effect was superior to that of
the same dose of simvastatin.
After confirming the anti-atherosclerosis effect of CACN136, we began
to predict its mechanism using bioinformatics. The network pharmacology
analysis indicated that CACN136 primarily exerted its effects on
LPS-induced inflammation and lipid metabolism in the pathological
process of atherosclerosis. RAW264.7 macrophage cells are commonly used
in atherosclerosis-related inflammation and lipid metabolism studies.
Therefore, transcriptomic analysis was conducted to further validate
the predictions made by network pharmacology. Among the differentially
expressed genes obtained through intersecting analysis, many were
related to inflammation and immunity, while fewer were related to lipid
metabolism. This could be attributed to the fact that both the control
and treatment groups of cells were stimulated with LPS, which primarily
mediated the production of inflammation in RAW264.7 cells. However, the
KEGG pathway enrichment analysis of the transcriptomic data revealed
that lipid and atherosclerosis signaling pathways, in addition to the
inflammation signaling pathway still ranked high. Meanwhile, curcumin
also has a regulatory effect on inflammation and lipid metabolism
([161]Ji et al., 2021; [162]Hasanzadeh et al., 2020), suggesting that
the anti-atherosclerosis mechanism of CACN136 is closely associated
with macrophage inflammation and lipid metabolism.
In vitro, CACN136 significantly modulates LPS-induced RAW264.7 M1/M2
polarization fractionation and inhibits inflammatory responses. M1
macrophages are a subtype of macrophages in an immunologically
activated state. They secrete inflammatory factors such as TNF-α and
IL-1β, and exhibit increased expression of iNOS, which is closely
associated with promoting inflammatory responses ([163]Wu et al.,
2019). iNOS is an important enzyme in M1 macrophages. When macrophages
are stimulated by LPS, they produce and release the iNOS protein. In M1
macrophages, the expression of iNOS and the production of NO are
significantly increased, thereby promoting inflammatory responses and
antimicrobial abilities. M2 macrophages, on the other hand, are a
subtype of macrophages in an immunosuppressive state. They secrete
immunosuppressive and reparative factors and are closely involved in
immune regulation and tissue repair processes. Arg1 is a specific
molecular marker in M2 macrophages. The expression and activity of Arg1
increase in M2 macrophages, which can inhibit inflammatory responses,
facilitate tissue repair, and regulate immune responses ([164]Luo et
al., 2020). Arg1 competitively inhibits the availability of arginine
required for the iNOS enzyme, thereby reducing NO synthesis and
decreasing inflammatory responses. Some studies have shown that,
curcumin can significantly reduce the expression of inflammatory
factors in the supernatant of LPS-stimulated raw264.7 cells and
inhibits iNOS protein expression with a Macrophage-inducible C-type
lectin dependent pattern ([165]Tan et al., 2019). Whereas CACN136, a
curcumin analog, both decreased the mRNA and protein levels of IL-1β
and iNOS, and increased the mRNA and protein levels of TGF-β and Arg1.
This indicates that CACN136 can significantly reduce the number of
M1-type RAW264.7 cells and increase the number of M2-type RAW264.7
cells, thereby suppressing inflammatory responses.
In vitro, CACN136 inhibits macrophage formation of foam cells while
inhibiting RAW264.7 uptake of cholesterol and promoting RAW264.7 efflux
of cholesterol. CD36 is capable of binding to LDL, ox-LDL, and other
lipid substances, promoting their uptake and conversion into foam
cells. Similarly, SRA1 can also bind to ox-LDL and mediate its uptake
and internalization. ABCG1 protein is predominantly expressed in
macrophages, dendritic cells, and endothelial cells, and it promotes
the efflux of cholesterol and lipids ([166]Cheng et al., 2016). ABCA1
protein is mainly expressed in macrophages and endothelial cells, and
it plays a role in regulating the transport and metabolism of
cholesterol and phospholipids ([167]Chen et al., 2021). ABCA1
facilitates the efflux of cholesterol and phospholipids from the
intracellular space to the extracellular space, forming HDL.
Additionally, SR-B1 has a high affinity for binding to HDL particles
and is involved in the reverse transport and efflux of cholesterol. HDL
is considered a “beneficial” cholesterol carrier that promotes reverse
transport and efflux of cholesterol. Therefore, CACN136 can reduce the
expression of CD36 and SRA1, inhibiting cholesterol internalization,
while upregulating the expression of ABCA1, ABCG1, and SR-B1 to promote
cholesterol efflux. These findings are similar to curcumin-related
reports, such as curcumin enhances the expression of ABCA1 and ABCG1 in
foam cells, promotes cholesterol efflux ([168]Xu et al., 2025),
regulates oxLDL-induced CD36 expression by inhibiting p38 MAPK
phosphorylation in RAW 264.7 cells, and inhibits foam cell formation
([169]Min et al., 2013). This confirms that CACN136 contributes to the
reduction of lipid deposition in RAW264.7 macrophages and inhibits the
occurrence and development of atherosclerosis.
CACN136 reduced plasma inflammatory factor levels and plasma
cholesterol levels in atherosclerosis model mice. The growth curve of
mice showed a decrease in body weight in all treatment groups. This is
because the drug has a lipid-lowering effect, reducing the levels of
cholesterol and triglycerides in mice and subsequently reducing body
weight. The rate and extent of weight loss were lower in the positive
control group compared to the CACN136 group, indicating that CACN136
degrades mouse fat more rapidly and efficiently, an effect similar to
that of curcumin. The hypolipidemic effects of curcumin in rodents were
first reported nearly 50 years ago ([170]Rao et al., 1970).
Supplementation of curcumin (0.05% w/w) in hamsters fed a high-fat diet
significantly reduced plasma TG and free fatty acid concentrations by
approximately 25% and 18%, respectively, and increased HDL-C by 17%,
but their LDL-C levels did not change significantly ([171]Jang et al.,
2008). Another study showed that (0.02% w/w) curcumin-treated mice had
significantly lower plasma TC, LDL-C, and TG levels after 18 weeks
([172]Shin et al., 2011). In the present study of atherosclerotic mice
treated with CACN136 (0.001% w/w) for only 28 days, the levels of TC,
TG and LDL-C were significantly reduced in the treated group compared
to the model group. It indicated that the lipid-lowering level of
CACN136 was significantly better than curcumin. Measurement of mouse
cholesterol levels showed that the plasma HDL levels of AS mice in the
model group were higher than those in the treatment groups. This may be
due to two reasons ([173]Roth et al., 2020): the therapeutic drug may
inhibit the synthesis of high-density lipoprotein in the liver or
increase the metabolic rate of HDL, thereby reducing HDL levels in mice
([174]Kühnast et al., 2015; [175]Leão et al., 2019; [176]Tuteja and
Rader, 2014); ([177]The, 2023) CACN136 can slow down the progression of
atherosclerosis by inhibiting inflammatory responses. However,
inhibiting inflammatory responses may also have a negative impact on
the synthesis and function of HDL ([178]Van Lenten et al., 1995;
[179]Charles-Schoeman et al., 2007), leading to a decrease in HDL
levels.
As with the validation results at the cellular level, animal
experiments corroborated the specific mechanisms of CACN136
anti-atherosclerosis as: improvement of inflammatory response through
downregulation of iNOS and upregulation of Arg1; and improvement of
lipid metabolism through upregulation of ABCG1 and downregulation of
CD36.
5 Conclusion
Our team has identified a novel curcumin analog, CACN136, with superior
anti-atherosclerotic activity and safety profile compared to
simvastatin. The mechanism involves the regulation of macrophage
polarization and lipid metabolism. CACN136 is a promising new drug for
future anti-atherosclerosis. However, more in-depth mechanisms remain
to be investigated.
Funding Statement
The author(s) declare that financial support was received for the
research and/or publication of this article. This study was supported
by the National Natural Science Foundation of China (No. 82474105), the
Cooperation Projectors of Chunhui Plan of the Ministry of Education of
China (No. HZKY20220563), the Sichuan Natural Science Foundation (No.
2024NSFSC0049, 2024NSFSC0625, 25NSFSC0581), Sichuan Science and
Technology Program (No. 2022YFS0627, 2022YFS0607), the Key R&D Project
of Luzhou-Southwest Medical University (No. 2023LZXNYDHZ002,
2024LZXNYDJ110, 2024LZXNYDJ009), the Cooperation Projects of Sichuan
Credit Pharmaceutical CO., Ltd., Central Nervous System Drug Key
Laboratory of Sichuan Province (No. 210027-01SZ, 200017-01SZ,
230007-01SZ, 230008-01SZ), the Chongqing Traditional Chinese Medicine
Inheritance and Innovation Team Construction Project “Traditional
Chinese Medicine New Drug and Safety Research Inheritance and
Innovation Team” (No. 2022-8), Luzhou Science and Technology Plan
Project (No. 2023JYJ034, 2023JYJ022, 2022-SYF-85, 2024RCM243).
Data availability statement
The original contributions presented in the study are included in the
article/supplementary material, further inquiries can be directed to
the corresponding authors.
Ethics statement
The animal study was approved by Animal Welfare Ethics Review Committee
of Southwest Medical University. The study was conducted in accordance
with the local legislation and institutional requirements.
Author contributions
QZ: Data curation, Conceptualization, Project administration, Writing –
original draft, Writing – review and editing, Investigation. YuZ:
Writing – original draft, Formal Analysis, Data curation,
Investigation. ZL: Writing – original draft, Investigation, Formal
Analysis. JT: Data curation, Formal Analysis, Writing – original draft.
CP: Software, Writing – original draft, Methodology, Investigation. WZ:
Investigation, Writing – review and editing, Software. PS: Writing –
original draft, Software, Investigation. YiZ: Methodology, Formal
Analysis, Writing – review and editing. JJ: Supervision, Writing –
review and editing, Methodology. YY: Writing – review and editing,
Methodology. SC: Writing – review and editing, Project administration.
YW: Project administration, Funding acquisition, Writing – review and
editing. LZ: Project administration, Conceptualization, Writing –
review and editing, Supervision.
Conflict of interest
The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.
Generative AI statement
The author(s) declare that no Generative AI was used in the creation of
this manuscript.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or claim
that may be made by its manufacturer, is not guaranteed or endorsed by
the publisher.
References