Tibial bone marrow cells from 3-month-old C57BL/6 mice (Guangdong Medical Laboratory Animal Center, China) were flushed and suspended in MEM-α (Gibco, UAS). Cells were collected by centrifugating at 500 RCF for 5 min and then cultured in MEM-αcontaining 10% FBS (Gibco, UAS), 1% penicillin–streptomycin (Corning, United States) and 30 μg/ml macrophage-colony stimulating factor (M-CSF) (Proteintech, United States) in a CO₂ incubator overnight. The adherents were bone marrow-derived macrophages (BMMs), which were digested into cell suspensions by trypsin after successful culture and used in subsequent experiments.

BMMs cells were seeded in a 96-well culture plate with 5 × 10³ cells/well and cultured for 24 h. Then, BMMs in 96-well culture plates were treated with 50, 25, 12.5, 6.25, 3.13, and 1.56 μM of ICAC for 24 h. The cell viability was determined by the MTT assay as we previously described (Huang et al., 2020).

RAW264.7 5 × 10³ cells/well were diluted with complete medium and seeded on a 96-well culture plate for 24 h. After that, cells were treated with ox-LDL at a final concentration of 50 μg/ml to generate foam cells. In the meantime, the cells were indicated with or without compounds for 24 h. 0.1% DMSO was used as a control in the group that was without compound treatment. Foam cells were detected by oil red O staining (ORO) according to the reported method. Briefly, the supernatant was removed and the cells were fixed with 4% paraformaldehyde and stored at 4°C overnight. The cells were washed three times with PBS after the paraformaldehyde was discarded and stained with an oil red O staining kit (Nanjing Jiancheng Bioengineering Research Institute, China). The foam cell was observed under a microscope (Leica DMi8/DPC7000T, Germany). Intracellular lipids were extracted by adding 50 μl of isopropyl alcohol and then shaking for 10 min. The extracted lipids were detected at a 492 nm wavelength by an automatic microplate reader (Thermo Varioskan Flash, United States).

For RT-qPCR and analyses, BMMs cells were diluted with complete medium containing 10 μg/ml M-CSF and seeded on a 6-well culture plate. BMMs were treated with 10 μM ICAC for 24 h, after stimulated by ox-LDL (50 μg/ml) for 24 h. Then all the cells were washed twice by PBS and collected for RT-qPCR or Western bolt experiments.

RAW264.7 cells were plated according to the method mentioned above. After 24 h of culture, RAW264.7 was equilibrated with 2 μM 25 NBD-cholesterol for 24 h and the lipid overload model of RAW264.7 was obtained. Subsequently, the cells were washed with PBS twice, and then cultured with serum-free culture with ICAC at 25, 12.5, 6.25, 3.13, and 1.56 μM for 18 h. HDL (final concentration of 30 μg/ml) was added, while the control wells were left without HDL for 2 h. The fluorescence-labeled cholesterol was released from cells into the medium and then was measured using a multifunctional enzyme plate apparatus at 485 nm excitation and 535 nm emission light by an automatic microplate reader. The fluorescence cholesterol content in the cell supernatant of treatment minus the cholesterol in the supernatant of control well was taken as the index to measure the cholesterol efflux of cells. The higher the ratio was, the higher the external cholesterol efflux was.

All animal experiments were approved by the Animal Ethics Committee of the Guangdong Provincial Engineering Technology Institute of Traditional Chinese Medicine (Guangzhou, China). The 6-week-old male C57BL/6 mice weighing 18∼22 g were obtained from the Guangdong Medical Experimental Animal Center (Guangzhou, China). C57BL/6 mice were randomly separated into control group, high-fat diet (HFD) (SYSE Bio-tec Co., Ltd., China) group, HFD with 40 mg/kg/d atorvastatin group (AT; positive control drug), HFD with 2 mg/kg/d T0901317 (T090, APExBIO, United States) group, HFD with 20 mg/kg/d ICAC (Chengdu, China) group, and HFD with 10 mg/kg/d ICAC combined with 2 mg/kg/d T0901317 group. Except for the control group, the others were fed a high-fat diet supplemented with the corresponding drug for 12 weeks. Mice were sacrificed at the end of the treatment. The liver, brown adipose tissue, kidney, heart, and body weight were recorded. The serum was collected to detect the content of total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein cholesterol (LDL-C), and ox-LDL. Parts of livers and intestines were stored at −80°C for mRNA and lipid detection. Livers for hematoxylin-eosin (HE) staining and ORO staining were fixed in paraformaldehyde.

At the end of the treatment, the body weight was recorded. And all the organs were removed from the attached adipose and weighted with an electronic balance (Sartorius, Germany). The organ index (mg/g) was calculated as the following formula: Organ   coefficients = O r g a n   w e i g h t ( m g ) R a t   o r   m o u s e   t o t a l   w e i g h t ( g ) .

Liver TC and TG were extracted by using the organic solvent extraction method. 100 mg of liver were grinded with 1 ml of physiological saline. The homogenate was divided into two parts, one was used to detect BCA, and the other was added to 500 μl of organic solvent (methanol: chloroform = 2:3), 12,000 g, 10 min, to remove the clear. Volatilize the organic solvent overnight. TC and TG can both be detected by using commercial assay kits (Nanjing Jiancheng Bioengineering Research Institute, China). Serum TC, HDL-C, and LDL-C levels were measured by commercial assay kits (Nanjing Jiancheng Bioengineering Research Institute, China), ox-LDL levels were measured by ELISA assay according to the manufacturer’s protocol (Tianjin Anoric Bio-technology Co., Ltd., China).

Livers were fixed in 4% paraformaldehyde (Servicebio, China) and made into slices (Thermo) for HE staining and ORO staining. For HE, the paraffin sections were dewaxed to water, stained with hematoxylin (Servicebio, China) and eosin (Servicebio, China), then dehydrated and sealed. For ORO, reheat and dry the frozen slices, then fix them in the fixative solution for 15 min, wash with tap water, and dry. Stain sections with Oil Red solution for 8–10 min in the dark, and cover them with a lid during dyeing (Servicebio, China). Differentiation of background was followed by immersion in hematoxylin. Seal the slices with glycerin gelatin. HE and ORO staining were observed under the microscope.

Total RNA isolation and mRNA levels for specific genes in macrophages, livers and intestine were performed using a real-time quantitative polymerase chain reaction (RT-qPCR) assay. Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, United States). cDNA was synthesized with 0.25 μg total RNA by using the Evo M-MLV RT Premix for qPCR (Accurate Biology, China). DNA amplification was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, China) with 0.5 μl of ROX reference dye (Accurate Biology, China) and performed in a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, United States) using SYBR Green detection chemistry with the resulting cDNAs. The primers sequences used were shown in Table 1.

For the WB experiment, cells were lysed in RIPA Lysis buffer containing PMSF (Beibokit, China). SDS-polyacrylamide gel electrophoresis was used to separate equal amounts of proteins from whole-cell lysates and then transferred onto PVDF membranes (Bio-Rad, United States). The blots were probed with various primary antibodies: LXRα (Santa Cruz, United States), ABCA1 (Abcam, United Kingdom), ABCG1 (Abcam, United Kingdom), Cyclooxygenase 2 (COX2) (Abcam, United Kingdom), Interleukin 1 beta (IL-1β), SR-BI, inducible nitric oxide synthase (iNOS) (Abcam, United Kingdom), fatty acid translocase (also known as cluster of differentiation 36, CD36) (Affinity, Australia), and low density lipoprotein receptor (LDLR) (Affinity, Australia), after being blocked with 8% (w/v) skimmed milk and then the appropriate secondary antibodies (1:10,000). After washing the membrane, it is developed by chemiluminescence in the ECL reagents (Thermo Fisher Scientific, United States), and the gel imaging analysis system (Tanon 5220 multi, China) was used for camera analysis. The target band of the obtained protein image and the gray value of the corresponding internal reference band were measured by Image J software.

All experiments were performed independently at least three times. The results are shown as the mean ± SEM. A one-way ANOVA test was used to compare differences between groups in the various experiments. Differences with a p-value < 0.05 were considered statistically significant.

Foam cells are characterized by cholesterol overload. Excessive cholesterol in macrophages needs to be excreted from cells by the RCT process to avoid foam cells forming. In this study, NBD-cholesterol was applied to prepare cholesterol-overloaded macrophages (Sengupta et al., 2013). To figure out the potential contribution of ICAC (Figure 1A) to cholesterol efflux, we initially evaluated the cytotoxicity of ICAC on BMMs. The results suggested that ICAC had no significant cytotoxicity at a concentration lower than 50 μM (Figure 1B). Cholesterol uptake results show that macrophages uptake NBD-cholesterol. The NBD-cholesterol load-macrophages are presented by green fluorescence. (Figure 1C). In order to detect the cholesterol efflux ability of ICAC, NBD-cholesterol load-macrophages were treated with different concentrations of ICAC. ICAC significantly promoted cholesterol efflux in a dose-dependent manner. As shown in Figures 1C,D, NBD-cholesterol fluorescence in macrophages was significantly reduced when ICAC≧ 3.13 μM. In addition, the results also show that LXR agonist T0901317 also increased cholesterol efflux, while HMG-COA inhibitor AT had no significant effect (Figures 1C,D).

Due to the major regulatory functions of LXRs and their downstream genes on lipid homeostasis, especially cholesterol metabolism and efflux, we aimed to evaluate whether ICAC affected the LXR-mediated RCT signaling pathway. According to the results of RT-qPCR and Western blot assays, ICAC increased the expression of genes and proteins of the RCT signaling pathway, including LXRα, ABCA1 and ABCG1 in macrophages (Figures 1E–K). ABCA1 and ABCG1 are upregulated by the activation of LXR, which promotes the efflux of free cholesterol from foam cells (Guo et al., 2018). Hence, a reduction of intracellular NBD-cholesterol fluorescence was observed after ICAC treatment (Figures 1C,D). Meanwhile, ICAC suppressed the cholesterol uptake association genes CD36 and LDLR and increased SR-BI (Figures 1L–R). These data indicate that ICAC inhibits cholesterol absorption and enhances the RCT pathway.

Ox-LDL is characterized as a harmful type of cholesterol which leads to the formation of foam cells. Enhancing the cholesterol efflux capacity in macrophages is considered to play an important role in inhibiting the formation of foam cells as well as reducing the inflammatory response (Mitchell and Carmody, 2018). Hence, we first investigated the competence of macrophages’ foaming inhibition of ICAC at 25 μM, 12.5 μM, 6.25 μM, 3.13 μM , and 1.56 μM in ox-LDL-stimulated RAW264.7. ORO staining results showed that ICAC and T0901317 significantly prevented cell foaming and intracellular lipid accumulation (Figures 2A,B).

NF-κB is a crucial transcription factor in directing the initiation and progression of inflammation. It controls expression of genes including cytokines IL-1β, iNOS, COX2 (Mitchell and Carmody, 2018). BMMs obtained from C57BL/6 mice were used to induce inflammation in macrophages. Next, inflammatory pathway related genes and proteins were evaluated. After treating with ICAC in ox-LDL-induced inflammatory macrophages, the expression mRNA level of NF-κB (Figure 2C). Meanwhile, the mRNA and protein levels of NF-κB downstream inflammatory factors iNOS, COX2 and IL-1β were decreased significantly (Figures 2D–J). Further studies have shown that ICAC inhibits the phosphorylation of P65 (Figures 2K–M) and IKB (Figures 2N–P), which indicates that the suppressing of the NF-κB signaling pathway. These results suggest that ICAC plays a role in inhibiting ox-LDL-induced foam cell formation and NF-κB pathway related inflammation responses.

Next, the in vivo lipid-lowering effect of ICAC was evaluated by using HFD-induced hyperlipidemia in C57BL/6 mouse. Two positive drugs were carried out in this study. One is the cholesterol synthesis inhibitor atorvastatin (AT), and the other is the LXR agonist T090. Compared with the rodent chow diet group (control group), the body weight of mice in the HFD group was significantly increased. HFD mice lost their body weights significantly with the treatment of ICAC, T090, AT, and T090 + ICAC (Figures 3A,B). Besides, the brown adipose tissue (BAT) coefficients were increased (Figure 3C), which may explain the HFD mice weight reduction after treatment with ICAC, T090, AT, and T090 + ICAC. Additionally, ICAC reduced serum TC (Figure 3D) and LDL-C (Figure 3E) in hyperlipidemia mice, as well as improved serum HDL-C level as two positive drugs did (Figure 3F). The data also indicated that ICAC can reduce the content of ox-LDL (Figure 3G), a high-risk factor for atherosclerosis. Our research also found that a half dose of ICAC combined with T090 own equal therapeutic effective compared to a single high dose of themselves respectively (Figures 3A–G). Furthermore, groups of ICAC, T090 + ICAC, and T090 but not AT showed significantly ox-LDL lowering effects (Figure 3G). Meanwhile, kidney coefficient and heart coefficient had no significant change, that mean ICAC had no toxicity to these organs at 20 mg/kg dose (Figures 3H,I). In serum pharmacodynamics parameters, ICAC has no significant difference in therapeutic effect from AT and T090 (Figure 3). These results suggest that ICAC is a potential drug for RCT promoting that can be used to treat hyperlipemia.

To further investigate the therapeutic effect of ICAC on HFD-induced hyperlipidemia in mice, liver morphological observations and lipid and inflammatory factor detection were performed. In the HFD group, the livers were light brown and the edges were passive and greasy (Figure 4A). HE staining showed that liver cells in the HFD group were swollen, cytoplasm was loose, lipid droplets of different sizes were vacuolated, and cell boundaries were unclear (Figure 4A). The ORO staining assay further showed increased lipid staining area of liver cells in the HFD group (Figures 4A,B). Compared with the HFD group, the liver color of treatment groups (ICAC, AT, and ICAC + T090) was redder, with sharp edges and smooth surfaces. In addition, the vacuoles and ordered cells were reduced after drug treatment in HE staining (Figure 4A). Furthermore, the number of cells stained by ORO were also significantly reduced after treating with ICAC, AT, and T090 + ICAC (Figures 4A,B). Additionally, TC contents of the liver were all inhibited in all treatment groups (Figure 4C). But unlike AT and ICAC, T090 significantly increased liver vacuolar lesions, ORO-stained lipid area, and TG content (Figures 4A,B,D), which indicated T090 had induced liver steatosis. Interestingly, ICAC enabled to eliminate the undesired liver steatosis of T090 when T090 combination with ICAC was used in treating hyperlipemia mice (Figures 4A,B,D). With the decrease of lipid in the liver, we also found that groups of ICAC, T090, AT, and T090 + ICAC displayed inhibitory effects on the gene expression of iNOS and COX2 in the liver (Figures 4E,F). In this study, the concent of TG in serum in all groups did not Significant change (Figure3J).

Next, we investigated the effects of ICAC in regulating RCT and adipogenesis pathways in vivo. ICAC but not AT up-regulated the expression of RCT key genes, including ABCA1, ABCG1, and CYP7A1 in the liver (Figures 5A–C), as well as increased LXRα, ABCG5, and ABCG8 in the intestine (Figures 5G–I). In addition, another key gene in the RCT pathway, LDLR, was also observed to have a trend of up-regulation after treatment (Figures 5D–F). These results indicate that ICAC promotes RCT.

A previous study explained that T090 activates LXRα and follows by amplifying expression of its downstream target gene SREBP-1c, leading to hepatic and plasma triglyceride levels increasing (Kim et al., 2021). In our study, T090 significantly increased LXRα mRNA level (Figure 5J), and caused increasing of lipogenic genes SREBP-1c and ACC in the liver (Figures 5K,L). However, ICAC and AT did not increase the liver LXRα mRNA level (Figure 5J). Moreover, both ICAC and AT inhibited the expression of SREBP-1c (Figure 5K), as well as its downstream genes like FAS, ACC, and SCD-1(Figures 5L–N). In addition, the combination of T090 with ICAC also significantly reduced the expression of adipogenic genes, FAS, ACC, and SCD-1 (Figures 5L–N). In conclusion, ICAC performed multiple functions, such as RCT promoting, lipogenesis suppressing, as well as inflammatory inhibiting. Compared to AT, ICAC had the advantage of facilitating RCT. Compared to T090, ICAC did not cause undesirable liver adipogenesis.