Mulberry extract upregulates cholesterol efflux and inhibits p38 MAPK‐NLRP3‐mediated inflammation in foam cells

Abstract The accumulation of foam cells in arterial intima and the accompanied chronic inflammation are considered major causes of neoatherosclerosis and restenosis. However, both the underlying mechanism and effective treatment for the disease are yet to be uncovered. In this study, we combined transcriptome profiling of restenosis artery tissue and bioinformatic analysis to reveal that NLRP3 inflammasome is markedly upregulated in restenosis and that several restenosis‐related DEGs are also targets of mulberry extract, a natural dietary supplement used in traditional Chinese medicine. We demonstrated that mulberry extract suppresses the formation of ox‐LDL‐induced foam cells, possibly by upregulating the cholesterol efflux genes ABCA1 and ABCG1 to inhibit intracellular lipid accumulation. In addition, mulberry extract dampens NLRP3 inflammasome activation by stressing the MAPK signaling pathway. These findings unveil the therapeutic value of mulberry extract in neoatherosclerosis and restenosis treatment by regulating lipid metabolism and inflammatory response of foam cells.


| INTRODUC TI ON
Endovascular therapy has broad applications in clinical diagnosis and treatment of cardiovascular diseases (Sun, 2014). However, restenosis remains a major cause of target lesion failure after interventional therapy because 15.3% of patients with femoral popliteal artery disease develop in-stent restenosis after stent implantation (Qato et al., 2018) despite the rapid development of drug treatment and surgical technology.
Histology, angiography, and intravascular imaging data all suggest that neoatherosclerosis is an important contributing factor for restenosis after vascular stent treatment (Park et al., 2012).
Neoatherosclerosis is triggered by the activation of endothelial cells, followed by the recruitment of circulating monocytes which subsequently differentiate to monocyte-derived macrophages and then macrophages foam cells. Foam cells are characterized by high intracellular lipid content and usually reside in the intima of artery hyperplasia (Funk et al., 1993). The late stage of neoatherosclerosis is accompanied by thin-walled fibrous atherosclerosis and lipid-rich neointima (Jinnouchi et al., 2017). Activated neointima cells secrete enzymes and chemicals that modify LDL, which in turn activate neointima cells and trigger various inflammatory signals (Pentikäinen et al., 2000). Thus, neoatherosclerosis plaques are featured by inflammation and lipid metabolic disorder.
NLRP3 is a key component of this multi-protein complex because it interacts closely with apoptosis-related dot-like protein (ASC) and recruits the precursor of cysteinyl aspartate-specific proteinase (caspase-1; Zhen & Zhang, 2019). Previous studies revealed elevated expression of the NLRP3 inflammasome in human atherosclerotic arteries (Shi et al., 2021). In addition, a study from the Canakinumab Anti-Inflammatory Thrombosis and Outcomes Study (CANTOS) showed that inhibition of IL-1β induced by NLRP3 inflammation reduces the incidence of atherothrombotic events and inflammation in patients after myocardial infarction (Ridker et al., 2017), suggesting that NLRP3 inflammasome may be a promising therapeutic target for cardiovascular diseases including atherosclerosis and restenosis. However, the underlying mechanism remains unclear.
Foam cells play key roles in the development of neoatherosclerosis. They are macrophages with excessive influx of modified lowdensity lipoprotein (LDL) and high accumulation of cholesteryl ester (Javadifar et al., 2021). This biological process can be reversed by cholesterol efflux which utilizes ATP binding cassette transporter A1 (ABCA1), ATP binding cassette transporter G1 (ABCG1), and scavenger receptor B1 (SR-B1) to transport excess intracellular lipids to liver for degradation (Wang & Westerterp, 2020)
Mulberry extract, a nutrition-rich natural dietary supplement, has long been used in Chinese medicine (Jan et al., 2021). It has been shown to reduce lipid oxidative stress, inflammation, and lipids accumulation in liver and help ameliorate lipid metabolism disorders in a nonalcoholic fatty liver rat model (Memete et al., 2022). In addition, the ethanol extract of black mulberry reduces foam cell formation and inhibits the development of atherosclerotic plaque through the ox-LDL-PPARγ-CD36 feed-forward cycle (Liu et al., 2020). The total phenols and flavonoids in the ethanol extract of mulberry and its derivative components are sufficient to elicit protective effects on oxidative stress and inflammation of macrophages stimulated by lipopolysaccharide . Mulberry extract also contains a high concentration of biologically active compounds such as anthocyanins and flavonols, which inhibit the expression of inflammatory mediators induced by lipopolysaccharide in RAW264.7 cells and reduce the secretion of pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)α (Jung et al., 2019).
In this study, we revealed that mulberry extract not only inhibits the formation of foam cells by upregulating the cholesterol efflux to inhibit intracellular lipid accumulation but also dampens NLRP3 inflammasome activation by stressing the MAPK signaling pathway.
Collectively, our results underscore the therapeutic value of mulberry extract in restenosis treatment.

| Mulberry extract
Mulberry fruit, Morus nigra, was harvested at ripe stage from the Plantation of National Mulberry Orchard (Zhenjiang, Jiangsu province, China). Fruits were dried by freeze dryer (EYELA FDU-2100) and milled into powder for further use. Two grams of mulberry fruit powder were mixed with hexane (1:10 w/v) and shaken continuously in an ultrasonic bath at 55°C for 1 h to remove lipids and fatty acids. Under the same conditions, the pellets were sonicated with 80% ethanol (1:3 w/v) to prepare a polyphenol-rich extract. The extract was then incubated overnight at 4°C and sonicated again for 25 min. Next, the extraction was filtered through Whatman No. 1 filter paper. The residue was re-extracted in the same way, combining three filtrates (5 mL each). The ethanol from the three filtrates was combined and evaporated under vacuum at 40°C to obtain the dry extract. The extracts were placed in plastic bottles and then stored at −20°C until use. For quality control, we provided the table of the substances identified in the Morus nigra extract by liquid chromatography-mass spectrometry (LC-MS).

| Tissue collection
Arterial samples were taken from patients with amputation due to atherosclerotic disease, while control arteries were taken from healthy individuals after accidental amputation. All samples are from Shanghai Renji hospital affiliated to Shanghai Jiao Tong University School of Medicine and processed in the Biobank of Renji Hospital. All procedures were approved by the Research Ethics Committee of Renji Hospital.

| Cell cultures
RAW 264.7 cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) plus 1% streptomycin in a humidified incubator at 5% CO 2 air and 37°C. When the cells grow to 80%-90% of the area, subculture, and reserve.

| Quantitative reverse transcription PCR (RT-qPCR)
The total RNA of cells was extracted according to the instructions of Trizol kit, and the RNA concentration and A260/A280 ratio were measured by nano drop2000. cDNA was obtained by reverse transcription kit; the amount of mRNA was measured by RT-qPCR kit, and three replicates were set in each group. Reaction conditions: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing/extension at 60 for 30 s, a total of 40 cycles. use ΔΔC T to calculate the gene expression level. The specific formula is as follows: ΔC t = C t value of target gene -C t value of GAPDH Gene; ΔΔC t = experimental group ΔC t control group ΔC t ; relative gene expression in the experimental group = 2 − C t .

| Western blotting
The human artery or cultured cells were lysed in a lysis buffer containing RIPA and 1 mM PMSF for 30 min on ice. After centrifugation at 12,000 ×g for 15 min at 4°C, the supernatants were collected as total proteins in artery or cells. The protein concentrations were also determined with a BCA assay kit. Aliquots (50 μg) of protein samples were separated on 10% SDS-PAGE and electro-transferred to polyvinylidene fluoride membranes. The PVDF membranes were incubated with primary antibodies overnight at 4°C after being blocked with 5% nonfat milk for 1 h. The membranes were then washed with TBST (five times, 3 min each) and incubated with secondary antibodies for 1-2 h at 37°C. The protein bands were detected with an enhanced chemiluminescence system on Tanon 5200s. Densitometric analysis was conducted by ImageJ. Actin proteins were detected as a control.

| Immunofluorescence
Tissue sections embedded in paraffin were cut into 4 μm sections, deparaffinized in xylene, and rehydrated in graded alcohol solution.
Slides were blocked with 5% (v/v) donkey serum for 30 min. After washing with 1× PBS, cells were stained with rabbit anti-NLRP3 and rabbit anti-CD68 overnight at 4°C. After three washes in PBS, the immunoreactive products were visualized by incubation with an appropriate secondary antibody (1:400) and DAPI (1:500, 216276; Roche) to visualize cell nuclei. Confocal microscopy was conducted using an Olympus CX31 confocal microscope.

| Oil Red O (ORO) staining
Lipid content was histologically assessed using ORO staining.
RAW264.7 cells were plated in 24-well plates and incubated with 50 μg/mL ox-LDL with or without Mulberry extract for 24 h. The cells were washed two times with PBS slightly, fixed with 4% paraformaldehyde for 30 min and then stained with filtered ORO solution (in a 6:4 ratio of 0.5% ORO in ddH 2 O) for 60 min. Wash the cells with distilled water for 1-2 times, 1-2 min each time and rinse with 60% isopropyl alcohol. The lipid droplets in the macrophages were observed under the microscope and collected images.

| Cell counting kit-8 analysis
The cell counting kit-8 (CCK-8) assay was applied to measure cell viability. RAW264.7 cells were cultured in a 96-well plate at a cell density of 5000 cells/well for 24 h. They were added with different concentrations of ox-LDL or different concentrations of mulberry extract. After incubating for 24 h, 10 μL/well CCK-8 is dissolved in 90 μL/well culture medium, 100 μL/well CCK-8 working solution was added. The OD value at the wavelength of 450 nm was measured using the microplate reader 1 h later.

| Cellular cholesterol assay
Cells were grown in 6-well plate to approximately 2 × 10 6 cells. The cells were washed twice with PBS to remove medium serum and resuspended in 0.1 mL of lysate per 1 × 10 6 cells and incubated in room temperature for 10 min. Lysates were then heated at 70°C for 10 min followed by centrifugation at 2000 g for 5 min at room temperature.

| Cell apoptosis analysis
Cell apoptosis was measured by using Annexin V-FITC/PI apoptosis detection kit (BD556547). After treatment for 24 h, cells were collected, washed with PBS, and resuspended in binding buffer.
Then, cells were incubated with 5 μL Annexin V-FITC and 5 μL PI for 15 min at RT in the dark. A flow cytometer was used to analyze cell apoptosis. The rate of apoptosis is expressed as the percentage of annexin V cells (Annexin V + PI − ) that are individually stained.

| Metabolomics analysis
Accurately weigh 25 mg of sample and add 0.6 mL 2-chlorophenylalanine
Experimental data were presented as means ± SEM. n expresses the number of independent experiments or samples. Statistical analysis was performed by the one-way analysis of variance (ANOVA) with the significance level set at p < .05 (GraphPad Prism 8).

| Increased expression of NLRP3 in vascular tissues in restenosis
To understand the unique transcriptional network in restenosis, Heat map shows genes in cytokine-cytokine receptor interaction, such as interleukin-1 (IL-1), are upregulated ( Figure 1d). IL-1 is a common pro-inflammatory cytokine that plays a key role in the innate immune response (Dinarello, 2011).
Since the NLRP3 inflammasome is an innate immune signaling complex and a key mediator of IL-1 family cytokine production (Grebe et al., 2018), we tested its mRNA level in restenosis (n = 4) and normal artery (n = 3) tissues by RT-qPCR. The results showed that NLRP3, caspase-1, ASC, and IL-1β mRNA were all significantly increased ( Figure 1e). Moreover, the NLRP3 protein level also markedly increased upon restenosis treatment compared with normal control group (Figure 1f). Fluorescence in situ hybridization confirmed the elevated signal of NLRP3 in restenosis artery tissue accompanied by increase filtration of CD68 + macrophage. Importantly, the co-localization of NLRP3 and CD68 suggests that NLRP3 mainly expresses in neointima macrophages (Figure 1g). These results indicate a correlation between NLRP3 expression, especially in macrophages, and restenosis.

| Active compounds in mulberry extract target genes involving in lipid metabolism and foam cell formation
Mulberry leaf and fruit extracts have long been used as traditional Chinese medicines to improve liver health by regulating glucose and lipid metabolisms (Lim et al., 2013). To evaluate the benefit of mulberry fruit Morus nigra extract in restenosis, we first tested its cyto-  (Table 1). In PubChem database, which provides information on the activities of chemical molecules in biological assays, we found 196 potential targets of these compounds. Next, we established a compound-target network using Cytoscape software to illustrate the interaction between these active compounds and their targets. Our network consists of 203 nodes which represent active compounds or their potential targets and 231 edges which indicate the interaction between the nodes (Figure 2b). These targets were compared with the 3429 DEGs, and 50 overlapping genes ( Table 2) were chosen for further study to examine the mechanism and antirestenosis efficacy of mulberry extract (Figure 2c). We also applied KEGG pathway enrichment ( Figure 2d) and GO analysis (Figure 2e) on these anti-restenosis targets and found that they are mainly in- genes provided by the STRING database (https://strin g-db.org/). PPI is defined by interaction scores (medium: >0.4) ( Figure 2f).
Intriguingly, IL-1β is one of the key hubs in the cluster ( Table 3). The findings show that mulberry extract is non-toxic and could regulate inflammatory response and lipid metabolism in foam cells.

| Mulberry extract inhibits the expression of NLRP3 via the MAPK signaling pathway in foam cells
The accumulation of lipids in macrophages and foam cell formation are closely associated with the development of atherosclerosis . In order to study the effect of mulberry  (Figure 3b). We also observed that this inhibition is dose-dependent (Figure 3b, compare lanes 3 and 4).
These results indicate that mulberry extract has the ability to inhibit ox-LDL-induced RAW264.7 macrophage foam cell production by suppressing intracellular lipid accumulation.
Since the lipid content of foam cells is associated with inflammation, we aimed to explore whether mulberry extract inhibits the mulberry extract on this pathway. GO analysis revealed that biological processes enriched in anti-restenosis targets are also involved in positive regulation of MAPK cascade and KEGG pathway enrichment analysis confirmed that MAPK signaling pathway is the principal pathway on these targets (Figure 2d,e). To test the efficacy of mulberry extract on MAPK pathway, macrophages were pretreated with mulberry extract for 1 h, and after ox-LDL induction, protein level of p38-MAPK and MAPK were determined by Western blotting. The results demonstrate that cells treated with mulberry extract has reduced expression of p38-MAPK (Figure 3d). Therefore, mulberry extract may regulate NLRP3 inflammasome through p38 MAPK signaling pathway.

F I G U R E 3
Mulberry extract inhibits the expression of NLRP3 in foam cells via MAPK signaling pathway. Foam cells were induced by treating RAW264.7 macrophages with 50 μg/mL ox-LDL for 24 hours. The cells were then given mulberry extract at low (5 mg/mL) or high concentration (10 mg/mL) as indicated. Following foam cell features were monitored. (a). Intracellular lipid level measured by Oil Red O staining. (b). Cholesterol ester content. (TC, total cholesterol; FC, free cholesterol; CE, cholesterol ester) qualified by cellular cholesterol assay of foam cells with or without treatment of mulberry extract as indicated. (c). Induced foam cells were treated with different concentrations of mulberry extract (0, 1, 5, 10 mg/mL) for 24 h and protein levels of NLRP3, caspase-1, ASC, and IL-1 β were determined by Western blotting. (d) The protein levels of NLRP3, ASC, p-p38, p38 MAPK, and IL-1β protein levels of cells undergoing indicated treatments were determined by Western blotting. Graph bars represent qualification of flow cytometry results (Data shown are mean ± SEM. Statistical significance was determined by two-way ANOVA with correction for multiple comparisons. *p < .01, **p < .001, ***p < .0001, ##p < .01 and ###p < .001.

| Mulberry extract stimulates cholesterol efflux and inhibits apoptosis of foam cells
To further understand the mechanism of mulberry extract, we ap- The main mechanical feature of restenosis after endovascular treatment is insufficient stent expansion or fracture. Meanwhile, local inflammation leads to invasive neointimal hyperplasia and neoatherosclerosis (Shlofmitz et al., 2019). During the development of restenosis, macrophages infiltrate in neointima and eventually aggregate near the surface of the stent and cause neoatherosclerosis that is not related to the original atherosclerotic tissue (Otsuka et al., 2015). Subsequently, these fat-laden macrophages, or foam cells, form atherosclerotic plaques, which can evolve into thin-walled atherosclerotic plaques with the risk of plaque rupture and occasional calcification (Otsuka et al., 2015). The transformation of macrophages into foam cells is characterized by lipid droplet formation and massive lipid accumulation (Chistiakov et al., 2016). To reverse this biological process, cholesterol transporters such as ABCA1-and ABCG1-mediated cholesterol efflux from macrophages to extracellular lipid acceptors. ABCA1 and ABCG1 deficiency increases foam cell formation and accelerates the development of atherosclerosis in mice. Several Chinese medicines are shown to promote cholesterol efflux by regulating the PPARγ-LXRα-ABCA1/ABCG1 pathway including Chrysin (Wang et al., 2015), Yin-xing-tong-mai decoction (Zheng et al., 2021) and Qing-Xue-Xiao-Zhi formula .
However, it is difficult to identify the active ingredient in these multi-ingredient medicines and their anti-inflammatory mechanism remains to be elucidated. Statins, as cholesterol-lowering drugs, are effective in reducing cardiovascular disease and mortality in patients at high risk of cardiovascular disease. (Robson, 2008) Nevertheless, it may cause side effects including rhabdomyolysis and liver dysfunction (Ruscica et al., 2023). In contrast, mulberry, Morus nigra, is a nutrition-rich dietary source and mulberry extract has no adverse health effect on human. Mulberry has been used in traditional Chinese medicine for centuries and increasing number of recent studies also show that mulberry fruit or leaf extracts are effective in boosting immunity, lowering blood glucose, and promoting metabolism (Zhou et al., 2017). Our results, for the first time, show that mulberry extract treatment increases the expression of cholesterol efflux genes ABCA1/ ABCG1 and inhibits lipid droplet formation and lipid deposition in foam cells. Interestingly, our metabolic pathway analysis of mulberry targets in restenosis-related genes reveals a significant enrichment of arginine biosynthesis. L-arginine, as a substrate for intracellular NO synthesis, has a variety of biological functions such as improving endothelial dysfunction and treating atherosclerosis (Li et al., 2019). A study in diabetes shows that oral L-arginine promotes NO synthesis and enhances vascular function (Kohli et al., 2004). In the future, we will further explore the role of mulberry extract in improving the function of endothelial cells as a new avenue to treat restenosis.
There are studies indicate a link between lipid and inflammation (Li et al., 2005). The two factors may establish a positive feedback mechanism and synergistically contribute to disease progression.
For example, lipid accumulation in Abca1/g1-deficient myeloid cells promotes the activation of NLRP3 inflammasome (Westerterp et al., 2018). Abnormal lipid metabolism also causes increased expression of pro-inflammatory cytokines and activating inflammation in atherosclerotic plaques (Westerterp et al., 2013). Our study used bioinformatic analyses of restenosis-related genes and predicted mulberry extract targets to identify potential anti-restenosis genes regulated by mulberry extract. The most noteworthy of these targets include genes associated with inflammation such as IL-1β, IL-6, CD36, HIF-1α, MMP2, ICAM-1, VCAM-1, and VEGF. CD36 participates in phagocytosis of apoptotic cells, pathogen recognition, and regulation of low-density lipoproteins. It also contributes to inflammatory responses and thrombotic diseases (Silverstein & Febbraio, 2009). MMP-2 is a multifunctional protein which expression is elevated in many cardiovascular pathologies (e.g., myocardial infarction, hypertensive heart disease) where tissue remodeling and inflammatory responses are perturbed (Hardy et al., 2018). Among the targets are also IL-1β which plays important roles in angiogenesis by synergistically inducing the production of VEGF with TNF and IL6. The expression of IL-1β is dependent on the activation of NLRP3 because NLRP3 inflammasome cleaves pro-caspase-1 into the active form caspase-1, thereby promoting IL-1β maturation and secretion (Agostini et al., 2004). IL-1β in turn regulates the initiation and amplification of inflammatory process. Intriguingly, our RNA-seq and florescent imaging data reveal not only the increased levels of IL-1β and NLRP3 in restenosis artery tissue but also a co-localization of NLRP3 with CD68 + macrophages, suggesting the regulatory roles inflammasomes in foam cells play in the development of restenosis. We also found that mulberry extract is effective in suppressing the activation of NLRP3 inflammasome. The mRNA and protein expression of NLRP3 in mulberry extract-treated foam cells are significantly decreased in a dosage-dependent manner, making mulberry extract a promising therapeutic treatment for restenosis patients.
Protein kinases, including JNK, p38 MAPK, and ERK, are activated in response to various stresses (Kim & Choi, 2015). P38 MAPK pathway is usually involved in stress and inflammatory response (Coulthard et al., 2009) and has been shown to regulate NLRP3 inflammasome activation (Rajamäki et al., 2016). From the database of traditional Chinese medicine, we found that multiple target genes of mulberry active compounds are involved in the MAPK signal pathway. We speculated that mulberry extract may regulate the activation of NLRP3 inflammatory bodies of macrophages via p38 MAPK pathway. Indeed, our data supports this hypothesis by showing that the protein levels of NLRP3, phosphorylated p38 MAPK, and IL-1β all decrease upon treatment of mulberry extract, making mulberry extract a potential treatment for not only restenosis but also other inflammation-related diseases.
In summary, our study uncovers the elevated expression of NLRP3 in restenosis artery wall, making it a novel therapeutic target for the disease. More importantly, we found that mulberry extract plays multiple regulatory roles in the formation of restenosis. It not only inhibits the inflammation in foam cells by suppressing p38 MAPK signaling pathway mediated inflammasome activation but also stimulates the ABCA1/ABCG1 mediated cholesterol efflux of foam cells to decrease their formation. Therefore, mulberry extract is a promising all natural and highly effective treatment for neoatherosclerosis and restenosis.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data and materials could be acquired from the corresponding authors by reasonable request.