Huxin Formula Inhibits Oxidized low-Density Lipoprotein-Induced Foam Cell Formation in THP-1 Macrophages via the LOX-1/NF-κB Pathway

Abstract

Background

Macrophage-derived foam cells are essential in the progression of atherosclerosis (AS). Based on our previous study, the Huxin Formula (HXF), a traditional Chinese medicine formula, demonstrates potential in anti-atherosclerosis. Nevertheless, it is still unknown how HXF affects the formation of foam cells derived from THP-1 macrophages.

Purpose

This research aims to examine the preventive role of HXF in the development of foam cells and its underlying molecular mechanism.

Methods

THP-1 derived macrophages and THP-1 cells overexpressing LOX-1 (LV-OLR1) were exposed to ox-LDL to establish foam cell models, and then treated with HXF. Meantime, Oil red O staining was used to detect lipid droplet production. ELISA kit was performed to measure intracellular levels of IL-6 and TGF-β. RT-qPCR and Western Blot were then utilized to determine the LOX-1 and NF-κB mRNA/protein levels.

Results

The findings indicated that HXF treatment potently reduced the lipid accumulation, downregulated IL-6 levels and upregulated TGF-β levels. However, this impact was almost reversed when LOX-1 was overexpressed in THP-1 cells stimulated with ox-LDL. Moreover, THP-1 were treated with HXF markedly reduced the levels of LOX-1 and NF-κB mRNA/protein, whereas overexpressing LOX-1 significantly reversed this effect.

Conclusion

HXF reduced the formation of foam cell in ox-LDL-stimulated THP-1 macrophages via inhibiting lipid accumulation and inflammation through regulating the LOX-1/ NF-κB pathway. These present findings further indicate a potential beneficial role of HXF in ameliorating atherosclerosis and foam cell formation, while provide a novel potential therapeutic strategy for preventing atherosclerosis.

Introduction

Atherosclerosis is a chronic inflammation of the vascular walls resulting from macrophage foam cells and endothelial injury, leading to myocardial infarction, stroke, heart failure, and many other life-threatening clinical manifestations.^1,2 The development and buildup of foam cells in the lipid-filled subendothelial area are crucial in the pathogenesis of AS. A high level of cholesterol esters and modified low-density lipoproteins (LDLs) within the intima are induced foam cell formation.^3,4 Therefore, preventing or reversing foam cell formation is highly important for the prevention of atherosclerosis.

The main receptors responsible for capturing oxidized low-density lipoprotein (ox-LDL) in macrophages are scavenger receptors (SRs) including scavenger cluster of differentiation 36 (CD36), scavenger receptor class A1 (SR-A1), and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1 or OLR1), as discovered by researchers. ⁵ LOX-1, a 50 kDa transmembrane glycoprotein that has the ability to bind to and internalize ox-LDL, ⁶ and it is essential to atherosclerosis pathogenesis. The use of targeted-LOX-1 treatment has been suggested as a possible approach for the discovery of drugs that can prevent atherosclerosis. nuclear factor-κB (NF-κB), a downstream molecule of LOX-1, is well-established to be activated throughout different stages of AS. ⁷ Many inflammatory cytokines are regulated by the NF-κB signaling pathway, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β. ⁸ Consequently, vascular inflammation caused by NF-κB impacts the onset and advancement of atherosclerosis.

Multi-component synergies are hallmarks of traditional Chinese medicine (TCM), and TCM have been widely used as an important supplement and replacement therapies of AS.^9-11 Huxin Formula (HXF) is derived from an empirical prescription named “Deng Lao Guanxin Formula”, which was created by Deng Tie-tao, a famous Chinese doctor. It consists of five traditional Chinese herbs: Ginseng Radix et Rhizoma, Citri Exocarpium Rubrum, Notoginseng Radix et Rhizoma, Aurantii Fructus and Carthami Flo. According to our previous clinical investigation, it was observed that the modified Huxin Formula enhanced the recuperation of patients following CABG surgery and alleviated their clinical symptoms. ¹² Subsequently, using a multi-center randomized controlled trial with a large sample size, we evaluated Huxin Formula's effects and safety on patients undergoing PCI (percutaneous coronary intervention), our research has shown that Huxin Formula improves patients’ quality of life and relieves symptoms associated with cardiac function class II.^13,14 Besides this, Huxin Formula could significantly reduce the expression of reverse cholesterol transport (RCT) associated genes, inhibit atherosclerotic plaque formation in ApoE^−/− mice, and prevent the development of atherosclerosis. ¹⁵ Nevertheless, it is still unclear how HXF affects the formation of foam cells derived from THP-1 macrophages. This research utilized a widely accepted foam cell model induced by ox-LDL to examine HXF's protective role against foam cell formation and the underlying molecular mechanism.

Results

Identification of major Components of HXF-Containing serum

Both negative and positive ion modes of ESI were utilized for the analysis and identification of the components in the serum containing HXF. Figure 1 displays the chromatograms of the overall ion current at both ESI modes. A total of 48 compounds were identified in the HXF serum, including 17 constituents from Ginseng Radix et Rhizoma, 19 constituents from Aurantii Fructus, 8 constituents from Citri Exocarpium Rubrum, 4 constituents from Carthami Flos, and 7 unknown constituents. The retention times, MS data of the characterize specific components and secondary fragment are summarized in Supplementary Table 1.

Figure 1.

Total ion chromatograms of HXF-containing serum analyzed by UPLC-Q-Exactive Orbitrap MS. (A) Positive ion mode. (B) Negative ion mode.

HXF Attenuated Lipid Accumulation in ox-LDL-Induced THP-1 Macrophages

In order to investigate the accumulation of lipids during various phases of foam cell formation, a model of foam cells derived from macrophages induced by ox-LDL was created. Figure 2A and 2C demonstrate that THP-1 macrophages accumulate lipid droplets in a time-dependent manner when exposed to Ox-LDL. The accumulation of lipid droplets in macrophages is significantly increased by ox-LDL for 24, 36, and 48 h, resulting in red staining of the lipid droplets compared to untreated controls (P < 0.05 or P < 0.01). To evaluate the protective effects of HXF at different concentrations (5% and 10% HXF-containing serum) on the accumulation of lipid droplets in THP-1 macrophages induced by Ox-LDL, we performed Oil Red O staining. Supplementary Figure 1 illustrates that there was no significant difference between the ox-LDL group and 5% HXF- containing serum group (P > 0.05). Therefore, 10% HXF-containing serum was selected for subsequent mechanism studies. As shown in Figure 2B and 2D, a decrease in the quantity of red droplets in the HXF treatment after 24, 36, and 48 h (P < 0.05). Further detection by Western blot showed that HXF could evidently upregulate ABCG1 expressions in foam cells (P < 0.01) (Figure 2E).

Figure 2.

HXF attenuated lipid accumulation in ox-LDL-induced THP-1 macrophages. (A, B) Oil Red O staining, representative microscopic images (scale bar = 50 μm) are shown. (C, D) Statistical analysis of oil red O staining was shown. (E) The protein expression levels of ABCG1 in ox-LDL- stimulated THP-1 macrophages were evaluated by Western blotting. Data are expressed as mean ± SD (n = 3). *P < 0.05 versus ox-LDL 0 h group, **P < 0.01 versus ox-LDL 0 h group, #P < 0.05 versus BC group, ^^P < 0.01 versus BC + ox-LDL group. BC group, blank serum control group; HXF group, HXF-containing serum group.

Overexpression of LOX-1 Reversed the Protective Effect of HXF in ox-LDL-Induced THP-1 Macrophages

In order to clarify whether the protective effect of HXF was associated with LOX-1 in ox-LDL-induced THP-1 macrophages, we employed lentivirus to overexpress the expression of LOX-1 in THP-1 cells. Following Oil Red O staining, the cells exhibiting red intracellular lipid showed a positive reaction. The THP-1 + BC group had a lower number of positive cells, whereas positive cells were abundant in the ox-LDL group (P < 0.01). The THP-1 + ox-LDL + HXF group exhibited a notable reduction in the number of positive cells, which aligns with the findings from previous studies (P < 0.05). However, HXF showed no significant effect on ox-LDL-induced lipid droplet accumulation in THP-1 macrophages when overexpress LOX-1 (P > 0.05) (Figure 3A and 3B). Similar results were observed for intracellular total cholesterol levels, ox-LDL stimulation markedly increased intracellular total cholesterol contents compared with THP-1 + BC group (P < 0.01). The treatment with HXF significantly reduced the increase in cholesterol contents caused by ox-LDL (P < 0.05). Then, the effects of HXF were reversed by overexpressing LOX-1 (P < 0.05) (Figure 3C).

Figure 3.

Overexpression of LOX-1 reversed the protective effect of HXF in ox-LDL-induced THP-1 macrophages. (A) Oil Red O staining (scale bar = 50 μm). (B) Quantitative analysis of Oil Red O staining data. (C) A total cholesterol assay kit was used to measure the intracellular contents of total cholesterol. Data are expressed as mean ± SD (n = 3). **P < 0.01 versus THP-1 + BC group, #P < 0.05 versus THP-1 + ox-LDL + BC group, ^P < 0.05 versus THP-1 + ox-LDL + HXF group, BC, blank serum control; HXF, HXF-containing serum.

The Effect of HXF on ox-LDL-Induced Production of IL-6 and TGF-β in THP-1 Macrophages

We overexpress LOX-1 in THP-1 macrophages to investigate the effects of HXF on TGF-β and IL-6 contents in ox-LDL-stimulated THP-1 macrophages. As shown in Figures 4A and 4B, HXF treatment potently downregulated the levels of pro-inflammatory cytokines IL-6 and this effect was nearly reversed by LOX-1 overexpression in ox-LDL-stimulated THP-1 macrophages (P < 0.01). With regards to anti-inflammatory factor, however, compared with THP-1 + BC group, the TGF-β contents were significantly increased after treatment with HXF, and similarly reversed by LOX-1 overexpression (P < 0.05 or P < 0.01).

Figure 4.

The effect of HXF on ox-LDL-induced production of IL-6 and TGF-β in THP-1 macrophages. (A, B) The concentrations of IL-6 and TGF-β in the supernatants were measured by ELISA. Data are expressed as mean ± SD (n = 3). **P < 0.01 versus THP-1 + BC group, #P < 0.05 versus THP-1 + ox-LDL + BC group, ##P < 0.01 versus THP-1 + ox-LDL + BC group, ^P < 0.05 versus THP-1 + ox-LDL + HXF group, ^^P < 0.01 versus THP-1 + ox-LDL + HXF group, BC, blank serum control; HXF, HXF-containing serum.

HXF Inhibit Macrophage Foam Cell Formation by Regulating the LOX-1/ NF-κB Pathway

Further investigation was conducted by qPCR and Western Blot to determine the molecular mechanism responsible for HXF against lipid accumulation in ox-LDL-induced THP-1 macrophages. As shown in Figure 5A and 5B, compared with the THP-1+ BC group, the mRNA expression of LOX-1 was increased in the THP-1 + ox-LDL + BC group (P < 0.05). Furthermore, no differences were observed between the THP-1 + ox-LDL + BC group and LV-OLR1 + ox-LDL + BC group (not shown). HXF treatment significantly reduced LOX-1 and NF-κB mRNA expression in ox-LDL-induced THP-1 macrophages (P < 0.05 or P < 0.01), whereas overexpressing LOX-1 significantly reversed this effect (P < 0.01). Moreover, we found a similar expression pattern in the key molecules LOX-1 by western blot analysis (P < 0.05 or P < 0.01) (Figure 5C). As shown in Figure 5D, the expression of NF-κB is decreased in nucleoprotein after HXF treatment (P < 0.05), whereas overexpressing LOX-1 significantly reversed this effect (P < 0.05).

Figure 5.

HXF inhibits macrophage foam cell formation by regulating LOX-1/ NF-κB pathway. (A, B) The mRNA expressions of LOX-1 and NF-κB in ox-LDL- stimulated THP-1 macrophages were analyzed by qPCR. (C) The protein expression levels of LOX-1 in ox-LDL- stimulated THP-1 macrophages were evaluated by Western blotting. Relative protein expression was normalized to GAPDH expression. (D) The protein expression levels of NF-κB in ox-LDL- stimulated THP-1 macrophages were evaluated by Western blotting. Relative protein expression was normalized to Lamin B1 expression. Data are expressed as mean ± SD (n = 3). *P < 0.05 versus THP-1 + BC group, **P < 0.01 versus THP-1 + BC group, #P < 0.05 versus THP-1 + ox-LDL + BC group, ##P < 0.01 versus THP-1 + ox-LDL + BC group, ^P < 0.05 versus THP-1 + ox-LDL + HXF group, ^^P < 0.01 versus THP-1 + ox-LDL + HXF group, BC, blank serum control; HXF, HXF-containing serum.

Discussion

Foam cells appear to play a pathogenic role in creating atherosclerotic plaques. Macrophages are the primary source of foam cells, which can be activated by signals from damaged tissues. ¹⁶ Ox-LDL is believed to contribute to the progression of atherosclerosis, specifically foam cell formation. In this study, ox-LDL was used to establish a foam cell model in THP-1 macrophages. According to the results, ox-LDL accumulated lipid droplets in THP-1 macrophages in a time-dependent manner, and the lipid accumulation in macrophages is apparently enhanced by ox-LDL for 24, 36 and 48 h. These findings suggested that ox-LDL promotes macrophages-derived foam cell formation, which is in concurrence with the available literature. ¹⁷

An increasing number of Chinese herbal medicines have been widely applied to treat AS. The Huxin formula (HXF), as a traditional Chinese decoction, has improved patient recovery after CABG surgery and alleviated symptoms. ¹³ HXF consists of five herbs, each of which is recognized for their ability to provide protection against atherosclerosis. One of the isolated constituents from Ginseng Radix et Rhizoma, ginsenoside F, defends endothelial cells from inflammatory damage in ApoE^−/− mice by inhibiting of NF-κB and LOX-1. ¹⁸ Further, ginsenoside Rb1 was capable of decreasing lipid buildup in macrophages by promoting autophagy, ¹⁹ and previous studies by our team have confirmed that HXF reverses the ox-LDL-induced expression of autophagy-related proteins, mitigating excessive autophagy in atherosclerosis progression. ²⁰ According to another research, the protective effect of Panax notoginseng saponins on rabbit atherosclerosis can be attributed to its anti-inflammatory or antioxidant properties. ²¹ The extract of Carthami Flos has been reported to have therapeutic effects on atherosclerosis in vivo and in vitro through multiple mechanisms. ²² A TCM formula's therapeutic effect, however, comes from a complex process in vivo, which is much more than just its ingredients. Our research revealed that ox-LDL (50 μg/ml) had a notable impact on the buildup of lipids in macrophages, but this effect was counteracted by HXF treatment lasting 24, 36, and 48 h. Thus, HXF inhibits the accumulation of lipids.

Macrophages in atherogenesis generate lipid droplets enclosed in membranes through the uptake and processing of lipoproteins. Furthermore, the creation of foam cells plays a role in maintaining the stability of atheromatous plaques and fatty streaks. ²³ The formation of foam cells involves several intricate procedures, such as lipid consumption, cholesterol esterification within the cell, and cholesterol efflux externally.^24,25 It was found in this study that HXF inhibited ox-LDL-induced macrophage foam cell formation in THP-1 cells, mainly through suppressing lipid droplet accumulation and cholesterol uptake. The main transporters accountable for RCT in macrophages are ATP-binding cassette transporter A-1 (ABCA1) and ATP-binding cassette transporter G-1 (ABCG1). ²⁶ Our team's previous studies have confirmed that HXF can enhance the expression of ABCA1, consequently inhibiting the formation of foam cells. ²⁷ This study found that administering HXF significantly increased ABCG1 expression in foam cells, promoting cholesterol removal.

LOX-1 was first cloned from bovine endothelial cells. Subsequently, it was discovered in human macrophages as well, playing a crucial role in every inflammatory phase of atherosclerosis. ²⁸ LOX-1 can be used to identify initial atheromatous lesions in humans in the endothelium, and in more advanced lesions in macrophages, which leads to the transformation of these cells into foam cells. ²⁹ LOX-1 is up-regulated by many proatherogenic factors, including ox-LDL, tumor necrosis factor (TNF), high glucose, angiotensin-II, and C-reactive protein. ³⁰ In a high-fat diet experiment involving Apoe^−/− mice overexpressing LOX-1, showed that increased uptake of modified lipids and accelerated macrophage infiltration attributed to LOX-1.^31,32 The present study showed overexpression of LOX-1 reversed the protective effect of HXF in ox-LDL-induced THP-1 macrophages. Furthermore, there was a decrease in LOX-1 mRNA and protein expression in the presence of HXF, hinting that a downregulation of LOX-1 may be one of HXF's antiatherogenic mechanisms. Atherosclerosis is also considered a type of chronic inflammation. When exposed to ox-LDL, macrophages produce a large number of proinflammatory molecules such as TNF-α and IL-6. ³³ These pro-inflammatory cytokines enhanced LOX-1 expression and ox-LDL uptake in macrophages. ³⁴ Our present study, HXF treatment potently can inhibit the inflammatory response, the release of the pro-inflammatory factor IL-6 was reduced and the release of the anti-inflammatory factors TGF-β was promoted, and this effect was nearly reversed by LOX-1 overexpression in ox-LDL-stimulated THP-1 macrophages.

NF-κB, a well-known regulator, has the ability to regulate the expression of functional genes, which includes molecules involved in inflammation and adhesion. ³⁵ The activation of transcription starts when NF-κB, moves from the cytosol to the nucleus. Newly discovered evidence has unveiled the possible involvement of NF-κB in the development of atherosclerosis. For example, the NF-κB activation has been detected in macrophages and human atherosclerosis plaques. ³⁶ Furthermore, Y. Li et al ³⁷ reported that LPS (NF-κB activator) caused a decrease in cholesterol efflux and treatment with PDTC (NF-κB inhibitor) reversed the LPS-induced downregulation of cholesterol efflux. In this study, we observed the activation of NF-κB in THP-1 macrophages induced by ox-LDL, while HXF inhibited the expression of NF-κB. Otherwise, NF-κB play an important function in LOX-1 expression. ³⁸ HXF treatment significantly reduced LOX-1 and NF-κB mRNA and protein expression in ox-LDL-induced THP-1 macrophages, whereas overexpressing LOX-1 significantly reversed this effect. The findings indicated that the anti-atherogenic impact of HXF is a result of inhibiting the LOX-1/NF-B signaling pathway.

Apparently, the present study naturally includes some limitations. Researchers found that SRs such as CD36, SR-A1, and LOX-1 are primarily responsible for absorbing ox-LDL in macrophages. ⁵ It remains unknown whether HXF regulates CD36 and SR-A1 expression in foam cells. In addition, the upstream mechanism of LOX-1/ NF-κB pathway requires more in-depth exploration.

Conclusions

In summary, our study showed that HXF reduced the formation of foam cell in ox-LDL-stimulated THP-1 macrophages via inhibiting lipid accumulation and inflammation through regulating the LOX-1/ NF-κB pathway. These present findings further indicate a potential beneficial role of HXF in ameliorating atherosclerosis and foam cell formation, while provide a novel potential therapeutic strategy for preventing atherosclerosis.

Materials and Methods

Reagents

RPMI-1640 medium, and Fetal bovine serum (FBS) were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). Oil Red O and Phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Ox-LDL was purchased from Yiyuan Biotechnology (Guangzhou, China). The lentiviruses were purchased from Shanghai GeneChem (Shanghai, China). The BCA protein assay kit was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Total cholesterol assay kit was purchased from Applygen Technologies, Inc. (Beijing, China). Rabbit anti-NF-κB p65 (D14E12), Rabbit anti-GAPDH (D16H11) were purchased from Cell Signaling Technology (Boston, MASS, USA). Human LOX-1/OLR1 Antibody (AF1798-SP) was purchased from R&D Systems (Minneapolis, MN, USA). Rabbit anti-ABCG1 was purchased from Slarbio science & Technology (Beijing, China). Mouse anti-Lamin B1 was purchased from Proteintech (Wuhan, China). Human Interleukin 6 (IL-6) and Human Transforming Growth factor β1 (TGF-β1) ELISA kits were purchased from Cusabio (Wuhan, China). Nuclear Protein Extraction kit was purchased from Biyuntian (Wuhan, China).

Preparation of HXF-Containing serum

As shown in Table 1, HXF contains five Chinese medicinal herbs, Ginseng Radix et Rhizoma, Citri Exocarpium Rubrum, Notoginseng Radix et Rhizoma, Aurantii Fructus and Carthami Flos were purchased from Kangmei Pharmaceutical (Guangdong, China). These ingredients were combined and soaked in 1300 mL of water for 30 min. Subsequently, they were boiled for 30 min in the initial round, and the resulting liquid was collected. Once again, boil for 30 min with 1000 ml of water for the second round, then gather the resulting liquid and mix it with the previous portion. Finally, the herbal liquid underwent filtration, resulting in a final concentration of 2.1 g/mL was obtained from the evaporation of the crude drugs.

Table 1.

The Compositions of HXF.

Chinese name	Academic name	plant part(s)	Amount (g)
Renshen	Ginseng Radix et Rhizoma	Root and rhizome	10
Juhong	Citri Exocarpium Rubrum	Pericarp	6
Sanqi	Notoginseng Radix et Rhizoma	Root and rhizome	10
Zhiqiao	Aurantii Fructus	Fruit	6
Honghua	Carthami Flos	flower	10

Sprague-Dawley rats (male, weighing 300g ± 20 g), were acquired from the Experimental Animal Center of Guangdong Province. Rats were maintained in specific pathogen-free conditions. A three-day adaptive feeding period was followed by a random division of rats into two groups (n = 10), the HXF-containing serum group (HXF) was intragastrically administered HXF (21 g/kg) twice a day for seven consecutive days, while the blank serum control group (BC) was intragastrically administered an equal volume of distilled water. Blood was collected from rats under anesthesia two hours after the last administration and centrifuged for 15 min. The animal experiment was approved by the Animal Care and Use Committee of Guangdong Provincial Hospital of Chinese medicine (No.2019021).

UPLC-MS Analysis of HXF-Containing serum

The HXF-containing serum analysis was performed using UPLC-Q-Exactive Orbitrap MS (Thermo Fisher Scientific, USA) according to previously published methods. ³⁹ Chromatographic separation of serum samples was performed on Thermo scientific Hypersil GOLD C₁₈ column (2.1 mm × 100 mm × 1.9 μm). The mobile phase consisted of acetonitrile solution (A) and 0.1% formic acid solution (B) with the flow rate of 0.3 mL/min. The elution procedure was as follows: 0–2.5 min (95% B), 2.5–3 min (95%-85% B), 3–17 min (85%-73% B), 17–19 min (73%-60% B), 19–27 min (60%-5% B), 27–30 min (5% B).

The mass spectrometer equipped with an H-ESI source that operated in both positive (ESI+) and negative (ESI−) electrospray ionization modes. The voltage for spraying was adjusted to 3.5 kV (positive) and 3.0 kV (negative). The capillary and auxiliary heating temperatures were 325 °C and 350 °C.

Cell Culture and Treatment

Human THP-1 monocytic cells were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were cultured in RPMI 1640 medium supplemented with 10% FBS. Approximately every two to three days, the culture medium was changed.

THP-1 monocytes and LOX-1-overexpressing THP-1 cells were differentiated into adherent macrophages using RPMI 1640 medium supplemented with PMA (160 nM), following incubation with 50 μg/mL ox-LDL in the presence or absence of 10% HXF-containing serum or blank serum for 0, 12, 24, 36 and 48 h.

Cell Transfection

An LOX-1-encoding lentivirus labeled with GFP (LV-OLR1) and a negative control lentivirus (CON522) were purchased from GeneChem. 1 × 10⁶ cells were transfected with 100 μL of HitransG A infection enhancing solution, the lentivirus infection reagent must be proportioned to an MOI of 30 along with the appropriate volumes. After 16 h, the medium was substituted with a new complete medium, and fluorescence expression was observed in fluorescent microscopy after 72 h. For all experimental groups, cells were processed for further experimental assays after transfection.

Cell Oil Red O Staining

In order to stain with Oil Red O, the original solution was thinned with ddH2O and subsequently filtered to create the working solution. The cells that underwent treatment were immobilized in 4% paraformaldehyde at room temperature for a duration of 30 min. Following fixation, the cells were rinsed with 60% isopropanol for a duration of 5 min and subsequently treated with Oil Red O working solution for 20 min. The cells were washed three times with purified water after removing from oil red O solution. Images were taken under a microscope for evaluation. After extracting with 100% isopropanol, the automatic plate-reader (BioTek, Winooski, VT, USA) was used to measure the absorbance of Oil Red O in the intracellular solution at 492 nm.

Intracellular Cholesterol Measurement

In order to determine the cholesterol content in THP-1 macrophages, a total cholesterol assay kit was used. Briefly, protein concentration was determined by BCA assay after cellular lipids were extracted with lysis buffer. After centrifugation for 5 min at 2000× g, 10 μL of the resulting liquid were combined with working solution in a 96-well plate, it was incubated at 37 °C for 20 min. Next, it was measured at 550 nm for absorbance. Molecular cholesterol per milligram of cellular protein was calculated for each sample.

Enzyme-Linked Immunosorbent Assay (ELISA)

According to the guidelines provided by the manufacturer, ELISA kits were utilized for the identification of TGF-β and IL-6 levels in the supernatant of cultured cells. To measure TGF-β, the culture supernatants were treated with 1 N HCl for 10 min to activate the latent TGF-β, and then neutralized using 1.2 N NaOH. In a microplate spectrophotometer, absorbance was measured at 450 nm.

Quantitative Real-Time PCR (qPCR)

FastPure Cell/Tissue Total RNA Isolation Kit (RC101, Vazyme, Nanjing, China) was used for the isolation of total RNA from cells. The reverse transcribed into cDNA after the concentration was determined, and stored at −20 °C. All primers were synthesized by Sangon Biotechnology (Shanghai, China) as follows: LOX-1, forward, 5′- CTGGCATGGAGAAAACTGTTAC-3′, and reverse, 5′- CATCCAAAGACAAGCACTTCTC-3′; NF-κB p65, forward, 5′- TATTTGAAACACTGGAAGCACG-3′ and reverse, 5′- CCGGAAGAAAAGCTGTAAACAT-3′; and GAPDH, forward, 5′- CAACGTGTCAGTGGTGGACCTG-3′ and reverse, 5′- GTGTCGCTGTTGAAGTCAGAGGAG-3′, as a housekeeping gene. Quantitative PCR (qPCR) reactions involved use of ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China). An analysis of the relative mRNA expression levels was conducted using the comparison of 2^−ΔΔCt values against GAPDH levels. The results are presented as relative expressions (fold).

Western Blotting Analysis

RIPA lysis buffer was utilized to extract protein from the cells in every group. For the extraction of nuclear proteins, we used the Nuclear Protein Extraction kit. The protein was loaded onto gels containing 12% SDS-PAGE and subsequently transferred to a PVDF membrane using electro-transfer. Following, primary antibodies anti-LOX-1 (1:2000 dilution), anti-NF-κB (1:5000 dilution) and anti-ABCG1 (1:2000 dilution) antibodies were incubated overnight at 4 °C. Afterwards, we cleansed and cultured the membranes for 1.5 h using a secondary antibody. GAPDH (dilution 1:8000) or Lamin B1 (dilution 1:20000) was also used for loading controls. Finally, blots were detected by an ECL method.

Statistical Analysis

One-way ANOVA was used to conduct a statistical comparison of normally distributed variables across multiple groups. The data was analyzed using SPSS statistical software version 19.0 and presented as the mean ± SD Statistical significance was defined as P < 0.05.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X241282836 - Supplemental material for Huxin Formula Inhibits Oxidized low-Density Lipoprotein-Induced Foam Cell Formation in THP-1 Macrophages via the LOX-1/NF-κB Pathway

Supplemental material, sj-docx-1-npx-10.1177_1934578X241282836 for Huxin Formula Inhibits Oxidized low-Density Lipoprotein-Induced Foam Cell Formation in THP-1 Macrophages via the LOX-1/NF-κB Pathway by Qiaohuang Zeng, Xiaomin Ou, Jing Cai, Taohua Lan, Weihui Lu and Wei Jiang in Natural Product Communications

Supplemental Material

sj-docx-2-npx-10.1177_1934578X241282836 - Supplemental material for Huxin Formula Inhibits Oxidized low-Density Lipoprotein-Induced Foam Cell Formation in THP-1 Macrophages via the LOX-1/NF-κB Pathway

Supplemental material, sj-docx-2-npx-10.1177_1934578X241282836 for Huxin Formula Inhibits Oxidized low-Density Lipoprotein-Induced Foam Cell Formation in THP-1 Macrophages via the LOX-1/NF-κB Pathway by Qiaohuang Zeng, Xiaomin Ou, Jing Cai, Taohua Lan, Weihui Lu and Wei Jiang in Natural Product Communications