Opuntia dillenii Haw. Polysaccharide Promotes Cholesterol Efflux in THP-1-Derived Foam Cells via the PPARγ-LXRα Signaling Pathway

There is increasing evidence supporting a role for enhanced macrophage cholesterol efflux in ameliorating atherosclerosis. Opuntia dillenii Haw. polysaccharide (ODP-Ia), the most important functional component obtained from Opuntia dillenii Haw. stem, has anti-atherosclerosis effects. Therefore, we propose that ODP-Ia could promote cholesterol efflux via the PPARγ-LXRα signaling pathway. In this study, THP-1 foam cells derived from macrophages were treated with different concentrations of ODP-Ia, GGPP (antagonist of LXRα) and GW9662 (antagonist of PPARγ), with or without 15 nmol ODP-Ia. The total cholesterol content in the cells was measured. The mRNA of ABCA1, ABCG1, PPARγ, LXRα and their protein levels in the foam cells were detected by RT–PCR and Western blot, respectively. The results showed that ODP-Ia plays a role in significantly promoting cholesterol efflux (p < 0.05) by upregulating the expression of ABCA1, ABCG1, SR-BI, PPARγ, PPARα and LXRα. Meanwhile, PPARγ and LXRα antagonists dramatically interfered the cholesterol efflux mediated by ODP-Ia (p < 0.05) and dramatically inhibited the upregulating effect of ODP-Ia on the expression of PPARγ, LXRα, ABCA1 and ABCG1 at both protein and mRNA levels (p < 0.05). In conclusion, ODP-Ia promotes cholesterol efflux in the foam cells through activating the PPARγ-LXRα signaling pathway. This bioactivity suggested that ODP-Ia may be of benefit in treating atherosclerosis.


Introduction
The balance of cholesterol metabolism must be precisely modulated to maintain the homeostasis of the body in peripheral cells, especially in macrophages [1]. Macrophages can take in not only the remaining free cholesterol in endometrial cells, but also that from cell debris and lipoprotein substances, and this intake is not suppressed by the negative feedback regulation mechanism [2]. Once the intake exceeds the amount of outflow, macrophages with excess lipid will initiate pathological processes, foam or apoptosis. Monocytes in the blood circulation are transformed into macrophages to phagocytize lipids accumulated in the vascular wall, which can be regarded as a self-protection mechanism of the human body. However, when consumed in large quantities, the human body must have a strong ability to transport these lipids out in order to maintain the balance of lipid metabolism. Therefore, transporting lipids, that is, promoting cholesterol outflow, directly affects the survival and development of macrophages [3][4][5].
There are three main pathways of cholesterol efflux from peripheral cells: watersoluble diffusion, Cavenger receptor class B type 1 (SR-BI) mediation and ATP binding cassette transporter A1 (ABCA1) mediation, among which ABCA1 mediation is considered to be the most important [6][7][8]. In this pathway, ABCA1 mediates intracellular free cholesterol and phospholipids to Apolipoprotein A-I (apoA-I), and synthesizes immature

Effects of ODP-Ia and Ezetimibe on the Viability of THP-1 Macrophage-Derived Foam Cells
As shown in Figure 2A, no significant difference in relative cell viability was observed between the 5, 10 and 20 nmol/L ODP-Ia groups and the control group (without ODP-Ia). Therefore, 10 nmol/L was used as the optimal treatment concentration of ODP-Ia, and 5, 10 and 15 nmol/L were used as the low-, medium-and high-concentration doses.
In another study [27], the optimal treatment concentration of ezetimibe that had an effect on THP-1 macrophage-derived foam cells was 3 μmol/L. As shown in Figure 2B, ezetimibe had no significant inhibitory effect on cells at concentrations of <9 μmol/L (no significant difference in relative survival compared with the control group). Therefore, the optimal treatment concentration of ezetimibe was 3 μmol/L.

Effects of ODP-Ia on Cholesterol Outflow from Foam Cells during apoA-I-Mediated Cholesterol Efflux
As shown in Figure 3A,B, when treated with apoA-I, the total cholesterol content of the THP-1 macrophage-derived foam cells decreased in a time-and concentration-dependent manner. Compared with the control group, the reduction effect of the low dose of 5 mg/mL was already significant (p < 0.05). However, no significant difference was observed between the 10 and 15 mg/mL groups. To obtain a more significant effect, we selected 20 mg/mL as the optimal treatment dose. The total cholesterol content of the THP-

Effects of ODP-Ia and Ezetimibe on the Viability of THP-1 Macrophage-Derived Foam Cells
As shown in Figure 2A, no significant difference in relative cell viability was observed between the 5, 10 and 20 nmol/L ODP-Ia groups and the control group (without ODP-Ia). Therefore, 10 nmol/L was used as the optimal treatment concentration of ODP-Ia, and 5, 10 and 15 nmol/L were used as the low-, medium-and high-concentration doses.

Effects of ODP-Ia and Ezetimibe on the Viability of THP-1 Macrophage-Derived Foam Cells
As shown in Figure 2A, no significant difference in relative cell viability was observed between the 5, 10 and 20 nmol/L ODP-Ia groups and the control group (without ODP-Ia). Therefore, 10 nmol/L was used as the optimal treatment concentration of ODP-Ia, and 5, 10 and 15 nmol/L were used as the low-, medium-and high-concentration doses.
In another study [27], the optimal treatment concentration of ezetimibe that had an effect on THP-1 macrophage-derived foam cells was 3 μmol/L. As shown in Figure 2B, ezetimibe had no significant inhibitory effect on cells at concentrations of <9 μmol/L (no significant difference in relative survival compared with the control group). Therefore, the optimal treatment concentration of ezetimibe was 3 μmol/L.

Effects of ODP-Ia on Cholesterol Outflow from Foam Cells during apoA-I-Mediated Cholesterol Efflux
As shown in Figure 3A,B, when treated with apoA-I, the total cholesterol content of the THP-1 macrophage-derived foam cells decreased in a time-and concentration-dependent manner. Compared with the control group, the reduction effect of the low dose of 5 mg/mL was already significant (p < 0.05). However, no significant difference was observed between the 10 and 15 mg/mL groups. To obtain a more significant effect, we selected 20 mg/mL as the optimal treatment dose. The total cholesterol content of the THP-1 macrophage-derived foam cells decreased significantly with increasing treatment time In another study [27], the optimal treatment concentration of ezetimibe that had an effect on THP-1 macrophage-derived foam cells was 3 µmol/L. As shown in Figure 2B, ezetimibe had no significant inhibitory effect on cells at concentrations of <9 µmol/L (no significant difference in relative survival compared with the control group). Therefore, the optimal treatment concentration of ezetimibe was 3 µmol/L.

Effects of ODP-Ia on Cholesterol Outflow from Foam Cells during apoA-I-Mediated Cholesterol Efflux
As shown in Figure 3A,B, when treated with apoA-I, the total cholesterol content of the THP-1 macrophage-derived foam cells decreased in a time-and concentration-dependent manner. Compared with the control group, the reduction effect of the low dose of 5 mg/mL was already significant (p < 0.05). However, no significant difference was observed between the 10 and 15 mg/mL groups. To obtain a more significant effect, we selected 20 mg/mL as the optimal treatment dose. The total cholesterol content of the THP-1 macrophage-derived foam cells decreased significantly with increasing treatment time (6-36 h) after treatment with 20 mg/mL apoA-I. The maximum reduction effect was observed after 24 and 36 h of treatment, but no significant difference was observed between the 24 and 36 h groups. Therefore, 24 h was chosen as the optimal treatment time. As shown in Figure 3C, compared with the control group, the total cholesterol content of the THP-1 macrophage-derived foam cells in the apoA-I group significantly decreased (p < 0.05). After pretreatment of the foam cells with ODP-Ia or ezetimibe for 2 h, the effect of apoA-I-mediated cholesterol efflux became stronger, although this effect was less effective than the effect of ezetimibe in the positive control group. The aforementioned results suggested that ODP-Ia can enhance apoA-I-mediated cholesterol efflux from foam cells.
Molecules 2022, 27, x FOR PEER REVIEW 4 of 17 (6-36 h) after treatment with 20 mg/mL apoA-I. The maximum reduction effect was observed after 24 and 36 h of treatment, but no significant difference was observed between the 24 and 36 h groups. Therefore, 24 h was chosen as the optimal treatment time. As shown in Figure 3C, compared with the control group, the total cholesterol content of the THP-1 macrophage-derived foam cells in the apoA-I group significantly decreased (p < 0.05). After pretreatment of the foam cells with ODP-Ia or ezetimibe for 2 h, the effect of apoA-I-mediated cholesterol efflux became stronger, although this effect was less effective than the effect of ezetimibe in the positive control group. The aforementioned results suggested that ODP-Ia can enhance apoA-I-mediated cholesterol efflux from foam cells.

Effects of ODP-Ia on the Cholesterol Outflow in Foam Cells
As shown in Figure 4, the cholesterol content of the foam cells group was significantly higher than that of the blank group (p < 0.05), which means the THP-1 cells were foamed successfully. After ODP-Ia intervention, the foamed effect was inhibited, and the cholesterol content in the foam cells showed a downward trend. These results indicate that ODP-Ia can promote cholesterol efflux in foam cells, and that 15 nmol/L is an effective dose.

Effects of ODP-Ia on the Cholesterol Outflow in Foam Cells
As shown in Figure 4, the cholesterol content of the foam cells group was significantly higher than that of the blank group (p < 0.05), which means the THP-1 cells were foamed successfully. After ODP-Ia intervention, the foamed effect was inhibited, and the cholesterol content in the foam cells showed a downward trend. These results indicate that ODP-Ia can promote cholesterol efflux in foam cells, and that 15 nmol/L is an effective dose.

Effects of ODP-Ia on the mRNA Expression of ABCA1, ABCG1, SR-BI, PPARγ, PPARα and LXRα in Foam Cells
As shown in Figure 5, the expression of PPARγ, PPARα, LXRα, ABCA1, ABCG1 and SR-BI mRNA in THP-1-derived foam cells treated with 50 µ g/mL ox-LDL decreased significantly. ODP-Ia intervention (48 h) effectively increased the expression of these genes, and there was a significant dose effect (p < 0.05). However, the effect of this improvement was lower than that of the positive control group (p < 0.05).

Effects of ODP-Ia on the mRNA Expression of ABCA1, ABCG1, SR-BI, PPARγ, PPARα and LXRα in Foam Cells
As shown in Figure 5, the expression of PPARγ, PPARα, LXRα, ABCA1, ABCG1 and SR-BI mRNA in THP-1-derived foam cells treated with 50 µg/mL ox-LDL decreased significantly. ODP-Ia intervention (48 h) effectively increased the expression of these genes, and there was a significant dose effect (p < 0.05). However, the effect of this improvement was lower than that of the positive control group (p < 0.05).

Effects of ODP-Ia on the mRNA Expression of ABCA1, ABCG1, SR-BI, PPARγ, PPARα and LXRα in Foam Cells
As shown in Figure 5, the expression of PPARγ, PPARα, LXRα, ABCA1, ABCG1 and SR-BI mRNA in THP-1-derived foam cells treated with 50 µ g/mL ox-LDL decreased significantly. ODP-Ia intervention (48 h) effectively increased the expression of these genes, and there was a significant dose effect (p < 0.05). However, the effect of this improvement was lower than that of the positive control group (p < 0.05).

Effects of ODP-Ia on the Protein Expression of PPARγ, PPARα, LXRα, ABCA1, ABCG1 and SR-BI in Foam Cells
The Western blot results were captured by the gel imaging system, and the density values of each bands were quantified by the Quantity One analysis software of BioRad company. As shown in Figure 6, THP-1 cells could express PPARγ, PPARα, LXRα, ABCA1, ABCG1 and SR-BI after being induced by PMA, but the expression of these proteins was obviously weakened after ox-LDL treatment. After further intervention with ODP-Ia and Ezetimibe, the expression of these proteins increased gradually, and the enhancement was dosage dependent. The results of band-density quantification showed that the effect of high-dose ODP-Ia intervention was significantly higher than that of the low-and middledose groups (p < 0.05), although it was not as high as that of the positive control Ezetimibe group (p < 0.05). It is worth noting that the expression of ABCA1 protein and the effect of the ODP-Ia high-dose group were similar to that of the positive control group (no statistical difference).

Effects of ODP-Ia on the Protein Expression of PPARγ, PPARα, LXRα, ABCA1, ABCG1 and SR-BI in Foam Cells
The Western blot results were captured by the gel imaging system, and the density values of each bands were quantified by the Quantity One analysis software of BioRad company. As shown in Figure 6, THP-1 cells could express PPARγ, PPARα, LXRα, ABCA1, ABCG1 and SR-BI after being induced by PMA, but the expression of these proteins was obviously weakened after ox-LDL treatment. After further intervention with ODP-Ia and Ezetimibe, the expression of these proteins increased gradually, and the enhancement was dosage dependent. The results of band-density quantification showed that the effect of high-dose ODP-Ia intervention was significantly higher than that of the low-and middle-dose groups (p < 0.05), although it was not as high as that of the positive control Ezetimibe group (p < 0.05). It is worth noting that the expression of ABCA1 protein and the effect of the ODP-Ia high-dose group were similar to that of the positive control group (no statistical difference).

Effects of GGPP and GW9662 on the Cholesterol Outflow in ODP-Ia-Mediated Foam Cell Cholesterol Efflux
As shown in Figure 7A, the intervention of the LXRα antagonist GGPP significantly inhibited the enhancement of ODP-Ia on cholesterol efflux in foam cells. The previous results indicated that 15 nmol/L ODP-Ia promoted cholesterol efflux in foam cells, resulting in a significant reduction in cholesterol content in ox-LDL-induced foam cells. However, after GGPP intervention, the reduction effect was inhibited, and the cholesterol content in foam cells showed an upward trend. The pre-incubation with a high dose of GGPP (10 mol/L) could efficiently inhibit the effect of ODP-Ia-mediated enhancement, with no significant difference from the foam cell control group, and also no difference with GGPP treatment without ODP-Ia. These results indicate that ODP-Ia-enhancement of cholesterol efflux in foam cells through ODP-1a was suppressed after inhibition of LXRα, which means ODP-Ia needs to activate LXRα nuclear receptors to promote cholesterol efflux in foam cells.

Effects of GGPP and GW9662 on the Cholesterol Outflow in ODP-Ia-Mediated Foam Cell Cholesterol Efflux
As shown in Figure 7A, the intervention of the LXRα antagonist GGPP significantly inhibited the enhancement of ODP-Ia on cholesterol efflux in foam cells. The previous results indicated that 15 nmol/L ODP-Ia promoted cholesterol efflux in foam cells, resulting in a significant reduction in cholesterol content in ox-LDL-induced foam cells. However, after GGPP intervention, the reduction effect was inhibited, and the cholesterol content in foam cells showed an upward trend. The pre-incubation with a high dose of GGPP (10 mol/L) could efficiently inhibit the effect of ODP-Ia-mediated enhancement, with no significant difference from the foam cell control group, and also no difference with GGPP treatment without ODP-Ia. These results indicate that ODP-Ia-enhancement of cholesterol efflux in foam cells through ODP-1a was suppressed after inhibition of LXRα, which means ODP-Ia needs to activate LXRα nuclear receptors to promote cholesterol efflux in foam cells. Similarly, as shown in Figure 7B, the PPARγ antagonist GW9662 also significantly interfered with the promotion of ODP-Ia on cholesterol efflux in foam cells. After preincubation with a medium dose of 10 μmol/L GW9662 for 2 h, and then treatment the foam cells with ODP-Ia for 2 h, the enhancement on cholesterol efflux shown previously was significantly reduced. When the dose of GW9662 increased to 20 mol/L, the effect of ODP-Ia on promoting cholesterol efflux was completely inhibited, and there was no significant difference with the foam cell control group. These results suggest that cholesterol efflux function of ODP-Ia in foam cells requires activation of PPARγ nuclear receptors, too.

Effects of GGPP and GW9662 on the Expression of ABCA1, ABCG1, PPARγ and LXRα at mRNA and Protein Level in ODP-Ia-Mediated Foam Cell Cholesterol Efflux
LXRα and PPARγ are very sensitive to macrophage intracellular cholesterol levels. When intracellular cholesterol levels increase, LXRα and PPARγ are activated to regulate downstream-related genes to promote cholesterol excretion. Therefore, in this study, we wanted to clarify the following problems: whether the upregulation of ABCA1 induced Similarly, as shown in Figure 7B, the PPARγ antagonist GW9662 also significantly interfered with the promotion of ODP-Ia on cholesterol efflux in foam cells. After preincubation with a medium dose of 10 µmol/L GW9662 for 2 h, and then treatment the foam cells with ODP-Ia for 2 h, the enhancement on cholesterol efflux shown previously was significantly reduced. When the dose of GW9662 increased to 20 mol/L, the effect of ODP-Ia on promoting cholesterol efflux was completely inhibited, and there was no significant difference with the foam cell control group. These results suggest that cholesterol efflux function of ODP-Ia in foam cells requires activation of PPARγ nuclear receptors, too. LXRα and PPARγ are very sensitive to macrophage intracellular cholesterol levels. When intracellular cholesterol levels increase, LXRα and PPARγ are activated to regulate downstream-related genes to promote cholesterol excretion. Therefore, in this study, we wanted to clarify the following problems: whether the upregulation of ABCA1 induced by ODP-1a in THP-1 macrophage-derived foam cells is achieved by activating LXRα and PPARγ, and whether the activation is regulated in a cascade manner or activated alone.
As shown in Figure 8, compared with the foam cell control group, the expression of ABCA1, ABCG1, PPARγ and LXRα mRNA in Group ODP-Ia increased significantly (p < 0.05). However, when GGPP was added, the expressions of those genes were significantly decreased (compared with the ODP-Ia group, p < 0.05). Medium-concentration (5 mol/L) GGPP could significantly inhibit the promotion of ODP-Ia on ABCA1, LXRα and PPARγ mRNA expression (compared with the foam cell control group, p > 0.05). However, the inhibi-tion of ABCG1 expression only required a low concentration (1 µmol/L). There was no significant dose-dependent effect of GGPP on the expression of these four genes.
by ODP-1a in THP-1 macrophage-derived foam cells is achieved by activating LXRα and PPARγ, and whether the activation is regulated in a cascade manner or activated alone.
As shown in Figure 8, compared with the foam cell control group, the expression of ABCA1, ABCG1, PPARγ and LXRα mRNA in Group ODP-Ia increased significantly (p < 0.05). However, when GGPP was added, the expressions of those genes were significantly decreased (compared with the ODP-Ia group, p < 0.05). Medium-concentration (5 mol/L) GGPP could significantly inhibit the promotion of ODP-Ia on ABCA1, LXRα and PPARγ mRNA expression (compared with the foam cell control group, p > 0.05). However, the inhibi-tion of ABCG1 expression only required a low concentration (1 µ mol/L). There was no significant dose-dependent effect of GGPP on the expression of these four genes. In the above results, GGPP, an antagonist of LXRα, reduced the transcription levels of ABCA1 and ABCG1 genes, suggesting that LXRα regulates the expression of these two genes in the process of cholesterol efflux. The same regulatory effect was also observed in the experimental group treated with PPARγ antagonist GW9662. As shown in Figure 9, the expression of these four genes decreased significantly in all groups that were pre-incubated with PPARγ antagonist GW9662 (compared with the ODP-Ia group, p < 0.05). Low-concentration (5 mol/L) GW9662 could significantly inhibit the promotion of ODP-Ia on the expression of ABCG1 and PPARγ mRNA (compared with the foam cell control In the above results, GGPP, an antagonist of LXRα, reduced the transcription levels of ABCA1 and ABCG1 genes, suggesting that LXRα regulates the expression of these two genes in the process of cholesterol efflux. The same regulatory effect was also observed in the experimental group treated with PPARγ antagonist GW9662. As shown in Figure 9, the expression of these four genes decreased significantly in all groups that were pre-incubated with PPARγ antagonist GW9662 (compared with the ODP-Ia group, p < 0.05). Low-concentration (5 mol/L) GW9662 could significantly inhibit the promotion of ODP-Ia on the expression of ABCG1 and PPARγ mRNA (compared with the foam cell control group, p < 0.05). The inhibition of the expression of ABCA1 and LXRα required a medium concentration (10 µmol/L) and a high concentration (20 µmol/L), respectively. The decreased expression of LXRα mRNA indicated that LXRα was also regulated by PPARγ. group, p < 0.05). The inhibition of the expression of ABCA1 and LXRα required a medium concentration (10 µ mol/L) and a high concentration (20 µ mol/L), respectively. The decreased expression of LXRα mRNA indicated that LXRα was also regulated by PPARγ. As shown in Figure 10, the expression of ABCA1, ABCG1, PPARγ and LXRα protein in Group ODP-Ia was significantly enhanced. However, after pretreatment with different concentrations of GW9662 or GGPP, the electrophoretic bands of those four proteins expression became shallower. The statistical results after quantification of protein expression were basically consistent with the results of the banding analysis. ODP-Ia significantly enhanced the expression of ABCA1, ABCG1, PPAR and LXRα proteins in THP-1 macrophage-derived foam cells (SNK-q test, p < 0.05), but the expression of these proteins was significantly reduced after pretreatment with LXR antagonist GGPP and PPARγ antagonist (p < 0.05).When the GGPP intervention dose reached a high dose of 10 mol/L, the expression of these proteins all decreased significantly to even lower levels than that of the foam cell group (p < 0.05), regardless of whether ODP-Ia pretreatment was used in advance.
These results suggest that ODP-Ia can significantly enhance the expression of ABCA1, ABCG1, PPAR and LXRα proteins in THP-1 macrophage-derived foam cells, but this enhancement can be inhibited or blocked by LXRα antagonist GGPP and PPARγ As shown in Figure 10, the expression of ABCA1, ABCG1, PPARγ and LXRα protein in Group ODP-Ia was significantly enhanced. However, after pretreatment with different concentrations of GW9662 or GGPP, the electrophoretic bands of those four proteins ex-pression became shallower. The statistical results after quantification of protein expression were basically consistent with the results of the banding analysis. ODP-Ia significantly enhanced the expression of ABCA1, ABCG1, PPAR and LXRα proteins in THP-1 macrophage-derived foam cells (SNK-q test, p < 0.05), but the expression of these proteins was significantly reduced after pretreatment with LXR antagonist GGPP and PPARγ antagonist (p < 0.05).When the GGPP intervention dose reached a high dose of 10 mol/L, the expression of these proteins all decreased significantly to even lower levels than that of the foam cell group (p < 0.05), regardless of whether ODP-Ia pretreatment was used in advance.
These results suggest that ODP-Ia can significantly enhance the expression of ABCA1, ABCG1, PPAR and LXRα proteins in THP-1 macrophage-derived foam cells, but this enhancement can be inhibited or blocked by LXRα antagonist GGPP and PPARγ antagonist GW9662. PPARγ antagonist GW9662 can downregulate the expression of ABCA1, ABCG1 and LXRα at the same time. LXRα antagonist GGPP can downregulate the expression of ABCA1 and ABCG1, suggesting that PPARγ and LXRα regulate the expression of ABCA1 and ABCG1 in a cascade manner. antagonist GW9662. PPARγ antagonist GW9662 can downregulate the expression of ABCA1, ABCG1 and LXRα at the same time. LXRα antagonist GGPP can downregulate the expression of ABCA1 and ABCG1, suggesting that PPARγ and LXRα regulate the expression of ABCA1 and ABCG1 in a cascade manner.

Discussion
In this study, we observed that ODP-Ia could significantly upregulate the expression of PPARγ, PPARα, LXRα, ABCA1, ABCG1, SR-BI mRNA and protein, and promoted apoA-I-mediated reduction in cholesterol accumulation in foam cells, although the effect was not as good as that of the positive control, and required a higher dose. It is suggested that ODP-Ia can promote cholesterol efflux by upregulating the expression of these related genes and proteins. The efficiency of cholesterol efflux is an important basis for assessing RCT [28]. It is also believed that the promotion of cholesterol reverse transport by HDL is mainly mediated by promoting cholesterol efflux [29].
At present, domestic and foreign studies have found that a variety of natural active substances have the effect of promoting the outflow of cholesterol from foam cells [30][31][32][33].
There are many factors affecting cholesterol efflux. PPARγ and PPARα are more well-researched receptors that can upregulate the expression of ABCA1 and SR-B1 in foam cells and promote cholesterol efflux [34]. Our results also showed that ODP-Ia upregulated the expression of these two receptors while mediating cholesterol efflux. Hence, we speculate that these two receptors play a role in the regulation of ABCA1 and ABCG1 expression in the process of ODP-Ia-mediated cholesterol efflux. Our results confirmed the hypothesis that the effect of ODP-Ia upregulating the expression of ABCA1, ABCG1, PPARγ and LXRα mRNA and protein was inhibited or blocked by the LXRα antagonist GGPP and the PPARγ antagonist GW9662. In this study, we also observed that PPARγ antagonist GW9662 not only downregulated the expression of PPARγ mRNA and protein, but also downregulated the expression of LXRα mRNA and protein, confirming that PPARγ plays a role in the PPARγ-LXRα cascade by regulating the downstream LXRα gene. The PPARγ-LXRα pathway is an important pathway for the expression of ABCA1 and ABCG1. The study of genistein [35], soy isoflavones [36], Myristica fragrans [37], the dried pericarp of Citrus reticulata Blanco [38], Ginger extract [39] and traditional Chinese medicine Yinxing-tong-mai decoction [40] confirmed the existence of this pathway. The Tangier disease caused by ABCA1 mutation cannot express ABCA1 protein, and LXRα and PPARγ cannot regulate the reverse transport of cholesterol, which directly proves the regulation of ABCA1 by PPARγ-LXRα pathway [41][42][43].
There are two ways in which PPARγ regulates the expression of downstream genes. First, when PPARγ is activated by ligands, it binds to the specific peroxisome proliferator response element (PPRE) upstream of those genes to regulate their transcription, translation and biological activity. Second, PPARγ can also bind to LXR or RXR to form a dimer; the target gene is regulated by LXR. LXRα is a subtype of LXR receptor, and another subtype is LXRβ. LXRα is closely related to lipid metabolism. Therefore, only LXRα subtype is discussed in this study. The LXRα subtype is highly expressed in macrophages and liver, and was first discovered in the liver cDNA library. At that time, its natural ligand was not found, so it was classified as an orphan receptor. However, subsequent studies have found that LXR has a variety of ligands, including not only endogenous ligands, such as various hydroxylated cholesterols and epoxy cholesterol, but also physiological ligands, such as glucose and sterol. In recent years, a large number of functional substances of plants have been found to activate LXRα, but these substances also activate PPARγ expression at the same time [44]. Therefore, it is difficult to define whether direct activation of LXRα or activation of PPARγ promotes the binding of PPARγ and LXR to dimer.
In the present study, we found that PPARγ antagonist GW9662 not only inhibits PPARγ expression but also inhibits LXRα expression, while also reducing ODP-Ia-mediated intracellular cholesterol efflux and downregulating ABCA1 expression, indicating that PPARγ and LXRα act synergistically to regulate the expression of ABCA1. Studies have shown that LXRα is the target gene of PPARγ [45], especially in macrophage-derived foam cells. PPARγ and LXRα regulate the cholesterol efflux through cascade regulation. Our results also suggest that ODP-Ia cascades upregulate downstream ABCA1 and ABCG1 gene expression through the PPARγ-LXRα pathway, thereby promoting cholesterol outflow. It is suggested that ODP-Ia can be further studied as an activator of LXRα and PPARγ to promote its development and utilization. However, we also found that the LXRα antagonist GGPP downregulated the expression of PPARγ. It is suggested that there may be an interaction between PPARγ and LXRα, and the specific mechanism deserves further study.

THP-1-Derived Macrophages and Foam Cells
THP-1 cells were inoculated in fresh medium (3 mL). Three microliters of PMA (160 µmol/L) were added. After 24 h incubation at 37 • C, most of the cells transformed from suspension to adhesion, indicating the successful induction of macrophages. Subsequently, the cells were incubated with 1640 medium (serum-free) with 0.3% BSA and 50 µg/mL of ox-LDL in a six-well plate at 37 • C for 48 h after the supernatant was replaced. Oil red O staining was used to identify whether the cells were successfully foamed.

Cell Viability Assays
Cell viability was determined using the MTT assay. THP-1 macrophage-derived foam cells were treated with different concentrations of ODP-Ia (0, 5, 10, 20, 40 and 80 nmol/L), as well as ezetimibe (0, 1, 3, 9, and 100 µmol/L). Then, the cells were rinsed with PBS 3 times after the supernatant was removed by centrifugation (800 rpm, 5 min) 24 h later. Then, the cells were incubated with 200 µL of complete medium and 20 µL MTT solution (5 mg/mL of MTT in PBS) in 5% CO 2 at 37 • C for 4 h. The supernatant was discarded carefully before 150 µL dimethyl sulfoxide (DMSO) was added. After vibration, the absorbance was read at 490 nm using a plate reader ((Multiskan Skyhigh; Thermo Fisher Scientific, Inc., Shanghai, China). Relative cell viability (percent of control) was calculated as follows: (mean OD (490 nm) of treated cells/mean OD (490 nm) of control cells) × 100.

ApoA-I Treatment in ODP-Ia-Induced Foam Cell
After macrophages were induced, the THP-1 macrophage-derived foam cells (10 6 /mL) were treated with different apoA-I concentrations (0, 5, 10, 15, and 20 mg/L) for 24 h, and the total cholesterol content in the cells was measured to investigate the optimal concentration of apoA-I for treatment. Then, this optimal treatment concentration (20 mg/L) was used to treat the foam cells, and the cholesterol efflux of the foam cells was measured at different times (6, 12, 18, 24 and 36 h) to determine the optimal treatment time. Finally, the THP-1 macrophage-derived foam cells (10 6 /mL) were incubated with different ODP-Ia concentrations (0, 5, 10, and 15 nmol/L) for 2 h, and then, apoA-I treatment was continued for 24 h. The lever of cholesterol in the foam cells was measured to investigate the effects of ODP-Ia on cholesterol outflow from apoA-I-treated foam cells during cholesterol efflux.

Groups of the Effect of ODP-Ia on the Cholesterol Efflux and the Associated Gene Expression in Foam Cells
After induction of macrophages, the cells were randomly divided into 6 groups, as shown in Table 1, and each treatment was performed three times, undergoing three replicates each time. The cells in each treatment group were collected, total RNA was extracted and the expression levels of ABCA1, ABCG1, SR-BI, PPARγ, PPARα and LXRα mRNA were detected by RT-qPCR. Western blot was used to detect their protein expression. Table 1. Groups of the effect of ODP-Ia on the cholesterol efflux-associated gene expression in foam cells.

Group Treatment
Blank no ox-LDL is added.

PPARγ-LXRα Antagonist Treatment in ODP-Ia-Induced Foam Cell
Intervention of THP-1 -derived foam cells with 15 nmol ODP-Ia was performed with different concentrations of LXRα antagonist GGPP and PPARγ antagonist GW9662, respectively. The total cholesterol content in the cells was measured to investigate whether GGPP and GW9662 inhibited the promotion of ODP-Ia on total cholesterol efflux in foam cells. In order to examine whether GGPP and GW9662 could inhibit the upregulation of ABCA1, ABCG1, PPARγ and LXRα expression level induced by ODP-1a in the foam cells, the mRNAs were detected by RT-qPCR. Western blot was used to detect those protein expressions. The experimental design of the LXRα antagonist GGPP were shown in Table 2. The PPARγ antagonist GW9662 was designed in a similar way, but the concentrations of GW9662 were 5, 10 and 20 mol/L, respectively.

Determination of Cholesterol Content in THP-1 Macrophage-Derived Foam Cells
Total cholesterol content of the foam cells was measured through an enzymatic assay technique using a commercial cholesterol assay kit. All experiments were performed according to the manufacturer's recommended protocol.

Western Blot Analysis
After the cells were lysed with RIPA Lysis Buffer, and total protein concentrations in the cells were examined using BCA Protein Assay Kits. The total protein content of each sample was 10 µL. The sample was mixed with the sample buffer in proportion and boiled for 5 min. Then, 20 µL of the mixture was used for SDS-PAGE electrophoresis (the electrophoretic voltage was 80 V for the shrink gel and 120 V for the separation gel). Proteins separated by the separating gel were transferred onto PVDF membranes. Then, the membranes were blocked with free-fat milk in TBS at 4 • C for 1 h and incubated with primary antibodies for PPARγ (1:1000 dilution), PPARα (1:1000 dilution), LXRα (1:1000 dilution), ABCA1 (1:200 dilution), ABCG1 (1:1000 dilution), SR-B1(1:1000 dilution), and GAPDH (1:1000 dilution) at 4 • C overnight. At last, the membranes were incubated with HRP-conjugated antibodies for 1 h at room temperature. After incubation, the membranes were washed 3 times with TBST for 15 min each time. The ECL color exposure test was performed. The exposed film was scanned and analyzed using quantity one professional grayscale analysis software. Using GAPDH as the internal reference, histone expression of each treatment was calculated and compared.

RT-PCR Assays
Total RNA was extracted from macrophages using an RNeasy kit, and cDNA was prepared using a cDNA reverse transcription kit. Then, 10 µL of the reverse transcription product was used for the PCR cycle. Primer and probe designs are presented in Table S1. The reaction conditions were pre-denaturation at 95 • C for 10 min, denaturation at 95 • C for 15 s, and annealing extension at 60 • C for 60 s. This cycle was repeated 40 times. According to the experimental results, the amplification efficiencies of the target gene and the internal reference gene GAPDH were basically the same. Therefore, the experimental data could be analyzed using the 2 −∆∆Ct method [47] to calculate the expression levels of each gene relative to GAPDH.

Statistical Analysis
All experiments were repeated at least three times, and the values are expressed as the mean ± SD. The results were analyzed using one-way analysis of variance (ANOVA) with SNK-q test, and a p-value < 0.05 was considered statistically significant.

Conclusions
In conclusion, our data reveal a favorable role of ODP-Ia in promotion of cholesterol efflux in the foam cells. Mechanistically, ODP-Ia activates the PPARγ/LXRα pathway to enhance ABCA1-and ABCG1-dependent cholesterol efflux. These findings extend our understanding for the anti-atherogenic action of ODP-Ia, and further support the notion that ODP-Ia may be a promising drug candidate for therapeutic intervention of atherosclerotic cardiovascular disease.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.