Protective Effect of Shenqi Maixintong Capsule against ox-LDL-Induced Injury in RAW264.7 Macrophages

Shenqi Maixintong capsule (SMC), a traditional Chinese medicine compound, exerts various therapeutic effects, including nourishing one’s vitality, improving blood circulation, regulating vital energy, and promoting coronary circulation. Studies have demonstrated that SMC could effectively alleviate atherosclerosis (AS) and delay its development. However, the effect of SMC on lipid metabolism remains unclear. Herein, we investigated the mechanism of SMC on lipid metabolism of RAW264.7 cells induced by oxidized low-density lipoprotein (ox-LDL). Drug-containing serum was prepared by intragastric administration of SD (Sprague Dawley) rats. RAW264.7 cells were transformed into foam cells with 25 mg/L ox-LDL. SMC-treated rat serum increased the survival rate of ox-LDL-induced RAW264.7 cells. Oil Red staining, total cholesterol, and free cholesterol detection data showed that the intermediated dose of SMC had the best effect on reducing the lipid accumulation, lipid droplets, intracellular cholesterol, and total cholesterol content of RAW264.7 cells. The results of western blotting showed that the expression of ABCA1, ABCG1, LXRα, PPARγ, and peroxisome proliferation-activated receptor alpha (PPARα) was increased by SMC, and proprotein convertase subtilisin/kexin type 9 (PCSK9) was reduced, which promoted reverse cholesterol transport (RCT) of RAW264.7 cells and inhibited foam cell formation. Furthermore, SOD and MDA data indicated that SMC could reduce the senescence of RAW264.7 cells. These findings suggest that SMC might prevent ox-LDL-induced damage of macrophages in AS patients by improving cell viability and slowing down lipid accumulation and senescence.


Introduction
Atherosclerosis (AS) is a common disease and the major cause of morbidities and mortalities worldwide. is is a complex pathological process and is an abnormal regulation involving multiple factors. It is widely accepted that the aggregation and foaming of cholesterol and cholesterol esters in macrophages play an important role in the formation of AS [1][2][3], and elevated circulating low-density lipoprotein cholesterol (LDL-c) represents the major independent risk factor for AS [4]. erefore, lowering LDL levels and blocking foam cell formation may be the key to reducing AS [5].
Numerous studies have shown that the classical PPARα-LXRα-ABCA1, PPARc-LXRα-ABCA1, and LXRα-ABCA1 signaling pathways involved in nuclear receptors mediate oxidized lipids in foam cells [6][7][8][9][10]. e proteins involved in these signaling pathways transfer excess lipids in the cell to the high-density lipoprotein (HDL) in the periphery for reverse transport to intrahepatic metabolism [11]. ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation [12][13][14]. ABCA1 and ABCG1 can be highly induced in macrophages [15]. It was found that the expression of ABCA1 is mainly regulated by nuclear receptors LXRα and LXRβ, while LXRα is regulated by PPARc [16,17]. en, PPARc and LXRα can affect cholesterol transport by inducing the expression of ABCA1 [18][19][20]. Previous studies have shown that PCSK9 increases expression in macrophages, causing inflammation and cholesterol accumulation by inhibiting RCT action [21,22].
SMC is a Chinese herbal compound first developed by Guangzhou hospital of integrated traditional Chinese and Western medicine. It is prepared from the eight herbs of Radix Ginseng Rubra, Radix Notoginseng, Rhizoma Ligustici Chuanxiong, Radix Salviae Miltiorrhizae, Radix Astragali seu Hedysaari, Rhizoma Cyperi, Fructus Crataegi, and Fructus Lycii. with modern Chinese medicine preparation technology. It has been used in clinical application for more than 20 years and has a definite effect on atherosclerosis [23]. 40 patients with cervical atherosclerotic plaque were treated with SMC and observed changes in plaque after 2 courses of treatment. e total effective rate was 72.5% [24]. Another clinical trial of 70 patients showed that SMC can effectively lower blood lipid levels and alleviate the disease [25]. Other studies have also shown that SMC has the effect of lowering blood lipids, improving vascular endothelial function, and delaying atherosclerosis [26][27][28][29].
Although SMC has an active role in stabilizing atherosclerotic lipid plaques, its mechanism is not clear. It is necessary to further study the mechanism of SMC regulating lipid metabolism. erefore, ox-LDL was used to induce the RAW264.7 macrophage formation cell injury model, and the effect of SMC on cell lipid metabolism was evaluated.

2.2.
Animals. 40 male SD (Sprague Dawley) rats (180-220 g, 4-5 weeks old) were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., license number SCXK (Lu) 20140007. All SD rats were housed under standard conditions (room temperature 23 ± 2°C, humidity 60 ± 15%, 12 h/12 h light/dark cycles) and given free access to standard rodent chow and water. All experimental procedures were performed in accordance with the Guidelines for Animal Experiments from the Committee of Medical Ethics, National Health Department of China.

Drug Content
Determination. Radix ginseng rubra, Panax notoginseng, and Ligusticum chuanxiong in the Chinese herbal compound improve blood circulation and remove blood stasis, and these three are the major ingredients in the SMC compound. erefore, the main active ingredients of these traditional Chinese herbs, ginsenoside Rg1 and ferulic acid, were selected as the calibration materials, and the drug content was determined according to the Chinese Pharmacopoeia 2015 edition. To control the SMC quality, the high-performance liquid chromatography (HPLC) method was performed to establish the fingerprint spectrum. e analyses were performed with an Agilent 1260 system (Agilent Technologies, Hewlett-Packard-strasse-8, 76337 Waldbronn, Germany) equipped with a quaternary gradient pump. e components were eluted with a system consisting of water (A) and acetonitrile (B), and the elution was A (75%) and B (25%). e chromatographic column was Agilent ZORBA×SB-C18 (4.6 × 250 mm, 5 μm). e mobile phase flow rate was 0.8 mL/min, and the column temperature was maintained at 25 ± 1°C. e detection wavelength was 203 nm (ginsenoside Rg1) and 316 nm (ferulic acid) with an injection volume of 10 μL. Ginsenoside Rg1 and ferulic acid were dissolved in methanol as control and measured by the HPLC. e specificity, linearity, precision, repeatability, stability, and recovery were further tested.

SMC Sample Preparation with Ginsenoside Rg1.
Take 5 g of SMC powder, put it in a Soxhlet extractor, add chloroform, and heat to reflux for 3 h. Take out the medicine residue, put it into the Erlenmeyer flask, add 50 mL of watersaturated n-butanol, and let it stand overnight. Sonicate for 30 min, filter and accurately measure 25 mL, and evaporate the solution to dryness. e residue was dissolved in methanol, diluted to 5 mL, and filtered.

SMC Sample Preparation with Ferulic
Acid. Take 1 g of SMC powder and place in an Erlenmeyer flask, and then 10 mL of 70% methanol was added and sealed. Weigh and heat reflux for 30 min. After cooling, weigh again and make up the lost weight with 70% methanol. Mix and filter the supernatant.

Preparation of Drug-Containing
Serum. 40 SPF (specificpathogen-free) healthy SD rats, male, weighing 180-200 g, were used in this study. After 10 days of adaptive feeding, they were randomly divided into blank group, low-dose group, middle-dose group, high-dose group, and 10 per group. SMC adult dosage was 4 capsules/time, 3 times/day, i.e., a total of 12 capsules daily. e rat dose was calculated according to "equivalent dose ratio between human and animal converted by body surface area" [30]. After soaking for 1 h, the SMC were prepared by water extraction twice. e extract was filtered and condensed to make the concentration equivalent to 2.0 capsules per mL and then kept at 4°C. Also, the final concentration was 0.27 capsules per mL (low), 0.54 capsules per mL (middle), and 1.08 capsules per mL (high) and was administered at a dose of 1 mL/100 g. Every test group was administered SMC at 1 mL/100 g/d, and the blank control group was given normal saline once a day 1 ml/100 g. e rats were intragastrically administered for 8 days, fasted for 12 h before the last administration, and anesthetized with 0.3% sodium pentobarbital 1 h after the administration. Blood was taken from the femoral artery, and serum was separated after centrifugation and filtered for use.

RAW264.7 Macrophage Cell
Culture. RAW264.7 cells were cultured in a DMEM medium containing 10% FBS in a humidified atmosphere at 37°C, 5% CO 2 . en, the cells were passaged three times. In the logarithmic growth phase, the cells were randomly divided into five groups: control group, ox-LDL group, low-dose group, intermediated-dose group, and high-dose group, at least three replicates for each group, and cell counting was performed so that the number of cells in each group was the same. e cells were cocultured with 25 mg/L ox-LDL for 48 h, except the blank group, and then replaced with normal medium (vehicle medium), blank medium, low-dose medium, intermediated-dose medium, and high-dose drug-containing medium for 24 h. Subsequently, the cells were collected and extracted for analysis.
2.6. Cell Proliferation Assay. RAW264.7 cells were seeded into 96-well plates (10 3 /well), and 100 μL medium was added into each well. 96-well plates were cultured for 24 h in a cell culture incubator containing 5% CO 2 at 37°C. en, the cells in the 96-well plate are administered to different concentrations of ox-LDL (0, 20, 40, 60, or 100 mg/L), different concentrations of drug-containing serum, blank (vehicle medium), and a mixture of ox-LDL + drug-containing serum. After 24 h of culture, cell proliferation assays were performed using a CCK8 kit and a microplate reader (490 nm).

Oil Red O Staining.
e well-grown macrophages were seeded in a 6-well plate at a density of 1 × 10 5 /well, cultured for 24 h, and the cell culture medium was discarded after the cells were attached. ox-LDL was prepared in a RPMI-1640 medium, and the final concentration was 20-30 mg/L. e prepared ox-LDL was added to a 6-well plate for 48 h to establish a foam cell model. Identification of foam cells by Oil Red O staining was carried out. After gently washing the cells 3 times with PBS, 4% paraformaldehyde was fixed at room temperature for 30 min. After washing, the cells were again incubated at room temperature with Oil Red O solution for 30 min. Discard the staining solution, fix it with 60% isopropyl alcohol for 5∼10 s, and rinse immediately with PBS 2-3 times. e morphology of the foam cells was observed under a microscope, and it was found that the intracellular bright red lipid droplets were foam cells. Oil Red O imaging was taken using the ×40 objective lens. Also, quantitative analysis of intracellular lipid droplets was performed using integrated optical density (IOD) and Image J software (National Institutes of Health, Bethesda, MD, USA).

Total Cholesterol and Free Cholesterol Assays.
RAW264.7 cells were pretreated and cultured according to the abovementioned method. en, the kit instructions were followed. After treatment, the cells were washed once with PBS, and the cells were lysed by ultrasonic homogenization, heated at 70°C for 10 min, and centrifuged at 2000 rpm for 5 min at room temperature. e supernatant was removed, and total cholesterol and free cholesterol were measured using a microplate reader (550 nm). Protein quantification was performed using the BCA method. And samples of total cholesterol and free cholesterol were tested for protein quantification.

SOD Measurement.
e cells were cultured as described above, and each group of cells was collected and operated according to the SOD detection kit. e original cell culture medium was discarded, and PBS was washed 2-3 times, and then the cells were collected, centrifuged at 1500 rpm for 10 min, and precipitated. Buffer was added, and the cells were sonicated. After that, the reagent was added to the cells according to the kit instruction. en, the cells were incubated at 37°C for 20 min and the SOD activity was read at 450 nm.

MDA Measurement.
e pretreatment of the cells is the same as the treatment for the SOD measurement. Heating was then carried out at 95°C for 20 min. e reagents required for the experiment were prepared and tested according to the MDA kit instructions. e absorbance was read at 530 nm.
2.11. Western Blot Assay. RAW264.7 cells were treated as described and washed three times with PBS, and cell lysates were generated using RIPA lysis buffer with PMSF. Blow off the cells and transfer the solution into a new EP tube. e solution was lysed on ice for 30 min and centrifuged at 12,000 rpm for 30 min at 4°C. en, the supernatant was assayed for protein using a BCA kit. e protein concentration was adjusted with RIPA lysate, and 5x loading buffer was added and boiled for 5 min. en, the proteins were stored at −80°C. Protein lysate was loaded onto 10% SDS-PAGE gel, electrophoresed, and then transferred to the polyvinylidene fluoride membrane. e membranes were probed with antibodies against ABCA1, ABCG1, LXRα, PPARα, PPARc, and PCSK9, respectively. Signals were Evidence-Based Complementary and Alternative Medicine amplified and observed with the horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling, CA, U.S.A., 1 : 5000 dilution) and enhanced chemiluminescence. Densitometry was detected with an ECL Western Blot Detection System (4A Biotech, Beijing, China). All experiments were repeated at least three times.

Statistical
Analyses. Statistical analysis was performed using SPSS 22.0 software. Data are presented as mean-± standard deviation. Statistical analysis was performed using one-way ANOVA, and post-hoc comparisons were carried out using Duncan's multiple-range test. P < 0.05 was considered statistically significant.

Determination of Drug Content.
To determine the quality of the SMC, two major compounds were used as quality control standards for SMC. e HPLC chromatogram of SMC was achieved on a C18 column using a mixture of acetonitrile and water with 0.1% phosphoric acid as the mobile phase. e HPLC chemical fingerprint of SMC is shown in Figure 1.
e average ginsenoside Rg1 content of the three batches of SMC samples was 14.05 mg/g. e linear regression equation and correlation coefficient of ginsenoside Rg1 were calculated as follows: y � 514.53x + 355.21, R2 � 0.9996, and r � 0.9997, indicating that the linear relationship of ginsenoside Rg1 is good in the range of 5.39∼50.30 μg. e RSD (relative standard deviation) values of the precision, repeatability, and stability test results are less than 2%, and the RSD value of the recovery test is 2.53%, indicating that the experimental research method is accurate and reliable, and the recovery rate experiment results are shown in Table 1.
e average ferulic acid content of the three batches of SMC samples was 2.42 mg/g. e linear regression equation and correlation coefficient of ferulic acid are as follows: y � 4153.3x + 37.111, R 2 � 0.999, and r � 0.9995, indicating that the linear relationship of ferulic acid is good in the range of 0.046∼0.396 μg. Similarly, the RSD values of the methodological tests are less than 2%, and the recovery rate experiment results are shown in Table 2.

Improvement of ox-LDL-Induced RAW264.7 Cell Viability by SMC.
e cell viability of RAW264.7 cells was evaluated, and Figure 2(a) shows that ox-LDL reduced the cell viability of RAW264.7 cells in a dose-dependent manner. Figure 2(b) shows that half of the cells survived at 100 mg/L ox-LDL and SMC was not cytotoxic to RAW264.7 cells at intermediated dose. In Figure 2 e Oil Red O staining showed that the lipid droplets in the ox-LDL + SMC groups were significantly less than those in the ox-LDL group (Figure 3). ere was significant difference between control and ox-LDL groups (P < 0.001), and the p value of the ox-LDL + intermediated group was less significant than that of the ox-LDL group (P < 0.001).
e total cholesterol and free cholesterol levels showed that low and intermediated dose of SMC significantly reduced total cholesterol (P < 0.01) and free cholesterol (P < 0.01) induced by ox-LDL ( Figure 4). erefore, SMC could inhibit ox-LDL-induced lipid accumulation in RAW264.7 cells.

Effects of SMC on SOD Activity and MDA in RAW264.7
Cells. RAW264.7 cells were cultured separately by SMC and ox-LDL treated together and ox-LDL alone, and then the SOD and MDA contents were determined. e data in Figure 5(a) show that the SOD activity of ox-LDL was lower than that of the other groups and the difference was statistically significant (P < 0.01). Different dose groups of SMC increased the decrease in SOD value caused by ox-LDL to varying degrees, and the groups of SMC alone did not significantly reduce the SOD value. MDA in the ox-LDL group was higher than the other groups, which was statistically significant (P < 0.01) ( Figure 5(b)). Different dose groups of SMC reduced the increase in MDA values caused by ox-LDL to varying degrees, and every dose group did not significantly change the MDA value of each group. erefore, SMC increased the SOD activity after ox-LDL treatment and decreased the content of MDA.

Regulation of the Expression of ABCA1, ABCG1, LXRα,
PPARα, PPARc, and PCSK9 in RAW264.7 Cells. It has been shown that ABCA1, ABCG1, and other related proteins are related to the regulatory mechanism of cellular cholesterol transport. We were wondering whether SMC indeed rescues the macrophage survival by activating this blah, the blah pathway. To test this hypothesis, we treated the RAW cells in vitro with SMC and measured ABCA1, ABCG1, LXRα, PPARα, PPARc, and PCSK9 by western blotting. Our data show ( Figure 6) that ox-LDL decreased the expression of ABCA1, ABCG1, LXRα, PPARα, and PPARc but increased PCSK9 expression. After treatment with SMC, ABCA1, ABCG1, LXRα, PPARα,and PPARc expressions were upregulated and the expression of PCSK9 was downregulated. us, SMC can modify the expression of ABCA1, ABCG1, LXRα, PPARα, PPARc, and PCSK9 in RAW264.7 cells.

Discussion and Conclusions
Foam cell formation is the key step in the development of AS. More and more studies have shown that macrophage uptake of ox-LDL by scavenger receptors (SRs) leads to significant accumulation of cholesterol, transforming macrophages into foam cells and promoting the development of AS [31][32][33]. Here, we used an ox-LDL-induced    staining. e bar graph shows the percentage of Oil Red O staining in different groups. ere was significant difference between control and ox-LDL groups (P < 0.001). Lipid droplets in the ox-LDL + SMC groups were significantly less than those in the ox-LDL group, and the p value of the ox-LDL + intermediated group was less significant than that of the ox-LDL group (P < 0.001). Original magnification: 400x. Data are presented as mean ± standard deviation. * P < 0.05, * * P < 0.01, and * * * P < 0.001. ox-LDL, oxidized low-density lipoprotein; SMC, Shenqi Maixintong capsule; low, low dose of SMC; intermediated, intermediated dose of SMC; high, high dose of SMC.  Evidence-Based Complementary and Alternative Medicine macrophage lipid accumulation cell model, demonstrating that SMC can reduce lipids in macrophages. However, our data showed that the intermediated dose of SMC had the best effect on reducing the lipid accumulation, lipid droplets, intracellular cholesterol, and total cholesterol content of RAW264.7 cells. is possibly results because the high dose had a certain cytotoxic effect, which reduced the efflux of lipids in cells. e preparation has been used clinically for nearly 20 years and has good clinical anti-AS effect. According to the existing research literature, previous reports have shown that SMC treatment can effectively change the blood lipid composition by reducing serum TG, TC, and LDL, increasing HDL, and eventually delaying atherosclerotic plaque formation [23][24][25][26]. Moreover, studies have shown that SMC can prevent and treat atherosclerosis by regulating the inflammatory response mediated by the NF-κB signaling pathway [27]. However, this study is the first time to investigate the mechanism of action of the SMC from the perspective of in vitro cell research. Previous studies and conclusions are based on the results of clinical experiments, and the efficacy is usually judged by performing serum detection and measurement of atherosclerotic plaque size. Also, previous studies have shown that SMC has a good effect on atherosclerosis, but it has not specifically explored the molecular pathway mechanism that may have an effect. In this study, we explored a possible mechanism by which SMC reduces intracellular lipid accumulation by establishing ox-LDLinduced macrophage-derived foam cell models. In our study, treating cells with SMC can reduce intracellular lipid levels and promote cholesterol transport by affecting the expression of proteins related to cholesterol transport. e reverse transport of cholesterol is mainly mediated by ABCA1 and ABCG1 [34,35]. LXRα agonists upregulate the expression of ABCA1 and ABCG1 and inhibit the differentiation of macrophages into foam cells by promoting cellular efflux [36][37][38]. LXRα expression was modulated by nuclear receptors, particularly PPARs, and many studies have determined that PPARc agonists can induce LXRα expression in human and murine macrophages [39][40][41]. Based on our western blotting results, it can be speculated that SMC may reduce foam cell formation by increasing the expression of cholesterol efflux transporters. is may be related to the effective regulation of ABCA1-related pathways.
Our test results of SOD and MDA found that SMCs in different dose groups can increase the SOD value of cells treated with ox-LDL and decrease the MDA value. SOD and MDA are classic indicators of oxidative stress evaluation, and there are currently reports in the literature that oxidative stress can promote cellular senescence [42][43][44]. Studies show that oxidative stress accelerate telomere shortening or dysfunction and telomere shortening suppresses cancer by triggering senescence in normal cells [45][46][47]. erefore, our results can deduce that SMC has a possible role in delaying cellular senescence.
In conclusion, as a traditional Chinese medicine preparation, SMC can be used for treating AS, which can block ox-LDL-induced damage of RAW264.7 cells, improve cell viability, and reduce lipid accumulation and senescence.
is may be related to the effective regulation of ABCA1related pathways, PPARα-LXRα-ABCA1, PPARc-LXRα-ABCA1, and LXRα-ABCA1, and the expression of proteins such as ABCG1, LXRα, and PCSK9. Future research will continue to study the effects of SMC on the expression of RCT-related proteins.  Figure 6: Western blot analysis of the expression of ABCA1, ABCG1, LXRα, PPARα, PPARc, and PCSK9 in RAW264.7 cells. GAPDH was served as an internal control of the protein level. Determined expression of the protein was subsequently quantified by densitometric analysis with that of control being 1.00-fold. e expression of ABCA1, ABCG1, LXRα, PPARα, and PPARc was decreased, induced by ox-LDL, but the expression of PCSK9 was increased, induced by ox-LDL. After treatment with SMC, ABCA1, ABCG1, LXRα, PPARα, and PPARc expressions were upregulated and the expression of PCSK9 was downregulated. Results are representative of at least three independent experiments.

Data Availability
All data analyzed during this study are included in the manuscript. e raw data used and analyzed during the current study are available from the corresponding author upon reasonable request.

Ethical Approval
All procedures described in this study were performed in accordance with the guidelines of the Institutional Animal Ethical Committee (Jinan University of Experimental Animal Center).

Conflicts of Interest
e authors have no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Qianqian Ma was responsible for sourcing of research articles, designing and performing the experiments, writing original draft, reviewing, and editing. Min Liang carried out compiling and writing of original draft, reviewing, and editing. Tiange Cai did reviewing and cross verification of research articles, writing review, and editing. Yingjia Yu, Xinjian Liang, Qingzhuang Chen, and Manling Du were responsible for experiment and data arrangement. Yong Ouyang and Chaojun Chen made supervision and editing of the final draft. Yu Cai carried out supervision and final editing of review. Q. Ma, M. Liang, and T. Cai have contributed equally to this manuscript.