INTRODUCTION

Atherosclerosis (AS) is a common vascular disease characterized by lipid metabolism disorder and chronic inflammation.1 AS is characterized by progressive chronic inflammation with different stages distinguished by unique lesions. In the absence of effective treatment, AS development accelerates, resulting in more complicated lesions.2 AS initially develops following regional blood flow disturbance in large or medium arteries, triggered by subendothelial lipoprotein retention and endothelial cell injury.3,4 These abnormal responses cause the thickening of the arterial wall and the gradual development of atherosclerotic plaques.1,5 Rupture of these vulnerable plaques can trigger the formation of a thrombus, causing stenosis or occlusion of the lumens and, ultimately, acute malignant heart and cerebrovascular events.4,5

More specifically, macrophages become foam cells following the uptake of lipoproteins. Foam cells then produce reactive oxygen species and induce an inflammatory reaction, which induces the recruitment of additional macrophages to the endothelium and subsequently accelerates AS progression.3 Macrophages differentiate into two main phenotypes generally defined as proinflammatory M1 and anti-inflammatory M2 macrophages.6–10 Therefore, inhibiting the polarization of macrophages toward the M1 phenotype represents a key strategy in AS treatment and prevention.11

Autophagy can improve the regulation of macrophage functions.12 In fact, impaired macrophage autophagy may have a proinflammatory effect through increased macrophage polarization toward the M1 phenotype.13 Autophagy is induced by the classical phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. mTOR, a PI3K family member and the downstream target of the PI3K/AKT pathway, exerts pleiotropic cellular effects, and it can regulate cell growth and proliferation while also serving as a key regulator of the autophagy initiation phase.14 Several studies have revealed that atherosclerotic plaque formation in ApoE−/− mice can be significantly accelerated by downregulating macrophage autophagy through the activation of the PI3K/AKT/mTOR pathway.15 However, downregulation of this pathway may enhance atherosclerotic plaque stability and inhibit AS progression-activated macrophage autophagy.16

Several studies have confirmed that traditional Chinese medicine (TCM) effectively inhibits AS progression by regulating the PI3K/AKT/mTOR signaling pathway.17 Meanwhile, we previously found that San Jie Tong Mai Fang (SJTMF), a classical herbal formula for promoting blood circulation and dissipating phlegm, can significantly regulate macroautophagy-related proteins in ApoE−/− mice.18,19 SJTMF is largely composed of Dan Shen, Shui Zhi, San Qi, Bei Mu, Yin Chen, Fu Ling, Bai Shao, Ze Xie, Tan Xiang, Jiang Can, Chan Tui, Zhi Qiao, Chen Pi, and Zhu Ru. We found that SJTMF contains a variety of compounds, such as flavonoids (quercetin, isorhamnetin, etc), fatty acyls (azelaic acid, dodecanoic acid, etc), and stilbenes (resveratrol, etc.), after the analysis of the pharmaceutical components of SJTMF (see Schedule, Supplemental Digital Content 1, https://links.lww.com/JCVP/A962). The influence of specific components of SJTMF needs further study. In this study (Fig. 1), we sought to investigate whether SJTMF inhibits AS progression in ApoE−/− mice by enhancing macroautophagy through the PI3K/AKT/mTOR signaling pathway.

FIGURE 1.:

Graphical summary. SJTMF protects against AS progression by regulating macroautophagy through the PI3K/AKT/mTOR signaling pathway.

MATERIALS AND METHODS Materials

SJTMF was provided by the Experimental Research Center of the Affiliated Hospital of Changchun University of Traditional Chinese Medicine (Changchun, China). The high-fat feed (XT108C) was purchased from Jiangsu Synergy (Nanjing, China). In addition, the autophagy inhibitor 3-MA (HY-19312) and the PI3K inhibitor LY294002 (HY-10108) were purchased from MedChemExpress (China). Atorvastatin calcium tablets (DW8413) were obtained from Pfizer Pharmaceuticals Ltd (New York, NY).

SJTMF Simmering Method

SJTMF is largely composed of Dan Shen (30 g), Shui Zhi (10 g), San Qi (10 g, for decoction), Bei Mu (30 g), Yin Chen (10 g), Fu Ling (10 g), Bai Shao (10 g), Ze Xie (30 g), Tan Xiang (10 g; for decoction), Jiang Can (10 g), Chan Tui (10 g), Zhi Qiao (10 g), Chen Pi (20 g), and Zhu Ru (10 g). SJTMF (400 g), comprising all Chinese herbal medicines and was boiled to 99.4 mL at a concentration of 40.24 g/kg. We calculated that the equivalent dose of SJTMF in mice was about 17.29 g/kg/d. Set the dosage of SJTMF to 40.24 g/kg/d. Gavage once a day.

Experimental Animals

Specific pathogen-free C57BL/6J pure male mice (5 week old) were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd (Beijing, China; experimental animal use permit number. SCXK [Beijing] 2016-0006). The mice were housed in controlled environmental conditions (temperature: 22–26°C, humidity: 50%–60%) with a 12-h light/dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee of Changchun University of Traditional Chinese Medicine (Changchun, China).

Model Construction and Drug Intervention

The effects of SJTMF on autophagy and PI3K/AKT/mTOR signal pathway were investigated by introducing 3-MA and LY294002. The mice were randomly divided into 8 groups (n = 10/group). Except for the control group, all mice were fed a high-fat diet20,21 (containing 20% fat, 1.25% cholesterol) for 16 weeks. The groups received the following treatments: control group: after 16 weeks on a normal diet, an equal volume of saline was administered by means of oral gavage once per day for 4 weeks; model group: after successful modeling, an equal volume of saline was administered by means of oral gavage once per day for 4 weeks; SJTMF group: after successful modeling, SJTMF (40.24 g/kg/d) was administered by means of oral gavage once per day for 4 weeks; Ato group: after successful modeling, 1 atorvastatin calcium tablet (3 mg/kg/d) was administered by means of oral gavage once per day for 4 weeks; 3-MA group: after successful modeling, 3-MA (30 mg/kg) was injected intraperitoneally twice per week for 4 weeks; 3-MA+SJTMF group: after successful modeling, 3-MA (30 mg/kg) and SJTMF (40.24 g/kg/d) were administered in combination, based on the regimens described above for each; LY294002 group: after successful modeling, a PI3K inhibitor, LY294002 (0.3 mg/kg), was administered intraperitoneally twice per week for 4 weeks; LY294002+SJTMF group: after successful modeling, LY294002 (0.3 mg/kg) and SJTMF (40.24 g/kg/d) were administered in combination according to the regimens described above for each. At the end of the intervention, under inhalation anaesthesia, orbital blood was collected, the serum was separated, and the aorta (from the root of the aorta to the bifurcation of the common iliac artery) was removed and fixed at −80°C and 4% paraformaldehyde for analysis.

Hematoxylin–Eosin (HE) Staining

Wax-embedded mouse aortic tissue samples were sectioned into 3-μm slices. After dewaxing and dehydration, the sections were stained with hematoxylin (g1004; ServiceBio, Wuhan, China) solution for 3–6 minutes and 0.5% eosin solution (e8090; Solarbio) for 2–3 minutes. They were then sealed with neutral gum (g8590; Solarbio). The samples were photographed through a microscope (DM1000; Leica Microsystems Shanghai Ltd, China), and the Leica Application Suite image system was used to capture the relevant regions of the samples.

Oil Red O Staining

The aortic tissues were separated, placed in fixation solution (G1101; Servicebio) for 24 hours, and dissected. They were then treated with Oil Red O stain (G1016; Servicebio) at 37°C for 60 minutes and removed. Fractionation was then performed with 75% ethanol until the fatty plaque appeared orange or bright red, whereas the other parts were nearly colorless. Next, the stained bulk tissue was unfolded and fixed on white filter paper and foam board under good lighting conditions, and the focal length was adjusted appropriately to capture clear images. Quantitative analysis of images was performed using Image-Pro Plus 6.0.

Transmission Electron Microscopy Observation

Aortic samples were placed in fresh fixative, cut into 1-mm samples, and fixed with 2.5% glutaraldehyde (30092436; Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) at 4°C for more than 30 minutes for detection. Next, samples were washed with 0.1 mol/L PBS thrice for 10 minutes each time, followed by treatment with 1% osmium acid (18456; Ted Pella, Inc, CA) for 1 hour. Next, the samples were dehydrated. After permeabilization and embedding, the tissues were sectioned by an ultrathin sectioning machine (S6 E; Leica, Germany) into 60-nm slices, which were stained with uranyl acetate (1005-25g; Merck, Germany) for 20 minutes in the dark. The copper mesh was then clamped out and washed thrice with double-distilled water and aspirated. Next, the sections were stained with lead citrate for 15 minutes; the mesh was removed, and excess lead was washed with double-distilled water and blotted on filter paper. The samples were observed through transmission electron microscopy (HT7700; Hitachi, Tokyo, Japan).

Serum Lipid and Inflammatory Cytokine Detection

The levels of serum lipids, including total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected with the corresponding biochemical kits (BS-420; Mindray, Shenzhen, China) following the manufacturer's instructions. The levels of serum inflammatory cytokines, including IL-6, IL-10, TNF-α, monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interleukin-1β (IL-1β), and ox-LDL, were determined by enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted using the Trizol (15596026, Ambion, CN) method. The RNA was then reverse-transcribed into cDNA, which was amplified by PCR. The reaction procedure comprised a predenaturation step at 95°C for 3 minutes, 95°C for 5 seconds, 56°C for 10 seconds, and 72°C for 25 seconds (40 cycles). In addition, the 2−ΔΔCt was used to calculate the relative mRNA content using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as the internal reference gene. The primer sequences were as follows: iNOS (Nos2)-forward, 5′-CCACGGACGAGACGGATAG-3′; Nos2-reverse, 5′-CGGGCACATGCAAGGAA-3′; Il6-forward, 5′-GTTGCCTTCTTGGGACTGA-3′; Il6-reverse, 5′-CAACTCTTTTCTCATTTCCACG-3′; Arg1-forward, 5′-TGTGAAGAACCCACGGTCTGT-3′; Arg1-reverse, 5′-TGGTTGTCAGGGGAGTGTT-3′; Igf1-forward, 5′-TGGACCGAGGGGCTTTTAC-3′; Igf1-reverse, 5′-TAGAGCGGGCTGCTTTTGT-3′; Gapdh-forward, 5′-CCTTCCGTGTTCCTAC-3′; and Gapdh-reverse, 5′-GACAACCTGGTCCTCA-3′.

Immunofluorescence

Wax-embedded mouse aortic tissue samples were processed (3 μm), baked in a constant temperature oven at 65°C for 1 hour, soaked in xylene I (10023418; Sinopharm Chemical Reagent Co., Ltd) for 15 minutes, and then in xylene II (10023418, Sinopharm Chemical Reagent Co., Ltd) for 15 minutes. After hydration, the slices were repaired under high pressure (125°C, −103 kPa) in 1 mM Tris EDTA buffer solution for 18 minutes, cooled naturally, and washed with PBS. After natural cooling, antigens were washed in PBS and repaired. The samples were then incubated in 10% goat serum in a wet box for 30 minutes. Subsequently, primary antibodies (CD86 1:200, LC3-II 1:200) were added dropwise and incubated overnight at 4°C. After washing, secondary antibodies (fluorescent secondary antibody Cy3 goat antirabbit 1:300, fluorescent secondary antibody 488 goat antimouse 1:400) were added and incubated for 1 hour at 37°C. A drop (approximately 20 µL) of antifluorescence quenching blocking solution (containing DAPI) was then applied to the tissue. Samples were photographed through fluorescence microscopy (Olympus, Tokyo, Japan).

Western Blotting

Mouse aortic tissue was cut, lysate was added at a ratio of 200 μL per 20 mg of tissue to achieve complete lysis, and samples were centrifugated at 12,000g for 15 minutes at 4°C. A BCA kit (PC0020; SolarBio, Beijing, China) was used to quantify the total proteins in each sample. Subsequently, 20 μg of protein was added to the gel (prepared with 12% isolate and 5% concentrate) and run at 80 V for 40 minutes for the concentrate and 120 V for 50 minutes for the isolate. Afterward, the proteins were transferred to PVDF membrane (Millipore) through wet transfer at 90 V transmembrane voltage for 50 minutes. The membranes were then blocked with 5% milk for 2 hours at room temperature and incubated with primary antibodies against LC3-II/Ⅰ (PAB32560, 1:1000; Bioswamp, Wuhan, China), beclin-1 (PAB35215, 1:1000; Bioswamp), and p62 (PAB35470, 1:1000; Bioswamp) at 4°C overnight. The membranes were subsequently washed and incubated with a secondary antibody (SAB43714, 1:10,000; Bioswamp) for 1 hour at room temperature. After adding the ECL luminescence solution, the bands were visualized in a fully automated chemiluminescence analyzer (5200; Tanon, Shanghai, China), and the grayscale values were read by TANON GIS software.

Statistical Analysis

The results are expressed as means ± SD. One-way analysis of variance (ANOVA) was performed to compare multiple groups. A P value of <0.05 was considered statistically significant. GraphPad Prism 8.0 software was used for graphical presentation.

RESULTS SJTMF Inhibits Atherosclerotic Plaque Damage in Mice

Pathological histomorphological changes of aortic root plaques were observed by HE staining (Fig. 2A). Compared with the control group, the aorta of the model group showed obvious plaques, the lumen was narrowed, the intima was obviously thickened, and the smooth muscle cells in the media were arranged disorderly and atrophied. However, compared with the model group, the SJTMF and Ato groups had less aortic plaque and reduced intima-media thickness. By contrast, the 3-MA group had increased aortic plaques, narrowed lumen, and thickened intima-media. Compared with the 3-MA group, the plaques in the 3-MA+SJTMF group were significantly reduced, the lumen was larger, and the smooth muscle layer was regular. Oil Red O staining (Fig. 2B) revealed that the overall aortic plaque area was significantly increased in the model group compared with the control group (P < 0.01). In addition, the SJTMF and Ato groups had significantly reduced plaque area compared with the model group (P < 0.01), whereas that of the 3-MA group was significantly larger (P < 0.05). Moreover, the plaque area was significantly reduced in the 3-MA+SJTMF group compared with the 3-MA group (P < 0.01). These data demonstrate that SJTMF can effectively reduce the formation of aortic plaque and improve the aortic injury of AS mice.

FIGURE 2.:

Histopathological examination of the aorta. A, Hematoxylin–eosin (HE) staining of mouse aortic tissue (×400). B, Oil red O staining of atherosclerotic plaques. ##P < 0.01 versus control group, **P < 0.01 versus model group, *P < 0.05 versus model group, ^^P < 0.01 versus 3-MA group. n = 10.

SJTMF Reduces Serum Lipid Levels and Inflammation in Atherosclerotic Mice

Compared with the control group, TC, TG, and LDL-C levels were significantly increased (P < 0.01), whereas that of HDL-C was significantly decreased (P < 0.01) in the model group. Furthermore, compared with the model group, TC, TG, and LDL-C levels were significantly lower (P < 0.01), and HDL-C levels were significantly higher (P < 0.01) in the SJTMF and Ato groups, whereas the opposite trends were observed in the 3-MA group compared with the model group (P < 0.05). However, compared with the 3-MA group, the TC, TG, and LDL-C levels were significantly lower in the 3-MA+SJTMF group (P < 0.01), whereas HDL-C levels were significantly higher (P < 0.01). Compared with the control group, the serum levels of IL-10 were significantly lower (P < 0.01), whereas those of IL-6, TNF-ɑ, MCP-1, CRP, and IL-1β were significantly higher (P < 0.01) in the model group. Meanwhile, compared with the model group, the level of IL-10 in the SJTMF and Ato groups increased significantly (P < 0.01), whereas the proinflammatory indicators decreased significantly (P < 0.01). Following intervention with 3-MA, the abundance of IL-10 decreased (P < 0.05), whereas the other indicators increased significantly (P < 0.01) compared with the model group. By contrast, the combined application of 3-MA and SJTMF significantly increased IL-10 abundance (P < 0.01), while decreasing those of the proinflammatory indicators (P < 0.01) (Table 1). These data demonstrate the ability of SJTMF to reduce serum lipid levels and cellular inflammatory response in AS mice.

TABLE 1. - Serum Biochemical Indicators in Mice Items Control Model SJTMF Ato 3-MA 3-MA+SJTMF TC (mmol/L) 5.794 ± 0.28 12.490 ± 0.69## 8.988 ± 0.58** 8.475 ± 0.39** 14.40 ± 0.54** 11.07 ± 0.57^^ TG (mmol/L) 1.153 ± 0.16 5.527 ± 0.43## 3.414 ± 0.56** 3.133 ± 0.49** 7.480 ± 0.51** 4.253 ± 0.49^^ HDL-C (mmol/L) 2.193 ± 0.17 1.025 ± 0.07## 1.646 ± 0.21** 1.916 ± 0.22** 0.795 ± 0.10* 1.282 ± 0.14^^ LDL-C (mmol/L) 2.157 ± 0.16 5.617 ± 0.45## 3.641 ± 0.33** 3.129 ± 0.20** 7.114 ± 0.37** 6.162 ± 0.26^^ IL-6 (pg/mL) 49.43 ± 10.70 278.70 ± 31.61## 162.70 ± 15.96** 89.80 ± 7.94** 303.40 ± 16.34** 234.7 ± 10.29^^ IL-10 (pg/mL) 586.60 ± 55.40 183.70 ± 21.40## 388.00 ± 50.26** 463.50 ± 31.18** 165.20 ± 13.87* 228.40 ± 13.92^^ IL-1β (pg/mL) 120.60 ± 16.26 528.60 ± 33.09## 333.80 ± 28.64** 198.60 ± 20.68** 640.30 ± 30.75** 496.70 ± 34.89^^ TNF-α (pg/mL) 67.07 ± 16.58 342.60 ± 24.91## 216.50 ± 23.17** 126.40 ± 14.04** 408.60 ± 15.59** 320.30 ± 17.86^^ MCP-1 (pg/mL) 119.80 ± 14.12 590.50 ± 56.33## 345.00 ± 37.91** 185.20 ± 20.67** 628.90 ± 29.28** 500.60 ± 26.72^^ CRP (ng/mL) 3.632 ± 0.58 16.29 ± 1.66## 9.26 ± 1.02** 5.32 ± 0.48** 17.85 ± 0.81** 13.35 ± 0.71^^

The results are expressed as the mean ± SD of 10 mice per group.

##P < 0.01, compared with the control group;**P < 0.01, compared with the model group; *P < 0.05, compared with the model group; ^^P < 0.01, compared with the 3-MA group.

SJTMF Affects Macrophage Recruitment and M1/M2 Polarization

Expression of CD86 and CD206 in aortic tissue was detected by immunofluorescence (Figs. 3A, B). Compared with the control group, the fluorescence intensity of CD86 was stronger, whereas that of CD206 was weaker in the model group. Compared with the model group, the fluorescence intensity of CD86 was weaker and that of CD206 was stronger in the SJTMF and Ato groups; similar results were observed between the 3-MA group and 3-MA+SJTMF group. The expression of M1 and M2 macrophage-related markers in aortic tissues was detected by qRT-PCR (Figs. 3C, D). Compared with the control group, the expression of Nos2 and Il6 mRNA was significantly higher (P < 0.01), whereas that of Arg1 and Igf1 was significantly lower (P < 0.01) in the aortic tissue of the model group. However, compared with the model group, the expression of Nos2 and Il6 mRNA was significantly lower in the SJTMF and Ato groups (P < 0.01), whereas that of Arg1 and Igf1 mRNA was significantly higher (P < 0.01). Furthermore, compared with the model group, the expression of Nos2 and Il6 mRNA in the 3-MA group was significantly increased (P < 0.01), whereas that of Arg1 and Igf1 mRNA decreased, although not statistically (P > 0.05). Meanwhile, the opposite results were observed between the 3-MA group and 3-MA + SJTMF group (P < 0.01, P < 0.05, respectively). These data demonstrate that SJTMF increased the polarization of macrophages toward to the M2 phenotype.

FIGURE 3.:

Effects of SJTMF on macrophage recruitment and M1/M2 polarization. A, Fluorescence expression of CD86 was detected using immunofluorescence (200). B, Fluorescence expression of CD206 was detected using immunofluorescence (×200). C, Expression of M1 macrophage-associated markers in aortic tissue detected using qRT-PCR. D, Expression of M2 macrophage-associated markers in aortic tissues detected using qRT-PCR. ##P < 0.01 versus control group, **P < 0.01 versus model group, *P < 0.05 versus model group, ^^P < 0.01 versus 3-MA group. n = 10.

SJTMF Regulates Aortic Autophagy

The ultrastructure of macrophage autophagic vesicles, as well as changes in lipid droplets within the aortic plaque tissue, were observed using transmission electron microscopy (Fig. 4A). The macrophages in the aorta of the control group exhibited few visible autophagic vesicles and no lipid droplet formations. By contrast, no autophagic vesicles were seen in the model group, whereas many lipid droplets were present. Compared with the model group, more autophagic vesicles and fewer lipid droplets were detected in macrophages of the SJTMF and Ato groups, whereas no autophagic vesicle formation with many lipid droplets were observed in the 3-MA group. By contrast, autophagic vesicles were observed in the 3-MA+SJTMF group. Next, the expression levels of autophagy-related proteins, LC3-II/I, beclin-1, and p62 in macrophages, were detected in the aortic plaques of each group through western blotting (Fig. 4B). Compared with the control group, LC3-II/I and beclin-1 protein levels were significantly lower (P < 0.01), whereas that of p62 was significantly higher (P < 0.01) in the model group. By contrast, compared with the model group, LC3-II/I and beclin-1 abundances were significantly higher (P < 0.01), and p62 was significantly lower (P < 0.01) in the SJTMF and Ato groups; the opposite results were observed for the 3-MA group compared with the model group (P < 0.01, P < 0.05, respectively). Meanwhile, compared with the 3-MA group, LC3-II/I and beclin-1 abundances were significantly higher (P < 0.01), whereas that of p62 was significantly lower (P < 0.01) in the 3-MA+SJTMF group. These results were supported by the immunofluorescence findings (Figs. 4C,E). These data demonstrate that SJTMF effectively promoted the occurrence of macroautophagy.

FIGURE 4.:

SJTMF-induced regulation of aortic autophagy. A, Transmission electron microscopy of macrophage autophagic vesicles in aortic plaque tissue (×8000). B, Western blotting of autophagy-related proteins expression levels of LC3-II/I, beclin-1, and p62 in macrophages in aortic plaques. C, Fluorescence expression of LC3-II detected using immunofluorescence (×200), (D) Fluorescence expression of beclin-1 detected using immunofluorescence (×200), (E) Fluorescence expression of p62 detected using immunofluorescence. ##P<0.01 versus control group, *P<0.01 versus model group, **P < 0.01 versus model group, ^^P < 0.01 versus 3-MA group. n = 10.

SJTMF Regulates Aortic Autophagy and Macrophage Polarization through the PI3k/Akt/mTOR Signaling Pathway

PI3K, AKT, mTOR, and their phosphorylated protein expression levels were detected in aortic plaques by western blotting (Fig. 5A). The phosphorylation levels of PI3K, AKT, and mTOR proteins were significantly higher in the model group than in the control group (P < 0.01). However, compared with the model group, the phosphorylation levels of PI3K and mTOR proteins were significantly lower in the SJTMF and LY294002 groups (P < 0.01), whereas that of phosphorylated AKT was reduced in the SJTMF group, although not significantly (P > 0.05) and significantly reduced in the LY294002 group (P < 0.01). Compared with the LY294002 group, the phosphorylation levels of PI3K and mTOR were significantly lower in the LY294002+SJTMF group (P < 0.01) and that of AKT was significantly lower, although not significantly (P > 0.05). The expression of macrophage-associated markers in aortic tissues was detected by qRT-PCR (Figs. 5B, C). The expressions of Nos2 and Il6 mRNA were significantly higher in the model group than in the control (P < 0.01), SJTMF (P < 0.01), and LY294002 (P < 0.01) groups. Compared with the LY294002 group, the expression of Nos2 mRNA was significantly lower, whereas that of Il6 was slightly reduced (P > 0.05) in the LY294002+SJTMF group (P < 0.01). The expressions of Arg1 and Igf1 mRNA were significantly lower in the model group than in the control, SJTMF, and LY294002 groups (P < 0.01 for each). However, the expression of Arg1 and Igf1 mRNA was significantly higher in the LY294002+SJTMF group than in the LY294002 group (P < 0.01). Western blot results showed that, compared with the control group, LC3-II/I and beclin-1 protein abundance was significantly lower, whereas that of p62 protein was significantly higher in the model group (P < 0.01; Fig. 5D). Compared with the model group, SJTMF and LY294002 treatment significantly upregulated the abundance of LC3-II/I protein (P < 0.01). Similarly, beclin-1 protein levels increased in the SJTMF and LY294002 groups (P < 0.05 and P < 0.01, respectively), whereas that of p62 protein decreased (P < 0.05 and P < 0.01, respectively) in these groups. Moreover, LC3-II/I levels increased in the LY294002+SJTMF group, though not significantly (P > 0.05), whereas beclin-1 levels increased significantly (P < 0.01) and that of p62 decreased significantly (P < 0.01) compared with the LY294002 group. These results agreed with the immunofluorescence findings (Figs. 5E,G). In addition, compared with the control group, the fluorescence intensity of CD86 was stronger in the model group, whereas that of CD206 was weaker. Meanwhile, compared with the model group, the fluorescence intensity of CD86 was weaker in the SJTMF and LY294002 groups, whereas that of CD206 was stronger. Finally, compared with the LY294002 group, the fluorescence intensity of CD86 was weaker and that of CD206 was stronger in the LY294002+SJTMF group (Figs. 6A, B). These data demonstrate that SJTMF may regulate aortic autophagy and macrophage polarization through PI3K/AKT/mTOR.

FIGURE 5.:

SJTMF-induced regulation of aortic autophagy and macrophage polarization through the PI3K/AKT/mTOR signaling pathway. A, Western blotting of PI3K, AKT, mTOR, and phosphorylated protein expression in aortic plaques. B, Expression of M1 macrophage-associated markers in aortic tissues detected using qRT-PCR. C, Expression of M2 macrophage-associated markers in aortic tissues detected using qRT-PCR. D, Western blotting of autophagy-related proteins expression levels of LC3-II/I, beclin-1, and p62 in macrophages in aortic plaques. E, Fluorescence expression of LC3-II detected using immunofluorescence (×200). F, Fluorescence expression of beclin-1 detected using immunofluorescence (×200). G, Fluorescence expression of p62 detected using immunofluorescence (×200). ##P < 0.01 versus control group, **P < 0.01 versus model group, *P < 0.05 versus model group, ^^P < 0.01 versus LY294002 group. n = 10.

FIGURE 6.:

A, Fluorescence expression of CD86 was detected using immunofluorescence (×200). B, Fluorescence expression of CD206 was detected using immunofluorescence (×200).

DISCUSSION

Although the pathogenesis of AS remains unclear, previous studies have implicated dyslipidemia, endothelial dysfunction, oxidative stress, foam cell formation, apoptosis and necrosis, immune regulation disorders, and chronic inflammation of vascular walls as factors involved in AS.22,23 In this study, we investigated the involvement of SJTMF in inhibiting plaque formation, improving serum lipid levels, and fighting inflammation and found that SJTMF can promote macroautophagy, thereby affecting macrophage polarization and regulating the expression of autophagy markers. We also verified that SJTMF may play an antiatherosclerotic role by inhibiting the PI3K/AKT/mTOR signaling pathway to mediate macroautophagy.

SJTMF improved the levels of serum lipids and inflammatory cytokines in ApoE−/− mice and reduced the number of plaques, lipid deposition, and macrophage infiltration in the aortic tissue. Studies have found that AS is characterized by the formation of atherosclerotic plaques in the arterial intima, leading to stenosis or occlusion of the official lumen, which eventually leads to the occurrence of cardiovascular diseases.24 The lipids in the arterial intima can deposit with the appearance of arterial lesions and plaques. As a treatment strategy against AS, lowering lipids (especially limiting LDL-C) and reducing inflammation have shown efficacy,25 whereas targeted regulation of macrophage polarization is another potential therapeutic strategy. The proinflammatory cytokines secreted by M1 macrophages are involved in inflammatory response and can activate the differentiation of CD4+ T cells into different helper T-cell subsets,26 along with other immune cells, thereby amplifying inflammation. By contrast, M2 macrophages can inhibit the recruitment of inflammatory cells, promote the excretion of inflammatory cells, and accelerate the regression of inflammation.27,28 In this study, following SJTMF treatment, the mRNA expression of M1 macrophage-related genes significantly decreased, whereas that of M2 macrophage-related genes significantly increased, suggesting that SJTMF may reduce the inflammatory response by regulating the polarization of macrophages from the M1 to M2 subtypes.

The discovery of autophagy provides a new therapeutic target for the prevention and treatment of AS. Drugs can affect the mechanism of autophagy in the process of injury, thereby intervening in the process of AS or reversing the role of plaque. Macroautophagy is a “self-eating” behavior of most eukaryotic cells and is closely linked to lipid metabolism, oxidative stress, and the inflammatory response. A cross talk mechanism has been previously demonstrated between macroautophagy and M1/M2 polarization.29,30 Macroautophagy influences the development of AS.31 Autophagic cells formed during autophagy wrap cytosols and form autophagy lysosomes to degrade their contents.32 Phagocytosis of oxidized lipoproteins by macrophages can accumulate lipid droplets in cells, thus forming foam cells,33,34 whereas autophagy can promote lipid hydrolysis and cholesterol outflow of foam cells and effectively inhibit AS progression.35 In this study, compared with the model group, the autophagy corpuscles in macrophages in plaques following SJTMF treatment increased, lipid droplets decreased, the expression levels of autophagy-related proteins, LC3-II/I and beclin-1, significantly increased, and the expression level of p62 protein significantly decreased, suggesting that SJTMF promoted autophagy. SJTMF significantly improved the levels of blood lipids and proinflammatory factors in ApoE−/− mice following treatment with 3-MA, reduced the level of M1 macrophage-related genes, and regulated the expression of autophagy-related proteins. The above results suggest that SJTMF can effectively reverse the lipid metabolism disorder and inflammatory reaction caused by autophagy inhibition, which demonstrates that SJTMF promotes macroautophagy.

Previous studies have shown that the regulation of macroautophagy by means of the PI3K/AKT/mTOR pathway can effectively improve AS.36,37 PI3K, an upstream kinase of the mTOR pathway, plays an important role in immunity and inflammatory response.37 PI3K comprises 3 types, among which type I PI3K activates the main downstream molecules of AKT and mTOR to inhibit autophagy, whereas type III PI3K can enhance autophagy by inducing beclin-1.38 In this study, we selected a broad-spectrum PI3K inhibitor (LY294002) as a reference to determine whether SJTMF regulates macroautophagy through the PI3K/AKT/mTOR pathway and found that SJTMF significantly increased the LC3-II/I ratio and beclin-1 protein expression by inhibiting PI3K/AKT phosphorylation and mTOR activation, thus promoting autophagy.

SJTMF contains Salvia miltiorrhiza, leech, and Panax notoginseng, which can improve blood circulation. Previous studies have shown that S. miltiorrhiza has a protective effect on cardiovascular disease. Moreover, salvianolic acid B in S. miltiorrhiza can improve autophagy dysfunction by inhibiting the AKT/mTOR signaling pathway.39 Tanshinone IIA30 and S. miltiorrhiza extract40 have good antiatherosclerotic activity by regulating autophagy. Leech and its extract have also been used in the treatment of cardiovascular diseases in TCM and can significantly weaken the atherosclerotic lesion area of ApoE−/− mice by means of a mechanism that potentially involves the inhibition of monocyte invasion of the vascular wall and improvement of vascular endothelial function.41,42 In addition, various components in other Chinese herbal compounds, such as P. notoginseng saponins pns,43Poria cocos polysaccharide pcp,44 and Alisol B23 acetate,45 play an anti-AS role by reducing inflammation, inhibiting cholesterol synthesis, and regulating oxidative stress.

This study has some limitations. First, we did not clarify the potential molecular mechanism by which SJTMF regulates PI3K. In addition, the regulation of SJTMF on M1/M2 polarization of macrophages requires further investigation. In future research, we intend to explore other pathways and targets of SJTMF for the treatment of AS.

CONCLUSIONS

We demonstrated that SJTMF can reduce the serum lipid level of ApoE−/− mice and lipid deposition of atherosclerotic plaques. Our results also show that SJTMF is involved in the autophagy regulation of macrophages, thus inhibiting AS, possibly through the inhibition of the PI3K/AKT/mTOR signaling pathway. Therefore, our study suggests SJTMF as a promising new treatment strategy against AS. The mechanism of SJTMF in the treatment of AS remains to be elucidated to provide a basis for its clinical application and development.

REFERENCES