Anti-Atherosclerosis Effect of Angong Niuhuang Pill via Regulating Th17/Treg Immune Balance and Inhibiting Chronic Inflammatory on ApoE-/- Mice Model of Early and Mid-Term Atherosclerosis

Angong Niuhuang Pill (ANP) is a well-known patented Chinese medicine which is used for hundreds of years for treating the central nervous system diseases. Atherosclerosis is a poly-aetiological chronic inflammatory vascular disease. Preventing inflammation is fundamental for treating atherosclerosis in early stages. In this study, we investigated the protective effects and possible mechanisms of ANP action on a high-fat diet induced early and mid-term atherosclerosis ApoE-/- mice. The effects of ANP were compared with accepted drug simvastatin. Twelve male C57BL/6J mice were used as the control group, and 60 male ApoE-/- mice were randomly divided into five groups: Model group, Simvastatin group, Low-, Medium-, and High-dose ANP group these groups received, respectively, saline, simvastatin (3.0mg/kg), low-dose ANP (0.25 g/kg), medium-dose ANP (0.50 g/kg), and high-dose ANP (1.0 g/kg), once every other day for 10 weeks. After administration, serum biochemical indices were detected by the automatic biochemical analyzer, the concentrations of IL-6 and IL-10 in the serum were assayed by ELISA, expression levels of IL-1β, TNF-α, MMP-2, MMP-9, CCL2, and its receptor CCR2 in the full-length aorta, and expression levels of transcription factors Foxp3, RORγt in the spleen were assayed via western blotting and RT-qPCR. Flow cytometry was used to analyze Th17 cells and Treg cells. Pathological and histological analysis was completed on aortic root. ANP decreased LDL/HDL ratio, concentrations of IL-6 while increased IL-10 in serum. Moreover, ANP down-regulated the expression levels of IL-1β, TNF-α, MMP-2, MMP-9, CCL2, and CCR2 receptor in the full-length aorta. In addition, ANP decreased Th17 cells and expression levels of transcription factor RORγt, increased Treg cells and expression levels of transcription factor Foxp3. ANP decreased content of collagen fibers and infiltration of inflammatory cells in the aortic root. In conclusion, we demonstrated that ANP has anti-atherosclerosis effects on a high-fat diet induced ApoE-/- mice early and mid-term AS model via regulating Th17/Treg balance, inhibiting chronic inflammation, reducing plaque collagen fibers, and reducing inflammatory cells infiltration, to exert its multi-channel multi-target anti-early and mid-term AS effects.


INTRODUCTION
Cardiovascular diseases (CVD) and related chronic diseases are the number one cause of death. About 80% of CVD-related deaths occur in low-income and middle-income countries (Feigin et al., 2015). According to China Cardiovascular Disease Report 2017, the number of patients with CVD reached 290 million, and CVD deaths account for more than 40% of deaths. Since 2004, the average growth rate of CVD cost in China was much higher than the growth rate of GDP (Chen et al., 2018). The burden of China's CVD is increasing; it has become a major public health problem. It is well accepted that aterosclerosis (AS) is the primary cause of CVDs. Therefore, the search for effective therapy is of fundamental importance.
Pathogenesis of AS includes lipid infiltration, damage response, mononuclear macrophage invasion, and inflammation response. At present, it is generally accepted that AS is a chronic inflammatory disease (Ross, 1999), which involves cellular immune response, particularly of Th17 cells and CD4 + CD25 + regulatory T (Treg) cells (Xie et al., 2010;Wu et al., 2017). Treg and Th17 cells have recently been described as two distinct subsets distinct from Th1 and Th2 cells, while Th17/Treg balance is different from balance of Th1/Th2 (Frostegard et al., 1999;Cheng et al., 2008). Treg cells expressing the forkhead/winged helix transcription factor (Foxp3) and Th17 cells expressing retinoic acid-related orphan receptorgt (RORgt) are two important immune cells for controlling anti-atherogenic cytokines such as interleukin (IL)-10, transforming growth factor (TGF)-b1, and pro-atherogenic cytokines such as IL-6, IL-17 (Shimon et al., 2006;Bettelli et al., 2007). Above cytokines play important roles in maintaining the number and function of Treg cells and Th17 cells and participate in the pathogenesis of human AS. In previous study (Chai et al., 2019), we reported that the Th17/Treg imbalance existed in the ApoE -/mice AS model, which was induced by consecutive 8 weeks high-fat diet. Therefore, the Th17/Treg balance is critical for prevention of AS and autoimmune response.
Chronic inflammation represents the most basic pathogenetic factor of AS (Ross, 1999), Cytokines orchestrate the complex inflammatory response within the atherosclerotic plaque throughout the entire AS evolution (Tousoulis et al., 2016). At the early and middle stages of AS, endothelial dysfunction, secretion of chemokines and cytokines such as CCL2, IL-1b, TNF-a expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), accumulation of immune cells such as T lymphocytes, dendritic cells, macrophages to the AS plaques, and migration and proliferation of vascular smooth muscle cells (VSMCs) to these plaques further aggravate inflammatory response (Ramji and Davies, 2015). Furthermore, activation of matrix metalloproteinases (MMPs) instigates expansion and instability of atherosclerotic plaques (Ramji and Davies, 2015;Tousoulis et al., 2016). The expanding plaque once ruptured may cause arterial thrombosis, stenosis or blockage of the lumen, affecting the arterial supply of blood to organs and tissues, eventually leading to cerebral infarction, acute coronary syndrome, myocardial infarction, and peripheral vascular disease (Weber and Noels, 2011). In the previous study (Chai et al., 2019), we found that ApoE -/mice induced by 8 weeks high-fat diet demonstrate severe inflammatory response in the full-length aorta. Therefore, inhibiting this inflammatory response, reducing migration and adhesion of inflammatory cells, together with maintaining plaque stability are essential for the treatment of early and mid-stage AS.
Angong Niuhuang Pill (ANP) is a well-known patented Chinese medicine, used to treat stroke, encephalitis, and meningitis. Its main components include Bovis Calculus, Powerdered Buffalo Horn Extract, Artificial Moschus, Cinnabaris, Coptis chinensis Franch., Hyriopsis cumingii(Lea), Scutellaria baicalensis Georgi, Realgar, Gardenia jasminoides J.Ellis, Curcuma aromatica Salisb., and Borneolum Syntheticum (Editorial Committee of Pharmacopoeia of Ministry of Health PR China, 2015). The chemical structure of the known component in artificial Moschus were shown in Supplementary Material (Table 1). Pharmacological effects of ANP include: hepatoprotection, anti-inflammation, anti-viral action, antipyresis, and anticonvulsive effects. Our previous study also found that ANP has anti-atherosclerosis and cardio-protective effect on high-fat diet combined with vitamin D3 induced AS rats (Fu et al., 2017). In the present study, we combine the studies of Th17/Treg balance and inflammatory pathways to attempt to investigate the possible mechanisms of ANP relevant for treating the early and middle AS in the ApoE -/mice induced by 10-week high-fat diet (Nakashima et al., 1994;Gupta et al., 1997;Meir and Leitersdorf, 2004).
Cinnabaris, Hyriopsis cumi-ngii (Lea) and Realgar were grounded or pulverized to the very fine powders (200 mesh). Coptis chinensis Franch., Scutellaria baicalensis Georgi, Gardenia jasminoides J. Ellis, and Curcuma aromatica Salisb. were pulverized to a fine powder (100 mesh). Bovis Calculus, Powerdered Buffalo Horn Extract, Artificial Moschus, and Powerdered Buffalo Horn Extract were triturated with the above powders, sifted and well mixed. Refined honey was mixed to make 600 big honeyed pills, or alternately coated with a gold film (Editorial Committee of Pharmacopoeia of Ministry of Health PR China, 2015). HLPC was used to verify the formulation to guarantee the quality of the ANP, fingerprint of ANP please see the Supplementary Figure S1. The UPLC Fingerprint of 24 ANP Batches was shown in Supplementary Figure S3.

Model Establishment
After ten days of adaptive feeding, 60 SPF male ApoE -/mice were randomly divided into five groups, i) model group, ii) simvastatin group (3.0 mg/kg), iii-v) low-, ledium-, and highdose ANP groups (0.25 g/kg, 0.50 g/kg, 1.0g/kg, respectively) and given free access to high fat feed and tap water for 10 weeks. Each group contained twelve mice. Twelve SPF male C57BL/6J mice were used as controls and were given free access to standard laboratory mouse chow and tap water for 10 weeks. On the first day of high-fat diet administration, all experimental drug treatments (simvastatin and ANP) were administered intragastrically once every other day for 10 weeks. Drug dosages were titrated according to animal weight. Saline was given intragastrically in control group and model group. All drug treatments were prepared immediately before administration as a suspension by dissolving an appropriate amount of the drug in distilled water.

Sample Collection
After administration, serum was collected for biochemical analysis. The aortic root was separated for immunofluorescence staining and Masson staining. The full-length aorta was separated for Real-time quantitative polymerase chain reaction (RT-qPCR) and Western Blotting analysis. The liver, kidney, spleen, and thymus were separated for organ coefficient or organ index. Primary splenocytes were separated for flow cytometric analysis from the spleen.

Organ Coefficient and Index Analysis
Liver coefficient, kidney coefficient, thymus index, and spleen index were calculated according to corresponded calculation formula separately. The formula of liver and kidney coefficient: weight of liver and kidney (mg)/weight of animal (g). The formula of thymus and spleen index: weight of thymus and spleen (mg)/weight of animal (g)*10.

Flow Cytometric Analysis of Th17 and Treg Cells
The ratio of Th17 to Treg cells in splenocyte suspensions was analyzed by flow cytometry. The splenocyte suspensions were stimulated with 10 ml of Cell Activation Cocktail (with Brefeldin A) and mixed. After incubation at 37°C for 6 h, the sample was centrifuged, and the precipitate was suspended in 1 ml RBC. The suspensions were then centrifuged and the precipitate re-suspended in PBS twice. After antibody labeling of surface proteins, the sample was incubated in the dark for 20 min followed by centrifugation (1,500 rpm). The cells in the precipitate were then fixed with fixation buffer, followed by incubation in the dark for 20 min. The sample was again centrifuged and suspended in 1 mL Permeabilization Wash Buffer. The cells were then washed twice and labeled with an immunofluorescent antibody. After incubation in the dark for 20 min, the sample was washed using 1 ml Permeabilization Wash Buffer twice and suspended in PBS prior to flow cytometric detection.

Masson Staining
Masson staining was used to detect collagen fibers in the plaque of aortic root. According to kit for Masson staining manufacturer instructions, the aortic root fixed in 4% paraformaldehyde were dehydrated in alcohol, paraffin-embedded, sectioned and subjected to Masson staining. The stained sections were observed under the light microscope. Masson staining of the whole aorta was measured by Image Pro Plus 6.0. The area proportion of collagen fiber was calculated as the ratio of stained area to area of interest calculated by Image Pro Plus 6.0.

Immunofluorescence Staining
Immunofluorescence staining was used to detect inflammatory cells infiltration such as macrophages, DCs, and VSMCs in the plaque of aortic root. Anti-CD68, anti-CD11c, and anti-aSMA were respectively used as special marker in macrophages, DCs, and VSMCs. Frozen slices of aortic root were blocked in blocking buffer for 60 min, aspirated blocking solution, applied diluted primary antibody, incubated overnight at 4°C, rinsed three times in PBS for 5 min each, and then incubated specimen in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1-2 h at room temperature in the dark, rinsed in PBS, thereafter, coverslips stained with DAPI. The stained sections were observed under fluorescence microscope, and Image J was used to semi-quantitatively analyze positive cells.

Cytokines Detection by ELISA
The serum concentrations of IL-6 and IL-10 were measured using the IL-6 and IL-10 ELISA kits. In line with the manufacturer's instructions, standard, or sample (100 µl) was added to each well and incubated for 2 h at 37°C. Then, the medium in each well was discarded, and biotin antibody (100 µl) was added to each well, followed by incubation for 1 h at 37°C. The medium in each well was aspirated and the wells were washed three times. Thereafter, HRP-avidin (100 µl) was added to each well and incubated for 1 h at 37°C. The medium in each well was aspirated and the wells were washed five times, followed by addition of tetramethylbenzidine substrate (90 ml) and incubation for 20 min at 37°C in the dark. Finally, the stop solution (50 ul) was added to each well, and the absorbance of the wells was read at 450 nm within 5 min.

Real-Time Quantitative Polymerase Chain Reaction
The mRNA levels of Foxp3, RORgt in the spleen, as well as CCR2, CCL2, IL-1b, TNF-a, MMP-2, and MMP-9 in the fulllength aorta, were analyzed by RT-qPCR. Total RNA was extracted with Trizol reagent according to the manufacturer instructions from full-length aorta and spleen. The concentration and purity of RNA was analyzed using a micro-detector. Reverse transcription kit was used for reverse transcription reaction to synthesize cDNA, RT-qPCR was performed to determine mRNA levels of inflammatory mediators with the SYBR Green real-time PCR master mix kit. Each sample was analyzed in triplicate, normalized to GAPDH. All the PCR primers used are listed in Table 1. RT-qPCR conditions were 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec, 55°C for 30 sec, and 72°C for 60 sec.

Western Blotting Analysis
Protein levels of Foxp3, RORgt in the spleen, as well as CCR2, CCL2, IL-1b, TNF -a, MMP-9, and MMP-2 in the full-length aorta were detected by western blotting analysis. Total proteins were extracted from full-length aorta and spleen. The concentrations of protein were determined by BCA protein assay. Each sample containing 30 µg protein was separated, respectively, on 8%, 10%, and 15% SDS-PAGE gel electrophoresis and then transferred onto a polyvinylidene

Statistical Analysis
All data were performed as mean ± standard error of the mean (mean±SEM) and were analyzed by One-way ANOVA with Dunnett T3 test in SPSS Statistics 18.0. p < 0.05 were considered as statistically significant.

Body Weight, Liver and Kidney Coefficients
The body weight of ApoE -/mice and C57BL/6J mice over a 10week period was shown in Figure 1A and Table 2. The body weight of model group was higher than control group, and the body weight of model group was significantly increased at 3 rd , 5 th , and 10 th week (p < 0.05 or p < 0.01) compared to the control group. The body weight of ANP groups and simvastatin group was lower than model group, and a significant difference was noted at 5 th , 9 th , and 10 th week (p < 0.05 or p < 0.01), compared to the control group. Liver coefficient was significantly increased (p < 0.05) while kidney coefficient was significantly decreased (p < 0.01) in the model group. In medium-and high-dose ANP groups liver coefficient was significantly decreased (p < 0.01). Kidney coefficient was not altered in all ANP groups. These results were shown inFigure 1B-C.

Effect of ANP on Serum Concentrations of IL-6 and IL-10
Serum concentrations of IL-6 and IL-10 were shown in Figures  3A, B. When compared with control group, concentrations of pro-inflammation IL-6 were significantly increased (p < 0.05), and concentrations of anti-inflammation IL-10 were significantly decreased (p < 0.01). These data indicate indicated that ApoE -/mice of model group had a chronic inflammation at the early and mid-term AS. When compared with model group, medium-dose ANP significantly decreased concentrations of serum IL-6 (p < 0.01), and all ANP administrated groups significantly increased concentrations of serum IL-10.

Effect of ANP on Th17/Treg Balance in the Spleen
According to results of thymus index and spleen index ( Figures  4A, B), there was an upward trend in the ANP groups, and lowdose ANP significantly increased spleen index (p < 0.05). It suggests that ANP may affect the immune regulation of the spleen. In order to verify above results, splenocytes were used to analyze Th17/Treg balance in the spleen by flow cytometric analysis. In the model group, Th17/CD4 + T cells ratio, and Th17/ Treg ratio were significantly increased, however, Treg/CD4 + T cells ratio was significantly decreased (p < 0.05 or 0.01). After ANP administration, when compared with model group, it was shown that medium-dose ANP significantly decreased Th17/ CD4 + T cells ratio (p < 0.01), while low-dose and high-dose ANP significantly increased Treg/CD4 + T cells ratio (p < 0.01). It was also shown that all ANP administrated groups could decrease Th17/Treg ratio ( Figures 4C-G).

Effect of ANP on Expression of RORgt and Foxp3 in the Spleen
RORgt and Foxp3 are transcription factors for Th17 cells which regulate Th17/Treg balance. To evaluate effect of ANP on Th17/ Treg balance, expression levels of RORgt, and Foxp3 were detected in the spleen. In the model group, mRNA and protein expression levels of RORgt was significantly up-regulated (p < 0.05 or 0.01), and Foxp3 was significantly down-regulated (p < 0.05 or 0.01). After ANP administration, when compared with model group, it was shown that low-dose and high-dose ANP significantly down-regulated protein expression of RORgt (p < 0.05). Moreover, low-and medium-dose ANP significantly up-  Similarly, in all ANP groups mRNA expression levels of RORgt were down-regulated, while mRNA for Foxp3 was up-regulated (p < 0.05 or 0.01; Figures 5A-E).

Effect of ANP on Expression of Cytokines and Chemokines in the Aorta
Cytokines and chemokines play important roles in early and mid-term of AS and have a profound influence on the pathogenesis of AS. When compared to the control group, mRNA and protein expression levels of IL-1b, TNF-a, CCL2, CCR2 in the model group were significantly up-regulated (p < 0.05 or 0.01 or 0.001). In the medium-dose ANP mRNA and protein expression levels of IL-1b were significantly downregulated (p < 0.05), while in low-and medium-dose ANP mRNA and protein expression levels of TNF-a, CCL2, and CCR2 were also significantly down-regulated (p < 0.05 or 0.01). At the mRNA level, the anti-inflammatory effect of the ANP was better than the positive drug simvastatin. It is worth noting that low-and medium-dose ANP had better antiinflammatory effect, but there was no significantly difference in the high-dose ANP group. These results were shown in

Effect of ANP on Expression of MMP-2 and MMP-9 in the Aorta
MMPs are a group of proteases that degrade extracellular matrix, and they are mainly secreted by macrophages and VSMCs in atherosclerotic plaques. Overexpression of MMPs promotes the degradation of collagen and elastic fibers in plaque, destroying the stability of arterial plaque, which facilitate induction of cardiovascular risk events. As shown in Figures 8 A-E, both mRNA and protein expression levels of MMP-2 and MMP-9 in the model group were significantly up-regulated (p < 0.01 or 0.001). Administration of ANP significantly down-regulated mRNA and protein expression levels of MMP-2 and MMP-9 (p < 0.05 or 0.01).

Effect of ANP on Collagen Fibers in the Aortic Root
Masson staining was performed in the aortic root. The area of fibrosis which was stained blue covered a much larger area in the model group than in the control group (p < 0.01). Conversely, the area of collagen fiber in the medium-dose ANP were significantly lower than the model group (p < 0.05). These results were shown in Figures 9A, B.

Effect of ANP on Inflammatory Cells Infiltration in the Aortic Root
Immunofluorescence staining was performed in the aortic root. In the model group, the positive area of macrophages which were stained red was higher than in the control group (p < 0.01), and the positive area of VSMCs and dendritic cells, which was respectively stained green and red also was higher than that in the model group (p < 0.001). In low, or medium or high-dose ANP groups, the positive area of these inflammatory cells was significantly lower than in the model group (p < 0.01 or 0.001). These results were shown in Figures 10A, B and Figures 11A-C.

DISCUSSION
We found that treatment with ANP corrected the immune imbalance of Th17/Treg cells in the early AS ApoE -/mice model by regulating RORgt and Foxp3 expression; ANP suppressed the release of pro-inflammatory mediators, promoted the release of anti-inflammatory factors, downregulated the expression of chemokines and their receptors promoting thus an anti-inflammatory effects in the early AS. Furthermore, ANP down-regulated expression of MMP-2, Anti-Atherosclerotic ANP on ApoE -/-Mice MMP-9, reduce collagen fibers and decreased infiltration of macrophages, dendritic cells, and vascular smooth muscle cells into plaques thus facilitating stability of the latter. In this study, ANP was a kind of tawny powder, and distilled water was used as the solution medium. Due to the relatively complex composition of ANP, ANP was tawny suspension after dissolution. In order to maintain the stability of drug suspension, the ANP suspension was prepared before administration. In our next study, we will focus on the metabolic components of ANP in mice and the pharmacodynamic substance basis of ANP, and analyze the prescription.

Fan et al.
Anti-Atherosclerotic ANP on ApoE -/-Mice that LDL-C/HDL-C ratio in the high-dose ANP group (1.0 g/kg) was significantly lower than those in the model group. However, low-dose and medium-dose ANP groups did not show a decrease in LDL-C/HDL-C ratio, which indicates that only a high-dose ANP has a weak lipid-lowering effect.
The high-fat diet is a potential risk factor for the liver and kidney. Treatment with ANP significantly reduced the liver coefficient but did not improve the kidney coefficient; thus pointing on a certain hepatoprotective effect of ANP. This agreed with previous findings showing that ANP has a preventive and therapeutic effect on liver damage caused by bacterial toxins (He et al., 1992). The apparent changes of spleen index and thymus index suggest that ANP may have a regulatory effect on the immune system. The Th17/Treg is a pair of new balance which differs from Th1/Th2 balance in the immune system and plays an important role in the development of AS and plague rupture (Cheng et al., 2008;Xie et al., 2010). Many studies have confirmed that regulating Th17/Treg balance is a critical intervention target during development process of AS (Xie et al., 2010;Konstantinos et al., 2014;Tian et al., 2017;Wang et al., 2017). Treatment  Anti-Atherosclerotic ANP on ApoE -/-Mice with ANP corrected the Th17/Treg cellular balance, with high doses of ANP having more pronounced immunomodulatory effect. The Th17/Treg balance is regulated by RORgt and Foxp3 respective transcription factors (Xie et al., 2010). We found that ANP significantly down-regulated expression of RORgt, while significantly up-regulating expression of Foxp3; indicating the underlying mechanisms. Chronic inflammation is the key factor in AS pathogenesis, while inhibiting inflammatory response controls AS evolution (Tousoulis et al., 2016;Chistiakov et al., 2018). Inflammatory cells involved in the AS process can express different inflammatory factors, such as IL-1b, TNF-a, and IL-6, which are mainly secreted by macrophages, lymphocytes, and VSMCs (Tedgui and Mallat, 2006). Expression of IL-1b and TNF-a is regulated by the p38 MAPK/NF-kB pathway (Chan et al., 2000). Increased expression of IL-1b and TNF-a promotes overexpression of downstream ICAM-1 and VCAM-1, to further promote migration and adhesion of VSMCs and endothelial cells (Mackesy and Goalstone, 2014). Expression of IL-6 is regulated by IL-6 receptor and signal transducing protein gp130 (Ait-Oufella et al., 2011); IL-6 being a pro-atherogenic cytokine exacerbates the progression of atherosclerosis in ApoE -/mice. Clinical studies revealed that high levels of serum IL-6 represent a risk factor for coronary artery disease (Biasucci, 1999). The anti-atherogenic cytokine IL-10 is secreted by Treg cells; IL-10 not only down-regulates expression of TNF-a, but also reduces expression of ICAM-1 in endothelial cells (Lisinski and Furie, 2002;Rajasingh et al., 2006). Treatment with ANP significantly reduced serum IL-6 and increased serum IL-10, although the dose-dependence was not obvious. Expression of TNF-a was significantly down-regulated in low-dose and medium-dose ANP groups, and expression of IL-1b was significantly down-regulated in low-dose ANP group. Thus ANP (especially at low and medium doses) exhibits antiinflammatory capabilities.
Atherosclerotic plaque, the morphological substrate of AS, is induced by multiple factors, including accumulation of VSMCs, macrophages and T lymphocytes (Andrés et al., 2012;Moore et al., 2013); the proliferation of VSMCs and connective tissue matrix such as collagen fibers and elastic fibers; deposition of cholesterol crystals and free cholesterol. All these factors contribute to formation of fragile arterial plaques (Watson et al., 2018). Maintaining stability of plaques and arresting expansion of vulnerable plaques are in the focus of current treatment for AS which has already formed. It was reported that CCL2, CCR2, MMP-2, and MMP-9 are involved in impairing stability of arterial plaques, reduction in expression of plaques stability (Ishibashi et al., 2004;Krohn et al., 2007;Zhao, 2010;Cipriani et al., 2013). We found that ANP significantly down-regulated expression of CCL2, CCR2, MMP-2, and MMP-9. Masson staining showed that mediumdose ANP decreased plaques area in the aortic root when compared to the model group, similarly reduced was the content of collagen fibers. Treatment with ANP also reduced infiltration of macrophages, vascular smooth muscle cells and dendritic cells into the aortic root as evidenced by immunocytochemical analysis. These results indicate that ANP may improve the stability of arterial plaques and prevent their expansion, suppressing infiltration of inflammatory cells, and decreasing expression of CCL2, CCR2, MMP-2, and MMP-9. All these findings suggest that, ANP may exert an anti-atherosclerotic effect at the early and mid-term of AS.

CONCLUSION
In conclusion, ANP protects early and mid-term atherosclerotic ApoE -/mice by regulating Th17/Treg balance, inhibiting chronic inflammation, reducing plaque collagen fibers, and reducing inflammatory cells infiltration. Moreover, ANP has anti-inflammatory effects in small doses, and ANP has immunoregulation effects on high doses. Possible mechanisms are shown in Figure 12.