− mice

Journal Pre-proof Inflammation inhibition and gut microbiota regulation by TSG to combat atherosclerosis in ApoE−/− mice Fengjiao Li, Ting Zhang, Yanran He, Wen Gu, Xingxin Yang, Ronghua Zhao, Jie Yu PII:

S0378-8741(19)31574-0

DOI:

https://doi.org/10.1016/j.jep.2019.112232

Reference:

JEP 112232

To appear in:

Journal of Ethnopharmacology

Received Date: 20 April 2019 Revised Date:

20 August 2019

Accepted Date: 11 September 2019

Please cite this article as: Li, F., Zhang, T., He, Y., Gu, W., Yang, X., Zhao, R., Yu, J., Inflammation inhibition and gut microbiota regulation by TSG to combat atherosclerosis in ApoE−/− mice, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112232. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Inflammation Inhibition and Gut Microbiota Regulationof TSG on Atherosclerosis in ApoE-/- Mice, Fengjiao Li, Ting Zhang, Yanran He et al. JEP_112232

Inflammation Inhibition and Gut Microbiota Regulation by TSG to combat Atherosclerosis in ApoE-/- Mice

Fengjiao Li, Ting Zhang, Yanran He, Wen Gu, Xingxin Yang, Ronghua Zhao, Jie Yu* Yunnan University of Chinese Medicine, Kunming, 650500, Yunnan Province, China *

Corresponding author:

Jie Yu, Tel.: +86-8710-65933303; E-mail address: [email protected]

Abstract Ethnopharmacological relevance: 2,3,5,4’-Tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG) is the main active component of Polygoni Multiflori Radix, a root of the homonymous plant widely used in traditional Chinese medicine. TSG has protective effects on the liver, reduces cholesterol and possesses anti-oxidant, anti-tumor, and anti-atherosclerotic properties. However, the pharmacological effects and mechanisms of action of Polygonum multiflorum on atherosclerosis (AS) have not been studied yet. Purpose: The aim of this research was to study the effects of Polygoni Multiflori Radix Praeparata (PMRP) and its major active chemical constituent TSG on AS in ApoE-deficient (ApoE-/-) mice fed with high fat diets to provide a scientific basis in the use of PMRP and TSG against cardiovascular diseases. Methods: High fat diet induced AS in ApoE-/- mice were treated with PMRP, TSG (low and high doses), and simvastatin (SIM) for 8 weeks. At the end of the treatment, mouse serum lipid levels, triglycerides (TG), and total cholesterol (TC) were

measured by an oxidase method (other indicators were determined by ELISA), while the content in oxidized low density lipoprotein (ox-LDL) and the expression of inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemotactic protein-1 (MCP-1) in the serum and aortic samples were measured by ELISA. Atherosclerotic plaque morphology was evaluated by oil red O in thoracic aorta. In addition, 16S rDNA-V4 hypervariable region genome sequence of all microbes in the fecal sample from each group was analyzed to evaluate potential structure changes in the gut microbiota after treatment with PMRP and TSG. Results: TSG markedly inhibited AS plaque formation in ApoE-/- mice. Furthermore, PMRP and TSG improved lipid accumulation by reducing TG and ox-LDL levels. TSG inhibited inflammation by the down-regulation of IL-6, TNF-α, VCAM-1 and MCP-1 expression in serum, and PMRP inhibited inflammation by reducing VCAM-1, ICAM-1 and CCRA expression in aortic tissue. In addition, TSG reduced or prevented AS by the regulation of the composition of the overall gut microbiota, such as Firmicutes, Bacteroidetes, Tenericutes, Proteobacteria phyla, Akkermensia genera and Helicobacter pylori. Conclusion: PMRP and TSG improved lipid accumulation and inflammation, and regulated the intestinal microbial imbalance in ApoE-/- mice. TSG exerted a preventive effect in the development and progression of AS.

Keywords: Polygoni Multiflori Radix Praeparata; 2,3,5,4’-Tetrahydroxy-stilbene2-O-β-D-glucoside; Atherosclerosis; ApoE-/- mice; Inflammation; Gut microbiota

1. Introduction Arteriosclerotic vascular disease, usually termed as atherosclerosis (AS) in short, is a progressive disease characterized by the accumulation of lipids and fibrous

elements in the large arteries. AS is the leading cause of death and serious illness in developed countries and it is becoming a global health problem (Shi et al., 2014; Chinese Society of Endocrinolog., 2016; Guo et al., 2016). In 1970s and 1980s, the lipid metabolism disorder and proliferation of smooth muscle cells were considered as the major causes of AS. In these years, plenty of experimental and clinical relationships between hypercholesterolemia and atheroma were reported. In recent decades, a prominent role of inflammation in AS has been underlined. The current opinion that both inflammation and immune response contribute to atherogenesis aroused people's interest (Jiang 2016; Wang et al., 2009; Zhang et al., 2013; Liu et al., 2015; GE Jun-bo et al., 2013). AS is characterized by the recruitment of monocytes and lymphocytes into the artery wall. The entry of leukocytes into the artery wall is mediated by adhesion molecules and chemotactic factors, such as P-selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1). This process is triggered by the accumulation of oxidized LDL (ox-LDL). The understanding of the underlying mechanisms related to inflammation in the pathogenesis of AS raises questions and opens opportunities in the prevention and therapy of this disease. In a broader perspective, recent studies showed that change in the composition of the intestinal flora is also a crucial factor in the pathogenesis of cardiovascular disease. Metagenome data revealed that relative abundance of Collinsella genus in the gut of AS patients are higher than the abundance in healthy people, while that of Roseburia and Eubacterium are higher in the healthy ones (Karlsson et al., 2015). In addition, the amount of Actinobacteria in the intestine of children with type 1 diabetes has a significant increase, while the ratio of Firmicutes/Bacteroidetes decreased (Murri et al., 2013). Previous studies showed that the relative amount of Akkermansia genus can effectively improve the metabolic syndrome (Shin et al., 2014; Cani et al., 2014; Everard et al., 2013). Obese ossabaws have a significantly lower abundance of the genus Prevotella and higher abundance of Clostridium in their colon than the lean

ossabaws (Pedersen et al., 2013). Helicobacter pylori infection is highly correlated with AS incidence, thus, facilitating its occurrence (Li et al., 2013). Recent studies revealed that trimethylamine-N-oxide (TMAO) is also involved in the relationship between intestinal microbiota and adverse cardiovascular events in humans. Trimethylamine (TMA) is generated from choline by gut microbial metabolism. Once TMA is absorbed by the host, the liver converts it to TMAO. Increased TMAO in serum promotes the risk of AS (Romano et al., 2015; Tang et al., 2013). Some gut microbes belonging to both Firmicutes and Proteobacteria phyla, such as Anaerococcus hydrogenalis, Proteu spanner and Providencia rettgeri, are capable to produce TMA from choline, as shown by in vitro and in vivo studies (Romano et al., 2015). These gut microbiota species are all probably contributed to the occurrence of AS. Polygoni Multiflori Radix Praeparata (PMRP) is the dry root of Polygonum multiflorum Thunb. widely used in the traditional Chinese medicine as an excellent crude drug in the treatment of hyperlipemia (Commission of Chinese Pharmacopoeia, 2015). TSG is the main active chemical constituent of PMRP, which possesses a remarkable lipid regulation effect, as demonstrated by our studies (Wang et al., 2012; Yu et al., 2014; Lin et al., 2015; Lin et al., 2017) and some of other scientists (Fang et al., 2005; Liu et al., 2007; Wang et al., 2008; Wang et al., 2008; Xu et al., 2014). In addition, TSG showed high antioxidant and free radical scavenging effects (Wang et al., 2008; Wang et al., 2008). More interestingly, TSG possesses specific properties that can be potentially useful against AS. TSG inhibits VEGF expression, as shown by both in vitro (Xu et al., 2014) and in vivo (Wang et al., 2008) studies, Furthermore, TSG could up-regulate the eNOS mRNA, while down-regulate the iNOS mRNA expression. Moreover, TSG revealed beneficial effects on blood lipid and non-alcoholic fatty liver disease (NAFLD) as shown in our previous studies (Lin et al., 2017; Li et al., 2012; Lin et al., 2014; Wang et al., 2014). Therefore, the aim of this work was to evaluate the effect and mechanism of action of TSG on atherosclerotic plaques via inflammation inhibition and gut microbiota regulation in AS-prone

ApoE-/- mice.

2. Materials and methods

2.1 Chemicals and reagents

Polygoni Multiflori Radix (PMR) was collected by the authors from the Luquan County of Yunnan Province in China. The plants were identified as the root of P. multiflorum Thunb. by Prof. Ronghua Zhao, Yunnan University of Traditional Chinese Medicine. Some samples were deposited in the Herbarium of Pharmacognosy, Yunnan University of Traditional Chinese Medicine. PMPR was processed by block soybean decoction according to the procedure recorded in the Chinese Pharmacopoeia (Commission of Chinese Pharmacopoeia, 2015) TSG was purchased from Nanjing (Jingzhu Bio-technology Co., Ltd., China). The purity was at least 98%. Simvastatin (SIM, Hangzhou MSD Pharmaceutical Co., Ltd., China) was used as the positive control. Normal diets were provided by Suzhou (Shuangshi Laboratory Animal Feed Science Co., Ltd., China), while cholesterol and fat were purchased from Beijing (Biotopped Science & Technology Co., Ltd., China) and Sichuan (Green Land Oil Co., Ltd., China), respectively.

2.2 Water extraction of Polygoni Multiflori Radix Praeparata

Hundred grams PMRP power was decocted with water (1000 ml, 800 ml, 600 ml) for 3 times, respectively, each time for 1 h. These water extractions were combined, concentrated, and lyophilized. The final extraction rate of PMPR was 41.08% of crude drug.

2.3 Animals and diets

Eight-week-old male ApoE-/- mice were provided by the Vital River Laboratory Animal Technology Co. Ltd., Beijing, China. Mice in one group (n=7) were housed in a stainless-steel rat cage containing sterile rice husks and left in a ventilated animal room. All mice were acclimated in a controlled environment (temperature 22±1 °C, 60±10% humidity and a 12 h/12 h light/dark cycle) with free access to water and a high fat diet containing 0.15% cholesterol, 21% fat and 78.85% basic feed. The study was performed in compliance with the animal experimental ethics committee of Yunnan University of Traditional Chinese Medicine (R-062014014). All reasonable efforts were made to minimize animal suffering.

2.4 Animal groups and treatment

Thirty-five male ApoE-/- mice were randomly assigned to 5 groups. The model group (MOD) received distilled water daily (0.01 ml/g), the other groups received water extraction of PMRP (PMPR, 1.125 mg/g per day), low dose TSG (TSG.L, 0.035 mg/g per day), high dose TSG (TSG.H, 0.07 mg/g per day) and SIM (0.0025 mg/g per day). All mice were fasted for 2 h each day before administration. Body weight was recorded every three days, while food intake was measured every day till the end of the experiment.

2.5 Evaluation of atherosclerotic lesions

Samples of the proximal thoracic aorta were collected, and adherent connective tissue was removed and cleaned. Then, thoracic aorta samples were longitudinally open and stained with oil red O. The atherosclerotic plaque area was quantified by

analyzing the open luminal surface image of the formalin-fixed aortic arch and thoracic aorta.

2.6 Lipid and lipoprotein detection in the serum

Retro-orbital blood samples (0.8-1.2 mL) were collected at two hours after administration of the therapeutic agents in the morning, at the end of the 8th week. Serum was centrifuged at 12000 g and 4 °C for 15 min and immediately analyzed. Triglyceride (TG), total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels in serum were determined by enzymatic colorimetric method using commercial assay kits (Biosino Bio-technology and Science Inc., China). Concentrations of oxidized low density lipoprotein (ox-LDL) were evaluated using assay kits purchased from Cusabio (Biotech Co., Ltd, China).

2.7 IL-6, TNF-α, VCAM-1 and MCP-1 detection in the serum

As pro-inflammatory or risk factors of AS, serum interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1(MCP-1) contents were measured. These indexes were analyzed at the end of the 8th week by assay kits purchased from Cusabio (Biotech Co., Ltd, China). The blood sample was treated using the same procedure described in 2.6. All bioassays were carried out in duplicate.

2.8 Inflammatory factors detection in the aortic tissue

At the end of the experiment the ApoE-/- mice were sacrificed by intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight). The aortic samples were collected, precisely weighed and homogenized with phosphate buffer solution (PBS) and then stored overnight at -20 °C. Two freeze-thaw cycles were performed to break cell membranes, and the homogenates were then centrifuged for 15 minutes at 12000 g and 4 °C. The supernatant was collected for biochemical analysis. VCAM-1, intercellular cell adhesion molecule-1 (ICAM-1), C-C chemokine receptor type 2 (CCR2) and ox-LDL concentration in the aortic homogenate samples were determined by the correspondent assay kits purchased from Cusabio（Biotech Co.,Ltd, China）.

2.9 Sequencing of the V4 region 16S rDNA gene in mice feces.

Feces samples of each mouse were collected at the last day and placed in a sterile centrifuge tube. They were then evenly grinded on liquid nitrogen and stored at -80 ℃ for further analysis. All fecal samples of the same group were carefully blended and grinded until reaching a fine powder. DNA was extracted according to the instruction in the stool DNA kit (Omegea Bio-tek, USA). To determine the diversity and composition of the bacterial community in each feces sample, the protocol described in Caporaso et al. was used (Caporaso et al., 2015). PCR amplification was performed using the 515f/806r primer set that amplifies the V4 region of the 16S rDNA gene. The primer set was selected due to its exhibited few biases and could yield accurate phylogenetic and taxonomic information. The reverse primer contained a 6-bp error-correcting barcode unique to each sample. DNA was amplified according to the protocol previously described (Magoč et al., 2011). The sample was sequenced using an Illumina MiSeq platform.

2.10 Statistical Analysis

Statistical analysis was performed using SPSS software (13.0 for Windows; SPSS, Chicago, IL, USA). One-way analysis of variance (ANOVA) was performed when multiple group comparison was carried out. Results were expressed as mean ± SD. Results were classified into three significant levels using the p value of 0.05, 0.01 and 0.001. Results were classified into one significance level using the p value of 0.05. Graphics were presented using Origin 6.1. A 95% confidence interval (CI) was used as the threshold to identify potential outliers in all samples, clustering was analyzed by the software of TMEV Clustering.

3. Results 3.1 Basic pharmacological indicators in experimental animals

The initial mice body weight (24±1g) and the daily food intake were similar among all groups. Body weight in PMRP group increased to 28% after 8 weeks of high fat diet (Fig. 1A). Food intake also increased, and the highest growth rates were observed in the TSG.L group, approximately 58.8% (Fig. 1B). No significant difference was observed in both body weight and average food intake among all groups. Both TSG and SIM did not affect mice appetite and body weight gain. As shown in Fig. 1C, liver, lung and kidney indexes also displayed no significant changes among all treatment groups, while spleen indexes in TSG.L, TSG.H and SIM groups slightly increased (p<0.05), probably because of the mobilization of the immune system in the mice of these groups.

3.2 Effect of TSG on serum and aortic lipids in ApoE-/- mice

TG, TC, LDL-C and ox-LDL content in serum and aortic tissue of ApoE-/- mice are listed in Table 1. Serum TG in PMRP group (p<0.05) and of the groups of both the TSG doses (p<0.01) was significantly lower compared with MOD. TSG.H reduced TG to about 30%, and this effect was more remarkable than the one exerted by SIM. Because of the knockout of both alleles of the ApoE gene, serum TC and LDL-C content in ApoE-/- mice greatly differed from normal C57BL/6J mice. TC content in normal mice serum is approximately 2.41-3.63 mmol/L (Miao et al., 1997). In our study, TC content increased to approximately 30 mmol/L in the MOD group. Unfortunately, neither PMRP nor TSG inhibited this increase. Fortunately, PMRP and TSG.H reduced serum ox-LDL level by 59.34% and 29.25% compared with the model group, and TSG (p<0.05) and SIM (p<0.01) were the most significant. In addition, TSG.H exerted a remarkable inhibition on the ox-LDL/total serum LDL ratio. This result indicated that TSG.H prevented oxidation of serum LDL and the effect was similar to SIM used as a positive control. Therefore, TSG blocked the oxidation of serum LDL, which is critical in the prevention and treatment of AS. However, aortic ox-LDL content was not well controlled by TSG, potentially suggesting that accumulated ox-LDL could not be easily deleted in atherosclerotic plaques.

3.3 Effect of TSG on inflammatory mediators in the serum and aortic sample of ApoE-/- mice

As show in Fig. 2A, TSG.H and SIM significantly decreased serum IL-6 level (p ＜0.05) compared with MOD. PMRP and TSG.L also inhibited the expression of IL-6, although no significant difference was observed. None of the above-mentioned treatments altered the expression of TNF-α to a certain extent compared with MOD,

(Fig. 2B). PMRP and TSG.H Treatment reduced serum VCAM-1 level by 29.37% and 1.24%, respectively, compared with MOD (Fig. 2C). In addition, TSG.H inhibited serum MCP-1 expression, although without significant difference (Fig. 2D). As a result, both PMRP and TSG might suppress the typical inflammatory mediators in the serum of the ApoE-/- mice, with a more remarkable effect by TSG.H. Treatment with PMRP, TSG.L and SIM significantly reduced VCAM-1 concentration by 33.79%, 38.79% and 35.33% respectively, in the aortic sample compared with MOD. In addition, our results showed that aortic ICAM-1 and CCR2 were significantly reduced in the PMRP group (4.224±0.075 ng/mg and 1.733± 0.238 pg/mg, respectively) compared with the model group (7.233±1.524 ng/mg and 2.613±0.256 pg/mg, respectively) (Fig. 3). In conclusion, PMRP and TSG might suppress the typical inflammatory mediators in aorta of ApoE-/- mice.

3.4 Effect of TSG on gut microbiota of ApoE-/- mice

Biodiversity and balance of gut microbiota are critical in the occurrence and development of AS. Therefore, the alteration of gut microbiota at the phylum, genus and species level after PMRP and TSG treatment deserved our attention. Results of the microbial classification at the level of phylum showed that the amount of Bacteroidetes and Firmicutes in the model group were 85.38% and 9.82%, respectively, while, the amount of Bacteroidetes was significantly reduced after the treatment with PMRP (68.77%), TSG.L (55.83%) and TSG.H (81.10%). The amount of Firmicutes was significantly increased to 21.2%, 16.3% and 11.9% after the treatment with PMRP, TSG.H and SIM, respectively. Therefore, PMRP and TSG.H increased the ratio of Firmicutes/Bacteroidetes to 30.8% and 20.1%, respectively, which was only 11.5% in the model group. Furthermore, the amount of Proteobacteria and Tenericutes, which are usually considered as pathogens, increased to 4.38% and

0.10% in the model group. However, TSG.L, TSG.H and SIM treatment reduced the amount of both Proteobacteria (1.28%, 1.20% and 1.23%, respectively) and Tenericutes (0.067%, 0.068%, 0.070%, respectively) (Fig. 4A/B). These results suggested that PMRP and TSG could raise the ratio of Firmicutes/Bacteroidetes to relieve AS, and TSG could also decrease Proteobacteria and Tenericutes in intestinal, thus exerting a therapeutic effect. A study indicated that Helicobacter pylori is a pathogenic microorganism of highly relevance for AS (Li et al., 2013). In our study, the amount of this bacterium in the model group was 1.03%. Interestingly, TSG.L, TSG.H and SIM treatment reduced the amount by 99.67%, 99.65% and 99.34% (Table 1). These results indicated that PMRP and TSG alleviated AS by raising the abundance of Akkermansia and reducing the amount of Helicobacter pylori (Table 2). Cluster analysis results of the genus level showed that the structure of gut microbiota after TSG treatment was quite similar to the positive control group. The fartherst distance was observed between TSG group and MOD group, indicating that TSG regulated intestinal microbiota in atherosclerotic mice (Fig. 5).

4. Discussion Both systemic inflammation and gut microbiota can influence lipid metabolism and act as environmental factors triggering the development of metabolic and cardiovascular disease. Inflammation significantly contributes to the pathogenesis of obesity, insulin resistance, and AS. Different intestinal microbiota may have both proand anti-atherogenic effects. ApoE is an important component of plasma lipoproteins and plays an important role in lipid transport and metabolism. ApoE-/- mice have become one of the most important animal models and well recognized for studying the pathogenesis of AS in recent years (Zhang et al., 2012). Control groups were not necessary for comparison to show that the model was successful (Li et al., 2019; Wang et al., 2019; Li et al., 2015; Zhang et al., 2010). It is therefore accepted that AS

is developed after 8 weeks of high fat diet and its pathological characteristics are very similar to those of human being. In this study, the anti-atherogenic effect of PMRP and its major component TSG was evaluated for the first time. Although previous works already reported that 2, 3, 4’, 5-tetrahydroxystilbene-2-0-β-D glycoside attenuates AS in ApoE-/- mice (Fruchart et al., 2004), the detailed research design and protein quantification in these two studies are different. The atherosclerotic plaques stained by oil red on in the aorta of the MOD group were orange-red, suggesting that the high-fat feeding ApoE-/- mice were successful in developing AS. The results showed that PMRP and TSG reduced both lipids (TG, ox-LDL) and inflammatory mediators (IL-6, TNF-α) concentration, decreased the related protein (MCP-1, CCR2) and adhesion molecules (VCAM-1, ICAM-1) in the serum and aortic tissue. In addition, in aortic samples, AS-associated protein concentration of VCAM-1, ICAM-1, and CCR2 was measured, therefore, no sufficient aortic sample amount remained to carry out do Oil Red O staining or further integrated statistical analysis. However, we could also observe the basic situation in a limited number of samples. According to your suggestion, the figure below is included in the manuscript as a supplementary material (Fig. S1). More mechanisms will be investigated and more solid evidence including Oil Red O staining will be provided in our future research. TSG also remarkably inhibited plaque formation and alleviated the development of AS in ApoE-/- mice. Interestingly, PMRP and TSG also played an important role in the regulation of the intestinal microbiota balance. An early study suggested that AS is associated with lipid accumulation and smooth muscle proliferation. TG and TC are risk factors in the development of AS (Fruchart et al., 2004). A review proposed that currently, the recommended blood biomarkers for risk prediction are LDL-C, HDL-C and TG. However, high level of serum TC cannot be a risk prediction, because more than half of the vascular events occur in individuals with lower TC concentration compared to normal level (Packard et al., 2008). Our study found that PMRP and TSG effectively inhibited the increase

of TG in the blood. These results were similar with that of Shenab et al. (Shen et al., 2016), who observed that a high-cholesterol diet significantly increases the classic AS blood markers (LDL, CHO, TG), while treatment with omega-3 fatty acid attenuates blood lipids. Ox-LDL plays an important role in the development and progression of AS (Wang et al., 2013). Many laboratory and clinical results suggest that LDL modified by oxidation evokes an inflammatory response in the artery wall, triggering many of the biological processes participating in AS initiation, progression and complication (Packard et al., 2008). In the present study, both PMRP and TSG significantly reduced the level of ox-LDL, which was increased in the model group. We hypothesized that PMRP and TSG could block the oxidation of LDL, which is critical in the prevention and treatment of AS. Experimental models have indicated that inflammation and oxidative stress, which mutually amplify each other within the vasculature and in the visceral fat, are key processes driving the initiation, progression and subsequent rupture of atherosclerotic plaques. One endothelial–leukocyte adhesion molecule emerged as a particularly attractive candidate for the early adhesion of mononuclear leukocytes to arterial endothelium at sites of atheroma initiation (Libby., 2002). One study showed that at an AS early stage, ox-LDL induces the expression of MCP-1 in vascular smooth muscle cells and endothelial cells (Hajjar et al., 2013). MCP-1 is a typical inflammatory factor that promotes the occurrence of AS by mediating the monocyte migration (Hu et al., 2011). CCR2, a receptor for MCP-1, plays an important role in the development of AS. A study showed that the combination of CCR2 and MCP-1 promotes the transfer of mononuclear cells to the plaque (Apostolakis et al., 2013). Other scientific works (Nguyen., 2019; Grancieri., 2019; Moss., 2019; Ye., 2005) also concluded that the formation of AS is closely related to VCAM-1, ICAM-1 and CCR2 protein.

An appropriate investigation of inflammatory factors in the aortic tissues might provide more information in explaining the mechanism. Therefore, we used both the aortic tissues and serum as samples for the detection of some inflammatory factors. Since VCAM-1, ICAM-1 and CCR2 are adhesion proteins, they were detected in the aortic tissue (Fig 3). Other inflammatory factors, such as IL-6, TNF-α, VCAM-1 and MCP-1 were detected in the serum (Fig 2). We were surprised to find that PMRP, TSG.L and SIM significantly reduced VCAM-1 concentration in the aortic tissue (Fig. 3A). Also, TSG.H induced a decrease, although not significant. It is speculated that PMRP, TSG.L and SIM could inhibit the development of AS by significantly decreasing VCAM-1 protein expression in the aorta. In addition, ICAM-1 and CCR2 protein expression in the aorta of the PMRP group was significantly reduced compared with the MOD group, while TSG and SIM did not reduce the expression of these proteins (Fig. 3B, C). Thus, we believe that PMRP might alleviate the development of AS by reducing the expression of ICAM-1 and CCR2 in the aortic tissue of AS mice. In addition, the reduction of ICAM-1 and CCR2 level in the aorta was not the mechanism of the effect of TSG and SIM on AS. In summary, we concluded that PMRP might produce anti-AS effects by down-regulating of VCAM-1, ICAM-1 and CCR2 protein expression in the aorta. TSG might inhibit the development of AS by down-regulating VCAM-1 protein expression in the aorta. These results were similar and consistent with those of Ishibashi et al. who found that pioglitazone affects MCP-1/CCR2 pathway by inhibiting the expression of MCP-1 receptor CCR2 in circulating monocytes, ultimately inhibiting the development of AS. It is widely recognized that AS is associated with vascular inflammation. Studies demonstrated that irisin significantly reduces AS in ApoE-/- mice via suppressing ox-LDL induced vascular inflammation and endothelial dysfunction (Zhang et al., 2016). Endothelial dysfunction is considered an important early event in the pathogenesis of AS, and the mechanisms that participate in endothelial dysfunction include oxidative excess and upregulation of adhesion molecules, such as VCAM-1

and ICAM-1 (Kong et al., 2014). Wan et al examined the antiatherogenic effect of ginsenosides in ApoE-/- mice and investigated the anti-angiogenic molecular mechanisms of Panax notoginseng saponins on human coronary artery endothelial cells in vitro. They found that Panax notoginseng saponins significantly reduces IL-6 and TNF-α serum level, and inhibits the expression of endothelia adhesion molecules, such as ICAM-1 and VCAM-1 induced by TNF-α (Wan et al., 2009). In the present study, IL-6, TNF-α, VCAM-1, and MCP-1 induced by high-fat diet in ApoE-/- mice were evaluated in blood samples and compared with the MOD group, resulting in a significantly reduced serum IL-6 level by TSG.H and SIM (Fig. 2A). However, the results of inflammatory factors such as TNF-α, VCAM and MCP-1 in serum showed that the concentration of these inflammatory factors was not reduced by PMRP and TSG administration (Fig. 2B, C, D). This result suggested that TSG.H might prevent AS by reducing serum IL-6 levels, indicating that TSG might show antiatherogenic activity through its lipid-lowering and anti-vascular inflammatory mechanism, at least in part. The potentially pathogenic bacteria involved in AS are often referred as some specific strains, such as Proteobacteria, Tenericutes and Helicobacter pylori. Jacques et al assessed the association between blood microbiota and incident of cardiovascular disease, and they found that Proteobacteria are directly correlated with the onset of cardiovascular diseases, with Proteobacteria as an independent marker (Amar et al., 2013). Yan et al. found that at the level of phylum, obese rats present a higher amount of Frimicutes and Tenericutes and less amount of Bacteroidetes compared to their lean counterpart (Yan et al., 2015). A study showed that Helicobacter pylori may be a risk factor of AS. It can infect macrophages, promote cell aggregation and expression of mucosal molecules, induce hyperhomocysteinemia and participate in the formation and development of AS (Liu et al., 2015). Ibrahim et al. investigated the carotid intima-media thickness and lipid parameters in Helicobacter pylori-positive and negative subjects. They found that the mean and maximum values of internal and common carotid intima-media thickness in

Helicobacter pylori-positive subjects are significantly higher than the ones in Helicobacter pylori-positive subjects. The levels of LDL-C, TC and TG in Helicobacter pylori seropositive patients were lower than the ones in controls. Therefore, they believe that Helicobacter pylori-positive infection may play a role in AS (Ibrahim et al., 2014). A series of studies explored the relationship between gut flora and obesity, showing that high fat induces an imbalanced ratio of Firmicutes versus Bacteroidetes in the gut of germ-free mice and humans (He et al., 2008). In our study, TSG significantly reduced the amount of Proteobacteria, Tenericutes and Helicobacter pylori, which are usually considered as pathogens. In addition, TSG increased the amount of Firmicutes and Akkermansia at the level of phylum and genus, respectively. A previous study found that Akkermansia muciniphila attenuates atherosclerotic lesions by ameliorating metabolic endotoxemia-induced inflammation through restoration of the gut barrier (Lin et al., 2016). These studies clearly support our findings. It is still controversial whether the imbalance among intestinal microorganisms is the cause of AS or whether their imbalance is caused by AS. Some studies (Jeffrey B et al., 2019; Yan et al., 2019) showed that scientists are aware of this important issue, and our research did not reach a conclusion yet. We believed that gut microbiota alteration was the cause but not the effect of TSG in its activity against AS because we observed intestinal microorganisms changes compared to our previous studies (Wang et al., 2012; Yu et al., 2014; Lin et al., 2015; Lin et al., 2017) with TSG. In these works we demonstrated that TSG regulates the relative ratio of Firmicutes, Bacteroidetes and Proteobacteri, thus improving intestinal microecology. TSG effectively increases Prevotella, [Prevotella], CF231 and Paraprevotella to the normal level. In addition, TSG reduces the relative amount of some genera of Firmicutes phylum, such as Collinsella, Faecalibacterium and Coprobacillus, and reduced Sutterella, Bilophila and Oxalobacter of the Proteobacteria phylum. Therefore, TSG reduces the potential pathogenic bacteria in NAFLD.

Despite our encouraging results, we understand that they are not enough to prove that gut microbiota alteration was the cause but not the effect of TSG in its activity against AS. In our next study, we will consider the use of aseptic animals or fecal transplantation methods to confirm that gut microbiota alteration is the cause but not the effect of TSG in its activity against AS.

5. Conclusions

In conclusion, this systematic study evaluated the effect of PMRP and its major active chemical constituent TSG on lipid accumulation, inflammation status and intestinal microbial imbalance in AS using ApoE-/- mice in combination with high-fat diet feeding. Our results suggested that TSG could serve as a promising compound against AS.

Author contributions Fengjiao Li and Ting Zhang wrote the manuscript; Yanran He performed the experiments; Wen Gu processed the data; Feng-Jiao Li participated to the whole work; Xing xin Yang and Jie Yu approved the final version of the manuscript; Jie Yu revised the paper; Jie Yu and Ronghua Zhao ideated and designed the experiments.

Abbreviations ANOVA: One-way analysis of variance AS: Atherosclerosis CCR2: C-C chemokine receptor type 2 ELISA: Enzyme-linked immunosorbent assay

HDL-C: High density lipoprotein cholesterol ICAM-1: Intercellular cell adhesion molecule-1 IL-1β: Interleukin-1β IL-6: Interleukin-6 LDL-C: Low density lipoprotein cholesterol MCP-1: Monocyte chemotactic protein-1 NAFLD: Non-alcoholic fatty liver disease ox-LDL: Oxidized low density lipoprotein PBS: Phosphate buffer solution PMR: Polygoni Multiflori Radix PMRP: Polygoni Multiflori Radix Praeparata TC: Total cholesterol TG: Triglycerides TMA: Trimethylamine TMAO: Trimethylamine-N-oxide TNF-α: Tumor necrosis factor-α TSG: 2, 3, 5, 4’-Tetrahydroxy-stilbene-2-O-β-D-glucoside VCAM-1: Vascular cell adhesion molecule-1

Acknowledgements This research was supported by the National Natural Science Foundation of China (grants n. 81960710, 81760733, 81660596, 81660684 and 81560612), the Natural Science Foundation of Yunnan Province (2018FF001(-005) and 2016FD050),

the Southern Medicine Collaborative and Innovation Center (30270100500), and the Young and Middle-Aged Academic and Technological Leaders of Yunnan (2015HB053). The authors would like to thank Novogene for their technical assistance with 16S rDNA sequence and analysis.

Conflict of Interest The authors declare that there are no conflicts of interest. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that could be interpreted as influencing the work described in this manuscript.

References

Shi, X. B.; D.Y. Hu. Misunderstandings in the prevention and treatment of atherosclerotic cardiovascular disease. J. China Circulation Magazine. 2014, 29(2), 158–160. Chinese Society of Endocrinology. Grading prophylaxis of atherosclerotic cerebrovascular disease in Chinese adults with type 2 diabetes Diabetes World (clinical).2016, 10(12), 531–536. Guo, D.; Juan, N. W.; Xu, W. M. Study on influences of community normalized management on patients with cardiovascular disease of atherosclerosis. 2016, 13(12), 10–12. Jiang, T. The study of hesperidin on atherosclerosis associated proteoglycans based on the role of phlegm pathogenic theory. J. Guangzhou University of Chinese Medicine, 2016.

Wang,Y. Y.; Li, M; Kong, X. Z. et al.,Wang. Progress in Research on Relationship between CD40-CD40L-mediated Inflammatory Responseand Atherosclerotic PlaqueStability. J. Chinese General Practice. 2009, 12(24), 2273–2275. Zhang, N; Li, L. S. New congnization on relationship between inflammation and atherosclerosis and research progress in related Chinese materia medica. J. Drug Evaluation Research. 2013, 36(4), 302–306. Liu, J. T. Progress in inflammatory pathogenesis of atherosclerosis. J. Journal of Xi’an Jiaotong University (Medical Sciences). 2015, 36(2), 141–152. GE Jun-bo,Y. E.; Yao, K.; Zhang. The role of inflammation in atherothrombosis. J.Chinese Journal of Integrated Traditional and Western Medicine. 2013, 33(12),1589–1592. Karlsson, F. H.; Fåk, F.; Nookaew I. et al.. Symptomatic atherosclerosis is associated with an altered gut metagenome. J. Nature Communication. 2013, 3(1245). Murri, M.; Leiva, I.; Gomez-Zumaquero, J. M. et al. Gut microbiota in children with type 1 diabetes differs from that in healthy children: a case-control study. J. BMC Med, 2013,11(1), 1–12. Shin, N. R.; Lee, J. C.; Lee, H. Y. et al. An increase in the Akkermansia spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice. J. Gut, 2014, 63(5), 727–735. Cani, P. D.; Everard, A. Akkermansia muciniphila: a novel target controlling obesity, type 2 diabetes and inflammation? J. Med Sci (Paris), 2014, 30(2), 125–127. Everard, A.; Belzer, C.; Geurts, L. et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. J. Proceedings of the National

Academy

of

Science

of

the

United

States

of

America.

2013,110(22),9066–9071. Pedersen; Ingerslev, H. C.; Sturek, M. et al., Characterisation of gut microbiota in

Ossabaw and Göttingen minipigs as models of obesity and metabolic syndrome. J. PloS One. 2013, 8(2). Li, Q.; Zhang, S. Gut microbiota and atherosclerosis. J. Drug Evaluation.2013, 21, 11–14. Romano, K. A.; Vivas, E. I.; Amador-Noguez, D. et al. Intestinal microbiota composition modulates choline bioavailability from diet and accumulation of the proatherogenic metabolite trimethylamine-N-oxide. J. MBio.2015, 6(2), e02481. Tang W. H.; Wang, Z.; Levison, B. S. et al.Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. J. Journal of Vascular Surgery. 2013, 58(2), 549. Commission of Chinese Pharmacopoeia. Pharmacopoeia of the People’s Republic of China. China Medico-Pharmaceutical Science & Technology Publishing House.2015. Wang, M. J.; Zhao R. H.; G.W.; Wang et al.Lipid regulation effects of Polygoni Multiflori Radix, its processed products and its major substances on steatosis human liver cell line L02. J. 2012, 139(1), 287–293. Yu, J.; Lin, P. ; Lu, J. M. et al. Effects of stilbene glucoside on lipid synthesis, decomposition and transport in steatotic L-02 hepatocytes. J. Chinese Pharmaceutical Journal.2014, 49(23), 2077–2082. Lin, P.; Lu, J. M.; Wang, Y. F. et al. Effects of stilbene glucoside on lipid synthesis, decomposition

and

transport

in

steatotic

L-02

hepatocytes.

J.

PloS

One.2015,10(10), 1–14. Lin, P.; Lu, J. M.; Wang, Y. F. et al. Prevention mechanism of 2,3,5,4'-tetrahydroxy -stilbene-2-O-β-D-glucoside on lipid accumulation in steatosis hepatic L-02 cell. J. Pharmacognosy Magazine. 2017, 13(50), 245–253. Fang, W. ; Qin, Y. W.; Wang, L. Y. et al. The protecting effect of PMTG on the

Atherosclerotic Lesion Formation. J.Chinese Journal of Drug Application and Monitoring.2005, 2, 48–51. Liu,

Q.

L.;

Xiao,

J.

H.;

Ma,

R.

et

al.

Effect

of

2,

3,5,4’-tetrahydroxystilbene-2-O-beta-D- glucoside on lipoprotein oxidation and proliferation of coronary arterial smooth cells. J.Journal of Asian Natural Products Research. 2007, 9(8), 689–697. Wang, C. Y.; Zhang,L. T.; Yang, Z. F. et al. Blood lipid regulation of ethyl acetate extracting fraction and stilbene glycoside from tuber of Polygonum multiflorum. J.Chinese Traditional and Herbal Drugs. 2008, 39(1), 78–83. Wang, Y. Q.; Shen, Y.; Wang, C. H. et al. Effects of 2, 3, 4’, 5-tetrahydroxystilbene-2-O-β-D glucoside on expressions of intercellular adhesion molecule-1 and vascular endothelial growth factor in aorta of atherosclerotic rats. J.Chinese Journal of Pharmacology and Toxicology.2008, 22(5), 329–335. Xu, X. L.; Huang, Y. J.; Ling, D.

Y. et al.

Inhibitory Effects of

2,3,4',5-tetrahydroxystilbene -2-O-β-D-glucoside on angiotensin II-induced proliferation of vascular smooth muscle cells. J.Chinese Journal of Integrative. Zhang, L.; Rui, Y. C. ; Qiu,Y. et al. Expression of VEGF in endothelial cells and the effects

of

2,

3,

4’,

5-tetrahydroxystilbene-2-O-β-D

glucoside.

J.Acta

Pharmaceutica Sinica. 2004,39(6), 406–409. Li, N.; Chen, Z.; Mao, X. J. et al. Effects of lipid regulation using raw and processed radix polygoni multiflori in rats fed a high-fat diet. J.Evidence-Based Complementary

and

Alternative

Medicine.

2012,

1–10.

doi:

10.1155/2012/329171. Lin, P.; He,Y. R.; Lu, J. M. et al. In Vivo Lipid Regulation Mechanism of Polygoni Multiflori Radix in High-Fat Diet Fed Rats. J.Evidence-Based Complementary and Alternative Medicine. 2014, doi: 10.1155/2014/642058

Wang, W. G.; He,Y. R.; Lin, P. et al. In vitro effects of active components of Polygonum

Multiflorum

Radix

on

enzymes

involved

in

the

lipid

metabolism.J.Journal of Ethnopharmacology. 2014, 153(3),763–770. Caporaso, J.G.; Kuczynski, J.; Stombaugh, J. et al.QIIME allows analysis of high-throughput community sequencing data. J.Nature Methods. 2010,7(5),335– 336. Magoč,T. S.; Salzberg, L. FLASH: fast length adjustment of short reads to improve genome assemblies. J.Bioinformatics. 2011, 27(21), 2957–2963. Miao, M. S. Laboratory animal and animal experiment technology. J.China Traditional Chinese Medicine Press.1997. Zhang, X. M.; Yang, J. H. The Relationshio of Apolipoprotein E and Atherosclerosis and the Application of ApoE Knockout Mice on Research of Atherosclerosis. J.Journal of Kunming Medical University. 2012, 1B,169–172. Li O.; Xu H.; Yu C. A. et al. Effects of Telmisartan on the Stability of Aortic Atherosclerotic Plaque in ApoE-deficient Mice. Chinese Journal of Integrative Medicine on Cardio-Ce rebrovascular Disease, 2019, 10: 2408. 17(5), 678-682. Wang Z. Y.; Pan L. N.; Chen L. Y. et al. Effects of platelet microparticles on atherosclerotic plaque formation and stability in ApoE-/- mice. Journal of Third Military Medical University, 2019,41(6),516-520. Li X., Zhou X.; Ji W. J. et al. The Dynamic Profile of Circulating Monocyte Subsets and Intraplaque Proliferating Macrophages During the Development of Atherosclerostic Lesion in ApoE Knockout Mice. Chin J Arterioscler, 2015, 23(1),24-28. Zhang G. X.; Gai G. Z. Effects of total alkaloids of Semen Raphani on blood lipids in ApoE knockout mice. Chinese Journal of Gerontology, 2010, 30(6),844-845. Fruchart, J. C.; Niernam, M. C.; Stroes, E. S. G. et al. New Risk Factors for

Atherosclerosis and Patient Risk Assessment. J.Circulation. 2004, 109(23), 15– 19. Packard, R. R. S.; Libby, P. Inflammation in Atherosclerosis: From Vascular Biology to Biomarker Discovery and Risk Prediction. J.Clinical Chemistry. 2008, 54(1),24–38. Shen, J.; Haffez, A.; Stevenson, J. et al. Omega-3 Fatty Acid Supplement Prevents Development of Intracranial Atherosclerosis. J.Neuroscience.2016.doi:10.1016/j. neuroscience. 2016.08.013. Wang, G. F.; Shi, C. G.; Sun, M. Z. et al. Tetramethylpyrazine Attenuates Atherosclerosis Development and Protects Endothelial Cells from ox-LDL. J.Cardiovascular Drugs and Thefrapy. 2013, 27, (3), 100–210. Libby Peter. Inflammation in atherosclerosis. Nature. 2002, 420: 868-74. Hajjar, D. P.; Gotto, A. M. Biological relevance of inflammation and oxidative stress in the pathogenesis of arterial diseases. J.American Journal of Pathology. 2013,182(5), 1474–1481. Hu, M. M.; Zhang, J.; Wang,W. Y. et al. The inhibition of lipoprotein-associated phospholipase

A2

exerts

beneficial

effects

against

atherosclerosis

in

LDLR-deficient mice. J.Acta Pharmacologica Sinica. 2011, 32(10), 1253–1258. Apostolakis, S.; Spandidos, D. Chemokines and atherosclerosis: focus on the CX3CL1/CX3CR1 pathway. J.Acta Pharmacologica Sinica. 2013,34(10), 1251– 1256. Nguyen Mau T,Fernando Sanuja,Schwarz Nisha et al. Inflammation as a Therapeutic Target

in

Atherosclerosis.

J

Clin

Med.

2019,

8(1109).

DOI ：

10.3390/jcm8081109 Grancieri Mariana, Martino Hercia Stampini Duarte, Gonzalez de Mejia Elvira et al. Seed Total Protein and Protein Fractions Digests Reduce Biomarkers of

Inflammation and Atherosclerosis in Macrophages in vitro. Mol Nutr Food Res. 2019, undefined: e1900021. Moss M Elizabeth,Lu Qing,Iyer Surabhi L et al. Endothelial Mineralocorticoid Receptors Contribute to Vascular Inflammation in Atherosclerosis in a Sex-Specific Manner. Arterioscler. Thromb. Vasc. Biol. 2019, 39: 1588-1601. Ye L. S.; Zhu T.V. Clinical Evaluation and Management of Inflammation in Atherosclerotic Cardiovascular Disease. Advances in cardiovascular diseases. 2005,26(3): 271-2715. Zhang,Y. Z.; Mu, Q.; Zhou, Z. et al. Protective Effect of Irisin on Atherosclerosis via Suppressing Oxidized Low Density Lipoprotein Induced Vascular Inflammation and Endothelial Dysfunction. J.PloS One.2016, 11(6), 1–19. Kong, B. S.; Cho,Y. H.; Lee, E. J. G Protein-Coupled Estrogen Receptor-1 Is Involved in the Protective Effect of Protocatechuic Aldehyde against Endothelial Dysfunction. J.PloS One. 2014, 9(11), 1–11. Wan, J. B.; Lee, M. Y.; Wang, J. D et al. Panax notoginseng Reduces Atherosclerotic Lesions in ApoE-Deficient Mice and Inhibits TNF-α-Induced Endothelial Adhesion Molecule Expression and Monocyte Adhesion. J.Fournal of Agricultural and Food Chemistry.2009, 57(15), 6692– 6697. Amar, J.; Lange, C.; Payros, G. et al. Blood Microbiota Dysbiosis Is Associated with the Onset of Cardiovascular Events in a Large General Population: The D.E.S.I.R. Study PloS One. 2013, 8(1). Yan, X. F.; Feng, B.; Li, P. C. et al. Microflora Disturbance during Progression of Glucose Intolerance and Effect of Sitagliptin: An Animal Study. J.Journal of Diabetes Research, 2016. Liu, J. T. Progress in inflammatory pathogenesis of atherosclerosis. J.Journal of Xi,an Jiao tong University (Medical Science). 2015, 36(2),141–152.

Ibrahim, H. A.; Mohammed, M. O.; Dhahir, H. A. R. et al. Impact of Helicobacter pylori Infection on Serum Lipid Profile and Atherosclerosis of Carotid Artery. J.International Journal of Clinical Medicine. 2014,5(15),933–939. He, Z. Q.; Zhen,Y.; Liang, C. et al. Vicious cycle composed of gut flora and visceral fat: a novel explanation of the initiation and progression of atherosclerosis. J.Medical hypotheses. 2008, 70(4), 808–811. Lin, J.; Lin, S. Q.; Vanhoutte et al. Akkermansia Muciniphila Protects Against Atherosclerosis by Preventing Metabolic Endotoxemia-Induced Inflammation in Apoe-/- Mice. J. Criculation.2016,33,(24), 2434–2446. Jeffrey S. B.; Jethro J. S.; Jorge A. E. et al. Microbiome Signatures Associated with Steatohepatitis and Moderate to Severe Fibrosis in Children With Nonalcoholic Fatty Liver Disease. Gastroenterology. 2019. DOI ： 10.1053/j.gastro.2019. 06.028. Yan M.; Ma X. G; Kris S. et al. Sex-specific association between gut microbiome and fat distribution. Nat Commun. 2019, 10: 2408.

MOD PMRP TSG.L TSG.H SIM

32

4.0

Food intake(g/each mice)

30

Body weight(g)

MOD PMRP TSG.L TSG.H SIM

4.2

28

26

24

3.8 3.6 3.4 3.2 3.0 2.8 2.6

22 2.4 1D

10D

19D

28D

37D

46D

55D

1W

2W

3W

Days

4W

5W

6W

7W

8W

Weeks

(A)

(B)

MOD PMRP TSG.L TSG.H SIM

7 6

Viscer index (%)

5 4 3 2 1

* * *

*

0 heart index

liver index

splee index

lung index

kidney index

(C) Fig.1. Body weight (A), mean food intake of each mice (B), and organ index (C) of mice in different groups (n=7). Under general conditions, body weight was recorded every three days, while food intakes were measured every day and the organ indexes were recorded at the end of research. One-way analysis of variance (ANOVA) was performed when multiple group comparisons were carried. Symbol * indicates a significant difference compared with control group, *p<0.05.

*

450 400

content of IL-6 (pg/ml)

2.0

content of TNFa (pg/ml)

2.5

* *

1.5

1.0

350 300 250 200 150 100

0.5

50 0

0.0 MOD

PMRP

TSG.L

TSG.H

MOD

SIM

PMRO

(A)

TSG.H

SIM

(B) 1800

800

1600

700

* 600

1400

content of MCP-1 (pg/ml)

content of VCAM-1 (pg/ml)

TSG.L

500 400 300 200 100

**

1200 1000

* 800 600 400 200 0

0 MOD

PRMP

TSG.L

(C)

TSG.H

SIM

MOD

PMRP

TSG.L

TSG.H

SIM

(D)

Fig.2. Attenuation of inflammatory parameters IL-6 (A), TNF-α(B), VCAM-1 (C) and MCP-1 (D) by PMRP and TSG in blood samples in ApoE-/- mice with high fat diet induced AS. Treatment with TSG might suppress the typical inflammatory status of ApoE-/- mice. One-way analysis of variance (ANOVA) was performed when multiple group comparisons were carried. Values are means, with their standard errors represented by vertical bars. The * indicates a significant difference compared with model group (n=7). *P<0.05, **P<0.01.

450

35

400

30

350

content of ICAM-1 (ng/mg)

content of VCAM-1 (pg/mg)

***

300

*

* 250

*

200 150 100

25

20

**

15

10

*

5

50

0

0 MOD

PMRP

TSG.L

TSG.H

MOD

SIM

(A)

PMRP

TSG.L

TSG.H

SIM

(B)

24

**

22

content of CCR2 (pg/mg)

20 18 16 14

**

12 10

**

8 6 4

*

2 0 MOD

PMRP

TSG.L

TSG.H

SIM

(C) Fig.3. Reduction of the increased concentrations of VCAM-1(A), ICAM-1(B) and CCR2 (C) by PMRP and TSG in aortic tissue in ApoE-/- mice with high fat diet induced AS. PMRP and TSG might suppress the typical inflammatory mediators. One-way analysis of

variance (ANOVA) was performed when multiple group comparisons were carried. Values are means, with their standard errors represented by vertical bars. The * indicates a significant difference compared with model group (n=7). *P<0.05, **P<0.01, ***P<0.001.

1.0

Relative Abundance

0.8

0.6

0.4

0.2

0.0

0.30

The ratio of Firmicutes/ Bacteroidetes

Bacteroidetes Verrucomicrobia Firmicutes Proteobacteria Tenericutes Actinobacteria Fusobacteria Cyanobacteria Acidobacteria TM7 Others

0.25

0.20

0.15

0.10

0.05

0.00

MOD

PMRP

TSG.L

TSG.H

SIM

Phylum Level

(A)

MOD

PMRP

TSG.L

TSG.H

SIM

Groups

(B)

Fig.4. Relative abundance of gut microbiota at the level of phylum (A) and the ratio of Firmicutes/ Bacteroidetes at the level of phylum (B) in ApoE-/- mice with high fat diet induced AS (n=7). PMRP and TSG could raise the ratio of Firmicutes/ Bacteroidetes to relieve AS, meanwhile, TSG also could decrease Proteobacteria and Tenericutes in intestinal to produce a therapeutic effect.

Fig.5. Species abundance clustering figure on the level of genus (n=7). Note: Absissa axies represents sample information, corinate axis represents annotation information for species, clustering tree on the left side is species clustering. The values of the hot spot at the center are Z value which were standardized of species abundance each line.

Table 1. Serum and Aortic tissue lipid contents of TG, TC, LDL (mmol/L) and ox-LDL(nmol/ml) in mice（ x± SD , n = 7).

Serum

Groups

MOD

PMRP

TSG.L

TSG.H

SIM

TG

0.8916±0.08324

0.6780±0.08324*

0.6130±0.06043**

0.5882±0.04915**

0.6439±0.1229*

TC

29.64±0.9228

31.51±1.803

28.35±0.9678

32.54±2.206

26.94±0.6594*

LDL

8.021±1.953

13.60±0.7608**

15.97±0.3015**

17.22±0.6314**

15.52±0.5749**

ox-LDL

1.733±0.1660

1.460±0.1989

1.328±0.0450*

1.280±0.0618*

1.074±0.0856**

2.227×10-2

1.070×10-2

8.313×10-3

7.432×10-3

6.915×10-3

0.5032±0.0920

0.2046±0.6812*

0.7793±0.0641*

0.3560±0.0128

0.7119±0.023*

ox-LDL/L DL(%) Aorta

ox-LDL

The * indicates a significant difference compared with control group. *p<0.05, **p<0.01,

Table 2. Relative abundance of Helicobacter pylori in ApoE-/- mice (n=7).

Groups

MOD

PMRP

TSG.L

TSG.H

SIM

Helicobacter pylori

0.010267

0.013162

3.58E-05

3.40E-05

6.73E-05

Serum

Aorta

Groups

MOD

PMRP

TSG.L

TSG.H

SIM

TG

0.8916±0.08324

0.6780±0.08324*

0.6130±0.06043**

0.5882±0.04915**

0.6439±0.1229*

TC

29.64±0.9228

31.51±1.803

28.35±0.9678

32.54±2.206

26.94±0.6594*

LDL

8.021±1.953

13.60±0.7608**

15.97±0.3015**

17.22±0.6314**

15.52±0.5749**

ox-LDL

1.733±0.1660

1.460±0.1989

1.328±0.0450*

1.280±0.0618*

1.074±0.0856**

ox-LDL/LD L(%)

2.227×10-2

1.070×10-2

8.313×10-3

7.432×10-3

6.915×10-3

ox-LDL

0.5032±0.0920

0.2046±0.6812*

0.7793±0.0641*

0.3560±0.0128

0.7119±0.023*

Supplementary Material

MOD

TSG

SIM

Fig.S1 Atherosclerotic plaques stained by oil red O in the aorta wall. The atherosclerotic plaques stained by oil red O in aorta indicated that atherosclerosis model was successfully established. TSG effectively reduce the number of atherosclerotic plaques.