System-based DNA microarray analyses of different gene expression profile in spontaneously hypertensive rats treated with Songling Xuemaikang Capsule reveals underlying causal mechanisms. CURRENT

Background: Hypertension is considered the major risk factor for human health in the world. Songling Xuemaikang Capsule (SXC) is clinically used as a medicine for the prevention and treatment of cardiovascular and cerebrovascular diseases such as hypertension and hyperlipidemia. However, the underlying mechanisms have yet to be fully identified. Methods: Valsartan, as a positive control drug, high- and low-dose of SXC were orally administration with for 28 days to investigate the antihypertensive effect of SXC in spontaneously hypertensive rats (SHRs). The serum levels of aldosterone and Angiotensin II (Ang II) were detected. The gene expression profiling was performed in the thoracic aorta of SHRs using the Whole Rat Genome Oligo nucleotide Microarray. The integrated causal network analysis was performed to understand the mechanism of antihypertensive effect of SXC. Results: The results shown that the systolic and diastolic blood pressure were significant decreased in SXC low-dosage group and high-dosage group compared with the control group respectively. SXC low and high-dosage treatment decreased serum aldosterone levels significantly but increased serum Ang II compared with the control group respectively. Causal network analysis shown that treatment with SXC reversing the vascular remodeling process, inhibiting vascular inflammation and atherosclerosis, reversing endothelial cells dysfunction and likely reducing peripheral vascular resistance by down-regulated processes related to vascular remodeling, dyslipidemia, the complement system, leukocyte rolling, and endothelial dysfunction. In addition, SXC treatment may also activate fibrinolysis and regulate lipid and glucose metabolism. Conclusions: Those obtained data could help our understanding and potential utilization of SXC in the treatment or prevention of hypertension 11 Powdered nacre, of pearl, from Pinctada maxima, showed the of reducing body weight, fat amount, and blood triglyceride level influencing the food body or amount of muscular tissue been to that to control and slow the of end-organ damage in hypertension 13–16 remodeling actin polymerization as is prolonged dose SXC leukocyte finally Eosinophil-platelet adhesion, during thrombosis, also downregulated. Taken together we used SHRs to analyze the hypotensive effects of SXC and valsartan in vivo, and reported the overall gene expression pro-files from thoracic aorta of SHRs orally treated with SXC for the first time. Our results suggest that dietary intake of SXC had mild BP lowering effect. Both valsartan and SXC high-dose down-regulated processes related to vascular remodeling and dyslipidemia. Smooth muscle relaxation was unique key pathway to valsartan due to its action as an angiotensin receptor antagonist, while down-regulation of the complement system, leukocyte rolling, and endothelial dysfunction was unique key pathway to SXC. The effects of SXC high-dose treatment appear to be broader than valsartan and the benefits could be summarized as follows. SXC high-dose treatment reverses the vascular remodeling process, it inhibits vascular inflammation and atherosclerosis by inhibiting platelet activation, adhesion and activation, as well as adhesion between platelets, endothelial cells, and leukocytes, thus reversing endothelial dysfunction and likely reducing peripheral vascular resistance. It also appears to activate fibrinolysis thereby inhibiting thrombus formation. In addition, it regulates the metabolism of lipids and glucose, which are associated with multiple conditions such as obesity, diabetes, metabolic syndrome, and cardiovascular disorders. TCM, Traditional Chinese medicine; AGTR1, angiotensin 1; HIF1A, hypoxia-inducible factor 1A; VLDLR, very low density lipoprotein receptor; ABCG1, ATP binding cassette transporter G1; ABCA1, ATP-binding cassette, subfamily A, member 1; BMP4, bone morphogenetic protein 4; A-FABP, adipocyte fatty acid‐binding protein; PGAR, peroxisome proliferatoractivated receptor γ angiopoietin-related gene; PPARGC1, peroxisome proliferator‐activated receptor‐ γ co‐activator‐1; PAI–1, plasminogen activator inhibitor–1; PBEF, pre‐B cell colony‐enhancing factor; IL–1, Interleukin–1; IL–6, Interleukin–6; JAK/STAT, Janus kinase–signal transducer and activator of transcription; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MRLC, multi resolution land characteristics; LARG, leukemia‐associated Rho GEF; ROCK, Rho kinase; PLC, phospholipase C; IP3, Inositol 1, 4, 5‐trisphosphate; TRPC1, transient receptor potential 1; NCX1, Na+/Ca 2+ exchanger 1; MaxiK, Ca2+-activated K+; PPARGC1, peroxisome proliferator‐activated receptor‐gamma coactivator‐1; eNOS, endothelial nitric oxide synthase; VEGF-A, vascular endothelial growth factor A; PAI–1, plasminogen activator inhibitor–1; EMT, epithelial-to-mesenchymal transition; CRP, C-reactive protein; CVD, cardiovascular disease; VSMC, vascular smooth muscle cell (VSMC); ICAM1, intercellular adhesion molecule–1.


Introduction
Hypertension, a critical public health problem worldwide, is considered the major risk factor for ischemic and hemorrhagic stroke, myocardial infarction, heart failure, chronic kidney disease, peripheral vascular disease (PVD), cognitive decline, and premature death. Hypertension is the leading modifiable risk-factor for cardiovascular disease, which represents the top cause of death in China 1,2 . Hypertensive patients' blood pressure (BP) needs be controlled, to decrease the incidence of adverse sequelae. In China, the weighted prevalence of hypertension has been rising, with rates increasing from 18% in 2002 to 23.2% in 2015 3,4 . Among those with hypertension, 46.9% were aware of their condition, 40.7% were taking prescribed medication to lower their BP, but only 15.3% achieved BP control 4 .
Conventional antihypertensive agents ranging from diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptors blockers, sympathoplegic agents, calcium channel blocker, αadrenergic blockers and β-adrenergic blockers others are performed to control blood pressure levels in hypertensive patients 5 . Those agents are usually associated with many adverse effects such as hypokalemia, hyponatremia, severe dry cough, ankle edema, headache, facial flushing, polyuria and allergy. Traditional medicine is widely used, and is of increasing health and economic importance and often termed alternative or complementary medicine in many countries 6 . In some countries, the majority of the population continue to use traditional medicine to meet their health needs. This may, in part, be attributed to increasing concerns about the safety and approaches of Western medicine 6 .
Overall, this may explain the increasing interest in panning out the beneficial health effects of various plants and herbs in different diseases including hypertension 7 .
Songling Xuemaikang Capsule (SXC) is a traditional Chinese patent medicine which has been authorized recommended by Chinese Pharmacopoeia. Three widely used traditional herbal medicines, Puerariae Lobatae Radix., Pine needles (Pinus densiflora Lamb.), and powdered nacre are containing in SXC. Puerarin extracted from Puerariae Lobatae Radix. possesses effects of antihypertension and stroke prevention by improved microcirculation in spontaneously hypertensive rats (SHRs) through increasing in cerebral blood perfusion both by arteriole relaxation and p42/44 MAPKs-mediated angiogenesis 8 and protecting against endothelial dysfunction and end organ damage by anti-oxidant and upregulation of phosphor-eNOS 9 . Some components extracted from pine needle have been proved to have vasorelaxant effects by blocking the voltage-operated Ca 2+ channel (VOCC) and inhibiting Ca 2+ influx to the cytoplasmic 10 and play an important role in the prevention of oxidative damage in vascular endothelial cells in hypertension patients via the PI3K/Akt/Bad signaling pathway 11 . Powdered nacre, mother of pearl, from Pinctada maxima, was showed the effect of reducing body weight, visceral fat amount, and blood triglyceride level without influencing the food intake, body length, or amount of muscular tissue 12 .
SXC has been widely used to treat hypertension and hypertension related symptoms in China.
Preliminary clinical studies demonstrated that SXC could contribute to BP control and slow the progression of end-organ damage in hypertension [13][14][15][16] .
However, the molecular mechanisms involved in SXC treated hypertension remain largely undefined.
Therefore, this study was carried out to investigate the antihypertensive effect of SXC in SHRs established animal model of essential hypertension. Furthermore, to elucidate its underlying mechanism, DNA microarray analyses were performed to obtain gene expression profiles in thoracic aorta tissue of SHRs after repeated oral administration of high-or low-dose of SXC and valsartan.
The aim of this study was to identify target genes which expression were markedly changed after oral administration of SXC in an established animal model of essential hypertension, and reconstructed a causal network to understand the molecular mechanism of in vivo antihypertensive activities of SXC. is maintained at 20 to 24 ° C, and the humidity is maintained at 30 to 70%. The daylighting of the animal husbandry is controlled by an electronic timed lighting system that turns off the lights for 12 hours every day and on for 12 hours. During the experiment, the animals were kept in single cages.

SHRs and Measurement of BP
They were fed with standard laboratory diet and guaranteed free drinking water.
The rats were acclimatized in the above conditions for about a week before the experiment.
Compound anesthesia was performed with 3% sodium pentobarbital solution (60 mg•kg -1 , i.p.). HD-S10 Small Animal Implant (Data Sciences International, New Brighton, MN, USA) was implanted on Day-3 ~ Day-4. The procedure was as follows: the incision was performed on the abdomen of the rat.
The catheter of the implant was inserted into the separated abdominal aorta. Bio-gel was used to stop bleeding. The implant was fixed on the abdominal wall. Then, the muscle and skin were sutured and sterilized. Subcutaneous injection of ibuprofen for analgesia. Gentamicin (8 mg•kg -1 •day -1 , i.p.) was intraperitoneally administered three days after surgery to prevent infection.

Isolation of Animal Tissue
At 28 days, all rats were killed with intraperitoneal injection of 3% sodium pentobarbital solution (60mg•kg -1 ), and 3 mL blood samples were collected from the heart. These blood samples were transferred immediately into aseptic capped tubes and centrifuged at 3000 g for 4 min at 4℃. The plasma supernatant was collected and stored at -80℃ until further analysis. The thoracic aorta dissected from each rat was shredded and immediately stored in liquid nitrogen. The thoracic aorta of all animals was collected for DNA microarray analyses from each group. The levels of Angiotensin II (Ang II), Aldosterone in serum of SHRs from different groups were separately determined by the enzyme-linked immunoassay kit (Mybiosource, San Diego, CA, USA).

DNA Microarray Analysis
The following procedures for sample preparation and microarray analysis were done at Agilent Whole Genome Oligo Microarrays (Agilent, Santa Clara, CA, USA). Total RNA was extracted and purified from thoracic aorta tissue separately by TRIzol® Reagent (Invitrogen life technologies, Carlsbad, CA, USA) and Cleaned-up by RNasey Mini Kit (Qiagen, Valencia, CA, USA). Then, the Cy3-labeled cRNA was transcribed from 1 μg of RNA of each group using the Quick Amp Labeling Kit, One-Color (Agilent, Santa Clara, CA, USA). Cy3-labeled cRNA was hybridized to the Whole Rat Genome Oligonucleotide Microarray ver. 3.0 (4×44k, G2505C-028282) (Agilent Technologies), following the manufacturer's hybridization protocol. After the washing step, the microarray slides were scanned using Agilent Microarray Scanner (Agilent G2505C, Agilent Technologies, Santa Clara, CA, USA) according to the manufacture's protocol. Microarray expression data were analyzed using Agilent Feature Extraction software (Agilent Technologies, Santa Clara, CA, USA) with the default settings for all parameters. The raw data were firstly normalized with the quantile algorithm, and the probes that at least one out of all samples had fluorescence signals in detection were chosen for further analyses.
Pre-processing of microarray data and identification of differential expression genes The microarray data was processed using the R statistical language (R version 3.4.2) and the limma package 17 . Beginning from the raw Agilent data files, we background-corrected and normalized between arrays using the quantile normalization method 18 . The normalized log2 intensities were visualized using principal components analysis (PCA) and hierarchical clustering. Limma package was used to performed the differential expression analysis. The differential expression genes (DEGs) foldchanges, P-values, and adjusted P-values were calculated for multiple testing by using the Benjamini-Hochberg method 19 . Gene lists filtered for padj (adjusted p-value) < 0.01 were used for further analysis.
Gene ontology and pathway enrichment analyses

Integration of Causal Network Model analysis
The topologically significant genes (TSGs) were identified by overconnection, hidden nodes, and causal reasoning tests. An overconnectivity test 20,21 identifies regulators of a DEG list that are directly connected and statistically overconnected to these DEGs. Hidden nodes analysis, the second network analysis method, is applied to identify genes with a high level of connectivity within an unlimited number of steps away from the data. The score is calculated using a conditionspecific network that joins all of the differentially expressed genes together via shortest paths 21,22 . Causal Reasoning is a shortest-path search based method which predicts upstream regulators causal for gene expression changes observed in experimental data 23 . The method prioritizes nodes in a molecular network that are likely responsible for the observed modulations in an experiment.
A hypergeometric test was performed for the gene list of DEGs and TSGs for enrichment analysis.
Pathway maps simultaneously enriched with both the DEGs and the corresponding TSGs (P < 0.05. The pathways from the step above are tested for "synergistic" behavior of the DEGs and the TSGs with enrichment synergy method 24 . Then, the finale key pathways are those which are enriched not only with DEGs but also with their regulators and where these two enrichments are synergistic. All the causal networks were constructed through MetaCore (Thomsan Reuters, New York, NY, USA)

Statistical analysis
Data was analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA).
One-way analysis of variance followed by Tukey's post hoc test was used for comparisons among multiple groups. A value of P<0.05 was considered statistically significant.

SXC can lower blood pressure in SHRs
Systolic BP and diastolic BP of SHRs were measured on every Thursday, at 2 hours after SXC treatment and valsartan for 4 weeks before DNA microarray analyses. As the results shown in table 1, both the systolic BP and diastolic BP were well controlled both in SXC high-and low-dose groups from the beginning to the end of experiment. Valsartan treated group dramatic decreased the systolic BP and diastolic BP from the 1 st week (Table 1). While compared to the rats in the control group, the systolic BP and diastolic BP were significant decreased in the rats of SXC high-and low-dose groups from the 3 rd week (Table 1). At the end of the experiment, the systolic BP and diastolic BP were significant decreased by 13.50%, 29.57% in SXC low-dosage group and 6.82%, 26.82% in SXC highdosage group compared to the control group respectively (Table 1). These results demonstrated SXC could be potentially used in the treatment of hypertension.

SXC reduces serum aldosterone content
The content of AngII and aldosterone in serum were detected via ELISA. Valsartan and two dosage SXC treatment all increased serum Ang II levels ( Fig 1A). As the results shown in Fig 1B, the serum aldosterone levels were significantly decreased by 12.66% and 41.86% in SXC low and high-dosage administrated groups compared with the control group respectively, while those in valsartan group was increased by 74.03% compared with the control group.

Identification and Hierarchical Clustering of DEGs
We used 44K gene chip microarray to identify differentially expressed genes in the thoracic aorta of SHRs treated with SXC low-dose, SXC high-dose and valsartan compared with control SHRs respectively. Limma was used to calculate fold-changes, P-values, and adjusted P-values for each comparison. The PCA plot of all samples shows much more robust clustering by sample group (Fig   2A). The volcano plots shown below in summarize the corrected p-values and log2 fold-changes that were calculated for each treatment (Fig 2B-D found the frequency of down-regulated genes were involved in GO cellular compartments terms related to the extracellular region which highly overlapped between the three treatments. Synapse, up-regulated GO cellular compartment was commonly enriched in valsartan and SXC high-dose treated groups. Up-regulated GO cellular compartments potassium channel complex was mostly enriched only when treated with valsartan while high-density lipoprotein particle was enriched only when treated SXC low-dose. For the biological process, the enriched down-regulated GO Biological processes included metabolic processes and leukocyte mediated immunity.
When treated with SXC high-dose and valsartan synaptic signaling was the up-regulated GO biological processes. Regulation of Wnt signaling was the up-regulated GO biological processes only in SXC high-dose treated group. The down-regulated GO molecular functions were significantly ascribed to oxidoreductase activity, likely related to lipid metabolism in all the three treatment. The up-regulated GO molecular functions were primarily ascribed to gated channel activity in SXC high-dose and valsartan treated groups, but mostly enriched in molecular function regulator in SXC low-dose treated group.
After the Go enrichment analysis, to overview the function of differential expressed genes in three treatment, these DEGs were utilized in the focused analysis to identify the Clarivate pathways in the three treatments compared to the control group. These results are shown in

Identification of topologically significant genes
To identify topologically significant genes (Ts genes), network-based analyses including: overconnectivity test, hidden nodes analysis, and causal reasoning analysis were performed. The overconnected nodes, acted as upstream regulators of differential expression were tested in each sample group independently by an overconnectivity test which identifies regulators of a DEGs list that are directly connected and statistically overconnected to these DEGs. As shown in Table 2, SP1 is the top ranked overconnected gene for all treatments, which involved in many cellular processes, including cell growth and differentiation, apoptosis, immune responses, DNA damage response, and chromatin remodeling 25 . PPAR-alpha and PPAR-gamma, and HIF1A were also identified as the overconnected transcription factors for all the three treatment. PPARs, a steroid hormone receptor, is a member of the nuclear receptor superfamily. Isomers of PPARs include PPAR-alpha, PPAR-beta, and PPAR-gamma, regulating metabolic processes including lipid and glucose homeostasis, and inflammatory responses [26][27][28] .HIF1A, a master transcriptional regulator of the adaptive response to hypoxia, has a key role in cellular response to hypoxia, including the regulation of genes involved in energy metabolism, vascular remodeling, and atherosclerosis 29 .
Hidden nodes analysis is based on shortest-path method that identifies genes with a high level of connectivity within an unlimited number of steps away from the data. Highly significant genes from this analysis are likely to be functionally involved in the molecular changes, including a driver role, despite not being differentially expressed at the transcriptional level themselves. PLAUR (uPAR) was identified as the top hidden node for both doses of SXC treatments. This gene codes for the plasminogen activator, which is involved in blood coagulation, cell adhesion, and inflammation via the JAK-STAT pathway. Fibronectin, ADAM12, and c-Fos were also highly ranked hidden nodes for both doses of SXC treatments. Fibronectin and ADAM12 are both involved in cell adhesion and extracellular matrix, while c-Fos is a transcription factor that participates in many signaling transduction cascades, including some related to inflammation and immune response ( Table 2).
Causal reasoning analyses, another shortest-path search-based method, predicts upstream regulators causal for gene expression changes observed in experimental data. Causal reasoning analyses prioritizes nodes in a molecular network that are likely responsible for the observed modulations in an experiment and takes the directionality of the edges into account as well as the biological effects (activation or inhibition).
CNOT3 was a top-ranked causal reasoning regulator for all three treatments. This gene is a general transcription regulator and is involved in bulk mRNA degradation, miRNA-mediated expression, and translational repression 30 (Table 2).
Causal network models demonstrated the molecular mechanism of SXC for control blood pressure While DEGs and functional pathways affected in the thoracic aortas of SHRs with different treatment were identified, the complex nature of hypertension makes it impossible to elucidate whether these changes are directly contributed to the therapeutics treatment or simply a reflection of individual differences or loss of critical regulatory signals. To our best knowledge, this is the first attempt applying causal network models to analyses transcriptional changes present in the therapeutics treatment of hypertension thoracic aortas tissues. To shed light on the causal processes underlying the expression changes observed in SHRs thoracic aortas regions, affected genes which either differentially expressed or topologically significant were searched among triggers of the signaling pathways on the key maps. Next, signal transduction pathways were selected that were downstream of affected triggers and contained consecutive signaling molecules belonging to affected genes. The identified pathways down to the topologically significant transcription factors (TFs) and/or cellular functional effects were traced and shown on the map. Causal models reconstructed with such a procedure illuminate mechanisms linking the different levels of cell regulation into a meaningful integrated system and provide a roadmap to the relevant signaling mechanisms. As shown in Table 3, the top 10 enriched key maps in the SXC high dose compared to control group involve mostly immune response, cell adhesion, vascular damage and lipid metabolism processes. These maps, along with others including enriched pathway maps and maps related to vascular cells and smooth muscle contraction were used to reconstruct a causal network model. The network model is divided into 2 maps (Fig 6 A and B). Several functional areas are highlighted on the maps: Lipid metabolism, Dyslipidemia and Inflammation, Complement System and Immune Response, Leukocyte Rolling and Endothelial Dysfunction and Vascular Remodeling. As shown in Table 4, the top 10 enriched key maps in the valsartan treated group compared to control group involve mostly in lipid metabolism processes. These maps, along with others including enriched pathway maps and maps related to Gprotein coupled receptor signaling cascades, were used to reconstruct a causal network model. The network model is divided into 2 maps (Fig 7 A and B). Several functional areas are highlighted on the maps: Vascular remodeling, smooth muscle relaxation and lipid metabolism.

The comparison of key pathways different therapy treatment
In order to identify the difference between SXC high-dose treatment and valsartan treatment in SHRs, all the dysregulated key pathways in both two treatment were compared. In the analysis of key pathway maps, 16 maps were found to be shared between the SXC high-dose and valsarta treatments. These key pathway maps are shown below in Table 5, with the p-values from the hypergeometric test performed. The overlapping maps describe processes related to lipid metabolism and dyslipidemia, endothelial dysfunction, inflammation, and platelet activation. All processes seem equally distributed between the two drugs, except for endothelial dysfunction such as role of cell adhesion in vaso-occlusion in sickle cell disease and vascular endothelial cell damage in systemic lupus erythematosus (SLE), which perhaps shows a small trend toward being more enriched in SXC treatment. In both groups, the treatment down-regulates the immune response and associated inflammatory processes, as well as dyslipidemia and lipid metabolism processes. Both treatments also play a role in reversing the effects of the vascular remodeling that takes places during hypertension.
There are 30 key pathways that are unique to the SXC high dose group are shown below in Table 6.
These maps relate mostly to the immune response triggered by complement system, inflammatory cytokines, and detailed pathway maps involving platelet activation, leukocyte rolling and cell adhesion processes that play a role in vascular remodeling and cardiovascular disorders. In the case of valsartan treated groups, the 31 unique key maps, shown below in Table 7, relate mostly to the angiotensin system, further dyslipidemia, lipid-related maps and oxidative stress.

Discussion
Elucidating the mechanism of action of drugs especially the Traditional Chinese medicine (TCM) and the causal events after drug treatment are challenging, but they are one of the primary goals of medical research. Understanding the mechanism of action of a drug can prioritize drug candidates or help identify novel indications for existing drugs. Because the direct target of the SXC treatment for hypertension is unknown, understanding the physiological effects of the treatment provides further insights into disease pathways and how they can be rescued. Microarrays provides high-resolution data that can reveal much about the state of the cell, tissue, and organism during treatment. Clarivate Analytics performed differential expression analysis on microarray data obtained from thoracic aorta samples in SHR rats treated with valsartan, SXC high-dose, SXC low-dose or appropriate vehicle controls.
The microarray data was high quality and thousands of differentially expressed genes were identified in each drug-control comparison. There was a high degree of overlap in gene expression, both between valsartan and SXC high-dose, and the two SXC dosages. GO analysis and the Clarivate pathways were performed to overview the function of differential expressed genes in three treatment.
The up-regulated ontologies were not significant after corrected multiple testing for GO cellular compartments, GO biological processes and GO molecular functions. The down-regulated ontologies were significantly ascribed to oxidoreductase activity, likely related to lipid metabolism for GO molecular functions, metabolic processes and leukocyte mediated immunity for GO biological Treatment with high dose SXC appears to inhibit all these processes. In particular, platelet activation and aggregation are inhibited via C1q/PKC/P-selectin and collagen I/collagen III/FC gamma/RII alpha receptor. Also inhibited by treatment are platelet-neutrophil-endothelial cell aggregations within the vasculature, which are otherwise increased in CVD and even in risk factors such as hypertension and dyslipidemia. Binding of ICAM1 with alpha M/beta-2 integrin receptor (also downregulated in this dataset) permits interactions to occur between neutrophils and endothelial cells, which in turn cause leukocyte rolling, adhesion, and finally transmigration to the intima layer of the vasculature.
Eosinophil-platelet adhesion, which occurs during thrombosis, is also downregulated.
Taken together we used SHRs to analyze the hypotensive effects of SXC and valsartan in vivo, and reported the overall gene expression pro-files from thoracic aorta of SHRs orally treated with SXC for the first time. Our results suggest that dietary intake of SXC had mild BP lowering effect. Both valsartan and SXC high-dose down-regulated processes related to vascular remodeling and dyslipidemia. Smooth muscle relaxation was unique key pathway to valsartan due to its action as an angiotensin receptor antagonist, while down-regulation of the complement system, leukocyte rolling, and endothelial dysfunction was unique key pathway to SXC. The effects of SXC high-dose treatment appear to be broader than valsartan and the benefits could be summarized as follows. SXC high-dose treatment reverses the vascular remodeling process, it inhibits vascular inflammation and atherosclerosis by inhibiting platelet activation, adhesion and activation, as well as adhesion between platelets, endothelial cells, and leukocytes, thus reversing endothelial dysfunction and likely reducing peripheral vascular resistance. It also appears to activate fibrinolysis thereby inhibiting thrombus formation. In addition, it regulates the metabolism of lipids and glucose, which are associated with multiple conditions such as obesity, diabetes, metabolic syndrome, and cardiovascular disorders.

Conclusions
Those results provided valuable information for our understanding of the molecular mechanisms that underlie the potential antihypertensive activities of SXC, and will contribute towards increased valueadded utilization of SXC.