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 processes and extracellular region for GO cellular compartments in all the three treatment. The up-regulated enriched Clarivate pathway maps were also not significant after multiple comparison correction. The down-regulated Clarivate pathway maps were more ascribed in PPAR regulation of lipid metabolism and immune response in valsartan treated group. The previous studies demonstrated valsartan therapy significantly lowered total- and LDL-cholesterol levels and had significant anti-inflammatory efficacy in hypertensive diabetic patients with enhanced inflammatory burden31,32. The enrichment analysis indicated that SXC treated SHRs down-regulated Clarivate pathways not only including PPAR regulation of lipid metabolism, complement system induced immune response but also including inflammation and vascular and endothelial dysfunction in SXC high-dose and low dose treated group.
Additionally, common transcript changes with different treatment were assessed using causal network analyses. Mechanism reconstruction through causal network models provides an integrated view of how a drug effects the cell and insight into previously unrecognized therapeutic mechanisms of the drug. In-depth investigation revealed several common molecular drivers down-regulated in both SXC high-dose treated group and valsartan treated group. These changes were integrated and comprised our causal map. The causal maps reflected some functional areas were shared between SXC treatment and valsartan treatment but through differing pathways in each treatment. Those common functional areas including “lipid metabolism, dyslipidemia and inflammation” and “Vascular remodeling” are detailed below.
Multiple cohort studies have strongly indicated a causal relationship between dyslipidemia and risk of developing hypertension. One possible explanation for this relationship is that hypertension and dyslipidemia share common pathophysiological etiologies, such as obesity and the resulting dysregulation of adipocytokine release from adipose tissue. Furthermore, dyslipidemia adversely affects functional and structural arterial properties and promotes atherosclerosis. These changes may impair BP regulation, which, in turn, predisposes individuals with dyslipidemia to development of hypertension33. AGTR1 mediates the predominant actions of angiotensin II on blood pressure, and it is the target of a widely used class of antihypertensive drugs: the angiotensin receptor antagonists34. This is in line with multiple studies which show an association between AGTR1 blocker treatment and downregulated lipid metabolism35–37. In our results, lipid metabolism, dyslipidemia and inflammation are down-regulated processes both upon treatment with valsartan and high dose SXC. HIF1A and PPAR-gamma appear to act as transcriptional regulators of the process which both down-regulated in both two treatment. Angiotensin II, acting through AGTR1, increases HIF–1A nuclear localization and activity. However, AGTR1 is downregulated in valsartan treated group, thereby down-regulating HIF–1A and its downstream effects on lipid metabolism. Both two treatment down-regulated LPL, VLDLR, ABCG1 and ABCA1 which lead to decreased dyslipidemia. Low levels of adiponectin are associated with endothelial dysfunction, hypertension and cardiovascular disease 38–40. Curiously, adiponectin is downregulated in both two treatment but with different regulators. Downregulated HIF–1A is the main influence factor of down-regulated adiponectin which also leads to downregulated PPCKC playing a role in gluconeogenesis in treatment with valsartan. It seems that more than one regulator changed caused adiponectin down-regulated when treated with SXC high dose. It is possible that this is explained by the fact that treatment with high dose SXC causes downregulation of BMP4, which through SMAD and PPAR-gamma regulates the transcription of A-FABP, LPL, Perilipin, CIDEC and Factor D which down-regulated contributing for decreased expression and differentiation of white fat genes and release of adiponectin.by downregulated HIF–1A. Additional, high dose SXC treatment also downregulated perilipin, PGAR and PPARGC1 by decreasing lipolysis. Upon SXC high dose treatment, HIF1A also down-regulates the expression of various genes that are known to play a role in inflammation: IL–1 beta, PAI–1, PBEF and IL–6, the latter doing so through the JAK/STAT and ERK/MEK cascades. Heme oxygenase 1, however, was also downregulated.
Vascular remodeling is an active process of structural modification that involves changes in at least four cellular processes: cell growth, cell death, cell migration, and the synthesis or degradation of extracellular matrix. Vascular remodeling is dependent on dynamic interactions between local growth factors, vasoactive substances, and hemodynamic stimuli and is an active process that occurs in response to long-standing changes in hemodynamic conditions; however, it may subsequently contribute to the pathophysiology of vascular diseases and circulatory disorders. Likewise, vascular fibrosis entails accumulation of collagen, fibronectin, and another extracellular matrix
components in the vessel wall and is an important aspect of extracellular matrix remodeling in hypertension41,42. Treatment with valsartan and high dose SXC appears to induce changes in vascular remodeling through different regulatory pathways. Actomyosin, responsible for cytoskeleton remodeling and regulation of smooth muscle contraction, is highly upregulated via MRLC upon both treatment. In treatment with valsartan, downregulated AGTR1 caused a series of G protein-coupled receptors (GPCRs) regulated vascular tone was down-regulated which up-regulated actomyosin through Gq/G11/LARG/RhoA/ROCK/MRLV pathway, whereas in treatment with high dose SXC actomyosin was up-regulated by RhoA/ROCK/MRLV and Rac/PAK/MRLV pathways. Phosphorylation of myosin light chains by MLCK triggers cross-bridge cycling between actin (downregulated) and myosin (upregulated) filaments 43. Down-regulation of PLC leads to a decrease in IP3 production, which reduces the release of calcium ions from the endoplasmic reticulum into the cytoplasm reducing the expression of calmodulin and thus reducing the activation of MLCK in treatment with valsartan. Likewise, several channel pumps (TRPC1, NCX1 and MaxiK) are deregulated upon treatment with valsartan, generating an exchange of calcium, sodium and potassium ions between the various compartments and contributing to smooth muscle contraction regulation. Hypertension-induced mitochondrial structural abnormalities are often accompanied by alterations in mitochondrial metabolic and bioenergetic functions. Valsartan also downregulated mitochondrial biogenesis by down-regulated PPAGC1 which played a central role in the regulation of mitochondrial biogenesis. It could be explained by the down-regulated eNOS and VEGF-A by valsartan44,45. Mitochondrial biogenesis and oxygen consumption increase markedly during adipogenic differentiation, and reducing mitochondrial respiration by hypoxia or by inhibition of the mitochondrial electron transport chain significantly suppresses adipogenic differentiation46. In treatment with high dose SXC, cytoskeletal actin is slightly downregulated. Treatment with high dose SXC also alters vascular remodeling through other pathways. In vascular structures some results suggest that actin polymerization increases as vasoconstriction is prolonged 47. Treatment with high dose SXC also regulated IL–6 /JAK/STAT pathway which regulating the secretion of growth and migration factors like VEGF-A, Cathepsin B (both downregulated in this dataset), MMP–9 (regulator)/collagen and Stromelysin–2/Nidogen pathways which are involved in extracellular matrix and cytoskeleton remodeling. TGF-α release by ADAM–17 has also been implicated in hypertension-induced vascular remodeling in mouse carotid arteries48. Treatment with high dose SXC down-regulates ADAM–17, and there is evidence that ADAM–17 binds glycoprotein VI and triggers platelet aggregation and thrombus formation. Increased plasminogen activator inhibitor type 1 (PAI–1) levels and decreased tissue plasminogen activator activity resulting impaired fibrinolytic function have been found in patients with hypertension and may account in part for the increased risk of atherosclerosis49. Treatment with high dose SXC appears to activate of fibrinolysis via downregulated PAI–1 and upregulated plasminogen and fibrinogen. Downregulated PAI–1 also leaves thrombin to exert its fibrinolytic function via cleavage of fibrinogen in treatment of high dose SXC. Epithelial-to-mesenchymal transition (EMT) leading to fibrosis and vascular alterations appears to play a role in numerous chronic cardiovascular disease states such as heart failure, pulmonary hypertension and various forms of chronic vasculopathy50. Treatment with high dose SXC appears to down-regulate this process via IL–6/JAK/STAT pathway, N-cadherin and vimentin.
The mostly interesting finding in our study is that high dose of SXC treatment has more altered the complement-mediated immune response in vascular and reduced the enrichment of lymphocytes in hypertensive injured vascular. Treatment with high dose of SXC causes downregulation of the complement system. Tissue injury, the primary signal for launching the innate immune response and inflammation, initiates several signaling cascades that regulate changes in the microvasculature, including the activation of complement. Complement activation may drive the pathology of hypertension and hypertensive injury through its impact on innate and adaptive immune responses. In addition to the proinflammatory properties of complement, complement cleavage fragments of C3 and C5 (the latter upregulated in this dataset) can exert anti-inflammatory effects that dampen the inflammatory response to injury. Complement components are engaged in the regulation of multiple phases of an inflammatory reaction, including changes in vascular flow, increase in vascular permeability, extravasation of leukocytes, and chemotaxis51,52. IL–6 and C-reactive protein (CRP) are regulators of the complement system. IL–6 produces inflammatory effects by inducing the transcription of factors in multiple pathways of inflammation such as JAK/STAT pathway, which is downregulated upon treatment with high dose of SXC. Treatment with high dose of SXC causes downregulation of the leukocyte rolling and ultimately intima hyperplasia and endothelial dysfunction. A wealth of evidence indicates a fundamental role for inflammation in the pathogenesis of cardiovascular disease (CVD), contributing to the development and progression of atherosclerotic lesion formation, plaque rupture, and thrombosis. An increasing body of evidence also supports a functional role for complement activation in the pathogenesis of CVD through pleiotropic effects on endothelial and hematopoietic cell function and hemostasis. Complement activation contributes to endothelial cell activation; leukocyte and vascular smooth muscle cell (VSMC) migration; platelet adhesion, activation and aggregation; activation of coagulation; and impaired fibrinolysis 53,54. 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.