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REVIEW Traditional Chinese Medicine's Intervention in Endothelial Nitric Oxide Synthase Activation and Nitric Oxide Synthesis in Cardiovascular System ZHU Jin-qiang (朱金墙)1, SONG Wan-shan (宋宛珊)2, HU Zhen (胡 珍)1, YE Qiao-feng (叶乔峰)1, LIANG Yu-bin (梁钰彬)1, and KANG Li-yuan (康立源)1 ABSTRACT Cardiovascular disease (CVD) is one of the most dangerous diseases which has become a major cause of human death. Many researches evidenced that nitric oxide (NO)/endothelial nitric oxide synthase (eNOS) system plays a significant role in the occurrence and development of CVD. NO, an important signaling molecule, closely associated with the regulation of vasodilatation, blood rheology, blood clotting and other physiological and pathological processes. The synthesis of NO in the endothelial cells primarily depends on the eNOS activity, thus the exploration of the mechanisms and effects of the eNOS activation on NO production is of great significance. Recently, studies on the effects of traditional Chinese medicine (TCM) and its extracts on eNOS activation and NO synthesis have gradually attracted more and more attentions. In this paper, we reviewed the mechanisms of NO synthesis and eNOS activation in the vascular endothelial cells (VECs) and intervention of TCM, so as to provide reference and train of thought to the intensive study of NO/eNOS system and the research and development of new drug for the treatment of CVD. KEYWORDS traditional Chinese medicine, cardiovascular disease, endothelial nitric oxide synthase activation, nitric oxide synthesis

In the 1980s', Furchgott, et al (1) found out that acetylcholine (ACh) relied on the existence of vascular endothelium to relax blood vessels and speculated that the vascular endothelial cells (VECs) could synthesize and secrete active substances, which they called endothelium derived relaxing factor (EDRF). It was confirmed by Palmer, et al(2) that the chemical nature of the EDRF was nitric oxide (NO). Then NO immediately became one of the hotspots in pharmaceutical research. Numerous studies show that NO is one signaling molecule with strong activity and plays an important regulatory role in cardiovascular system, nervous system, circulatory system, respiratory system and other systems, especially those closely associated with the occurrence and development of hypertension, heart failure, shock, atherosclerosis and other cardiovascular diseases (CVD). Although NO has strong activity, studies on it mainly concentrated on the key enzyme in NO synthesis, because of its short half-life, particularly on "endothelium" NO synthase (eNOS) isoforms in VECs. The advances in researches of NO synthesis, eNOS activation mechanism in VECs and traditional Chinese medicine (TCM) interventions in both former aspects were summarized in this paper.

Origin and Physiological Characteristics of NO Now, it is a general knowledge that NO is synthesized from L-arginine and oxygen, which is catalysed by the NOS family through the reaction of multi-step oxidation-reduction. Currently, NOS is known to be generally divided into two types: constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS), while cNOS can be divided into two subtypes: eNOS and neural nitric oxide synthase (nNOS).(3) There are different types of NOS in different kinds of cells, and they can promote NO synthesis through different signal transduction mechanisms. eNOS and nNOS are mainly distributed in the endothelial cells and the nervous system respectively, which are in charge of

©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2015 Supported by the National Key Basic Research and Development Program of China (No. 2012CB518404) 1. Institute of Traditional Chinese Medicine, Tianjin Key Laboratory of Chinese Medical Pharmacology, Tianjin University of Traditional Chinese Medicine, Tianjin (300193), China; 2. Encephalopathy Department, the Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin (300150), China Correspondence to: Dr. KANG Li-yuan, Tel: 86-22-59596345, E-mail: [email protected] DOI: 10.1007/s11655-015-1964-1

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basic NO synthesis and regulate various physiological functions. eNOS and nNOS can be Ca2+-dependently activated by ACh or bradykinin: they could be activated and rapidly promote the synthesis of NO when the concentration of intracellular Ca 2+ is elevated, while decreasing NO production when the concentration of Ca2+ is decreased. Consequently, an undulatory property of NO release necessary for physical adjustment will be formed. eNOS is a kind of protein combined with membrane and has some effect on controlling blood vessel tension and platelet aggregation, while nNOS is a protein existing in cytosol and works as neurotransmitters. iNOS is not only expressed in immunoreactive cells (e.g. macrophages and neutrophils), but also expressed in fibroblast, keratinocyte cells, endothelial cells and vascular smooth muscle cells. iNOS doesn't possess Ca 2+ dependency and is not expressed in resting cells; while its activation is stimulated by cytokines or immune microorganisms, such as tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ), interleukin-1β (IL-1β) and lipopolysaccharide (LPS), and catalyse massive synthesis of NO above physiological concentration, which can cause a series of pathological impacts.(4,5) NO is extremely unstable in vivo due to its short half-life (only 1 to 5 s), and has unpaired electrons in the molecule, which can form free radicals and react with other molecules including oxygen, super-oxide radical and transition metals (e.g. iron combined with hemoglobin). It is fat-soluble, so that it could pass through the cell membrane and spread rapidly, and could be quickly inactivated by hemoglobin, oxygen free radicals or hydroquinone in vivo .(6) It is through the cyclical process of generation and inactivation that NO plays an important role in regulating a variety of physiological functions. Due to its unique physical, chemical properties and biological activity, NO has effect on both targeting other molecules by synthesizing them and also by circumambient specific receptors, meaning that NO plays a role of information transfer among cells and synapses in an autocrine and/or paracrine way, which is different from other common physical substances. Therefore, NO is a messenger molecule with both features of the first messenger and the second messenger. In addition, it has double regulatory effects. It can control blood tension, improve blood flow, inhibit platelet aggregation, regulate the non-adrenergic and non-

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cholinergic (NANC) neurotransmitter and improve memory when the concentration of NO is low, while relate to cell toxicity, inhibit tumor cells growth (antitumor), and increase the ability of defense to pathogen when its concentration is high.(7) Otherwise, NO as a water-soluble gas molecule in the cardiovascular system is not stored in the organelles like other signal molecules, and its production is mainly determined by the activity of eNOS.

eNOS Activation Mechanisms and NO Synthesis eNOS activity is closely related to NO synthesis in the VECs. eNOS has a homologous dimer structure consisting of two identical subunits, and each subunit contains an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain contains binding sites for heme, L-arginine, and tetrahydrobiopterin (BH4), and the reductase domain contains binding sites for flavine mononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADPH) and calmodulin (CaM). In the catalytic reaction of eNOS, NADPH-derived electrons pass through the reductase domain flavins and then must be transferred to the heme located in the oxygenase domain, so that the heme iron could bind with O2 and stepwise catalyse NO synthesis from L-arginine (Figure 1). As electrons need transfer from the reductase domain of an eNOS monomer to the oxygenase domain of another eNOS monomer, the dimerization of eNOS monomer is the precondition for expressing its activity.(8,9) eNOS is a Ca2+/CaM-dependent enzyme, which is regulated by Ca2+/CaM and phosphorylation, and its main activation pathways include Ca 2+ transient-induced isolation of caveolin/eNOS combination, the regulation of heatshock protein 90 (Hsp90) and the phosphorylation of eNOS.(10)

Caveolin-1 Regulates eNOS Activity In VECs, eNOS activity is mostly related to the interaction with caveolin-1 (Cav-1). The caveolin scaffolding domain (CSD) will restrict eNOS trafficking and activation after binding to eNOS. (11) Two cytoplasmic domains of Cav-1 are documented to interact with eNOS: the N-terminal oligomerization domain and a lesser extent C-terminal tail. (12) The sites of Cav-1 binding to eNOS are not only positioned in the oxygenase domain of eNOS but also located in its reductase domain, and the binding of Cav-1 and eNOS can play a synergistical role in modulating

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NADPH HOOC

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NADP++H+ -

e →FAD→FMN

CaM L-Arg

Heme

NH2

BH4 BH4 H2N

Heme

L-Arg CaM

FMN←FAD←eNADP++H+

reductase domain

COOH NADPH

oxygenase domain

Figure 1. Model for an eNOS Dimer Including Domain Swapping and Electron Transfer Pathway Notes: eNOS has a homologous dimer structure consisting of two identical subunits, and each subunit contains an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain contains binding sites for heme, L-arginine (L-Arg), and tetrahydrobiopterin (BH 4 ), and the reductase domain contains binding sites for flavine mononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADPH) and calmodulin (CaM). e-=electron

regulate Cav-1 expression in endothelial cells so as to promote vasodilation.(22) Furthermore, Cav-1 can activate phosphatidylinositol-3-kinase (PI3K) signal pathway via promoting the binding of estrogen receptor α (ERα) to plasma membrane, leading to eNOS activation and NO synthesis.(23) In intact microvessel, Cav-1 and endothelial [Ca 2+ ] i antagonistically regulate eNOS activity, and increase NO production, which is the key determinant of the degree of permeability increasing during inflammation. (24) Recent studies suggested that Ser activation induced the phosphorylation of protein kinase B (Akt), Cav-1 and eNOS, and facilitated dissociation of eNOS from Cav-1, thereby causing eNOS activation. On the contrary, activation of eNOS promoted Src-dependent Cav-1-Tyr-14 phosphorylation and eNOS/Cav-1 binding, thereby depressing eNOS activity, which is the feedback inhibition of eNOS.(25,26)

Hsp90 and eNOS Activity the catalytic activity of eNOS. Cav-1 combination with the reductase domain of eNOS inhibits heme iron reduction and NO synthesis, which is primarily responsible for antagonizing CaM binding to its consensus sequence in the "hinge'' region and for slowing electron transfer from the reductase. Also, the Cav-1 binding motif in eNOS lies between the heme and the CaM binding domains adjacent to a glutamate residue (Glu-361), which is necessary for the binding of L-arginine. (13-15) Experiments in vitro and in vivo show that, in the absence of Cav-1, eNOS presents the state of hyperactivity.(16,17) However, whether this ability is due to the lack of a direct interaction between eNOS and Cav-1, or interference in the caveolar signal is not yet clear. Two new proteins named NOSIP (an eNOS interacting protein) and NOSTRIN (an eNOS traffic inducer) specifically modulate the caveolin/eNOS interaction. Overexpression of either protein in eNOS-expressing cells has similar effects on the translocation of eNOS away from the plasma membrane and uncoupling of eNOS from the signaling platforms concentrated in plasma membrane caveolae, which leads to reduce NO production.(18-20) Previous research reported that insulin can not only promote the interaction between eNOS and Cav-1, but also induce eNOS/Cav-1 to transfer and bind to plasma membrane, thereby regulating eNOS activity and NO production. (21) The interaction of eNOS and Cav-1 is also regulated by the estrogen. Estrogen can up-regulate the expression of eNOS and down-

Hsp90, an allosteric modulator of eNOS, can facilitate CaM-induced dissociation of eNOS from Cav-1, and modulate heme insertion and the formation of active dimeric enzyme form, thus affecting eNOS proteolysis. Therefore, it will enhance the catalytic activity of eNOS and NO production after binding to eNOS.(27,28) It is reported that eNOS is a highly sensitive calpain substrate. In the presence of Ca2+, calpain can be recruited in a ternary complex containing eNOS, Hsp90, and the protease, in which Hsp90 and eNOS become resistant to calpain digestion. Moreover, the level of Hsp90 expression is directly correlated with the extent of eNOS degradation. Accordingly, in the presence of the Hsp90 inhibitor geldanamycin (GA), NO production was largely decreased, and eNOS was extensively degraded. Otherwise, in rat aorta, under conditions of calpain activation, eNOS is much more vulnerable to proteolytic degradation than nNOS, since Hsp90 is more abundant in brain, which also demonstrates that Hsp90 operates in protecting eNOS from calpainmediated degradation in the course of the intracellular dynamic redistribution that accompanies the eNOS activation and NO production.(29,30) Aschner, et al(31) also found that Hsp90 is a modulator of eNOS activity and vascular reactivity in the newborn piglet pulmonary circulation. Uncoupling of eNOS with Hsp90 inhibitor GA or redicicol can inhibit ACh-induced vasorelaxation, and this action is associated with generation. Hsp90 also can bind with other regulatory

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molecules (such as kinases and phosphatases, etc.) to form a complex as scaffold proteins, thereby affecting the eNOS activity.(32) In addition, carboxyterminal region residues S-nitrosylation of Hsp90 can also regulate eNOS phosphorylation.(33)

Effects of eNOS Phosphorylation on Its Activity eNOS phosphorylation can regulate its activity and endothelium NO synthesis through the influence on the binding of eNOS and CaM. eNOS can be phosphorylated on serine, threonine and tyrosine residues, which highlight the potential role of phosphorylation in regulating eNOS activity. It was confirmed that there are numerous phosphorylation sites in eNOS, but mostly known the functional consequences of phosphorylation of a serine residue (human eNOS sequence: Ser1177) in the reductase domain and a threonine residue (human eNOS sequence Thr495) within the CaM-binding domain. In unstimulated endothelial cells, Ser1177 is not phosphorylated but is rapidly phosphorylated after the stimulation of fluid shear stress, vascular endothelial growth factor (VEGF) or bradykinin via protein kinase A (PKA) or Akt pathway;(34-37) Ser633 is situated in a recently defined CaM autoinhibitory domain within the flavin-binding region of eNOS, which can be phosphorylated in vitro by PKA and PKG pathways in order to facilitate the binding of eNOS and CaM and enhance its activity, but the speed of Ser633 phosphorylation is much lower than that of Ser1177 and Thr495;(38) Thr495, phosphorylated most probably by PKC pathway, is a negative regulatory site phosphorylation, and is associated with a decrease in enzyme activity. (39) Indeed, in VECs, substantially more CaM binds to eNOS when Thr495 is dephosphorylated stimulated with agonists such as bradykinin, histamine, or a Ca 2+ ionophore, (34) which also elevate endothelial [Ca2+]i and increase eNOS activity by ten- to twenty- fold over basal level. Furthermore, the dephosphorylation of Thr495 even more associates with eNOS uncoupling in vitro . (40) However, it remains to be investigated whether this occurs in vivo and relates to a decrease in BH4 and/or L-arginine availability. All of Ser1177, Ser633 and Thr495 phosphorylation are regulated by adenosine 5'-monophosphate (AMP)activated protein kinase (AMPK). Numerous data suggested that the AMPK-dependent phosphorylation

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of eNOS (on Ser1177) follows endothelial cells stimulation with VEGF, hypoxia, peroxisome proliferatoractivated receptor-agonists, and adiponectin.(41) After stimulation with atorvastatin, the AMPK phosphorylate eNOS on Ser633 following endothelial cells and no such observation could be detected in endothelial cells from AMPKα2–/– mice.(42) It is worth mentioning that, under normal conditions, the AMPK is unlikely to play a major role in the regulation of eNOS, but Akt, PKA, and CaMKⅡ would be expected to have dominant effect in this process. Additionally, the tyrosine kinases inhibitors can reduce the endothelium NO releasing and fluid-induced vasorelaxation, which suggest that tyrosine plays a certain role in the regulation of eNOS activity.(43-45)

Main Factors Affecting NO Synthesis Factors affecting NO synthesis are very complicated, mainly including CaM, BH4/dihydrobiopterin (BH2), platelet/endothelial cell adhesion molecule-1 (PECAM-1), shear stress, angiotensin Ⅱ (AngⅡ) and so on. CaM is the first reported eNOS-associated protein, whose association with the CaM-binding domain within eNOS is determined by multiple molecular interactions as well as by Thr495 phosphorylation/ dephosphorylation.(34,46) Besides, Hsp90 and Ser1177 phosphorylation can also influence the interaction between these two proteins.(47) In cultured VECs, augment in BH 4 levels can improve NO output, and the lack of BH4 results in the uncoupling of NOS, which causes the generation of reactive oxygen species, NO bioavailability decrease and endothelial dysfunction. BH2 and BH4 bind eNOS with equal affinity and it can efficiently replace BH4 to bind to eNOS causing eNOS uncoupling.(48) BH4/BH2 ratio plays a potent role in the regulation of eNOS activity. Dihydrofolate reductase (DHFR) can reduce BH2 and regenerate BH4. Indeed, DHFR expression is reduced by AngⅡ, a stimulus that also decreases BH4 levels and elicits eNOS uncoupling.(49) PECAM-1 contains two intracytoplasmic immunoreceptor tyrosine-based inhibitory motifs.(50) It plays the role of mechanotransduction in endothelial cells when it is rapidly tyrosine-phosphorylated following the application of fluid shear stress

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under static conditions. (51) In human endothelial cells, downregulation of PECAM-1 significantly attenuated the shear stress-induced Akt and eNOS phosphorylation and decreased eNOS activity.(52)

In vitro experiment results suggested that laminar shear stress could increase the enzymatic activity of guanosine triphosphate cyclohydrolase and the subsequent generation of BH4.(53) Shear stress-induced NO increase is related to eNOS phosphorylation and enhance sensitivity of the enzyme for Ca2+, so that eNOS can be activated at resting level.(54) In a word, the signaling pathways involved in the regulation of eNOS expression are wondrous complex, mainly attribute to the transcription factors nuclear factor-κB (NF-κB) and Kruppel-like factor-2 (KLF-2), and eventually act on forkhead box protein O1 (FoxO1) and specific microRNAs.(8)

Intervention of TCM or Its Extract in eNOS Activation and NO Synthesis in Cardiovascular System As mentioned above, signal transduction pathways of eNOS activation mechanism are so extremely complex that they can form a complex network. A large number of researches demonstrated that TCM or its extract containing complex compositions can intervene in eNOS activation and NO synthesis, which plays a great and potent role in prevention and treatment of CVD. Ginsenoside Rg1 and Rb1 can indeed serve as an agonist ligand for glucocorticoid receptors (GR) causing eNOS activation and rapid NO production via the non-transcriptional PI3K/Akt pathway and/or mitogen-activated protein kinase kinase (MEK)/extracellular singal-regulated kinase (ERK) pathway, and it is interesting that androgen receptor is involved in the regulation of acute eNOS activation by Rb1. (55,56) Two metabolites of ginsenoside, protopanaxadiol (g-PPD) and protopanaxatriol (g-PPT), are functional ligands for both GR and ERβ, and can increase [Ca2+]i, eNOS phosphorylation and NO production in human umbilical vein endothelial cells (HUVECs). (57) Moreover, different extract from the ginseng affect NO synthesis and vascular endotheliumdependent relaxation through multiple signaling pathways. Protopanaxatriol-enriched extract (TE) has the highest potency in NO production, followed by crude

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extract (CE), protopanaxadiol-enriched extract (DE), and Rg1. eNOS activation and NO production immediately appear in a linear increase in HUVECs treated with TE via rapid activation of intracellular signaling pathways. TE-induced activation of eNOS is abolished by pretreatment with PI3K-Akt inhibitor wortmannin, AMPK inhibitor compound C or NOS inhibitor NG-nitro-Larginine methyl ester (L-NAME), whereas Rg1-induced eNOS phosphorylation is only partially attenuated. Further analysis revealed that TE, but not Rg1, results in AMPK phosphorylation at Thr172.(58) In human endothelial cells (ECV304), Rg3 increases both the phosphorylation and the expression of eNOS in a concentration-dependent manner, and enhances the enzyme activities of PI3K, c-Jun N-terminal kinase (JNK), and p38 kinase as ERand GR-dependent.(59) Puerarin has the similar action with Rg3 as it activates the ER-mediated PI3K/Akt- and CaMKⅡ/ AMPK-dependent pathway to stimulate eNOS phosphorylation and NO production. (60) Saponins derived from the roots of platycodon grandiflorum (CKS) stimulate eNOS phosphorylation and NO production via the activation of PI3K/Akt, p38/ mitogen-activated protein kinase (MAPK), AMPK, and CaMK Ⅱ.(61) Syringaresinol induces vasorelaxation by two distinct mechanisms involving PI3K/Akt- and phospholipase C (PLC)/ Ca 2+/CaMKKβ-dependent eNOS phosphorylation and Ca2+-dependent eNOS dimerization to enhance NO production in endothelial cells.(62) Ginkgo biloba extract EGb761 increases NO production contributing to NO-dependent vasorelaxation and reduces blood pressure by enhancing eNOS promoter activity, eNOS expression and Akt-induced phosphorylation of eNOS at the Ser 1177. (63) Qing Huo Yi Hao (清 活1号), a Chinese herbal formula, and its active components ligustrazine stimulate uncoupling protein 2 mRNA and protein expression, and reverse the down-regulation of eNOS Ser1177 and Akt/eNOS phosphorylation and the reduction of NO generation in high glucose-induced brain microvascular endothelial cells (bEnd.3), HUVECs and human aortic endothelial cells (HAECs), thereby expressing the antioxidant and endothelial protective effects.(64) Rhizoma coptidis rhizome extract berberine enhances phosphorylation of eNOS at Ser1177 and promotes the association of eNOS with Hsp90 to increase NO production in a dose dependent manner.(65) Resveratrol, a grapederived polyphenolic phytoalexin produced by several plants, enhances phosphorylation of eNOS at Ser1177

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and AMPK at Thr172 to increase NO output, elicites endothelium-dependent vasodilatations and alleviates high glucose-mediated endothelial dysfunction in a dose dependent manner.(66) Another compound from vitis amurensis grapes amurensin G enhanced NO production via eNOS phosphorylation in endothelial cells, and ER-dependent AMPK/PI3K pathways are required.(67) In ECV304, nectandrin B isolated from Myristica fragrans as a potent AMPK activator can increase eNOS phosphorylation and NO production in a concentration-dependent manner, and ERαdependent PI3K activity is required.(68) Onychin has a protective action and antagonizes the growth inhibition of endothelial cells injured by menadione though increasing eNOS activity and up-regulating phosphERK1/2 expression. (69) Depside salt from Salvia Miltiorrhiza can increase the platelet eNOS activity evidently at the concentration below 10 mg/L to inhibit platelet aggregation.(70) An animal experiment showed that extracts from leaves of Apocynum venetum L. had significant anti-hypertensive properties. In vitro experiments showed that it increased NO yield and eNOS activities, and enhanced expression of eNOS, p-eNOS, p-PI3K and p-Akt in EA hy 926 cells, which suggested that its mechanisms may be associated with activation of PI3K/Akt pathway in endothelium. (71) Osthole, a natural coumarin compound isolated from Angelica pubescens Maxim, was dependent on endothelial integrity and NO production, and was mediated by endothelial PI3K/Akt-eNOS-NO pathway.(72)

Summary and Prospect Due to the high morbidity and mortality of CVD, operative medicine development as well as other intervention strategies has become a hot and difficult point. Plenty of researches show that NO/eNOS system plays an important role in CVD such as hypertension, atherosclerosis, shock, heart failure, and so on. Therefore, strengthening eNOS activity and promoting the NO synthesis has become one of the effective means of prevention and treatment of CVD. eNOS activation mechanism is a complex and precise regulation network, for example, phosphorylation at Serl179/Ser635 sites enhances affinity between eNOS and CaM, so that eNOS can even be activated by [Ca2+]i at resting level.(73,74) The clarifications of these mechanisms will help for developing new drugs for the prevention and control of CVD. Thus, CaMKⅡ/ AMPK and PI3K/Akt signal transduction pathway,

Cav-1, Hsp90, eNOS phosphorylation loci, which are associated with eNOS activation, and CaM, BH4/BH2, PECAM-1, shear stress, AngⅡ, which are associated with the NO synthesis, may become targets for clinical intervention (Figure 2). Recent studies showed that TCM is characterized by multiple components, multiple targets, multiple approaches, and its active ingredients combined with each other in a reasonable and complex manner to act on multiple targets associated with the diseases in the body through multiple pathways, which plays the role of the body's overall regulation, and it is the advantage of TCM for treatment of diseases.(75) As mentioned above, TCM or its extract can activate eNOS through several multiple pathways, such as PI3K/Akt, MEK/ERK or AMPK signaling pathways, which may adjust eNOS activation and the NO synthesis at the same time to play more effective role in prevention and treatment of CVD (Figure 3). Research on this field, selecting active ingredients of Chinese herbal medicine about this regulatory mechanism could be of a great importance to the prevention and treatment of CVD. Drugs

Drugs L-Arginine

ER GR

NADPH BH

4

MAPK Ca2+

Ang Ⅱ

P

P Hsp90

eNOS

Cav-1

PI3K

L-Citrulline ER GR NO CaMK Ⅱ

Akt

Ca2+ AMPK

CaM translation

P

NF-κB eNOS gene AP-1 KLF2 Foxo-1 P Inhibitory site P Stimulatory site

Cav-1

Figure 2. Regulation of eNOS Activation and NO Synthesis Notes: eNOS activation mechanism is a complex and precise regulation network. It include CaMKⅡ/AMPK and PI3K/Akt signal transduction pathway, Cav-1, Hsp90, eNOS phosphorylation loci, which are associated with eNOS activation, and CaM, BH4/BH2, PECAM-1, shear stress, AngⅡ, which are associated with the NO synthesis. TCM

TCM ER GR + + TCM

ER GR

NO

p38

MAPK +

L-Citrulline

L-Arginine P

+

PI3K P Akt P

eNOS –

AMPK P

P translation NF-κB eNOS gene AP-1 KLF2 Foxo-1

P Inhibitory site

CaMK Ⅱ Ca2+

P Hsp90

P Stimulatory site

+

+ Stimulation

TCM – Inhibition

Figure 3. Intervention of TCM or Its Extract in eNOS Activation and NO Synthesis in Cardiovascular System Notes: TCM or its extract can activate eNOS through several multiple pathways, such as PI3K/Akt, MEK/ERK or AMPK signaling pathways, to increase eNOS phosphorylation at the Ser 1177 and promote the association of eNOS with Hsp90, which may stimulate eNOS activation and the NO synthesis.

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Conflict of Interest The authors declare that there is no conflict of interest.

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