Therapeutic Potential of Hydroxysafflor Yellow A on Cardio-Cerebrovascular Diseases

The incidence rate of cardio-cerebrovascular diseases (CCVDs) is increasing worldwide, causing an increasingly serious public health burden. The pursuit of new promising treatment options is thus becoming a pressing issue. Hydroxysafflor yellow A (HSYA) is one of the main active quinochalcone C-glycosides in the florets of Carthamus tinctorius L., a medical and edible dual-purpose plant. HSYA has attracted much interest for its pharmacological actions in treating and/or managing CCVDs, such as myocardial and cerebral ischemia, hypertension, atherosclerosis, vascular dementia, and traumatic brain injury, in massive preclinical studies. In this review, we briefly summarized the mode and mechanism of action of HSYA on CCVDs based on these preclinical studies. The therapeutic effects of HSYA against CCVDs were presumed to reside mostly in its antioxidant, anti-inflammatory, and neuroprotective roles by acting on complex signaling pathways.


INTRODUCTION
Cardio-cerebrovascular diseases (CCVDs) are characterized by ischemic or hemorrhagic lesions of the heart, brain, and peripheral circulatory tissues . It is the high incidence, recurrence, and disability rates of CCVDs that directly aggravate the global burden of public health and hinder socio-economic development (Minno et al., 2019). Although much progress has been made in understanding the pathological mechanisms of CCVDs, there is still no effective therapy to prevent or stop the epidemic trend of CCVDs, resulting in the urgent need to identify novel therapeutic options (Kazantsev and Outeiro, 2010;Upadhyay, 2014).
Traditional Chinese medicine (TCM), a cost-effective and safe remedy, has been widely used in China and surrounding countries (including Japan and Korea) for the treatment and management of CCVDs with exact and prominent efficacy. Carthamus tinctorius L. (Compositae) ( Figure 1A) seeds are known to be rich in a-linoleic acid and have been used since ancient times as a source of cooking oil. Meanwhile, its flowers are widely used for coloring and flavoring foods and manufacturing dyes Guo et al., 2019). Notably, the medical use of Carthami Flos ( Figure 1B, the dried florets of C. tinctorius) was first documented in the Golden Chamber Synopsis (Han Dynasty,~2000 years ago) (Ma et al., 2014), and also described in the Compendium of Materia Medica (Ming Dynasty,~500 years ago) as being able to "invigorate the blood circulation", suggestive of its potential uses against circulatory system diseases. In modern Chinese clinic, Honghua injection (made from the water extract of Carthami Flos) and Danhong injection (extracted and refined from Salviae miltiorrhizae Radix et Rhizoma and Carthami Flos herb pair) are widely used for the treatment of coronary heart disease, angina pectoris, myocardial infarction, ischemic encephalopathy, and cerebral thrombosis (Fan et al., 2019;Zhang et al., 2019).
The chemical constituents of Carthami Flos are plentiful, and include flavonoids (e.g. quinochalcone C-glycosides), alkaloids, phenolic acids, fatty acids, and more (Yue et al., 2013). Among them, hydroxysafflor yellow A (HSYA, Figure 1C) is both a representative water-soluble quinochalcone C-glycoside pigment and the quality marker of Carthami Flos. It produces remarkable pharmacological activities in CCVDs that have aroused great interest worldwide (Zhang et al., 2019), and massive preclinical studies have aimed to prove the pharmacological effects and dissect the mechanisms of actions of HSYA in treating CCVDs. Safflower yellow injection, a purified yellow pigment extract from Carthami Flos containing no less than 70% of HSYA, is commercially available for stable exertional angina pectoris of coronary heart disease with a marked curative effect in Chinese clinic Xuan et al., 2018). Here, we briefly summarize the existing evidence to provide valuable references and implications for the clinical uses of HSYA.

THERAPEUTIC POTENTIAL OF HSYA ON CCVDS Effects on Myocardial Ischemia (MI)
It is acknowledged that MI results from insufficient bloodoxygen supply (Thiagarajan et al., 2017) and the improved vasomotor and circulatory functions exert beneficial effects on MI (Ribeiro et al., 2019). The vasoconstrictor endothelin and the vasodilator nitric oxide (NO) are known as two common regulators modulating vasomotor function. HSYA can reverse the circulating levels of both in acute MI animals (e.g., dogs and rats), thereby elevating myocardial blood-oxygen supply and reducing myocardial injury and apoptosis (Li et al., 2006;Wang et al., 2007). Other vasomotor function-related factors, such as 6-keto-prostaglandin F1a, thromboxane B2, and angiotensin II (Ang II), were also of great importance for HSYA (Wang et al., 2007). Angiogenesis participates in the circulatory function recovery from MI, and HSYA exerts the pro-angiogenic effects in two main ways: (1) nucleolin-mediated post-transcriptional regulation of vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase (MMP) -9 expressions (Zou et al., 2018); and (2) the up-regulation of heme oxygenase-1 (HO-1)/VEGF-A/stromal cell-derived factor-1a cascade .
A specialized piece of in vivo research demonstrated that the antioxidant effect of HSYA was involved in the prevention of Ang II-induced myocardial hypertrophy (a compensatory response to MI), which may act through the activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)/NAD(P)H: quinone oxidoreductase 1/HO-1 signaling pathway (Ni et al., 2018). Nrf-2, as the main regulator of the antioxidant system present on the cardiovascular system, is becoming a very promising pharmacological target for cardiovascular diseases (McSweeney et al., 2016). Our research group has found that HSYA possessed significant antioxidant activity in vitro (Yue et al., 2014). Thus, the antioxidant effect of HSYA may be essential to improve the outcomes of cardiovascular diseases.
Beside inflammation and apoptosis, MI/R damages cardiomyocytes in part via the opening of the mitochondrial permeability transition pore (mtPTP), a non-selective pore that penetrates the inner and outer mitochondrial membranes (Bhosale and Duchen, 2019). HSYA has the capability to enter the cardiomyocytes and then inhibit mtPTP opening to alleviate H/R-induced myocardial injury through the enhanced endothelial nitric oxide synthase (eNOS)-produced NO Huber et al., 2018).

Effects on Hypertension
Hypertension is a major global health challenge and an important risk factor of CCVDs. The blood pressure control rate of hypertensive patients in developing countries remains at unacceptably low levels (Mills et al., 2016). There is evidence that the conspicuous antihypertensive effect of HSYA is attributed to the inhibition of voltage-gated channels, the renin-angiotensinaldosterone system, and the sympathetic nervous system. Specifically, HSYA inhibited the endotehlin-independent contraction of the thoracic aorta rings of rats through the blockade of inositol 1,4,5-triphosphate receptor in vascular smooth muscle cells (VSMCs), leading to the decrease of extracellular Ca 2+ influx . Beside VSMCs, endothelial cells participate in vasoconstriction and relaxation. Yang et al. found that oral HSYA has a concentration-dependent antihypertensive effect. It reversed the constriction of mesenteric arteries induced by a thromboxane A2 mimetic agent, the potential mechanism of which might be associated with the TRPV4 channel-dependent Ca 2+ influx, protein kinase Adependent eNOS phosphorylation, and NO production (Yang et al., 2020). A further study disclosed that HSYA could normalize blood pressure and heart rate dose-dependently in spontaneously hypertensive rats, which might be related to activating K ATP and BK Ca channels, inhibiting L-type Ca channels, decreasing Ca 2+ influx, and subsequently inhibiting cardiac contractility (Nie et al., 2012;Wang et al., 2020). However, HSYA reduced blood pressure and heart rate in normotensive rats, on which more focus needs to be placed in the future. Moreover, HSYA can increase the reduced diastolic response of the thoracic aortic to acetylcholine and sodium nitroprusside, and thus attenuate the vascular contractile effect of phenylephrine (Jin et al., 2013). HSYA can also inhibit proliferative activity and collagen synthesis of Ang II-induced adventitial fibroblasts, reduce the expressions of MMP-1, transforming growth factor-b1, a-smooth muscle actin, and NF-kB p65, and thereby decreasing vascular adventitia proliferation and hyperplasia during vascular remodeling (Yuan et al., 2014). Obviously, it could be drawn that HSYA might be potentially useful as an antihypertensive drug via multiple mechanisms mainly involved in both cardiac output and peripheral resistance.
Hypertension may cause ventricular hypertrophy, which produces mechanical stimulation to the heart, further causing arrhythmia, heart failure, coronary occlusion, and sudden death (Pearson et al., 1991). A study by Wang et al. indicated that oral HSYA exhibited anti-apoptotic effects on hypertensive ventricular hypertrophy in rats by increasing the B-cell lymphoma-2 (Bcl-2)/ Bcl-2 associated X protein (Bax) ratio and blocking serum MMP-2 and MMP-9 levels . Moreover, pulmonary arterial hypertension is a common combination of congenital heart disease with systemic-to-pulmonary artery shunt diseases, leading to right ventricular heart failure and premature death . Voltage-gated K + channel (K V channel) is an important channel for maintaining normal membrane potential and muscle tension of VSMCs, while HSYA could activate the Kv channel of pulmonary artery VSMCs to reduce vascular tension, suggesting that HSYA may be a potential medication for pulmonary arterial hypertension (Bai et al., 2012).

Effects on Atherosclerosis (AS)
AS could precipitate the onset of myocardial infarction and inflammation and is being increasingly recognized as the main pathogenic mechanism through the narrowing and blockage of arteries and the increased risk of blood vessel rupture (Kotla et al., 2017). HSYA could inhibit ROS-induced inflammation in THP-1 macrophages (Jiang et al., 2017), but could also suppress tumor necrosis factor-a (TNF-a)-induced inflammatory responses through inhibiting the TNF-a receptor type 1mediated classical NF-kB pathway in arterial endothelial cells (Wang H. F. et al., 2016). Jiang et al. discovered that HSYAmediated sonodynamic therapy induced an autophagic response to inhibit inflammation via the PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway in THP-1 macrophages (Jiang et al., 2017). Nevertheless, it is very interesting to justify the potential biphasic effect of HSYA under excessive macrophage autophagy, which can drive the instability of atherosclerotic plaque (Petrovski et al., 2011).
In fact, the oxidation of low-density lipoprotein (LDL) and oxidized LDL (ox-LDL)-induced vascular damage are key events in early AS. Although its LDL-lowering effect remains unknown, HSYA was able to reduce the susceptibility of LDL to copperinduced lipid peroxidation in vitro (Bacchetti et al., 2020). It is interesting to identify the effect of HSYA against ox-LDL formation in vivo. In ox-LDL-induced foamy macrophages, HSYA displayed obvious repairing effects on the de novo fatty acid biosynthesis pathway, among which oleoyl-(acyl-carrier-protein) hydrolase was postulated to be a target of HSYA (Wei et al., 2018). HSYA also exerted protective effects against ox-LDL-induced VSMCs proliferation via increasing mitogen-activated protein kinase phospholipase-1 expression and the proportion of cells in the G0/ G1 phase, followed by reducing p-extracellular regulated protein kinases activity (Sheng et al., 2012). Moreover, HSYA has been shown to significantly improve ox-LDL-induced human endothelial injury, partially via the anti-apoptotic effect of the mitochondrial membranous voltage-dependent anion-selective channel protein 2 (Ye et al., 2017). Recently, Miao et al. revealed that HSYA could inhibit the high ox-LDL-induced human coronary artery endothelial cell injury, possibly via increasing eNOS expression and NO release, while inhibiting LDL receptor-1 expression and lactate dehydrogenase release (Miao et al., 2019).
During the development of AS, platelets can accelerate activation and release a variety of active substances, such as platelet-activating factor (PAF) and thromboxane B2, conversely promoting platelet adhesion and aggregation, and even damaging vascular endothelial cells . HSYA was able to inhibit PAF-induced platelet aggregation in rabbits by blocking PAF-mediated washed rabbit platelets (WRPs) and polymorphonuclear leukocyte aggregation (Zang et al., 2002).
Collectively, the above in vitro studies have manifested the potential anti-AS effects of HSYA, which should go through additional in vivo studies to determine its clinical implications.

Effects on Vascular Injury and Remodeling Diseases
The vascular endothelium plays an important role in modulating numerous aspects of vascular homeostasis (Scarabelli et al., 2002). HSYA was capable of promoting the survival and proliferation of vascular endothelial cells under both normoxic and hypoxic conditions, and its effect was stronger under hypoxia via upregulating the Bcl-2/Bax ratio and accumulating hypoxiainducible factor-1 (HIF-1) a, which was related to VEGF and its receptor system (Song et al., 2005;Ji et al., 2008). Also, HSYA could protect human umbilical vein endothelial cells (HUVECs) from hypoxia-induced injury by reducing p53 expression in the cell nucleus and up-regulating eNOS expression to produce NO in cell supernatant (Ji et al., 2009).
Abnormal proliferation of VSMCs is a crucial cytopathological basis for the development and progression of vascular remodeling diseases (Ivey et al., 2008). HSYA could inhibit platelet-derived growth factor-BB induced VSMCs proliferation by decreasing proliferating cell nuclear antigen expression and blocking mitogen-activated protein kinase/extracellular regulated protein kinases and Akt signaling pathways (Song et al., 2014;Zhao et al., 2015). In the LPS-induced VSMCs proliferation and migration model, HSYA inhibits the up-regulation of TLR4 expression and the activation of Ras-related C3 botulinum toxin substrate 1/Akt pathway (Yang et al., 2015).

Effects on Cerebral Ischemia (CI)
CI is one of the leading causes of death worldwide, and patients who survive CI often experience paralysis, impaired speech, or loss of vision (Moskowitz et al., 2010). HSYA appears to treat focal CI injury in rats through its anti-coagulation effects on thrombosis formation and platelet aggregation, as well as beneficial regulation on prostacyclin/thromboxane and blood rheological changes . HSYA could also preserve cortex mitochondrial function of CI rats via scavenging free radicals, reducing lipid peroxides, and antagonizing Ca 2+ (Tian et al., 2004). Importantly, HSYA possessed a better effect on cerebrovascular vasodilatation than on cardiovascular vasodilatation , but the differential molecular mechanism remains to be discovered.
The blood-brain barrier (BBB) essentially maintains a stable cerebral homeostasis, while the destruction or increased permeability of BBB are common pathological processes during many serious cerebrovascular diseases . A study by Lv et al. revealed that HSYA significantly attenuated BBB dysfunction in anti-inflammatory patterns in ischemia stroke via the tight junction pathway, especially the NMMHC IIA, TLR4/ PI3K/Akt/Jun N-terminal kinase 1/2/14-3-3ϵ pathway while inhibiting the expressions of occludin, claudin-5, and zonula occludens-1 (Lv and Fu, 2018). Since MMPs are the main endoproteinases involved in BBB destruction (Romanic et al., 1998), the prominent inhibitory effects of HSYA on MMP-2 and MMP-9 mentioned in cardiovascular diseases may also contribute to BBB improvement.
It is worth mentioning that CI plays a causal role in facilitating neuronal death (Martin and Wang, 2010). A metabonomic study revealed that HSYA could attenuate excitatory amino acid-induced neurotoxicity, at least partially, through inhibiting the NF-kB pathway in the cerebral tissues of the middle cerebral artery occlusion (MCAO) model rats (Liu et al., 2013). Other protective mechanisms of HSYA against excitotoxic neuronal death include the inhibition of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) expression and the Bcl-2 family regulation in cortical cultures, and the inhibition of the N-methyl-d-aspartate-induced and NMDARs-mediated intracellular Ca 2+ increase in hippocampal cultures (Yang et al., 2010;Wang X. T. et al., 2016). In oxygenglucose deprivation (OGD)-induced BV2 microglia, the neuroprotective action of HSYA involves the decreased expressions of pro-inflammatory cytokines, including interleukin (IL) -1b, TNF-a, inducible nitric oxide synthase, cyclooxygenase-2, and monocyte chemotactic protein-1, as well as the reserved phosphorylation of p38 and nuclear translocation of p65 . Peroxynitrite-mediated protein tyrosine nitration and nitrosative stress represent the crucial pathogenic mechanisms of CI, while the anti-nitrative pathway might contribute to the neuroprotective efficacy of HSYA. Specifically, Sun et al. discovered that HSYA blocked authentic peroxynitrite-induced tyrosine nitration in primary cortical neurons by the reduction of inducible nitric oxide synthase expression and NO content, suggestive of its peroxynitrite scavenging abilities . They further reported that HSYA prevented peroxisome proliferator-activated receptor g nitrative modification in primary neurons and resumed eroxisome proliferator-activated receptor g activity stimulated by either 15-deoxy-delta prostaglandin J2 or rosiglitazone .

Effects on Cerebral Ischemia/Reperfusion (CI/R) Injury
A growing body of research has evidenced that oxidative stress is implicated in the pathogenesis of CI/R injury. Wei et al. showed that HSYA might oppose CI/R injury of MCAO rats through attenuating the elevation of malondialdehyde (MDA) level and decreasing superoxide dismutase (SOD) activity in the ipsilateral hemisphere and serum (Wei et al., 2005). HSYA could also reduce CI/R-induced protein oxidation and nitration, attenuate BBB destruction, and importantly inhibit the up-regulation of 12/15-lipoxygenase, which is implicated in the oxidative stress of CI/R (Sun et al., 2012). In an in vitro assay, HSYA was shown to block OGD/reoxygenation (OGD/R)-induced PC12 cells apoptosis through the suppression of intracellular oxidative stress (Fan et al., 2011).
An inflammatory reaction is a recognized player in CI/R damage. Through suppressing TLR4 pathway-mediated signaling responses, HSYA could up-regulate brain-derived neurotrophic factor (BDNF) in MCAO mice at post-ischemia/ reperfusion (Lv et al., 2015), but also exert neurotrophic and anti-inflammatory functions in LPS-activated co-existence systems for microglia and neurons (Lv et al., 2016). In the microglia of the ischemic cortex after acute CI/R, HSYA exerted anti-inflammatory effects by activating the TLR9 signaling pathway and suppressing the NF-kB pathway (Gong et al., 2018). Further studies demonstrated that HSYA significantly inhibited NF-kB p65 nuclear translation and p65 binding activity, both mRNA and protein levels of intercellular adhesion molecule 1, and the infiltration of neutrophils .
Cognitive impairment is becoming a serious mental deficit that severely affects the life quality of patients following CI/R (Jokinen et al., 2006). HSYA has the capacity to improve neurological deficit scores and increase the surviving hippocampal CA1 pyramidal cells in focal CI/R rats . HSYA injected via the common carotid artery significantly rescued the neurological and cognitive functional deficits of MCAO rats against CI/R injury. Meanwhile, HSYA could markedly down-regulate JAK2-mediated signaling, while promoting the expression of the suppressor of cytokine signaling protein 3 (SOCS3) Yu et al., 2020). Furthermore, the neuroprotective effect of HSYA against CI/R injury might be conferred through activating the Akt-dependent autophagy pathway (Qi et al., 2014). In both OGD/R-induced primary mouse neurons and PC12 cells, HSYA inhibited phenylalanine biosynthesis to enhance mitochondrial function and biogenesis for neuroprotection (Chen S. N. et al., 2019).
PI3K-mediated signaling pathways are also involved in the protective effects of HSYA against apoptosis and autophagy during CI/R. Chen et al. reported that HSYA critically reduced CI/R-mediated apoptosis through the PI3K/Akt/glycogen synthase kinase 3b signaling pathway .
HSYA protected the cerebral microvascular endothelial cells against OGD/R-induced injury by inhibiting autophagy via the Class I PI3K/Akt/mTOR signaling pathway . Proteomic analysis showed that mTOR, Eftud2, Rab11, Ppp2r5e, and HIF-1 signaling pathways were the key hub proteins and pathways in HSYA against CI/R injury . Therefore, the PI3K/Akt/mTOR signaling pathway needs to be further studied to clarify the mechanisms of actions of HSYA against CI/R injury.
Similar to MI/R, CI/R also results in mtPTP opening. Mechanically, HSYA could inhibit mtPTP opening by inhibiting Ca 2+ -induced ROS generation and H 2 O 2 -induced swelling of mitochondria isolated from rat brains, improving mitochondrial energy metabolism and enhancing ATP levels and the respiratory control ratio in the ischemia brain (Tian et al., 2008).

Effects on Vascular Dementia (VaD)
VaD is characterized by pathological damage and a decline in intelligence resulting from hypoxic-ischemic or hemorrhagic brain injury . As mentioned before, HSYA has the capacity to improve cognitive impairment. In VaD rats, Zhang et al. revealed that HSYA promoted angiogenesis and increased synaptic plasticity via up-regulating the hippocampal expressions of VEGF-A, NMDAR type-1, BDNF, and GluN2B (a subunit of NMDAR), thus improving spatial learning and memory Xing et al., 2016). Although no drug is approved, the above findings may shed light on the therapeutic potential of HSYA for managing the progress of VaD.

Effects on Traumatic Brain Injury (TBI)
TBI refers to the injury of cerebral tissue structure/function caused by various kinds of mechanical violence in the outside world (Xiu et al., 2018). HSYA has the potential to be utilized as a neuroprotective agent in cases of TBI. Firstly, TBI enables HSYA to distribute in the cerebral tissues of rats (Bie et al., 2010). Then, the antioxidant effect of HSYA in the brain of the TBI rats could explain the TBI improvement via increasing SOD, catalase and glutathione levels, while reducing MDA and oxidized glutathione (GSSG) levels (Bie et al., 2010;. Lastly, HSYA could increase mitochondrial ATPase (i.e., Na + , K + -ATPase, Ca 2+ -ATPase, and Mg 2+ -ATPase) and tissue plasminogen activator activities, while decreasing plasma plasminogen activator inhibitor-1 activity and MMP-9 expression in the hippocampus of TBI rats .
In summary, the above findings buttress the assertion that HSYA exerts cardio-cerebrovascular protective activities through complex pathways and exhibits a definite curative effect in the application of CCVDs (Table 1 and Figure 2).

CONCLUSIONS AND PROSPECTS
It is becoming clear that the important mechanisms by which HSYA exerts extensive biological activities in CCVDs are through its antioxidant, anti-inflammatory, and neuroprotective effects.
And there is no doubt that HSYA is a promising lead drug candidate in designing new multi-targeted therapeutic agents against CCVDs. The other analogues of HSYA, safflor yellow A  and safflor yellow B (Wang et al., 2009;Wang et al., 2013), showed similar protective effects against CCVDs. Further structural modification of HSYA should be extensively made and coupled with quantitative structure-activity relationship studies to develop more selective and safe drugs.
The oral bioavailability of HSYA is extremely low (~1.2%) (Ekin, 2005) and oral administration of HSYA accounts for about 0.9% of all in vivo experiments from Table 1. However, among many administration routes, oral administration is of great significance in drug formation because of its convenience and safety. Thus, the microemulsion, nanoemulsion, and nanoparticles of HSYA have been developed to overcome the low oral bioavailability Qi et al., 2011;Lv et al., 2012;Shi et al., 2018;Zhao B. X. et al., 2018). Considering the weak ability of HSYA to penetrate the BBB in physiologic condition (He et al., 2008), Borneolum Syntheticum and Acori Talarinowii Rhizoma were used to enhance its BBB permeability . Nevertheless, more attention should be payed to the overall efficacy and safety evaluation of HSYA before and after improving oral bioavailability and BBB permeability.
In the past decade, cohesive evidence showed that gut microbiota may serve as a therapeutic target of natural compounds derived from TCM (Yue et al., 2019a;Yue et al., 2019b). Oral HSYA is mostly retained in the intestinal tract as its prototype, which inevitably interacts with gut microbiota. Obesity is considered to be one of the most important risk factors of CCVDs. Our research group reported that HSYA mediated its anti-obesity effects by reversing gut microbiota dysbiosis in obese mice, followed by increasing short-chain fatty acid (SCFA)producing bacteria . For SCFAs, it has a wellestablished role in maintaining host immune function after MI (Tang et al., 2019). The potential for microbial synthesis of SCFAs, including propionate and butyrate, was low in patients with atherosclerotic cardiovascular disease (Jie et al., 2017). Recently, the microbiota-gut-brain axis has shown to influence BBB permeability and the pathological process of TBI (Braniste et al., 2014;Ma et al., 2017). Thus, it is feasible to unveil the underlying mechanisms of oral HSYA on CCVDs from the new perspective of gut microbiota modulation.
Carthami Flos is a common part of preparations used in TCM and other traditional medicinal systems. It is necessary to strengthen the compatibility research of HSYA and other TCM-derived components. As an example, HSYA and Danshensu synergistically enhanced the antioxidant defense system and anti-apoptotic effects on MI/R injury through the Akt/Nrf2/HO-1 signaling pathway (Hu et al., 2016). Their combination further achieved enhanced neuroprotective effects on CI/R injury by alleviating pro-inflammatory and oxidative stress reactions via the TLR4/NF-kB and Nrf2/HO-1 pathways (Xu et al., 2017). On the other hand, combinations with existing western medications may also provide new therapy options for CCVDs patients. For example, HSYA as an add-on therapy to acetylglutamine could synergistically modulate the neuronal  (Li et al., 2006) Acute MI model in male Wistar rats 5, 10, 20 mg·kg -1 i.v. Increases the activity of serum NO synthase, the content of NO and 6-keto-prostaglandin F1a, and decreases the levels of creatine kinase-MB, lactate dehydrogenase, thromboxane B2 and Ang II (Wang et al., 2007) Acute MI model in male C57 mice 25 mg·kg -1 i.p. Promotes the migration and tube formation of HUVECs, enhances the expressions of nucleolin, VEGF-A and MMP-9 (Zou et al., 2018) MI model in male C57BL/6 mice 15, 30, 60 mg·kg -1 i.v. Promotes endothelial progenitor cells function through the HO-1/VEGF-A/stromal cell-derived factor-1a signaling cascade  MI model in male SD rats 2, 5 mg·kg -1 i.p. Activates the Nrf2/NAD(P)H:quinone oxidoreductase 1/HO-1 signaling pathway (Ni et al., 2018) Ang II-induced H9c2 cells 80 mmol·L -1 / Increases the cell viability, however, reduces protein synthesis rate, mitigates cell surface area and decreases the expression of brain natriuretic factor and b-myosin heavy chain (Ni et al., 2018) Myocardial ischemia/ reperfusion (MI/ R) injury MI/R model in male SD rats 5 mg·kg -1 i.p. Decreases JAK2/signal transducer and activator of transcription 1 activity, enhances antioxidant capacity and decreases apoptosis (Zhou et al., 2019) Hyperlipidemia combined with MI/R model in male Wistar rats 8, 16, 32 mg·kg -1 i.p. Suppresses the over-expression of TLR4 (Han et al., 2016) H/R and LPS-induced neonatal rat ventricular myocytes 1, 3, 10 mmol·L -1 / Decreases excessive secretion of inflammatory cytokines, down-regulates over-expression of TLR4 and NF-kB (Han et al., 2016) H/R-induced H9c2 cells 6.25, 12.5, 25 mmol·L -1 / Improves cardiomyocyte viability, maintains mitochondrial membrane potential, reduces apoptotic cardiomyocytes, decreases Caspase-3 activity, and inhibits NLRP3 inflammasome activation (Ye et al., 2020) H/R-induced H9c2 cells 1.25, 5, 20 mmol·L -1 / Activates the hexokinase II proteins, restores mitochondrial energy, reduces ROS generation
The randomized controlled clinical trial (RCT) is an essential step in confirming the efficacy and safety of drugs. In contrast with the large numbers of preclinical experiments, only a few completed RCTs of HSYA were reported, which were mainly reflected in evaluating the efficacy and safety of HSYA injection in the treatment of acute ischemic stroke with blood stasis syndrome (Qin et al., 2016;Hu et al., 2020), followed by a currently ongoing phase III RCT (No. CTR20150839, http://www.chinadrugtrials.org.cn/).  (Lv et al., 2015) OGD/R induced human brain microvascular endothelial cells 10-80 mmol·L -1 / Inhibits autophagy via the Class I PI3K/Akt/mTOR signaling pathway  LPS-stimulated non-contact transwell co-culture system comprised microglia and neurons 50, 100 mmol·L -1 / Exerts neurotrophic and anti-inflammatory functions in response to LPS stimulation by inhibiting TLR4 pathwaymediated signaling (Lv et al., 2016) OGD-induced PC12 cells 10, 100 mmol·L -1 / Suppresses the intracellular oxidative stress and mitochondria-dependent caspase cascade (Fan et al., 2011) OGD/Reduced primary neurons and PC12 cells 1, 10 mmol·L -1 / Reduces phenylalanine level, promotes mitochondrial function and biogenesis for neuroprotection (Chen S. N. et al.,
However, none of the existing RCTs were of high methodological quality, and the conclusions need to be further verified by large sample, multicenter, and double-blind RCTs (as compared to traditional treatment regimens). In addition, clinical evidence supporting the application of HSYA for the management of CCVDs other than acute ischemic stroke with blood stasis syndrome is still lacking.