Pharmacodynamic effects of Dan-hong injection in rats with blood stasis syndrome

Biomedicine & Pharmacotherapy 118 (2019) 109187

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Pharmacodynamic effects of Dan-hong injection in rats with blood stasis syndrome Cong Bi, Pan-Lin Li, Yan Liao, Hong-Yu Rao, Pei-Bo Li, Jing Yi, Wei-Yue Wang, Wei-Wei Su

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Guangdong Engineering & Technology Research Center for Quality and Efficacy Reevaluation of Post-Market Traditional Chinese Medicine, Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, No. 135, Xingang Xi Road, Guangzhou 510275, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Chinese herbal formula Prescription compatibility connotation Cardiovascular diseases Inflammation Immune response Endothelial function Oxidative stress Myocardial energy metabolism Platelet-activating factor Liver function Renal function

Dan-hong injection (DHI) is extracted from Salvia miltiorrhiza (SM) and Carthamus tinctorius (CT) and is widely used for the treatment of cardiovascular diseases. Our previous results showed DHI could improve hemorheology in rats. Since complex cellular interactions such as inflammation and oxidative stress are believed to be implicated in the pathogenesis of cardiovascular events, investigation of such pathological factors will contribute substantially to the understanding of the features and mechanisms of DHI. Therefore, in this study we used a rat model of blood stasis to explore the overall effects of DHI by detecting twenty three indexes, which were related to inflammation, immune response, vascular endothelial function, myocardial energy metabolism, oxidative stress, platelet aggregation, liver and renal function. Meanwhile, the interaction between SM and CT was discussed by comparing the effects of each single herb. DHI could significantly decrease the serum contents of IL-1β, TNF-α, IL-6, IL-8, IgM, IgG, IgA, MPO, hs-CRP, MDA, LDH, CK-MB, PAF, ALP and Cr, while elevate NO, SOD, TP and UA levels, indicating that DHI could inhibit inflammation and platelet aggregation, thereby relieving immune response and peroxidation, protecting vascular endothelial and organ function, and then prevent and treat cardiovascular diseases. In terms of compatibility, SM and CT showed complementary effects on markers of inflammatory and oxidative status, vascular endothelial damage and myocardial energy metabolism. On the other hand, they counteracted each other and SM reduced the side effects of creatinine caused by CT. This study contributes to comprehensively understand the pharmacodynamics effects and mechanism of DHI.

1. Introduction At present, cardiovascular disease is still the most important cause of death in the world. According to TCM theory, blood stasis is the pathological key of cardiovascular disease and is closely related to coronary heart disease [1]. Blood stasis is a complex pathological system, accompanied by vascular endothelial damage, free radical accumulation, inflammation, liver and kidney injury, which in turn further promotes the occurrence of blood stasis [2,3]. Therefore, it is necessary to carry out the treatment and mechanism research of blood stasis at various aspects. TCM formulas are developed through thousands of years of clinical examination and refinement, and has attracted more attention over the

past decade [4,5]. Salvia miltiorrhiza Bunge (SM, Dan-shen in Chinese) and Carthamus tinctorius L. (CT, Hong-hua in Chinese) are TCM pairs for promoting blood circulation to remove blood stasis. Dan-hong injection (DHI) is extracted from SM and CT with a ratio of 3:1, which is the integration of traditional Chinese medicine and modern formulations. DHI has been clinically proven to be effective in the treatment of coronary heart disease, angina pectoris and cerebral thrombosis, and in the prevention of coronary stent restenosis and late thrombosis as well [6–8]. Compared with chemical drugs, DHI has the characteristics of multi-component interacting with multiple targets, and can exert a holistic therapeutic effect. In our previous studies, the material basis of DHI was investigated and 82 chemical constituents and nine core bioactive components were identified [9]. Meanwhile, DHI could

Abbreviations: TCM, traditional Chinese medicine; SM, Salvia miltiorrhiza Bunge; CT, Carthamus tinctorius L; DHI, dan-hong injection; SMI, Salviae miltiorrhizae injection; CTI, Carthamus tinctorius injection; HPLC, high performance liquid chromatography; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-8, interleukin 8; TNF-α, tumor necrosis; IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; MPO, myeloperoxidase; NO, nitric oxide; hs-CRP, high sensitive C-reactive protein; SOD, superoxide dismutase; MDA, methane dicarboxylic aldehyde; LDH, lactic dehydrogenase; CK-MB, creatine kinase-MB; α-HBDH, α-hydroxybutyric dehydrogenase; PAF, platelet activating factor; ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; TP, total protein; Cr, creatinine; UA, uric acid; BUN, blood urea nitrogen; ROS, reactive oxygen species ⁎ Corresponding author. E-mail address: [email protected] (W.-W. Su). https://doi.org/10.1016/j.biopha.2019.109187 Received 1 April 2019; Received in revised form 14 June 2019; Accepted 28 June 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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(A). The column temperature was set at 40 °C, and the wavelength was at 288 nm. The injection volumes of samples were 10 μL. Chromeleon 7.2 Chromatograph Data System carried out the calculation of peak area.

improve the blood circulation by reducing blood viscosity and blood coagulation. However, it is not clear what role DHI plays in multiple pathological conditions such as inflammation and oxidative stress, and how these effects are related to blood circulation. On the other hand, interactions between herbs may occur at the levels of pharmaceutics, pharmacokinetics, and pharmacodynamics [10]. Our previous studies have found that the ratio of CT and SM could significantly affect hemorheology parameters [9]. Therefore, further experiments are needed to clarify what roles SM and CT play in other aspects of pharmacodynamics and what interaction they have. The purpose of this study is to elucidate the comprehensive pharmacodynamic effects on blood stasis rats and the contribution of each herb. Effects of DHI on immunity, inflammation, oxidative stress, myocardial energy metabolism, platelet aggregation, liver and renal function were investigated via establishing a rat model of blood stasis and setting up three DHI dose groups. At the same time, in order to clarify the scientific connotation of DHI prescription, the therapeutic effects of SM, CT and DHI were compared and the contribution of each herb was contrasted. This study will help to understand the pharmacodynamic effects of DHI comprehensively and lay a foundation for the follow-up study of pharmacological mechanism.

2.3. Animals Eighty male Sprague-Dawley rats, specific-pathogen-free (SPF), 63–70 days old, weighing 180–220 g, were provided by Guangdong Medical Experimental Animal Center (License No: SCXK- (Yue) 20130002) and kept in the SPF laboratory of Ocean and Traditional Chinese Medicine Laboratory of Sun Yat-sen University (License No. SYXK-(Yue) 2014-0020). The temperature in the SPF laboratory was maintained at 20 °C–23 °C, and the relative humidity was kept at 50%–65%. The rats were fed with pellet feed (10 g/100 g weight/d) and housed under a 12 h light-dark cycle. The experiments were carried out after the animals adapted to the new environment for one week and appropriate measures were taken to reduce the damage to the rats during the experiments. All animal experiments were approved by Animal Ethics Committee of the School of Life Sciences, Sun Yat-sen University. 2.4. Establishment of blood stasis rat model and mode of administration

2. Materials and methods

The raw herbs Salviae Miltiorrhizae (Lot. 170701) and Carthamus tinctorious (Lot. 170701) were provided by Shandong Buchang Pharmaceutical Co., Ltd. The herbs were identified by Professor Wenbo Liao (School of Life Sciences, Sun Yat-Sen University, China) and the voucher specimens have been deposited in our laboratory. The preparation process of DHI was as follows. SM 300 g and CT 100 g were decocted together for two times with water, 1 h each time. And the decoctions were mixed, filtered, and concentrated. Then ethanol was added and the supernatant was filtered and collected. After properly concentrated, active carbon was added to the supernatant, stirred and filtered. The filtrate was diluted to 400 mL with water for injection after the ethanol was recovered and adjusted to pH 6.5 with NaOH solution. At last, the DHI was encapsulated and sterilized. The preparation process of SM injection (SMI) and CT injection (CTI) were same as DHI by using 300 g SM and 100 g CT respectively. Aspirin Enteric-coated Tablets (Bayer, Germany), adrenaline hydrochloride injection (Hengtong, Sichuan, China), chloral hydrate (Guangzhou Chemical Reagent Factory, Guangzhou, China) were purchased from market. Danshensu, protocatechualdehyde, p-coumaric acid, salvianolic acid D, lithospermic acid, salvianolic acid B and salvianolic acid A (purity≥98%) were purchased from Shanghai Yuanmu Biotechnology Co., Ltd. Rosmarinic acid (purity = 98.5%) were purchased from National Institutes for Food and Drug Control.

Eighty rats were randomly divided into eight groups with ten rats each: control group, model group, aspirin group (10 mg/kg/d), DHI low-dose group (0.75 mL/kg/d, the clinical dosage in human), DHI intermediate-dose group (1.5 mL/kg/d), DHI high-dose group (3 mL/ kg/d), SMI group (3 mL/kg/d) and CTI group (3 mL/kg/d). The lowand intermediate-dose of DHI were diluted with normal saline, so that the volume of administration was the same as the high-dose of DHI. The amounts of herbs used to prepare SMI, CTI and DHI high-dose is same. All rats received administrations once a day for ten consecutive days. The aspirin group was administered by gavage while the other groups were administered by intramuscular injection. The control group and the model group were intramuscular injected with the same volume of normal saline. Thirty minutes after the tenth administration of drug, the control group was subcutaneously injected with normal saline while other groups were subcutaneously injected with adrenaline (0.8 mg/ kg). Two hours later all groups except control group were immersed in ice-water (0–4 °C) for 5 min, and after another 2 h, were injected again with adrenaline (0.8 mg/kg). All groups were fasted for 12 h next, and then received the last administration. Fifteen minutes later, the rats were intraperitoneally injected with 10% chloral hydrate (v/v) (0.35 mL/100 g). Blood was drawn into plastic vacuum tubes from heart. Then the serum was separated from blood by centrifugation. All blood samples were used to detect pharmacodynamic indexes. The collection and detection of blood were processed according to standard operating procedures. The daily weight of rats was shown in the supplement.

2.2. High performance liquid chromatography (HPLC) analysis of samples

2.5. Detection of pharmacodynamic indexes in serum

According to our previous results, the essential components of DHI related to the efficacy mainly include salvianolic acids from SM, pcoumaric acid and hydroxysafflower yellow A from CT [9]. In order to ensure the efficacy, safety and consistent quality of DHI, it is necessary to control the quality of samples by detecting the components with HPLC analysis [11]. DHI, SMI and CTI samples were diluted to 40% with 10% methanol solution (v/v) containing 0.2% glacial acetic acid (v/v), and filtered through 0.45 μm membrane filters before injection. HPLC analysis was performed by using Ultimate 3000 DGLC system (Dionex, USA) with a diode array detector (DAD). The chromatograms were obtained on a Welch Ultimate XB-C18 column (4.6 × 250 mm, 5 μm) using acetonitrile (A) (HPLC) and 0.05% trifluoroacetic acid (B) (HPLC). The flow rate was 1 mL/min and the gradient elution was as follows: 0–65 min, 2–30% (A); 65–75 min, 30% (A); 75–76 min, 30–2%

The characteristic pharmacodynamic indexes in serum were selected to evaluate the effects of DHI on blood stasis rats. Inflammation response in rats was assessed by proinflammatory factors including interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8) and tumor necrosis factor α (TNF-α) while immune response was evaluated by detecting immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) levels, and those indexes were determined by ELISA kits (Nanjing Jiancheng Bioengineering Institute, Lot. 201709). The concentrations of myeloperoxidase (MPO) and nitric oxide (NO) were measured by colorimetric kits, high sensitive C-reactive protein (hs-CRP) content was determined by ELISA kits (Nanjing Jiancheng Bioengineering Institute, Lot. 201709), and they were used to estimate vascular endothelial function. Oxidative stress was assessed by testing superoxide dismutase (SOD) and methane dicarboxylic aldehyde (MDA)

2.1. Preparation of samples and reagents

2

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content using colorimetric kits. The levels of lactic dehydrogenase (LDH), creatine kinase-MB (CK-MB) and α-hydroxybutyric dehydrogenase (α-HBDH) which can reflect the degree of myocardial injury were detected by Automatic Biochemistry Analyzer (Roche, MODULAR P800). Platelet activating factor (PAF) is involved in many processes of cardiovascular disease and its content is estimated by ELISA kits (Nanjing Jiancheng Bioengineering Institute, Lot. 201709). Alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total protein (TP), creatinine (Cr), uric acid (UA) and blood urea nitrogen (BUN) are commonly used indexes for testing liver and kidney function and were assayed using Automatic Biochemistry Analyzer (Roche, MODULAR P800).

Table 1 Concentrations of the eight components in the samples. Components

danshensu protocatechualdehyde p-coumaric acid salvianolic acid D rosmarinic acid lithospermic acid salvianolic acid B salvianolic acid A

Concentration (mg/ml) DHI

SMI

CTI

1.37 0.18 0.03 0.21 0.21 0.06 0.67 0.76

1.35 0.16 – 0.23 0.22 0.06 0.69 0.77

– – 0.03 – – – – –

2.6. Statistical analysis Statistical analysis was performed with SPSS 19.0 software (IBM, Armonk, NY, USA). One-way analysis of variance and t-test were used to compare the results among groups. A P-value of less than 0.01 or 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. HPLC fingerprints of samples The HPLC fingerprints of SMI, CTI and DHI are shown in Fig. 1. The peaks were identified by comparing the retention times with those of the reference substances. Danshensu, protocatechualdehyde, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B and salvianolic acid A were detected in SMI and DHI. p-coumaric acid were detected in CTI and DHI. The contents of each component in DHI, SMI and CTI samples are shown in Table 1. It indicated that DHI components had good correlation with SMI and CTI components, wherefore the samples were reliable for subsequent experiments.

Fig. 2. The daily body weight (g) of rats over 10 days experiments (n = 10). Data are mean ± SD (n = 10).

3.3. Effects of DHI, SMI and CTI on inflammation Inflammation and immune are involved in many processes of cardiovascular disease and has become new targets to prevent cardiovascular disease [12]. Evidences have indicated that the substances form in the process of blood stasis can cause inflammation [13]. The inflammatory mediators and cytokines in turn facilitate endothelial dysfunction and induce platelet adhesion, which promote plaque formation and further lead to atherosclerosis [12]. IL-1β, TNF-α, IL-6, and IL-8 are important proinflammatory factors, which could promote the accumulation of immune complexes on endothelial cells, thereby increasing the risk of thrombus [14,15]. TNF-α is also a key on triggering cascades of inflammation. The results are shown in Fig. 3. The levels of all these indexes were significantly different between the control group and the model group. (P < 0.01), suggesting that the model rats had excessive inflammation. In the three doses of DHI groups and aspirin group, IL1β, TNF-α, IL-6, and IL-8 levels were significantly lower compared with the model group (P < 0.05, P < 0.01). The contents of the four indexes in the high-dose group were lower than those in the aspirin group (P < 0.01). In addition, IL-1β, TNF-α, IL-6, and IL-8 contents were significantly decreased in SMI group compared with the model group (P < 0.01). CTI decreased significantly TNF-α and IL-8 levels (P < 0.01) but had no significant effects on the level of IL-1β and IL-6. It is noteworthy that the reducing effects of DHI on TNF-α and IL-6 were better than SMI and CTI (P < 0.01) at the same dose, and the levels of IL-1β, TNF-α and IL-6 in SMI were lower than those in CTI. In summary, DHI, SMI and CTI could reduce inflammatory response in vivo, and the effect of DHI was similar to that of aspirin. The same results have been reported in the treatment of cerebral infarction and myocardial ischemia-reperfusion by DHI [16,17]. Studies have also shown that salvianolic acid B from SM could activate SIRT1-mediated autophagy [18] and salvianolic acid A could inhibit NF-κB and PI3K/ Akt signaling pathway [19,20], which can depress the expression of downstream pro-inflammatory factors. Safflower yellow from CT

3.2. The daily body weight of rats The daily weight of the rats over the 10 days experiments is shown in Fig. 2. The results showed that the trend of body weight gain in each group was basically the same, indicating that DHI may have little effect on metabolism.

Fig. 1. HPLC fingerprints of the samples. (A) reference substances, (B) SMI, (C) CTI, (D) DHI. Peak 1- danshensu, 2- protocatechualdehyde, 3- p-coumaric acid, 4- salvianolic acid D, 5- rosmarinic acid, 6- lithospermic acid, 7- salvianolic acid B, 8- salvianolic acid A. 3

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Fig. 3. Effects of SMI, CTI and DHI in rats with blood stasis on (A) IL-1β, (B) TNF-α, (C) IL-6 and (D) IL-8. Data are mean ± SD (n = 10). #P < 0.05 and ##P < 0.01 compared with control group, *P < 0.05 and **P < 0.01 compared with model group, ▲P < 0.05 and ▲▲P < 0.01 are the comparison between the two groups.

significantly decrease IgM, IgA and IgG levels (P < 0.05, P < 0.01), SMI significantly decreased the contents of IgM, IgA and IgG (P < 0.05, P < 0.01) while CTI significantly decreased IgM (P < 0.01). The result suggested that DHI could inhibit the levels of immunoglobulin and reduce immune response, both CT and SM contributed to the efficacy. This effect has not been reported.

regulates inflammation by directly acting on BV2 microglia [21]. The present study demonstrated this effect by detecting pharmacodynamic indexes. 3.4. Effects of DHI, SMI and CTI on immune response Both adaptive and innate immunity participate in blood stasis. As activated, immune cells, including T cells and macrophages, release proinflammatory factors and proteases which attack collagenous cap and suppress collagen formation. Then the plaque stability is reduced and thrombosis is formed [22]. IgM, IgA and IgG are the main components of serum immunoglobulin and reflect the degree of immune response [23,24]. The effects of DHI on immune globulin have not been reported. As shown in Fig. 4, IgM, IgA and IgG levels were significantly increased in the model group compared with the control group (P < 0.01). It indicated that acute immune response occurred in the model rats under the stimulation of cold coagulation. Compared with the model group, the aspirin group and all the three doses of DHI could

3.5. Effects of DHI, SMI and CTI on vascular endothelial function Almost all risk factors for atherosclerosis and vascular inflammation may affect vascular endothelial function. Thrombin which produced at the site of vascular inflammatory activates platelets, endothelial cells and macrophages can activate inflammatory signals and promote leukocyte adhesion [25]. MPO and hs-CRP are the markers of vascular inflammation, which are regarded as predictive factors for myocardial infarction [26]. NO synthesized and released by vascular endothelial cells participate in vascular tone regulation and platelet aggregation, it also maintain the balance between smooth muscle cell growth and

Fig. 4. Effects of SMI, CTI and DHI in rats with blood stasis on (A) IgM, (B) IgA and (C) IgG. Data are mean ± SD (n = 10). #P < 0.05 and ##P < 0.01 compared with control group, *P＜0.05 and **P＜0.01 compared with model group, ▲P < 0.05 and ▲▲P < 0.01 are the comparison between the two groups. 4

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Fig. 5. Effects of SMI, CTI and DHI in rats with blood stasis on (A) hs-CRP, (B) MPO and (C) NO. Data are mean ± SD (n = 10). #P < 0.05 and ##P < 0.01 compared with control group, *P < 0.05 and **P < 0.01 compared with model group, ▲P < 0.05 and ▲▲P < 0.01 are the comparison between the two groups.

3.6. Effects of DHI, SMI and CTI on oxidative stress

differentiation [27,28]. The results are shown in Fig. 5. Compared with the control group, the concentrations of three indexes were significantly different in the model group (P < 0.01), suggesting that the blood stasis model rats were accompanied with vasoconstrictive dysfunction and acute vascular inflammation. Compared with model, DHI low-dose group significantly decreased hs-CRP level (P < 0.01), DHI low- and intermediate-dose groups significantly reduced MPO level (P < 0.05, P < 0.01), and three dose groups of DHI increased NO content (P < 0.05, P < 0.01). Besides, the levels of MPO and hs-CRP in lowdose group were lowest, while NO content in high-dose group was highest in the three dose groups of DHI. Aspirin could significantly reduce MPO level (P < 0.01) but had no significant effect on the contents of hs-CRP and NO (P > 0.05). On the other hand, CTI was more effective than SMI and DHI in inhibition of hs-CRP level, and SMI was more potent than CTI in suppression of MPO level. NO content was significantly increased in SMI, CTI and DHI, suggesting that both SMI and CTI could dilate blood vessels and regulate the growth of smooth muscle cell. In general, DHI could inhibit vascular inflammation and expand the blood vessels, therefore protecting vascular endothelial function in rats with blood stasis. The consequence is consistent with clinical trials, which have testified that DHI is effective for vascular endothelial repair in patients with coronary heart disease after percutaneous coronary intervention, and its mechanism may be mobilization of endothelial progenitor cells and inhibition of endothelial cell exfoliation [29]. Besides, the reference also proves the role of CT in elevating NO [30] and SM in enhancing vasodilation [31].

Growing evidence indicates that overproduction of reactive oxygen species (ROS) may contribute to cardiovascular disease [32]. In pathological conditions, excessive ROS promotes the growth and migration of vascular smooth muscle and inflammatory cells. At the same time, it promotes the formation of atherosclerotic plaques, which leads to myocardial metabolic disorders and oxygen supply reduction [33–35]. Using 2,2-diphenyl-1-picrylhydrazyl spectrophotometric assay, the free radical scavenging capacity of DHI has been proved and protocatechuic acid showed the best free radical scavenging activity [36]. In addition, Studies found that several salvianolic acid components in SM and safflower yellow in CT have protective effects against peroxidation damage in cell experiment [37,38]. But the compatibility effects of the two herbs have not been reported. In this study, the influence of DHI on oxidative stress in rats was evaluated by SOD and MDA at the pharmacodynamics level. SOD is an important protective enzyme which can resist the superoxide anion, and its decreased levels may aggravate oxidative stress in patients with coronary heart disease [39]. MDA content can indirectly reflects the severity of free radical attack on cells [40]. The results are shown in Fig. 6. Significant differences were observed on SOD and MDA levels between control and model (P < 0.01). In the process of scavenging free radicals in rats with blood stasis, over-consumption of SOD might trigger free radical reaction on membrane structural lipids, further leading to cell damage. MDA and phospholipid proteins could form insoluble metabolic end products, which accumulate in cells and affect the functions. Compared with model group, aspirin and the low- and intermediate-dose of DHI

Fig. 6. Effects of SMI, CTI and DHI in rats with blood stasis on (A) SOD and (B) MDA. Data are mean ± SD (n = 10). #P < 0.05 and ##P < 0.01 compared with control group, *P < 0.05 and **P < 0.01 compared with model group, ▲P < 0.05 and ▲▲P < 0.01 are the comparison between the two groups. 5

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Fig. 7. Effects of SMI, CTI and DHI in rats with blood stasis on (A) α-HBDH (B) LDH and (C) CK-MB. Data are mean ± SD (n = 10). #P < 0.05 and ##P < 0.01 compared with control group, *P < 0.05 and **P < 0.01 compared with model group, ▲P < 0.05 and ▲▲P < 0.01 are the comparison between the two groups.

ATP reserve and decrease AcCoA/CoA ratio, thus improving myocardial energy metabolism efficiency [45]. Furthermore, safflower yellow in CT can reduce LDH leakage, and relieve ATP content decrease and ultrastructural injury in ventricular myocardium, thus alleviating myocardial hypoxic injury [46]. To sum up, SM and CT can alleviate myocardial energy metabolism disorder from different aspects.

could significantly reduce MDA level (P < 0.01), the three dose groups of DHI could increase SOD activity (P < 0.05, P < 0.01). MDA level in the aspirin group was significantly lower than that in DHI groups (P < 0.01). These data indicated that maybe DHI could reduce the extent of oxidative damage, in addition, the efficacy was dose-independent. For another, SOD content was significantly increased and MDA level was reduced in SMI and CTI compared with model (P < 0.05, P < 0.01). The effects of SMI and CTI on SOD and MDA concentrations were more significant than DHI (P < 0.05, P < 0.01). This result showed that SMI and CTI could protect the body from oxidative damage, and the way SM and CT work together may not be synergy but competition.

3.8. Effects of DHI, SMI and CTI on platelet-activating factor PAF is a kind of endogenous phospholipid. It can promote platelet aggregation, increase capillary permeability, stimulate the production of vasoactive substances and inflammatory mediators, and then induce chain reaction and amplification effect, thereby aggravating the damage to tissues [47–49]. It participates in a wide range of pathophysiological conditions of cardiovascular disease including atherosclerosis, coronary heart disease, and heart failure [50]. At present, studies have reported that DHI can reduce PAF level in patients with cerebral infarction [51] and myocardial infarction [52], but there is no study in patients with blood stasis. Less research has been done on the effects of SM and CT on PAF. In this study, PAF was significantly higher in the model group compared with the control group after modeling (P < 0.01) (see Fig. 8), suggesting that platelet aggregation might increase. The three doses of DHI, SMI and CTI significantly reduced PAF level (P < 0.01). Moreover, the reducing effect of SMI was more

3.7. Effects of DHI, SMI and CTI on myocardial energy metabolism On myocardial energy metabolism, myocardial enzymes including α-HBDH, LDH and CK-MB show the degree of myocardial injure [41], and they are usually used in the diagnosis of myocardial infarction [42]. When myocardial cells are damaged, the permeability of myocardial cell membrane changes and the intracellular CK-MB leaks which leads to the increase of serum CK-MB content. As is shown (Fig. 7), αHBDH, LDH and CK-MB content were significantly raised in the model group compared with the control group (P < 0.01), probably because the energy of body was seriously consumed under the stimulation of ice water and adrenaline. Myocardial cells were damaged, and myocardial tissue was in a state of energy metabolism disorder. After preventive administration, aspirin significantly decreased α-HBDH, LDH and CKMB levels (P < 0.05, P < 0.01), while the three doses of DHI significantly decreased LDH and CK-MB levels (P < 0.01). Besides, the high-dose DHI had a better effect than aspirin in reducing of CK-MB content (P < 0.01). In conclusion, DHI could not only reduce the disorder of myocardial energy metabolism, but also protect myocardial cells and reduce the risk of myocardial infarction. These results were in good agreement with clinical trials which demonstrate that DHI could significantly decrease the risk of heart failure, recurrent angina and arrhythmia [43]. Besides, studies have also found that the mechanism of DHI on protecting myocardial tissue may be anti-inflammatory, antioxidative and activation of risk pathways, [44]. In the aspect of single herbs, SMI significantly decreased CK-MB level (P < 0.01), while CTI significantly decreased LDH content (P < 0.05). SMI, CTI and DHI all had no effect on the content of α-HBDH. Pharmacological studies indicate that water-soluble components in SM can increase myocardial

Fig. 8. Effects of SMI, CTI and DHI in rats with blood stasis on PAF. Data are mean ± SD (n = 10). #P < 0.05 and ##P < 0.01 compared with control group, *P < 0.05 and **P < 0.01 compared with model group, ▲P < 0.05 and ▲▲P < 0.01 are the comparison between the two groups. 6

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significantly decreased in SMI compared with model (P < 0.05, P < 0.01), suggesting that SMI maybe reduce hepatocyte damage, while CTI may be conducive to the synthesis and metabolism of hepatocytes.

significant than CTI (P < 0.01). As is clear from the above analysis, DHI can reduce PAF level and this may be one of the mechanisms of anti-thrombosis of DHI. SM in DHI maybe contribute more effectively to the reduction of PAF level than CT. Aspirin showed no significant effect compared with model, probably because aspirin inhibits platelet aggregation by inactivating cyclooxygenase rather than reducing PAF [53].

3.10. Effects of DHI, SMI and CTI on renal function Cr, BUN and UA are the three indicators which commonly used for testing renal function in clinical. Cr and BUN which excreted by kidney can evaluate the glomerular filtration. UA is associated with the secretory function of renal tubules and is deemed to one of the causes of kidney damage and renal function progression [62]. All three indexes content in the model group were significantly different from the control group (P < 0.01) (see Fig. 10), suggesting that stimulation of cold and adrenaline might lead to abnormal glomerular filtration and tubular secretion in rats. After treatment, aspirin had no significant effect on three indexes levels. Compared with model, DHI intermediate- and high-dose groups could increase UA content (P < 0.05, P < 0.01), DHI high-dose group could decrease Cr concentration at the same time (P < 0.01), indicating that DHI could relief kidney injury caused by blood stasis. DHI can also reduce renal blood flow resistance and relieve clinical symptoms in patients with chronic kidney disease [63]. SM could decrease Cr level while CT could increase UA and decrease BUN (P < 0.05, P < 0.01). It turned out that CTI and SMI could ameliorate glomerular injury and kidney tubular secretion respectively. It is remarkable that Cr content was increased in CT and was decreased in SM compared with model. When glomerular filtration function is impaired, Cr accumulates to become a harmful toxin, so the data suggested SM could inhibit CT damage to the glomerulus, help DHI regulate organism stability and reduce side effects. The above indexes are interrelated. Studies have shown that coronary thrombosis usually begins with the rupture of atherosclerotic plaques, leading to abnormal platelet activation with consequent formation of thrombus [64]. Abnormally activated platelets can accelerate the release of proinflammatory factors and induce vascular inflammation which in turn promotes platelet aggregation and thrombosis formation [12]. Inflammatory factors such as IL-1β and TNF-α can activate NOX expression. NOX, a key gene of HIF-1 signaling pathway and fluid shear stress and atherosclerosis pathway, can product ROS and further increasing oxidative stress level [65]. Excessive production of ROS leads to eNOS uncoupling, thereby reducing NO level and causing

3.9. Effects of DHI, SMI and CTI on liver function Blood stasis can reduce the blood flow of liver, further inhibit pathogen clearance, and enhance hepatic inflammation or fibrosis-related injury [54]. Hence, the drugs of activating blood circulation to remove blood stasis are usually useful for protecting liver. Both SM and CT can inhibit liver fibrosis, and SM is widely used in clinical treatment of liver diseases, one of its protective mechanisms is to inhibit the activity of discoidin domain receptor 2 tyrosine kinase in liver stellate cells [55]. Studies have proved that DHI can improve liver function in patients with hepatic veno-occlusive disease and liver cirrhosis in clinic [56,57], but we don't know what effect of DHI on liver injury caused by blood stasis. In this study, the function of liver is assessed by ALT, AST, TP and ALP. ALT and AST are sensitive biomarkers for liver injury [58]. They exist in hepatocytes, and are released into the blood when hepatocytes are damaged, causing the increase of blood ALT and AST. An increase in ALP reflects the degree of cholestasis [59]. TP reflects the protein synthesis function of liver [60]. As demonstrate in -Fig. 9, there were significant difference between control and model for all four indexes levels (P < 0.05, P < 0.01). It suggests that the hepatocytes of blood stasis rats were damaged, and the secretory function of liver was abnormal. After administration, neither aspirin nor DHI showed significant difference on ALT or AST content compared with model. These results are different with those of previous studies [61], probably because the liver injury caused by blood stasis is not as serious as hepatic failure, and the improving effect of DHI is not obvious. TP content was increased significantly in aspirin group and DHI three doses groups (P < 0.01), the level of ALP was decreased significantly in the aspirin group as well as DHI intermediate- and high-dose groups (P < 0.05, P < 0.01), indicating that DHI and aspirin might relieve the abnormal of the secretion and metabolism function of liver to a certain extent. In terms of single herbs, TP level was significantly increased and ALP level was significantly decreased in CTI (P < 0.05) and AST content was

Fig. 9. Effects of SMI, CTI and DHI in rats with blood stasis on (A) ALT, (B) AST, (C) TP and (D) ALP. Data are mean ± SD (n = 10). #P < 0.05 and ##P < 0.01 compared with control group, *P < 0.05 and **P < 0.01 compared with model group, ▲P < 0.05 and ▲▲P < 0.01 are the comparison between the two groups. 7

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Fig. 10. Effects of SMI, CTI and DHI in rats with blood stasis on (A) UA, (B) Cr and (C) BUN. Data are mean ± SD (n = 10). #P < 0.05 and ##P < 0.01 compared with control group, *P < 0.05 and **P < 0.01 compared with model group, ▲P < 0.05 and ▲▲P < 0.01 are the comparison between the two groups.

characteristic makes SM and CT complement the pharmacodynamics of DHI. The present study also found that there is not only synergism between herbs, but also counteraction which can reduce side effects such as the effect on Cr. In conclusion, the efficacy of DHI is not a simple superposition of the efficacy of the two herbs, but a result of the interaction between the components. And the compatibility of the DHI prescription is scientific and effective. It should be pointed out that this study is carried out on animals, not on human, and the pharmacological mechanism and targets of DHI are not clear. However, this study could relatively fully evaluate the pharmacodynamic effects of DHI in rats with blood stasis, and may provide scientific experimental evidence for the clinical application of DHI. What’s more, this study could lay the foundation for follow-up studies on mechanism.

endothelial dysfunction [66]. Besides, IL-1β has been indicated to induce apoptosis [67]. Apoptosis, inflammation and oxidative stress may further injure organ such as heart, liver and kidney. In summary, blood circulation interacts with multiple pathological conditions such as inflammation, oxidative stress, apoptosis and vascular endothelial function. In this study, based on “fury” and “cold pathogen” in the etiology of qi-stagnation and blood stasis syndrome in TCM theory, ice-water bath and adrenaline were used to copy the classical rat model of blood stasis [9,68]. Previous studies have proved that this model is abnormal in blood rheology and coagulation function, which can reproduce the blood stasis syndrome [9]. This study found that the blood stasis rats showed abnormalities in inflammatory response, vascular endothelial function, oxidative stress, immune response, liver and renal function, platelet aggregation, and myocardial energy metabolism. Aspirin has multiple pharmacological effects and usually used for antipyretic, analgesic and anti-platelet aggregation in clinic [69], so it was selected as a positive control. The results of administration showed that DHI could significantly improve many aspects of pathological indexes in vivo. The effect of DHI on inflammation and immunity was as good as that of aspirin, DHI and aspirin had respective advantages in oxidative stress and myocardial energy metabolism. DHI was significantly more effective in reducing PAF than aspirin. Furthermore, Statistics showed the adverse drug reaction rate of DHI is 3.5% [70].The present results also suggested that DHI might have advantages in protecting renal function compared with aspirin. In general, both DHI and aspirin have multiple pharmacological effects and significant treatment in rats with blood stasis, but they are quite different in clinical indications. Aspirin is usually used for antipyretic and antithrombotic while DHI is mainly for the patients with chest discomfort and apoplexy caused by obstruction of blood stasis and is used under the guidance of TCM theory. In the aspect of prescription compatibility, the contribution of each herb to the DHI efficacy of blood stasis was mainly concerned in this study. SM and CT could collectively reduce the level of inflammatory factors, scavenge oxygen free radicals, relieve vascular endothelial damage and improve myocardial energy metabolism, but they also have different emphasis on the efficacy indicators and direction. SM plays an important role in the improvement of inflammation, immune response, platelet aggregation, hepatocyte injury and glomerular filtration function, while CT have a major role in the improvement of tissue necrosis, synthesis and metabolism of liver and kidney tubular secretion. This

4. Conclusion In conclusion, this study expounds the pharmacodynamic profile of DHI on blood stasis rats from multiple systems, and proves the scientificity of DHI prescription compatibility. The experiments demonstrate that DHI can significantly inhibit inflammatory factors and platelet aggregation, and then alleviate peroxidation injury, regulate immune function and vascular function, protect organ function (heart, liver and kidney). The prevention and treatment of cardiovascular diseases by DHI may be related to the above pharmacodynamics effects. Besides, CT and SM have different emphasis on efficacy, and work synergistically or oppositely, which make them not only complement each other in function but also counteract a little side effects.

Author contributions Cong Bi wrote the main paper and completed the experiments, WeiWei Su and Pan-Lin Li conceived the project and designed the experiments, Yan Liao provided technical support in animal experiments, Hong-Yu Rao contributed to the analysis of data, Pei-Bo Li played a role in conception and language of the paper, Jing Yi and Wei-Yue Wang implement the experiments. All the authors approved the final manuscript.

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