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The mixture of Salvia miltiorrhiza-Carthamus tinctorius (Danhong injection) alleviates low-dose aspirin induced gastric mucosal damage in rats.

ABSTRACT

Background: Danhong injection (DHI) is quite often used in combination with low-dose aspirin (ASA, 75-325 mg daily) in clinic, particularly for the treatment of cardiovascular diseases. Exploring their interaction profile is of great clinical importance.

Purpose: The current study aims to explore the interaction between DHI and low-dose ASA in rats. Methods: Sixty four rats were randomly divided into eight groups. Stomach and other four vital organs were collected for histological evaluation. Organs which exhibited histological changes were selected for a further study to evaluate the damage score and mode of action. We tested the protective effect of DHI on gastric mucosal damage in different regimes of administration. COX activity, gastric mucus secretion, pepsin activity, antioxidant activity and ROS level were assayed to reflect the protective effect of DHI on gastric mucosal damage induced by ASA.

Results: Stomach was the target organ of interaction when DHI and ASA were used in combination. DHI alleviated gastric mucosal damage by 55.8% when DHI was injected before ASA (Group E) and by 53.5% when DHI was injected 2 h after ASA administration (Group F). Additionally, if DHI treatment was appended to the long-term administration of ASA, DHI still decreased the gastric mucosal damage score in 52.0% from 2.50 to 1.20. DHI improved gastric mucus secretion, as well as decreased pepsin activity to maintain the integrity of gastric mucosal barrier (P < 0.05). Furthermore, DHI recovered antioxidant activity which was impaired by ASA. In details, DHI decreased gastric mucosal ROS level, increased CAT, GSH-Px and SOD activity, and reduced MDA concentration (P <0.05). When ASA (71.9 [micro]M) was used in combination with DHI (23-fold dilution, presented in terms of concentrations of DSS, PA, SaD RA, SaB and SaA were 6.45-6.92, 1.10-1.14, 1.09-1.10, 0.86-0.90, 16.76-19.38 and 1.83-1.94 [micro]g/ml, respectively) in vitro, the inhibition rate of ASA increased from 38.6% (ASA alone) to 62.8% (ASA-DHI) on COX-1 and from 28.9% (ASA alone) to 38.8% (ASA-DHI) on COX-2 (P < 0.05). DHI strengthened the inhibition activity of ASA on both COX-1 and COX-2, which showed that DHI alleviated ASA induced gastric mucosal damage but not antagonized anti-COX effect of ASA.

Conclusions: Gastric protective benefits were clearly produced when DHI and ASA were used in combination, which provided rational guidance for clinical combined application of DHI and ASA.

Keywords:

Danhong injection

Aspirin

Drug interaction

Cyclooxygenase

Drug combination

Gastric mucosal damage

Introduction

Danhong injection (DHI) is composed of two typical Chinese species, the radix and rhizome of Salvia miltiorrhiza Bunge (Labiatae) and the dry flower of Carthamus tinctorius L. (Asteraceae). In China, DHI is widely applied for the treatment of cardiovascular and cerebrovascular diseases, such as coronary heart disease, angina pectoris, myocardial infarction, cerebral thrombosis and ischemic encephalopathy. It also has significant effect on nephropathy, ophthalmopathy, osteopathy and pulmonary diseases (Tang and Li 2011). In addition to its outstanding efficacy, DHI possesses reliable safety in clinic (Li et al. 2011). The incidence of adverse reactions or events of DHI is 0.638%, and most of the adverse reactions would be recovered or improved without sequelae or death. In China, the annual sale of DHI was 800 million dollars in 2014, which has made DHI one of the most promising drugs to be a "blockbuster".

Aspirin (ASA), a classic analgesic and anti-inflammatory agent, is one of the most prescribed medicines worldwide. At doses higher than 325 mg daily, ASA is used as an effective pain reliever by inhibiting both COX-1 and COX-2. At low-doses (75-325 mg daily), ASA predominantly inhibits the COX-1 isoform to inactive platelets while its measurable anti-inflammatory effect is virtually deprived (Gaetano et al. 2003). In recent years, the major indication for ASA is cardiovascular disease at doses of 75-100 mg daily in most patients (Sostres and Lanas 2011). In United States, there are more than 50 million people (nearly 36% of the population) taking ASA at low doses to prevent cardiovascular diseases.

Multi-target combination is one of the most promising ways in treating multi-factorial diseases, such as diabetes, neurodegenerative disorders, cardiovascular diseases and cancers (Hopkins 2008). In China, India and other oriental countries, herbal medicines are widely used with conventional drugs in clinic (Patwardhan and Mashelkar 2009; Qiu 2007). In China, DHI and ASA are quite often used in combination, particularly for the treatment of cardiovascular diseases. Based on the hospital information system (HIS), the frequency of DHI and ASA combination could reach 90% in patients with coronary heart disease (Du J et al. 2011), which means that if 100 patients are treated with DHI, then 90 of them would take ASA at the same time. In our previous study, we have revealed that drug interaction is clearly produced in the combination of DHI and ASA by a metabolomics strategy (Li et al. 2015). The DHI-ASA interaction affects some key endogenous biomarkers and metabolic pathways, which would not happen when DHI or ASA is used alone. However, the substantive benefits or risks caused by this kind of interaction still remain unknown. In the present study, we have explored the target organs of interaction between DHI and ASA. The results showed that in different regimes of administration, DHI (4.16ml/kg daily, presented in terms of DSS, PA, SaD RA, SaB and SaA were 0.62-0.66, 0.10-0.11, 0.10-0.11, 0.082-0.087, 1.60-1.85 and 0.17-0.18 mg/kg daily) could lessen gastric mucosal damage caused by ASA (10.41 mg/kg daily) during their concurrent treatment (14 or 28 days). Our results confirmed the DHI-ASA interaction and pointed out that this interaction would lead to protective effect on stomach during the drug combination.

Materials and methods

Materials and animals

DHI was provided by Buchang Pharma Co., Ltd., China (Lot Number: 13011002, 13071011, 13042014, 14022016, and 14031018), and DHI in batch 13042014 was administrated in animal experiment. ASA (purity [greater than or equal to] 99%) was purchased from Aladdin chemistry Co., Ltd., China (Lot Number: 27383). Five standards about S. miltiorrhiza: (R)-3, 4-dihydroxyphenyllactic acid sodium salt (danshensu, DSS), procatechuic aldehyde (PA), salvianolic acid D (SaD), rosmarinic acid (RA), salvianolic acid B (SaB) and salvianolic acid A (SaA) (Fig. 1) were all purchased from Chinese materials research center, China. Stock solutions of DSS, PA, SaD, RA, SaB and SaA were prepared by dissolving accurately weighed standards in 10% methanol. The concentrations of DSS, PA, SaD, RA, SaB and SaA were 1.12, 1.30, 1.27, 1.08, 2.66 and 1.35mg/ml, respectively. ASA was dissolved in water (3.2mg/ml) every day before administration. Formaldehyde solution was diluted with water to a concentration of 10% (v/v). The concentrations of chloral hydrate solution and sodium citrate solution were 10% and 3.8% (w/v), respectively. Cyclooxygenase (COX) (ovine) Inhibitor Screening Assay Kit (Item No. 560101), indomethacin (Item No. 70270) and NS-398 (Item No. 70590) were all purchased from Cayman Chemical, USA. Pepsin assay kit (Item No. A080-1), reactive oxygen species (ROS) assay kit (Item No. E004), catalase (CAT) assay kit (Item No. A007-1), glutathione peroxidase (GSH-Px) assay kit (Item No. A005), superoxide dismutase (SOD) assay kit (Item No. A001-3) and malondialdehyde (MDA) assay kit (Item No. A003-1) were all purchased from Nanjing Jiancheng Bioengineering Institute, China.

SPF male Sprague-Dawley rats were purchased from Vital River Laboratory Animal Technology Co. Ltd., Beijing, China (license number: SCXK (Beijing) 2013-0001). All the rats were kept in Drug Safety Evaluation Center of Nanjing University of Chinese Medicine, Nanjing, China. All studies on animals were in accordance with the guidelines of the Animal Ethics Committee of Nanjing university of Chinese medicine.

Characterization of DHI

Ultra-high-performance liquid chromatography coupled with photo-diode array and quadrupole time of flight mass spectrometry (UPLC-PDA-OTOF/MS; Waters Corp., Milford, MA, USA) was used to establish the chromatography fingerprint of DHI and quantify the contents of DSS, PA, RA, SaD, SaB and SaA. The separation was performed on an Acquity UPLC BEH [C.sub.18] column (100 mm x 2.1 mm, 1.7 [micro]m; Waters) using water-formic acid (A; 100:0.1, v/v) and acetonitrile (B) as mobile phase at a flow rate of 0.4ml/min. The conditions of gradient eluting were optimized as follows: 5-40% B (0-9.0 min), 40-80% B (9.0-10.0 min), 80-80% B (10.0-12.0 min), 80-5% B (12.0-12.5 min). The column temperature was maintained at 35 [degrees]C and the injection volume was 1 [micro]1. The analytical method of quantification was validated by linearity, recovery, precision and stability. To evaluate the linearity, stock solution was diluted with 10% methanol to obtain working solutions with six different concentrations: calibration curve, correlation coefficient and linear range of each analyte were calculated. Recovery was determined by six replicate analyses of samples containing known amount of analytes. For intra-day and inter-day precision, working solution was analyzed for six times on the same day and three consecutive days. Stability was evaluated by injecting DHI at 0, 2, 4 and 6h (three samples per time point), and presented as relative standard deviation (RSD).

Experimental design

Sixty four rats weighing 200-250 g were fasted overnight with free access to water before the first administration. The daily dose of DHI and ASA for rats was 4.16ml/kg (presented in terms of DSS, PA, SaD RA, SaB and SaA were 0.62-0.66, 0.10-0.11, 0.100.11, 0.082-0.087, 1.60-1.85 and 0.17-0.18 mg/kg, respectively) and 10.41 mg/kg, respectively, which was equivalent to a 60 kg person taking 40 ml DHI and 100 mg ASA daily. The daily doses were set in consideration of a fact that the doses of 40 ml and 100 mg daily were most prescribed in clinic for DHI (Chen et al. 2011) and ASA (Silagy et al. 1993), respectively. The main route of DHI administration in clinic is intravenous drip (Chen et al. 2011), thus we injected DHI into rats through caudal vein in this animal experiment. There were eight groups (A, B, C, D, E, F, G and H). All rats were administrated daily at scheduled time (9:00-11:30 a.m.). Animals of group A (CTL): we supplied water and food regularly; animals of group B (ASA): we gave ASA solution intragastrically (10.41 mg/kg); animals of group C (DHI): we injected DHI through caudal vein (4.16 ml/kg); animals of group D (NS-ASA): first of all we injected saline (4.16 ml/kg) and immediately after we gave ASA solution (10.41 mg/kg); animals of group E (DHI-ASA): first of all we injected DHI (4.16 ml/kg) and immediately after we gave ASA solution (10.41 mg/kg): animals of group F (ASA-DHI): first of all we gave ASA solution (10.41 mg/kg) and 2h later we injected DHI (4.16 ml/kg). Animals in the above-mentioned groups (A, B, C, D, E and F) were administrated daily for 14 consecutive days. Animals of group G ([ASA.sub.long]): we gave ASA solution (10.41 mg/kg) daily for 28 days; animals of group H ([ASA.sub.long]-DHI): we gave ASA solution (10.41 mg/kg) daily in the first 14 days and we gave ASA solution (10.41 mg/kg) right after DHI injection (4.16 ml/kg) daily in the following 14 days. On the 15th (A, B, C, D, E and F) or 29th day (G and H), all the rats were sacrificed. Lung, heart, liver, stomach and kidney were collected from each rat and fixed in 10% buffered formalin for hematoxylin-eosin staining (HE). Pathological evaluation was performed by pathologists in Medical School of Southeast University who were unaware of the treatments of animals. Damaged organs were selected for a further step for determining damage score and mode of action (Fig. 2).

Damage score of gastric mucosa

The pathological sections of gastric mucosa were examined under optical microscope to observe: epithelial cells with degeneration, necrosis or pycnosis; lamina propria with edema, hyperemia or inflammatory infiltration; submucosa with edema, hyperemia or inflammatory infiltration. Damage in each part was graded and scored as follows: intact = 0, slight = 0.5, mild = l, moderate = 2, severe = 3. The scores of the three parts were added to obtain damage score of gastric mucosa.

Inhibitory effects on COX-1 and COX-2

The inhibitory effects of DHI (23-fold dilution, presented in terms of concentrations of DSS, PA, SaD RA, SaB and SaA were 6.45-6.92, 1.10-1.14, 1.09-1.10, 0.86-0.90, 16.76-19.38 and 1.83-1.94 [micro]g/ml, respectively), ASA (71.9 [micro]M) and DHI-ASA (23-fold dilution- 71.9 [micro]M) on COX-1 and COX-2 were assayed by using the COX (ovine) Inhibitor Screening Assay Kit. The assay directly measured PG[F.sub.2[alpha]] by Sn[Cl.sub.2] reduction of COX-derived PG[H.sub.2] produced in the COX reaction. The prostanoid product was quantified via enzyme immunoassay (E1A) using a broadly specific antiserum that bound to all the major prostaglandin compounds. Indomethacin and NS-398 were used to standardize the assay for COX-1 and COX-2, respectively.

Evaluation of gastric mucus secretion

Periodic acid-Schiff staining (PAS) was used to evaluate the secretion of gastric mucus, which was also performed by pathologists in Medical School of Southeast University, China. Area and integrated optical density (IOD) of positive staining were used to reflect the secretion level of gastric mucus, which was calculated by Image-Pro Plus V6.0 (for windows).

Pepsin activity of gastric mucosa and gastric juice

Rats in group A (CTL), D (NS-ASA) and E (DHI-ASA) were anesthetized with chloral hydrate solution (0.3ml/100g) before they were sacrificed on the 15th day. Pylorus of each rat stomach was ligated. After 4 h, all rats were sacrificed. Fresh glandular stomach and gastric juice were collected on ice. Pepsin activities of gastric mucosa and gastric juice were determined by using Pepsin assay kit.

ROS level of gastric mucosa

A portion of fresh glandular stomach was cut into pieces and digested by Trypsin to obtain single cell suspension. Flow cytometry was used to determine ROS level of gastric mucosa on the same day of sampling. DCFH-DA (2, 7-dichlorofuorescin diacetate) was added as fluorescence probe. Excitation and emission wavelengths were set at 488 nm and 525 nm, respectively. The fluorescence intensity was proportional to the level of ROS, and ROS level was presented as mean fluorescence intensity and positive incidence.

At the end of 4h ligation, blood was sampled from carotid artery (the volume of blood to sodium citrate solution was 9 to 1), and centrifuged at 13,000 x g for 10 min to get plasma. Fresh glandular stomach and plasma were prepared for the determination of antioxidant activity. Activity of CAT, GSH-Px and SOD, as well as concentration of MDA were assayed by using CAT assay kit, GSH-Px assay kit, SOD assay kit and MDA assay kit, respectively.

Statistical analysis

All data were expressed as means [+ or -] S.E.M. Significant differences between groups were performed by nonparametric Mann-Whitney test. Statistical significant was set at P < 0.05.

Results

Characterization of DHI

The typical HPLC-PDA fingerprint chromatograms of DHI in five batches (Lot Number: 13011002, 13071011, 13042014, 14022016, and 14031018) showed a batch-to-batch similarity of DHI (Fig. 3A). Six components were identified as DSS, PA, SaD RA, SaB and SaA (Fig. 3B) by comparing retention time, UV spectrum and MS fragment of each peak with those of DSS, PA, SaD RA, SaB and SaA standards.

Phenolic acids have been reported to be the main bioactive compounds of DHI and used as marker components for quality control (Liu et al. 2013a, 2013b). Thus we determined the contents of DSS, PA, SaD, RA, SaB and SaA in DHI by UPLC-PDA. The validation results showed that the developed method was reliable for simultaneous determination of these phenolic acids in DHI (Supplementary Table). The concentration ranges of DSS, PA, SaD RA, SaB and SaA in DHI of five batches were 148.46-159.27, 25.27-26.22, 25.07-25.33, 19.77-20.87, 385.39-445.73 and 42.03-44.54 [micro]g/ml, respectively.

Protective effect of DHI on ASA-induced gastric mucosal damage during different regimens of administration

Stomach was the target organ of interaction between DHI and ASA. DHI lessened gastric mucosal damage due to ASA when they were used in combination (Fig. 4A). However, DHI and ASA had no side effect on rat lung, heart, liver or kidney whether they were used alone or in combination. Histological examination showed that administration of ASA (10.41 mg/kg) for 14 consecutive days induced gastric mucosal damage in rats (P<0.05). When ASA (10.41 mg/kg) was administrated in combination with DHI (4.16ml/kg, presented in terms of DSS, PA, SaD RA, SaB and SaA were 0.62-0.66, 0.10-0.11, 0.10-0.11, 0.082-0.087, 1.60-1.85 and 0.17-0.18 mg/kg), the damage score was significantly decreased in 55.8% from 2.56 to 1.13 (P<0.05) (Fig. 4B).

The mucosal damage induced by ASA is mainly marked by mucosal epithelial damage, which appeared as cell degeneration of covering and glandular epithelia. Gastric mucosal damage mainly appeared at the area of glandular stomach, therefore this part of tissue was used for the determination of pepsin activity, ROS level, antioxidase activity and MDA concentration.

According to the clinical experience, DHI and ASA are mainly administrated simultaneously in their combined therapy in clinic. For some patients, they used to take ASA when they wake up, and there will be a time interval about 2 h to be injected with DHI by nurses. Accordingly, we set Group E and F to test the influence of both the sequence and the time interval on gastric mucosal damage. The results showed that DHI alleviated gastric mucosal damage by 55.8% when DHI was injected before ASA (Group E) and by 53.5% when DHI was injected 2 h after ASA administration (Group F) (Fig. 4A, B), which indicated that the sequence and time interval of the drug combination had no significant influence on the protective effect of DHL

Although administration of DHI could range from 1 to 96 days, 14 or 15 days are the most commonly used ranges (Chen et al. 2011). Thus, 14 days were chosen in this study. On the other hand, ASA at low doses are mainly prescribed for 3 months in the clinic (Sorensen et al. 2000). In consideration of the medication time of DHI and ASA, we designed an additional regimen of drug combination in which DHI treatment was appended to the long-term administration of ASA (Group G and H). In this regimen, DHI alleviated the gastric mucosal damage with a damage score reduction of 52.0% from 2.50 to 1.20 (P < 0.05) (Fig. 4A, B).

Enhancement of DHI on the inhibitory effect of ASA on COX-1 and COX-2

At the concentration of 71.9 |xM, ASA inhibited COX-1 and COX-2 activity with inhibition rates of 38.6% and 28.9%, respectively. When ASA (71.9 [micro]M) was used in combination with DHI (23-fold dilution, presented in terms of concentrations of DSS, PA, SaD RA, SaB and SaA was 6.45-6.92, 1.10-1.14, 1.09-1.10, 0.86-0.90, 16.76-19.38 and 1.83-1.94 [micro]g/ml, respectively), the inhibition rates were increased by 62.8% and 38.8%, respectively (P<0.05) (Table 1). Drug combination of DHI and ASA strengthened the inhibition activity of ASA on both COX-1 and COX-2. Indomethacin and NS-398 were used as reference standards for COX-1 and COX-2, respectively. In the COX inhibitory assay, indomethacin and NS-398 yielded approximately 50% inhibition on COX-1 and COX-2 at a final concentration of 0.22 [micro]M and 0.18 [micro]M, respectively, which was concordant with the reference values provided by the kit and indicated that this assay had essential accuracy and repeatability.

It has been reported that the therapeutical effect of DHI is mainly derived from its platelet inhibition by reducing the expression of CDP26, as well as activating the GP IIb/IIIa receptor (Wang et al. 2014). In our study, DHI has shown potent inhibitory effect on COX-1 with a percent inhibition of 61.4%, which proposed that the inhibition effect of DHI on COX-1 might contributed to its platelet inhibition.

Promotion of DHI on gastric mucus secretion during its concurrent treatment with ASA

Gastric mucus is essential in gastric mucosal barrier as the first line of defense. The results of PAS staining showed that administration of ASA decreased the secretion of gastric mucus (P<0.05) (Fig. 5A, B). which was in agreement with previous reports (Asada et al. 1990; Sarosiek et al. 1986). When ASA was administrated in combination with DHI, there were 3.4- and 3.8-fold increase in the Area and IOD of positive staining, respectively (P<0.05) (Fig. 5A, B), which showed that the secretion of gastric mucus was significantly improved due to the drug combination.

Down regulation of DHI on pepsin activity during its concurrent treatment with ASA

Administration of ASA for 14 days increased pepsin activity both in the gastric mucosa (Fig. 6A) and the gastric juice (Fig. 6B) (P<0.05). When ASA was administrated in combination with DHI, the pepsin activity of gastric mucosa (Fig. 6A) and gastric juice (Fig. 6B) was significantly decreased by 23.1% and 32.4%, respectively (P< 0.05).

Recovery effect of DHI on antioxidant activity during its concurrent treatment with ASA

The generation of ROS plays an important role in the pathogenesis of experimental gastric mucosal damage induced by water immersion-restraint stress, gastric ischemia-reperfusion and 100% ethanol (Kwiecien et al. 2002). The increase of ROS could break the normal antioxidant and oxidative balance, which leads to the oxidative stress and cell damage (Oh et al. 2001). ASA was also a forceful risk factor for gastric mucosa and our study of ROS supported evidences that increase of ROS level was involved in the gastric mucosal damage caused by ASA (P< 0.05). When ASA was administrated in combination with DHI, the fluorescence intensity was decreased by 38.8% from 46.81 to 28.66 (P<0.05) (Table 2; Fig. 7A, B), which showed that ROS level was significantly decreased due to drug combination.

CAT, GPx and SOD are major antioxidative enzymes to scavenge ROS and MDA is generated when ROS reacts with cellular lipids. In gastric mucosa, administration of ASA decreased the activity of CAT and GPx, as well as increased the MDA concentration (P<0.05). When ASA was administrated in combination with DHI, CAT and GPx activities were significantly increased by 1.86- and 1.97-fold, and MDA concentration was significantly decreased in 46.4% from 0.84 to 0.45 (P<0.05). In rat plasma, DHI increased activity of CAT and SOD by 61.2% and 24.1%, respectively, and decreased MDA concentration from 4.28 to 3.24 (P<0.05) (Table 3). These results showed that DHI could recover antioxidant activity impaired by ASA and decrease the ROS level of gastric mucosa.

Discussion

The emptying of prostaglandins caused by inhibition effect of ASA on COX could lead to the gastric mucosal damage (Fig. 8). Prostaglandins, an important factor in gastric mucosal protection, could stimulate gastric mucus secretion, regulate pepsin activity and enhance gastric mucosal blood flow (Asada et al. 1990). In our study, DHI increased gastric mucus secretion, as well as decreased pepsin activity thus maintaining the integrity of the gastric mucosal barrier. Furthermore, DHI showed significant effect in protecting gastric mucosa by decreasing ROS level in stomach through antioxidant activity recovery, and disrupting the vicious circle between gastric mucosal blood flow and ROS level (Duan et al. 2004; Holzer et al. 1991; Said and El-Mowafy 1998) (Fig. 8).

Two isoforms of COX (COX-1 and COX-2) are essential in gastric mucosal damage caused by ASA and have been particularly proposed in recent years. Constitutive COX-1 is thought to produce protective prostaglandins for physiological functions, whereas inducible COX-2 mainly mediates the inflammatory process, and is stimulated by inflammatory conditions, lipopolysaccharide, growth factors and cytokines (Mahmud et al. 1996). COX-1 is stably expressed in both the platelets and the stomach. Inhibition of COX-1 could decrease the production of thromboxane [A.sub.2] (TX[A.sub.2]) in platelets and thus bring about the antithrombotic effect. Simultaneously, the decrease of protective prostaglandins caused by inhibition of COX-1 in stomach could lead to gastric damage. On the other hand, more evidence has revealed that only inhibition of both COX-1 and COX-2 would induce gastric damage (Wallace et al. 2000). In the process of gastric ulcer, the rapid induction of COX-2 expression and extra production of prostaglandins could contribute to the ulcer repair (Mizuno et al. 1997; Schmassmann et al. 1998). In consideration of the essential roles of COX-1 and COX-2 in ASA-induced gastric damage, we hypothesized that DHI might antagonize the inhibitory effect of ASA on COX to produce the protective effect. However, our results showed that DHI alleviated gastric mucosal damage due to ASA but not through modulating COX activity. The combination of DHI and ASA strengthened the inhibition extent on both COX-1 and COX-2. Consequently DHI might protect gastric mucosal damage through a non-prostaglandins way. Protease-activated receptor 2 (PAR-2), abundantly expressed in stomach, is a protective factor in gastric mucosa by triggering the secretion of gastric mucus, increasing the gastric mucosal blood flow, and suppressing the secretion of gastric acid (Kawabata 2002). DHI might protect the gastric mucosal damage through the PAR-2 way, which would be further studied in our next step.

Gastrointestinal complications seem to be the most common side effects of ASA. In the clinic, enteric-coated ASA is used to avoid this risk, however, a series of pharmacoepidemiological studies have pointed out that the risk of upper gastrointestinal bleeding associated with coated ASA is similar to that caused by non-coated ASA (Abajo and Garcia 2001; Derry and Loke 2000; Garcia et al. 2001; Kelly et al. 1996). Enteric-coated ASA could not reduce the risk of gastrointestinal damage. In consideration of its outstanding effects in analgesic, anti-inflammatory and antiplatelet, ASA is still popular for having benefits as well as risks. For instance, in 1000 patients who have taken ASA for 5 years, 12-40 patients would benefit from its antiplatelet effect, and 24 patients would suffer from its gastrointestinal complications. The severity of cardiovascular diseases varies case by case and different patients have different desires for the antiplatelet benefit of ASA. Accordingly, the benefit-risk ratio would be different depending on patients. For some elderly patients with history of ulcers who have taken ASA, the risk of gastrointestinal bleeding might overcome the potential cardiovascular benefits. In our study, we found out that DHI could significantly alleviate the gastric mucosal damage caused by ASA when they were used in combination, which could contribute to the benefits of ASA administration.

Conclusion

Stomach was the target organ of interaction between DHI and ASA during their concurrent treatment. DHI could alleviate ASA-induced gastric mucosal damage in different regimes of administration. DHI could improve gastric mucus secretion, as well as decrease pepsin activity to maintain the integrity of gastric mucosal barrier. Furthermore, DHI could recover antioxidant activity and the ROS level which were impaired by ASA to protect the gastric mucosa damage. Drug combination with DHI strengthened the inhibition activity of ASA on both COX-1 and COX-2. DHI alleviated ASA-induced gastric mucosal damage but not antagonized anti-COX effect of ASA. Benefits of protective effect on stomach were clearly produced when DHI and ASA were used in combination.

http://dx.doi.org/10.1016/j.phymed.2016.03.006

ARTICLE INFO

Article history:

Received 10 September 2015

Revised 2 March 2016

Accepted 9 March 2016

Abbreviations: ASA, aspirin; CAT, catalase; COX, cyclooxygenase; DCFH-DA, 2, 7-dichlorofuorescin diacetate; DHI, Danhong injection; DSS, danshensu; EIA, enzyme immunoassay; GSH-PX, glutathione peroxidase; HE, hematoxyiin-eosin staining; HIS, hospital information system; IOD, integrated optical density; MDA, malondialdehyde; PA, procatechuic aldehyde; PAR-2, protease-activated receptor 2; PAS, periodic acid-Schiff staining; PDA, photo diode array; QTOF/MS, quadrupole time of flight mass spectrometry; RA, rosmarinic acid; ROS, reactive oxygen species; RSD, relative standard deviation; SaA, salvianolic acid A; SaB, salvianolic acid B; SaD, salvianolic acid D; SOD, superoxide dismutase; TX[A.sub.2], thromboxane [A.sub.2]; UPLC, ultra high performance liquid chromatography.

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgments

This work was supported by National Basic Research Program of China (973 Program) (2011CB505300, 2011CB505303), Key Research Project in Basic Science of Jiangsu College and University (14KJA360001), National Technology Major Project of China (2015ZX09501004001006), Graduate Research Innovation Program of Jiangsu College (KYLX_0968) and National Natural Science Foundation of China (81573714).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.03.006.

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Jian-ping Li (a,b,1), Jian-ming Guo (a,b,1), Yong-qing Huaa (a,b), Kevin Yue Zhu (a,b), Yu-ping Tang (a,b), Bu-chang Zhao (c), Li-fu Jia (c), Jing Zhao (c), Zhi-shu Tang (d), Jin-ao Duan (a,b), *

(a) Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China

(b) Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China

(c) Buchang Pharma, Xi'an 710000, China

(d) Shanxi University of Chinese Medicine, Xianyang 712000, China

* Corresponding author at: Jiangsu Key Laboratory for High Technology Research of TCM Formulae and Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China. Tel./fax: +86 25 85811116.

E-mail address: dja@njucm.edu.cn, dja@njutcm.edu.cn (J.-a. Duan).

(1) These authors contributed equally to this work.

Table 1
Inhibitory effect of ASA, DHI and DHI-ASA
on COX-1 and COX-2 (mean [+ or -] S.E.M.).

Inhibitory effect on COX-1

Name               Concentration          Inhibition (%)

Indomethacin (a)   0.22 [micro]M          50.5 [+ or -] 1.5
ASA                71.9 [micro]M          36.8 [+ or -] 0.2
DHI                23 fold dilution (b)   61.4 [+ or -] 3.2
DHI-ASA (c)        23 fold dilution-      62.8 [+ or -] 1.7 *
                     71.9 [micro]M

Inhibitory effect on COX-2

Name               Concentration (pM)     Inhibition (%)

NS-398 (a)         0.18 [micro]M          49.3 [+ or -] 2.8
ASA                71.9 [micro]M          28.9 [+ or -] 2.2
DHI                23 fold dilution (b)   1.02 [+ or -] 0.1
DHI-ASA            23 fold dilution-      38.8 [+ or -] 1.9 *
                     71.9 [micro]M (c)

* Indicates significant (P < 0.05) change compared to ASA.

(a) Indomethacin and NS-398 were used as reference standards
for COX-1 and COX-2, respectively.

(b) DHI was diluted by 23 fold (presented in terms of
concentrations of DSS, PA, SaD RA, SaB and SaA were 6.45-6.92,
1.10-1.14, 1.09-1.10, 0.86-0.90, 16.76-19.38 and 1.83-1.94
[micro]g/ml, respectively) in the COX assay.

(c) DHI (23 fold dilution) and ASA (71.9 [micro]M) were
administrated in combination in the COX assay.

Table 2
ROS level of gastric mucosa determined by flow cytometry.

Group         Fluorescence intensity   Positive incidence (%)

CTL (a)       27.22 [+ or -] 5.62      65.08 [+ or -] 9.77
NS-ASA (b)    46.81 [+ or -] 13.63 *   81.24 [+ or -] 6.86 *
DHI-ASA (c)   28.66 [+ or -] 0.99 *    69.71 [+ or -] 0.42 *

* Indicates significant (P < 0.05) change compared to CTL.
(#) Indicates significant (P< 0.05) change compared to NS-ASA.

(a) Animals of group A (CTL): we supplied water and food
regularly for 14 consecutive days.

(b) Animals of group D (NS-ASA): first of all we injected
saline (4.16 ml/kg) and immediately after we gave ASA solution
(10.41 mg/kg) daily for 14 consecutive days.

(c) Animals of group E (DHI-ASA): first of all we injected DHI
(4.16 ml/kg, presented in terms of DSS, PA, SaD RA, SaB and
SaA were 0.62-0.66, 0.10-0.11, 0.10-0.11, 0.082-0.087, 1.60-1.85
and 0.17-0.18 mg/kg, respectively) and immediately after we gave
ASA solution (10.41 mg/kg) daily for 14 consecutive days.

Table 3
Antioxidant activity of rat gastric mucosa
and plasma (mean [+ or -] S.E.M.).

Group         Gastric mucosa

              CAT activity           GPx activity
              (U/mg prot)            (U/mg prot)

CTLa          1.47 [+ or -] 0.23     216.97 [+ or -] 36.94
NS-ASA (b)    0.65 [+ or -] 0.14 *   154.97 [+ or -] 28.14 *
DHi-ASA (c)   1.21 [+ or -] 0.25 *   305.25 [+ or -] 70.07 *

Group         Gastric mucosa

              SOD activity            MDA concentration
              (U/mg prot)             (nmol/mg prot)

CTLa          165.04 [+ or -] 15.16   0.61 [+ or -] 0.17
NS-ASA (b)    177.50 [+ or -] 16.42   0.84 [+ or -] 0.11 *
DHi-ASA (c)   178.87 [+ or -] 11.51   0.45 [+ or -] 0.09 *

Group         Rat plasma             Rat plasma

              CAT activity           GPx activity
              (U/ml)                 (U/ml)

CTLa          0.72 [+ or -] 0.19     2899.22 [+ or -] 309.85
NS-ASA (b)    0.49 [+ or -] 0.08 *   3337.67 [+ or -] 465.97
DHi-ASA (c)   0.79 [+ or -] 0.05 *   3270.70 [+ or -] 264.88

Group         Rat plasma

              SOD activity            MDA concentration
              (U/ml)                  (nmol/ml)

CTLa          31.44 [+ or -] 3.37     2.86 [+ or -] 0.49
NS-ASA (b)    23.21 [+ or -] 3.67 *   4.28 [+ or -] 0.77 *
DHi-ASA (c)   28.81 [+ or -] 2.14 *   3.24 [+ or -] 0.48 *

* Indicates significant (P < 0.05) change compared to CTL. #
Indicates significant (P < 0.05) change compared to NS-ASA,

(a) Animals of group A (CTL): we supplied water and food regularly
for 14 consecutive days.

(b) Animals of group D (NS-ASA): first of all we injected saline
(4.16 ml/kg) and immediately after we gave ASA solution (10.41
mg/kg) daily for 14 consecutive days.

(c) Animals of group E (DHI-ASA): first of all we injected DH1
(4.16 ml/kg, presented in terms of DSS, PA, SaD RA, SaB and SaA
were 0.62-0.66, 0.10-0.11, 0.10-0.11, 0.082- 0.087, 1.60-1.85 and
0.17-0.18 mg/kg, respectively) and immediately after we gave ASA
solution (10.41 mg/kg) daily for 14 consecutive days.
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Author:Li, Jian-ping; Guo, Jian-ming; Hua, Yong-qing; Zhu, Kevin Yue; Tang, Yu-ping; Zhao, Bu-chang; Jia, L
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 1, 2016
Words:6666
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