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Abstract

Danshen injection, a pharmaceutical dosage form of Danshen, has been widely used in the treatment of coronary heart diseases, myocardial infarction, and hypertension. With more and more adverse drug reactions linked with Danshen injection, its safety comes under suspicion. To evaluate its safety, mice were divided into four groups: vehicle, low-, middle-, and high-Danshen group, and each group was intravenously administered with Danshen injection at a dose of 0, 0.64, 1.55, and 5.76 g/kg/day for 5 days, respectively (the low dosage was the recommended clinical dosage, the middle dosage was the most commonly used higher dosage, and the high dosage was the highest dosage used in clinic). Peripheral vascular toxicity wasn’t observed in the low-dosage group, elevated serum endothelin-1 (ET-1) was observed in the middle-dosage group; and more peripheral vascular toxicities like increased vascular leakage, elevated serum nitrate and ET-1, and vascular endothelial cells apoptosis were detected in the high-dosage group. In vitro study, low-concentration Danshen injection showed protective effect to human umbilical vein endothelial cells (HUVECs), while high concentration displayed strong cytotoxic effects, including increase in nitric oxide and ET-1 production, inhibition of cell viability, and apoptosis induction. Further, the HUVECs’ apoptosis induced by high-concentration Danshen injection was found along with the induction of reactive oxygen species. In conclusion, these results suggest that Danshen injection is nontoxic in its recommended clinical dosage, and the 2.4-fold as the recommended clinical dosage might be the highest safety dosage in clinic treatment. In addition, Danshen injection is a potential vascular toxic drug in its high dosage and shouldn’t be used far beyond its recommended dosage in clinic treatment.

Keywords

Danshen injection adverse drug reactions peripheral vascular toxicity vascular endothelial cells apoptosis

Introduction

Danshen, the root of a plant called Salvia miltiorrhiza Bunge, has been widely used in China for over a thousand years to treat various diseases. Based on the traditional Chinese medicine (TCM) theory, Danshen belongs to the class of medicine that ‘promotes circulation and dissipates blood stasis and its extract products are commercially available for clinical treatment, especially in Asia.¹ In China, numerous pharmaceutical dosage forms of Danshen are commercially available, including tablets, capsules, granules, injections, oral liquids, sprays, and dripping pills of either Danshen or Fufang Danshen.^1,2 The application of traditional Chinese medicine injections (CMIs) including Danshen injection has changed the traditional delivery methods of Chinese medicine and broadened the clinical application of Chinese medicine. Now Danshen injection has been widely used in the treatment of coronary heart diseases, myocardial infarction, and hypertension.^3,4 Moreover, it has showed beneficial effects as an anti-dementia, antioxidant, and antitumor agent.^5
–7

From TCMs’ safety perspective, some clinical studies point out that the incidence of adverse drug reactions (ADRs) in CMIs is higher than the average in TCM drugs, and three-quarters of CMI-linked ADRs are serious.⁸ Therefore, the State Food and Drug Administration of China declares that the CMIs must be reevaluated, and the technical process, quality standard, clinical efficacy, and safety of injections should be included. As for the safety of Danshen injection, most part of the studies is about ADRs in case-report and the rest is about the acute and sub-chronic toxicity. The ADRs of Danshen injection include allergic reactions, like anaphylactic shock, skin rashes, fever, and asthma, which are the most common acute toxicities of Danshen,⁹ while others include cardiovascular system events (bradycardia, tachycardia, and elevation of blood pressure), liver injury (elevation of direct bilirubin, indirect bilirubin, and alanine aminotransferase), kidney damage (naked eye hematuria and hemolytic uremic syndrome), respiratory tract hemorrhage, facial angioneurotic edema, phlebitis, and even death.^10
–12 An overview about adverse events of traditional CMIs shows that the proportion of ADR articles of Danshen injection is in the 8th place among 33 CMIs on the National Essential Medicines List of China, while the top four CMIs are suspended from sales or removed from the market,⁸ and another epidemiological investigation finds that the proportion of ADR articles of Danshen injection is in the first place among Chinese activating blood herbal injection.¹³ Although laboratory research about the safety of Danshen injection is scanty, there are the same results in the acute and sub-chronic toxicity studies: Danshen injection is nontoxic in its recommended clinical dosage and its toxicity can be observed in extremely high dosage (100 times as the recommended clinical dosage in acute study and 20 times in sub-chronic toxicity study).^14,15 But the majority of herbs in TCM appears to have a wide range of dosages in clinical treatment, in which Danshen injection can be used up to eight fold higher than the recommended clinical dosage in liver fibrosis, preeclampsia, and cirrhosis.^16

–19 Thus, people are wondering whether Danshen injection is really safe enough and its conventional dosage used in clinical treatment is safe too?

So, it is really necessary to assess the safety of CMIs including Danshen injection when being used within or beyond the recommended clinical dosage by further pharmaceutical, experimental, and clinical research. One category of the ADRs of Danshen, namely hematuria, hemolytic uremic syndrome, respiratory tract hemorrhage, edema, and phlebitis, seems associated with peripheral vascular injury. Vascular injury can be caused by drugs in clinical treatment, which is called drug-induced vascular injury (DIVI). The risk assessment of DIVI is difficult to detect or monitor in patients,²⁰ but it has been demonstrated that some drugs are associated with vascular injury in animals including interleukin-2 in mice and type III phosphodiesterase in rats.^21,22 Could Danshen injection be a potential vascular toxic drug? And that ADRs associated with peripheral vascular injury of Danshen injection are related with DIVI?

In the present study, we firstly ascertained the safety of Danshen injection in mice at the recommended clinical dosage, the most commonly used higher dosage, and the highest dosage in clinical treatment, and then we tested the toxicity of Danshen injection to human umbilical vein endothelial cells (HUVECs) in vitro studies.

Materials and methods

Materials

Danshen injection was obtained from Chiatai Qingchunbao Pharmaceutical Co., Ltd (Hangzhou, China). Evan’s blue, formamide, 3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl tetrazoliumbromide (MTT), and N-acetylcysteine (NAC) were produced by Amresco Inc. (Solon, Ohio, USA).

Animals and treatments

One hundred and twenty pathogen-free Kunming mice, half male and half female, weighing 20–25 g were obtained from Experiment Animal Center of Liaoning University of Traditional Chinese Medicine (Shenyang, China). The animals were acclimatized to standard laboratory conditions (temperature 22–25°C, relative humidity 50–60%, and 12-h photoperiod lights on 07:00–19:00). This study was approved by the Institutional Animal Ethics Committee of Liaoning University of Traditional Chinese Medicine, and the study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

Briefly, mice were randomly divided into four groups (n = 30/group), including vehicle group, low-Danshen group, middle-Danshen group, and high-Danshen group. The mice of low, middle, and high Danshen groups were intravenously administered Danshen injection at the dose of 0.64, 1.55, and 5.76 g/kg/day, respectively (the concentration of dosing solutions in low group was the recommended clinical dosage; the dose of middle group was the most commonly used higher dosage in clinical treatment, which was 2.4 times the recommended clinical dosage; and the dose of high group was the highest dosage used in clinical treatment, which was 9 times the recommended clinical dosage and was regarded below the toxic level in the previous study).^11,14 Mice of the vehicle group were intravenously administered saline at the same volume one time per day. Each group of mice was administered or killed differently within 102 h (Figure 1), and lungs and livers were collected for microscopic examination, and serum was collected for serological analysis.

Figure 1.

Mice treatment. Mice were divided into four groups: vehicle group (30), low-Danshen group (30), middle-Danshen group (30), and high-Danshen group (30). Each group mice has three endings: A, B, and C. The A ending: mice were intravenously administered Danshen or saline five times, at 0, 24, 48, 72, and 96 h respectively. Two hours after the last injection (in 98 h), the mice were killed and lungs and livers were collected for histological observation. The B ending: mice were administered Danshen or saline five times. Two hours after the last Danshen or saline injection (in 98 h), mice were injected Evan’s blue, intravenously. Another 2 h later, lungs and livers were harvested for macroscopic observation (in 100 h). The C ending: mice were first administered Danshen or saline at 0 h, and blood was collected from the hearts at 6 and 24 h. Then the remaining mice were administered second, third, and fourth injection at 24, 48, and 72 h, respectively. Before and 6 hours after the fifth injection (in 96 and 102 h, respectively), blood was again collected from the mice. All the blood samples were collected for serological detection. The numbers 30, 25, 20, 15, and 5 represent the number of remaining or killed mice in each administered group.

Detection of vascular injury

On day 5, the mice received the last saline or Danshen injection and 2 h later were injected 0.1 ml of 1% Evan’s blue in phosphate-buffered saline (PBS), intravenously. After 2 h, mice were exsanguinated under anesthesia, and the lungs and livers were harvested to observe pathology damage and then placed in formamide at 37°C overnight. The Evan’s blue in the organs was quantified by measuring the absorbance of the supernatants at 650 nm with a spectrophotometer. Each mouse was individually analyzed for vascular leakage, and data from 5 mice of each group were expressed as mean ± SD.²¹

Detection of serum NO and ET-1

Blood samples were collected from hearts under anesthesia at 6, 24, 96, and 102 h, respectively, after they were administered with Danshen or saline (Figure 1 ). Serum was stored at −70°C until used.

Serum nitrite: The measurement of serum nitrite was performed using the total nitric oxide (NO) assay (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions. The determination of total serum nitrate and nitrite concentration in this assay provides a quantitative measurement of NO generated by NO synthesis. The serum samples were pretreated and then optical density (OD) was measured at 540 nm.¹³ Each experiment was performed in triplicate.

Serum ET-1: Serum endothelin-1 (ET-1) levels were assayed with a commercially available Mouse ET-1 enzyme-linked immunosorbent assay (ELISA) kit (USCN, China). Measurement of sandwich ELISA was performed according to the manufacturer’s instructions, and the absorbance at 570 nm was measured using a microplate reader (Bio-Rad, Hercules, California, USA). Each experiment was performed in triplicate.

Histopathological studies

Lungs and livers were fixed in 10% formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histopathological evaluation. The cleaved-caspase-3 antibody and UltraSensitive™ SP IHC kit (Maixin Biotech, China) were used for immunohistochemistry. The results were reviewed by two independent researchers and analyzed using the MetaMorph/DP10/BX41 image analyzing system (UIC/Immunohistochemistry Olympus, US/Japan).

TUNEL assay of cell apoptosis

Cell apoptosis was evaluated with conventional TUNEL assay using DeadEnd Colorimetric TUNEL System (Beyotime Institute of Biotechnology, China) by following the manufacturer’s instructions. The karyotin of apoptosis nucleus (Nu) was pitchy, while that of the non-apoptosis Nu was calamine blue. Numbers of TUNEL-positive stained endothelial cells (ECs) were counted in 10 different high-magnification views of each lung slide, and the number of positive cells was counted for every 1000 cells.

The apoptosis rate (%) = \frac{number of positive apoptosis cell}{1000} \times 100 % .

Transmission electron microscopic study

Lungs were fixed, postfixed, and embedded as described previously.²³ Ultrasections were observed under a transmission electron microscope (model RILI H-7500; Hitachi, Japan) at 80 kV.

Cell culture and treatment

HUVECs were obtained from School of Basic Medical Sciences, China Medical University, China. Cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Invitrogen), 100 mg/ml heparin and penicillin/streptomycin (100 U/mL and 100 μg/mL, Invitrogen) at 37°C, in a humidified atmosphere containing 95% air and 5% carbon dioxide. Experiments were performed with cells grown to a confluency of 80%. For experiments, cells were divided into four groups, vehicle (Danshen 0), Danshen 2, Danshen 10, and Danshen 50. The vehicle group was treated by PBS and Danshen groups were treated by 2, 10, and 50 μg/mL Danshen injection, respectively.²⁴

Culture medium nitrite and ET-1 detection

After cells were treated with various concentrations of Danshen injection or PBS for 12 h, 200 μL culture medium of each well was collected and then nitrite and ET-1 expression were measured according to the instructions given in the total NO assay kit or ET-1 ELISA kit. OD was read in a microplate reader at 540 or 570 nm. Each experiment was performed in triplicate.

Cell viability assay

Cell viability was evaluated by the MTT assay. Briefly, 4 h after being seeded in a 96-well plate, HUVECs were treated with Danshen injection or PBS for 24 h, or pretreated with 50 μM NAC for 12 h. Then, 10 μL MTT (5 mg/ml) was added to each well and incubated at 37°C for 4 h, and the absorbance was measured using a microplate reader (Bio-Rad) at 570 nm.

Cellular apoptosis detection by flow cytometry

HUVECs were seeded in 6-well plates and incubated according to the aforementioned method for 24 h. The cells were then collected and treated as the protocol in annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (SAB, Pearland, Texas, USA) and the percentages of apoptotic cells were determined by a flow cytometer (BD, Franklin Lakes, New Jersey, USA). Results were analyzed by Cell Quest Pro software (BD).

Western blotting analysis

HUVECs, pretreated or non-treated with NAC, were treated with Danshen or PBS for 24 h. Then, the cells were lysed and subjected to Western blot analysis as described previously.²⁵ The blots were probed with antibodies against glyceraldehyde 3-phosphate dehydrogenase, cleaved caspase-3, and cleaved poly(adenosine diphosphate-ribose)polymerase (PARP; SAB). Horseradish peroxidase-conjugated secondary antibodies (ZSGB BIO, China) were used in conjunction with an enhanced chemiluminescence detection system (Amersham, UK). Staining was quantified by scanning densitometry.

Measurement of ROS production

To evaluate reactive oxygen species (ROS) production induced by Danshen, intracellular oxidative stress ROS assay kit (GENMED, China) was used as previously described.²⁶ HUVECs were treated with various concentrations of Danshen injection for 12 h. The cells were collected and treated with dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 30 min at 37°C as the protocol. Next, the 2′,7′-dichlorofluorescein (DCF) fluorescence stained cells were visualized under a fluorescence microscope at the excitation wavelength of 540 nm and an emission wavelength of 590 nm (Olympus, Japan). Alternatively, the ROS content was measured by FACSCalibur flow cytometer (BD) and analyzed on Cell Quest software (BD FACSCalibur Flow Cytometer Cell Quest Software). ROS was determined by comparing the changes in fluorescence intensity with that of the control.

Statistical analysis

Each experiment was carried out at least three times separately. Data are expressed as means ± SD, Statistical comparison between different groups was done using one-way analysis of variance, followed by Fisher post hoc test to detect the difference between various groups. The value of p < 0.05 was considered to be significant. Statistical analyses were conducted with Statistical Package for the Social Sciences 15.0.

Results

High-dosage Danshen injection induced peripheral vascular function damage

At necropsy, no treatment-related macroscopic findings were identified in different Danshen dosage groups. Further, we tested the integrity of peripheral vessels through the evaluation of vascular pathology damage by injecting Evan’s blue into mice vein after Danshen treatment.²¹ The increase of vascular leakage was observed in all mice of high-dosage Danshen group in which macroscopic blue was observed in eyelids, irises, claws, the skin around nose, and even the tissue of lungs and livers. However, these phenomena weren’t observed in the vehicle, low-dosage, and middle-dosage group mice (Figure 2(a) and (b); Table 1). Besides, quantifying the Evan’s blue leakage into the tissues, the Evan’s blue levels significantly increased in lungs and livers of high-dosage Danshen group (p < 0.01 and p < 0.05, respectively). Also there wasn’t significant difference observed among vehicle, low-dosage, and middle-dosage groups (Figure 2(c)). These results indicated that low- and middle-dosage Danshen injection didn’t affect the peripheral vascular integrity, but high-dosage Danshen injection induced peripheral vascular function damage.

Table 1.

Peripheral vascular integrity test of Danshen injection toxicity in Evan’s blue.

Group	Dose of Danshen (g/kg/day BW)	Times of the clinical concentration for human use	Macroscopic observed (eyelids, irises, claws, and skin around nose in blue)	Tissue observed
Group	Dose of Danshen (g/kg/day BW)	Times of the clinical concentration for human use		Lung in blue	Liver in blue
Vehicle group	0	0	0/5	0/5	0/5
Low-Danshen group	0.64	1	0/5	0/5	0/5
Middle-Danshen group	1.55	2.4	0/5	0/5	0/5
High-Danshen group	5.76	9	5/5	5/5	5/5

BW: body weight.

Figure 2.

High-dosage Danshen injection damaged peripheral vascular integrity. Danshen injection was given to mice via tail vein for 5 days constantly. Two hours after the last Danshen or saline injection, mice were treated with Evan’s blue via tail vein. (a) Mouse of the high-Danshen group. (b) Histopathology of lung and liver with pulmonary vessels (black asterisk), hepatic portal vessels (white asterisk), and local lung tissue (arrow) in blue; and all liver tissue in dark. (c) Quantity of Evan’s blue leakage outside vessels of the lung and liver.

In addition, we tested serum nitrate and ET-1, two biomarkers correlated with vascular EC function to observe the effect of high-dosage Danshen injection on microvessels. Significant elevation of serum nitrate was seen at 6 h and beyond in high-dosage Danshen injection group compared with the vehicle group, but the values of low- and middle-dosage group were roughly as those of the vehicle group. As for serum ET-1, besides the significant elevation in high-dosage Danshen group at all time points, there was significant elevation in middle-dosage Danshen group at 102 h compared with the vehicle group (Figure 3).

Figure 3.

High-dosage Danshen injection induced serum nitrate and ET-1 elevations. Mice were intravenously administered Danshen injection at the dose of 0, 0.64, 1.55, and 5.76 g/kg/day, respectively. At the 6, 24, 96, and 102 h, serum nitrate and ET-1 were analyzed. (a) Serum nitrate levels. (b) Serum ET-1 levels. ET-1: endothelin-1.

High-dosage Danshen injection induced peripheral vascular pathology damage

We investigated the reason for high-dosage Danshen-mediated peripheral vascular function damage. Lung tissues stained with H&E showed widespread alveolar wall thickness and severe hemorrhage in the high-dosage Danshen group, while little pathology damage was seen in vehicle, low-dosage, and middle-dosage Danshen groups (Figure 4(a)). In the liver tissues, there was no obvious difference observed in high-dosage Danshen group and the other groups (data not supplied). To further examine this pathological effect, an in situ TUNEL assay was used to detect apoptotic cells in lung sections. The results demonstrated that lung EC apoptosis occurred in the high-dosage Danshen group, whereas few apoptotic cells were found in vehicle, low-dosage, and middle-dosage Danshen groups (Figure 4(b)). We further investigated high-dosage Danshen injection-mediated apoptosis on ECs by electron microscopic studies. As shown in Figure 4(c), the ECs of vehicle, low-dosage, and middle-dosage Danshen groups showed normal morphological features, and integrated tight junctions (TJs) of the air–blood barriers were observed in the vehicle group. In contrast, ECs not only revealed nonintegrated TJs, but extensive cell membrane break and cytoplasm damage in the high-dosage Danshen group. Tested with cleaved caspase-3, one of the apoptosis proteins, the high-dosage Danshen showed a stronger immunoreactivity (p < 0.05), which was consistent with the result of TUNEL and electron microscopic studies (Figure 4(d)).

Figure 4.

High-dosage Danshen injection induced vascular EC apoptosis in mice. (a) Lung sections stained with H&E revealed pulmonary histopathological changes. Widespread alveolar wall thickness and severe hemorrhage are shown in high-dosage Danshen group (black asterisk). (b) Detection of apoptotic ECs in lung tissue by TUNEL staining. Apoptotic cells are depicted by brown-stained nuclei in high-dosage Danshen group (black arrow). (c) The ultrastructure of air–blood barriers in lung tissue. The EC of the vehicle group showed normal morphology with intact Nu, cell membrane, and integrated TJs (long arrowhead). The ECs of low-dosage and middle-dosage Danshen group showed intact Nu and cell membrane, too. The EC of high-dosage of Danshen group showed significant damage with cell membrane break (short white arrowhead) and cytoplasm damage (short black arrowhead), and didn’t reveal integrated TJ either. RBCs lay within the blood vessel lumen in three group photos (original magnification ×5000). (d) The immunoreactivity for cleaved caspase-3 in lung. The black arrows pointed to the positive results. Scale bar: 80 μm. ET-1: endothelin-1; H&E: hematoxylin and eosin; Nu: nucleus; TJ: tight junction; RBC: red blood cells.

High-concentration Danshen injection induced NO and ET-1 elevation in HUVECs in vitro

To further determine the effect of high-dosage Danshen injection on ECs, we used the HUVECs in vitro and observed the culture medium nitrite and ET-1 under Danshen exposure. As shown in Figure 5, the NO production in HUVEC culture medium was slightly decreased after incubation for 12 h in the Danshen 2 group compared with the vehicle group, while high-concentration Danshen increased NO production. Specially, the level of NO production was notably higher in the Danshen 50 group than that of the vehicle group (p < 0.05). These results indicated that 2 μg/mL Danshen injection inhibited the NO production, while high-concentration Danshen injection increased NO production in a concentration-dependent manner in HUVECs. The same results were observed in culture medium ET-1 analysis.

Figure 5.

High-concentration Danshen injection induced NO and ET-1 elevation in HUVECs. HUVECs were treated with Danshen injection (0, 2, 10, and 50 μg/mL) for 24 h, and culture medium was collected. (a) Culture medium nitrate levels. (b) Culture medium ET-1 levels. NO: nitric oxide; HUVEC: human umbilical vein endothelial cell; ET-1: endothelin-1.

High-concentration Danshen injection induced HUVECs’ apoptosis in vitro

We investigated the effect of Danshen injection on cell viability. The number of HUVECs in Danshen 2 group was significantly increased as compared to the vehicle group (p < 0.05). But in Danshen 10 and 50 groups, Danshen injection reduced HUVECs’ viability, and the statistical difference only exists between the Danshen 50 group and vehicle group (p < 0.01; Figure 6(a)). Next, the apoptotic activity was measured via annexin V- FITC/propidium iodide apoptosis detection using flow cytometry. After being treated with Danshen injection for 24 h, low-concentration Danshen injection (2 μg/mL) slightly reduced the rate of apoptotic and death cells. On the contrary, high-concentration Danshen (10 and 50 μg/mL) increased the apoptosis and total cell death rate, special in the Danshen 50 group (Figure 6(b)). Last, we analyzed the activation of caspase-3 characteristic for induction of apoptosis as well as the inactivation of PARP, a DNA repair factor, by immunoblotting. Caspase 3 activation and PARP inactivation/cleavage were observed in Danshen 50 group, which was similar to that of the flow cytometry test (Figure 6(c)).

Figure 6.

High-concentration Danshen injection induced HUVEC apoptosis. HUVECs were treated with Danshen injection (0, 2, 10, and 50 μg/mL) for 24 h. (a) The cell viability was measured with MTT assay. Data are expressed as the mean of the triplicate determinations (X ± SD) compared with vehicle cells (100%). (b) Cell death rate was analyzed by the annexin V-FITC/PI assay by flow cytometry. (c) Cell lysates were prepared and subjected to immunoblotting with antibodies to cleaved caspase 3, cleaved PARP, and GAPDH. Data are means ± SD of three independent experiments. HUVEC: human umbilical vein endothelial cell; FITC: fluorescein isothiocyanate; PI: propidium iodide; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

ROS involved in high-concentration Danshen injection induced HUVECs’ apoptosis in vitro

With regard to the apoptotic mechanism, we analyzed the intracellular ROS production with DCFH-DA staining. At 12 h after treatment, about 1.8, 2.1, 5.6, and 27.6% of cells in 0, 2, 10, and 50 μg/mL Danshen-treated groups showed bright DCF fluorescence, respectively (Figure 7(a)). When the DCF fluorescence-stained cells were observed under a fluorescence microscope, the ROS generation similarly increased in the high-dosage Danshen (50 μg/mL) group by direct visual judgment of the fluorescence intensity (Figure 7(b)).

Figure 7.

High-concentration Danshen injection induced production of ROS in HUVECs. (a and b) Cells were treated with Danshen injection for 12 h and labeled with DCFH-DA, and the fluorescence intensity of the oxidized product DCF in individual cells was detected by flow cytometry and fluorescence microscopy. (c and d) HUVECs were pretreated with 50 μM NAC for 12 h, followed by 50 μg/mL Danshen injection for another 12 h. MTT assay and Western blotting were performed as described in the legend to Figure 6. ROS: reactive oxygen species; HUVEC: human umbilical vein endothelial cell; DCFH-DA: dichloro-dihydro-fluorescein diacetate; DCF: 2′,7′-dichlorofluorescein; NAC: N-acetylcysteine.

To find out whether the ROS production was related to the increased cell apoptosis, HUVECs were pretreated with ROS scavenger NAC (50 μM), followed by the high-dosage Danshen (50 μg/mL) exposure. We found that NAC indeed recovered survival of high-dosage Danshen-treated cells (Figure 7(c)) and diminished cell apoptosis (Figure 7(d)), which suggested a role of ROS in high-dosage Danshen-induced cell apoptosis.

Discussion

DIVI is a common finding in preclinical toxicity testing of drugs in animals, and similar findings have not been documented in clinical trials or in the clinical histories of patients treated with drugs that demonstrated this effect preclinically, making the relevance of this finding unclear with respect to human risk.²⁷ For the majority of TCMs, the efficacy and toxicity assessments are based on traditional knowledge and clinical experience rather than evaluation in a laboratory, so the DIVI of TCMs has been overlooked in traditional herb treatment. Vascular injury is the main pathological phenomenon in DIVI, which is characterized by the damage of peripheral vascular integrity and increased vascular permeability, and damage and dysfunction to the ECs are the major features of the vascular pathology.^21,28 In the process of vascular injury, ECs may undergo apoptosis or necrosis and ultimately lead to endothelial dysfunction and accelerate the pathological progress.²⁹ ECs are capable of producing a large variety of different molecules, in which the main endothelium-derived relaxing factor and potent vasoconstrictor are NO and ET-1.²⁸ ET-1 has been suggested as a potential marker of endothelial dysfunction for its pathophysiologic role in various forms of cardiovascular disease.^30,31 ET-1 binding to its receptor subtype B on the ECs’ surface can induce NO release in acute period of vascular injury.^32,33 NO is oversupplied in some pathologic conditions,^34,35 and excessively elevated levels of NO can cause vascular injury by reacting with ROS/reactive nitrogen species (RNS) and superoxide dismutase.³⁶

In our study, low-dosage Danshen injection (0.64 g/kg, the recommended clinical dosage) and middle-dosage Danshen injection (1.55 g/kg, the 2.4-fold as the recommended clinical dosage) didn’t show peripheral vascular toxicities in mice except the elevated serum ET-1 in middle-dosage group in 102 h. While the pPeripheral vascular injury events happened when administered with high-dosage Danshen injection (5.76 g/kg, the nine fold as the recommended clinical dosage), including the elevated serum nitrite and ET-1, increase of vascular leakage and apoptosis of lung tissue vascular ECs’ occurred. These results indicate that Danshen injection could be toxic to peripheral vessels when used in high dosage, and the 2.4-fold as the recommended clinical dosage might be the highest safety dosage in clinical treatment. The toxic dosage seems lower than the results of previous studies that toxic effects could be observed at the dosage as 100 times as the recommended clinical dosage in acute study and 20 times in sub-chronic toxicity study.^14,15 This could be explained that only macroscopic observation but no functional assessment and microscopic observation were done in those previous studies. Furthermore, our findings only observed pathological changes in lung and liver, while up to twenty fold higher than the recommended clinical dosage could cause lung, liver, and kidney hemorrhage or congestion in mice.¹⁵ This interesting result may hint that lung might be the most vulnerable organ to vascular toxicity caused by high-dosage Danshen injection.

In order to confirm the effects of high-dosage Danshen injection on vascular ECs’ apoptosis, in vitro test of incubated HUVECs were used. Based on the knowledge from the published article that 2 and 10 μg/mL Danshen could promote endothelial progenitor cells’ augmentation and enhance its functional activity, while 50 μg/mL Danshen restrains it,²⁴ we treated HUVECs with three concentration regimens, 2, 10 and 50 μg/mL, too. In our results, low-concentration Danshen injection showed protective effect, such as slight decrease in the NO, ET-1 production, and the rate of apoptotic and death cells and significantly increase in cell viability, while high-concentration Danshen injection displayed strong toxicity to HUVECs, including significant increase in the NO and ET-1 production, inhibition of cell viability, and apoptosis induction. These findings are in line with some previous reports about the anticancer properties of some Danshen compounds through the induction of apoptosis, like Salvianolic acid B, isolated and purified from Danshen, reduce cancer cells’ growth via induction of apoptosis.³⁷ High-concentration Danshen injection contains high-level cytotoxic components, which are equally toxic to normal cells and could be the reason of HUVECs’ apoptosis.

Some compounds of Danshen, such as Salvianolic acid B and cryptotanshinone, induce cancer cells’ apoptosis by ROS-mediated kinase activation signaling pathway.^37

–40 Cell apoptosis protein caspases are sensitive to redox changes in the ECs, and processing and activation of caspase 3 are regulated by the increase of intracellular ROS and its downstream intermediary proteins. In our research, we also found that ROS could be stimulated in HUVECs’ apoptosis induced by high-concentration Danshen injection; conversely, this wasn’t observed in the low-concentration group. To further elucidate the apoptosis induced by high-concentration Danshen injection, we checked the effect of NAC, selective inhibitors of ROS. Results showed that NAC almost completely inhibited the activation of caspase-3 and the inactivation of PARP and recovered the survival of treated cells. These results suggested that high-concentration Danshen injection could induce ROS activation, which leads to caspase-dependent cell death in HUVECs.

It is well known that the combinations of NO with ROS can induce cytotoxicity, and ROS-mediated vascular injury has been shown to be augmented by high levels of endogenous NO.⁴¹ Our results found that ROS generation was followed by NO increase in HUVECs’ apoptosis induced by high-concentration Danshen injection, which further confirmed the toxicity of Danshen injection to mice peripheral vascular when subjected to high dosage. Taken together, our findings suggest that peripheral vascular injury induced by high-dosage Danshen injection was mediated by ROS/RNS generation.

Based on the toxicological parameters measured and conditions of these studies, our data demonstrated that Danshen injection couldn’t induce any signs of toxicity when administered intravenously in mice in the recommended clinical dosage, but its vascular toxicity could been observed when used in higher dosage, elevated serum ET-1 in 2.4-fold as the recommended dosage, increased vascular leakage, elevated serum nitrate and ET-1, and ECs apoptosis in 9-fold as the recommended dosage. In summary, this result suggests that Danshen injection is nontoxic in its recommended clinical dosage, and the 2.4-fold as the recommended clinical dosage might be the highest safety dosage in clinic treatment. In addition, Danshen injection could cause vascular toxicity when used in high dosage, which might be a warning to doctors that Danshen injection is a potential vascular toxic drug in its high dosage and shouldn’t be used far beyond its recommended dosage in clinic treatment.

Footnotes

Authors’ Contribution

The authors CW and RZ contributed equally to this work.

Conflict of interest

The authors declared no conflicts of interest.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Xinglin scholars Project of Liaoning University of Traditional Chinese Medicine.

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An in vivo and in vitro study

Abstract

Keywords

Introduction

Materials and methods

Materials

Animals and treatments

Detection of vascular injury

Detection of serum NO and ET-1

Histopathological studies

TUNEL assay of cell apoptosis

Transmission electron microscopic study

Cell culture and treatment

Culture medium nitrite and ET-1 detection

Cell viability assay

Cellular apoptosis detection by flow cytometry

Western blotting analysis

Measurement of ROS production

Statistical analysis

Results

High-dosage Danshen injection induced peripheral vascular function damage

High-dosage Danshen injection induced peripheral vascular pathology damage

High-concentration Danshen injection induced NO and ET-1 elevation in HUVECs in vitro

High-concentration Danshen injection induced HUVECs’ apoptosis in vitro

ROS involved in high-concentration Danshen injection induced HUVECs’ apoptosis in vitro

Discussion

Footnotes

Authors’ Contribution

Conflict of interest

Funding

References