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Introduction

Ischemic heart disease is a serious condition due to its high morbidity and mortality, which is responsible for ~40% deaths associated with cardiovascular diseases worldwide (1). At present, timely restoration of blood flow is a generally accepted strategy that is effective for improving ischemic heart injury (2,3). However, reperfusion may trigger several adverse responses, including calcium overload, oxidative-nitrosylation stress, endoplasmic reticulum stress, mitochondrial dysfunction and inflammation, leading to cell death and heart dysfunction (2,3). Therefore, myocardial ischemia/reperfusion injury is an essential clinical problem worldwide that has attracted increasing attention from clinicians. An increasing number of studies have focused on strategies for alleviating this condition.

A number of studies have previously demonstrated that cardiomyocyte autophagy is increased in both acute ischemia-reperfusion (4-6) and chronic ischemia models (7,8). Autophagy is increasingly recognized as a potential target for the treatment of myocardial ischemia-reperfusion (9,10) and is generally activated by oxidative stress, hypoxia and starvation, which is physiologically a cell defense and adaption mechanism to promote cell survival (11). However, excessive and uncontrolled autophagy can result in cell death (12). Therefore, maintaining autophagy at a reasonable level is essential for determining cell fate. Tert-butylhydroperoxide (t-BHP) is an organic peroxide compound that is used widely as an alternative to H2O2 in oxidative stress studies to mimic cellular oxidative damage (13-15). In addition to the increased production of reactive oxygen species (ROS), t-BHP-induced cell death can also result from the activation of autophagy (16,17). Therefore, inhibition of autophagy may protect against t-BHP-induced H9c2 cardiomyocyte death (15).

Traditional Chinese medicine provide an important platform for the discovery of novel therapeutic agents. Danshen and Chuanxiong are two common traditional Chinese medicines that have been used widely for myocardial ischemia therapy (18,19). Danshensu (DSS) and tetramethypyrazin (TMP) are the major active components present within Danshen and Chuanxiong, which exert various pharmacological properties on the cardiovascular system (18,19). In particular, compound DT-010 (Fig. 1) is a patented compound derived from DSS and TMP that was previously synthesized (20). A previous study demonstrated the protective activities of DT-010 against myocardial ischemia and reperfusion injury in a rat model, where DT-010 treatment reduced the infarct size (20). The cardioprotective effect of DT-010 is more potent compared with that of DSS and TMP (21). This previous study also revealed that DT-010 markedly increased cell viability and suppressed cell apoptosis in t-BHP-treated H9c2 cells via activation of the peroxisome proliferator-activated receptor γ coactivator (PGC)-1/nuclear factor erythroid 2-related factor 2 (Nrf2)/transcription factor A and the mitochondrial/heme oxygenase-1 (HO-1) signaling pathway (20). In addition, DT-010 has also been reported to prevent doxorubicin-induced cardiotoxicity by inhibiting apoptosis, ROS generation and autophagosome formation (21).

Figure 1 Structural formula of DSS, TMP and DT-010. DSS, danshensu; TMP, tetramethypyrazin.

It was speculated that autophagy may also be involved in the protective effects of DT-010 against myocardial ischemia-reperfusion injury. Therefore, the present study established a t-BHP-induced oxidative injured cell model using H9c2 cardiomyocytes and evaluated the effect of DT-010 on autophagy and its underlying regulatory signaling mechanism.

Materials and methods

Materials

t-BHP, acridine orange (AO), monodansylcadaverine (MDC), MTT, rapamycin and hydroxy-chloroquine were obtained from Sigma-Aldrich, Merck KGaA. Hoechst 33342 was obtained from Molecular Probes, Thermo Fisher Scientific, Inc. The primary antibody against microtubule-associated protein 1A/1B-light chain 3 (LC3; cat. no. L8918) was purchased from Sigma-Aldrich, Merck KGaA. Primary antibodies against β-actin (cat. no. 4967), p62 (cat. no. 8025), mTOR (cat. no. 2983), 5'-AMP-activated protein kinase (AMPK; cat. no. 5831), unc-51 like autophagy activating kinase 1 (Ulk1; cat. no. 8054), phosphorylated (p)-mTOR (cat. no. 5536), p-AMPK (cat. no. 2535) and p-Ulk1 (cat. no. 14202) were obtained from Cell Signaling Technology, Inc. All primary antibodies were rabbit anti-rat antibodies (dilution, 1:1,000). Anti-rabbit IgG, horseradish peroxidase-conjugated antibody (cat. no. 7074) was purchased from Cell Signaling Technology, Inc. Monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP)-LC3 adenovirus (cat. no. HB-AP21000001; https://www.hanbio.net/cn/services/item/7) was purchased from Hanbio Biotechnology Co., Ltd. The lactate dehydrogenase (LDH) kit (cat. no. C0016) was obtained from Beyotime Institute of Biotechnology. DMEM and FBS were obtained from Thermo Fisher Scientific, Inc. The purity of DT-010 was >95% (22).

Cell culture

The rat myocyte cell line H9C2 was obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences. H9c2 cardiomyocytes were maintained in DMEM containing 10% FBS. A humidified atmosphere containing 5% CO2 at 37˚C was required for cell growth and cell treatment. Cells were passaged or treated when they reached ~80% confluence.

MTT and LDH measurement

Cell survival was detected using MTT and LDH kits. To assess cytotoxicity, cells were seeded into 96-well plates at a density of 1x104 cells/well and cultured at 37˚C. After 24 h, DT-010 was added alone for 1 h at 37˚C. The concentration of DT-010 was set based on a previous study (20), which showed that 300 µM DT-010 treatment alone may exert cytotoxicity. Therefore, a dose range of DT-010 between 10 and 100 µM was chosen for the present study.

Cell viability was measured using MTT assay. To detect the effects of DT-010 on t-BHP-induced injury, DT-010 was first added and the cells were incubated for 1 h at 37˚C. Subsequently, cell supernatant was removed, and the cells were washed twice with Hanks' balanced salt solution (HBSS) to avoid drug interaction. After 6 h exposure to 300 µM t-BHP at 37˚C, the cell supernatant was then removed before the cells were incubated with 100 µl 0.5 mg/ml MTT in the dark at 37˚C for 4 h. The crystals were dissolved by DMSO and the optical density at 570 nm was measured in each well. Cell viability was determined as previously described (20).

The activity of LDH in the cell supernatant was measured using the LDH kit according to the manufacturer's protocol.

For autophagy agonist and blocker treatment, cells were first pretreated with the autophagy agonist rapamycin (500 nM) (23) or the autophagy blocker hydroxy-chloroquine (50 µM) (24,25) for 1 h before being treated with DT-010 for 1 h and t-BHP for a further 6 h. All treatments were performed at 37˚C. In between each treatment, cells were washed twice with HBSS to avoid drug interaction.

Cell apoptosis analysis

Hoechst 33342 nuclear staining was performed to assess cell apoptosis detection as described previously (20). Following DT-010 treatment for 1 h, the cell supernatant was removed, and cells were washed twice with HBSS. Subsequently, the cells were treated with 300 µM t-BHP for another 2 h at 37˚C. H9c2 cells were subsequently dyed with Hoechst 33342 at a final concentration of 5 µg/ml at 37˚C in the dark for 20 min and then observed under an inverted fluorescence microscope (magnification, x400). After Hoechst staining, normal cells stain dark blue with homogeneous fluorescence intensity whereas apoptotic cells are characterized by shrunken and irregular nuclear shapes, in addition to broken and condensed chromatin. Cell apoptosis measurement was repeated three times independently, with 6-8 fields of view in each group randomly photographed. The apoptosis rate was calculated using ImageJ software version 1.44p (bundled with Java v1.6.0_20; National Institutes of Health) using the following formula: Apoptotic rate=(number of apoptotic cells/number of total cells) x100%.

Autophagic flow analysis

Autophagic flow was evaluated using the mRFP-GFP-LC3 assay. H9c2 cells were first seeded onto the laser confocal culture dishes at a density of 5x103 cells/dish. After reaching 30% confluence, cells were transfected with the mRFP-GFP-LC3 adenovirus (1.58x1010 pfu/ml) at 1,500 MOI for 36 h. After another 48 h of culture in complete DMEM, the cells were treated with DT-010 for 1 h and 300 µM t-BHP for 2 h at 37˚C. Finally, cells were fixed with 4% paraformaldehyde at room temperature for 15 min and then observed under a laser confocal microscope (magnification, x630). Autophagic flux was visualized and analyzed via fluorescence imaging of autophagosomes and autolysosomes. Yellow (GFP+/mRFP+) and red (GFP-/mRFP+) puncta indicate the presence of autophagosomes and autolysosomes, respectively.

AO and MDC staining

AO and MDC staining are commonly used to measure autophagosome vesicle formation (26,27). After 1 h treatment with 30 µM DT-010 and another 2 h 300 µM t-BHP treatment at 37˚C, cells were gently rinsed with HBSS and stained with 1 µM AO and 50 nM MDC for 10 min in the incubator at 37˚C. Cells were observed under an inverted fluorescence microscope (magnification, x200), where six-eight fields of view were randomly imaged in each group.

Western blotting

Proteins were extracted from H9c2 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Proteins were quantified with a BCA protein assay kit (Beyotime Institute of Biotechnology) and 30 µg denatured protein from each sample was subjected to 10 or 12% SDS-PAGE. Proteins were then transferred onto PVDF membranes. Following blocking of the membrane with 5% skimmed milk at room temperature for 2 h, immunoblotting was performed by incubation with specific primary antibodies (1:1,000) at 4˚C overnight. After washing, horseradish peroxidase-labeled secondary antibodies (1:2,000) were added and the membranes were incubated at room temperature for 2 h. An ECL detection kit (cat. no. P0018S; Beyotime Institute of Biotechnology) was utilized for protein detection. The optical density of the protein bands was analyzed using the Carestream MI SE system (4,000 MM PRO system; Carestream Health, Inc.).

Statistical analysis

Results are presented as the mean ± SD of at least three experimental repeats. All data were analyzed using GraphPad Prism software (version 6.01; GraphPad Software, Inc.). Significant differences were determined using one-way ANOVA followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

Results

DT-010 protects cardiomyocytes against oxidative stress damage

H9c2 cardiomyocytes were first exposed to t-BHP to mimic ischemia-reperfusion-induced cellular oxidative stress damage. DT-010 alone did not induce cytotoxicity in H9c2 cells (Figs. 2A, B and S1). Compared with cells in the control group, t-BHP exposure significantly decreased cell viability whilst significantly increasing LDH release (Fig. 2A and B). DT-010 pretreatment dose-dependently restored cell viability and reduced LDH release compared with cells in the t-BHP-only group (Fig. 2A and B), suggesting a cardioprotective effect of DT-010 which was observed at 30 and 100 µM. Subsequently, Hoechst 33342 staining was performed for cell apoptosis detection. Normal cells were stained dark blue with homogeneous fluorescence intensity whereas apoptotic cells were characterized by shrunken and irregular nuclear shapes, coupled with condensed chromatin (Fig. 2C and D). The level of apoptosis in H9c2 cells increased significantly following t-BHP exposure compared with that in the control group, but was significantly lower in cells treated with DT-010. This observation suggests a protective effect of DT-010 against apoptosis induced by t-BHP (Fig. 2C and D).

Figure 2 DT-010 protects cardiomyocytes against t-BHP-induced injury. (A) Cell viability and (B) lactate dehydrogenase release were measured. Data are presented as the mean ± SD from six experimental repeats. (C) Percentage of apoptotic cells was measured, which was calculated from (D) representative Hoechst 33342 staining images. Yellow and red arrows indicate normal and apoptotic cells, respectively. Scale bars, 20 µm. Data are presented as the mean ± SD from three experimental repeats. ###P<0.001 vs. Ctrl; *P<0.05, **P<0.01 and ***P<0.001 vs. t-BHP. t-BHP, tert-butylhydroperoxide; Ctrl, control; LDH, lactate dehydrogenase.

DT-010 inhibits oxidative stress-induced autophagy in cardiomyocytes

LC3 protein is a hallmark protein of autophagy induction (26). When autophagy is initiated, LC3-I, the cytosolic form of LC3 is converted into the autophagosomal membrane LC3-II form via enzymatic digestion (26). Therefore, increased LC3-II/LC3-I ratios can be used to indicate autophagosome formation (26). t-BHP treatment significantly increased the LC3-II/LC3-I ratio compared with cells in the control group, which was significantly prevented by DT-010 pretreatment (Fig. 3A), suggesting that DT-010 can inhibit oxidative stress-induced autophagy in cardiomyocytes. Furthermore, AO and MDC staining demonstrated that DT-010 may inhibit autophagy induced by oxidative stress (Fig. 3B). AO and MDC are two commonly used specific agents for the assessment of autophagosomes, acidic endosomes and lysosomes. Acidic vesicular organelles including autophagolysosomes can be stained by AO, while MDC represents widely used fluorescent probes that preferentially accumulate in autophagic vacuoles (26-28). Following t-BHP treatment, the number of orange-red AO stained particles and the extent of MDC green fluorescence were observed to be increased. By contrast, DT-010 pre-treatment markedly reduced the formation of autophagosome vesicles as indicated by AO and MDC staining.

Figure 3 DT-010 inhibits oxidative stress-induced autophagy in cardiomyocytes. (A) Conversion of LC3-I into LC3-II in H9c2 cells was detected by western blotting and quantified. (B) H9c2 cells were stained using AO and MDC. Magnification, x200. Red arrows indicate acidic vesicular organelles including autophagolysosomes stained by AO. (C) H9c2 cells were transfected with the mRFP-GFP-LC3 adenovirus and LC3 puncta were observed. Magnification, x630. Data are presented as the mean ± SD from four experimental repeats. ###P<0.001 vs. Ctrl; **P<0.01 and ***P<0.001 vs. t-BHP. t-BHP, tert-butylhydroperoxide; AO, acridine orange; MDC, monodansylcadaverine; GFP, green fluorescent protein; mRFP, monomeric red fluorescent protein; LC3, microtubule-associated protein 1A/1B-light chain 3.

Autophagic flux is characterized by sequential conversion of autophagosomes into autolysosomes (26). The number of autolysosomes (red puncta; GFP-/mRFP+) were found to be markedly increased in cells in the t-BHP-treated group compared with that in the control group, while autophagosomes (yellow puncta; GFP+/mRFP+) exhibited no difference between the two groups. However, in cells pre-treated with DT-010, the numbers of autolysosomes (GFP-/mRFP+) were decreased compared with that in the t-BHP-treated group (Fig. 3C). Therefore, these data suggest that DT-010 may inhibit autophagic flow induced by t-BHP in cardiomyocytes.

DT-010 prevents oxidative damage induced by t-BHP by inhibiting of autophagy

MTT assay results revealed that t-BHP treatment significantly decreased cell viability (Fig. 4). Treatment with the autophagy agonist rapamycin treatment significantly aggravated cell injury induced by t-BHP further (Fig. 4A), whilst autophagy blocker chloroquine treatment significantly attenuated t-BHP-induced cell injury (Fig. 4B). These data indicate that autophagy may be involved in t-BHP-induced cardiomyocyte damage. DT-010 pretreatment significantly prevented t-BHP-induced cell damage (Fig. 4), which was partially but significantly abolished by rapamycin (Fig. 4A) and significantly improved by hydroxy-chloroquine treatment (Fig. 4B). These results suggest that the cardioprotective effects of DT-010 could be mediated by inhibiting autophagy.

Figure 4 DT-010 protects cardiomyocytes from oxidative damage by inhibiting autophagy. (A) Autophagy agonist rapamycin and (B) autophagy blocker CQ were used to investigate the role of autophagy in the cardioprotective effects of DT-010. MTT assay was used for cell viability determination. Data are presented as the mean ± SD from four experimental repeats. ###P<0.001 vs. Untreated; *P<0.05, **P<0.01 and ***P<0.001 vs. t-BHP; &&P<0.01 and &&&P<0.001 vs. t-BHP + DT-010. CQ, hydroxy-chloroquine; RAPA, rapamycin; t-BHP, tert-butylhydroperoxide.

DT-010 attenuates autophagy via the AMPK-mTOR-Ulk1 signaling pathway

t-BHP treatment increased the LC3-II/LC3-I ratio whilst downregulating the expression levels of p62 compared with those in cells in the control group (Fig. 5). However, DT-010 pretreatment significantly reduced the LC3-II/LC3-I ratio and significantly increased the expression levels of p62 compared with those in the t-BHP group (Fig. 5A). AMPK-mTOR-Ulk1/2 has been previously reported to be one of the signaling pathways that regulate autophagy (29-32). Therefore, the present study further measured the expression levels of key components of the AMPK/mTOR/Ulk1 signaling pathway. t-BHP treatment significantly increased the levels of AMPK phosphorylation whilst downregulating the expression of total AMPK. By contrast, AMPK phosphorylation was significantly decreased whereas the total expression of AMPK was significantly increased in cells pre-treated with DT-010 compared with those in cells treated with t-BHP alone (Fig. 5B). The expression of total mTOR and Ulk1 proteins were not observed to be altered after t-BHP treatment or DT-010 pretreatment (Fig. 5B). However, t-BHP treatment significantly decreased the levels of mTOR and Ulk1 phosphorylation compared with cells in the control group (Fig. 5B), both of which were significantly prevented by DT-010 pretreatment (Fig. 5B). These results suggested that DT-010 may inhibit t-BHP-induced intracellular autophagy by inhibiting the AMPK-mTOR-Ulk1 signaling pathway.

Figure 5 DT-010 inhibits autophagy via the AMPK/mTOR/Ulk1 signaling pathway. (A) LC3-I to LC3-II conversion and p62 expression were measured and quantified by western blotting. (B) Expression of total AMPK, mTOR and Ulk1 protein and their corresponding phosphorylation were measured by western blotting. Data are presented as the mean ± SD from three experimental repeats. ##P<0.01 and ###P<0.001 vs. Ctrl; *P<0.05, **P<0.01 and ***P<0.001 vs. t-BHP. AMPK, 5'-AMP-activated protein kinase; Ulk1, unc-51 like autophagy activating kinase 1; Ctrl, control; LC3, microtubule-associated protein 1A/1B-light chain 3; t-BHP, tert-butylhydroperoxide; p-, phosphorylated.

Discussion

Based on the previously reported properties of DSS and TMP on the cardiovascular system, a large number of DSS and TMP conjugates was synthesized previously, following which their cardiovascular effects were screened in different models in vivo and in vitro (21). Previous structure-effect studies demonstrated that DT-010 is a compound that constitute DSS and TMP linked via an ester bond (22) and displayed better cardioprotective effects compared with DSS, TMP or a combination of the two (20-22,33-35). However, the mechanism underlying the cardioprotective effects of DT-010 remain unclear. Therefore, the present study explored the involvement of autophagy in the cardioprotective effect of DT-010 against oxidative stress injury. DT-010 may facilitate cardiomyocyte survival following t-BHP insult, implicating the cardioprotective effects of DT-010 reported in a previous study (20). DT-010 may be considered as a candidate for myocardial ischemia and reperfusion injury treatment.

Autophagy has been considered to be an important target for treating ischemia-reperfusion injury (5). A large number of studies have previously revealed that myocardial ischemia and reperfusion causes mass generation of ROS in cells, which triggers autophagy (5,36,37). t-BHP is commonly used as an oxidative stress inducer due to its ability of producing free radicals (15). The present study demonstrated that t-BHP exposure stimulated autophagy formation in a manner that was associated with marked increases in the LC3-II/LC3-I ratio and autophagic flux. Furthermore, the expression of p62, an autophagy-specific metabolic substrate, was decreased following t-BHP treatment. DT-010 pretreatment was found to inhibit t-BHP-induced autophagy. Subsequently, rapamycin treatment prior to t-BHP exposure was observed to aggravate t-BHP-mediated cell damage and autophagy. By contrast, hydroxy-chloroquine treatment attenuated cell damage and autophagy induced by t-BHP treatment, suggesting that t-BHP induced H9c2 cell damage by activating autophagy. DT-010 attenuated t-BHP-induced cardiomyocyte injury by at least in part inhibiting autophagy, which was partially but significantly abolished by rapamycin and significantly improved by hydroxy-chloroquine treatment. At present, different physiological consequences of autophagy have been observed in myocardial ischemia reperfusion injury. Short-term and moderate activation of autophagy may confer beneficial effects by degrading dysfunctional or damaged proteins and organelles to promote cell survival, whilst a persistent elevation of autophagy may promote cell death (38-42). The duration and degree of ischemia and reperfusion are essential for the modulation of autophagy as a pharmacological strategy (43,44). Therefore, a number of studies have reported that activation of autophagy could mitigate cardiac ischemia/reperfusion injury (45-47). However, other studies have also reported that inhibition of autophagy could alleviate cardiac ischemia/reperfusion injury (42,48,49), which were consistent with results from the present study. In the future, the optimal time course of DT-010 treatment following myocardial ischemia and reperfusion injury in animal models should be investigated, such that more data is required prior to its proposed clinical use.

The regulation of signal transduction molecules and pathways involved in autophagy is highly complex. The AMPK-mTOR-Ulk1/2 pathway is an important signaling pathway that regulates autophagy (29-32,50-54). AMPK is a pivotal energy sensor for maintaining metabolic homeostasis, whereby AMPK is activated in cells suffering from ischemia and reperfusion during starvation (29). t-BHP-induced ROS accumulation causes oxidative stress and dysfunction in mitochondria and results in an energy crisis (16,55,56), which can lead to the activation of AMPK. AMPK activation may result in the inactivation of mTOR and activation of Ulk1 via the phosphorylation of the Ser317 and Ser777 residues, thus promoting autophagy (57). Phosphorylation of Ulk1 Ser757 prevents Ulk1 activation and inhibits autophagy (57). It was previously reported that AMPK inhibitor Compound C could suppress AMPK/mTOR-mediated autophagy in a rat myocardial infarction model (58). The present study revealed that the levels of p-AMPK were increased whereas total AMPK protein expression was inhibited after t-BHP treatment. Following DT-010 pre-treatment, phosphorylation of AMPK was decreased, whilst total expression of AMPK was enhanced compared with cells treated with t-BHP alone. A previous study reported that the expression of total AMPK protein can be altered by various factors, such as menadione-induced oxidative stress (59). In the present study, it was therefore speculated that the expression of total AMPK protein may be inhibited by severe oxidative stress. However, the underlying mechanism of this phenomenon need to be investigated further. In addition, it was also found that the levels of p-mTOR and p-Ulk1 (Ser757) were decreased in cells in the t-BHP group, which could be prevented by DT-010 pre-treatment. Rapamycin treatment attenuated the cardioprotective effects of DT-010 against t-BHP-induced toxicity in H9c2 cells. This observation suggested that DT-010 stimulated mTOR activity, leading to the suppression of autophagy initiation. A previous study revealed that DT-010 markedly elevated Akt phosphorylation, followed by the reduction in AMPK phosphorylation (inhibition) and subsequent phosphorylation and activation of mTOR within 10 min after treatment (21). Therefore, it can be concluded that DT-010 may inhibit t-BHP-induced autophagy in cardiomyocytes by inhibiting the AMPK-mTOR-Ulk1 signaling pathway, thereby preventing t-BHP-induced cardiomyocyte injury. Previous studies also revealed the potent antioxidative effects of DT-010 (20,22). DT-010 was found to eliminate a number of free radical species (•O2-, •OH and ONOO-) induced by t-BHP and increases the expression levels of cellular redox-related proteins, including PGC-1α, Nrf2, and HO-1(20). Since oxidative stress caused by ROS is a potent inducer of autophagy (16), scavenging of ROS and alleviation of oxidative stress by DT-010 could also contribute to its inhibitory effects on t-BHP-induced autophagy.

In summary, DT-010 was found to protect cardiomyocytes against oxidative stress injury by inhibiting autophagy through the AMPK-mTOR-Ulk1 signaling pathway. The findings of the present study provided evidence for the use of autophagy regulators in therapeutic strategies for myocardial ischemia and reperfusion injury. DT-010 may be a promising candidate for myocardial ischemia-reperfusion injury therapy.

Supplementary Material

DT-010 alone does not induce cytotoxicity in H9c2 cells. H9c2 cells were treated with DT-010 for 1 h. Cell viability was detected using MTT assay. Data are presented as the mean ± SD from six experimental repeats.

Acknowledgements

Not applicable.

Funding

The present study was supported by grants from the National Natural Science Foundation of China (grant no. 81703509), and Science and Technology Planning Project of Guangdong Province, China (grant no. 2015A020211015).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. Compound DT-010 is available with the agreement of the corresponding author.

Authors' contributions

LW and LS conceived, designed and supervised the whole study. JL, CX and HH performed the experiments, and further analyzed and interpreted the data. YW, PY, YS, and LX provided some professional help during the experiment. CX, LW, YW and LS wrote and revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

Yuqiang Wang, Pei Yu, Luchen Shan and Yewei Sun are inventors of the patent covering DT-010 (patent no. CN105294666A) and have financial interest in Changzhou Magpie Pharmaceuticals, Inc. (Changzhou, China), which is developing DT-010 as a therapeutic agent.

References