Radical-Scavenging and Anti-Oxidative Activities Of TBN in Cell-Free System and Murine H9c2 Cardiomyoblast Cells

Reactive oxygen species (ROS) and reactive nitrogen species are believed to be one of the most important culprits in the pathogenesis of cardio/cerebrovascular diseases. Intensive researches have been conducted to target free radicals as potential treatment for cardio/cerebrovascular diseases. The 2-[[(1,1-dimethylethyl) oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a novel nitrone derivative of tetramethylpyrazine, has been demonstrated to exhibit significant therapeutic effects in ischemic stroke and Parkinson’s models due to its multiple functions, including calcium overload blockade and free radical-scavenging activity. In the present study, we found that TBN had significant radical trapping effect in cell-free assays. Additionally, TBN effectively blocked tert-butylhydroperoxide (t-BHP)-induced murine H9c2 cardiomyoblast cell death, suppressed H9c2 cell apoptosis and reversed the decrease in mitochondrial membrane potential. Furthermore, TBN markedly inhibited t-BHP-induced ROS generation and free radical NO and ONOO.Taken together, these results suggest that TBN might be a potential candidate for the treatment of ischemic cardio/ cerebrovascular diseases by targeting free radicals. DOI : 10.14302/issn.2471-2140.jaa-15-765 Corresponding author : Zaijun Zhang, Institute of New Drug Research and Guangzhou Key Laboratory of Innovative Chemical Drug Research in Cardio-cerebrovascular Diseases, Jinan University College of Pharmacy, Guangzhou, 510632, China. E-mail: zaijunzhang@163.com Keyword : Tetramethylpyrazine, Nitrone, Radical scavenger, Reactive oxygen species. Received : Oct 09, 2015 Accepted : Dec 14, 2015 Published: Dec 22, 2015; Freely Available Online www.openaccesspub.org | JAA CC-license DOI : 10.14302/issn.2471-2140.jaa-15-765 Vol-1 Issue 1 Pg. no.56 Introduction Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are a family of molecules that include molecular oxygen and its derivatives produced in all aerobic cells. They usually act as second messengers in cell signaling that are essential for various biological processes in normal cells . It has been well documented that they regulate many signal transduction pathways by directly reacting with and/or modifying the structures of proteins, enzymes, transcription factors and genes to modulate their functions. Reactive oxygen species ROS, which are mainly generated in mitochondria, includes three types: superoxide anion radical (O2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH), Approximately 1-3% of the oxygen taken up by the cell escapes from the mitochondrial electron transport chain, and is constitutively present in the form of O2 . O2 is subsequently converted to H2O2 by dismutation of O2 – or directly from the action of oxidase enzymes. Hydroxyl radical a(OH), a highly reactive species that can be converted from H2O2 by Fenton reaction, will modify base pairs and cause strand breaks and result in DNA damage. RNS, often refers to nitric oxide (NO) and peroxynitrite (ONOO). Nitric oxide radical (NO) can arise from L-arginine catalyzed by cytosolic or mitochondrial nitric oxide synthases (NOS) , while in the presence of O2 , NO will react with O2 – instantly to form peroxynitrite (ONOO). It has been demonstrated that ischemia is a restriction of blood supply generally due to congestion, as the limitation of blood flow to the tissue eventually culminates cell damage. However, the re-introduction of oxygen to the ischemic tissues results in a burst of ROS and RNS production, which can subsequently cause fatal damage in cellular components. This type of tissue damage is referred to as ischemia reperfusion injury . Any aberrance in reactive species, in particular those derived from NO and O2 , have been shown to cause cellular oxidative damage and trigger specific signaling events that culminate in altered cellular physiology 7, . Normal tissues have a defense system against these toxic ROS and RNS, however, ischemia reperfusion injury overwhelms the protective mechanisms and results in ROS and RNS burst, which is the culprit of the pathogenesis and/or progression of ischemic cardio/ cerebro vascular disease. Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one, Figure 1), designed and marketed as a neuroprotectant by Mitsubishi Tanabe Pharma Corporation (Tokyo, Japan), acts as a potent antioxidant and effective free radical scavenger against oxidative stress and neuronal apoptosis, and is used for the purpose of aiding neurological recovery following acute ischemic cerebral infarction 10, . Besides, it also exhibits preventive effects on myocardial injury in patients with acute myocardial infarction 14, . Tetramethylpyrazine (2,3,5,6-tetramethylpyrazine, TMP, Figure 1) is the main active ingredient of traditional Chinese medicine Ligusticum wallichii Franchat (Chuan Xiong). It has been demonstrated that TMP exerts potential radical-scavenging and antioxidative activities in vitro , and has been used in the therapy of cerebral ischemic disease 19, 20 and myocardial ischemia-reperfusion injury 21, . Figure 1 The chemical structures of Edaravone, TMP, NXY-059, and TBN. Freely Available Online www.openaccesspub.org | JAA CC-license DOI : 10.14302/issn.2471-2140.jaa-15-765 Vol-1 Issue 1 Pg. no.57 Disodium (tert-butylimino) methyl) benzene-1, 3disulfonate N-oxide (NXY-059, Figure 1), a disulfonyl derivative of spin trap α-phenyl-tert-butyl nitrone (PBN) 23, 24 that has potent radical-trapping property, was designed as a neuroprotective compound for ischemic stroke . Although the SAINT III (Stroke-Acute Ischemic NXY Treatment III) clinical trial failed in 2006 , the concept of using radical-trapping property of nitrone moiety as neuroprotective agents for ischemic reperfusion injury therapy remains viable. TBN (2-[[(1, 1-dimethylethyl) oxidoimino]-methyl]-3, 5, 6-trimethylpyrazine, Figure 1), a TMP derivative armed with a powerful nitrone moiety, was designed as a dual-functional agent targeting overload of calcium and free radicals by our group . We had previously demonstrated that TBN possessed significant free radical -scavenging activity against various radicals, including hydroxyl (OH), superoxide (O2 ) and peroxynitrite (ONOO) 29, . Furthermore, we have also revealed that TBN remarkably protects neuronal cells from oxidative injury in vitro 29 and rats from ischemic stroke 29, . Due to the high reactivity of reactive species with the surrounding biological macromolecules and relatively short duration of existence, the determination of ROS and RNS scavenging effects of target compounds in intracellular compartments is difficult . Up to now, there were very few systematic evidence demonstrated the radical scavenging effectiveness among edaravone, TMP and NXY-059. Herein, we determine the radicalscavenging and antioxidant effects of TBN in comparison with edaravone, TMP and NXY-059 in vitro, providing experimental evidence for identifying TBN as a potential therapeutic agent of ischemic cardio/cerebrovascular disease. Materials and Methods Chemicals and Reagents TBN and NXY-059 were synthesized and purified in our laboratory as described previously . TMP, edaravone, 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N-dimethyl-4-nitrosoaniline (p-NDA), H2O2, pyrogallol, 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), 3-aminophthalhydrazide (luminol), tert-butyl hydroperoxide (t-BHP), 3-morpholinosydnonimine hydrochloride (SIN-1) and diethylamine NONOate diethylammonium salt (DEA NONOate) were purchased from Sigma Aldrich (St Louis, Mo, USA). Hoechst 33342, MitoProbe JC-1 assay kit, MitoSOXTM Red Mitochondrial Superoxide Indicator, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), dihydrorhodamine 123 (DHR 123), 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 0.25% Trypsin-EDTA, Penicillin-Streptomycin (10,000 U/ml), phosphate buffered saline (PBS), were obtained from Invitrogen, Life technologies (Carlsbad, CA, USA). All other reagents were purchased from Sigma Aldrich unless otherwise specified. 2.2. Determination of radical-trapping activity against DPPH, OH, O2 − and ONOO by cell-free assays The comparison of free radical-trapping activities among TBN, TMP, NXY-059 and edaravone (5, 20, 80, 320 μM used for all compounds) against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), hydroxyl radical (OH), superoxide anion (O2 ) and peroxynitrite (ONOO) were determined by cell-free assays according to the previously published procedures with minor modification 31, . DPPH Radical-Scavenging Activity. One hundred microliter methanol (control) or a methanolic solution of each test compound was added in 96-well plates, and then 100 μl methanolic solution of DPPH (final concentration 50 μM) was added into each well. The plates were incubated in the dark at room temperature for 50 min. The measurement at 517 nm was measured Freely Available Online www.openaccesspub.org | JAA CC-license DOI : 10.14302/issn.2471-2140.jaa-15-765 Vol-1 Issue 1 Pg. no.58 using a plate reader (BioTek Synergy 4, Winooski, Vermont, USA). The clearance of the DPPH radical was calculated as follows: Clearance (%)= [(Actrl–At) /Actrl] × 100. Where Actrl was the absorbance of the control, and At was the absorbance of each sample solution at the time t = 50 min. Hydroxyl Radical-Scavenging Activity. The pNDA, FeSO4 and H2O2 were freshly prepared in N2purged, double-distilled H2O (ddH2O) to get a concentration of 1.0 mM, 2.0 mM and 1.0 mM respectively. While all of the test compounds were freshly dissolved in ddH2O before the experiment, then 300 μl ddH2O (control) or different concentration of each test compound, 50 μl p-NDA, 125 μl H2O2 and 125 μl Fe 2+ were added to 48-well plate in order. Hydroxyl free radical was generated by the Fenton reaction (Fe + H2O2 → Fe 3+ + OH + OH). The bleaching of p-NDA was monitored as the loss in absorbance at 440 nm for 100 s on a BioTek Synergy 4. T


Introduction
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are a family of molecules that include molecular oxygen and its derivatives produced in all aerobic cells. They usually act as second messengers in cell signaling that are essential for various biological processes in normal cells 1  It has been demonstrated that ischemia is a restriction of blood supply generally due to congestion, as the limitation of blood flow to the tissue eventually culminates cell damage. However, the re-introduction of oxygen to the ischemic tissues results in a burst of ROS and RNS production, which can subsequently cause fatal damage in cellular components. This type of tissue damage is referred to as ischemia reperfusion injury 6 .
Any aberrance in reactive species, in particular those derived from NO and O 2 -, have been shown to cause cellular oxidative damage and trigger specific signaling events that culminate in altered cellular physiology 7,8 .
Normal tissues have a defense system against these toxic ROS and RNS, however, ischemia reperfusion injury overwhelms the protective mechanisms and results in ROS and RNS burst, which is the culprit of the pathogenesis and/or progression of ischemic cardio/ cerebro vascular disease.

Statistical analysis
Each assay was carried out three times in order to determine the reproducibility. Data were expressed as mean ± standard deviation (SD) and statistical calculations were performed using prism 6.0 GraphPad software (GraphPad, San Diego, CA, USA). Comparisons between the different groups were performed by one-way analysis of variance (ANOVA) followed by Turkey's test. p < 0.05 were considered significant.

Free radical-scavenging effect in cell-free assays
The results showed that the effect on various radicaltrapping among four different compounds was concentration-dependent from 5 to 320 μM. The radicalscavenging effects of TBN were more potent than TMP and NXY-059 against DPPH ( Fig. 2A), O 2 - (Fig. 2B) and ONOO - (Fig. 2C) at the same concentration, even close to edaravone. However, TBN was slightly better than TMP, while weaker than NXY-059 and edaravone in terms of OH-trapping activity (Fig. 2D). t-BHP on H9c2 cells (Fig. 3B). Compared to TMP and NXY-059, TBN was more efficient than TMP and NXY-059 against t-BHP induced cell death. Edaravone (5 μM)

Effects of t-BHP
served as a positive control, also mildly increased the cell viability.  induced by t-BHP at the same concentration, it was even better than the effect of edaravone at higher concentration ( Fig. 6A and B).  (Fig. 7A). In the ONOOtrapping activity, the fluorescent intensity of the SIN-1 treated alone group increased 287.6 ± 12.1% compared to the   the NO-scavenging activity, TBN was also weaker to edaravone but stronger than TMP and NXY-059 in ONOO --trapping activity (Fig. 7B).

Discussion
In the present study, we first compared the free As suppression of excessive production of intracellular NO and ONOO -, TBN will benefit the cardio/ cerebrovascular system.
In conclusion, the radical-trapping and neuroprotective effects of TBN described herein and previously 29,31,35 suggest that TBN might be a promising candidate for the treatment of ischemic cardio/cerebrovascular diseases.

Acknowledgements
This work is partially supported by grants from China's