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Научни трудове на Съюза на учените в България-Пловдив. Серия Г. Медицина, фармация и дентална медицина т. XXII. ISSN 1311-9427 (Print), ISSN 2534-9392 (On-line). 2017. Scientific works of the Union of Scientists in Bulgaria-Plovdiv, series G. Medicine, Pharmacy and Dental medicine, Vol.XXII. ISSN 13119427 (Print), ISSN 2534-9392 (On-line). 2018.
ИЗСЛЕДВАНЕ ЧРЕЗ ДИФЕРЕНЦИАЛНА СКАНИРАЩА КАЛОРИМЕТРИЯ НА ЕФЕКТА НА МИРТЕНАЛ ПРОТИВ СКОПОЛАМИН-ИНДУЦИРАНА ДЕМЕНЦИЯ ПРИ ГРИЗАЧИ
Силвия Абарова1, Румона Койнова2, Стела Захоринова1,
Стела ДрагомановаЗ, Любка Танчева4, Борис Тенчов1 1Катедра Медицинска Физика и Биофизика, Медицински Университет-София, 1431 София, България 2Ohio Sltatii University College ofPharmacy, Columlbiis, OH 43210, USA ЗМедицински Университет, 9000 Варна, България 4Институт по Невробиология, БАН, 1113 Софиа, България
DIFFERENTIAL SCANNING CALORIMETRY INVESTIGATION OF DRUG EFFICACY OF MTYRTENAL AGAINST SCOPOLAMINE-INDUCED DEMENTIA IN RODENTS
SilviyaAbarova1, Rumiana Koynova2, Stella Zahai"inova1, Stella Dragomanova3, Lyubka Tandieva4, Boris Tenc0ov1 1 Dept.Med. Phys. Biophys., Medical University - Sofia, 1431 Sofia, Bulgaria 2 Ohio State University Сollege ofPharmacy, Columbus, OH 43210, USA
3 Medical University, 9000 Varna, Bulgaria 4 Inst. Neurobiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
ABSTRACT
In this study, we employed differential scanning calorimetry (DSC) to characterize the changes in the denaturational profiles of the brain proteome of mice associated with scopolamine-induced cognitive neurodegenerative disorder (dementia) induced by Scopolamine (Sc) and to evaluate the efficacy of a preventive treatment with Myrtenal (Myr), a natural product of plant origin expected to hinder dementia development. The DSC measurements performed on supernatants of brain tissue homogenates revealed large differences between the heat capacity profiles for healthy animals and for animals with scopolamine-induced dementia. The heat capacity profiles of brain tissue supernatants from healthy animals displayed well expressed low-temperature exothermic
transitions peaking in the range 35-45oC, thus preceding in temperature the endothermic denaturational transitions. The exothermic transitions were only observed in supernatants of brain tissue homogenates, and not in other samples from the same animals, e.g., centrifugation sediments of brain tissue homogenates, liver homogenates, blood plasma. Remarkably, the low-temperature exotherms were completely eliminated by the scopolamine treatment and replaced with high-temperature exothermic transitions. A most notable result of this study was that treatment with Myr applied simultaneously with the Sc treatment, neutralized the Sc effect and resulted in preservation of the low-temperature exothermic transitions. In principle, exothermic transitions might result from processes of protein aggregation or fibrillization, or from reversal of protein cold denaturation processes. The enthalpy (area) of the exothermic transitions is similar in magnitude to that of the endothermic denaturational transitions, thus suggesting that a substantial portion of the brain proteins was involved in the exothermic processes. These experiments demonstrated that DSC is an appropriate method with great potential for detection and characterization of brain proteome changes taking place in brain tissues affected by neurodegeneration.
Key words: DSC, dementia, myrtenal
INTRODUCTION
Alzheimer's disease (AD) is a progressive brain disorder characterized by the memory and cognitive impairments. Presently, there is no unified theory of AD pathogenesis and available treatments are only symptomatic, mainly with acetylcholinesterase inhibitors such as memantine, antioxidants, etc. Various plant extracts are used for therapeutic purposes because they contain several active substances, some of which are used to treat central nervous system diseases.
Studies provided evidence that monoterpenes, in particular myrtenal (Myr), have the potential to affect AD. Myr is a terpenoid aldehyde, related to myrtenol, a bicyclic monoterpene found in many plants (Myrtus communis, Hyssopus officinalis and others). The natural compound Myr combines in its in vitro effects both antioxidant and anti-acetylcholinesterase activity [Kaufmann et al., 2011]. Our previous data revealed significant improving effect of Myr on cognitive function of experimental rodents [Dragomanova et al., 2015]. The mechanism of the preventive effects Myr on the processes of neurodegeneration in mice is less well studied and data are primarily from the last few years.
In this study we used an experimental animal (mice) model of drug-induced dementia, by means of manipulation of the central neuronal/neurotransmitter pathways by a chemical substance, scopolamine (Sc). Sc blocks the binding sites of the muscarinic acetylcholine receptors in the cerebral cortex and causes a strong release of acetylcholine, which impairs hippocampal nerves and causes a reduction in the memory in a dose-dependent manner in mice [Riedel et al., 2009]. As highlighted in a recent study, the Sc-induced dementia is an appropriate dementia model to reveal AD-type cognitive impairments and for planning therapeutic strategies against cognitive dysfunctions [Haider et al., 2016].
The idea of the present DSC study is to show that distinct differences exist between the characteristic heat capacity profiles of brain tissue supernatants isolated from healthy animals and from demented animals. Our aim was to utilize these differences in the signature thermograms as a tool for assessment of the efficiency of preventive treatments with biologically active substances, such as Myr.
MATERIALS AND METHODS
Scopolamine animal model of dementia. For the induction of dementia, the experimental animals (Albino male, sexually mature ICR mice, 18-20 g) were treated daily for 11 days with Sc
(1 mg/kg b.w., i.p.). Controls (healthy animals) were injected daily (i.p.) with 0.1 ml of physiological saline per 10 g b.w.
Treatment with Myrtenal (Myr). Simultaneously with the treatment with Sc, the animals were injected with Myr in doses of 20 mg per 1 kg b.w. [Kaufmann et al., 2011]). Control animals received physiological saline in the same volume (0.1 ml/10 g b.w.). The experiments with animals were conducted according to the ethical principles and professional responsibility of scientists established by the Ethics Commission at the Institute of Neurobiology, Bulgarian Academy of Sciences.
Preparation of brain tissue homogenates and supernatants. The experimental animals were decapitated under light ether anesthesia 24 h after the last treatment. Brains were removed rapidly on ice and homogenized in phosphate buffer at 10-fold dilution with PBS and centrifuged at 3000 g to obtain the 10% supernatant.
DSC measurements. The DSC measurements were performed using a Nano DSC from Thermal Analysis Instruments with 300 ^l measuring cell volume. Heating and cooling scans were performed at 1 K/min scan rate in the range 20-110 °C.
RESULTS AND DISCUSSION
In order to determine whether the induction of dementia by Sc results in detectable changes in the thermal denaturation profile of affected by the disease tissues we measured the heat capacity profiles of mice brain supernatants from healthy and animals with dementia (Fig. 1.)
Fig. 1. DSC heat capacity profiles of brain supernatants derived from mice brain homogenates.
(A) Samples from healthy animals; (B) Samples from animals treated with scopolamine.
The supernatants of brain tissue homogenates showed large differences between the heat capacity profiles for healthy animals and for animals with scopolamine-induced dementia: the heat capacity profiles of brain homogenates from healthy animals displayed well expressed exothermic transitions with peak temperatures typically at 40-45°C (Fig. 1A), which exceed by few degrees the mice body temperature (36.9°C) but still precede in temperature the endothermic denaturational transitions; the low-temperature the low-temperature exotherms were completely abolished by the Sc treatment (Fig. 2B).
This notable result indicates that the scopolamine-induced neurodegenerative process is associated with the water-soluble protein fractions, which exhibit the exothermic transitions.
Another interesting result of the present study relates to the healing effects of protectant Myr when this protectant is administered concurrently with Sc to the test animals. DSC measurements showed that upon co-treatment of mice with Sc and Myr, the exothermic transitions, typical for healthy animals, were preserved to significant extent (Fig. 2). This result demonstrates a well expressed neuroprotective effect of the above compounds, which partially or completely blocked the action of Sc.
The present DSC results well correlate with the results obtained from a behavioral step-through test as shown in our previous work [Abarova et al., 2017].
Fig. 2. DSC heat capacity profiles of brain supernatants from mice treated simultaneously with Sc
and Myr (see Materials and Methods).
CONCLUSION
The present DSC study reports the characteristic heat capacity profiles upon thermal denaturation of tissue homogenates. The results presented here provide evidence that DSC can be used for detecting changes in the brain proteome accompanying the development of neurodegenerative disorders. Another important outcome of the data shown is that DSC provides new means to characterize and verify the protective effects of promising substances, such as Myr, potentially useful as a protectant in the treatment of cognitive diseases such as Alzheimer's disease.
Acknowledgement. Support of the Medical University - Sofia, Bulgaria, grant 8-C/2016, and of the Bulgarian National Science Research Fund, NSRF grant DN03/13/2016, is acknowledged.
References
[1] Kaufmann D, Dogra AK, Wink M. 2011. Myrtenal inhibits acetylcholinesterase, a known Alzheimer target, J Pharm Pharmacol 63(10):1368-71.
[2] Dragomanova S, Tancheva L, Georgieva M, Georgieva A, Dishovsky C, Kalfin R, et al., 2015. Preventive effect of the natural monoterpene myrtenal on cognitive disorders in dement mice, Eur Neuropsychopharmacol 25:S578-S9.
[3] Riedel G, Kang SH, Choi DY, Platt B. 2009. Scopolamine-induced deficits in social memory in mice: Reversal by donepezil, Behav Brain Res 204(1):217-25.
[4] Haider S, Tabassum S, Perveen T. 2016. Scopolamine-induced greater alterations in neurochemical profile and increased oxidative stress demonstrated a better model of dementia: A comparative study, Brain Res Bull 127:234-47.
[5] Abarova S, Koynova R, Tancheva L, Tenchov B. 2017. A Novel DSC Approach for Evaluating Protectant Drugs Efficacy against Dementia, Biochimica et Biophysica Acta (BBA) -Molecular Basis of Disease 1863:2934-41.
