Novel effect of Arthrocen (avocado/soy unsaponifiables) on pentylenetetrazole-induced seizure threshold in mice: Role of GABAergic pathway
Ramin Goudarzi, Golnaz Zamanian, Alireza Partoazar, Ahmadreza Dehpour
a Division of Research and Development, Pharmin USA, LLC, SanJose, California, USA.
b Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
c Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran
a b s t r a c t
Arthrocen, an avocado/soy unsaponifiable (ASU)-containing agent, is now used in the clinic and has potentially to decrease joint inflammation and pain associated with mild to severe osteoarthritis. Phytosterols are the major component of Arthrocen with documented anti-inflammatory properties, antioxidant, and analgesic effects.
Here, we evaluated ASU anticonvulsant effect by its oral administration in pentylenetetrazole (PTZ)-induced sei- zure threshold and Maximal Electroshock Seizure (MES) Models. Also, the involvement of N-methyl-D-aspartate (NMDA) receptor, benzodiazepine receptor, and nitric oxide (NO) pathway were studied in anticonvulsant effect of ASU in male NMRI mice. Acute administration of Arthrocen (150, 75, 30, 10 mg/kg) by oral gavage significantly (p b 0.001) increased the clonic seizure threshold induced by intravenous administration of PTZ. Nonspecific in- ducible NO synthase (NOS) inhibitor L-NAME (10 mg/kg) and a specific NMDA receptor antagonist MK-801 (0.05 mg/kg) did not affect the anticonvulsant effect of Arthrocen, while pretreatment with flumazenil (0.25 mg/kg), a selective benzodiazepine receptor antagonist, reversed this effect (p b 0.01). Also, Arthrocen treated mice did not affect tonic hindlimb extension in the MES model. The data showed that Arthrocen might produce its anticonvulsant effect by enhancing GABAergic neurotransmission and/or action in the brain.
1. Introduction
Seizure is the clinical manifestation of an abnormal, excessive, hypersynchronous discharge of a population of cortical neurons [1,2]. Among the different cascades of molecular and cellular processes that maybe involved in epileptogenesis and the development of seizure- generating neuronal current, neuroinflammation appears as one of the most critical neurological disorders [3–8]. Also, there are many mecha- nisms by which seizures can develop. Three common mechanisms include the following: 1) Reduction of the inhibitory mechanism (espe- cially synaptic inhibition due to g-aminobutyric acid [GABA]), 2) Im- provement of the excitatory synaptic mechanism (especially those mediated by N-methyl-D-aspartate [NMDA]), 3) Improvement of endogenous neuronal burst firing (usually by enhancing voltage- dependent calcium currents). Various forms of human seizure may be caused by any one or combination of the above mechanisms [9].
Despite experimental models of epilepsy have been identified widely to understand the neurobiology of disease and development in new antiepileptic medications, the attempt for the progression in anti- epileptic drugs with the least side-effect profiles are needed yet [10,11]. Arthrocen is an alternative Avocado/Soybean Unsaponifiable (ASU) formulation-containing supplement. The major ingredients of Arthrocen are composed essentially of dihydrocholesterol, campesterol, phytos- terols, triterpene alcohols, lipophilic vitamins, triterpenoids, stigmastanol, β-sitosterol, and its sterol composition compared to other formulas of ASU extracts, which are swiftly agglutinated into cells. Although the rec- ognition of the active components is unclear, Arthrocen is categorized as a human prescription medication in some countries, while in the United States, is known as a food supplement [12–14].
Additionally, in vitro and in vivo laboratory studies have illustrated that ASUs have positive effects on osteoarthritis by inhibiting a number of molecules and pathways that interfere with osteoarthritis; ASU stim- ulates the synthesis of collagen and aggrecan by inhibiting inflamma- tory cytokines such as IL-1, IL-6, IL8, TNF, and PGE2 via modulation of NF-kappaB. Based on this subject, studies suggest that ASU has anti- inflammatory effects in mice [15–17].
The previous studies have been indicated that avocado (100–800 mg/kg) significantly (p b 0.05–0.001) delayed the onset of, and pentylenetetrazole (PTZ)-induced seizures. In addition, avo- cado has been relatively more anticonvulsant in PTZ- and picrotoxin (PCT)-induced convulsions than in bicuculline (BCL)-induced sei- zure in mice. In general, it was concluded that the average onset and duration of convulsion were markedly delayed and reduced, re- spectively, by avocado consumption [18]. In addition, in other previ- ous experimental studies, various parts of avocado have been used as an effective anticonvulsant and anti-inflammatory remedy [19–24]. Also, the chronic administration of soybean extracts has exhibited an anticonvulsant property [25] in maximal electroshock model on seizure of mice, although the exact mechanisms are unknown yet.
The PTZ is a GABA-A receptor antagonist as a convulsant that causes intense seizures when administered to rodents [26,27]. Moreover, PTZ acts as a high-performance model for the recognition of novel antiepi- leptic molecules or drugs that mainly process through GABA, but the mechanism that PTZ evokes its function is not extremely well-under- stood [28–30].
However, regarding the potential benefit of understanding the anti- convulsant mechanisms, the purpose of the present study was to inves- tigate the underlying mechanisms underpinning ASU efficacy and anticonvulsant property of ASU in animal models of seizure in mice, with a view of providing pharmacological justification.
2. Methods
2.1. Chemicals
The drugs used are as follows: Arthrocen samples were acquired from Pharmin USA, LLC (USA), and pentylenetetrazole (PTZ), MK-801, L-NAME, flumazenil, were purchased from Sigma (U.K). All drugs were dissolved in physiological saline solution to such concentrations that requisite doses were administered in a volume of 10 ml/kg. In all experiments, Arthrocen was administrated by oral gavage, but PTZ was administered intravenous (i.v.); MK-801, flumazenil, and L-NAME were administered intraperitoneal (i.p.).
2.2. Subjects
In this study, male NMRI mice weighing 23–30 g were used at the time of the acute administration. The animals were housed in a temperature-controlled room (24 ± 1 °C) in standard polycarbonate cages, four to five mice in each cage, on a 12-hour light/dark cycle with free access to food and water. All procedures were carried out in accordance with institutional procedures for animal care and use. Addi- tionally, all efforts were made to reduce animal suffering and to use only the number of animals necessary to produce reliable scientific data.
2.3. Determination of clonic seizure threshold
Animals in the experiment received different doses of drugs and sei- zure; the threshold of PTZ-induced seizures was determined via a sy- ringe pump, by inserting a 30 gauge butterfly needle into the tail vein of the mouse and the infusion of PTZ (0.5%) at a constant rate of 1 ml/min. The infusion was halted when forelimb clonus followed by full clonus of the body occurred. A minimal dose of PTZ (mg/kg of mice weight) needed to induce a clonic seizure was measured as an index of seizure threshold.
2.4. Electroshock test
Maximal electroshock seizure (MES) in mice is typically induced using electroshock by passing alternating current (50 Hz, 50 mA, and 0.2 s) via ear electrodes. In order to improve electrode contact, the elec- trodes were moistened with normal saline before being attached to the ears of the mouse. Electroshock induces a continuum of motor convul- sions that are dependent on the intensity of the electrical stimulation current. The most common endpoint for anticonvulsant drug activity in MES is the inhibition of tonic hindlimb extension (THE). Data were expressed as the duration of THE after electroshock test.
2.5. Treatment
In experiment 1, animals received acute ASU oral gavage (300 mg/kg) at different times (60, 90, 120 min). In experiment 2, animals received acute oral gavage of different doses of ASU or saline (0 [saline], 1, 10, 30, 75,150, 300 mg/kg) 60 min before determination of the clonic seizure threshold. In experiment 3, animals received NO synthase (NOS) inhibi- tor doses of L-NAME (10 mg/kg), and another group received inhibitor NMDA-receptor (NMDAR) MK-801 (0.05 mg/kg) acutely administered alone or 15 min before ASU (10 mg/kg) and 60 min before PTZ-induced seizure threshold. In experiment 5, animals received GABA-A receptor antagonist flumazenil (0.25 mg/kg), which was acutely administered alone or 5 min before ASU and 60 min before PTZ-induced seizure thresh- old. In the other experiments, saline or ASU (10 mg/kg, oral gavage) were given 60 min before seizure induction by maximal electroshock.
2.6. Statistical analysis
Data are presented as means ± standard error of the mean (S.E.M.) of PTZ’s minimal dose (mg/kg) and analyzed using the SPSS statistical soft- ware package (version 15). One-way analyses of variance (ANOVA) followed by posthoc Tukey’s tests were used to analyze the data where appropriate. Tests of homogeneity of variance were used to ensure nor- mal distribution of the data. A p-value of less than 0.05 was defined as statistically significant.
3. Results
3.1. Effect of ASU on PTZ-induced seizures
As given, Fig. 1 shows the time-course of the anticonvulsant effect of ASU. The single doses of acute ASU (300 mg/kg, oral gavage) at different times (60, 90, 120 min) significantly increased the anticonvulsant effect the PTZ-induced seizure threshold compared to saline-treated control group (**p b 0.01).
3.2. Effects of different doses of ASU in acute study
Fig. 2 shows that the effect of acute ASU administration of different doses (0 [saline], 1, 10, 30, 75,150, 300 mg/kg, oral gavage) on PTZ- induced seizure threshold. Although, one-way ANOVA revealed a signif- icant effect for ASU with different dosage (150, 75, 30, 10 mg/kg, ***p b 0.001; 1 mg/kg, *p b 0.05), another dose (300 mg/kg) showed no signif- icant effect on seizure threshold compared to the saline group.
3.3. Effect of NOS inhibitor on the anticonvulsant effect of ASU
3.3.1. Effect of L-NAME on the anticonvulsant effect of ASU
In experiment 3, we evaluated the effect of acute administration of L- NAME with ASU on the anticonvulsant effect of ASU. Fig. 3 shows that acute i.p. administration of NOS inhibitor L-NAME at doses used (10 mg/kg, i.p.) did not significantly effect the PTZ-induced seizure threshold compared to the saline group. Also acute administration at doses of L-NAME (10 mg/kg) with ASU (10 mg/kg, oral gavage), which did not affect the anticonvulsant effect of ASU.
3.4. Effect of NMDA antagonist on the anticonvulsant effect of ASU
3.4.1. Effect of MK-801 on the anticonvulsant effect of ASU
In experiment 4, the acute administration of NMDAR antagonist af- fects the anticonvulsant property of ASU. Fig. 4 shows that acute i.p. ad- ministration of NMDAR antagonist MK-801 with dose of (0.05 mg/kg) did not affect the PTZ-induced seizure threshold compared to the saline group. Also acute administration at dose of MK-801 (0.05 mg/kg) showed no significance on the anticonvulsant effect of ASU (10 mg/kg, oral gavage.).
3.5. Effects of flumazenil on anticonvulsant effects of ASU
Results from Fig. 5 showed that acute i.p. administration of GABA-A receptor antagonist at flumazenil doses used (0.25 mg/kg) did not affect the PTZ-induced seizure threshold compared to saline group. Also, acute administration at doses of flumazenil (0.25 mg/kg) approximately inhibited the anticonvulsant effect of Arthrocen (10 mg/kg, oral gavage).
3.6. Effects of ASU on tonic hindlimb extension (MES model)
In our experiment, different doses of ASU (0 [saline], 10, and 20 mg/kg) were evaluated orally on tonic hindlimb extension of mice using MES model. Data described that ASUs were not affected signifi- cantly on the MES model (data were not shown).
4. Discussion
The results in this study indicate that the acute oral gavage of Arthrocen possesses anticonvulsant activity in PTZ-induced seizure threshold in NMRI male mice. This anticonvulsant activity change sig- nificantly by administration of neither L-NAME, nonspecific inhibitor NOS, nor MK-801, specific NMDAR antagonist. While it reversed signif- icantly by using flumazenil, a GABA-A receptor antagonist. Hence, we proposed that GABA-A receptors but not NO/NMDA pathways are in- volved in anticonvulsant activity of Arthrocen.
Brain inflammation is considered as a vital etiopathogenetic mecha- nism of epilepsy that could be targeted to control seizures; specific in- flammatory mediators overexpressed in human epileptogenic foci are known to promote seizures in animal models [31]. All common risk fac- tors for epilepsy like traumas, malignancies, and infections are accompa- nied by different levels of central nervous system (CNS) inflammation which in turn have been associated with the occurrence of seizures [32,33]. Besides, previous studies have indicated that ASU and its com- pounds show anti-inflammatory effects [19], which may include altered Na+-K+ ATPase expression [34], pyridoxamine-5′-phosphate (PMP) metabolism [35], and inhibition of expression of inducible nitric oxide [36].
In the present study, we demonstrated that NO inhibitor L-NAME (10 mg/kg), NMDAR antagonist MK-801 (0.05 mg/kg) in combination with Arthrocen agent (10 mg/kg, oral gavage), did not affect the PTZ- induced seizure threshold. The MES and PTZ are the most commonly used preliminary tests for screening of potential anticonvulsant drugs. The PTZ test is considered to be a predictor of likely therapeutic efficacy against generalized clonic seizure.
Studies have demonstrated several biochemical hypothesis involv- ing inhibitory GABAergic system, and the system of excitatory amino acids glutamate and aspartate have been reported in epilepsy. A convul- sive state is induced by the direct blockade of GABA-A mediated open- ing of Cl- ion channels of three receptors of glutamate [37].
However, our present state of knowledge of the chemical constitu- ents of the ASU is limited.
It is, therefore, impossible for us at this stage, to identify with cer- tainty, the anticonvulsant constituent/s of ASU.
However, experimental evidence obtained in the present animal study shows that the Arthrocen significantly delayed the onset of sei- zures induced by PTZ, whereas it did not affect the tonic hindlimb exten- sion in the MES model. The previous studies showed that flumazenil is a benzodiazepine antagonist, which inhibits the central effects of benzodi- azepine agonists by competing with these drugs for the benzodiazepine receptor.
The inhibitory effect of flumazenil is specific for central benzodiaze- pine receptors [38] and drugs that enhance GABA-A receptor neuro- transmission, such as Benzodiazepines (BZDs) [39,40] that can block seizures induced by PTZ.
Seizures induced with PTZ have been shown to be due to the inhibi- tion and/or attenuation of GABAergic neurotransmission [41–43]. The findings in this study tend to suggest that ASU agents might have inhibited and/or attenuated PTZ-induced seizures of the mice used by enhancing, or in some ways interfering with, GABAergic action and/or neurotransmission, so it is not unreasonable to speculate that ASU agents probably produces its anticonvulsant activity by enhancing GABAergic neurotransmission and/or action.
To conclude, the results of this study illustrate that Arthrocen had anticonvulsant property in PTZ-induced seizure threshold. This activity could not be reversed by NO inhibitor and NMDAR antagonist, but the GABA-A receptor antagonist could completely block the anticonvulsant effect of Arthrocen. Our findings reveal that GABA-A receptor may be contributing to the anticonvulsant effect of ASU.