Part 2:Echinacoside Inhibits Glutamate Release By Suppressing Voltage-Dependent Ca2+ Entry And Protein Kinase C in Rat Cerebrocortical Nerve Terminals

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Cheng Wei Lu 1,2, Tzu Yu Lin 1,2, Shu Kuei Huang 1 and Su Jane Wang 3,*

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3.2 Therapeutic Implications

Excitotoxicity, a pathological process caused by excessive glutamate release and glutamate receptor activation, is the major cause of neuronal death in acute and chronic brain disorders such as stroke, traumatic brain injury, Parkinson’s, and Alzheimer’s diseases [13,41], and therapeutic strategies involving glutamate release inhibition may be promising neuroprotective strategies for treating such diseases. Echinacoside has been confirmed to penetrate the blood-brain barrier (BBB) and exhibits neuroprotective effects in various in vivo models of neurotoxicity [8,10–12,42]. Although the mechanism of these neuroprotective effects is not completely understood, several possible mechanisms have been reported including inflammatory response inhibition, mitochondrial function stabilization, antioxidation, free radical scavenging, and neurotrophic function mimicking [5,9,12,42]. In the current study, the ability of echinacoside to reduce glutamate release from nerve terminals may also partly explain its neuroprotective mechanism. However, whether this effect contributes to the apparent therapeutic potential of echinacoside in brain disorders associated with glutamate excitotoxicity warrants further research.

Neuroprotective effects of cistanche echinacoside

4.Materials and Methods

4.1. Chemicals

Fura-2-acetoxymethyl ester (Fura-2-AM) and 3’,3’,3’-dipropylthiadicarbocyanine iodide [DiSC3(5)] were purchased from Invitrogen (Carlsbad, CA, USA). o-conotoxin MVIIC, rottlerin, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X), 5,6,7,13-tetrahydro- 13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Go6976) and N-[2-(p- bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) were purchased from Tocris Bioscience (Bristol, UK). Echinacoside, dantrolene, DL-threo-beta-benzyl-oxyaspartate (DL-TBOA), 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157), 2-(2-amino-3- methoxyphenyl)-4H-1-benzopyran-4-one) (PD98059), ethylene glycol bis(β-aminoethyl ether)- N,N,N/,N/-tetraacetic acid (EGTA) and all other reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

4.2. Animals

Two-month old male Sprague–Dawley rats were used. Animals were housed under standardized environmental conditions (22 ± 1 oC; 50% relative humidity; 12 h light/dark cycle) and allowed unlimited access to food and water. The animals were killed by decapitation and the cerebral cortex was rapidly removed at 4 oC. The experimental procedures were approved by the Fu Jen Institutional Animal Care and Utilization Committee (A10259), in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and to use a minimum number of animals necessary to produce reliable results.

4.3. Synaptosomal Preparations

Synaptosomes were purified from the cerebral cortex of rats on discontinuous Percoll gradients as described previously [43,44]. Briefly, the tissue was homogenized in medium containing 0.32 M sucrose (pH 7.4), the homogenate was centrifuged for 10 min at 3000× g (5000 rpm in a JA 25.5 rotor; Beckman Coulter, Inc., Miami, FL, USA) and 4 oC, and the supernatant was centrifuged again for 12 min at 14,500× g (11,000 rpm in a JA 25.5 rotor). The pellet was gently resuspended in 0.32 M sucrose (pH 7.4), and an aliquot of this synaptosomal suspension (2 mL) was placed onto a 3 mL Percoll discontinuous gradient containing 0.32 M sucrose, 1 mM EDTA, 0.25 mM DL-dithiothreitol, and 3%, 10%, and 23% Percoll (pH 7.4). After centrifugation at 32,500× g (16,500 rpm in a JA 20.5 rotor) for 7 min at 4 oC, the synaptosomes were recovered from between the 10% and the 23% Percoll bands, and they were diluted in a final volume of 30 mL of HEPES buffer medium (140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM MgCl2﹒ 6H2O, 1.2 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES (pH 7.4)). Following further centrifugation at 27,000× g (15,000 rpm in a JA 25.5) for 10 min, the synaptosome pellet was resuspended in 3 mL of HEPES buffer medium, and the protein content was determined using a Bradford assay. Finally, 0.5 mg of the synaptosomes suspension was diluted in 10 ml of HEPES buffer medium and centrifuged at 3000× g (5000 rpm in a JA 20.1 rotor) for 10 min. The supernatant was discarded, and the pellets containing the synaptosomes were stored on ice and used within 4–6 h.

cistanche herb

4.4. Glutamate Release

Glutamate release was assayed by online fluorimetry as described previously [45,46]. Synaptosomal pellets were resuspended in HEPES buffer medium (0.5 mg/mL) and preincubated at 37 oC for 10 min in the presence of 16 uM bovine serum albumin to bind any free fatty acids released from synaptosomes during preincubation. A 2-mL aliquot of the synaptosomes was transferred to a stirred cuvette containing 2 mM NADP+, 50 units of glutamate dehydrogenase, and 1.2 mM CaCl2, and the fluorescence of NADPH was measured in a Perkin-Elmer LS-55 spectrofluorimeter (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) at excitation and emission wavelengths of 340 and 460 nm, respectively. As synaptosomes are not amenable to electrical stimulation, the potassium channel blocker 4-aminopyridine was used to stimulate glutamate release. 4-aminopyridine destabilizes the membrane potential and is thought to cause repetitive spontaneous Na+ channel-dependent depolarization that closely approximates in vivo depolarization of the synaptic terminal that leads to the activation of voltage-dependent Ca2+ channels and neurotransmitter release [47]. Data were obtained at 2 s intervals. A standard of exogenous glutamate (5 nmol) was added at the end of each experiment. The value of the fluorescence change produced by the standard addition was used to calculate the released glutamate as nanomoles of glutamate per milligram of synaptosomal protein (nmol/mg). Release values quoted in the text are levels attained at steady-state after 5 min of depolarization (nmol/mg/5 min). Cumulative data were analyzed using Lotus 1-2-3 spreadsheets (IBM, White Plains, NY, USA) and MicroCal Origin (OriginLab Corporation, Northampton, MA, USA).

4.5. Plasma Membrane Potential

The plasma membrane potential was determined with a membrane-potential-sensitive dye, DiSC3(5) [48]. Synaptosomes were resuspended in HEPES buffer medium, and 2 mL aliquots were transferred to a stirred cuvette containing 5 uM DiSC3(5) at 37 oC in a Perkin–Elmer LS-55 spectrofluorometer (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). After allowing the mixture to equilibrate for 3 min, the fluorescence was determined at excitation and emission wavelengths of 646 and 674 nm, respectively. Data were collected at 2 s intervals. Cumulative data were analyzed using MicroCal Origin (OriginLab Corporation, Northampton, MA, USA) and expressed in fluorescence units.

4.6. Cytosolic Ca2+ Concentration ([Ca2+]C)

The [Ca2+]C was measured with the Ca2+ indicator fura-2. Synaptosomes (0.5 mg/mL) were preincubated in HEPES buffer medium containing 5 uM fura-2 and 0.1 mM CaCl2, for 30 min at 37 oC in a stirred test tube. After fura-2 loading, synaptosomes were centrifuged in a microcentrifuge for 30 s at 3000× g (5000 rpm). The synaptosomal pellets were resuspended in HEPES buffer medium, and the synaptosomal suspension was stirred in a thermostatted cuvette in a Perkin-Elmer LS-55 spectrofluorometer (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). CaCl2 (1 mM) was added after 3 min and further additions were made after an additional 10 min. Fluorescence data were accumulated at excitation wavelengths of 340 and 380 nm (emission wavelength 505 nm) at 2 s intervals. [Ca2+]C (nM) was calculated using calibration procedures [49] and equations described previously [50]. Cumulative data were analyzed using MicroCal Origin (OriginLab Corporation, Northampton, MA, USA).

4.7. Western Blotting

Synaptosomes were homogenized in a lysis buffer (10 mM HEPES buffer, pH 7.4), 1% Triton X-100, and protease inhibitor mixture. Lysates were clarified by centrifugation, and protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane. The membranes were blocked with Tris-buffered saline that contained 5% low-fat milk and incubated with appropriate primary antibody (phospho-protein kinase C (pan), 1:3000, NOVUS Biologicals Inc., Beverly, MA, USA) overnight at 4 oC. After three washes in Tris-buffered saline, the membrane was then treated with the secondary horseradish peroxidase-conjugated antibody (1:3000) for 1 h at room temperature. The membranes were then washed at least three times with Tris-buffered saline and visualized using the enhanced chemiluminescence system (Amersham, Buckinghamshire, UK). An aliquot of samples was loaded and probed with an anti-PKC antibody for detection of PKC as a loading control. The level of expression or phosphorylation was assessed by band density, which was quantified by densitometry. Densitometric quantification of bands was analyzed using Syngene software (Synoptics, Cambridge, UK).

4.8. Statistical Analysis

Data were obtained from a single synaptosomal preparation and were not independent of one another. To test the significance of the effect of a drug versus control, a two-tailed Student’s t-test was used. When an additional comparison was required (such as whether a second treatment influenced the action of echinacoside), a one-way ANOVA followed by Tukey’s test was used. Analysis was completed via software SPSS (17.0; SPSS Inc., Chicago, IL, USA). Data are expressed as mean ± S.E.M.; significance was evaluated at p < 0.05 for all statistical measures.

cistanche

5. Conclusions

This is the first study demonstrating that echinacoside inhibits glutamate release from rat cerebrocortical synaptosomes by reducing Ca2+ influx through Cav2.2 and Cav2.1 channels, and this release inhibition is likely dependent on the suppression of the protein kinase C pathway, at least in part. The present finding is valuable because it provides a novel insight into the mechanisms of action of echinacoside in the brain.

Acknowledgments: This work was supported by a grant from the Ministry of Science and Technology (MOST 103-2320-B-030-001 MY3).

Author Contributions: Tzu Yu Lin and Su Jane Wang conceived and designed the experiments; Cheng Wei Lu performed the experiments; Cheng Wei Lu and Shu Kuei Huang analyzed the data; Su Jane Wang wrote the paper.

Conflicts of Interest: The authors declare no conflict of interest.

cistanche herb extract

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