• Users Online: 91
  • Print this page
  • Email this page

 
Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 62  |  Issue : 1  |  Page : 17-26

Possible nitric oxide mechanism involved in the protective effect of L-theanine on haloperidol-induced orofacial dyskinesia


1 Department of Neurosurgery, Mackay Memorial Hospital, Taipei, Taiwan, ROC
2 Department of Anesthesia, En Chu Kon Hospital, Sanshia District, New Taipei City, Taiwan, ROC
3 Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan, ROC
4 Department of Psychiatry, Yuan-Shan Branch of Taipei Veteran General Hospital, Yilan County; Department of Biomedical Engineering, National Defense Medical Center, Taipei, Taiwan, ROC
5 Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei; School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan, ROC

Date of Submission19-Apr-2018
Date of Decision13-Jan-2019
Date of Acceptance24-Jan-2019
Date of Web Publication22-Feb-2019

Correspondence Address:
Dr. Hsiang-Chien Tseng
Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, No. 95, Wenchang Road, Shilin Dist., Taipei 11101, Taiwan
ROC
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_8_19

Rights and Permissions
  Abstract 

Having powerful antioxidative properties, L-theanine (LT), one of the major amino acid components in green tea, has potent anti-oxidative and neuroprotective effects. In this study, we examined the potential protective effects of LT on haloperidol (HAL)-induced orofacial dyskinesia (OD) in rats. HAL treatment (1 mg/kg intraperitoneally for 21 days) induced OD; significant increases (P < 0.001) in the frequency of vacuous chewing movement and tongue protrusion as well as the duration of facial twitching. LT treatment (100 mg/kg orally for 35 days, starting 14 days before HAL injection) was able to prevent most of the HAL-induced OD. LT treatment was also able to reduce the lipid peroxidation production and nitric oxide (NO) level, and enhance the antioxidation power in striatum from rats with HAL treatment. In order to examine the implication of NO pathway activity in HAL treatment, either NO precursor (L-arginine) or NO synthase inhibitor (L-NAME) was co-pretreated with LT; NO precursor treatment eliminated the protective effect of LT, in contrast to that NO synthase inhibitor treatment significantly potentiated the LT effects on behavioral and biochemical protection in HAL-treated rats. These results suggested that the NO pathway was implicated, at least in part, in the HAL-induced OD, as well as in the protective effect of LT in treating HAL-induced OD. The above evidence provides a clinically relevant value for LT in delaying or treating tardive dyskinesia.

Keywords: Haloperidol, L-theanine, nitric oxide, orofacial dyskinesia, striatum


How to cite this article:
Tsai CC, Wang MH, Chang KC, Soung HS, Yang CC, Tseng HC. Possible nitric oxide mechanism involved in the protective effect of L-theanine on haloperidol-induced orofacial dyskinesia. Chin J Physiol 2019;62:17-26

How to cite this URL:
Tsai CC, Wang MH, Chang KC, Soung HS, Yang CC, Tseng HC. Possible nitric oxide mechanism involved in the protective effect of L-theanine on haloperidol-induced orofacial dyskinesia. Chin J Physiol [serial online] 2019 [cited 2019 May 22];62:17-26. Available from: http://www.cjphysiology.org/text.asp?2019/62/1/17/252838

Hung-Sheng Soung, Chih-Chuan Yang and Hsiang-Chien Tseng contributed equally to this work.



  Introduction Top


Tardive dyskinesia (TD), an iatrogenic, hyperkinetic movement disorder characterized by choriform, athetoid, and rhythmic involuntary movements usually involving the mouth, face, and tongue, is considered as a late-onset adverse effect of prolonged administration of neuroleptic drugs.[1] TD has been considered an important clinical problem in the treatment of schizophrenia because it may persist for months or years after the drug withdrawal and about 2% of patients are irreversible.[2] Long-term treatment with haloperidol (HAL), a first-generation neuroleptic drug used for the treatment of certain mental/mood disorders (e.g., schizophrenia and schizoaffective disorders), has been recognized as valuable tool to study the neuropathology of TD and behavioral symptoms of Parkinson's disease in animal models. Rats treated with HAL chronically, block dopamine (DA) receptors, produce neurotoxicity, and develop orofacial dyskinesia (OD) which is characterized by increased vacuous chewing movement (VCM), tongue protrusion (TP), orofacial burst, and cataleptic behavior.[3],[4],[5],[6],[7] Similar to the clinical findings, these symptoms and oxidative stress seem to be related to OD; the development of HAL-induced OD has been associated to oxidative stress in striatum; an increased production in striatal lipid peroxidation (LPO) byproduct and nitric oxide (NO) levels, together with a rapid reduction in antioxidative enzyme activity.[8],[9] NO functions as a neuronal mediator in the central nervous system, physiologically, but excess production of NO is neurotoxic by damaging critical metabolic enzymes. NO could further react with superoxide anion to form an even more potent oxidant, peroxynitrite (ONOO−), to lead to more severe damage to cellular components such as proteins, DNA, and lipids.[10] Since the fundamental characteristics of biochemistry and physiology of brain are high lipid concentration and high energy consumption, therefore, it is particularly susceptible to free radical and oxidant-mediated insult. These previous findings in animal models support the idea of using antioxidants to treat TD may be crucial.[3],[4],[5],[6],[7]

L-Theanine (LT), an analogous chemical structure to glutamate and glutamine, comprises 0.5%–2% of the dry weight of tea, is synthesized from ethylamine and glutamate in tea leaves. It is the main nonprotein amino acid component responsible for the flavor and taste of green tea, and thus determines the quality of green tea. LT is rapidly absorbed into the blood from the intestinal tract through cotransport with Na +, and is then further redistributed to other organs, including the brain.[11] It is stated that LT has not shown toxic effect either on animals or on humans. Due to the taste-enhancing property and promising health benefits, LT has been approved as generally regarded as safe (GRAS) ingredient by the Food and Drug Administration and widely used in the food and pharmaceutical industry.[11],[12],[13] Accumulating evidence has shown that LT has numerous pharmacological properties such as sedative, hypotensive, anti-obesity, anti-inflammatory, and anti-tumorigenesis effects.[14],[15],[16],[17],[18] LT is also able to scavenge reactive radicals and decrease the peroxidative condition.[12],[19],[20] Furthermore, it has been anticipated to have neuroprotective properties to treat behavioral impairments induced by ischemia, toxins, and stress.[15],[19],[20],[21] Although the previous report in this study has indicated that LT administration is able to prevent the HAL-induced OD in rats,[4] up to date, the reports regarding its protective effect on HAL-induced OD are still limited. With LT's powerful anti-oxidative properties, we hypothesize that LT is able to neutralize the oxidative damage induced by HAL, and sequentially disrupt the OD development in animals. In this study, we examined the possible therapeutic effects of LT and its implication with the NO mechanism in preventing the HAL-induced OD in rats. OD is characterized by the increases of the frequency in VCMs and TP, as well as the duration of the facial twitching (FT). Since it has been previously reported that the altered striatal oxidative state, antioxidation power, and NO concentration were tightly correlated with OD development in animal models,[3],[4],[5],[6],[7] the LPO production (oxidative status), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels (antioxidation power), and striatal NO level were also examined in both control and HAL-treatment rats either with or without LT treatment.


  Materials and Methods Top


Animals

All the experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the U. S. National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University College of Medicine (IACUC Approval No: 20150217). Male Wistar rats weighing 300–320 g (about 3-month of age) were used for the study. A group of three animals was kept in Plexiglas cages with free access to food and water in a room with controlled temperature (22°C ± 3°C) and in 12-h light/dark cycle with lights on at 7:00 a.m. In order to minimize animal anxiety and discomfort, every rat was gently handled for 20 min/day for 4 days before starting the experimental procedures.

Experimental groups and drug treatment

The animals were randomly divided into two main groups as follows: control group (C; vehicle treated) and HAL treatment group (H), and these two groups were further divided into several treatment subgroups (n = 8 for each subgroup); treatments included LT 30 or 100 mg/kg treatment (T30 and T100), NO precursor; L-arginine (LA) 50 mg/kg treatment (LA50) or NO synthase inhibitor; L-NAME (LN) 10 mg/kg treatment (LN10) as well as combined treatments of LT with LA or LN [Table 1]. LT or its vehicle (distilled water) was orally administrated for 35 days. However, LA, and LN or their vehicle (normal saline) was injected for 35 days and HAL (1 mg/kg) was injected on the 15th day after the administration of LT or distilled water. The injection of LA or LN preceded the LT or distilled water administration by 60 min. From the 15–35th day, the administration of LT or distilled water preceded the HAL injection by 30 min. On the 36th day, 24 h after the injection of HAL, all animals were observed for the quantification of OD behavior in medication withdrawal [Figure 1]. Animals were sacrificed about 1 h after behavioral measurements. During this study, six rats died naturally for unknown reasons.
Table 1: Experimental Groups

Click here to view
Figure 1: Experimental design and drug treatment paradigm

Click here to view


Drugs

HAL (4­-[4-(4-Chlorophenyl)­-4­-hydroxy­-1­-piperidinyl]­-1­-(4-fluorophenyl)-1-butanone), LT (N-ethyl­-L-glutamine ≥98%), LA and L-NAME were from Sigma, St. Louis, Missouri, USA and they were prepared using normal saline. HAL, LA and L-NAME were administered intraperitoneally. However, LT, prepared using distilled water, was administrated by gavage during the period of housekeeping and animal nursing. All the drug dosages were adapted from the previous publications,[4],[20],[22] and administrated in the volume of 1.0 ml/kg body weight.

Behavioral assessment of orofacial dyskinesia

On the 36 day, 24 h after the injection of HAL, animals were observed for the quantification of OD behavior.[3] Animals were placed separately in the cage (20 cm × 20 cm × 19 cm), equipped with mirrors underneath the floors, to permit behavioral quantification even when the animal was facing away from the observer. To quantify the occurrence of OD, the events of VCM and TP, and the duration of FT were recorded 5 min in each section after a period of 2 min adaptation. In order to eliminate subjective bias on the part of experimenters, each animal arbitrarily assigned a number and all behavioral assessments were performed independently by two well experienced coworkers who were blinded to all treatments in all experiments. Neither experimenters knew what animals were treated or what the animal groups were. Each experimenter independently performed the tests and the clinical assessment evaluation. All behavioral experiments were conducted between 09:00 a.m. and 11:00 a.m.

Biochemical measurement

Rats were sacrificed 1 h after behavioral quantification. Brains were immediately taken out and washed with ice cold saline to remove blood and kept at −80°C. The part of striatum was rapidly dissected from the intact brain carefully on ice plate according to the stereotaxic atlas of Budantsev et al.[23] The dissected out striatum tissue was rinsed with isotonic saline and weighed, and then it was homogenized with 0.1N HCl. A 10% (w/v) tissue homogenate was prepared in a 0.1 M phosphate buffer (pH 7.4); the post nuclear fraction for CAT assay was obtained by centrifugation of the homogenate at 1000 g for 20 min at 4°C. For other enzyme assays, it was centrifuged at 12,000 g for 60 min at 4°C.

Assessment of lipid peroxidative indices

Lipid peroxide concentration was measured by the thiobarbituric acid reactive substance (TBARS) assay adapted from Ohkawa et al.[24] according to the procedures described by Hashimoto et al.[25] The concentration was measured in nanomoles malondialdehyde/mg protein. Malondialdehyde levels were then further normalized to a standard preparation of 1, 1, 3, 3-tetraethoxypropane.

Measurement of reduced glutathione

GSH was determined by the method of Ellman.[26] To the homogenate, 10% trichloroacetic acid was added, centrifuged followed by addition of 1.0 mL Ellman's reagent (19.8 mg of 5, 5-0-dithiobisnitro benzoic acid in 100 mL of 1.0% sodium citrate and 3 mL of phosphate buffer [pH 8.0]). The final developed product was measured at 412 nm. The results were expressed as nanomole GSH per milligram wet tissue.

Measurement of superoxide dismutase activity

The assay to determine SOD activity was based on the ability of SOD to inhibit spontaneous oxidation of adrenaline to adrenochrome.[27] To 0.05 mL supernatant, 2.0 mL of carbonate buffer and 0.5 mL of ethylenediaminetetraacetic acid were added. The reaction was started by the addition of 0.5 mL of epinephrine, and the auto-oxidation of adrenaline (3 × 10−4 M) to adrenochrome at pH 10.2 was measured the optical density at 480 nm. The changes in optical density every min were measured at 480 nm normalized to a blank reagent. The results are expressed as units of SOD activity (milligram per protein). One unit of SOD activity induced approximately 50% inhibition of adrenaline. The results were expressed as nmol SOD U per mg wet tissue.

Measurement of catalase activity

The CAT activity assay was adapted from Beers and Sizer.[28] The reaction mixture consisted of 2 mL phosphate buffer (pH 7.0), 0.95 mL of hydrogen peroxide (0.019 M), and 0.05 mL supernatant with a total volume of 3 mL. Absorbance was recorded at 240 nm every 10 s for 1 min. One unit of CAT was defined as the amount of enzyme required to decompose 1 mmol of peroxide per min, at 25°C and pH 7.0. The results were expressed as units of CAT activity (milligram per protein). Units of activity were determined from the standard graph of H2O2. The results were expressed as CAT U per mg wet tissue.

Determination of nitrites concentration

The level of nitrites (the final products of NO metabolism) was determined using Roche's test “NO colorimetric assay.”[29] The method relies on the following reaction: nitritessulfanilamideN-(1-naphthyl)-ethylenediamine dihydrochloride and produces a reddish–violet diazo dye with the result read at 540 nm. To determine the nitrite level in the tissue, a mixture of 100 μL homogenate and 400 μL redistilled water was incubated in a hot water bath (100°C) for 15 min to stop all enzymatic processes. After cooling, 30 μL Carrez I reagent (0.36 M K4[Fe (CN)6] ×3 H2O) and 30 μL Carrez II reagent (1 M ZnSO4× 7 H2O) were added to each sample. Next, the samples were alkalized to pH 8 by adding 4 μL of 10 M NaOH and centrifuged at 10,000 ×g before further use. For nitrite determination, 75 μL of supernatant and 75 μL of redistilled water were added in microplate wells. In blank samples, redistilled water was used instead of supernatant. The samples were incubated at room temperature for 30 min, and then the absorbance was read at 540 nm. Finally, the tested samples were color developed by adding 50 μL of 1% solution of sulfanilamide in 2.5% H3 PO4 and 50 μL of 0.1% solution of N-(1-naphthyl)-ethylenediamine dihydrochloride in 2.5% of H3 PO4. After mixing, microplates were placed in the dark for 15 min and absorbance was again measured at 540 nm. The results were calculated according to the standard curves obtained from different concentrations of sodium nitrite solutions (6–600 μM), using the change in absorbance measured before and after incubation with sulfanilamide and N-(1-naphthyl)-ethylenediamine dihydrochloride. Concentration of nitrite was used as an index for the NO level in the tested tissue sample and it was expressed as nmol per mg protein.

Statistical analysis

Data were expressed as mean ± standard deviation t-test was used to determine the difference between C and H groups. Data were subsequently analyzed using two-way ANOVA, with the factors group (C vs. H) and treatment (saline, LT, LA and LN), followed by one-way ANOVA and Tukey pair-wise post hoc test. Statistical significance was defined when P < 0.05.


  Results Top


Effects of L-theanine and nitric oxide modulators on haloperidol-induced Increase in the frequency of vacuous chewing movement and tongue protrusion as well as the duration of facial twitching

The effects of HAL on the VCM, TP and FT were presented in [Figure 2]a, [Figure 2]b, [Figure 2]c. HAL (1 mg/kg for 21 days) induced significant increases in the VCM, TP, and FT (t-test, C vs. H; P < 0.001). HAL treatment increased VCM to 21.79 times from 1.9 to 43.3 (times/5 min); TP to 8.24 times from 2.1 to 19.4 (times/5 min); FT to 7.15 times from 2.6 to 21.2 (seconds/5 min). These results indicated that H had OD development. The two-way ANOVA revealed significant main effects of groups (C vs. H) (VCM: F (1,36) = 67.52, P < 0.001; partial Eta squared = 0.614), (TP: F (1,36) = 72.85, P < 0.001; partial Eta squared = 0.611), (FT: F (1,36) = 76.69, P < 0.001; partial Eta squared = 0.682) and LT treatment (VCM: F (2,36) = 25.32, P < 0.001, partial Eta squared = 0.593), (TP: F (2,36) = 29.45, P < 0.001, partial Eta squared = 0.681) (FT: F (2,36) = 24.22, P < 0.001, partial Eta squared = 0.526) and a significant group × treatment interaction (VCM; F (2,36) = 36.53, P < 0.001, partial Eta squared = 0.856), (TP; F (2,36) = 27.23, P < 0.001, partial Eta squared = 0.64), (FT; F (2,36) = 30.26, P < 0.001, partial Eta squared = 0.709). Post hoc analysis showed that LT treatment had no significant effect on VCM, TP, and FT in the C, but the increased VCM, TP and FT induced by HAL were dose-dependently inhibited by LT. These results suggested that LT was able to reduce the increased VCM, TP and FT induced by HAL. However, dosages of 100 mg/kg LT treatment was not able to completely block the increased VCM, TP and FT induced by HAL; 100 mg/kg LT treatments still had significant higher VCM (P < 0.001), TP (P < 0.01) and FT (P < 0.01) compared to the C. In order to examine the implication of NO pathway activity in HAL-induced increased VCM, TP and FT, NO precursor LA (50 mg/kg) or NO synthase inhibitor LN (10 mg/kg) was co-pretreated with LT. Two-way ANOVA showed that the results of LA co-pretreated with LT had a significant main effect of groups (C vs. H) (VCM: F (1,48) = 856.54, P < 0.001; partial Eta squared = 0.956), (TP: F (1,48) = 428.18, P < 0.001; partial Eta squared = 0.921) and (FT: F (1,48) =765.83, P < 0.001; partial Eta squared = 0.854) but nonsignificant effects for LA/LT treatment (VCM: F (3,48) = 1.63, P = 0.295, partial Eta squared = 0.086), (TP: F (3,48) = 0.89, P = 0.764, partial Eta squared = 0.052), (FT: F (3,48) = 1.84, P = 0.324, partial Eta squared = 0.084) and group x treatment interaction (VCM: F (3,48) = 1.51, P = 0.464, partial Eta squared = 0.071) (TP: F (3,48) = 0.47, P = 0.789, partial Eta squared = 0.031) (FT: F (3,48) = 2.88, P = 0.063, partial Eta squared = 0.139). These results indicated that NO precursor LA nullified most of the beneficial effects of LT on reducing the increased VCM, TP, and FT induced by HAL. On the other hand, the results of LN co-pretreated with LT had significant main effects on groups (C vs. H) (VCM: F (1,48) = 150.92, P < 0.001; partial Eta squared = 0.848), (TP: F (1,48) = 152.73, P < 0.001; partial Eta squared = 0.681), (FT: F (1,48) = 198.94, P < 0.001; partial Eta squared = 0.851) and LN/LT treatment (VCM: F (3,48) = 56.38, P < 0.001, partial Eta squared = 0.731) (TP: F (3,48) = 52.78, P < 0.001, partial Eta squared = 0.694) (FT: F (3,48) = 88.37, P < 0.001, partial Eta squared = 0.845) as well as a significant group x treatment interaction (VCM: F (3,48) = 58.96, P < 0.001, partial Eta squared = 0.787) (TP: F (3,48) = 54.76, P < 0.001, partial Eta squared = 0.722) (FT: F (3,48) = 82.12, P < 0.001, partial Eta squared = 0.831). These results indicated that NO synthase inhibitor LN pretreatment with LT proved to be significantly more effective than LT treatment alone in reducing the increased VCM, TP, and FT in H (2a, 2b, 2c). Of note, LA or LN treatment alone did not produce any significant effect on VCM, TP, and FT in C and H.
Figure 2: (a-c) Effects of L-theanine and nitric oxide modulators (L-arginine and L-NAME) on haloperidol-induced increase in (a) the frequency of vacuous chewing movement, (b) tongue protrusion and (c) the duration of facial twitching. Vacuous chewing movement (a), tongue protrusion (b) and facial twitching (c) were used as an output measurement to assess haloperidol-induced orofacial dyskinesia. Data were presented as mean ± standard error of the mean (n = 8). Data were presented as mean ± standard error of the mean (n = 8). Data were analyzed using two-way ANOVA, post hoc with one-way ANOVA and Tukey pairwise tests (**P < 0.01, ***P < 0.001 by comparing within the group; ##P < 0.01, ###P < 0.001 by comparing to the C)

Click here to view


Effects of L-theanine and nitric oxide modulators on the increase of striatal lipid peroxide production in H

The results of HAL treatment affecting the production of TBARS in rat striatum are shown in [Figure 3]. HAL treatment (1 mg/kg for 21 days) induced a significant increase in the TBARS level in rat striatum (t-test, P < 0.001). The two-way ANOVA revealed significant main effects of groups (C vs. H) (F (1,36) =78.35, P < 0.001; partial Eta squared = 0.716) and LT treatment (F (2,36) = 25.86, P < 0.001, partial Eta squared = 0.604) and a significant group x treatment interaction (F (2,36) = 33.24, P < 0.001, partial Eta squared = 0.691). Post hoc analysis showed that LT treatment had no significant effect in the C, but LT dose-dependently decreased TBARS levels in H. These results suggested that LT was able to decrease the oxidative stress in striatum of H. We also examined the effects of NO pathway modulators on TBARS production; Two-way ANOVA showed that the results of LA co-pretreated with LT had a significant main effect of groups (C vs. H) (F (1,48) = 293.35, P < 0.001; partial Eta squared = 0.872) but nonsignificant effects for LA/LT treatment (F (3,48) = 2.52, P = 0.131, partial Eta squared = 0.134) and group x treatment interaction (F (3,48) = 1.52, P = 0.292, partial Eta squared = 0.087). These results indicated that NO precursor LA abolished most of the beneficial effect of LT on TBARS level reduction. On the other hand, the results of LN co-pretreated with LT had significant main effects of groups (C vs. H) (F (1,48) = 132.65, P < 0.001; partial Eta squared = 0.786) and LN/LT treatment (F (3,48) = 29.52, P < 0.001, partial Eta squared = 0.689) and a significant group x treatment interaction (F (3,48) = 26.46, P < 0.001, partial Eta squared = 0.690). These results indicated that NO synthase inhibitor LN pretreatment with LT further reduced the TBARS level compared to that of LT-treated alone in H [Figure 3]. However, LA or LN treatment alone did not produce any significant effect on changing TBARS levels in C and H.
Figure 3: Effects of L-theanine and nitric oxide modulators (L-arginine and L-NAME) on the increase of striatal lipid peroxide production induced by haloperidol. Thiobarbituric acid reactive substance level in rat striatum was used as an output measurement to assess haloperidol-induced dysfunctions. Data were presented as mean ± standard error of the mean (n = 8). Data were analyzed using two-way ANOVA, post hoc with one-way ANOVA and Tukey pairwise tests (***P < 0.001 by comparing within the group; ##P < 0.01, ###P < 0.001 by comparing to the C)

Click here to view


Effects of L-theanine and nitric oxide modulators on the decrease of striatal antioxidation power in H

The data of HAL treatment affecting on the levels of GSH and protective enzymes such as SOD and CAT in rat striatum are presented in [Figure 4], [Figure 5], [Figure 6]. HAL treatment (1 mg/kg for 21 days) significantly decreased the levels of GSH, SOD, and CAT in rat striatum (C vs. H, t-test, P < 0.001). Two-way ANOVA followed by one-way ANOVA post hoc revealed that the decreased levels of GSH, SOD, and CAT caused by HAL were partially and almost restored by 30 and 100 mg/kg LT, respectively. These results suggested that LT was able to enhance the antioxidation power that was decreased in striatum of H. To examine the NO pathway implication on current HAL/LT treatment paradigm, NO precursor LA (50 mg/kg) or NO synthase inhibitor LN (10 mg/kg) was co-pretreated with LT. Two-way ANOVA showed that the results of LA co-pretreated with LT had a significant main effect of groups (C vs. H) (GSH: F (1,48) =617.15, P < 0.001; partial Eta squared = 0.909), (SOD: F (1,48) = 376.65, P < 0.001; partial Eta squared = 0.913) and (CAT: F (1,48) = 827.12, P < 0.001; partial Eta squared = 0.922) but nonsignificant effects for LA/LT treatment (GSH: F (3,48) = 1.47, P = 0.537, partial Eta squared = 0.084), (SOD: F (3,48) = 0.47, P = 0.541, partial Eta squared = 0.029), (CAT: F (3,48) = 1.64, P = 0.232, partial Eta squared = 0.093) and group x treatment interaction (GSH: F (3,48) = 1.43, P = 0.552, partial Eta squared = 0.0840) (SOD: F (3,48) = 0.28, P = 0.588, partial Eta squared = 0.017) (CAT: F (3,48) = 3.47, P = 0.077, partial Eta squared = 0.186). These results indicated that NO precursor LA nullified most of the beneficial effects of LT on increasing GSH, SOD, and CAT levels. On the other hand, the results of LN co-pretreated with LT had significant main effects on groups (C vs. H) (GSH: F (1,48) =148.72, P < 0.001; partial Eta squared = 0.836), (SOD: F (1,48) = 117.54, P < 0.001; partial Eta squared = 0.784), (CAT: F (1,48) = 203.52, P < 0.001; partial Eta squared = 0.848) and LN/LT treatment (GSH: F (3,48) = 52.23, P < 0.001, partial Eta squared = 0.915) (SOD: F (3,48) = 40.32, P < 0.001, partial Eta squared = 0.715) (CAT: F (3,48) = 66.83, P < 0.001, partial Eta squared = 0.775) as well as a significant group x treatment interaction (GSH: F (3,48) = 49.30, P < 0.001, partial Eta squared = 0.871) (SOD: F (3,48) = 34.25, P < 0.001, partial Eta squared = 0.766) (CAT: F (3,48) = 69.92, P < 0.001, partial Eta squared = 0.782). These results indicated that NO synthase inhibitor LN pretreatment with LT proved to be significantly more effective than LT treatment alone in increasing the levels of GSH, SOD, and CAT in H striatum [Figure 4], [Figure 5], [Figure 6]. However, LA or LN treatment alone did not produce any significant effect on changing GSH, SOD, or CAT levels in C and H.
Figure 4: Effects of L-theanine and nitric oxide modulators (L-arginine and L-NAME) on the on the decrease of striatal glutathione induced by haloperidol. The levels of glutathione in rat striatum were used as output measurements to assess haloperidol-induced dysfunctions. Data were presented as mean ± standard error of the mean (n = 8). Data were analyzed using two-way ANOVA, post hoc with one-way ANOVA and Tukey pairwise tests (***P < 0.001 by comparing within the group; ##P < 0.01, ###P < 0.001 by comparing to the C)

Click here to view
Figure 5: Effects of L-theanine and nitric oxide modulators (L-arginine and L-NAME) on the on the decrease of striatal superoxide dismutase induced by haloperidol. The levels of superoxide dismutase in rat striatum were used as output measurements to assess haloperidol-induced dysfunctions. Data were presented as mean ± standard error of the mean (n = 8). Data were analyzed using two-way ANOVA, post hoc with one-way ANOVA and Tukey pairwise tests (***P < 0.001 by comparing within the group; ##P < 0.01, ###P < 0.001 by comparing to the C)

Click here to view
Figure 6: Effects of L-theanine and nitric oxide modulators (L-arginine and L-NAME) on the on the decrease of striatal catalase induced by haloperidol. The levels of catalase in rat striatum were used as output measurements to assess haloperidol-induced dysfunctions. Data were presented as mean ± standard error of the mean (n = 8). Data were analyzed using two-way ANOVA, post hoc with one-way ANOVA and Tukey pairwise tests (**P < 0.01, ***P < 0.001 by comparing within the group; ###P < 0.001 by comparing to the C)

Click here to view


Effects of L-theanine and nitric oxide modulators on the increase of striatal nitric oxide in H

The effects of HAL on the NO production in rat striatum are shown in [Figure 7]. HAL treatment (1 mg/kg for 21 days) significantly increased the NO concentration in rat striatum (t-test, P < 0.001). The two-way ANOVA revealed significant main effects of groups (C vs. H) (F (1,36) =168.92, P < 0.001; partial Eta squared = 0.851) and LT treatment (F (2,36) = 55.72, P < 0.001, partial Eta squared = 0.801) and a significant group x treatment interaction (F (2,36) = 48.12, P < 0.001, partial Eta squared = 0.757). Post hoc analysis showed that LT treatment had no significant effect in the C, but LT dose-dependently decreased NO levels in H. These results suggested that LT had the effect to decrease the NO production that was found increased in striatum of H. We also tested the effects of NO pathway modulators on NO production; Two-way ANOVA showed that the results of LA co-pretreated with LT had a significant main effect of groups (C vs. H) (F (1,48) = 345.35, P < 0.001; partial Eta squared = 0.898) but nonsignificant effects for LA/LT treatment (F (3,48) =1.49, P = 0.172, partial Eta squared = 0.084) and group x treatment interaction (F (3,48) = 0.179, P = 0.905, partial Eta squared = 0.014). These results indicated that NO precursor LA abolished the beneficial effect of LT on NO level reduction. On the other hand, the results of LN co-pretreatment with LT had significant main effects of groups (C vs. H) (F (1,48) = 136.32, P < 0.001; partial Eta squared = 0.774) and LN/LT treatment (F (3,48) = 47.35, P < 0.001, partial Eta squared = 0.776) and a significant group x treatment interaction (F (3,48) = 45.72, P < 0.001, partial Eta squared = 0.754). These results indicated that NO synthase inhibitor LN pretreatment with LT further reduced the NO level in H [Figure 7]. Similar to the findings from aforementioned parameters, LA or LN treatment alone did not produce significant effect on changing NO levels in C and H.
Figure 7: Effects of L-theanine and nitric oxide modulators (L-arginine and L-NAME) on the increase of striatal nitric oxide induced by haloperidol. The levels of nitric oxide in rat striatum were used as output measurements to assess haloperidol-induced dysfunctions. Data were presented as mean ± standard error of the mean (n = 8). Data were analyzed using two-way ANOVA, post hoc with one-way ANOVA and Tukey pairwise tests (**P < 0.01, ***P < 0.001 by comparing within the group; ##P < 0.01, ###P < 0.001 by comparing to the C)

Click here to view



  Discussion Top


The results of the present study highlighted the neuroprotective potential of LT, probably via its potent antioxidative activity and NO pathway modulation, against HAL induced OD in rats. The behavioral dysfunction, the elevated NO level and oxidative status induced by HAL were prevented by LT treatment. In addition, NO precursor and NO synthase inhibitor co-treatment with LT significantly decreased and intensified LTs protective effects respectively. These results suggested a protecting effect of LT to protect from HAL-induced OD or clinical human TD.

Our result of OD caused by HAL was consistent with others' and our previous findings; HAL significantly increased the frequency of VCM and TP as well as the duration of FT indicating a neurotoxicity effect produced by HAL.[3],[4],[5],[6],[7] In the current study, we also found that HAL increased NO and LPO levels in rat striatum, suggesting that NO and free radicals may be implicated with the development of OD. HAL treatment also decreased the levels of GSH (an endogenous antioxidant), SOD, and CAT (cellular antioxidant enzymes), further supporting the implication of NO/free radical toxicity in HAL-induced OD. It has been reported that HAL-induced OD is strongly associated with the status of striatal oxidative stress.[4],[5],[30] HAL, as a DA antagonist, blocks the DA receptors and increase metabolism of DA. The accelerated DA metabolism would facilitate the formation of reactive metabolites and hydrogen peroxide, which has been linked to the increased oxidative stress in dopaminergic neurons.[3],[4],[5],[6],[7] Furthermore, the O-quinone aminochrome produced from autoxidation of DA could further lose one electron to form the leukoaminochrome O-semiquinone radical which has been considered to be one of the primary sources of endogenous reactive species implicated with many neurodegenerative diseases.[31],[32],[33] Since monoamines are abundant in the basal ganglia including striatum, more susceptible to free radical damage in striatum would be anticipated. Furthermore, NO has been shown to be harmful under pathological conditions that involve the production of ROS including superoxide anions and the formation of more potent oxidant ONOO − to create even more damage to cellular components such as proteins, DNA and lipids.[10],[34],[35] With the fundamental properties of high lipid cell architecture and high energy consumption of brain cells; they are particularly more susceptible to free radical and oxidative stress insults. These evidence support that NO may also play a crucial role in the HAL-induced pathophysiology. Taken together, the above evidences supported the implication of both striatal nitrosative and oxidative stresses in the development of OD.

In this study, we showed that the levels of NO and TBARS were increased, at the same time, the levels of GSH, SOD and CAT were decreased in striatum of rats after HAL treatment. These results suggested that striatum was one of the primary targets in HAL-treated rats susceptible to NO neurotoxicity and oxidative stress. Therefore, effectively getting rid of the excessive NO and free radicals or boosting up the anti-oxidative factors in striatum would ameliorate the development of the OD caused by HAL.[4],[5],[6],[7],[29] LT, a natural analog of glutamate, the major and potent anti-oxidative amino acid components in green tea, has been demonstrated to have strong anti-oxidative effects and is able to clear excessive free radicals.[4],[12],[19],[20] Therefore, LT would be expected to have the ability to reduce nitrosative and oxidative damage to the striatum. In this study, HAL treatment significantly caused nitrosative and oxidative damage (increased the levels for LPO and NO and decreased the levels for SOD, GSH and CAT) in rat striatum, and these changes were significantly improved by LT pretreatment and modulated by NO pathway modulators. In addition to its powerful antioxidant effects, our findings also suggest that LT could exert its neuroprotective effecting by modifying NO pathway activity; It has been reported that NO can be produced by inducible NO synthase (iNOS) and neuronal NO synthase (nNOS) and it plays a crucial biological role in pathophysiological processes, including neurotoxicity.[10],[22],[34],[35] LT also decreased the production of NO induced by glutamate by downregulation of iNOS and nNOS protein activity in vitro, and in vivo in quinolinic acid-treated rats to reduce oxidative stress, thus preventing and the subsequent cytotoxic injury.[36],[37] The nNOS isoform of NOS produces toxic effect through NO. Therefore, LT's inhibition on nNOS may be one of its neuroprotective mechanisms. In addition to this, LT has been reported to attenuate glutamate-induced cytotoxicity by inhibition of glutamate transporter and decreasing calcium influx, and it blocked the activation of NF-κB, thus preventing iNOS induction and the subsequent cytotoxic injury;[12],[37],[38] these previous reports together with the current findings, LT treatment is expected to have the effects of reducing striatal NO and oxidative susceptibility in HAL-treated rats.

LT has been reported to increase CAT activity and attenuated NO as well as LPO levels,[4],[12],[37] suggesting that LT could directly reduce NO level and produce antioxidant action as well as indirectly modulate other antioxidant enzyme activities. In this study, our results were consistent with these previous findings; LT was able to diminish the increased level of NO as well as LPO and up-regulate the levels of GSH, SOD, and CAT those were found reduced in rat striatum. Moreover, LT was able to prevent HAL-induced OD with the evidence of preventing most typical behavior of OD. GSH and protective enzymes, such as SOD and CAT have been demonstrated to have protective effects in treating OD development induced by HAL;[4],[5],[6],[7] these previous results may underlie, at least in part, the anti-OD developed property of LT in HAL-treated rats by taking out excessive NO/free radicals and increasing anti-oxidative factors in rat striatum.

Findings from behavioral and biochemical parameters in the present study, LA co-treatment with LT eliminated the protective effect of LT, in contrast to that LN co-treatment with LT significantly potentiated the LT effects on behavioral and biochemical alterations induced by HAL. However, LA or LN treatment alone did not produce significant effect on changing VCM, TP, FT, TBARS, GSH, SOD, CAT, and NO levels in C and H groups; These results suggested that NO pathway modulators might not be able to act alone to change OD development and nitrosative/oxidative levels and they needed to work together with the mechanisms of LT on improving the HAL-induced OD as well as on reducing nitrosative/oxidative damage those were observed increased in rats treated with HAL.

Our findings provide evidence of LT's therapeutic value in treating OD in animal model, presumably also a beneficial effect in treating clinical relevant human TD, probably via its potent antioxidative activity and modification of NO pathway activity.

Acknowledgements

The authors thank Persistent BioMed Editing Service for their critical review and English copyediting of the current manuscript.

Financial support and sponsorship

This study was supported by the Yuan-Shan Branch of Taipei Veteran General Hospital (VGH-106-2), Mackay Memorial Hospital (MMH-105-67) and Shin Kong Wu Ho-Su Memorial Hospital (SKH-8302-105-DR-22).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Kane JM, Smith JM. Tardive dyskinesia: Prevalence and risk factors, 1959 to 1979. Arch Gen Psychiatry 1982;39:473-81.  Back to cited text no. 1
    
2.
Glazer WM, Morgenstern H, Schooler N, Berkman CS, Moore DC. Predictors of improvement in tardive dyskinesia following discontinuation of neuroleptic medication. Br J Psychiatry 1990;157:585-92.  Back to cited text no. 2
    
3.
Burger ME, Fachinetto R, Zeni G, Rocha JB. Ebselen attenuates haloperidol-induced orofacial dyskinesia and oxidative stress in rat brain. Pharmacol Biochem Behav 2005;81:608-15.  Back to cited text no. 3
    
4.
Chen CN, Chang KC, Wang MH, Tseng HC, Soung HS. Protective effect of L-theanine on haloperidol-induced orofacial. Chin J Physiol 2018;61:35-41.  Back to cited text no. 4
    
5.
Kamyar M, Razavi BM, Hasani FV, Mehri S, Foroutanfar A, Hosseinzadeh H, et al. Crocin prevents haloperidol-induced orofacial dyskinesia: Possible an antioxidant mechanism. Iran J Basic Med Sci 2016;19:1070-9.  Back to cited text no. 5
    
6.
Nade VS, Kawale LA, Yadav AV. Protective effect of Morus alba leaves on haloperidol-induced orofacial dyskinesia and oxidative stress. Pharm Biol 2010;48:17-22.  Back to cited text no. 6
    
7.
Patil R, Hiray Y, Shinde S, Langade P. Reversal of haloperidol-induced orofacial dyskinesia by Murraya koenigii leaves in experimental animals. Pharm Biol 2012;50:691-7.  Back to cited text no. 7
    
8.
Hanff TC, Furst SJ, Minor TR. Biochemical and anatomical substrates of depression and sickness behavior. Isr J Psychiatry Relat Sci 2010;47:64-71.  Back to cited text no. 8
    
9.
Zhang XY, Zhou DF, Shen YC, Zhang PY, Zhang WF, Liang J, et al. Effects of risperidone and haloperidol on superoxide dismutase and nitric oxide in schizophrenia. Neuropharmacology 2012;62:1928-34.  Back to cited text no. 9
    
10.
Calabrese V, Mancuso C, Calvani M, Rizzarelli E, Butterfield DA, Stella AM, et al. Nitric oxide in the central nervous system: Neuroprotection versus neurotoxicity. Nat Rev Neurosci 2007;8:766-75.  Back to cited text no. 10
    
11.
Türközü D, Şanlier N. L-theanine, unique amino acid of tea, and its metabolism, health effects, and safety. Crit Rev Food Sci Nutr 2017;57:1681-7.  Back to cited text no. 11
    
12.
Kakuda T. Neuroprotective effects of theanine and its preventive effects on cognitive dysfunction. Pharmacol Res 2011;64:162-8.  Back to cited text no. 12
    
13.
Wang D, Gao Q, Wang T, Qian F, Wang Y. Theanine: The unique amino acid in the tea plant as an oral hepatoprotective agent. Asia Pac J Clin Nutr 2017;26:384-91.  Back to cited text no. 13
    
14.
Pérez-Vargas JE, Zarco N, Vergara P, Shibayama M, Segovia J, Tsutsumi V, et al. L-theanine prevents carbon tetrachloride-induced liver fibrosis via inhibition of nuclear factor κB and down-regulation of transforming growth factor β and connective tissue growth factor. Hum Exp Toxicol 2016;35:135-46.  Back to cited text no. 14
    
15.
Yin C, Gou L, Liu Y, Yin X, Zhang L, Jia G, et al. Antidepressant-like effects of L-theanine in the forced swim and tail suspension tests in mice. Phytother Res 2011;25:1636-9.  Back to cited text no. 15
    
16.
Yoto A, Motoki M, Murao S, Yokogoshi H. Effects of L-theanine or caffeine intake on changes in blood pressure under physical and psychological stresses. J Physiol Anthropol 2012;31:28.  Back to cited text no. 16
    
17.
Zhang G, Miura Y, Yagasaki K. Effects of dietary powdered green tea and theanine on tumor growth and endogenous hyperlipidemia in hepatoma-bearing rats. Biosci Biotechnol Biochem 2002;66:711-6.  Back to cited text no. 17
    
18.
Zheng G, Sayama K, Okubo T, Juneja LR, Oguni I. Anti-obesity effects of three major components of green tea, catechins, caffeine and theanine, in mice. In Vivo 2004;18:55-62.  Back to cited text no. 18
    
19.
Sumathi T, Shobana C, Thangarajeswari M, Usha R. Protective effect of L-theanine against aluminium induced neurotoxicity in cerebral cortex, hippocampus and cerebellum of rat brain – Histopathological, and biochemical approach. Drug Chem Toxicol 2015;38:22-31.  Back to cited text no. 19
    
20.
Thangarajan S, Deivasigamani A, Natarajan SS, Krishnan P, Mohanan SK. Neuroprotective activity of L-theanine on 3-nitropropionic acid-induced neurotoxicity in rat striatum. Int J Neurosci 2014;124:673-84.  Back to cited text no. 20
    
21.
Zukhurova M, Prosvirnina M, Daineko A, Simanenkova A, Petrishchev N, Sonin D, et al. L-theanine administration results in neuroprotection and prevents glutamate receptor agonist-mediated injury in the rat model of cerebral ischemia-reperfusion. Phytother Res 2013;27:1282-7.  Back to cited text no. 21
    
22.
Chen CN, Chang KC, Lin RF, Wang MH, Shih RL, Tseng HC, et al. Nitric oxide pathway activity modulation alters the protective effects of (-) Epigallocatechin-3-gallate on reserpine-induced impairment in rats. Behav Brain Res 2016;305:198-211.  Back to cited text no. 22
    
23.
Budantsev A, Kisliuk OS, Shul'govskiĭ VV, Rykunov DS, Iarkov AV. The brain in stereotaxic coordinates (a textbook for colleges). Zh Vyssh Nerv Deiat Im I P Pavlova 1993;43:1045-51.  Back to cited text no. 23
    
24.
Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8.  Back to cited text no. 24
    
25.
Hashimoto M, Tanabe Y, Fujii Y, Kikuta T, Shibata H, Shido O, et al. Chronic administration of docosahexaenoic acid ameliorates the impairment of spatial cognition learning ability in amyloid beta-infused rats. J Nutr 2005;135:549-55.  Back to cited text no. 25
    
26.
Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7.  Back to cited text no. 26
    
27.
Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972;247:3170-5.  Back to cited text no. 27
    
28.
Beers RF Jr., Sizer IW. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 1952;195:133-40.  Back to cited text no. 28
    
29.
Bilska A, Dubiel M, Sokołowska-Jezewicz M, Lorenc-Koci E, Włodek L. Alpha-lipoic acid differently affects the reserpine-induced oxidative stress in the striatum and prefrontal cortex of rat brain. Neuroscience 2007;146:1758-71.  Back to cited text no. 29
    
30.
Thakur KS, Prakash A, Bisht R, Bansal PK. Beneficial effect of candesartan and lisinopril against haloperidol-induced tardive dyskinesia in rat. J Renin Angiotensin Aldosterone Syst 2015;16:917-29.  Back to cited text no. 30
    
31.
Cho CH, Lee HJ. Oxidative stress and tardive dyskinesia: Pharmacogenetic evidence. Prog Neuropsychopharmacol Biol Psychiatry 2013;46:207-13.  Back to cited text no. 31
    
32.
Lohr JB, Kuczenski R, Niculescu AB. Oxidative mechanisms and tardive dyskinesia. CNS Drugs 2003;17:47-62.  Back to cited text no. 32
    
33.
Raudenska M, Gumulec J, Babula P, Stracina T, Sztalmachova M, Polanska H, et al. Haloperidol cytotoxicity and its relation to oxidative stress. Mini Rev Med Chem 2013;13:1993-8.  Back to cited text no. 33
    
34.
Kumar P, Kumar A. Effect of lycopene and epigallocatechin-3-gallate against 3-nitropropionic acid induced cognitive dysfunction and glutathione depletion in rat: A novel nitric oxide mechanism. Food Chem Toxicol 2009;47:2522-30.  Back to cited text no. 34
    
35.
Kumar P, Kumar A. Protective effects of epigallocatechin gallate following 3-nitropropionic acid-induced brain damage: Possible nitric oxide mechanisms. Psychopharmacology (Berl) 2009;207:257-70.  Back to cited text no. 35
    
36.
Di X, Yan J, Zhao Y, Zhang J, Shi Z, Chang Y, et al. L-theanine protects the APP (Swedish mutation) transgenic SH-SY5Y cell against glutamate-induced excitotoxicity via inhibition of the NMDA receptor pathway. Neuroscience 2010;168:778-86.  Back to cited text no. 36
    
37.
Jamwal S, Kumar P. L-theanine, a component of green tea prevents 3-nitropropionic acid (3-NP)-induced striatal toxicity by modulating nitric oxide pathway. Mol Neurobiol 2017;54:2327-37.  Back to cited text no. 37
    
38.
Kim TI, Lee YK, Park SG, Choi IS, Ban JO, Park HK, et al. L-theanine, an amino acid in green tea, attenuates beta-amyloid-induced cognitive dysfunction and neurotoxicity: Reduction in oxidative damage and inactivation of ERK/p38 kinase and NF-kappaB pathways. Free Radic Biol Med 2009;47:1601-10.  Back to cited text no. 38
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed344    
    Printed38    
    Emailed0    
    PDF Downloaded65    
    Comments [Add]    

Recommend this journal