Effects of combined use of tryptophan on fluoxetine in depression and anxiety-like behavior in rats with depression

【Abstract】 Objective To investigate the effects of combined use of tryptophan on fluoxetine in depression and anxiety-like behaviors in rats with depression. Methods Sixty male Wistar rats were randomly divided into control group, tryptophan group, fluoxetine group and tryptophan + fluoxetine group, 15 each. Rats in the control group were given 9 g/L sodium chloride injection (5 mtAg), the tryptophan group was given 50 mg/kg tryptophan, and the fluoxetine group was given 10 mg/kg fluoxetine, tryptophan. The rats in the fluoxetine group were given 50 mg/kg tryptophan and 10 mg/kg fluoxetine. All groups of rats were given 21 days of chronic, moderate and unpredictable stress stimulation to establish a depression model. The modified forced swimming test was used to detect the stationary time, swimming time and climbing time of the four groups of rats. The elevated plus maze test was used to detect the time when the rats in the 4 groups entered the open arm and closed the arm, and the time to enter the central area and the number of open arms exceeded the total number of times. The levels of tryptophan in cerebrospinal fluid of 4 groups of rats were determined by high performance liquid chromatography. Results (1) There were significant differences in static time, swimming time and climbing time between the 4 groups (P<0.05). Both fluoxetine and tryptophan had significant effects on the experimental behavioral indicators of modified forced swimming in rats. (2) There were significant differences in the time between the 4 groups of rats entering the open arm time and entering the closed arm and the central area (衅0.05). Fluoxetine had a significant effect on the experimental behavioral indicators of the rat elevated plus maze. Tryptophan had no significant effect on the number and timing of rats entering each arm and central zone. There was no significant interaction between the effects of fluoxetine and tryptophan on the experimental behavioral indicators of the rat elevated plus maze. (3) There was a statistically significant difference in the determination of tryptophan levels in the cerebrospinal fluid of the 4 groups (F_6.874, P=-O.002). The tryptophan level in the cerebrospinal fluid of the tryptophan + fluoxetine group was significantly higher than that of the control group and the tryptophan group, and the difference was statistically significant (0.05). Conclusion (1) Tryptophan can aggravate the depression-like behavior of rats, and has no obvious effect on anxiety behavior; (2) Fluoxetine can reverse the depression-like behavior of rats, but cause anxiety; (3) Tryptophan and fluoxetine The combined use had no significant effect on the treatment of anxiety-like behavior and depression-like behavior in rats.

[Key words] tryptophan; fluoxetine; depression; anxiety; interaction

Depression is an emotional disorder characterized by significant and persistent low mood. It has the characteristics of high prevalence, high recurrence rate, high disability rate and high suicide rate, which seriously endangers human body and mind health. Studies have shown that 5-hydroxytryptamine (5-HT) is an important regulator of emotional behavior, and its dysfunction may be involved in the pathogenesis of depression. Currently. Selective 5-HT reuptake inhibitors (SSRIs), such as fluoxetine, are the drugs of choice for the treatment of depression; however, such drugs have poor efficacy, long treatment cycles, multiple side effects, and high recurrence rates. Therefore, it is urgent to develop new drugs for the prevention and treatment of depression. Recent studies have shown that oral tryptophan can increase the synthesis of 5-HT. Promote the recovery of 5-HT energy system functions. Therefore, this study investigated the behavior and biochemical changes of tryptophan combined with fluoxetine in rats with depression to investigate the efficacy of tryptophan combined with fluoxetine in the treatment of depression.

Materials and Methods

First, experimental animals and reagents, instruments

Sixty healthy adult male Wistar rats, 8 weeks old, weighing 230-280 g, were provided by the Experimental Animal Center of North China Coal Medical College. Nested in cages, room temperature (22 ± 1) ° C, humidity (55 ± 5)%, light and dark cycle 12 / 12h, free to eat, drink. Rats were adaptively housed for 1 week before the experiment. Rats were divided into control group, tryptophan group, fluoxetine group and tryptophan + fluoxetine group according to random block method, 15 rats each. L. Tryptophan was purchased from Shanghai Kangda Amino Acid Factory: Fluoxetine hydrochloride raw material was purchased from Xi'an Wanchang Biotechnology Co., Ltd.; Supelcosil LC. 18 columns (250 mm x 4.6 mnl, 5 buckets m) were purchased from Shanghai Chuding Analytical Instruments Co., Ltd.; Eppendorf 5408R desktop high speed refrigerated centrifuges were purchased from Eppendorf, Germany.

Second, the establishment of depression model rats

Long-term chronic, moderate and unpredictable stress stimuli were given to the rats in the 4 groups for 21 days, including electric shock (40V, 20 s/time, interval 50 S, total 20 times), heat stress (45 ° C, 5 min) Ice swimming (4 ° C, 5 min), tail (1 min), day and night reversal, fasting (48 h), water ban (24 h). One stimulus is given daily, and the same stimulus does not appear continuously.

Third, the method of administration

Rats were also given intragastric administration during the stimulation period, once a day for 42 days. Rats in the control group were given 9 g/L sodium chloride injection (5 mE&g), 50 mg/kg tryptophan in the tryptophan group, and 10 mg/kg fluoxetine and tryptophan in the fluoxetine group. The fluoxetine group was given 50 mg/kg tryptophan and 10 mg/kg fluoxetine simultaneously.

Fourth, behavioral indicators observation

1. Improved forced swimming test (provided by Shanghai Xinsoft Co., Ltd.): After 42 days of continuous gavage, the rats were fasted for 12 h and placed in a transparent cylindrical swimming bucket with a diameter of 21 cm and a height of 50 cm. Water temperature (25) ±1) ° C, water depth 30cm, forced to swim 15 minutes after taking it out of the pool. Dry and put back in the cage. After 24 h, the rats were placed in a swimming bucket again, and observed continuously for 5 min to analyze the time of the rats standing still, swimming and climbing.

2. Elevated Cross Maze Experiment (supplied by Shanghai Xinsoft Co., Ltd.): The experiment was performed on the second day after the last administration. The device consists of two opposite closed arms (50 cm × 10 cm) and two opposite open arms (50 cm × 10 cm) to form a square structure with a central zone (10 cm x 10 cm). All four arms are black. 43 cm from the ground. The rats were placed in a quiet, dark open box for 5 min, then placed from the central area toward the open arm. Continuous observation for 5 min, recording the time and number of access to the open arm and the time and number of times to enter the closed arm.

Five, cerebrospinal fluid sample collection

After 4 hours of the first administration, the rats were intraperitoneally injected with 20% urethane (1.2g/kg) anesthetized, placed prone on the animal fixation frame, and curved in the headline of the rat's occipital foramen. Angle, a 1 mL syringe was inserted into the cisterna magna of the cerebellum, and 50-60 μL of cerebrospinal fluid was aspirated and centrifuged at 10 000 x g for 5 min. The supernatant was stored in a 70 ° C environment.

6. Determination of tryptophan concentration by high performance liquid chromatography

Cerebrospinal fluid samples were derivatized with o-phthalaldehyde. Separated by high performance liquid chromatography. The column was Supelcosil LC-18 (250 mm x 4.6 mm, 5 ̈m), and the mobile phase A was 0.04 mol/L sodium dihydrogen phosphate monohydrate (pH 5.5) containing 20% ​​by volume of methanol; Mobile phase B was 0.01 mol/L sodium dihydrogen phosphate monohydrate (pH 5.5), containing 80% by volume of methanol; eluted with a binary gradient, flow rate 1.4 mL/min, column temperature 24 ° C, The injection volume is 50 buckets L. The fluorescence detector excites a wavelength of 360 nln and an emission wavelength of 455 nl TI, which is quantified by external standard method.

Seven, statistical analysis

Analysis was performed using SPSSl3.0 statistical software. The measurement data is expressed as mean ± standard deviation 6 spider). The behavioral index and cerebrospinal fluid tryptophan levels were statistically analyzed by one-way ANOVA. If there is a significant interaction between tryptophan and fluoxetine in the treatment of depression and anxiety-like behavior in rats, ANOVA simple effect test is performed, and LSD is used for comparison between groups. t method; if the interaction is not significant, the independent sample t test was used to compare the difference between the tryptophan + fluoxetine group and the control group. The difference was considered statistically significant at P < 0.05.

discuss

5-HT is an important neurotransmitter in the central nervous system and participates in mood regulation, learning and memory. Low function of 5-HT system is an important mechanism of depression, and tryptophan is a limiting factor for 5-HT synthesis. Tryptophan cannot pass freely through the blood-brain barrier. It needs to be transported to the brain via a non-specific amino acid transporter, but peripheral tryptophan concentration can affect the availability of tryptophan in the central nervous system. Therefore, based on the hypothesis that the 5-HT energy system regulates mood and behavior, El administration of tryptophan can increase 5-HT levels in the brain. This study was to observe the effect of combined use of tryptophan on fluoxetine in depression and anxiety-like behavior in rats with depression.

According to the results of the modified forced swimming experiment, tryptophan significantly increased the resting time of the rats, reduced the climbing time, and showed a tendency to reduce swimming time, thus having the effect of aggravating the depression-like behavior of rats, which may be related to tryptophan. The dose is used. 5-HT synthesis can be restricted when the tryptophan concentration is not reached, resulting in the inability of 5-HT levels in the brain to increase hippocampal neural precursor cells or enhance neuronal regeneration and aggravate depression. Studies have shown that restricted tryptophan intake will increase the resting time of rats in modified forced swimming experiments. And this depression-like behavior may be associated with decreased activity of the 5-HT system. The results of this study are also shown. Without limiting diet and not consuming endogenous 5-HT. Oral tryptophan in rats had no positive effect on 5-HT energy system activity. Fluoxetine significantly reduced resting time and increased swimming time. It has the effect of reversing the depression-like behavior of rats, which can inhibit the reuptake of 5-HT by presynaptic membrane and increase the level of 5-HT in synaptic cleft of nerve cells. Enhance its emotional regulation.

According to the results of the elevated plus maze experiment, tryptophan had no significant effect on anxiety behavior in rats, while fluoxetine had an anxiogenic effect. Most related studies have shown that fluoxetine can increase extracellular 5-HT levels in the brain. The results of this study on the effects of fluoxetine on reversing depression-like behavior and anxiety-causing were consistent with those of other investigators. However, other studies have shown that fluoxetine has an anxiolytic effect. The reasons for the inconsistency between the conclusions of the study and the paper may be related to the different doses administered. According to the therapeutic effect of these studies. In this study, the dose of fluoxetine was chosen to be 10 mg/(kg·d), which is a higher dose. At present, there is no consistent conclusion about the study of tryptophan to alleviate depression. The combined use of tryptophan had no significant effect on the reversal of depression-like behavior by fluoxetine. However, the results of this study found that there is a certain interaction between tryptophan and fluoxetine on the resting time of rats. It is suggested that elevated tryptophan levels in cerebrospinal fluid may offset the effect of fluoxetine on reversing depression-like behavior. In addition, combined use of tryptophan and fluoxetine did not significantly improve the anxiety behavior of rats. Animal experiments showed that the improvement of depression and anxiety-like behaviors in rats with depression was not stable with tryptophan combined with fluoxetine, simelistidine, clomipramine or amitriptyline. But studies by Cervo et al. Two citalopram-resistant mice were sensitized to citalopram by pre-administration of tryptophan.

This study found that the use of tryptophan or fluoxetine alone had no significant effect on the level of tryptophan in rat cerebrospinal fluid; however, the level of tryptophan in the cerebrospinal fluid of rats fed with tryptophan and fluoxetine was significantly higher than that of the control group. . It is speculated that there is a significant interaction between the effects of tryptophan and fluoxetine on tryptophan levels in rat cerebrospinal fluid. Tryptophan is converted to 5-hydroxytryptophan (5.HTP) by tryptophan hydroxylase (TPH) and converted to 5-HT by 5-HTP decarboxylase. Fluoxetine not only increases the 5-HT concentration in the nerve cell gap. At the same time, Shishkina and other studies showed that fluoxetine can up-regulate rat brain TPH. 2 enzyme mRNA levels, can increase the level of tryptophan non-specific amino acid transporter, and its transport to the brain increased concentration, so the concentration of tryptophan in the rat cerebrospinal fluid increased in combination. however. The results of this study indicate that the combination of tryptophan and fluoxetine has no synergistic effect in reversing depression in depressed model rats. It is speculated that long-term use of drugs and improved forced swimming experiments, elevated plus maze experiments affect the expression and activity of enzymes involved in 5-HT synthesis and metabolic processes.

In summary. The combination of tryptophan and fluoxetine did not significantly promote the depression-like behavior of rats, and did not significantly improve the anxiety-like behavior of rats.

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