Application of Klebsiella oxytoca

Abstract

The present invention discloses the use of Klebsiella oxytoca. The present invention provides the use of Klebsiella oxytoca in the preparation of a medicament for reducing latent psychological craving after methamphetamine addiction and / or treating methamphetamine addiction. The Klebsiella oxytoca of the present invention can reduce methamphetamine craving. Experiments have shown that it can significantly inhibit latent psychological craving in rats 30 days after abstinence following methamphetamine self-administration training. The present invention regulates the composition of the intestinal flora after addiction by gavage, gradually reducing addicts' craving for drugs and effectively improving drug abstinence rates.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the application of Klebsiella oxytoca. Background Art

[0002] Currently, treatments for substance addiction primarily rely on psychological intervention and rehabilitation, supplemented by medication. However, individual variability is significant, and overall effectiveness is unsatisfactory. Furthermore, most medications are neurotransmitter antagonists or agonists, which can produce significant side effects. Therefore, developing an effective therapeutic bacterium that can colonize the intestine and exert its therapeutic effects has profound implications for the treatment of drug addiction. Summary of the Invention

[0003] The present invention aims to provide a use of Klebsiella oxytoca. The Klebsiella oxytoca of the present invention can be used to reduce the latent psychological craving after methamphetamine addiction, treat methamphetamine addiction, and effectively improve drug withdrawal rates.

[0004] The present invention provides Klebsiella oxytoca used in the preparation of a drug for reducing latent psychological craving after methamphetamine addiction.

[0005] The present invention also provides the use of Klebsiella oxytoca in the preparation of a medicine for treating methamphetamine addiction.

[0006] In the present invention, the Klebsiella oxytoca has a strain number of (China Industrial Culture Collection Center CICC21518).

[0007] The present invention also provides the use of Klebsiella oxytoca in reducing latent psychological cravings after methamphetamine addiction and / or treating methamphetamine addiction.

[0008] The present invention further provides a drug for reducing latent psychological craving after methamphetamine addiction and / or treating methamphetamine addiction, comprising an active ingredient of Klebsiella oxytoca and an auxiliary agent.

[0009] In the above-mentioned medicine, the auxiliary agent is physiological saline and / or pharmaceutically acceptable excipients, specifically physiological saline.

[0010] Among the above-mentioned drugs, the dosage form of the drug is a dosage form for administration through the gastrointestinal tract.

[0011] The present invention provides a method for treating methamphetamine addiction using the drug for reducing latent psychological cravings after methamphetamine addiction and / or treating methamphetamine addiction or the physiological saline solution of Klebsiella oxytoca, comprising the following steps:

[0012] The drug for reducing latent psychological craving after methamphetamine addiction and / or treating methamphetamine addiction or the physiological saline solution of Klebsiella oxytoca is orally administered to an individual who is withdrawing from methamphetamine to reduce the individual's craving after withdrawal from methamphetamine and treat methamphetamine addiction.

[0013] The present invention has the following beneficial effects:

[0014] The Klebsiella oxytocaine of the present invention can reduce methamphetamine craving and treat methamphetamine addiction. Experiments have shown that it can significantly suppress latent psychological craving in rats after 30 days of abstinence following methamphetamine self-administration training. The present invention gradually reduces drug craving in addicts through oral administration, thereby treating methamphetamine addiction and effectively improving drug abstinence rates. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the experimental process of the present invention.

[0016] Figure 2 These are the results of methamphetamine self-administration training in rats.

[0017] Figure 3 This is a 30-day withdrawal extinction test for rats (number of effective nose touches).

[0018] Figure 4 This is a 30-day withdrawal extinction test for rats (number of invalid nose touches).

[0019] Figure 5 Open field test for rats. DETAILED DESCRIPTION

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] In the following examples, unless otherwise specified, the results of each experiment are the results of three parallel experiments.

[0023] The following examples of the present invention show that Klebsiella oxytoca can reduce methamphetamine craving or treat methamphetamine addiction. Figure 1 The experimental process shown is as follows:

[0024] 1) Rats self-administration (methamphetamine administration) training;

[0025] 2) Klebsiella oxytoca intervention and testing.

[0026] Example 1

[0027] Rat self-administration training

[0028] (1) Male Sprague-Dawley (SD) rats (SPF grade) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Animals were group-housed 5 per cage and acclimated for 7 days. Rats were housed in a standardized specific pathogen-free (SPF) animal laboratory with a temperature (22 ± 2°C), humidity (50 ± 10%), and lighting schedule (12-hour light / dark cycle, 8:00–20:00). Rats had free access to food and water throughout the experiment. Rats weighed approximately 320 g for the experiment.

[0029] Methamphetamine hydrochloride was purchased from the Narcotics Control Intelligence Technology Center of the Ministry of Public Security and dissolved in 0.9% saline to prepare a methamphetamine hydrochloride solution with a concentration of 0.75 mg / mL.

[0030] (2) The rat self-administration box and software were purchased from Ningbo Anlai Software Technology Co., Ltd. The rat self-administration training box had a plate-shaped bottom, transparent plexiglass front and back walls, and a gray aluminum side wall. Two nose-poke devices were located 9 cm above the bottom of the box.

[0031] The rats were intubated in advance, and the self-administration cannula fixed on the skull was connected to the drug administration pump through a rubber tube. The hose was protected by a metal spring and the top was connected to a rotating bearing to ensure that the animals could move freely in the self-administration training box.

[0032] During the experiment, when an animal touched the active nose-poke device, intravenous drug administration was triggered, accompanied by a light-sound conditional cue, which lasted for 5 seconds. A computer simultaneously recorded the number of times the animal touched the active nose-poke and the inactive nose-poke.

[0033] (3) Rat self-administration surgery: After the rat is anesthetized with isoflurane, the right neck opening is opened and the jugular vein is separated by about 1 cm. The previously prepared sharp intravenous cannula is buried in the right jugular vein, with the cannula tip located at the right atrium opening. After the intravenous cannula is fixed, it is passed subcutaneously through the ear and placed above the skull (the scalp is disinfected with alcohol and iodine in advance), and connected to the intravenous self-administration cannula. After the intravenous cannula is completed, the rat is placed on a brain stereotaxic instrument (prone position), and the rat is fixed in the horizontal direction with ear bars. The skull is exposed with an artery clamp, and the skull position is adjusted so that the anterior and posterior fontanelles are at the same level (the height difference is less than 0.1 mm). The skull connective tissue is removed with a cotton swab, and the cannula is fixed to the skull with a screw and mixed dental cement. The stainless steel inner core is inserted into the cannula to keep it unobstructed and prevent infection. After the cement solidifies, the rats are removed from the positioning apparatus and housed individually for 5-7 days of recovery. Methamphetamine self-administration training is performed after the rats have returned to their pre-operative weight. Penicillin and sodium heparin are administered daily to prevent clogging or infection of the intravenous cannula. Penicillin administration is discontinued after the rats begin self-administration training.

[0034] (4) Methamphetamine self-administration training in rats: Rats were trained in methamphetamine self-administration for 10 days (0.1 mg / kg / infusion after each effective nose contact), with 6 hours of training per day and a 5-minute rest period between each hour. Self-administration training was conducted during the rats' night cycle. The training used a fixed ratio 1 reinforcement program with a 20-second refractory period between each administration. Each training period was signaled by the cage light turning on. After the animal touched the effective nose contact, the cage light turned off, and methamphetamine was automatically administered, accompanied by a sound and light cue lasting 5 seconds. After the 20-second refractory period, the cage light turned on again. To prevent animals from overdosing and dying, the number of methamphetamine administrations per hour was limited to 15 times. After the training, the animals were grouped according to the number of administrations during the training period. There was no statistical difference in the mean number of administrations for each group of animals.

[0035] (5) Natural withdrawal: During the natural withdrawal process, the animals were kept in normal cages without exposure to methamphetamine or drug-related cues. They were kept in an animal room with a fixed temperature (22±2°C), humidity (50±10%), and lighting schedule (12-hour light / dark cycle, lights off at 8 am and lights on at 8 pm), with free access to food and water. During the withdrawal period, the rats were petted every 2-3 days. Rats that had completed self-administration training were obtained.

[0036] Example 2

[0037] Bacterial intervention after self-administration training in rats

[0038] (1) Bacterial gavage: Klebsiella oxytoca was purchased from China Industrial Microbiology Culture Collection Center (No.: CICC21518) and cultured in nutrient broth. After enrichment by high-speed centrifugation (5000×g, 20 minutes), the bacteria were washed twice with physiological saline and collected (2×10 8 CFU / time), and bacterial gavage was performed on the second day of withdrawal for 28 days, twice a day (once in the morning and once in the evening), and a disappearance test was performed on the 30th day of withdrawal.

[0039] The rats treated with Klebsiella oxytocae by oral gavage were the bacteria intervention group, and the rats in the bacteria intervention group were the rats that completed the self-administration training in Example 1 of the present invention;

[0040] Rats gavaged with normal saline were used as a control group, recorded as the normal saline intervention group. The rats in the normal saline intervention group were the rats that completed the self-administration training in Example 1 of the present invention.

[0041] (2) Extinction test: On the second day after the methamphetamine self-administration training in Example 1 of the present invention (i.e., the first day of withdrawal) and the 30th day of withdrawal, the rats were placed in the self-administration training box again for extinction test. During the extinction test, the cage light was turned on as a signal during the test period. After the animal touched the effective nose touch, the cage light was turned off, accompanied by sound and light cues with a duration of 5 seconds. However, each time the effective nose touch was touched, it was no longer accompanied by methamphetamine. Each test time was 30 minutes. The number of times the animals in the bacteria intervention group and the normal saline intervention group touched the effective or invalid nose touch during the test was recorded by computer synchronously.

[0042] (3) Open field test: The spontaneous activity box is made of black homemade plexiglass with a digital camera 1 meter above. The experiment is conducted in a quiet environment. The rat is placed in a corner, and the video is collected and timed. After 5 minutes of observation, the next rat is replaced for testing. The box needs to be cleaned in the middle to prevent the rat feces and odor from affecting the next behavioral test. The behavioral data are analyzed using an animal motion trajectory tracking system (EthoVision XT 10) to count the distance the rat moves in the open field.

[0043] result:

[0044] 1. After 10 days of methamphetamine self-administration training, the results are as follows Figure 2 As shown by Figure 2 As can be seen from the figure, both curves are rising, proving that the rats have learned significant self-administration, indicating that both groups of rats have been successfully modeled and can be used for subsequent drug intervention.

[0045] 2. After 28 days of oral gavage intervention with Klebsiella oxytoca, a regression test was performed on the 30th day of withdrawal, e.g. Figure 3The results showed that rats in the Klebsiella oxytoca intervention group showed reduced craving compared with the saline intervention group.

[0046] 3. If Figure 4 As shown in the figures, on the 30th day of withdrawal, there was no significant difference in the number of ineffective nose touches between the Klebsiella oxytoca intervention group and the saline intervention group, indicating that the Klebsiella oxytoca intervention of the present invention can eliminate the methamphetamine craving of rats after 30 days of withdrawal.

[0047] 4. The spontaneous activity of rats was analyzed at the same time (test time 5 minutes). The total movement distance of the Klebsiella oxytoca intervention group was 1353 cm, while the total movement distance of the saline intervention group was 1438 cm. Figure 5 As shown, the results showed that there was no significant difference between the Klebsiella oxytoca intervention group and the normal saline intervention group (control), further indicating that the Klebsiella oxytoca intervention of the present invention can reduce the methamphetamine craving of rats after 30 days of withdrawal.

[0048] Therefore, the results of the above embodiments of the present invention show that after the intervention of Klebsiella oxytoca in rats, the rats' craving for methamphetamine can be significantly reduced, and the rats' addiction to methamphetamine can be treated, providing a new method and idea for the treatment of methamphetamine addiction.

Claims

1. Use of Klebsiella oxytoca in the preparation of a medicament for reducing latent psychological cravings following methamphetamine addiction.

2. Use of Klebsiella oxytoca in the preparation of medicines for treating methamphetamine addiction.

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