Application of ethyl pyruvate in preparing drugs for treating or preventing methamphetamine addiction

Abstract

The present invention discloses the use of ethyl pyruvate in the preparation of a drug for treating or preventing methamphetamine addiction. The present invention belongs to the field of pharmaceutical technology and provides the use of ethyl pyruvate in the preparation of a drug for treating or preventing methamphetamine addiction. Ethyl pyruvate inhibits the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway, reduces neuroinflammatory responses, and significantly reduces addictive behaviors and nerve damage caused by methamphetamine. This provides a new therapeutic strategy for the treatment of methamphetamine addiction and has broad clinical application prospects. Ethyl pyruvate has significant neuroprotective effects, can reduce nerve damage, and promote nerve repair.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the use of ethyl pyruvate in preparing a drug for treating or preventing methamphetamine addiction. Background Art

[0002] Methamphetamine (METH), a commonly abused substance, is highly addictive. Long-term use can lead to severe neurological damage and psychological dependence, impacting patients' social functioning and quality of life. Existing treatments primarily focus on withdrawal therapy and psychological interventions, but these approaches have limited effectiveness in alleviating addiction and neurological damage, and are difficult to fundamentally improve addictive behaviors.

[0003] High-mobility group protein B1 (HMGB1), a molecule closely associated with immune and inflammatory responses, has been shown to play a crucial role in the development of various neurodegenerative diseases and drug addiction. Studies have shown that HMGB1 binds to the TLR4 receptor, activating downstream signaling pathways, inducing neuroinflammatory responses and promoting addictive behaviors.

[0004] Ethyl pyruvate (EP) is a molecule with significant anti-inflammatory and antioxidant effects that has shown potential in multiple neuroprotection studies in recent years. However, no studies have demonstrated the application of EP in the treatment of methamphetamine (METH) addiction. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes the use of ethyl pyruvate in the preparation of drugs for treating or preventing methamphetamine addiction. Ethyl pyruvate inhibits the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway, reduces neuroinflammatory responses, and significantly reduces addictive behaviors and neural damage caused by METH, providing a new therapeutic strategy for the treatment of METH addiction and has broad clinical application prospects.

[0006] To achieve the above object, the present invention provides the use of ethyl pyruvate in the preparation of a drug for treating or preventing methamphetamine addiction.

[0007] Preferably, the ethyl pyruvate reduces methamphetamine-induced nerve damage, promotes nerve repair, and inhibits methamphetamine addiction behavior by targeting and regulating the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway.

[0008] Preferably, the ethyl pyruvate exerts a neuroprotective effect by inhibiting the HMGB1-TLR4 pathway, alleviating methamphetamine-induced nerve damage, promoting nerve repair, and inhibiting methamphetamine addiction behavior.

[0009] Preferably, the ethyl pyruvate is administered via intraperitoneal injection at a dosage of 50-80 mg / kg.

[0010] The present invention also provides a medicine for treating or preventing methamphetamine addiction, wherein the active ingredient of the medicine is the ethyl pyruvate.

[0011] The present invention also provides the use of the ethyl pyruvate in preparing a medicine for repairing nerve damage caused by methamphetamine.

[0012] Preferably, the ethyl pyruvate exerts a neuroprotective effect by inhibiting the HMGB1-TLR4 pathway, alleviating nerve damage caused by methamphetamine and promoting nerve repair.

[0013] The present invention also provides a medicine for repairing nerve damage caused by methamphetamine, wherein the active ingredient of the medicine is the ethyl pyruvate.

[0014] The present invention also provides the use of the ethyl pyruvate in preparing a drug for inhibiting methamphetamine addiction behavior.

[0015] Preferably, the ethyl pyruvate inhibits methamphetamine addiction behavior by targeting and regulating the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] This study investigates the neuroprotective effects of ethyl pyruvate in a rat methamphetamine self-administration model and its regulation of the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway. Ethyl pyruvate inhibits the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway, reducing neuroinflammatory responses and significantly alleviating methamphetamine-induced addictive behaviors and neural damage. This study provides a novel therapeutic strategy for methamphetamine addiction and has broad clinical application prospects. Ethyl pyruvate exhibits significant neuroprotective effects, alleviating neural damage and promoting neural repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1The statistical chart of the number of active nose-poking and METH injection times of METH self-administered rats in different experimental groups, where A is the number of active nose-poking and B is the number of METH injection times. " represents p ≤ 0.001," ” represents p ≤ 0.0001;

[0020] Figure 2 The statistical graphs of the breakpoints and final ratios of METH self-administered rats in different experimental groups, where A is the breakpoint and B is the final ratio. In the figure, "ns" means p>0.05, ” represents p ≤ 0.0001;

[0021] Figure 3 This is a dose-response curve of METH self-administration. ” represents the significant difference in the rat group injected with 80 mg / kg Ep, p≤0.05, “#” represents the significant difference in the rat group injected with 50 mg / kg Ep, p≤0.05;

[0022] Figure 4 Figure 2 is the protein analysis of METH self-administered rats in different experimental groups, where A is the protein expression diagram of HMGB1-TLR4 pathway in each group, control represents the complete blank control group, Meth represents the METH self-administered rat group injected with normal saline intraperitoneally, B is the relative content of HMGB1 protein in each group, control represents the complete blank control group, Vehicle represents the METH self-administered rat group injected with normal saline intraperitoneally, C is the relative content of TLR4 protein, control represents the complete blank control group, Vehicle represents the METH self-administered rat group injected with normal saline intraperitoneally, D is the relative content of MyD88 protein, control represents the complete blank control group, Vehicle represents the METH self-administered rat group injected with normal saline intraperitoneally. The rat group of METH self-administered with saline, E is the relative content of PI3K protein, control represents the complete blank control group, Vehicle represents the rat group of METH self-administered with intraperitoneal injection of normal saline, F is the relative content of p-AKT protein, control represents the complete blank control group, Vehicle represents the rat group of METH self-administered with intraperitoneal injection of normal saline, G is the relative content of p-NFKB protein, control represents the complete blank control group, Vehicle represents the rat group of METH self-administered with intraperitoneal injection of normal saline, H is the relative content of IL-6 protein, control represents the complete blank control group, Vehicle represents the rat group of METH self-administered with intraperitoneal injection of normal saline, represents p≤0.05, represents p≤0.01, represents p≤0.001, represents p ≤ 0.0001;

[0023] Figure 5 This is an analysis of apoptosis and pyroptosis in the medial prefrontal cortex of rats with METH self-administration in different experimental groups, where A is the expression of caspase3 and caspase1, representative proteins of the apoptosis and pyroptosis pathways, control represents a complete blank control group, Meth represents the group of rats with METH self-administration injected intraperitoneally with normal saline, B is the protein content of activated Cleaved-caspase3 relative to total caspase3 (inactive precursor), control represents a complete blank control group, Meth represents the group of rats with METH self-administration injected intraperitoneally with normal saline, 50EP represents the group with 50 mg / kg ethyl pyruvate injected intraperitoneally, C is the protein content of activated Cleaved-caspase1 relative to total caspase1 (inactive precursor), control represents a complete blank control group, Meth represents the group of rats with METH self-administration injected intraperitoneally with normal saline, represents p≤0.05, represents p≤0.01, represents p ≤ 0.001;

[0024] Figure 6 TLR4 expression in rats with METH self-administration in different experimental groups + The number of neurons is counted. In the figure, control represents the complete blank group, and Meth represents the METH self-administered rat group injected with normal saline intraperitoneally. represents p ≤ 0.0001;

[0025] Figure 7 The number of microglia co-localized with neurons in rats self-administered with METH in different experimental groups is statistically analyzed. In the figure, control represents the complete blank group, and Meth represents the METH self-administered rat group injected with normal saline intraperitoneally. represents p ≤ 0.001;

[0026] Figure 8 METH self-administration rats and SST + The number of microglia co-localized with neurons is counted. In the figure, control represents the complete blank group, and Meth represents the METH self-administered rat group injected with normal saline intraperitoneally. represents p ≤ 0.001;

[0027] Figure 9 TLR4-positive SST in rats with METH self-administration in different experimental groups +The number of neurons is counted. In the figure, control represents the complete blank group, and Meth represents the METH self-administered rat group injected with normal saline intraperitoneally. represents p ≤ 0.0001;

[0028] Figure 10 SST of HMGB1 nucleocytoplasmic translocation in rats with METH self-administration in different experimental groups + The proportion of neurons, control represents the complete blank group, Meth represents the METH self-administration rat group injected with normal saline intraperitoneally, represents p≤0.05, Represents p ≤ 0.01. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] 1. Animals

[0036] Adult male Sprague-Dawley rats (8 weeks old, weighing 280–300 g) were purchased from the Zhejiang Provincial Laboratory Animal Center (Hangzhou, China). The rats were housed in standard plastic rodent cages with controlled temperature and humidity under a 12-h light / 12-h dark cycle (lights on at 8:00 AM and off at 8:00 PM) with free access to food and water.

[0037] 2. Drugs

[0038] Methamphetamine was provided by the Narcotics Intelligence and Forensic Medicine Center of the Ministry of Public Security (Beijing, China) and dissolved in 0.9% sterile saline. Ethyl pyruvate was purchased from Sigma-Aldrich and diluted in 0.9% sterile saline.

[0039] 3. Jugular vein catheterization and METH self-administration

[0040] Rats underwent surgical implantation of chronic indwelling intravenous catheters under anesthesia with Zoletil® 50 (50 mg / mL, intraperitoneal injection, Vic, France). To maintain catheter patency, the catheters were flushed daily with 0.1 mL of heparinized saline (heparin to saline at a volume ratio of 1:100). For infection prevention, penicillin was administered intramuscularly at a dose of 30 mg / kg / day.

[0041] After a week of recovery, rats began METH self-administration training in the second week using a training cage equipped with two nose-pokes. Training utilized a fixed-ratio 1 (FR1) reinforcement schedule. Following active nose-pokes, rats received a single METH injection (0.05 mg / kg). Each injection consisted of a discrete conditioned cue (CS) consisting of a 5-second light and a noise from an injection pump. Following the injection, a 20-second pause period was imposed, during which responses did not produce any programmed consequences, but were recorded. Training continued for 4 hours daily for 10 days to achieve a stable level of responding activity.

[0042] 4. Impact of EP on METH Strengthening

[0043] The purpose of this study was to evaluate the effects of EP on METH reinforcement under a FR1 schedule. After 10 days of training, rats self-administering METH were randomly divided into three groups of six. 30 minutes before the start of the experiment, EP was administered at three doses: 0 mg / kg, 50 mg / kg, and 80 mg / kg, all via intraperitoneal injection.

[0044] like Figure 1 China A and Figure 1As shown in middle B, intraperitoneal injection of 50 mg / kg and 80 mg / kg EP reduced the number of active nose pokes (F(2,15)=25.36, p<0.0001) and the number of injections (F(2,15)=34.67, p<0.0001) in rats self-administering METH.

[0045] 5. Impact of EP on METH Reward Motivation

[0046] This experiment aimed to assess METH motivation by determining breakpoints using a progressive ratio (PR) procedure, which measures the maximum effort rats are willing to exert in pursuit of a reward. Training continued under a FR1 schedule until behavioral responses stabilized. Rats were randomly assigned to receive an intraperitoneal injection of 0, 50, or 80 mg / kg of EP. Thirty minutes later, motivation was tested for 4 hours under a PR schedule.

[0047] The test results under PR program are as follows Figure 2 China A and Figure 2 As shown in Figure B, 80 mg / kg EP inhibited METH craving, reducing the breakpoint (F(2,15)=23.78, p<0.0001) and the final ratio (F(2,15)=12.30, p=0.0007). The reward motivation test employed a progressive ratio (PR) procedure, which measures reward motivation and craving by increasing the difficulty of obtaining the drug. Specifically, the rats were required to make an exponentially increasing number of valid touches to obtain a single methamphetamine injection. The final ratio was calculated as: response ratio = [5e(number of injections × j)] - 5. A higher proportionality factor (j) indicates a higher number of nose touches required to obtain a single methamphetamine injection. For example, the first dose required one valid nose touch, the second required two valid nose touches, and the third required four valid nose touches. That is, according to the increasing ratio, the rat needed to complete a corresponding number of valid nose touches to receive one dose of methamphetamine: 1, 2, 4, 5, 8, 11, 15, 20, 24, 32, 40, 49, 62, 77, 95, 116, 145, 178, 209, 268, 329, 402, 482, 603, etc. During the testing period, if the rat did not reach the required number of valid nose touches within one hour to receive one dose of the drug, the program would automatically terminate. The number of methamphetamine doses the rat received at the end of the program was called the breakpoint, and the number of valid nose touches the rat completed before receiving the last dose of methamphetamine was called the final ratio.

[0048] 6. Effects of EP on Multiple-Dose METH Self-Administration

[0049] Rats continued METH self-administration training under a FR1 schedule until their behavioral responses stabilized. Subsequently, self-administration training with different doses of METH (ranging from 0 to 0.2 mg / kg, in increments of 0.003125, 0.00625, 0.0125, 0.025, 0.05, and 0.1 mg / kg) was performed. Training under the multiple-dose METH schedule began with a 25-minute extinction trial (0 mg / kg / METH), followed by seven 25-minute trials in which different doses of METH were administered in ascending order; each trial was separated by a 5-minute interval, for a total duration of 4 hours. After self-administration behavior stabilized again, the rats were randomly divided into three groups of six. They were then given an intraperitoneal injection of EP at doses of 0, 50, or 80 mg / kg, and behavioral testing began 30 minutes later.

[0050] The results are as follows Figure 3 As shown in the figure, it was confirmed that EP could significantly shift down the dose-response curve of METH (F(2,120)=1.845, p=0.0395).

[0051] VII. Western Blot

[0052] Brain tissue protein was extracted using lysis buffer at 4°C, and protein concentration was determined using the BCA assay (Beyotime). Proteins were then separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 3% BSA-TBST for 1 hour and then incubated overnight at 4°C on a shaker. Primary antibodies included: β-actin (1:5000; AC026; ABclonal), rabbit monoclonal HMGB1 (1:1000; ab79823; Abcam), rabbit polyclonal TLR4 (1:250; PA5-23124; Invitrogen), mouse monoclonal MyD88 (1:400; sc-74532; Santa Cruz), rabbit monoclonal Phospho-NF-κB p65 (1:500; MA5-15160; Invitrogen), rabbit monoclonal NF-κB p65 (1:500; 8242; CST), rabbit PI3K (1:1000; 4292; CST), rabbit monoclonal Phospho-AKT (1:2000; 4060; CST), rabbit monoclonal AKT (1:1000; 4685; CST), mouse monoclonal IL-6 (1:1000; ab9324; Abcam), rabbit monoclonal Caspase-1 (1:1000; 83383; CST), and rabbit monoclonal Caspase-3 (1:2000; ab184787; Abcam). The cells were washed three times with TBST for 10 minutes each. Secondary antibodies were incubated at room temperature for 1 hour. Protein bands were detected using a chemiluminescence imaging system (Tanon-5300M), and protein analysis and quantification were performed using ImageJ.

[0053] The results are as follows Figure 4 and Figure 5As shown in the results, it was found that intraperitoneal injection of 50 mg / kg and 80 mg / kg EP reduced the expression levels of HMGB1 (F(3,8)=12.15, p=0.0024), TLR4 (F(3,8)=98.98, p<0.0001), MyD88 (F(3,8)=19.93, p=0.0005), PI3K (F(3,8)=9.075, p=0.0059), p-AKT (F(3,8)=19.22, p=0.0005), p-NFKB (F(3,8)=17.22, p=0.0008) and pro-inflammatory cytokine IL-6 (F(3,8)=134.7, p<0.0001) in the mPFC of rats self-administered with METH. At the same time, intracavitary injection of 50 mg / kg and 80 mg / kg EP reduced the protein levels of cleaved-caspase1 (F(3,8)=16.18, p=0.0009) and cleaved-caspase3 (F(3,8)=26.87, p=0.0002) in the mPFC of rats self-administered with METH, inhibiting pyroptosis and apoptosis.

[0054] 8. Immunofluorescence

[0055] Rats were deeply anesthetized and transcardially perfused with pre-cooled saline followed by 4% paraformaldehyde. The brains were post-fixed and sectioned into 5 μm thick paraffin sections for further processing. After dewaxing and antigen retrieval, the sections were blocked with 3% bovine serum albumin and then incubated with primary antibodies overnight at 4°C. The primary antibodies included rabbit polyclonal TLR4, mouse polyclonal NeuN, mouse monoclonal CaMKII α, mouse monoclonal Parvalbumin, rat monoclonal somatostatin SST, mouse monoclonal IBA1, rabbit monoclonal IBA1, and rabbit monoclonal HMGB1. Secondary antibodies were Alexa Fluor TM 488 goat anti-mouse IgG (H+L), Alexa Fluor TM 594 goat anti-rabbit IgG (H+L) and Alexa Fluor TM Plus 488 goat anti-rat IgG (H+L). Use DAPI to label cell nuclei.

[0056] The results are as follows Figures 6 to 10 As shown in Figure 3, it was confirmed that intraperitoneal injection of 50mg / kg and 80mg / kg EP significantly reduced TLR4 + The number of neurons (e.g. Figure 6 (F(3,8)=85.47, p<0.0001); reversed the METH-induced crosstalk between microglia and neurons (as shown in Figure 7 (F(3,8)=27.96, p=0.0001); reversed the METH-induced microglial and SST+ Neuronal crosstalk (F(3,8)=30.25, p=0.0001)) (e.g. Figure 8 Reversal of TLR4-positive SST + The increase in neurons (F(3,8)=111.6, p<0.0001) (e.g. Figure 9 reversed METH-induced SST + Increased nuclear translocation of HMGB1 in neurons (e.g. Figure 10 This indicates that EP exerts neuroprotective effects by inhibiting the HMGB1-TLR4 pathway.

[0057] All measurements in this paper were performed using independent samples. Data are presented as mean ± SEM (standard error of the mean). Statistical analysis was performed using GraphPad Prism version 9. The Shapiro-Wilk test was used to assess normal distribution of the data. If the data followed a normal distribution, the means of the independent sample groups were compared using a t-test or one-way analysis of variance (ANOVA); otherwise, nonparametric tests were used. The significance level for all tests was set at 0.05.

[0058] The present invention provides a study on the neuroprotective effect of EP in a rat METH self-administration model and its regulation of the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway. It is demonstrated that EP has strong targeting properties. By targeting the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway, EP regulates neuroinflammatory responses and precisely intervenes in the neural mechanisms of METH addiction. It is demonstrated that EP has a significant neuroprotective effect. EP has a significant neuroprotective effect, can reduce nerve damage, and promote nerve repair. It is demonstrated that EP has a significant inhibitory effect on addictive behavior. By regulating the neuroinflammatory response, EP significantly inhibits the addictive behavior caused by METH, and has great therapeutic potential.

[0059] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. The use of ethyl pyruvate in preparing a drug for treating or preventing methamphetamine addiction, characterized in that: The ethyl pyruvate reduces methamphetamine-induced neural damage, promotes neural repair, and inhibits methamphetamine addiction behavior by targeted inhibition of the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway. The ethyl pyruvate exerts a neuroprotective effect by inhibiting the HMGB1-TLR4 pathway, reduces methamphetamine-induced neural damage, promotes neural repair, and inhibits methamphetamine addiction behavior.

2. The application according to claim 1, characterized in that The ethyl pyruvate is administered via intraperitoneal injection at a dosage of 50-80 mg / kg.

3. The use of ethyl pyruvate in the preparation of a medicament for repairing nerve damage caused by methamphetamine, characterized in that: The ethyl pyruvate exerts a neuroprotective effect by inhibiting the HMGB1-TLR4 pathway, alleviating nerve damage caused by methamphetamine and promoting nerve repair.

4. The use of ethyl pyruvate in the preparation of methamphetamine addiction behavior inhibitory drugs, characterized in that: The ethyl pyruvate inhibits methamphetamine addiction behavior by targeting and inhibiting the HMGB1-TLR4-MyD88-PI3K-AKT-NF-KB signaling pathway.

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