Application of inhibiting outer GABAergic neurons to improve elemene-induced pain and anxiety

Abstract

A behavioral test proves that treatment of a chemotherapeutic drug elemene can cause hyperalgesia of a mouse along with anxiety-like mood; according to the present invention, it is found that the elemene treatment activates several brain regions including a lateral septum (LS), a cingulate cortex (ACC), a paraventricular nucleus (PVT), and a hypothalamic dorsal medial nucleus (DMH), the most significant one of the GABA energy neurons of the LS, the most significant one of the GABA energy neurons of the LS, the most significant one of the GABA energy neurons of the LS, the most significant one of the GABA energy neurons of the LS, the most significant one of the GABA energy neurons of the LS, and the most significant one of the GABA energy neurons of the LS, the most significant one of the GABA energy neurons of the LS, the most significant one of the GABA energy neurons of the LS, the Chemical genetic inhibition and apoptosis of LS neurons significantly reduce mouse pain and anxiety behaviors caused by elemene treatment, and a foundation is laid for clinical application. According to the application, the influence of elemene-induced chemotherapy-induced peripheral neuropathy and anxiety is researched from the perspective of relieving the side effect of the medicine, and the research finds that inhibition of LS brain region GABAergic neurons has an improvement effect in the aspects of chemotherapy-induced peripheral neuropathy and anxiety, and the life quality of cancer patients after treatment can be better improved possibly.

Description

Technical Field

[0001] The present invention relates to the application of inhibiting lateral septal GABAergic neurons to improve pain and anxiety-like behaviors induced by elemene. Background Art

[0002] Pain is defined as unpleasant sensory and emotional experiences associated with or similar to possible or actual tissue damage. Among them, pain can be divided into two different types: acute pain, which lasts less than three months and is usually triggered by harmful stimuli caused by injury, disease process, or abnormal function of muscles or internal organs; chronic pain, which lasts longer than the expected course of the acute disease process and is usually characterized by pain lasting more than six months. According to the "China Pain Medicine Development Report (2020)", the number of chronic pain patients in China exceeds 300 million, and is increasing at a rate of 10 to 20 million per year. As the third largest health problem after cardiovascular and cerebrovascular diseases and tumors, pain significantly affects the health and quality of life of individuals. However, there is still no treatment that can eliminate chronic pain for everyone.

[0003] Cancer-related pain has been widely studied, but the discomfort caused by tumor treatment (such as chemotherapy) is often overlooked. Even after the tumor is cured or in remission, this discomfort may last longer or even become chronic. The most common and significant adverse effect of chemotherapy is chemotherapy-induced peripheral neuropathy (CIPN). About 20% of patients receiving standard-dose chemotherapy drugs and almost 100% of patients receiving high-dose chemotherapy drugs will develop CIPN. CIPN is a common complication of complex anti-tumor treatment. The neuropathic pain secondary to it is the main source of influence on patients' daily life and significantly reduces the quality of life of cancer patients. Long-term use of chemotherapy drugs such as platinum, paclitaxel and vinblastine can cause CIPN. These chemotherapy drugs cause CIPN related to specific brain regions, including the periaqueductal gray matter, thalamus, anterior cingulate cortex (ACC), thalamus, secondary somatosensory cortex, and sometimes less clear areas such as the "frontal cortex" or the entire cortex. For example, studies have found that paclitaxel treatment activates the prefrontal cortex, amygdala, hippocampus, hypothalamus, and striatum. Although some potential causes have been discovered, there is still no quick solution or effective drug. Creating neuroprotective drugs and innovative therapies with reduced toxicity profiles will help to better understand the pathophysiology of chemotherapy pain and the regulatory brain regions associated with chemotherapy pain propensity.

[0004] Elemene is the main active ingredient extracted from the rhizome of Curcuma australis and is a representative natural product with broad antitumor activity. Elemene injection is a type II non-cytotoxic antitumor drug independently developed in my country, with significant antitumor efficacy and mild side effects. It has been reported that elemene injection can cause adverse reactions during treatment, including pain, fever, severe chest pain, dyspnea, and wheezing, among which pain and fever are the most common. However, whether elemene can cause CIPN in tumor treatment like other chemotherapy drugs has not been reported yet.

[0005] CIPN is a common complication of anti-tumor treatment, which seriously affects the quality of life of patients and there are currently no effective treatment drugs and methods. Understanding the pathophysiology of chemotherapy pain and the regulatory brain areas related to chemotherapy pain tendency and their specific mechanisms of action on pain will help to discover specific drugs and therapies. As an anticancer drug with good anti-tumor effects and few side effects, inhibiting its side effects can help to better improve the cancer treatment effect and quality of life of patients after treatment, but its side effects in CIPN are not clear and the treatment methods in this regard are still unclear. ① The present invention proves through behavioral tests that the treatment of the chemotherapy drug elemene can cause hyperalgesia in mice accompanied by anxiety-like emotions;

[0006] ② The present invention found that elemene treatment activated several brain regions including the lateral septum (LS), cingulate cortex (ACC), paraventricular nucleus of the thalamus (PVT) and dorsomedial hypothalamic nucleus (DMH), among which the most significant was the GABA neurons of the LS; ③ Chemical genetic inhibition and apoptosis of LS neurons significantly reduced the pain and anxiety-like behaviors of mice induced by elemene treatment, laying the foundation for clinical application. From the perspective of alleviating drug side effects, the present invention clarifies the role of inhibiting GABA neurons in the LS brain region in alleviating CIPN, so as to better improve the quality of life of cancer patients after treatment. Summary of the invention

[0007] The first objective of the present invention is to address the deficiencies of the prior art and provide an application of elemene as a CIPN drug or treatment method by inhibiting GABAergic neurons in the LS region of the brain.

[0008] The second objective of the present invention is to provide the use of GABAergic neurons in the LS region of the brain as a target-specific drug or therapeutic method for preventing and treating elemene CIPN.

[0009] The third object of the present invention is to provide a drug or treatment method for treating or preventing elemene CIPN, including GABAergic neurons in the LS region of the brain as target specificity.

[0010] Preferably, the dosage form of the drug is any medically approved dosage form.

[0011] Preferably, a pharmaceutically acceptable carrier is also included.

[0012] The invention injects elemene into the abdominal cavity of mice, measures the pain domain by mechanical pain and thermal pain to evaluate the pain hypersensitivity of the mice, and evaluates the anxiety of the mice by open field and cross maze experiments. The invention treats Fos-Cre with tamoxifen and elemene. ERT2 :Ai14;GAD67-GFP mice were used and immunofluorescence was used to explore the brain areas and specific neuron types of neuron activation; Gi(hM4Di) virus or Casp3 virus was injected into the LS stereotaxically to inhibit or apoptose the GABAergic neurons of the LS through chemical genetic means, and pain hypersensitivity and anxiety were detected behaviorally.

[0013] Preferably, mice are housed under standard laboratory conditions (temperature, 22±2°C; humidity, 50-60%) at the Experimental Animal Center of Hangzhou Normal University (HSD 20210903).

[0014] Preferably, the experimental mice are all 6-8 weeks old adult mice.

[0015] The present invention studies the effect of elemene on hyperalgesia and anxiety in mice and its inhibitory mode. The study found that after intraperitoneal injection of elemene, the hyperalgesia and anxiety of mice increased and this aggravation activated the GABAergic neurons of LS. In addition, the hyperalgesia and anxiety of mice can be alleviated by chemical genetic inhibition or apoptosis of such neurons in the LS brain region. Therefore, the present invention can provide a new target for elemene CIPN, and can also provide new ideas for the development of drugs and treatment methods for CIPN of other drugs and even chronic pain.

[0016] Therefore, the present invention has the following beneficial effects:

[0017] (1) It inhibits GABAergic neurons in the LS brain region and has a short onset of action;

[0018] (2) High success rate and good results;

[0019] (3) This study not only provides a new target for the treatment of CIPN caused by elemene, but also provides new ideas for the development of drugs for CIPN and chronic pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The effect of intraperitoneal injection of elemene on hyperalgesia and anxiety in mice. Figure A is a schematic diagram of the experimental process; Figures B and C are the latency and threshold analysis of abnormal pain and heat pain paw withdrawal in the control group and elemene group mice (n=13, two-way RM ANOVA); Figure D is a display of the results of the open field test in the control group and elemene group mice; Figure E is a statistical diagram of the time in the center of the open field test (n=12, two-way RM ANOVA); Figure F is the analysis and statistics of the distance to the center area of ​​the open field test (n=12, t-test); Figure G is the analysis and statistics of entering the center area in the open field test; Figure H is the analysis and statistics of the total mileage in the open field test (n=12, t-test); Figure I is a representative trajectory of the elevated plus maze test; Figure J is the analysis and statistics of the open arm time in the elevated plus maze test (n=12, t-test); Figure K is the analysis and statistics of the closed arm time in the elevated plus maze test (n=12, t-test); Figure L is the analysis and statistics of entering the open arm in the elevated plus maze test (n=12, t-test); Figure M is the analysis and statistics of the total mileage in the elevated plus maze test (n=12, t-test). Data are mean ± standard error, bw, body weight; ip, intraperitoneal injection; ns, not statistically significant. *P<0.05, **P<0.01.

[0021] Figure 2 Figure 1 shows the activated brain regions and specific neuron types in mice treated with the chemotherapy drug elemene. ERT2 : Typical diagram of Ai14 mouse gene identification; Figure B shows Fos-Cre in control and elemene-treated mice ERT2 : Representative images of c-fos (red) in the ACC and LS of Ai14 mice. Scale bars, 1 mm (left), 50 μm (right); Panel C shows Fos-Cre in control and elemene-treated mice. ERT2: Representative images of c-fos (red) in the PVT and DMH of Ai14 mice. Scale bar, 1 mm (left), 50 μm (right); Panel D is the density analysis of c-fos+ cells in ACC, LS, PVT and DMH (n=7, two-way RM ANOVA); Panel E is the representative immunostaining image of c-fos in the LS of wild-type mice treated with control or elemene. Scale bar, 1 mm (left), 50 μm (right); Panel F is the representative immunostaining image of c-fos (green) and GAD 67 (magenta) or c-fos (green) and CaMK IIα (magenta) in the LS of elemene-treated mice. Scale bar, 1 mm (left), 50 μm (right); Figure G is a statistical analysis of the density of CaMKⅡα / c-fos+ and GAD 67+ / c-fos+ cells in LS (n=6, t-test); Figure H is a representative image of the genotyping of GAD 67-GFP mice; Figure I is a representative image of Fos-Cre treated with elemene ERT2 : Representative immunostaining images of c-fos (red), GAD 67 (green) and CaMK IIα (magenta) in the LS of Ai 14:GAD 67-GFP mice; Figure J is a density analysis statistical graph of CaMK IIα / c-fos+ and GAD 67+ / c-fos+ cells in the LS of Fos-CreERT 2:Ai 14:GAD 67-GFP mice (n=9, t-test). Data are mean ± standard error, **P<0.01, ****P<0.0001.

[0022] Figure 3Chemical genetic inhibition or apoptosis of LS neurons alleviated elemene-induced pain and anxiety-like behavior in mice. Wherein: Figure A is a schematic diagram of the experimental process; Figure B is a typical diagram of viral expression and c-fos activation after the control virus pAAV-hsyn-MCS-EGFP-3FLAG, neuron inhibition virus pAAV-hsyn-HA-hM 4D(Gi)-IRES-mCitrine-WPRE and apoptosis virus pAAV-hsyn-taCasp 3-T2 A-TEVP-P2A-EGFP-3FLAG were injected into the LS. Scale bar, 1mm (left), 50μm (right); Figure C, D are allodynia and heat pain paw withdrawal threshold and latency analysis of control mice, Gi-injected mice and Casp3-injected mice (n=5, two-way RM ANOVA); Figure E is a representative track of the open field test of control mice, Gi-injected mice and Casp3-injected mice. Figure F is a statistical graph of the time spent in the center area of ​​the open field test (n=9, ordinary one-way ANOVA); Figure G is an analytical statistical graph of the distance to the center area of ​​the open field test (n=9, ordinary one-way ANOVA); Figure H is an analytical statistical graph of entering the center area in the open field test; Figure I is an analytical statistical graph of the total mileage in the open field test (n=9, ordinary one-way ANOVA); Figure J is a representative trajectory of the elevated plus maze test of control mice, Gi-injected mice, and Casp3-injected mice; Figure K is an analytical statistical graph of the open arm time in the elevated plus maze test (n=9, ordinary one-way ANOVA); Figure L is an analytical statistical graph of the closed arm time in the elevated plus maze test (n=9, ordinary one-way ANOVA); Figure M is an analytical statistical graph of entering the open arm in the elevated plus maze test (n=9, ordinary one-way ANOVA); Figure N is the total mileage analysis statistics in the elevated plus maze test (n = 9, ordinary one-way ANOVA). Data are mean ± standard error, *P < 0.05, **P < 0.01. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. The following description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without having to go through creative work. Therefore, the present invention is not limited to the following embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

[0024] Example 1: Effects of intraperitoneal injection of elemene on hyperalgesia and anxiety in mice

[0025] 1) In order to detect the effect of elemene on pain perception, a mechanical allodynia test was performed. The specific operation is as follows: the test animal is placed on a raised grid and covered with a transparent plastic box. The calibrated von Frey filament stimulates the middle of the plantar surface of each mouse paw until the filament bends to 45°. A rapid withdrawal or withdrawal is considered a positive response. Paw lifting caused by normal motor behavior is ignored. In the absence of a response, the next force is applied. In the case of a response, the next lower force filament is used. The tactile stimulus that produces a 50% probability of withdrawal is calculated and used as the paw withdrawal threshold (PWT);

[0026] 2) In order to detect the effect of elemene on pain perception, a thermal allergy test was performed. The specific operation is as follows: Before the experiment, each mouse was adapted to a plexiglass cube for 30 minutes for 3 days. When the animals were calm and awake, the thermal pain threshold was measured. Focal radiant heat (20W power) was applied to the central area of ​​the hind paw to measure the withdrawal latency (PWL) of the hind paw under thermal stimulation. The PWL of each mouse was measured three times with a minimum interval of 5 minutes, and the average value was recorded. In order to avoid tissue damage, the cutoff time was 20s;

[0027] 3) In order to further detect the effect of elemene on anxiety, an open field test was performed. The specific operation was as follows: each mouse was placed in an open field chamber of 50cm×50cm×50cm and allowed to explore freely for 10min. The area was divided into 9 quadrants (1 central quadrant and 8 peripheral quadrants). The time, entry, center area distance, and total travel distance during the test were recorded using a digital camera and measured using the Animal Movement Tracking System XT 12 (Noldus). After each exploration, the device was cleaned with 75% ethanol;

[0028] 4) In order to further detect the effect of elemene on anxiety, an elevated plus maze test was performed. The specific operation is as follows: the maze apparatus consists of a "+"-shaped maze located above the floor, two closed arms in opposite positions, two open arms in opposite positions, and a central area (each arm is 5 cm × 30 cm, and the wall height of the closed arm is 15 cm). Each mouse was placed in the central area and allowed to freely explore the maze for 5 minutes, which was recorded with a digital camera. The movement was tracked and analyzed using ANY-maze software (Stoelting, Wooddale, IL 60191, USA). After each exploration, the device was cleaned with 75% ethanol.

[0029] Example 2: Activated brain regions and specific neuronal types in mice treated with the chemotherapy drug elemene

[0030] 1) Genotype identification Determine the desired transgenic mice: The genotype of Fos-CreERT2: Ai 14 mice was determined by PCR using the following oligonucleotides: forward primer for wild-type fos gene: 5′-GTCCGGTTCCTTCTATGCAG-3′, primer for normal fos gene: 5′-GAACCTTCGAGGGAAGACG-3′, forward primer for mutant fos gene: 5′-CCTTGCAAAAGTATTACATCACG-3′. This primer amplifies a 357-bp fragment specific to the wild-type allele and generates a 232-bp fragment specific to the mutant allele. The forward primer for the wild-type Ai14 gene is 5′-AAGGGAGCTGCAGTGGAGTA-3′, and the forward primer for the mutant Ai14 gene is 5′-CTGTTCCTGTAGGGCATGG-3′. The primers amplified a 297-bp fragment specific for the wild-type allele and produced a 196-bp fragment specific for the mutant allele. The genotype of the GAD 67-GFP mouse was determined by PCR using the following oligonucleotides: primer #1 (5′-GGCACAGCTCTCCCTTCTGTTTGC-3′) corresponding to intron 0b of the GAD 67 gene, primer #2 (5′-GCTCTCCTTTCGCGTTCCGACAG-3′) corresponding to intron 1 of the GAD 67 gene, and primer #3 (5′-CTGCTTGTCGGCCATGATATAGACG-3′) corresponding to the EGFP gene. Primer sets #1 and #2 amplified a 265-bp fragment specific for the wild-type allele, and primer sets #1 and #3 produced a 564-bp fragment specific for the mutant allele.

[0031] 2) Immunofluorescence experiments to detect activated brain areas and neuronal types: Mice were deeply anesthetized by inhalation of isoflurane and perfused transcardially with 0.9% saline and then 4% paraformaldehyde (PFA) in 0.01M phosphate buffer (PBS) (pH 7.4). After removal from the skull, the brain was postfixed in 4% PFA for 4-6 hours and dehydrated in a graded sucrose series (20% to 30%). After embedding in the optimal cutting temperature compound, the brain was coronally sliced ​​to 20 μm using a freezing microtome (Thermo, CryoStar NX 50OP). For c-fos, CaMKIIα and GAD 67 immunofluorescence staining, the sections were rinsed with 0.5% Triton-X in 0.01M PBS and blocked with 5% BSA for 1 h at room temperature. The sections were then incubated with primary antibodies (rabbit anti-c-fos, 1:500, ab 222699; mouse anti-CaMKIIα, 1:200, MA 1 -048; mouse anti-GAD 67, 1:500, MAB 5406) ​​in 5% BSA at 4°C overnight. The sections were then rinsed and incubated with secondary antibodies for 1 hour at room temperature. After three washes in PBS, the slides were mounted and covered with coverslips. Images were captured using a fluorescence microscope (Olympus VS200);

[0032] like Figure 2 , elemene treatment activated GABAergic neurons including LS, ACC, PVT, and DMH, and mainly activated LS.

[0033] Example 3: Chemical genetic inhibition or apoptosis of LS neurons alleviates elemene-induced pain and anxiety-like behaviors in mice

[0034] In order to detect the effect of inhibiting GABAergic neurons in the LS brain region on elemene-induced hyperalgesia and anxiety in mice, the neurons in the LS brain region were inhibited by chemical genetics and behavioral tests were performed on mice. The specific operations were as follows:

[0035] 1) Stereotaxic injection of virus: Virus injection was performed as described previously

[49] . Briefly, for intracranial virus injection, mice were anesthetized with isoflurane, placed in a stereotaxic apparatus (RWD, Shenzhen, China), and injected with 0.20 μL of pAAV-SYN-MCS-EGFP-3FLAG (2.43E+13 V.G. / mL), pAAV-SYN-taCasp3-T2A-TEVP-P2A-EGFP-3FLAG (5.25E+12 V.G. / mL), pAAV-hSYN-HA-hM4D(Gi)-IRES-mCitrine-WPRE (9.40 x 10E13 VG / mL) virus solution was injected into the LS (0.7 mm anterior-posterior (AP), ±0.5 mm medial-lateral (ML), ~3.5 mm dorsal-ventral (DV) from bregma) at a rate of 0.02 μL / min through a 1 μL Hamilton microinjector (Hamilton, NV, USA) for 10 min. After injection, the needle was left in place for another 10 minutes and then slowly withdrawn to minimize the spread of the virus solution. Three weeks later, clozapine N-oxide (CNO) was injected intraperitoneally to activate the Gi virus. All viruses were purchased from Oobio Corporation (Shanghai, China) and histological verification was performed at the injection site after behavioral experiments.

[0036] 2) After the virus took effect, mice were intraperitoneally injected with elemene and their hyperalgesia and anxiety were tested by mechanical allodynia test, thermal allodynia test, open field test, and elevated plus maze;

[0037] like Figure 3 As shown, after inhibiting the neurons in the LS brain area, the hyperalgesia and anxiety caused by elemene in mice were improved.

[0038] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0039] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention. In summary, the present invention found that GABA neurons in the LS brain region play a promoting role in the hyperalgesia and anxiety induced by elemene in mice, providing a new possible target for the treatment of CIPN, which can provide new ideas for the treatment of CIPN and chronic pain.

Claims

1. Application of LS brain region neurons as targets in the preparation of drugs or treatment methods for the treatment of elemene-induced peripheral neuropathy and anxiety.

2. The use according to claim 1, characterized in that Inhibition of GABAergic neurons in the LS brain region can alleviate elemene-induced peripheral neuropathy and anxiety.

3. Application of GABAergic neurons in the LS brain region as targets in the preparation of drugs or treatments for preventing and treating elemene-induced peripheral neuropathy and anxiety.

4. A drug or method for treating or preventing elemene-induced peripheral neuropathy and anxiety, characterized in that Including targeting GABAergic neurons in the LS brain area.

5. A drug or method for treating or preventing elemene-induced peripheral neuropathy and anxiety according to claim 1, characterized in that The dosage form of the drug is any dosage form recognized medically.

6. A drug or method for treating or preventing elemene-induced peripheral neuropathy and anxiety according to claim 1, characterized in that Also included are pharmaceutically acceptable carriers.

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