阅读说明：本技术 Bapta-am在制备镇痛药物中的应用 (Application of BAPTA-AM in preparing analgesic ) 是由 曹君利 杨俊霞 丁海雷 张红星 姜彦羽 于 2021-04-08 设计创作，主要内容包括：本发明公开一种BAPTA-AM在制备镇痛药物中的应用,属于医药技术领域,所述的镇痛药物是抑制神经病理性疼痛的药物。本发明将BAPTA-AM定位注射于神经病理性疼痛小鼠前扣带回,可明显缓解疼痛。本发明采用行为学实验和分子生物学实验相结合的方法,充分证明BAPTA-AM具有明显镇痛作用。(The invention discloses an application of BAPTA-AM in preparing analgesic drugs, belonging to the technical field of medicines. The BAPTA-AM is injected into the anterior cingulum of a mouse with neuropathic pain in a positioning way, so that the pain can be obviously relieved. The invention adopts a method combining behavioral experiments and molecular biological experiments to fully prove that the BAPTA-AM has obvious analgesic effect.)

1. An application of BAPTA-AM in preparing analgesic is provided.

2. The use according to claim 1, wherein said analgesic agent is an agent that inhibits neuropathic pain.

3. The use of claim 2, wherein the medicament is a medicament prepared from BAPTA-AM as an active ingredient, together with pharmaceutically acceptable excipients.

4. The use according to claim 3, wherein said medicament is an injectable formulation.

5. The use of claim 4, wherein the injectable formulation comprises a solution of BAPTA-AM for administration by injection.

6. The use according to claim 5, wherein the BAPTA-AM solution has a concentration of 1 mM.

7. Use according to claim 3, characterized in that the adjuvant used is DMAO.

Technical Field

The invention relates to an application of BAPTA-AM in preparing analgesic drugs, belonging to the technical field of medicines.

Background

Pain is a protective response of the body caused by external noxious stimuli, but after noxious stimuli such as inflammation, local nerve compression, trauma, etc. are removed, the pain symptoms are not necessarily removed, and even aggravated. Neuropathic pain, which is recognized as the king of chronic pain, is the most clinically intractable and almost insensitive to existing analgesics, and neuropathic pain of either peripheral or central origin continuously imposes a great burden on individuals and society, especially in terms of increased disability rate, decreased productivity and quality of life, and continuously increased medical and health investment. Therefore, the mechanism of the occurrence, development and maintenance of neuropathic pain is deeply researched, a theoretical basis can be provided for diagnosis and treatment of the neuropathic pain, and a new target point is searched for research and development of new drugs.

Clinical and basic neuroimaging have demonstrated that the Anterior Cingulated Cortices (ACCs) are involved in pain regulation. Whether it is noxious stimulation or is told that noxious stimulation is likely, ACC brain regions are activated. The ACC participates in addressing the unpleasant sensations of pain and facilitating the choices of pain-related emotions, cognition and reactions, in encoding pain sensation mood information, and combining pain sensation and cognitive information with mood information to form a complete pain experience. In the ACC region of neuropathic pain animals, the frequency and amplitude of EPSCs are increased, and the research of a subject group of the applicant shows that the scaffold protein caveolin 1(Cav-1) mediated NMDA receptor 2B subunit translocation (Trafficking) of an ACC neuron postsynaptic membrane and a related intracellular signaling pathway play an important role in the high excitability and pain regulation of ACC neurons in neuropathic pain (Yang JX, et. al., JNeurosci.2015). From the above studies, it was shown that the anterior cingulate gyrus has a very important role in the regulation of pain.

Studies have shown an increase in prokyglumic acid when the body is in a state of pain. Intracellular calcium increases effect exocytosis of synaptic vesicles and neurotransmitter release. Intracellular calcium ion elevation plays an important role in neurotransmitter release. BAPTA-AM is an intracellular calcium ion chelating agent, and the current research shows that the liposome loaded with BAPTA-AM can relieve the symptoms of liver failure by clearing intracellular calcium overload, and the nanoparticle of BAPTA-AM can be used for treating acute kidney injury caused by ischemia-reperfusion.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an application of BAPTA-AM in preparing analgesic drugs.

In order to achieve the purpose, the invention adopts the application of BAPTA-AM in preparing analgesic drugs.

As an improvement, the analgesic drug is a drug for inhibiting neuropathic pain.

As an improvement, the medicament is prepared by taking BAPTA-AM as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

As a refinement, the medicament is an injection preparation.

As a refinement, the injection preparation contains BAPTA-AM solution and is used for injection administration.

As a refinement, the concentration of the BAPTA-AM solution is 1 mM.

As an improvement, DMAO is adopted as an auxiliary material.

The principle of the invention is as follows: when the pain is neuropathic, the release of the glutamic acid of the anterior cingulum of the body is increased, the release of the glutamic acid needs to be triggered and maintained by the increase of intracellular calcium ions, and the release of the glutamic acid is reduced by the administration of the calcium ion chelating agent BAPTA-AM to the anterior cingulum, so that the aim of relieving the pain is fulfilled.

Compared with the prior art, the BAPTA-AM is locally injected into the anterior cingulum of a mouse with neuropathic pain, so that the pain can be obviously relieved. The invention adopts a method combining behavioral experiments and molecular biological experiments to fully prove that the BAPTA-AM has obvious analgesic effect.

Drawings

FIG. 1 is a graph showing the effect of BAPTA-AM of the present invention on the thermal pain behavioral characteristics of CCI mice (expressed as heat-shrinkable paw latency);

FIG. 2 shows the change of glutamate release after the local injection of BAPTA-AM of the present invention into CCI mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It should be understood, however, that the description herein of specific embodiments is only intended to illustrate the invention and not to limit the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in the specification of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention.

Example 1

1.1 reagents used in the invention:

BAPTA-AM; dimethyl sulfoxide (DMSO); balanced salt solution sigma company.

Glutamic acid detection kit: shanghai initiating test reagents, Inc.

Other chemicals were purchased from sigma, Inc., unless otherwise noted.

Experimental animals: the male Kunming mouse is a clean-grade mouse, 20-25 g and is provided by the experimental animal center of Xuzhou medical college.

1.2 preparation of neuropathic pain (CCI) model:

CCI surgery was performed according to the methods described by Bennett and Xie: mice were anesthetized with pentobarbital (40mg/kg, i.p.), the middle upper part of the right sciatic nerve was exposed (in all cases, the right sciatic nerve was ligated in this study), and three lines (1 mm apart) were ligated with 5-0 silk at the bifurcation of the sciatic nerve. The control group (sham) was exposed only and not ligated. The incision was sutured layer by layer and the wound treated with antibiotics. Previous studies have shown that pain caused by CCI can last at least months.

1.3 mouse ACC region stereotactic injection:

the mouse is anesthetized by pentobarbital (40mg/kg, i.p.), fixed on an electronic stereotaxic instrument special for the mouse, the position of an ear bar is noticed, the depth of the ear bars on two sides is fixed to enable a crisp click sound to be heard, and the height of the ear bar and the height of two incisors are adjusted to be on the same plane. Shearing hair, disinfecting head skin with iodophor, cutting skin at the position 0.5cm from the center of the back of two eyes, and dipping 3% H with cotton swab₂O₂Repeatedly wiping incision to destroy fascia and expose skull, finding bregma, adjusting needle position at 1.11mm before bregma and 0.25mm away, and calibrating to 0.5 μ lPrepared 0.1% DMAO and 0.1% DMAO solution containing 1mM BAPTA-AM are slowly injected into a position with the depth of 2.10mM by a micro-injection needle (the injection duration is 30 seconds), the needle is left for 10 minutes, the needle is slowly pulled out, the skin is sutured, iodophor is disinfected, and an antibiotic liquid is coated on the incision. The whole process is carried out until the animals recover, and the room temperature is kept at about 26 ℃.

1.4 thermal hyperalgesia assay:

mice were injected with 0.1% DMAO and 0.1% DMAO solution containing 1mM BAPTA-AM in a targeted manner in the ACC zone of sham and CCI groups for 7 days, and were subjected to behavioral tests before (0h) injection, 15min after injection, 0.5h, 1h, 2h and 4h, as follows: placing an organic glass box on a glass plate with the thickness of 3mm, and irradiating the sole of a rat to be tightly attached to the glass plate by using a thermal radiation stimulator according to a Hargreaves method. The time from the start of irradiation until the mice developed a paw lifting (cut-off time of 20s), i.e. paw withdrawal latency (longer paw withdrawal latency), indicating better pain relief. Each animal was assayed 3 times, and the mean value was taken, with 5min intervals between each assay. The results show that BAPTA-AM is effective 15 minutes after injection, with a significant pain relief effect within 2h, as shown in figure 1.

1.5 detection of glutamate Release from ACC region in neuropathic pain mice:

directly decapitating each group of mice, peeling off brain tissue on ice, taking 2.34mM before bregma and 0.22mM after bregma, bypassing 0.6mM cerebral cortex, namely brain tissue in an ACC area, washing the brain tissue for 3 times by using normal saline, hardening the brain tissue in the ice-cold normal saline for 3 minutes, cutting the brain tissue into 300 um/sheet by using a microtome, putting the cut brain tissue into prepared artificial cerebrospinal fluid containing 37 ℃ and 100mM Kcl, collecting supernatant after 30 minutes, centrifuging (12000rpm for 2 minutes), detecting the detection, and drying the tissue to be used as internal reference.

The glutamic acid assay was performed strictly according to the instructions of the Biovision kit K629-100, namely:

A) preparation of reagents: before detection, GlutamateAssay Buffer (glutamic acid detection Buffer) is put at room temperature, so that the detection result is prevented from being influenced by the temperature; glutamate Enzyme Mix (glutaminase Mix) was dissolved in 220. mu.l assay Buffer and 820. mu.l ofddH was added₂O-soluble Glutamate developer) Repeatedly sucking for dissolving, avoiding vortex oscillation, and putting on ice for later use; add 10. mu.l 0.1M glutamic acid standard solution into 990. mu.l assay Buffer to obtain 1mM glutamic acid standard solution;

B) adding 0, 2, 4, 6, 8 and 10 mu l of glutamic acid standard solution into each well of a 96-well plate to obtain the amount of 0, 2, 4, 6, 8 and 10 nmol/empty; complementing the volume of each hole to 50 mu l by using GlutamateAssay Buffer solution to obtain the concentration of 0, 0.04, 0.08, 0.12, 0.16 and 0.2 nmol/mu l of each hole, making a standard curve, and adding 50 mu l of a sample to be detected into each hole;

C) preparing reaction liquid according to the proportion: mu.l assay Buffer, 8. mu.l Glutamate Developer, 2. mu.l Glutamate Enzyme Mix (Glutamate Developer) per well; blank control was added 92. mu.l of assay Buffer, 8. mu.l of Glutamate Developer;

D) keeping away from light, detecting absorbance value at 450nm with enzyme labeling instrument at 37 deg.C for 30 min;

E) the glutamic acid content of each sample was calculated from the standard curve.

As demonstrated by the results in FIG. 2, the targeted injection of 0.1% DMAO solution containing 1mM BAPTA-AM in the pre-cingulated gyroglobus region of neuropathic pain mice reduced glutamate release.

The invention proves the application of BAPTA-AM in preparing the neuropathic pain analgesic drug, and the analgesic effect is realized by reducing the release of the proglenoid glutamic acid of a mouse with neuropathic pain.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.