阅读说明：本技术 一种clp257在制备防治焦虑的药物中的应用 (Application of CLP257 in preparation of anxiety prevention and treatment medicines ) 是由 丁海雷 房谈谈 杨俊霞 任昆鹏 银梦冰 林治华 李贺 江维 于 2021-10-26 设计创作，主要内容包括：本发明公开一种CLP257在制备防治焦虑的药物中的应用。本发明通过抑制发育期氯胺酮多次暴露小鼠腹侧海马CA1(vCA1)锥体神经元的兴奋性,从而发挥缓解焦虑的作用,且CLP257能够缓解焦虑行为是采用行为学实验、细胞学和分子生物学实验相结合的方法得到充分证明的。(The invention discloses an application of CLP257 in preparing a medicament for preventing and treating anxiety. The invention plays a role in relieving anxiety by inhibiting excitability of the pyramidal neurons of the ventral hippocampus CA1(vCA1) of mice exposed by ketamine for multiple times in the development period, and the fact that the CLP257 can relieve the anxiety behavior is fully proved by adopting a method of combining behavioral experiments, cytology and molecular biology experiments.)

1. An application of CLP257 in preparing medicine for preventing and treating anxiety is disclosed.

2. The use of CLP257 of claim 1, wherein the medicament is for the prevention and treatment of anxiety, resulting from multiple exposures of ketamine during development.

3. The use of CLP257 in preparing a medicament for preventing and treating anxiety according to claim 1, wherein the medicament is prepared from CLP257 as an active ingredient, and pharmaceutically acceptable adjuvants.

4. The use of CLP257 of claim 3, wherein the medicament is an injectable formulation.

5. The use of CLP257 of claim 4 in the preparation of a medicament for preventing or treating anxiety, wherein the injectable formulation comprises a solution of CLP 257.

6. The use of CLP257 of claim 5 in the preparation of a medicament for the prevention or treatment of anxiety, wherein the concentration of the CLP257 solution is 500. mu.M.

7. The use of CLP257 in preparing a medicament for preventing and treating anxiety according to claim 3, wherein the adjuvants used are physiological saline and dimethyl sulfoxide mixed at a volume ratio of 10: 1.

Technical Field

The invention relates to an application of CLP257 in preparing a medicament for preventing and treating anxiety.

Background

General anesthesia has the advantages of analgesia, sedation, anxiety relief and the like, and more infants and young children are subjected to operations under the general anesthesia condition globally. With the progress of research in recent years, it has been found that exposure to anesthesia during development may lead to impaired nerve function due to the particularity of the developing brain (neuronal division and differentiation, synaptogenesis, low rate of neuronal maturation, etc.). Ketamine (KTM) is widely used in anesthesia, analgesia and sedation of pediatric patients due to its characteristics of low cost, fast onset of action, long action time, and little influence on respiration and circulatory system. With the widespread clinical use of ketamine, multiple studies have found that developmental ketamine exposure may lead to anxiety.

Anxiety disorders are the most common psychopsychological disorders in clinical settings, and are characterized by widespread and persistent anxiety states, recurrent panic attacks, autonomic dysfunction, and motor restlessness, with moderate anxiety causing rapid response to possible risks, but persistent anxiety that is not proportional to actual risks may cause normal functional disturbances and result in significant social and personal burdens. At present, the medicines for treating anxiety disorder are few, the action targets are mainly concentrated on a catecholamine system, a serotonin (5-HT) system, a Norepinephrine (NE) system and the like, the treatment effect of the medicines aiming at the targets is not ideal, and a series of adverse reactions can be generated after long-term administration. To improve the treatment strategy for anxiety disorders, a thorough understanding of the underlying mechanisms of anxiety is needed.

The hippocampus, which is located between the thalamus and the medial temporal lobe, is part of the mesolimbic system and can be divided into CA1, CA2, CA3 and the dentate gyrus according to cell morphology and fibrous connectivity. The hippocampus can be divided into Dorsal (DH) and Ventral (VH) hippocampus according to their dorsal-ventral axes, which have been shown by previous anatomical studies to have large differences in input and output nerve projections, with the dorsal hippocampus projecting widely to the combined cortical areas, and the ventral hippocampus projecting to brain areas associated with autonomic nerves, neuroendocrine and mood regulation, such as the prefrontal cortex, amygdala and hypothalamus, among others, with several studies demonstrating that the ventral hippocampus CA1(vCA1) pyramidal neurons are positively correlated with anxiety.

Na⁺-K⁺-2Cl^-Cotransporter 1 (Na)⁺–K⁺–2Cl^-cotransporter isoform1, NKCC1) are widely distributed in a variety of tissues, including stomach, heart, skeletal muscle, lung, brain, and kidney, mediating Na⁺、K⁺And Cl^-Transport into the cell. K⁺-Cl^-Cotransporter 2 (K)⁺–Cl^-cotransporter isoform 2, KCC2) is abundantly expressed in most mature mammalian central neurons, mediating K⁺And Cl^-Transport to the outside of the cell. Multiple studies found that NKCC1 and KCC2 together modulate GABA function in the central nervous system. GABA_AReceptor (GABA)_ARs) are widely distributed among nerve receptors and are the major inhibitory receptors of the central nervous system. GABA_AThe receptor being ligand-gated Cl^-Channel, GABA agonizing the receptor causing Cl^-The channel is open. Activation of NKCC1 with intracellular Cl^-Increased concentration, and activation of KCC2 can result in intracellular Cl^-The concentration is reduced. Normal mature central neuron NKCC1/KCC2 ratio is less, and neuron intracellular Cl is less^-Lower concentration of GABA_AReceptor-modulated Cl^-Opening of the channel causes Cl^-Influx (neuronal hyperpolarization), GABA acts as an inhibitory neurotransmitter. Intracellular Cl when the NKCC1/KCC2 ratio is large^-Higher concentration of GABA_AReceptor-modulated Cl^-Cl when channel is open^-Efflux (neuronal depolarization), GABA converts excitatory neurotransmitters and it has been found that an increase in NKCC1/KCC2 leads to an increase in hippocampal pyramidal neuron excitability.

Multiple exposures of ketamine during development can cause an increase in the ventral hippocampus NKCC1/KCC2, and NKCC1/KCC2 lead to anxiety by increasing the excitability of vCA1 pyramidal neurons.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an application of CLP257 in preparing a medicament for preventing and treating anxiety.

In order to achieve the purpose, the invention adopts an application of CLP257 in preparing a medicament for preventing and treating anxiety.

As an improvement, the medicament for preventing and treating anxiety is a medicament for preventing and treating anxiety caused by multiple exposures of ketamine in a developmental period.

As an improvement, the medicament is prepared by taking CLP257 as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

As a refinement, the medicament is an injectable formulation.

As a refinement, the injectable formulation contains a CLP257 solution.

As a refinement, the concentration of the CLP257 solution is 500. mu.M.

As an improvement, the adopted auxiliary materials are normal saline and dimethyl sulfoxide which are mixed according to the volume ratio of 10: 1.

The principle of the invention is as follows: multiple exposures of ketamine during development can cause an increase in the ventral hippocampus NKCC1/KCC2, and NKCC1/KCC2 lead to anxiety by increasing the excitability of vCA1 pyramidal neurons. The KCC2 agonist CLP257 alleviates anxiety caused by multiple exposures of ketamine during development by inhibiting vCA1 pyramidal neurons.

Compared with the prior art, the method has the advantages that excitability of the hippocampus CA1(vCA1) pyramidal neurons of the ventral side of the mice exposed by ketamine for multiple times in the development period is inhibited, so that the effect of relieving anxiety is achieved, and the fact that the CLP257 can relieve the anxiety behavior is fully proved by adopting a method combining behavioral experiments, cytology experiments and molecular biology experiments.

Drawings

FIG. 1 shows the effect of vCA1 brain region administration of the KCC2 agonist CLP257 on anxiety-like behavior in mice exposed multiple times to ketamine during development;

FIG. 2 shows Westernblot to detect changes of mouse ventral hippocampus NKCC1/KCC2 after multiple exposure of ketamine in development period;

FIG. 3 shows the developmental time ketamine multiple exposure of vCA1 pyramidal neuronal excitability changes in mice;

FIG. 4 shows the effect of chemogenetic inhibition vCA1 of pyramidal neurons on anxiety-like behavior of mice exposed multiple times to ketamine during development;

FIG. 5 is a graph showing the effect of KCC2 agonist CLP257 on the excitability of vCA1 pyramidal neurons in mice exposed multiple times with ketamine during development.

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.

The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

Study animals: adult Kunming mice, offered by the laboratory animal center, Xuzhou university of medical. Mating the female mouse and the male mouse in a cage-combining way according to the male-female ratio of 2: 1-3: 1 to make the female mouse pregnant, and checking whether vaginal emboli exist in the morning on 2-6 days after cage-combining; the vaginal opening of the female mouse can see a milky vaginal suppository after pregnancy, and the pregnancy is recorded as 0.5(embryonic 0.5, E0.5); pregnant mice are naturally delivered, namely the birth day of newborn mice is marked as postnatal day 0 (postnatal day 0, P0), the newborn mice are fed with mother mice before weaning (namely P21-P24), and are fed with common feed after weaning.

Multiple ketamine exposure dosing method during development period: randomly dividing the Hokkenminn mice with P6 days into a normal saline group and a ketamine group according to a random digital table method, wherein the ketamine group is administrated by intraperitoneal injection (i.p), and the single dose is 100 mg/kg; the normal saline group is injected with normal saline with the same volume as the ketamine group into the abdominal cavity every time, and the injection is performed once at 12: 00-14: 00pm every day for six days continuously. After each injection, two groups of animals are respectively raised by using independent mouse cages, placed in a quiet air-conditioned room, and are enhanced to keep the temperature at 25 +/-1 ℃, the color change of the skin of the mouse is observed to prevent hypoxemia, and the mice are placed back to the original mouse cages after the positive reflection of the mouse is recovered.

Placing a tube in a brain area: the ketamine group and the normal saline group are quickly fixed on a Stoelting stereotaxic apparatus after anesthesia when the mice are P32 days. The criteria for fixation completion were judged to be no movement of the tail-lifted or moderate swing of the mouse brain sack and cranial level. Zero point was determined using bregma as a standard and the dosing cannula was implanted in a position according to the coordinates of region vCA1 (AP: -2.90, ML: + -3.25, DV: -4.20) of Kunming mouse day P32.

Elevated plus maze experiment: the brain was cannulated for 7 days prior to the elevated plus maze procedure, 1h prior to which KCC2 agonist CLP257(400nl, 500. mu.M, 100. mu.l/min) was cannulated. The experiment was carried out in an elevated plus maze 75cm from the ground, consisting of a pair of open arms (30cm long x 5cm wide x 18cm high), a pair of closed arms (30cm long x 5cm wide x 18cm high) and a central zone (5cm long x 5cm wide) where the two arms intersect. The experimental time is 08: 00-12: 00am in the morning of the test day, and the mice are placed in a quiet experimental environment to adapt for 30-60 minutes before the experiment. The mice were placed in the central area facing the open arm and allowed to move freely for 5 minutes during the test. The anxiety-like mood of the mice was evaluated by recording the number of times the mice entered the open arms (requiring both forepaws to enter them), the number of times the mice entered the closed arms, the open arm dwell time and distance, the closed arm dwell time and distance. After each mouse is finished, the bottom surface and the wall of the maze are wiped by 75% alcohol, so that the residual smell of the mouse is prevented from influencing the test result of the next mouse. As shown in FIG. 1, the percentage of open arm exploration time increased 107.14% (P < 0.01) and the percentage of entry times increased 38.19% (P < 0.05) in the KTM + CLP257 mice group, compared to the KTM + Vehicle group, suggesting that the KCC2 agonist CLP257 plays a role in anxiety reduction.

Western blot detection is carried out according to the conventional steps: p35 extracts mouse ventral hippocampus brain tissue protein, and the detection result is shown in figure 2, compared with the normal saline group, the expression of KCC2 of the ventral hippocampus of the ketamine group mice is reduced (P is less than 0.05), NKCC1 has no obvious change (P is more than 0.05), and NKCC1/KCC2 are increased (P is less than 0.01).

The complete operation flow of the immunofluorescence detection signal molecule: p35 day mice were anesthetized by intraperitoneal injection with chloral hydrate (350mg/kg), the limbs were fixed, the chest was opened to expose the heart, a scalp needle was inserted from the left ventricular apex to the ascending aorta, the right auricle was excised, and physiological saline (about 10ml) was infused until the liver became white and the fluid from the right auricle became clear, and then infused with 4% paraformaldehyde until the lower limbs and the tail became hard (about 20 ml). Cutting off the head, opening the skull, taking the whole brain, placing the whole brain in 4% paraformaldehyde at 4 ℃, fixing for 6-8 h, and then placing the brain tissue at 4 ℃. Transferring whole brain into phosphate buffer solution containing 20% and 30% sucrose in sequence for gradient dehydration, and placing in refrigerator at 4 deg.C until the tissue blocks sink, which can reduce ice crystal formation during rapid freezing and improve slicing quality. Trimming the brain tissue after sugar precipitation, placing on a precooled section base, embedding with OCT embedding gel, freezing and fixing at-20 ℃ (about 30min), adjusting the section thickness to 30 μm, and taking coronal continuous section. One section was selected and separated by two and one section was collected and placed in 0.01mol/L PBS. Washing the cut brain slices with 0.01M PBS on a shaker for three times (5 min/time); and sealed overnight. The cells were washed three more times with 0.01M PBS (5 min/time). Adding primary antibody at 4 deg.C for 48-72 hr. Wash three times with 0.01M PBS (5 min/time). Adding secondary antibody, and keeping the temperature at room temperature for 2 h. 0.01MPBS was washed three times (5 min/time). And sealing and taking a picture. The results are shown in FIG. 3A: compared with the saline group, the c-Fos expression positive neurons in the mouse pyramidal neurons of the ketamine group are increased by 83.33 percent (P is less than 0.0001), and multiple exposures of ketamine in the development stage result in the increase of the activation rate of the pyramidal neurons in the distant vCA1 area of the mouse.

In vitro patch clamp recording: after anesthesia of P35 day, the mouse was decapitated to take out brain, and the dissected brain tissue was placed in ice-cold high-sugar section solution and frozen for 5 min. Cutting the brain tissue containing vCA1 area into 300 μm thin slices with a vibrating microtome, placing the slices in a chamber with 95% O₂+5％CO₂Incubating the mixed gas high sugar slice solution in a water bath at 37 deg.C for 15min, and balancing in ACSF at room temperature for 30 min. The neuron spontaneous discharge frequency in the I-0 recording mode is measured by using drawn glass microelectrodes (7-12M omega) close to nerve cells (sealing resistance 80-120M omega) in the pyramidal cell layer in the vCA1 area. The multiclad 700B patch clamp amplifier is used for signal filtering and acquisition, and the Digidata 1440A digital-to-analog converter and pClamp 10.2 software are used for data acquisition and analysis. The results are shown in FIG. 3B: compared with the normal saline group, the spontaneous discharge frequency of the cone neurons in vCA1 area of the ketamine group mice is increased by 92.25% (P < 0.001), and multiple exposure of ketamine in the developmental stage leads to vCA1 area of cone neurons in the advanced stage of the miceThe excitability is increased.

Chemical genetic inhibition: mice in the ketamine group were quickly fixed on a Stoelting stereotaxic apparatus after anesthesia for day P25. The criteria for fixation completion were judged to be no movement of the tail-lifted or moderate swing of the mouse brain sack and cranial level. The zero point is determined by taking bregma as a standard, and chemogenetic suppression virus (rAAV2/9-CaMK IIalpha-hM 4Di-mCherry) or control virus (rAAV2/9-CaMK IIalpha-mCherry) is injected in a positioning way according to the coordinates (AP: -2.90, ML: +/-3.20, DV: -4.15) of vCA1 region of Kunming mouse at P25 day. Mice were raised to P46 days (3 weeks were required for full expression of the virus) for the elevated plus maze experiment, 30 minutes before which mice were injected intraperitoneally with CNO 1mg/kg in a single dose. The results are shown in figure 4, the pyramidal neurons in vCA1 region were chemogenetically inhibited, the percentage of time spent in the development period of ketamine multiple exposure mice entering the open arm exploration is increased by 143.36% (P < 0.01), the percentage of entering times is increased by 57.21% (P < 0.05), and the anxiety behavior of the mice is reduced, which indicates that the excitability and anxiety of the pyramidal neurons in vCA1 region are positively correlated.

Day P35 of ketamine group, isolated patch clamp recorded the spontaneous firing frequency of the pyramidal neurons in region vCA1, and was incubated with KCC2 agonist CLP257(50 μ M, 1h) before recording, and the results are shown in fig. 5, where KCC2 agonist CLP257 resulted in 42.26% reduction in spontaneous firing frequency of the pyramidal neurons in region vCA1 of mice after multiple exposure of ketamine during development (P < 0.01), and CLP257 reversed the increase in excitation of the pyramidal neurons in region vCA1 of mice during the progression due to multiple exposure of ketamine during development.

Taken together, the KCC2 agonist CLP257 plays an anxiolytic role by inhibiting the multiple exposure of ketamine during development to the excitability of mouse vCA1 brain region pyramidal neurons.

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.