CN117815247A - Application of ZL006 in preparation of antidepressant drugs - Google Patents

Abstract

The invention discloses application of ZL006 in preparation of antidepressant drugs. ZL006 can obviously improve the depression-like behavior of rats after cerebral apoplexy, including increasing the preference of syrup, enhancing the desire to live, improving the movement and exploring ability disorder of PSD rats, can obviously alleviate brain tissue nerve cell injury, improve the antioxidant capacity at the same time. ZL006 has obvious treatment effect on post-stroke depression, and can be used for preparing medicaments for treating depression.

Description

Application of ZL006 in preparation of antidepressant drugs

Technical Field

The invention belongs to the field of medicines, and relates to application of ZL006 in preparation of antidepressant drugs.

Background

Post-stroke depression (Post Stroke Depression, PSD) is a psychological disorder and cognitive dysfunction which occur after cerebral apoplexy, is one of main complications of cerebral apoplexy, remarkably increases the death rate and disability rate of cerebral apoplexy patients, reduces the life quality of patients, and brings heavy burden to patients and families and even society thereof. PSD is a common, treatable condition, and early detection and proper treatment is critical for PSD patients. Regarding the pathogenesis of PSD, the research shows that the glutamate system may become a break-through for the treatment of PSD.

Under cerebral ischemia conditions, excitatory amino acids (e.g., glutamate) are over-released, causing NMDAR to be over-activated, activating nNOS and coupling with PSD95, forming NMDAR-PSD95-nNOS macromolecular complexes, initiating the production of downstream signaling molecules NO, causing apoptosis and nerve damage. Blocking the coupling of downstream PSD95-nNOS is an ideal drug action target, and is expected to develop a safe and effective anti-cerebral-stroke therapeutic drug without obvious side effects (Zhou, li et al, "Treatment ofcerebral ischemia by disrupting ischemia-induced interaction ofnNOS with PSD-95." Nature medium vol.16,12 (2010): 1439-43.Doi: 10.1038/nm.2245).

Decoupling agent ZL006 of the formulaIt can target and destroy PSD95-nNOS interaction, and the neuroprotection effect of ZL006 has been effectively verified on nerve disease models such as cerebral apoplexy, chronic pain and the like at present (Lee, wan-Hung et al, "Small molecule inhibitors ofPSD-nNOS protein-protein interactions as novel analysis science vol.97 (2015): 464-75.Doi:10.1016/j. Neuropharm.2015.038). However, no report has been made on the use of the compounds in the treatment of post-stroke depression.

Disclosure of Invention

The invention aims to provide application of ZL006 in preparation of antidepressant drugs.

Further, the antidepressant is a drug for treating post-stroke depression.

Further, the antidepressant also contains pharmaceutically acceptable auxiliary materials or auxiliary medicinal components.

Further, the antidepressant is in the form of an oral preparation or an injection.

Further, the administration amount of the antidepressant is 10-20 mg ZL006/kg.

According to the invention, animal experiments show that ZL006 can obviously improve the depression-like behavior of PSD rats, including increasing sugar preference, enhancing survival desire, improving the movement and exploration ability disorder of PSD rats, and simultaneously can obviously reduce brain tissue nerve cell damage and improve oxidation resistance. The invention discovers that ZL006 has obvious treatment effect on post-stroke depression for the first time.

Drawings

FIG. 1 is a graph showing the results of the experiments of the behavioural assay for each group of rats;

FIG. 2 is a diagram showing pathological sections of brain tissue of rats in each group.

FIG. 3 is a serum biochemical marker of each group of rats.

Detailed Description

The invention is further described below with reference to specific embodiments and figures. In the examples described below, the experimental reagents or materials employed are all commercially available.

EXAMPLE 1 therapeutic Effect of ZL006 on post-stroke depression

1. Experimental materials

(1) Laboratory animals and groups

60 adult SPF-grade male rats Sprague-Dawley (SD) rats were purchased from Nanjing Qinghai Longshan laboratory animal farm. SD rats were kept under standard living conditions, the ambient temperature was maintained at 25+ -1deg.C, the relative humidity was 50+ -10%, and the day and night cycle was continued for 12 hours with food and water available for free intake, and experiments were conducted after 7 days. Rats were divided into 4 groups: sham surgery (Sham) group, model (Model) group, ZL006 low dose-10 mg/kg (ZL) group, ZL006 high dose-20 mg/kg (ZH) group.

(2) Experimental medicine and reagent

ZL006 (SML 0146), penicillin (1.00263), absolute ethanol (abm.g490) were purchased from microphone; cell culture grade dimethyl sulfoxide (D8371) was purchased from beijing solebao technologies limited; isoflurane (R510-22-10) is purchased from ravode life technologies limited; the detection kit for MDA (A003-1-2), SOD (A001-1-2) and GSH (A006-1-1) is purchased from Nanjing to build a bioengineering institute.

(3) Experimental instrument

Multichannel small animal anesthesia machine: product number F710, vendor EZVET.

Multifunctional enzyme-labeled instrument: number 2108171E, vendor BioTek.

Low temperature high speed centrifuge: fresco17, supplier Thermo Fisher scientific.

2. Experimental method

(1) Construction of rat brain ischemia/reperfusion injury (MCAO/R) model

The MCAO/R model is constructed by the method described in the literature [ protection of brain ischemia reperfusion injury by Jinran. Phellodendrine [ D ]. Nanjing university of chemical industry, 2021.DOI:10.27241/d.cnki.gnjgu.2021.002643 ]:

before the experiment starts, rats are placed on the back after being anesthetized with isoflurane, the neck is shaved and disinfected, an incision of 1-2cm is cut along the middle of the neck, subcutaneous muscle tissue is separated, a Common Carotid Artery (CCA) is seen, mucous membrane and vagus nerve around the CCA are carefully separated, an Internal Carotid Artery (ICA) and an External Carotid Artery (ECA) are continuously separated towards the distal end along the CCA, the CCA is clamped by a micro vascular clamp, after the ECA is ligated twice with 4-0 suture, a blood vessel is cut in the middle of the ligation, and a small opening of 45 degrees is cut on the cut ECA bifurcation by ophthalmology, so that the blood vessel is gradually pushed into the ICA from the opening in a natural state, when the black mark of the special line plug reaches the vicinity of the Y-shaped bifurcation opening and the insertion of the line plug is completed when the pushing is continued, the line plug and the ECA stub are tensioned and fixed by the ligation line to prevent bleeding, and the micro vascular clamp is taken out. Cleaning neck blood trace with cotton ball, suturing wound skin and sterilizing. After 1h, the wire bolt is gently pulled to have resistance, and the redundant wire bolt is cut off. After waking, rats were observed for symptoms of neurological deficit and scored for neurological scores, with neurological score criteria shown in table 1. Neurological scores ranging from 1 to 3 indicated successful modeling of the MCAO/R model. Sham rats were sutured to the wound after isolation of only the common carotid artery. Rats were given penicillin (ip, 10 ten thousand U/dose) daily for 3 consecutive days after surgery. Rats that die or have insufficient scores during the course of the experiment will be complemented by the same batch of rats.

TABLE 1Longa neuro-behavioural scoring system

(2) Superposition of depression models: chronic Unpredictable Mild Stimulus (CUMS)

And (3) constructing a PSD model by using a CUMS modeling and solitary culture method after 3 days of (MCAO/R) modeling. The method specifically comprises the following steps: (1) inverting the day and night for 36 hours; (2) water is forbidden for 18h; (3) fasted for 24 hours; (4) a humid environment for 24 hours; (5) tilting the cage for 18h at 45 °; (6) forced swimming for 5min at 4 ℃; (7) heat stimulation at 45 ℃ for 5min; (8) clamping tail for 1min. The rats were stimulated for a fixed time daily and randomly selected one of the methods, each method was used no more than 4 times for 21 days. The Sham group rats are bred in groups, and 4-6 rats are bred in each cage; the other three groups of rats were bred in a single cage.

(3) Experimental administration

After the 21 st day test, the administration was by intraperitoneal injection. The specific preparation method of the mixed liquid medicine comprises the following steps: an appropriate amount of ZL006 was dissolved in DMSO to form ZL006-DMSO at a volume ratio of 1:1:8, mixing ZL006-DMSO with a solvent according to a mass ratio of 1:9, and finally adding 2% of a surfactant Tween-80.ZL 006 was administered to the ZL rats at 10mg/kg, ZH 006 was administered to the ZL rats at 20mg/kg, and ZL 006-free mixed liquor was administered to the Sham group and Model group at the same volume. After 24h, the behavioural experiments were performed in the order described below.

(4) Behavioural experiments

Sugar water preference experiment (Sucrose Preference Test, SPT): animals were required to be sugar trained three days in advance before the start of the experiment, with 2 bottles of 1% sucrose water given to each cage of rats on the first day, during which time they were free to eat. The following day, 1 bottle of 1% sucrose water and 1 bottle of deionized water were given to each cage of rats during which time they were free to eat. On the third day, rats in each group had fasted and had been kept water-out for 24 hours. On the fourth day, 100mL deionized water and 1% sucrose water were prepared in 1 mL bottles each, the consumption of deionized water and sugar water was measured for 3 hours, the positions of the sugar water bottles and deionized water bottles were changed every 1 hour, and finally the sugar water consumption rate (%) = sugar water consumption (mL)/(deionized water+sugar water consumption) ml×100% was calculated.

Open Field Test (OFT): the specification of the rat open field experiment box is 80cm multiplied by 40cm (length multiplied by width multiplied by height); the inner wall is blackened, and the bottom surface is divided into 25 small square grids of 16cm multiplied by 16cm, and the small square grids are divided into a central area and a peripheral area. The animals are brought into the laboratory to adapt to the environment 3 hours before the experiment starts, the experiment box is ensured to be clean, the rats are gently grasped from the raising cage and immediately leave after being placed into the central area of the experiment box, the camera is opened to record the movement track of the rats within 5 minutes, the environment is kept to be weak light irradiation and quiet and comfortable as much as possible in the experiment process, and after the experiment is finished, the trace left by the last rat is wiped by 75% alcohol, so that the interference is eliminated. The index was recorded after the experiment: the number of horizontal exercises, the number of vertical stands, the residence time in the central area, etc., and each experimental index was independently evaluated by a researcher outside of the two experimental designs and the average value was taken.

Forced swimming experiment (Forced Swimming Test, FST): the diameter of the forced swimming bucket of the rat is 20cm, and the height is 50cm; the water depth is the basis that the tail of the rat cannot be supported at the bottom of the barrel; the water temperature was set at 24-26 ℃. The total FST time is 6min, the first 2min is the adaptation time, and the camera is used for recording the motion of the last 4 min. After the experiment is finished, the rat hair is wiped, a heating lamp is arranged to help the rats to recover the body temperature, and water in the water bucket is replaced after each rat experiment is finished. The experimental indexes are swimming time and immobility time (movement of forelimbs immobility and hindlimbs to avoid upward water-drawing when submerged), each index is evaluated by researchers outside two experimental designs, and the average value is taken.

(5) HE staining, nissl staining

Rats were sacrificed the day after the end of the behavioural experiment and quick material was obtained. After the brain is completely taken out, the brain is washed by pre-cooled normal saline and is fixed in 4% paraformaldehyde for 48 hours. The fixed brains were embedded in paraffin for histopathological examination. 4 μm serial thin sections of tissue were taken for hematoxylin-eosin (HE) staining and Nissl staining. And observing pathological changes of the brain tissue slices of each group by adopting a panoramic scanner, and intercepting corresponding main pathological change parts.

(6) Serum biochemical index

The fresh blood sample obtained by taking materials is kept stand for 30min at room temperature, and then is put into a centrifuge for centrifugation at 3000rpm for 10min, and the supernatant is sucked. The obtained serum sample is stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃, melted on ice before being taken, experimental operation is carried out strictly according to the specification of a biochemical kit, and the SOD activity, GSH and MDA content in the serum are measured.

3. Experimental results

(1) Sugar water preference experimental results

Sugar water preference experiments are commonly used to assess the level of depression in animals, which exhibit reduced interest in sucrose water and reduced consumption of sugar water. As shown in fig. 1A, the sugar water consumption was significantly reduced in Model rats compared to Sham group (< 0.01). The sugar water consumption of PSD rats was increased in a dose-dependent manner after ZL006 administration, and in the high dose ZH group, the sucrose consumption was significantly increased (< p < 0.01) and restored to Sham group levels. SPT results show that ZL006 can improve the dietary interest loss of PSD rats and improve the dietary preference of the PSD rats.

(2) Forced swimming test results

The forced swimming experiment is an animal model with the effect of despair depression. In this test, animals forced to swim in a closed cylinder initially struggle to swim vigorously, then stay in a floating state, and finally give up the hope of escaping. The longer the immobility time, the more severe the depression. The Model group showed a significant increase in immobility time (< p < 0.01) compared to Sham group, confirming depression-like behavior in this Model. Notably, the immobility time was significantly reduced in the treatment group compared to the Model group (< 0.0001) even lower than that in the Sham group (fig. 1B). FST results indicate that ZL006 significantly improves depressive-like behavior.

(3) Open field experimental results

Open field tests are used to assess the motor activity and curiosity of animals against external stimuli. The main indexes of the method comprise the total running distance (crossing number) and the number of vertical standing times, and the method can well reflect the activity capability and exploring behavior of animals. As shown in fig. 1C, the number of transverse lattices of the Model group of rats was significantly reduced (p < 0.001) compared with that of the Sham group, and after administration, the number of transverse lattices of the ZL group was increased (p < 0.05), and no significant change was observed in the ZH group. As shown in fig. 1D, model group rats were significantly reduced in number of vertical stands compared to Sham group (p < 0.01), and after administration, the number of vertical stands in the treatment group was restored to the level for Sham group (p < 0.01). The OFT results show that ZL006 can improve the movement and exploration ability of PSD rats to a certain extent.

(4) HE staining, nissl staining results

Cerebral ischemia reperfusion can lead to neuronal apoptosis, exacerbating brain injury. To analyze the damage of PSD rat neurons, we used HE staining and Nissl staining of brain histopathological sections (FIG. 2). HE staining results show that the cells of the Sham group are full in morphology, complete in structure and compact in arrangement. The Model group nerve cell nuclei were stained and contracted, the cells were loosely arranged, and the surrounding was vacuolated. The ZL006 treatment group has reduced necrotic neurons, reduced cavity and more complete tissue structure. Nissl staining results show that the Sham group neurons have complete structure, clear nucleolus and rich Nishi bodies. The Model group neurons were not uniformly shrunken, the cell contours were not clear, and the cytoplasmic Nib bodies were reduced. The ZL006 treatment group had partial cell nucleus shrinkage and increased overall Nib number. The results of HE staining and Nissl staining are consistent, and show that ZL006 administration has a certain protection effect on PSD rat nerve cells, and can effectively relieve the damage of neurons.

(5) Serum biochemical index results

SOD, GSH and MDA are commonly used to evaluate oxidative stress injury following cerebral ischemia. SOD is used as an important antioxidant enzyme and can directly remove harmful free radicals. An increase in MDA levels indicates an exacerbation of lipid peroxidation. GSH plays an important role in maintaining the redox balance of neuronal cells, and it can further decompose toxic peroxidic materials, enhancing antioxidant capacity. As shown in fig. 3, ZL006 significantly increased the antioxidant enzyme SOD activity and GSH levels in a dose-dependent manner, showing a certain antioxidant effect, compared to the model group. Notably, ZL006 did not exert a restorative effect on MDA levels at high doses, but rather enhanced the levels. Overall, the antioxidant mechanism of ZL006 needs further research to better alleviate oxidative stress injury after cerebral ischemia.

4. Experimental nodule

In conclusion, the invention successfully constructs the rat model which embodies the main behavior characteristics and pathological characteristics of PSD by combining MCAO cerebral ischemia reperfusion with CUMS stimulation. The model is proved to have typical depression-like behaviors through the behavioral detection after 24 hours by giving high and low doses of ZL006 or solvent carrier to PSD rats, and the syrup preference rate of the PSD rats is reduced, the immobility time, the external interest is reduced and the like, and the model is consistent with typical depression conditions. The performance of ZL006 in SPT, FST and OFT is corrected after administration, and the rapid antidepressant effect of ZL006 on PSD rats is proved. Meanwhile, ZL006 can also obviously lighten the damage of nerve cells, improve the SOD activity and GSH level of antioxidant enzyme, reduce the oxidation pressure of brain tissues, and preliminarily show that one of possible protection mechanisms is through the antioxidant effect. In addition, it was found by histopathological examination that the brain tissue nerve cells of the rats of the ZL006 treatment group were less damaged compared to the model group, indicating that ZL006 might alleviate PSD symptoms by protecting nerve cells.

The invention constructs a rat model with depression after cerebral apoplexy by combining MCAO, CUMS and solitary culture, and preliminarily verifies that the drug ZL006 targeting PSD95-nNOS has obvious anti-depression effect on PSD rats, and the ZL006 can generate neuroprotection and anti-depression effects on PSD through oxidation resistance and other ways.

Claims (5)

1. The application of ZL006 in preparing antidepressant, wherein the structural formula of ZL006 is shown in the specification.

2. The use according to claim 1, wherein said antidepressant is a medicament for the treatment of post-stroke depression.

3. The use according to claim 1, wherein the antidepressant comprises a pharmaceutically acceptable adjuvant or adjuvant drug ingredient.

4. The use according to claim 1, wherein the antidepressant is in the form of an oral or injectable formulation.

5. The use according to claim 1, wherein the antidepressant is administered in an amount of 10-20 mg zl006/kg.