Application of HCN1 as target spot of post-ischemic stroke depression in drug screening and treatment

Abstract

The invention discloses application of HCN1 as a target spot of post-cerebral arterial thrombosis depression in drug screening and treatment, and belongs to the technical field of biological medicine. Researches show that the depression behavior occurs after ischemia-reperfusion injury, meanwhile, expression of brain tissue HCN1 and inflammatory protein is up-regulated, and knock-down of the HCN1 is beneficial to reduction of brain tissue inflammation and improvement of the depression behavior after cerebral apoplexy. The invention provides a new research thought and direction for clinical treatment of post-stroke depression, and provides powerful evidence for the effect of HCN1 in nerve injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of polarization-activated cyclic nucleotide-gated ion channel HCN1 as a target for post-ischemic stroke depression in drug screening and disease treatment. Background Art

[0002] Stroke is the leading cause of death among Chinese residents, of which ischemic stroke accounts for about 80%, with the characteristics of high morbidity, high disability rate, high mortality rate and high recurrence rate. However, less than 10% of ischemic stroke patients can receive thrombolysis or interventional treatment in the acute phase, and the vast majority of patients will have residual neurological dysfunction. Among them, the incidence of post-stroke depression (PSD) is about 30%-60%, which is one of the common complications after stroke. PSD patients have a long recovery period and low quality of life, which brings a heavy burden to patients' families and society.

[0003] At present, some studies have shown that ischemia causes brain tissue necrosis and atrophy, leading to disorders in the monoamine neurotransmitter system such as serotonin and dopamine, which may be one of the mechanisms that induce PSD. However, antidepressant drugs targeting neurotransmitters have poor therapeutic effects on PSD, suggesting that the mechanism of PSD may be different from traditional depression. Therefore, in-depth exploration of the pathogenesis of PSD and finding specific therapeutic targets are of great significance for promoting the rehabilitation of patients with post-stroke depression.

[0004] Hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN) are special voltage-gated ion channels that are activated when the membrane potential is hyperpolarized, resulting in Na + Inflow and K + Outflow. There are 4 subtypes of HCN channels, namely HCN1-4, of which HCN1 and HCN2 are widely and highly expressed in the cerebral cortex, hippocampus and other brain regions, while HCN3 and HCN4 are only expressed in small amounts in specific brain regions such as the cerebellum and olfactory bulb. HCN channels play an important role in physiological processes such as neuronal rhythmic regulation, transmitter release, and synaptic plasticity. Their abnormal expression or function is closely related to a variety of neurological diseases. At present, the role of HCN channels in post-stroke depression is still unclear. Summary of the invention

[0005] The present invention shows that depressive behavior occurs after ischemia-reperfusion injury, and the expression of HCN1 and inflammatory proteins in brain tissue is upregulated, while knocking down HCN1 helps reduce brain tissue inflammation and improve post-stroke depressive behavior. Based on the above findings, the present invention proposes a scheme for treating post-ischemic stroke depression using HCN1 as a target, providing a new research idea and direction for the clinical treatment of post-stroke depression, and providing strong evidence for the role of HCN1 in neural injury.

[0006] In a first aspect, the present invention provides the use of an HCN1 expression inhibitor in the preparation of a drug for treating depression after ischemic stroke. Experimental data show that the HCN1 expression inhibitor reduces brain tissue inflammation and improves post-stroke depressive behavior by inhibiting HCN1 expression.

[0007] In one embodiment of the present invention, the HCN1 expression inhibitor is the HCN channel blocker ZD7288. In another embodiment of the present invention, the HCN1 expression inhibitor is a gene containing a silencing gene HCN1 shRNA adenovirus; mouse HCN1 Taking the gene (NM_010408.3) as an example, the sequence of shRNA can be designed as 5'-GCTGGTTTGTGGTGGACTTCA-3' (SEQ ID NO.1), 5'-GGGAAGAGATATTCCACATGA-3' (SEQ ID NO.2) or 5'-GGATTACCATGCTGAGCATGA-3' (SEQ ID NO.3), and the resulting adenovirus can all downregulate the expression of HCN1; it is understandable that for different subjects, according to their HCN1 Gene design corresponding shRNA to inhibit HCN1 expression.

[0008] In a second aspect, the present invention provides the use of HCN1 as a target in screening drugs for treating post-ischemic stroke depression, specifically, screening out substances that inhibit up-regulated expression of HCN1 in an animal model of post-ischemic stroke depression as candidate drugs.

[0009] Preferably, in the above application, the method of constructing an animal model of depression after ischemic stroke is: firstly, the animal is subjected to MCAO modeling, and then the obtained MCAO model is subjected to rotating rod, open field, social and sugar water preference experiments in sequence, and the animal model that exhibits depressive-like behavior in both sugar water preference and social experiments is screened. Compared with direct drug screening with the MCAO model, the animal model provided by the present invention is more accurate and the results obtained are more reliable.

[0010] In a third aspect, the present invention provides a drug for treating depression after ischemic stroke, wherein the drug has an HCN1 expression inhibitor as one of its active ingredients.

[0011] The above-mentioned medicines may also contain pharmaceutically acceptable excipients; and the medicines may be prepared into different dosage forms according to actual needs.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time the association between HCN1 and post-ischemic stroke depression, and reveals the finding that knocking down HCN1 helps reduce brain tissue inflammation and improve post-stroke depression behavior, thereby clarifying that HCN1 can be used as a therapeutic target for post-ischemic stroke depression. The present invention provides a new approach for the clinical treatment of post-stroke depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 This is a diagram showing the TTC staining results of the brain tissue of MCAO ischemia-reperfusion mice in Example 1 of the present invention; Figure 2 This is a graph showing the results of behavioral experiments on the post-stroke depression-like affective disorder animal model established in Example 1 of the present invention; Figure 3 This is a graph showing the results of detecting HCN expression in the ischemic hippocampus of different types of mice in Example 1 of the present invention; Figure 4 This is a graph showing the test results of the effect of administration of the HCN channel blocker ZD7288 on the behavior of post-stroke depression mice in Example 2 of the present invention; Figure 5 This is a diagram showing the detection result of down-regulating HCN1 expression by the HCN1 knockdown virus in Example 3 of the present invention; Figure 6 This is a graph showing the test results of the effect of down-regulating HCN1 expression by the HCN1 knockdown virus on the behavior of post-stroke depression mice in Example 4 of the present invention; Figure 7 This is a graph showing the expression of NLRP3 inflammation-related pathway proteins in ischemic hippocampal tissue after the HCN1 knockdown virus down-regulates HCN1 expression in Example 4 of the present invention. DETAILED DESCRIPTION

[0015] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the description and claims of this application and any variations thereof are intended to cover non-exclusive inclusions.

[0017] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0018] Example 1 By establishing and studying an animal model of post-stroke depressive-like affective disorder, we found that the expression of HCN1 in the ischemic hippocampus of post-stroke depressed mice was significantly increased.

[0019] The method for establishing an animal model of post-stroke depressive disorder-like affective disorder includes the following steps: (1) MCAO modeling.

[0020] Male SPF C57BL / 6J mice, 8-10 weeks old, weighing 18-25 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. No more than 5 mice were kept in each cage, the room temperature was 20℃-26℃, the humidity was 40%-70%, the light was 12h / 12h day and night, and the mice were allowed to take feed and drinking water freely. Modeling began after 7 days of adaptive feeding.

[0021] After the mice were anesthetized with a mixture of 1.5% isoflurane and oxygen, the body temperature was maintained at 37℃±0.5℃ during the operation. The hair on the neck of the mice was cleaned with scissors, disinfected with iodine, and the skin was cut along the midline of the neck, with a length of about 1 to 2 cm. The subcutaneous submandibular gland tissue of the mice was separated, and the common carotid artery, external carotid artery and internal carotid artery on the left were bluntly separated. The common carotid artery and external carotid artery were threaded for use. The external carotid artery was ligated, and a slipknot was tied near the bifurcation of the neck in the external carotid segment for use. The artery clamp temporarily clamped the common carotid artery and the internal carotid artery. A small opening was made on the external carotid artery segment with microscissors, and the embolism line wrapped with one end of the silicone head was inserted from the external carotid opening to the common carotid artery. After cutting the distal end of the external carotid artery, the vascular clamp on the internal carotid artery was opened, the insertion direction of the embolism line was adjusted, and the embolism line was slowly inserted into the internal carotid artery and blocked the middle cerebral artery. The insertion depth was about 9.5 mm from the bifurcation of the common carotid artery. After feeling the resistance, it was stopped, and the silk thread for use in the neck was ligated tightly. The wound was closed and sutured, and the mice were placed in an animal incubator. After the mice fully woke up, they were placed in a clean cage to wait for the suture to be removed. After 1.5 hours of ischemia, the suture was gently pulled out to restore blood perfusion, the excess sutures were cut off and the surgical incision was sutured. The mice after modeling were placed back in a clean cage and given sufficient feed and water. The corresponding blood vessels of the mice in the sham operation group were isolated and the external carotid artery was cut, but no suture was inserted.

[0022] TTC staining was performed on MCAO ischemia-reperfusion mice and sham-operated mice. The specific operation was as follows: after the mice were anesthetized, the brain tissue was removed by decapitation, and the brain tissue was frozen at -20°C for 10 minutes. The brain coronal slices were cut into 2mm slices using a mouse brain mold; the brain slices were placed in a 1% TTC solution (dissolved in PBS), and placed in a 37°C incubator for 30 minutes away from light, and the brain slices were shaken halfway to ensure uniform staining; the stained brain slices were then placed in a 4% paraformaldehyde solution for fixation. After fixation and preservation, photos were taken to observe the infarction condition. The brain tissue in the ischemic area was stained white. The TTC staining results of MCAO ischemia-reperfusion for 24 hours showed that the mouse hippocampus was obviously ischemic ( Figure 1 ).

[0023] (2) Behavioral experiments.

[0024] Four weeks after MCAO modeling, the rotarod, open field, social interaction and sucrose preference tests were performed in sequence to detect depression-related behaviors.

[0025] Rotarod test: The mice were placed on the fixed rod of the rotarod instrument (Shanghai Xinruan XR-6C) for pre-adaptation; then, during the test, the rotation speed of the rotarod was set to 30 rpm, and the time each mouse fell within 180 seconds was recorded. After each test, the rotarod was cleaned with alcohol and dried. The time the mouse stayed on the rotarod was used as an indicator to evaluate the motor balance ability.

[0026] Open field test: Place the mouse with its back facing the experimenter in the center area at the bottom of the open field test box and allow it to explore freely for 10 minutes. Open Shanghai Xinruan SuperMaze animal behavior video analysis software to record the mouse's activities in the open field for 10 minutes, including average movement speed, central area movement time, etc. Ensure a quiet environment during the experiment. After the experiment, put the mouse back into the cage. After each experiment, clean the entire open field test box with 75% alcohol to prevent residual odor from affecting the next test.

[0027] Social experiment: The sociability test used a three-box social instrument (XR-XJ117, Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China); in the pre-adaptation phase, two small empty cages were placed in the left and right boxes, and the experimental animals were allowed to freely explore the instrument from the middle box for 10 minutes; in the test phase, a strange mouse was placed in the small cage in the right box, and then the experimental mouse was placed in the instrument from the middle box, allowing it to freely explore the device for 10 minutes, and the time the mouse spent in each of the three boxes was recorded. The time spent in the right box with the strange mouse was used to evaluate the mouse's social ability.

[0028] Sugar water preference experiment: Mice were housed in individual cages and fed freely. Two bottles of water were provided in each cage for adaptation. After 24 hours of adaptation, one of the water bottles was replaced with a 1% sucrose solution, and the weights of the two bottles were weighed. After 24 hours, the positions of the two water bottles were swapped, and the weights of the two bottles were weighed after another 24 hours of observation. The intake of 1% sucrose and water by mice within 48 hours was calculated. The calculation of the sucrose preference index was: (1% sucrose intake) / (1% sucrose intake + water intake).

[0029] By comparing with the mean of the sham operation group, the mice that showed depressive-like behaviors in both the sugar water preference and social experiments were divided into the post-stroke depression group (PSD group), and the mice that did not show depressive-like behaviors in both experiments were divided into the post-stroke non-depressive group (Non-PSD group).

[0030] The results are as follows Figure 2 As shown in the figure, there was no significant difference between the mice in the sham operation group and the mice in the rotarod test and the average speed in the open field test 4 weeks after MCAO modeling, indicating that the motor function of the mice was not significantly impaired 4 weeks after MCAO ( Figure 2 C, E). The time spent in the central area of ​​the open field by mice in the PSD group was significantly reduced, indicating that the mice showed anxiety-like behavior ( Figure 2 D). In addition, the sugar water preference level of mice in the PSD group ( Figure 2 A) Social level is significantly reduced ( Figure 2 B), showing that mice exhibit depressive-like behaviors.

[0031] Furthermore, Western Blotting protein detection was performed on the ischemic hippocampal regions of the sham-operated mice, PSD group mice and Non-PSD group mice. The specific operation was as follows: the mice were anesthetized and decapitated, the brain tissue was quickly removed on ice, and the left ischemic hippocampal tissue was taken; the sample was homogenized with tissue lysis buffer and centrifuged to obtain the supernatant, which was the protein sample. After BCA protein quantification, the loading buffer was added for denaturation, and the sample was frozen in a -80℃ refrigerator for later use; 30μg protein sample was subjected to 10% SDS / PAGE gel electrophoresis, and the membrane was transferred to PVDF membrane at 280mA on ice for 2h, blocked at room temperature for 1h, and incubated with a certain dilution ratio of primary antibody at 4℃ overnight; on the next day, the PVDF membrane was rinsed with TBST (5min×3 times), and incubated with the corresponding HRP-labeled secondary antibody (1:3000) at room temperature for 1h; TBST was rinsed 3 times, the PVDF membrane was exposed after being poured with ECL developer, and the grayscale value of the band was analyzed by NIH Image J to detect the changes in the expression of HCN1, HCN2, and NLRP3 inflammatory pathway proteins.

[0032] The results are as follows Figure 3 As shown, the expression of total protein and membrane protein of HCN1 in the CA1 region of the ischemic side of the hippocampus of mice in the PSD group was significantly increased, while the expression of HCN2 did not change significantly.

[0033] Example 2 Based on the model mice established in Example 1 and the findings thereof, this example further demonstrated the association between up-regulated HCN1 channel expression and anxiety-depression-like behaviors of PSD mice by administering HCN channel blockers into the lateral ventricles of mice.

[0034] Intracerebral ventricular administration: After the mice were anesthetized with a mixture of 1.5% isoflurane and oxygen, they were fixed on a brain stereotaxic instrument; the hair on the mouse head was removed and the surgical area was cleaned and disinfected; the scalp was incised with a scalpel to expose the anterior fontanel, and the thin film on the surface of the skull was gently removed with a sterile cotton swab; according to the brain atlas, the zero point was set based on the anterior fontanel and the interaural line, and the injection coordinates were determined to be 0.3mm behind the anterior fontanel, 1mm to the side, and 3mm deep; each mouse was injected with 200μM ZD7288 3μl (5mg dissolved in 170μl DMSO, and then diluted to 200μM with saline) at a rate of 0.3μl / min; after the injection, wait for about 2 minutes to ensure that the drug was fully absorbed; after the injection, slowly lift the syringe, suture the scalp and disinfect it.

[0035] Behavioral testing was performed 3 days after administration, and the testing method was the same as in Example 1. The test results are shown in Figure 4As shown, intracerebroventricular injection of HCN channel blocker ZD7288 can increase the activity distance of PSD mice in the central area of ​​the open field (A, B), and increase the social behavior of PSD mice (A, C), indicating that inhibition of HCN1 can improve the anxiety-depression-like behavior of PSD mice. n=8, *P<0.05, compared with Sham+Con group. #P<0.05, compared with PSD+Con group. Sham+Con is a sham-operated group of mice without drug administration, and PSD+Con is a PSD mouse without drug administration.

[0036] Example 3 In this case, adeno-associated virus (AVV) was used to prepare HCN1 knockdown virus, which can significantly reduce HCN1 expression.

[0037] In this case, rAAV9-ZsGreen-shRNA-mHcn1 adeno-associated virus was prepared using the shRNA shown in SEQ ID NO.1, which acts on the mouse Hcn1 (NM_010408.3) gene. The brain stereotaxic instrument locates the CA1 region of the hippocampus, and 1 μl of HCN1 knockdown virus is slowly injected into the CA1 region of the left hippocampus via a microinjection pump ( Figure 5 A), the specific method is as follows: PSD mice were anesthetized with a mixture of 1.5% isoflurane and oxygen and fixed on a brain stereotaxic instrument; the hair on the head of the mice was removed, the scalp was incised after cleaning and disinfection, the bregma was exposed, and the thin membrane on the surface of the skull was removed; according to the brain atlas, the injection coordinates were determined to be 2.5 mm behind the bregma, 1.8 mm away from the side, and 1.5 mm deep; each mouse was injected with 1 μl of rAAV9-ZsGreen-shRNA-mHcn1 adeno-associated virus (1×10 13 vg / ml) at a rate of 0.1μl / min. After the injection, wait for about 5 minutes to ensure that the drug is fully absorbed. After the injection, slowly lift the syringe, suture the scalp and disinfect it. Another PSD mouse was injected with 1μl of control virus rAAV9-ZsGreen-shRNA (1×10 13 vg / ml) as a control.

[0038] Four weeks after virus injection, mice were sampled for western blot to detect changes in HCN1 protein expression, and brain tissue frozen sections were used to observe virus transfection. Figure 5 As shown, there is a large amount of fluorescence expression in the hippocampus at the virus injection site, indicating that the injection site is transfected with the virus (B); in addition, injection of HCN1 knockdown virus into the ischemic hippocampus can significantly reduce HCN1 expression (C, D).

[0039] Example 4 Using the HCN1 knockdown virus, control virus and virus injection method prepared in Example 4, this example demonstrates the positive effect of down-regulating HCN1 expression on improving the depressive behavior of PSD mice.

[0040] The specific process is as follows Figure 6 As shown in A, PSD mice were obtained and injected with HCN1 knockdown virus, and molecular biology and behavioral tests were performed four weeks after virus injection.

[0041] Behavioral test results Figure 6 As shown in the figure, injection of HCN1 down-regulating virus Hcn1 shRNA into the CA1 region of the ischemic hippocampus can increase the time that stroke mice spend in the central area of ​​the open field, indicating that it can improve the anxiety-like behavior of stroke mice (B, C, F). At the same time, HCN1 down-regulating virus Hcn1 shRNA can increase the social behavior of stroke mice, indicating that it can improve the depressive-like behavior of stroke mice (E, G). In addition, the results of molecular biological tests are shown in the figure. Figure 7 As shown, HCN1 knockdown virus can significantly reduce the expression of NLRP3 inflammation-related pathway proteins in ischemic hippocampal tissue. The above results show that HCN1 downregulation virus can reduce brain tissue inflammation and improve anxiety-depression-like behaviors in stroke mice. *P<0.05, **P<0.01, compared with the SHAM+Control group. #P<0.05, ##P<0.01 compared with the PSD+Control group.

[0042] In summary, both the HCN1 channel inhibitor ZD7288 and the HCN1 knockdown virus can improve the depressive behavior of PSD mice by downregulating HCN1 expression in hippocampal neurons, providing a new approach for the clinical treatment of post-stroke depression.

[0043] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. Application of HCN1 expression inhibitors in the preparation of drugs for the treatment of post-ischemic stroke depression.

2. The use according to claim 1, characterized in that: The HCN1 expression inhibitor reduces brain tissue inflammation and improves post-stroke depressive behavior by inhibiting HCN1 expression.

3. The use according to claim 1, characterized in that: The HCN1 expression inhibitor is ZD7288.

4. The use according to claim 1, characterized in that: The HCN1 expression inhibitor contains a silencing gene HCN1 shRNA adenovirus.

5. The use according to claim 4, characterized in that When gene HCN1 When derived from mouse, the sequence of the shRNA is shown in any one of SEQ ID NO.1-3.

6. Application of HCN1 as a target in screening drugs for the treatment of post-ischemic stroke depression.

7. The use according to claim 6, characterized in that: Substances that inhibit the up-regulated expression of HCN1 in animal models of post-ischemic stroke depression are selected as candidate drugs.

8. The use according to claim 7, characterized in that: The method for constructing the animal model of post-ischemic stroke depression is as follows: firstly, the animal is subjected to MCAO modeling, and then the obtained MCAO model is subjected to rotating rod, open field, social and sucrose preference experiments in sequence, and the animal model showing depressive-like behavior in both sucrose preference and social experiments is screened.

9. A drug for treating depression after ischemic stroke, characterized in that: An inhibitor of HCN1 expression is one of the active ingredients.

10. The drug according to claim 9, characterized in that It also contains pharmaceutically acceptable excipients.

Images