Application of SPAK inhibitor ZT-1a in preparation of medicine for treating epilepsy

Abstract

The invention provides application of an SPAK inhibitor ZT-1a in preparation of a medicine for treating epilepsy, the structural formula of the SPAK inhibitor ZT-1a is shown in the specification, the activity of a # imgabs0 # WNK-SPAK-KCC2 / NKCC1 signal channel is increased in the first week after embryos and newborns are born, the activity of the # imgabs0 # WNK-SPAK-KCC2 / NKCC1 signal channel is remarkably reduced from brain maturation to adult, but the channel is reactivated in the epilepsy pathology state. The phosphorylation level of the Thr1007 residue of KCC2 is increased, and the function of KCC2 is reduced, so that the epilepsy symptom is aggravated. According to the invention, the novel SPAK inhibitor ZT-1a activates a transfer function of multiple regulation and control of KCC2 and NKCC1 by inhibiting the pathway, and effectively inhibits epilepsy behaviors induced by an epileptic agent and occurrence of epilepsy-like discharge of cultured neurons. The invention provides a brand new treatment means for treating epilepsy based on a neuron cation-chloride ion transporter regulation mechanism, and changes the current anti-epilepsy drug target screening which focuses on a sodium ion channel and a GABA inhibition function, so that the application has great drug research and development value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a SPAK inhibitor ZT-1a, specifically to the use of the SPAK inhibitor ZT-1a in the preparation of a drug for treating epilepsy. Background Art

[0002] Epilepsy is a syndrome characterized by recurrent brain dysfunction caused by large-scale abnormal synchronous discharges of brain neurons, affecting about 50 million people worldwide. In China, epilepsy is also one of the most common neurological diseases, with an overall prevalence of 7.0‰ and an annual incidence of 28.8 / 100,000. There are currently about 9 million epilepsy patients. Epilepsy not only seriously affects the quality of life of patients, but also brings huge economic burdens to society and families, and also brings huge challenges to the national health care industry. Currently, most anti-epileptic drugs that can be used for clinical treatment are sodium ion channel blockers or GABAergic system regulators such as phenobarbital and carbamazepine. Long-term use has great side effects and is prone to drug resistance. Therefore, it is urgent to find an effective intervention mechanism and develop new anti-epileptic drugs for the long-term treatment of epilepsy patients.

[0003] The potassium-chloride cotransporter KCC2 is essential for maintaining Cl in mature neurons - It is necessary for homeostasis and normal inhibitory function of GABA. Partial knockout of KCC2 in mice, fruit flies, and nematodes all increases the intracellular Cl - Ion concentration ([Cl - ] i ), depolarizing GABA (E GABA ), destroying the inhibitory effect and causing neuronal overexcitation. Complete knockout will lead to respiratory failure and death. The occurrence and development of epilepsy have confirmed that KCC2 expression levels and functions are downregulated. The researchers used electrophysiological and molecular biological methods to confirm that KCC2 downregulation precedes seizure activity induced by convulsions in vitro and in vivo. The reduction of KCC2 expression and function promotes the development of grand mal epileptic seizures. Therefore, KCC2 is expected to be a new target for epilepsy treatment, aiming to improve KCC2 function and promote neuronal Cl - Squeeze to restore GABA A Inhibitory effect of GABA after R opening.

[0004] WNK is a newly discovered class of serine / threonine protein kinases that can sense intracellular Cl -Changes in concentration, extracellular osmotic pressure, and cell volume regulate the activity of cation-chloride cotransporters (CCCs) by phosphorylating downstream SPAKs to maintain the homeostasis of cells and organisms, affecting cell morphology and behavior. The activity of the WNK-SPAK-CCCs signaling pathway increases in the first week after birth of embryos and neonates, and decreases significantly as the brain matures into adulthood. However, under pathological conditions, this pathway is reactivated. At present, research on the multiple regulatory mechanisms of the WNK-SPAK pathway on transporters is mainly focused on the field of ischemic stroke, and only focuses on the regulation of the activity of the potassium-sodium-chloride cotransporter NKCC1. Pan-protein kinase inhibitors (Staurosoprine & NEM) and SPAK-specific inhibitors Closantel and STOCK1A-14279 / 50699 have been shown to regulate NKCC1 activity by inhibiting the WNK-SPAK pathway, but these drugs have produced adverse reactions such as weakness, visual impairment, and even blindness in clinical trials. In response to the above side effects, Zhang Jinwei's research group improved and synthesized a new SPAK-specific inhibitor ZT-1a, and used ZT-1a to successfully inhibit the activation of NKCC1 by SPAK kinase after MCAO ischemia to improve brain damage and brain edema area in mice.

[0005] More and more studies have shown that in the pathological state of epilepsy, the activated WNK-SPAK pathway simultaneously enhances the phosphorylation levels of the corresponding KCC2 (Thr1007) and NKCC1 (Thr212) residues, which can increase the level of neuronal [Cl - ] i , making neurons GABA A After R is opened, the inhibitory effect of GABA is weakened, aggravating epileptic symptoms. In this process, it is very worthy of attention whether the new SPAK inhibitor ZT-1a can treat epilepsy by inhibiting the activation of the WNK-SPAK-KCC2 / NKCC1 pathway. The related research of the present invention starts from a variety of epilepsy models in vivo and in vitro, and studies the inhibitory effect of ZT-1a on cultured hippocampal neurons and epileptic discharges in rat epilepsy models by inhibiting the upstream SPAK kinase activation to multiple regulate downstream KCC2 and NKCC1 protein transport functions. The determination of the inhibitor's regulatory effect on epilepsy will provide a new research and development direction for the future research and development of anti-epileptic drugs with the WNK-SPAK-KCC2 / NKCC1 pathway as the target. Summary of the invention

[0006] In view of the above technical problems in the prior art, the present invention provides the use of a SPAK inhibitor ZT-1a in the preparation of a drug for treating epilepsy, and the application is intended to solve the technical problem that the drugs in the prior art are not effective in treating epilepsy.

[0007] The present invention provides the use of a SPAK inhibitor ZT-1a in the preparation of a drug for treating epilepsy, and its structural formula is shown below:

[0008]

[0009] The present invention also provides the use of potassium ion-chloride ion co-transporter (KCC2) and potassium-sodium-chloride ion co-transporter (NKCC1) as targets in screening drugs for treating epilepsy.

[0010] The present invention clarifies a mechanism mediating the occurrence and development of epilepsy pathology, establishes the correlation between the WNK-SPAK-KCC2 / NKCC1 signaling pathway expressed by central hippocampal neurons and epilepsy, and that abnormal upregulation of SPAK is one of the important mechanisms for inducing epilepsy, and establishes the regulatory effect of ZT-1a on epilepsy as a SPAK inhibitor.

[0011] The present invention first studies the whole PTZ-induced epilepsy mouse model to explore whether intracerebroventricular injection of ZT-1a affects PTZ-induced epileptic behavioral seizures; the results of the study clearly show that ZT-1a can effectively reduce the susceptibility of mice to epilepsy. Western Blot experiments on the hippocampal membrane / whole protein of modeled mice further proved that the anti-epileptic effect of ZT-1a was achieved by inhibiting the activation of SPAK after convulsive stimulation, thereby revealing the regulatory mechanism of ZT-1a and WNK-SPAK-KCC2 / NKCC1 involved in epilepsy. Subsequently, cell electrophysiological experiments were performed on primary hippocampal cultured neurons at DIV14. On the basis of incubating neurons with 20uM epileptogen CTZ for 3h, the spontaneous epileptiform discharges of each neuron were detected using whole-cell patch clamp after 30min 3uM ZT-1a and 30min 10uM NKCC1 inhibitor Bumetanide (BUM) were given respectively. The experimental results showed that ZT-1a showed an inhibitory effect on epileptic burst discharges of hippocampal cultured neurons after CTZ epilepsy modeling, similar to the positive control group BUM. After adding ZT-1a, the proportion of epileptic neurons in primary cultured hippocampal neurons decreased, and the duration, frequency and intensity of epileptic neuron seizures were weakened, further indicating the anti-epileptic effect of ZT-1a.

[0012] Compared with the prior art, the technical effect of the present invention is positive and obvious. Different from the traditional treatment strategy of simply improving the inhibitory function of GABA, the present invention uses KCC2 as a new target for epilepsy treatment, finds its upstream regulatory signal pathway WNK-SPAK-KCC2 / NKCC1, and provides the use of SPAK inhibitor ZT-1a in the preparation of anti-epileptic drugs. ZT-1a can achieve multiple regulation of downstream KCC2 and NKCC1 proteins by inhibiting the activation of upstream SPAK kinase, reducing or even reversing the damage of the disease to the inhibitory function of GABA, and can effectively prevent the occurrence and development of epilepsy. Compared with traditional pan-protein kinase inhibitors, ZT-1a has a stronger inhibitory effect on SPAK protein and fewer side effects, so it is of great value in drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The effect of ZT-1a on epileptic behavioral seizures in PTZ-induced C57 / BL6 mice; 20mM1uL ZT-1a or an equal amount of DMSO was injected into the lateral ventricle of the mice for PTZ cumulative modeling experiments. Compared with the control group, ZT-1a significantly reduced the susceptibility of mice to epileptic seizures. Figure a shows that ZT-1a inhibited the proportion of IV / V grade epileptic seizures in mice at various doses of PTZ, Figure b shows that the cumulative PTZ dose required for mice in the ZT-1a experimental group to reach IV / V grade seizures was significantly higher than that in the control group, Figure c shows that ZT-1a inhibited the intensity of epileptic behavior in mice at various doses of PTZ, and Figure d shows that ZT-1a inhibited the epileptic behavior of mice at an initial PTZ concentration of 30mg / kg.

[0014] Figure 2The effect of ZT-1a on the expression of specific membrane proteins in the hippocampus. 24 hours after the acute PTZ epilepsy modeling experiment, the membrane proteins of the left and right hippocampus of the ZT-1a experimental group and the DMSO control group were extracted, and the blank control group and the group only given ZT-1a were added for Western Blot experiments. Figure a shows the original bands of membrane protein immunoblotting of WNK-SPAK-KCC2 / NKCC1 pathway proteins KCC2, SPAK, NKCC1 and p-KCC2, p-SPAK, p-NKCC1 in the DMSO+Saline group, DMSO+PTZ group, ZT-1a group and ZT-1a+PTZ group. Figure b shows the statistical graph of KCC2 membrane protein expression. The expression of KCC2 membrane protein in the DMSO+PTZ group was significantly reduced compared with that in the DMSO+Saline group, while the administration of ZT-1a before convulsive stimulation could alleviate the PTZ-induced decrease in KCC2 expression level, and the administration of ZT-1a alone had little effect on the expression of KCC2 membrane protein. Figure c shows the statistical graph of SPAK membrane protein expression. After PTZ convulsive stimulation, the expression of SPAK membrane protein in the DMSO+PTZ group increased significantly, and ZT-1a could reverse the PTZ-induced increase in SPAK protein level. Figure d is a statistical graph of NKCC1 membrane protein expression. Compared with the DMSO+Saline group, the NKCC1 membrane protein expression in the DMSO+PTZ group increased significantly, and ZT-1a could also reverse the PTZ-induced increase in NKCC1 membrane protein expression. Figure e is a statistical graph of p-KCC2 membrane protein expression. In the statistics of the ratio of KCC2p-T1007 phosphorylated protein to the total expression of membrane KCC2, the phosphorylation level of KCC2p-T1007 in the DMSO+PTZ group increased significantly, and the increase in phosphorylation level could be inhibited by ZT-1a. Figure f is a statistical graph of p-SPAK membrane protein expression. After PTZ convulsive stimulation, the phosphorylation level of SPAKp-S373 increased significantly, which could also be inhibited by ZT-1a. Figure g is a statistical graph of p-NKCC1 membrane protein expression. Similarly, the phosphorylation level activated by convulsive stimulation in the DMSO+PTZ group could be inhibited by the early administration of ZT-1a.

[0015] Figure 3The effect of ZT-1a on the expression of specific whole proteins in the hippocampus. The sample source of whole proteins is the same as that of membrane proteins. Figure a shows the original bands of whole protein immunoblotting of WNK-SPAK-KCC2 / NKCC1 pathway proteins KCC2, SPAK, NKCC1 and p-KCC2, p-SPAK, p-NKCC1 in the DMSO+Saline group, DMSO+PTZ group, ZT-1a group and ZT-1a+PTZ group. Figure b shows the statistical graph of KCC2 whole protein expression. Similar to membrane proteins, the expression of KCC2 whole protein in the DMSO+PTZ group was significantly reduced compared with the DMSO+Saline group, and the reduced KCC2 expression could be alleviated by ZT-1a. Figure c shows the statistical graph of SPAK whole protein expression. After PTZ convulsive stimulation, the expression of SPAK protein in the DMSO+PTZ group was significantly increased, and ZT-1a could reverse the increase in SPAK protein level induced by PTZ. Figure d shows the statistical graph of NKCC1 whole protein expression. Compared with the DMSO+Saline group, the NKCC1 protein expression in the DMSO+PTZ group was significantly increased, and ZT-1a could also reverse the PTZ-induced increase in NKCC1 protein expression. Figure e is a statistical chart of p-KCC2 protein expression. In the statistics of the ratio of KCC2p-T1007 phosphorylated protein to the total expression of membrane KCC2, the phosphorylation level of KCC2p-T1007 in the DMSO+PTZ group was significantly increased, and the increase in phosphorylation level could be inhibited by ZT-1a. Figure f is a statistical chart of p-SPAK protein expression. After PTZ convulsive stimulation, the phosphorylation level of SPAKp-S373 was significantly increased, which could also be inhibited by ZT-1a. Figure g is a statistical chart of p-NKCC1 protein expression. Similarly, the phosphorylation level activated by convulsive stimulation in the DMSO+PTZ group could be inhibited by the early administration of ZT-1a.

[0016] Figure 4 The effect of ZT-1a on abnormal epileptiform discharges of cultured hippocampal neurons induced by CTZ. Figure a shows the original spontaneous discharge activity of neurons in the normal untreated, CTZ, CTZ+ZT-1a, and CTZ+BUM experimental groups clamped at -70mV under the current clamp mode, and the right side is an enlarged view of the relevant area on the left. Figure b shows that ZT-1a and the positive control group BUM can significantly reduce the proportion of CTZ-induced epileptiform neurons. Figure c shows that ZT-1a can significantly reduce the duration of a single burst of CTZ-induced epileptiform neurons. Figures d and e show that compared with the control group neurons induced by pure CTZ, the discharge frequency and the number of action potentials per unit burst time of CTZ-induced epileptiform neurons after incubation with ZT-1a and BUM were significantly reduced. DETAILED DESCRIPTION

[0017] In order to better understand the essence of the present invention, the application of ZT-1a in regulating the WNK-SPAK-KCC2 / NKCC1 pathway in anti-epileptic applications was explored. An epilepsy model was established in an in vitro cultured hippocampal neuron cell model and an in vivo experimental animal model to explore the mechanism by which the WNK-SPAK-KCC2 / NKCC1 pathway affects the abnormal discharge of hippocampal neurons.

[0018] Experimental materials and equipment

[0019]

[0020]

[0021]

[0022] Example 1 Cultivation of hippocampal neurons

[0023] After deep anesthesia of female SD rats at 17-19 days of pregnancy, cut the abdomen in a V shape, remove the embryo, and quickly place it on pre-cooled dissection fluid. Under a microscope, peel off the hippocampus tissue of the fetus in a frozen environment. Transfer the hippocampus tissue to 0.05% trypsin-EDTA, digest it with trypsin in an incubator at 37°C for 15-20 minutes, add D12F medium containing 10% serum to terminate the reaction, and repeatedly wash the enzyme solution with D12F to count the cells. Select 40k / cm according to experimental requirements. 2 The cells were seeded at a density of 100 μg / mL on a glass slide containing 0.1 mg / mL Poly-D-lysine. The medium was replaced halfway the next day and 3 days later. About 6 days later, the glial cells were checked to see if they covered the glass slide. After this operation, half the medium was replaced every three days. The cells cultured for DIV13-17 were used for electrophysiological patch clamp recording.

[0024] Example 2 After cultured hippocampal neurons were incubated with CTZ (20 μM for 3 h), the electrical activity of neurons was recorded using a patch clamp:

[0025] In the whole-cell recording of primary cultured hippocampal neurons, Axonpatch 700B data acquisition amplifier and AxonDigidata 1440A digital-to-analog converter were used to collect data, and pClamp 10.3 was used to analyze the collected data. The glass electrode used in the recording process was made by a P-97 microelectrode puller, and the temperature control and rate were set to keep the resistance of the glass electrode at about 3-5MΩ after filling the electrode with liquid. The cell sheet was taken out of the cell room and placed in the extracellular fluid, and then placed in the chamber of the Leica inverted phase contrast microscope. The hippocampal neurons adhered to the cell sheet can be seen by the naked eye, and cells with transparent cell bodies and triangular shapes were selected as recording objects. After the glass electrode is filled with the internal solution and is ready to enter the water, a certain positive pressure is applied through the needle to prevent the tip of the electrode from being blocked by foreign matter. Then the lens is raised to find the electrode, and the electrode and the lens are moved down together to move the electrode to the top of the selected cell. Then, by observing the electrode resistance approaching the cell, the resistance value rises. After the value rises by about 0.2-0.3MΩ, the positive pressure is removed and a negative pressure is applied to the electrode to form a high-resistance seal with the cell. The high-resistance seal must reach more than 2GΩ. In the state of high-resistance seal, fast and slow capacitance compensation is applied, and then the cell is gently sucked to form a whole-cell recording. First, a CTZ-induced (20μM 3h) epileptic discharge model is established. 3uM ZT-1a or 10uM Bumetanide is added to the extracellular fluid 30min before recording. When recording the action potential discharge of hippocampal neurons, the cell membrane potential was clamped at -70mV by injecting current in the current clamp mode, and its spontaneous action potential was recorded in the gap-free mode. Referring to the previous published papers, this type of discharge pattern with the following characteristics is defined as epileptic discharge: in a burst discharge unit, the single depolarization contained in it exceeds 10mV and the duration exceeds 300ms and the number of action potentials exceeds five. If there are more than two burst discharge units when recording spontaneous action potentials for 10 minutes, the cell is considered to be an epileptic burst discharge cell.

[0026] Cell electrophysiological experiments were performed on primary hippocampal cultured neurons at DIV14. After incubating the neurons with 20uM epileptogen CTZ for 3h, the spontaneous epileptiform discharges of each neuron were detected using whole-cell patch clamp after 30min 3uM ZT-1a and 30min 10uM NKCC1 inhibitor Bumetanide were given. After clamping the cells and breaking the membrane to wait for the cells to stabilize for 3min, they were stably clamped at -70mV by injecting current in the current clamp mode, and then continuously recorded for 10min in the gap-free mode to observe the changes in the spontaneous epileptiform burst discharges of neurons.

[0027] The experimental results showed that ZT-1a exhibited an anti-epilepsy effect similar to that of the positive control group Bumetanide. After adding ZT-1a, the proportion of epileptiform neurons in primary cultured hippocampal neurons decreased (CTZ+ZT-1a, n=10 v.s. CTZ n=10: 40% vs 90%, P=0.027). At the same time, we further analyzed and counted the frequency and intensity of burst attacks of epileptiform neurons, and found that the average burst attack frequency of epileptiform neurons incubated with ZT-1a in the extracellular solution was significantly lower than that of epileptiform neurons incubated with CTZ alone (CTZ+ZT-1a, n=10 v.s. CTZ n=10: 0.012±0.005Hz vs 0.028±0.008Hz, P=0.049). In addition, the number of action potentials contained in the burst release per unit time was also reduced (CTZ+ZT-1a, n=10 v.s. CTZ n=10: 1.26±0.52 vs 2.40±0.354, P=0.09).

[0028] The above results indicate that ZT-1a can significantly inhibit the epileptic-like burst discharge phenomenon of hippocampal cultured neurons after CTZ epilepsy modeling, and reduce the duration, frequency and intensity of epileptic-like neurons (such as Figure 4 shown).

[0029] Example 3: Implantation of catheters in mice and acute PTZ (pentylenetetrazole) epilepsy modeling

[0030] First, C57 / BL6 mice weighing about 23-25g were subjected to lateral ventricle catheterization surgery, and an injection catheter (outer diameter 0.48mm, inner diameter 0.34mm, depth 2.2mm) was implanted using a brain stereotaxic instrument (AP: -0.5mm, ML: 1.0mm, DP: -2.1mm). After fixing the catheter with dental cement, the catheter needle and catheter cap need to be screwed in to protect the catheter to prevent food residues and bedding in the animal cage, as well as cerebrospinal fluid and brain tissue hyperplasia from clogging the catheter. After surgery, the animals need to recover in the animal room for at least 4 days before subsequent experiments can be carried out. On the day of PTZ epilepsy modeling, ZT-1a (20mM 1μL) was administered to the experimental group animals by intracerebroventricular catheterization half an hour in advance, and the control group was given an equal amount of DMSO solution. After the administration, the needle was left in place for 10 minutes to wait for drug absorption. The timing started from the completion of the administration. PTZ was administered intraperitoneally 30 minutes later. The animals injected with PTZ were observed behaviorally, and the seizure level of each animal was assessed according to the Racine standard. PTZ was administered cumulatively, with an initial dose of 30 mg / kg, and 10 mg / kg was added every 10 minutes until the mice reached Racine seizure level IV / V.

[0031] Racine Scoring Criteria:

[0032] Grade 1: Facial twitching, facial muscle tremors, accompanied by chewing movements;

[0033] Grade 2: Nodding movements, wet-dog shaking, and twitching of the neck muscles;

[0034] Grade 3: Paroxysmal spasms of unilateral forelimbs, with arched back and upright tail behavior;

[0035] Grade 4: hind limb standing and bilateral forelimb paroxysmal spasms;

[0036] Level 5: The limbs twitch violently, the whole body loses balance, and the patient rolls over.

[0037] In the whole PTZ epilepsy mouse model, ZT-1a and DMSO were injected into the lateral ventricle to verify the effect of ZT-1a on PTZ-induced epileptic behavior. The experimental results showed that at the initial dose of 30 mg / kg and the cumulative doses of 40, 50, 60, and 70, the proportion of mice reaching grade IV / V seizures in the ZT-1a group was lower than that in the DMSO control group (DMSO, n=13v.s.ZT-1a, n=15: 30 mg / kg: 15.38% vs 0.00%, P=0.087; 40 mg / kg: 38.46% vs 6.67%, P=0.051; 50 mg / kg: 76.92% vs 20.00%, P=0.003; 60 mg / kg: 92.31% vs 80.00%, P=0.289). After statistical analysis of the cumulative doses of PTZ required for the two groups of mice to reach IV / V seizures, it was found that the cumulative dose of PTZ required for the mice in the ZT-1a experimental group to reach IV / V seizures was significantly higher than that in the DMSO control group (DMSO, n = 13 v.s. ZT-1a, n = 15: 49.23 ± 3.70 vs 62.67 ± 1.94, P = 0.001). Then, we performed statistical analysis on the highest level of seizures at each cumulative PTZ dose, which showed that the highest seizure level in the ZT-1a group at 30, 40, 50, and 60 PTZ cumulative doses was significantly lower than that in the DMSO group (DMSO, n = 13 v.s. ZT-1a, n = 15: 30 mg / kg: 3.23 ± 0.19 vs 0.67 ± 0.25, P = 1.07 × 10 -8; 40mg / kg: 3.5±0.26 vs 2.4±0.23, P=0.003; 50mg / kg: 4±0.30 vs 3±0.11, P=0.001; 60mg / kg: 4.67±0.33 vs 4.14±0.22, P=0.22). And within 10 minutes after the initial injection of 30mg / kg, all 15 mice in the ZT-1a group had seizures at grade II or below, while correspondingly, 11 of the 13 mice in the DMSO group had seizures at grade III, and two even had high-grade epileptic seizures at grade IV / V.

[0038] The above results indicate that intracerebroventricular injection of ZT-1a can significantly reduce the susceptibility to epileptic seizures and the intensity of induced epileptic behavior.

[0039] The results of behavioral experiments showed that intracerebroventricular injection of ZT-1a can significantly reduce the susceptibility to epileptic seizures and the intensity of induced epileptic behavior. In order to explore the essential mechanism behind the phenomenon, we performed Western Blot experiments on the hippocampus of mice in the ZT-1a experimental group and DMSO control group 24 hours after acute PTZ epilepsy modeling, as well as the blank control group and the group only given ZT-1a, to detect the expression and phosphorylation levels of WNK-SPAK-KCC2 / NKCC1 related pathway proteins (such as Figure 1 shown).

[0040] Total / membrane protein extraction from hippocampal tissue:

[0041] 24 hours after the acute PTZ epilepsy modeling experiment, the mice were anesthetized, and the flexor reflex of the mice was tested to determine whether they were deeply anesthetized. The chest cavity was cut open to expose the heart, and a perfusion circuit was established. Pre-cooled artificial cerebrospinal fluid was used for slow perfusion to flush the blood in the brain blood vessels. After the perfusion was completed, the mice were quickly decapitated, the left and right hippocampi were removed, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator for long-term storage.

[0042] When extracting tissue protein, pre-mix the Homo buffer in the protein extraction kit with the protease / phosphatase inhibitor cocktail at a ratio of 1:500. Cover the bottom of the homogenization tube with homogenization beads and pre-cool it on ice. Add the tissue and 800uL pre-mixed lysate to the homogenization tube, quickly homogenize it in an automatic homogenizer at a speed of 42r / min for 10s, immediately insert it back to ice to cool, and then replace the homogenization, allowing twice. Let it stand for 5min, draw the supernatant and foam into a new EP tube, take 300uL of it and centrifuge it at 4℃, 12000rpm for 15min, and take the supernatant as the extracted whole protein. The remaining supernatant and foam are first centrifuged at 4℃, 700rcf for 10min, and after extracting the supernatant, centrifuge it at 4℃, 10000rcf for 30min. Keep the precipitate and mix it with 220uL PBST to obtain the extracted membrane protein. Take out 10uL of the extracted whole protein and dilute it 4 times with HomoBuffer lysis buffer, and use the BCA kit together with the membrane protein stock solution to quantify the protein, and calculate the concentration and the loading volume of 10ug according to the standard protein curve. After adding 1 / 4 of the volume of 5×Loading Buffer to the remaining whole protein, the whole protein is inactivated in a 95℃ water bath for 5min, and the membrane protein is inactivated in a 45℃ water bath for 45min, and stored in a -80℃ refrigerator for a long time.

[0043] Western Blot Protein Immunoblotting:

[0044] (1) Electrophoresis: First, prepare 8% polyacrylamide gel in advance according to the recipe in the Bio-Tech gel kit. Assemble the gel plate into the electrophoresis tank and pour in the diluted electrophoresis solution. Take out the protein and centrifuge it. Carefully pull out the comb and add the sample to the comb hole. First set the constant voltage to 70V for electrophoresis. After the protein is compressed into a straight line and runs through the concentrated gel, adjust the voltage to 100V until it runs to the bottom of the gel.

[0045] (2) Transfer: During electrophoresis, prepare 1 L of transfer buffer and refrigerate at 4°C. The transfer buffer formula is as follows: 3.03 g Tris, 14.4 g Glycine, 200 mL methanol, 800 mL ddH 2 O. Cut the corners of the PVDF membrane (pore size 0.45um) and place it in methanol to activate it until it is translucent (about 3 minutes), then soak it in 4°C transfer solution together with the sponge and filter paper for at least 10 minutes. Assemble it in the order of black side of the transfer clip-sponge-filter paper-glue-PVDF membrane-filter paper-sponge-white side of the transfer clip, place it in the transfer tank, pour in the transfer buffer, put it in the built-in ice box, and set the parameters to a constant current of 300mA for 90 minutes.

[0046] (3) Blocking: Use TBST (1×TBS+0.5‰ Tween20) to prepare 5% skim milk powder as the blocking solution, and use 5% fetal bovine serum BSA to block phosphorylated proteins. After taking out the membrane from the transfer tank, place it in the blocking solution and shake it slowly at room temperature for 2 hours.

[0047] (4) Incubation: Primary antibody: After blocking, dilute the primary antibody with the corresponding 5% skim milk powder / BSA, soak the membrane in it, and incubate overnight at 4°C. Secondary antibody: On the second day, place the strips in TBST buffer and wash them quickly on a shaker three times, each time for 15 minutes. Dilute the secondary antibody with 5% skim milk powder / BSA (ratio of 1:5000), place the washed strips in it, and shake slowly at room temperature for 1.5 hours.

[0048] (5) Development: After the secondary antibody incubation, wash the strips quickly in TBST for 3 times, 15 min each time. After washing, place the membrane face up on a clean surface, take 200uL of developer and evenly drop it on the strips. After reacting in the dark for 2 min, place it in a developer for exposure.

[0049] like Figure 2 , 3 As shown in the figure, the experimental results show that PTZ convulsive stimulation can significantly increase the phosphorylation activation levels of hippocampal membrane / whole protein pSPAK (Ser373), pKCC2 (Thr1007) and pNKCC1 (Thr212), and the activation of pSPAK (Ser373) and pNKCC1 (Thr212) can enhance the function of the corresponding proteins. On the contrary, the activation of pKCC2 (Thr1007) inhibits the transport capacity of KCC2. At the same time, the overactivation of the WNK-SPAK-KCC2 / NKCC1 pathway can be significantly inhibited by ZT-1a. From the perspective of protein expression level, 24 hours after PTZ convulsive stimulation, the expression levels of SPAK and NKCC1 proteins increased significantly, while KCC2 decreased significantly, and ZT-1a can also reverse this change.

[0050] The above results show that ZT-1a can not only reverse the excessive activation of pKCC2 (Thr1007) and pNKCC1 (Thr212) caused by the activation of pSPAK (Ser373) after PTZ convulsive stimulation, improve the transport capacity of KCC2, but also alleviate the decrease in KCC2 expression level. ZT-1a can enhance the function of KCC2 under pathological conditions with impaired Cl- transport and alleviate the decrease in KCC2 expression level, alleviate the damage of convulsive stimulation to the inhibitory function of GABA, and achieve the purpose of treating epilepsy symptoms.

[0051] ZT-1a reduced the phosphorylation activation level of the WNK-SPAK-KCC2 / NKCC1 signaling pathway in the hippocampus of mice with acute epilepsy induced by PTZ.

[0052] The present invention clarifies a mechanism that mediates the occurrence and development of epilepsy pathology, verifies one of the important mechanisms of epilepsy induction when the WNK-SPAK-KCC2 / NKCC1 signaling pathway expressed by central hippocampal neurons is activated, and establishes the therapeutic effect of the novel SPAK inhibitor ZT-1a on epilepsy. The present invention is studied on the overall PTZ-induced epilepsy mouse model, and the research results clearly show that lateral ventricular injection of ZT-1a can significantly reduce the susceptibility to epileptic seizures and reduce the intensity of induced epileptic behavior. Subsequently, two groups of experimental hippocampal samples 24 hours after acute PTZ epilepsy modeling were extracted for Western Blot experiments, and the molecular regulatory mechanism behind the behavioral differences was clarified: after PTZ convulsive stimulation, the phosphorylation activation levels of pSPAK (Ser373), pKCC2 (Thr1007) and pNKCC1 (Thr212) of the mouse hippocampal membrane / whole protein can be significantly inhibited by ZT-1a, enhancing the transport function of KCC2 and alleviating the decline in the expression level of KCC2, alleviating the damage of convulsive stimulation to the inhibitory function of GABA, and achieving the purpose of treating epileptic symptoms. Further, using the in vitro cultured neuron model, a CTZ-induced epileptiform discharge model was established. 30 minutes before recording, 3uM ZT-1a was added to the extracellular fluid, the cell membrane potential was clamped at -70mV, and its spontaneous action potential was recorded to observe the changes in the proportion of epileptiform neurons and the frequency and intensity of power generation. The experiment proved that ZT-1a can significantly inhibit the epileptiform burst discharge phenomenon of hippocampal cultured neurons after CTZ epilepsy modeling, and reduce the duration, frequency and intensity of epileptiform neurons.

[0053] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and equivalent changes made by using the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Application of SPAK inhibitor ZT-1a in the preparation of drugs for treating epilepsy, the structural formula of which is shown below:

2. Use of potassium-chloride co-transporter and potassium-sodium-chloride co-transporter as targets in screening drugs for the treatment of epilepsy.

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