Application of Tertiapin-Q in improving memory impairment of mouse with postoperative cognitive impairment

Abstract

The invention discloses an application of Tertiapin-Q in improving memory impairment of a mouse with postoperative cognitive impairment, and relates to the technical field of medicines, Tertiapin-Q is a melittin derivative and is the most specific G protein activated inward rectifying potassium (GIRK) channel inhibitor so far; researches find that Tertiapin-Q can improve excitability of neurons of mice with postoperative cognitive impairment during memory coding and improve learning and memory ability impairment caused by the postoperative cognitive impairment; the research is helpful to reveal the specific effect of the GIRK channel in memory coding and extraction, and provides a new thought and method for prevention and treatment of postoperative cognitive impairment.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of Tertiapin-Q in improving memory impairment in mice with postoperative cognitive dysfunction. Background Art

[0002] Postoperative cognitive dysfunction is a common and important complication in patients after anesthesia and surgical procedures, especially in elderly patients. Its core pathological features include glial cell activation, decreased neuronal excitability, and cognitive impairment. With the aging process, the occurrence of postoperative cognitive dysfunction is becoming more and more common, causing many medical and social problems. Therefore, finding effective prevention and treatment methods has important medical value and social significance.

[0003] G protein-activated inward rectifier potassium (GIRK) channels are widely expressed in the nervous system, among which GIRK1 / 2 subtypes are mainly distributed in the hippocampus and participate in the regulation of cognitive function. After GIRK channels are activated by G protein-coupled receptors, potassium ions are allowed to flow out, leading to cell membrane hyperpolarization, reducing neuronal excitability, and helping to maintain the excitation-inhibition balance and the stability of neural networks. Studies have shown that overactive GIRK channels cause hippocampal pyramidal neurons to show enhanced GIRK currents, leading to defects in contextual fear learning and recall in mice, causing cognitive dysfunction.

[0004] Tertiapin-Q (TPNQ) is a melittin derivative extracted from bee venom and a specific blocker of GIRK channels. Studies have shown that after TPNQ is used to inhibit GIRK channels, the number of discharges of excitatory neurons during depolarization increases, accompanied by a decrease in rheological base and an increase in membrane output impedance, which enhances the intrinsic excitability of neurons.

[0005] In summary, it is necessary to clarify the role of GIRK channel in postoperative cognitive dysfunction and to find potential therapeutic targets for the prevention and treatment of postoperative cognitive dysfunction. Summary of the invention

[0006] In order to solve the above problems, the present invention provides the use of Tertiapin-Q in improving memory impairment in mice with postoperative cognitive dysfunction.

[0007] To achieve the above object, the present invention provides the following technical solutions: Application of Tertiapin-Q (HY-P1275, MCE) in improving memory impairment in mice with postoperative cognitive dysfunction.

[0008] Furthermore, the Tertiapin-Q is used to increase the excitability of neurons during memory encoding in mice with postoperative cognitive dysfunction.

[0009] Furthermore, the Tertiapin-Q is used to improve the impaired learning and memory ability of mice with postoperative cognitive dysfunction.

[0010] Furthermore, the active ingredient of the drug is Tertiapin-Q.

[0011] Furthermore, the administration concentration of Tertiapin-Q is 0.25 mM.

[0012] Furthermore, the administration method of Tertiapin-Q is intracerebroventricular cannula administration.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The results of the present invention indicate that Tertiapin-Q has a potential improvement effect on postoperative cognitive dysfunction and memory impairment. Tertiapin-Q inhibits G protein activation of inward rectifier potassium channels, increases the excitability of neurons during memory encoding in mice, promotes the formation of engram complexes, and improves learning and memory abilities. These results provide a theoretical basis for GIRK channels as a potential therapeutic target for postoperative cognitive dysfunction (POCD).

[0014] The Tertiapin-Q of the present invention can increase the excitability of neurons during memory encoding in mice with postoperative cognitive dysfunction, improve the impaired learning and memory ability caused by postoperative cognitive dysfunction, help reveal the specific role of GIRK channels in memory encoding and retrieval, and provide new ideas and methods for preventing and treating postoperative cognitive dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Histological verification of the catheter placement in the right lateral ventricle of mice; Figure 2 The total distance traveled in the open field experiment; Figure 3 The movement speed for the open field experiment; Figure 4 This is the heat map of the motion trajectory of the open field experiment; Figure 5 It is the percentage of freezing reaction time in the training phase of the conditioned fear experiment; Figure 6 It is the percentage of freezing reaction time in the test phase of the conditioned fear experiment; Figure 7 This is the c-fos fluorescence staining image of the hippocampal CA1 area; Figure 8 Quantitative analysis of the number of c-fos positive neurons in the CA1 region of the hippocampus; Fig. 9This is the fluorescence staining image of GIRK channel in hippocampus; Fig.10 Quantitative analysis of the mean fluorescence intensity of GIRK channels in the hippocampus. DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example 1

[0017] method 1.1 Mouse modeling and grouping The mice selected in the present invention were purchased from Changzhou Cavens Experimental Animal Co., Ltd. (Changzhou, China). Thirty 15-month-old male C57BL / 6J mice were randomly divided into a control group (CV), a POCD group (PV), and a POCD+TPNQ group (PT), with 10 mice in each group. All mice underwent right lateral ventricle catheterization. The PV and PT groups were subjected to POCD modeling, while the CV group was not treated. One hour before the conditioned fear test, the PT group was given TPNQ via the lateral ventricle catheter, and the CV and PV groups were given an equal volume of control agent.

[0018] All mice underwent right lateral ventricle catheterization. Mice were anesthetized with isoflurane, and their heads were fixed on a stereotaxic instrument after the righting reflex disappeared. A heating blanket was used to maintain the body temperature of the mice during the operation, and respiration was monitored. Erythromycin ointment was applied to the eyes of the mice. The head was prepared and disinfected, and then cut open along the midline. The subcutaneous fascia was cleaned with a cotton swab to expose the anterior and posterior fontanelles and sutures. The skull was leveled, and the anterior fontanelle was used as the coordinate origin. The needle point was located 1.8 mm to the right and 1.0 mm to the back. The skull was drilled and the meninges were pierced. The injection cannula was vertically lowered 1.3 mm into the lateral ventricle with the anterior fontanelle as the Z-axis origin (AP: 1.0 mm, ML: 1.8 mm, DV: 1.3 mm). The cannula was fixed with dental cement, and skull nails were buried in the skull for reinforcement. After the operation, the needle core was inserted to prevent the cannula from being blocked. After the mice woke up on the heating plate, they were put back in the cage to recover, and the model was established one week later.

[0019] The control group was not treated, and the mice in the other groups underwent laparotomy for postoperative cognitive dysfunction modeling. The mice were anesthetized with isoflurane and placed in a supine position on a heating blanket after the righting reflex disappeared. The abdomen was prepared and disinfected, and a 2-cm incision was made along the midline of the abdomen to expose the abdominal cavity. The intestine was explored, and the cecum and the end of the ileum were left open for 10 minutes before being retracted. The peritoneum, muscles, and skin were sutured in layers using 4-0 sterile silk sutures. The total operation time was about 15 minutes.

[0020] 1.2 Open field experiment On the fifth day after modeling, the three groups of mice underwent an open field test to assess their autonomous movement ability and anxiety level. In a quiet room with appropriate lighting, the mice were placed in the center of the bottom of a 40cm x 40cm x 40cm white acrylic box without a top, and allowed to explore freely for 10 minutes. The total movement distance and movement speed of the mice were recorded using a video tracking system directly above the box. After the experiment, the box was wiped with alcohol to avoid odor interference.

[0021] 1.3 Fear Conditioning Six to seven days after modeling, the three groups of mice underwent conditioned fear experiments to evaluate their learning and memory performance. First, a training experiment was conducted. The mice were placed in a 30cm x 30cm x 40cm experimental box and explored freely for 180 seconds. They were given 70dB, 3kHz sound stimulation for 30 seconds, and combined with a 0.7mA foot shock for 2 seconds. After staying for 30 seconds, the mice were taken out and put back into the cage. After the experiment, the box was wiped with alcohol to avoid odor interference. After an interval of 24 hours, a test experiment was conducted. The mice were placed in the same experimental box for 5 minutes without any stimulation. The freezing reaction time of the mice was recorded, which was defined as no other autonomous activities except breathing. After the experiment, the box was wiped with alcohol to avoid odor interference.

[0022] 1.4 Drug intervention Before the conditioned fear training experiment, the injection tube was inserted into the cannula, and 3 µl of Tertiapin-Q (0.25 mM, HY-P1275, MCE) or control agent (PBS) was slowly injected into the lateral ventricle at a rate of 0.5 µl / min using a microinjection system. After 5 minutes, the injection tube was slowly removed to allow sufficient drug infiltration, and the conditioned fear training experiment was performed 1 hour later.

[0023] 1.5 Immunofluorescence staining After the behavioral experiment, brain tissue samples were collected. Under isoflurane anesthesia, the diaphragm and ribs were cut from the xiphoid process, the chest cavity was opened to expose the heart, the right auricle was cut open, and the scalp needle was inserted from the apex to the left ventricle, and 20 ml of pre-cooled PBS buffer was perfused. After the liver turned white, 40 ml of pre-cooled 4% paraformaldehyde was continued to be perfused, and the brain was removed by decapitation. The brain tissue was placed in 4% paraformaldehyde and fixed overnight at 4°C. The brain tissue was then transferred to 30% sucrose solution for dehydration. After sinking to the bottom, the brain tissue was wrapped with an embedding agent and placed at -80°C to prepare frozen sections for subsequent staining.

[0024] The frozen hippocampal sections were placed at room temperature to dry, washed with PBS buffer and then permeabilized, blocked with 10% calf serum at room temperature for 2 hours, incubated with primary antibody at 1:500 at 4°C overnight, washed with PBS buffer, incubated with secondary antibody at room temperature for 1 hour, washed with PBS and sealed, photographed with an Olympus laser confocal microscope, and analyzed with ImageJ software.

[0025] 1.6 Statistical analysis Graphpad Prism 9.0 software (Graphpad Software Inc.) was used for data statistics and analysis. Measurement data were expressed as mean ± standard error (mean ± SEM). Student's t test was used for pairwise comparisons, and one-way analysis of variance was used for comparisons among the three groups. Turkey multiple comparisons were used for post-tests. Differences were considered statistically significant when P value < 0.05.

[0026] 2. Results 2.1 Tertiapin-Q improves learning and memory abilities in mice with postoperative cognitive dysfunction The location of the intracerebroventricular catheter in mice Figure 1 As shown, the tip of the cannula passes through the brain tissue and enters the lateral ventricle. Figure 2 , Figure 3 and Figure 4 As shown in the figure, we tested the voluntary movement ability and anxiety level of mice. There was no statistical difference in the total movement distance and movement speed of the three groups of mice. The movement trajectory images showed that the mice in each group had similar exploratory behaviors in the central and peripheral areas, indicating that the application of Tertiapin-Q in vivo did not affect the movement and anxiety levels of mice.

[0027] like Figure 5 and Figure 6 As shown in the figure, during the conditioned fear training phase, there was no significant difference in the percentage of basal freezing reaction time of the three groups of mice during the exploration period before the foot click, eliminating the interference of the baseline. In the conditioned fear experimental test phase, compared with the CV group, the percentage of freezing reaction time of mice in the PV group was significantly reduced, indicating that the modeling caused cognitive dysfunction and affected the learning and memory ability of mice. After the administration of Tertiapin-Q, the percentage of freezing reaction time of mice in the PT group was significantly increased compared with the PV group, indicating that Tertiapin-Q has a significant improvement effect on the cognitive ability of mice with postoperative cognitive dysfunction.

[0028] 2.2 Tertiapin-Q increases neuronal excitability during memory encoding in mice with postoperative cognitive impairment like Figure 7 and Figure 8As shown, compared with the CV group, the number of c-fos-positive neurons in the CA1 region of the hippocampus in the PV group decreased, indicating that surgical anesthesia reduced the excitability of hippocampal pyramidal neurons in mice; compared with the PV group, the number of c-fos-positive neurons in the CA1 region of the hippocampus in the PT group increased, suggesting that the in vivo application of Tertiapin-Q improved the excitability of hippocampal pyramidal neurons in mice with postoperative cognitive dysfunction.

[0029] like Fig. 9 and Fig.10 As shown, there was no significant difference in the expression of GIRK2 protein in the molecular layer of the hippocampus of the three groups of mice, indicating that a single administration of GIRK channel inhibition would not change the overall expression level of the channel protein in the hippocampus.

[0030] In summary, the Tertiapin-Q used in the present invention has a positive effect on the learning and memory ability of mice with postoperative cognitive dysfunction, improves the excitability of hippocampal pyramidal neurons, and promotes the formation of memory encoding and engram complexes. At the same time, it provides a theoretical basis for GIRK channels as a potential therapeutic target for POCD.

[0031] No matter from which aspect, the above embodiments are only for the purpose of illustrating the present invention, rather than limiting it. Those skilled in the art can modify and change the embodiments without departing from the principle and spirit of the present invention, and the scope of the present invention is limited by the appended claims and their equivalents.

[0032] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0033] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. Application of Tertiapin-Q in the preparation of drugs for treating memory impairment caused by postoperative cognitive dysfunction.

2. The use according to claim 1, characterized in that: The Tertiapin-Q is used to enhance the excitability of neurons during memory encoding in mice with postoperative cognitive dysfunction.

3. The use according to claim 1, characterized in that: The Tertiapin-Q is used to improve the impaired learning and memory abilities of mice with postoperative cognitive dysfunction.

4. The use according to claim 1, characterized in that: The active ingredient of the drug is Tertiapin-Q.

5. The use according to claim 1, characterized in that: The administration concentration of Tertiapin-Q was 0.25 mM.

6. The use according to claim 1, characterized in that: The administration method of Tertiapin-Q is intracerebroventricular cannula administration.

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