Application of luteolin in preparation of medicine for improving cognitive impairment induced by corticosterone

Abstract

The invention discloses an application of luteolin in preparation of a medicine for improving cognitive impairment induced by corticosterone (Cort), and relates to a medicine for improving cognitive impairment induced by corticosterone (Cort). By constructing a mouse cognitive impairment model induced by chronic Cort, research proves that the luteolin can remarkably improve the spatial learning and memory ability of a model mouse. The action mechanism is related to a multi-target synergistic effect, and mainly comprises the steps of relieving oxidative stress injury, inhibiting hippocampal neuron apoptosis, adjusting the autophagy level and enhancing synaptic plasticity. The new application and multi-mechanism synergistic effect of the luteolin for improving the Cort-induced cognitive impairment are defined, and a new technical scheme is provided for developing safe and effective cognitive impairment treatment medicines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a new use of luteolin in improving Cort-induced cognitive impairment in mice, and an action mechanism thereof for reducing oxidative stress, inhibiting central neuron apoptosis and excessive autophagy, and improving synaptic plasticity. BACKGROUND

[0002] Cognitive impairment is a kind of nervous system disease with learning and memory ability decline and cognitive function decline as core features, covering stress-related cognitive impairment, senile dementia, vascular cognitive impairment and other types, which seriously affects the daily life ability and quality of life of patients. The currently clinically used cognitive improvement drugs have defects such as limited efficacy, obvious side effects, and narrow application range, and most of the drugs only target a single pathological link, which is difficult to cope with the complex pathogenesis of cognitive impairment. Therefore, it has important clinical value and application prospect to develop new drugs with safe and effective multi-target synergistic effect.

[0003] Corticosterone (Cort) as the main glucocorticoid in mammals plays a key role in regulating stress response, metabolism and immune function. Under normal physiological concentration, Cort is of great significance to the development and function maintenance of the nervous system, but long-term or excessive Cort can cause central nervous system dysfunction, especially selective damage to the hippocampus, and then induce cognitive impairment.

[0004] Luteolin (LUT) is a small molecule flavonoid compound, and its structure is as follows:

[0005]

[0006] Luteolin has anti-inflammatory, antioxidant, antitumor and other pharmacological activities. LUT belongs to a small molecule compound with high fat solubility, which can easily penetrate the blood-brain barrier. Studies have shown that LUT can relieve cognitive dysfunction in epileptic model mice by inhibiting neuroinflammation and reducing oxidative stress. However, there is no report that Luteolin has an improving effect on Cort-induced cognitive impairment. SUMMARY

[0007] In view of the problems in the prior art that Cort-induced cognitive impairment lacks multi-target synergistic intervention drugs, the treatment effect is limited, and the side effects are obvious, and the application scene of luteolin has not been expanded to the field of cognitive impairment, the present application provides an application of luteolin in preparing a drug for improving Cort-induced cognitive impairment. Further, the present application also provides a drug for improving Cort-induced cognitive impairment with luteolin as an effective ingredient.

[0008] This invention, through extensive modern pharmacological research, pharmacodynamic experiments, and behavioral tests, demonstrates that luteolin has the following effects:

[0009] 1. By synergistically alleviating oxidative stress, inhibiting neuronal apoptosis, regulating autophagy levels, and enhancing synaptic plasticity, Cort-induced cognitive impairment is improved;

[0010] 2. It can alleviate oxidative stress, including increasing SOD activity and reducing MDA content;

[0011] 3. It can inhibit neuronal apoptosis, including by increasing the Bcl-2 / Bax ratio;

[0012] 4. It can regulate autophagy, including reducing the LC3-II / LC3-I protein ratio in the hippocampus and increasing the expression of p62 protein;

[0013] 5. It can enhance synaptic plasticity, including upregulating the protein expression of SYP and PSD-95.

[0014] The behavioral testing methods used to assess the effect of luteolin on cognitive impairment described in this invention include one or more of the Morris water maze test and the new object recognition test.

[0015] Based on the above research findings, it has been demonstrated that luteolin can significantly improve the application value of Cort-induced cognitive impairment. At the same time, it has been revealed that luteolin can exert its effects by simultaneously regulating multiple pathological pathways such as oxidative stress, apoptosis, autophagy and synaptic plasticity, providing a new approach for developing safe, effective and multi-targeted drugs for the treatment of cognitive impairment.

[0016] Furthermore, the administration method of luteolin in the drug for improving Cort-induced cognitive impairment described in this invention is by gavage.

[0017] Furthermore, the dosage form of the drug for improving Cort-induced cognitive impairment described in this invention is any one of an oral formulation or an injectable formulation suitable for animals or humans.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention reveals for the first time that luteolin has a significant ameliorative effect on Cort-induced cognitive impairment, expanding its application areas. Specifically, at the behavioral level, it shortens the escape latency of Cort-induced mice, increases the number of times mice cross platforms, improves the time and percentage of distance traveled in the target quadrant, and enhances spatial learning and memory abilities. At the cellular and molecular level, it effectively increases SOD activity, reduces MDA content to inhibit oxidative stress, upregulates the Bcl-2 / Bax ratio to inhibit neuronal apoptosis, reduces the LC3-II / LC3-I ratio and increases p62 protein expression to restore autophagy function, and upregulates SYP and PSD-95 protein expression to improve synaptic plasticity. This invention, through systematic experiments, clarifies the novel uses and multi-mechanism synergistic effects of luteolin in improving Cort-induced cognitive impairment, demonstrating broad application prospects and providing a new and effective means for the prevention and treatment of cognitive impairment. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the escape latency of mice during the navigation phase in a water maze experiment. Figure 1 A is a bar chart showing the escape latency of mice. Figure 1 B is a representative trajectory diagram of the positioning navigation period.

[0021] Figure 1 The results showed that, compared with the control group, the escape latency of mice in the Cort group was significantly prolonged. ## P<0.01), while the escape latency of mice in the LUT group was significantly shorter than that in the Cort group (**P<0.01).

[0022] Figure 2 This is a schematic diagram of the spatial exploration experiment in the water maze experiment, in which... Figure 2 A is a bar chart showing the average swimming speed of mice. Figure 2 B is a bar chart showing the number of mouse platform crossings. Figure 2 C is a bar chart showing the percentage of time mice spent swimming in the target quadrant. Figure 2 D is a bar chart showing the percentage of swimming distance of mice in the target quadrant.

[0023] Figure 2 A showed that there was no significant difference in swimming speed among the groups of mice, and their motor abilities were not affected. Figure 2 BD showed that, compared with the control group, the number of platform crossings was significantly reduced, and both the percentage of time spent and the percentage of distance traveled in the target quadrant were significantly lower. ## P<0.01), while the latency period of mice in the LUT-treated group was significantly shorter than that in the Cort group, the number of crossings was significantly increased, and the percentage of time spent in the target quadrant and the percentage of distance traveled were significantly increased (*P<0.05, **P<0.01).

[0024] Figure 3This is a schematic diagram showing the MDA content and SOD activity in the hippocampus of mice in each group. Figure 3 A is a bar chart showing the MDA content in the mouse hippocampus. Figure 3 B is a bar chart of SOD activity in mouse hippocampus tissue.

[0025] Figure 3 The results showed that, compared with the control group, the MDA content in the hippocampus of mice in the Cort group was significantly increased. ## P<0.01), SOD activity decreased significantly ( ## P<0.01), while the MDA content in the hippocampus of mice in the LUT-treated group was significantly reduced (**P<0.01), and the SOD activity was significantly increased (**P<0.01).

[0026] Figure 4 This diagram illustrates the expression of Bcl-2 and Bax proteins in the hippocampus of mice in each group. Figure 4 A is the Western blot image of the protein. Figure 4 B is a bar chart showing the relative expression levels of proteins.

[0027] Figure 4 The results showed that, compared with the control group, the ratio of the related protein Bcl-2 / Bax in the hippocampus of mice in the Cort group was significantly reduced. ## P<0.01). Meanwhile, the Bcl-2 / Bax ratio in the hippocampus of mice in the LUT-treated group was significantly increased (*P<0.05, **P<0.01).

[0028] Figure 5 This diagram illustrates the expression of p62 and LC3 proteins in the hippocampus of mice in each group. Figure 5 A is the Western blot image of the protein. Figure 5 B and C are bar charts showing the relative expression levels of proteins.

[0029] Figure 5 The results showed that, compared with the control group, the expression of p62 protein in the hippocampus of mice in the Cort group was significantly reduced. ## P<0.01), the ratio of LC3Ⅱ / LC3Ⅰ increased significantly ( ## P<0.01), while the expression level of p62 protein in the hippocampus of mice in the LUT-treated group was significantly increased (**P<0.01), and the ratio of LC3Ⅱ / LC3Ⅰ was significantly decreased (**P<0.01).

[0030] Figure 6 Images show the expression of SYP and PSD-95 proteins and Golgi staining in mouse tissues from each group. Figure 6 A is the Western blot image of the protein. Figure 6 B and C are bar charts showing the relative expression levels of proteins. Figure 6D is the Golgi staining result of the hippocampus of each group of mice, which shows the panoramic view of Golgi staining in the mouse hippocampus, and the dendritic spine density of the CA1, CA3 and DG regions of the mouse hippocampus, respectively.

[0031] Figure 6 The results showed that, compared with the control group, the expression of synaptic-related proteins SYP and PSD-95 in the hippocampus of Cort group mice was significantly reduced, the density of dendritic spines in CA1, CA3 and DG regions was decreased, and the branches were thinner and fewer. ## P<0.01), while in the LUT-treated group, the expression of SYP and PSD-95 proteins in the hippocampus of mice was significantly increased, the density of dendritic spines in the CA1, CA3 and DG regions was increased, and the branches were thicker and more numerous (*P<0.05, **P<0.01). Detailed Implementation

[0032] To further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention. They should not be construed as limiting the present invention.

[0033] The following are some of the pharmacodynamic tests and results of this invention:

[0034] Part 1: Effects of LUT on Cort-induced cognitive impairment in mice

[0035] Eighty-four male SPF-grade C57BL / 6J mice, 6 weeks old and weighing 18-20g, were purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd. After one week of acclimatization, the mice were randomly divided into six groups of 14 mice each: a control group (Ctrl), a Cort group, low, medium, and high dose LUT groups (10, 20, and 40 mg / kg), and a fluoxetine (Flu) group. Except for the control group, the other five groups received subcutaneous injections of Cort solution (20 mg / kg) for 21 consecutive days. Starting on day 8 of Cort injection, LUT solution (10, 20, and 40 mg / kg) was administered by gavage, and Flu solution (10 mg / kg) was administered intraperitoneally for 21 consecutive days.

[0036] After drug administration, behavioral experiments were conducted using the Morris water maze test to determine if cognitive impairment occurred in the mice. The water temperature in the pool was maintained at 20±2℃. Before the experiment, 0.5g / L of titanium dioxide powder was added to the water to make the maze background white and opaque, facilitating the tracking of the mice's movements. The maze was divided into four quadrants, with different shaped patterns evenly distributed on the inner wall of the pool as spatial references. In the spatial positioning training, a hidden platform was fixed at the center of the target quadrant. Mice entered the water from preset points in the four quadrants, 1cm below the water surface, and were allowed to freely explore and find the platform within 60 seconds. The experiment ended when the mouse stayed on the platform for 5 seconds; the time from entry into the water to landing on the platform was called the escape latency. If the platform was not found within 60 seconds, the mice were manually guided and allowed to stay for 10-20 seconds; this latency was recorded as 60 seconds. Each mouse underwent training 4 times a day, with 20-minute intervals between each training session, for 5 consecutive days. After the experiment, the mice were dried and placed in cages lined with heating pads to prevent hypothermia. The SmartV3.0 water maze image acquisition system was used to track and record the movement trajectory of mice through a camera, and the data analysis system calculated the average escape latency of the daily experiments.

[0037] In the spatial memory test phase, mice were placed in a pool without hidden platforms for a single exploration experiment. Mice entered the water from a pre-defined position in the quadrant opposite to the target quadrant, initially facing the pool wall, and were allowed free exploration for 60 seconds. A behavior tracking system automatically collected data on the animals' swimming path, quadrant dwell time distribution, total swimming distance, frequency of entry into the target quadrant, and number of virtual platform crossings during the test, used to quantitatively assess the mice's spatial memory retention ability. Experimental results are as follows: Figure 1 , 2 As shown.

[0038] Experimental results: Compared with the control group, the escape latency of mice in the Cort group was significantly prolonged, the number of platform crossings was significantly reduced, and the percentage of time spent and distance traveled in the target quadrant was significantly decreased. ## P<0.01). Mice in the LUT-treated group had a significantly shorter latency period and a significantly increased number of crossings compared to the Cort group, with a significantly higher percentage of time spent in the target quadrant and a significantly higher percentage of distance traveled (*P<0.05, **P<0.01). In the behavioral assessment of the spatial exploration experiment, there were no significant differences in swimming speed among the groups, and motor ability was not affected.

[0039] Experimental conclusion: Cort administration can impair spatial learning and memory abilities in mice, while LUT can improve Cort-induced spatial learning and memory impairment in mice.

[0040] Part 2: Effects of LUT on Cort-induced cognitive impairment and oxidative stress in mice

[0041] Immediately after the behavioral tests were completed, tissue sampling was performed. One mouse from each of the Ctrl, Cort, LUT (40 mg / kg), and Flu groups was selected. Blood was collected, and the mice were euthanized by cervical dislocation. The whole brain was then removed and fixed in fixative for at least 48 hours, followed by Golgi staining. The remaining mice were euthanized by cervical dislocation, and the hippocampuses were immediately removed and stored at -80°C for subsequent SOD, MDA, and Western blot analysis.

[0042] Hippocampal tissue was removed from an ultra-low temperature freezer (-80℃) and diluted to nine times its total volume with physiological saline at a 1:9 ratio of mass (g) to volume (mL). The tissue was then subjected to ultrasonic lysis to promote disruption, followed by centrifugation at low temperature, and the supernatant was collected for later use. MDA and SOD assays were performed according to the procedures outlined in the MDA and SOD kits (Nanjing Jiancheng Bioengineering Institute, catalog numbers: A003-1, A001-3). Protein concentrations in the hippocampal samples were measured, and the MDA content and SOD activity were calculated based on these concentrations. The experimental results are as follows: Figure 3 As shown.

[0043] Experimental results: Compared with the control group, the MDA content and SOD activity in the hippocampus of mice in the Cort group were significantly increased and significantly decreased. ## P<0.01), while the MDA content in the hippocampus of mice in the LUT-treated group was significantly reduced and the SOD activity was significantly increased (**P<0.01).

[0044] Experimental conclusion: LUT can improve Cort-induced cognitive impairment in mice by improving hippocampal peroxidation.

[0045] Part 3: Effects of LUT on apoptosis, autophagy, and synaptic plasticity in Cort-induced cognitively impaired neurons in mice.

[0046] Hippocampal tissue was removed from an ultra-low temperature freezer (-80℃). A lysis buffer was prepared using a mixture of RIPA:protease inhibitor:phosphatase inhibitor (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P1081) at a ratio of 100:1:1. The lysis buffer was added at a ratio of tissue weight to lysis buffer volume of 1:9. Two grinding balls were added to the grinding tube, which was then placed in a pre-chilled tissue homogenizer for grinding at 50 Hz for 5 min. All the ground tissue was transferred to a new pre-chilled EP tube and centrifuged at 13,000 rpm for 15 min at 4℃. The protein supernatant was collected and protein quantification was performed using the BCA method. Loading buffer was added to the protein sample according to the specified ratio, and after mixing, the sample was denatured by boiling in a metal bath at 97℃ for 10 min. Load 30-50 μg of protein onto a sample for SDS-PAGE electrophoresis. Prepare a 5% stacking gel (deionized water, 30% acrylamide, 1.0 M Tris-HCl pH 6.8, 10% SDS, 10% ammonium persulfate, TEMED) and a 12% separating gel (deionized water, 30% acrylamide, 1.0 M Tris-HCl pH 6.8, 10% SDS, 10% ammonium persulfate, TEMED). Proteins were separated using Tris-HCl (pH 8.8), 10% SDS, 10% ammonium persulfate, and TEMED. The protein gel was then transferred to a methanol-activated PVDF membrane for transfer. The membrane was blocked with 5% milk powder at room temperature for 2 hours, followed by incubation with diluted Bcl-2 (1:1000), Bax (1:500), p62 (1:1000), LC3 (1:1000), PSD-95 (1:1000), SYP (1:50000), and β-Actin (1:1000) antibodies at 4°C overnight on a shaker. The next day, the corresponding secondary antibody (1:1000) was added and incubated at room temperature for 1 hour. The membrane was then washed with TBST and exposed using a gel imaging analyzer. Finally, the grayscale values ​​were analyzed using ImageJ software, and the relative expression level was calculated using β-actin as an internal reference.

[0047] The fixed brain tissue was selected from specific regions of mouse brain tissue according to experimental requirements, cut into 2-3 mm thick samples, rinsed with pre-cooled physiological saline, and transferred to 45 mL round-bottom centrifuge tubes. Golgi staining solution (Beijing Solarbio Science & Technology Co., Ltd., catalog number: G1061) was injected to immerse the samples. Staining was carried out for 14 days in a cool, dark, and well-ventilated environment, with the solution changed every 48 hours thereafter to ensure uniform and reliable staining, providing technical support for subsequent neuroanatomical analysis. After rinsing the tissue samples three times with distilled water, they were immersed in glacial acetic acid overnight to soften, rinsed again, and finally stored in sucrose solution. The tissue was cut into 100 μm pieces using a microtome, mounted on gelatin-coated slides, and air-dried overnight in the dark. The dried tissue slides were treated with concentrated ammonia for 15 min, washed with distilled water for 1 min, treated with acidic hardening fixer for 15 min, washed with distilled water for 3 min, air-dried, and mounted with glycerol gelatin. A panoramic multi-slice scan was used to obtain a panoramic image of the brain tissue. Experimental results are as follows: Figures 4-6 As shown.

[0048] Experimental results: Western blot results showed that, compared with the control group, the ratio of apoptosis-related protein Bcl-2 / Bax, the expression of autophagy-related protein p62, and synapse-related proteins SYP and PSD-95 were significantly decreased in the hippocampus of mice in the Cort group, while the ratio of LC3Ⅱ / LC3Ⅰ was significantly increased. # P<0.05, ## P<0.01), while in the LUT-treated group, the Bcl-2 / Bax ratio, p62 protein, and SYP and PSD-95 protein expression were significantly increased in the hippocampus, and the LC3Ⅱ / LC3Ⅰ ratio was significantly decreased (*P<0.05, **P<0.01). Golgi staining results showed that, compared with the control group, the density of dendritic spines in the CA1, CA3, and DG regions of the hippocampus of mice in the Cort group was decreased, and the branches were thinner and fewer. In contrast, the density of dendritic spines in the CA1, CA3, and DG regions of the hippocampus of mice treated with LUT and Flu was increased, and the branches were thicker and more numerous.

[0049] Experimental conclusion: LUT can improve Cort-induced cognitive impairment in mice. The mechanism may be related to inhibiting excessive neuronal apoptosis and autophagy and improving synaptic plasticity.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of luteolin in the preparation of a drug for improving Cort-induced cognitive impairment, wherein the structure of the luteolin is as follows:

2. Use according to claim 1, characterized in that, The luteolin improves Cort-induced cognitive impairment through the synergistic effects of relieving oxidative stress, inhibiting neuronal apoptosis, regulating autophagy level and enhancing synaptic plasticity.

3. Use according to claim 2, characterized in that, The relieving oxidative stress includes increasing the activity of superoxide dismutase (SOD) and reducing the content of malondialdehyde (MDA).

4. Use according to claim 2, characterized in that, The inhibiting neuronal apoptosis includes increasing the ratio of Bcl-2 / Bax.

5. Use according to claim 2, characterized in that, The regulating autophagy includes reducing the protein ratio of LC3-II / LC3-I in hippocampal tissue and increasing the expression of p62 protein.

6. Use according to claim 2, characterized in that, The enhancing synaptic plasticity includes up-regulating the protein expression of synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95).

7. A medicament for ameliorating Cort-induced cognitive impairment, characterized by The luteolin of claim 1 is used as an active ingredient.

8. The medicament according to claim 1, characterized in that, The administration mode is intragastric administration.

9. The medicament according to claim 7, characterized in that, The dosage form of the drug is any one of oral preparations and injection preparations suitable for animals or humans. The dosage form of the drug is any one of oral preparations and injection preparations suitable for animals or humans.

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