Use of wumei pill in the preparation of a drug for treating diabetic encephalopathy

Abstract

The application discloses application of a Fructus Mume pill in preparation of a medicine for treating diabetic encephalopathy, and finds that there is a clear correlation between the Fructus Mume pill and the diabetic encephalopathy. Research results show that the Fructus Mume pill can improve the abnormal metabolic state of a diabetic model mouse, reduce learning and memory dysfunction and cognitive impairment under a diabetic state, and has a good prevention and treatment effect on the diabetic encephalopathy. The application clearly defines the pharmacological effect of the Fructus Mume pill in preventing and treating the diabetic encephalopathy and a possible action mechanism, and provides an experimental basis for TCM intervention on the diabetic encephalopathy. The application provides a new train of thought for research and development of a medicine related to the diabetic encephalopathy, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Wumei Pill in the preparation of drugs for the treatment of diabetic encephalopathy. Background Technology

[0002] Diabetic encephalopathy (DE) is a central nervous system complication caused by long-term metabolic disorders in diabetic patients. It is mainly characterized by decreased learning and memory abilities, cognitive impairment, and emotional and behavioral abnormalities, resulting in a high disability rate and socioeconomic burden. Epidemiological studies show that diabetic patients have a significantly higher risk of cognitive impairment and dementia than non-diabetic individuals, with a markedly increased incidence of Alzheimer's disease and vascular dementia. Patients with diabetic encephalopathy often exhibit pathological changes such as hippocampal neuronal damage, synaptic dysfunction, and enhanced neuroinflammatory responses, severely impacting their quality of life. However, a unified diagnostic standard for diabetic encephalopathy is currently lacking, and its pathogenesis is not fully understood. It is generally believed to be closely related to multiple factors, including long-term hyperglycemia, insulin resistance, oxidative stress, and neuroinflammation.

[0003] Currently, the selection of drugs for treating diabetic encephalopathy is relatively limited. Clinically, treatment mainly focuses on controlling blood sugar and improving symptoms, with a lack of specific drugs targeting central nervous system damage. In some patients, simple blood sugar-lowering therapy is insufficient to effectively improve cognitive dysfunction, and long-term use of certain drugs may produce adverse reactions. For example, while insulin and oral hypoglycemic agents can improve metabolic status, their effects on existing neuronal damage and neuroinflammation are limited. Therefore, developing safe and effective novel treatments that can simultaneously regulate metabolic disorders and central nervous system damage has become an urgent problem to be solved in the prevention and treatment of diabetic encephalopathy. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides the use of Wumei pills in the preparation of a drug for treating diabetic encephalopathy.

[0006] As a preferred embodiment of the application described in this invention, the drug is used to improve abnormal glucose and lipid metabolism caused by diabetic encephalopathy.

[0007] As a preferred application of the present invention, the drug is used to improve cognitive impairment in diabetes.

[0008] As a preferred embodiment of the application described in this invention: the drug is used to alleviate hippocampal neuronal damage caused by diabetic encephalopathy and / or promote synaptic plasticity.

[0009] As a preferred embodiment of the application described in this invention: the drug is used to inhibit the abnormal activation of the CXCL1 / CXCR2 signaling pathway and reduce neuroinflammatory response.

[0010] As a preferred embodiment of the application described in this invention, the Wumei Pill is made from the following raw materials in parts by weight: 25-35 parts of dried plum, 3-9 parts of asarum, 8-12 parts of dried ginger, 3-9 parts of aconite, 2-6 parts of Sichuan pepper, 3-9 parts of cinnamon twig, 12-20 parts of coptis, 3-9 parts of phellodendron bark, 3-9 parts of ginseng, and 2-6 parts of angelica.

[0011] As a preferred embodiment of the application described in this invention, the Wumei Pill is made from the following raw materials in parts by weight: 30g of dried plum, 6g of asarum, 10g of dried ginger, 6g of aconite, 4g of Sichuan pepper, 6g of cinnamon twig, 16g of coptis, 6g of phellodendron bark, 6g of ginseng, and 4g of angelica.

[0012] As a preferred embodiment of the application described in this invention, the dosage form of the Wumei Pill is an oral preparation.

[0013] As a preferred embodiment of the application described in this invention, the drug comprises a pharmaceutically acceptable carrier and / or excipients for Wumei pills.

[0014] Beneficial effects of this invention: This invention has discovered a clear correlation between Wumei pills and diabetic encephalopathy. Research results show that Wumei pills can improve the metabolic abnormalities in diabetic model mice, alleviate learning and memory impairments, and reduce cognitive impairment in diabetic states, thus exhibiting a good preventive and therapeutic effect on diabetic encephalopathy.

[0015] While improving metabolic abnormalities, Wumei pills can alleviate hippocampal neuronal damage caused by diabetes, promote the expression of synapse-related proteins, and enhance synaptic plasticity, thus improving the pathological changes of diabetic encephalopathy from the perspectives of neural structure and function. Furthermore, Wumei pills can reduce the level of inflammation in brain tissue under diabetic conditions. This effect is related to inhibiting inflammation-related signaling pathways and reducing neuronal damage mediated by inflammatory factors, thereby playing a positive role in the prevention and treatment of diabetic encephalopathy.

[0016] This invention clarifies the pharmacological effects and possible mechanisms of action of Wumei Pill in the prevention and treatment of diabetic encephalopathy, providing experimental evidence for traditional Chinese medicine intervention in diabetic encephalopathy. This invention offers new insights for the research and development of drugs related to diabetic encephalopathy and has promising application prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein: Figure 1The results of metabolic-related indicators in each group of mice are shown.

[0018] Figure 2 The changes in cognitive function in each group of mice are shown.

[0019] Figure 3 The histopathological changes and synaptic protein expression in the hippocampus of mice in each group were analyzed.

[0020] Figure 4 The study investigated the expression levels of CXCL1 and CXCR2, as well as the changes in inflammatory factors TNF-α and IL-1β, in the hippocampus of mice in each group. Detailed Implementation

[0021] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0022] Experimental reagents and materials: Dapagliflozin (catalog number D126800) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). Mouse CXCL1 (GRO alpha) recombinant protein (250-11-5UG) was purchased from Thermo Fisher Scientific (USA). Herbal medicines were purchased from Xuzhou Hanbang Pharmaceutical Chain Co., Ltd. Antibodies including: PSD-95 (20665-1-AP), BDNF (66292-1-Ig), GFAP (60190-1-Ig), and β-Actin (66009-1-Ig) were purchased from Proteintech (China); Synapsin I (AF6201), GAP43 (DF7766), CXCR2 (DF7095), and CXCL1 (AF5403) were purchased from Affinity Biosciences (China). Secondary antibodies, including HRP-labeled goat anti-mouse IgG (H+L) (A0216) and HRP-labeled goat anti-rabbit IgG (H+L) (A0208), a mouse TNF-α ELISA kit (MB-2868A), and a mouse IL-1β ELISA kit (MB-2776A), were purchased from Jiangsu Enzyme-Label Biotechnology Co., Ltd. (China). All reagents were used strictly according to the instructions, and freshly prepared solutions were used for each experiment to ensure consistency and reproducibility.

[0023] Example 1: Preparation method of Wumei Pill: 30g dried plum, 6g asarum, 10g dried ginger, 6g aconite, 4g Sichuan pepper, 6g cinnamon twig, 16g coptis, 6g phellodendron bark, 6g ginseng, and 4g angelica. Soak the ten herbs in distilled water and then decoct. Specific preparation steps are as follows: First, add water to the aconite and asarum and decoct for 60 minutes to reduce toxicity. The water volume should be twice the weight of the herbs. Then add the other herbs and decoct for 40 minutes. Collect the decoction, add water to the dregs again and decoct for another 40 minutes. Combine the two decoctions. Filter the combined decoction through gauze, pour it into a pot, and heat over low heat to concentrate it to one-quarter of its volume to obtain the extract. Place the extract in a freeze dryer and pre-freeze at -40℃ for 4 hours. Transfer it to the freeze-drying chamber, start the vacuum pump, adjust the vacuum degree to 20 Pa, maintain the temperature at -30℃, and continue drying for 18 hours. Then, increase the temperature to 20℃ at a rate of 2℃ / h and continue drying for 6 hours to obtain the freeze-dried powder of Wumei Pill (WMP).

[0024] Experimental method of this invention: WMP Chemical Identification Based on LC-MS / MS: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used for sample quality control and compound identification. LC-MS / MS analysis was performed using a Thermo Fisher Scientific ultra-high performance liquid chromatography system (Vanquish), coupled with a Phenomenex Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm) and an Orbitrap Exploris 120 mass spectrometer. Mobile phase A was 0.01% aqueous acetic acid solution, and mobile phase B was a mixture of isopropanol and acetonitrile (1:1, v / v). The autosampler temperature was set to 4 °C, and the injection volume was 2 μL.

[0025] Mass spectrometry data were acquired using data-dependent acquisition (DDA) mode under Xcalibur software control, resulting in MS / MS spectra. Electrospray ionization (ESI) source parameters were set as follows: sheath gas flow rate of 50 Arb, auxiliary gas flow rate of 15 Arb, capillary temperature of 320 °C; full scan resolution of 60,000, MS / MS resolution of 15,000; stepped normalized collision energy (SNCE) of 20, 30, and 40; and spray voltages of 3.8 kV and -3.4 kV in positive and negative ion modes, respectively. Compound structures were identified by comparison with high-resolution mass spectrometry data, standards, and published literature.

[0026] Laboratory animals and grouping: 8-week-old males db / m and db / dbMice were purchased from Jiangsu Cavens Laboratory Animal Co., Ltd. (China). Mice were housed under SPF conditions at a temperature of 22 ± 3 ℃ and a humidity of 50 ± 5%, with a 12 / 12-hour light / dark cycle, and were given free access to standard feed and water. db / db Mice were randomly divided into four groups (n = 8 per group): 1. Model group: orally administered an equal volume of purified water; 2. Low-dose WMP group (WMP-L): orally administered WMP 3 g / kg; 3. High-dose WMP group (WMP-H): orally administered WMP 6 g / kg; 4. Positive control group (dapagliflozin): orally administered dapagliflozin 20 mg / kg. Age-matched mice were also included. db / m Mice (n = 8) served as the normal control group and were orally administered an equal volume of purified water. Umeboshi pills were suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) and administered via gavage at a dose of 0.1 mL / 10 g body weight. Mice were administered the pills once daily for 8 weeks, and body weight (BW) and fasting blood glucose (FBG) were recorded at 8, 10, 12, and 16 weeks of age. After the experiment, behavioral tests were performed, and the mice were euthanized for whole brain tissue collection. The experiment was approved by the Animal Ethics Committee of Xuzhou Medical University.

[0027] Morris Water Maze Experiment: The Morris water maze was used to assess spatial learning and memory. A circular pool was used at 25°C, divided into four quadrants, with a hidden platform submerged 1 cm below the surface. Mice were trained after one day of acclimatization. For four consecutive days, mice were placed at a random starting point and given 90 seconds to find the platform. If they failed to find it, they were guided to the platform and remained there for 20 seconds. On day 5, a probe experiment was conducted; the platform was removed, and the time spent in the target quadrant and the number of times the mice traversed the platform were recorded. After the experiment, the mice were dried and kept warm.

[0028] New and Old Object Recognition Experiment: During the training phase, two identical objects (A and B) were placed on one side of an open field. Mice were placed in the objects from behind, and each mouse was observed for 5 minutes. The exploration time and number of contacts were recorded. After 24 hours, the mouse was tested by replacing object B with a new object C, and its exploration behavior was recorded. Recognition index = (exploration time of new object) / (exploration time of new object + familiar object) × 100%.

[0029] Histopathological analysis: After the mice were sacrificed, the brain tissue was fixed in 4% paraformaldehyde for 48 hours, dehydrated and embedded in paraffin, sectioned at 3 μm, and stained with HE and Nissl stain to observe the pathological morphology of neurons in the CA1 and CA3 regions of the hippocampus.

[0030] Western blotting: Hippocampal tissue was lysed, and protease / phosphatase inhibitors were added to RIPA buffer. The mixture was centrifuged at 12,000 rpm for 15 minutes at 4 °C, and quantified using BCA. After separation by SDS-PAGE, the tissue was transferred to an NC membrane, blocked for 20 minutes, and incubated overnight at 4 °C with primary antibody (Synapsin I, GAP43, PSD-95, BDNF, CXCL1, CXCR2 1:1000; β-Actin 1:5000). The next day, the tissue was incubated with secondary antibody at 37 °C for 1 hour, developed by ECL, and quantitatively analyzed using ImageJ.

[0031] ELISA: The levels of TNF-α and IL-1β in mouse hippocampus tissue were detected according to the instructions.

[0032] Statistical analysis: Data are expressed as mean ± SEM and analyzed using GraphPad Prism 10. After verifying normality and homogeneity of variance, one-way ANOVA was used for comparisons among the three groups, and Tukey post-hoc test was used for multiple comparisons. P < 0.05 was considered statistically significant.

[0033] Example 2: Example 1 Experimental Results: Changes in body weight and fasting blood glucose levels in each group of mice: Figure 1 The results of metabolic-related indicators for each group of mice are shown. In this table, A represents the change in body weight of mice in each group, and B represents the change in fasting blood glucose levels of mice in each group. Figure 1 As shown, week 0 is the starting point of the experimental treatment (the actual age of the mice was 8 weeks). (Compared to...) db / m Compared with the control group, db / db The body weight (BW) and fasting blood glucose (FBG) levels of mice in the model group were significantly increased (P < 0.001). db / m Compared with the control group, both high-dose WMP treatment and the dapagliflozin positive control significantly reduced [the risk of infection]. db / db The levels of BW and FBG in mice (P < 0.01 or P < 0.001) indicate that WMP has a significant ameliorative effect on diabetes-related metabolic abnormalities.

[0034] Changes in cognitive function in mice of different groups: Figure 2The following charts illustrate changes in cognitive function in each group of mice: A shows the change in escape latency during Morris water maze training; B shows representative swimming trajectories during the Morris water maze training phase; C shows the average swimming speed in the Morris water maze experiment; D shows the number of platform crossings in the Morris water maze probe experiment; E shows the dwell time in the target quadrant during the Morris water maze probe experiment; F shows representative exploration trajectories during the training and testing phases of the new and old object recognition experiment; G shows the recognition index of familiar object 1 during the training phase of the new and old object recognition experiment; H shows the recognition index of familiar object 2 during the training phase of the new and old object recognition experiment; and I shows the recognition index of new objects during the memory retention phase of the new and old object recognition experiment. The Morris water maze experiment showed that, during a continuous 4-day training period, the escape latency of mice in all experimental groups gradually decreased with the extension of training time. db / m Compared with the control group, db / db The escape latency of the model group mice was significantly prolonged (P < 0.001), suggesting impaired learning and memory abilities. Conversely, the escape latency of the WMP-treated groups, especially the high-dose WMP group, and the dapagliflozin-treated group was significantly shortened (P < 0.05 or P < 0.01). Representative swimming trajectories showed reduced exploration behavior in the target quadrant of db / db mice, while WMP and dapagliflozin treatments significantly enhanced orientation to the target area. Comparison of average swimming speeds among the groups showed a slight decrease in motor ability in the model group, but there were no statistically significant differences between the WMP dose groups and the dapagliflozin group and the model group, indicating that the experimental results were not significantly affected by differences in motor ability. Further analysis revealed that, compared with the model group, high-dose WMP and dapagliflozin treatments significantly increased the number of platform crossings and the time spent in the target quadrant (P < 0.05 or P < 0.01).

[0035] In the experiment of recognizing familiar and unfamiliar objects, there was no significant difference in the recognition index of familiar objects among the mice in the training phase; however, in the memory retention phase, db / db Mice had a significantly lower recognition index for new objects than db / m Control group (P < 0.001). Compared with the model group, high-dose WMP and dapagliflozin treatment significantly improved the recognition index of new objects in mice (P < 0.05 or P < 0.01), suggesting that they can improve the recognition and memory ability of diabetic mice.

[0036] Pathological changes in hippocampal tissue of mice in each group: Figure 3The histopathological changes and synaptic protein expression in the hippocampus of mice in each group were analyzed. A shows representative HE-stained images of hippocampal tissue from each group of mice, displaying morphological changes in neurons in the CA1 and CA3 regions; B shows a statistical chart of the number of neurons in the CA1 region of the hippocampus from each group of mice; C shows a statistical chart of the number of neurons in the CA3 region of the hippocampus from each group of mice; D shows representative Nissl-stained images of hippocampal tissue from each group of mice, displaying the distribution of Nissl bodies in the CA1 and CA3 regions; E shows a statistical chart of the Nissl-positive area (or Nissl body level) in the CA1 region of the hippocampus from each group of mice; F shows a statistical chart of the Nissl-positive area (or Nissl body level) in the CA3 region of the hippocampus from each group of mice; G shows the Western blot images and grayscale statistics of the presynaptic marker protein Synapsin-1 in the hippocampus from each group of mice; H shows the Western blot images and grayscale statistics of the GAP43 protein in the hippocampus from each group of mice; I shows the postsynaptic dense protein PSD-95 in the hippocampus from each group of mice. Western blot band diagrams and grayscale statistics of BDNF protein in the hippocampus of each group of mice; J represents the Western blot band diagrams and grayscale statistics of BDNF protein in the hippocampus of each group of mice. HE staining results showed that, compared with db / m Compared with the control group, db / db In the model group mice, neurons in the CA1 and CA3 regions of the hippocampus were disordered and the number of cells was significantly reduced. After WMP intervention, especially high-dose WMP treatment, the neuronal structure in the CA1 and CA3 regions of the hippocampus of mice tended to be intact, the cell arrangement was more regular, and the number of neurons increased significantly.

[0037] Nissl staining results further indicate that, with db / m Compared with the control group, db / db The number of Nissl body positive cells in the hippocampus of mice was significantly reduced (P < 0.001), indicating impaired neuronal integrity; while intervention with WMP and dapagliflozin could significantly reverse the above changes, resulting in a significant increase in the number of Nissl body positive cells in the CA1 and CA3 regions of the hippocampus (P < 0.05 or P < 0.01).

[0038] Expression of synapse-related proteins in the hippocampus of mice in each group: Western blotting analysis showed that, compared with... db / m Compared with the control group, db / db The expression levels of presynaptic marker protein Synapsin-1, synaptic plasticity-related protein GAP43, and postsynaptic density protein PSD-95 in mouse hippocampus were significantly decreased (P < 0.001 or P < 0.01). Compared with the model group, high-dose WMP and dapagliflozin treatment significantly upregulated the expression levels of the above proteins (P < 0.05 or P < 0.01).

[0039] In addition, with db / m Compared with the control group, db / db The expression of brain-derived neurotrophic factor (BDNF) in the hippocampus of mice was significantly downregulated; while high-dose WMP and dapagliflozin treatment significantly increased the expression level of BDNF (P < 0.05 or P < 0.01), suggesting that WMP helps improve diabetes-related synaptic plasticity disorders.

[0040] Changes in the CXCL1 / CXCR2 axis and inflammatory factors in the hippocampus of mice in each group: Figure 4 This chart shows the expression levels of CXCL1 and CXCR2, as well as the changes in inflammatory factors TNF-α and IL-1β, in the hippocampus of mice in each group. Specifically: A shows the expression of CXCL1 protein in the mouse hippocampus; B shows the expression of CXCR2 protein in the mouse hippocampus; C shows the statistical results of TNF-α level detection in the mouse hippocampus; and D shows the statistical results of IL-1β level detection in the mouse hippocampus.

[0041] Western blotting results showed that, compared with db / m Compared with the control group, db / db The expression level of CXCL1 protein in mouse hippocampus was significantly increased (P < 0.001 or P < 0.01), while WMP intervention significantly downregulated its expression (P < 0.05 or P < 0.01). As a specific receptor for CXCL1, CXCR2... db / db The expression in mouse hippocampus was also significantly increased and was significantly inhibited after WMP treatment (P < 0.001 or P < 0.01).

[0042] ELISA test results further indicate that, compared with db / m Compared with the control group, db / db The levels of pro-inflammatory cytokines TNF-α and IL-1β in mouse hippocampus tissue were significantly increased (P < 0.001); while WMP intervention significantly reduced the expression levels of the above inflammatory factors (P < 0.001 or P < 0.05).

[0043] The experimental results above show that WMP can significantly improve db / db It reduces metabolic abnormalities and cognitive dysfunction in mice, alleviates hippocampal neuronal damage, improves synaptic plasticity, and reduces neuroinflammatory response by inhibiting abnormal activation of the CXCL1 / CXCR2 signaling axis, thus exerting a significant protective effect against diabetes-related brain injury, with effects comparable to those of the dapagliflozin positive control.

[0044] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of Wumei Pill in the preparation of drugs for the treatment of diabetic encephalopathy.

2. The application according to claim 1, characterized in that: The drug is used to improve abnormal glucose and lipid metabolism caused by diabetic encephalopathy.

3. The application according to claim 1 or 2, characterized in that: The drug is used to improve cognitive impairment in people with diabetes.

4. The application according to claim 1 or 2, characterized in that: The drug is used to reduce hippocampal neuronal damage and / or promote synaptic plasticity caused by diabetic encephalopathy.

5. The application according to claim 1 or 2, characterized in that: The drug is used to inhibit the abnormal activation of the CXCL1 / CXCR2 signaling pathway and reduce neuroinflammatory responses.

6. The application according to claim 1 or 2, characterized in that: The Wumei Pill is made from the following raw materials in parts by weight: 25-35 parts of dried plum, 3-9 parts of asarum, 8-12 parts of dried ginger, 3-9 parts of aconite, 2-6 parts of Sichuan pepper, 3-9 parts of cinnamon twig, 12-20 parts of coptis, 3-9 parts of phellodendron bark, 3-9 parts of ginseng, and 2-6 parts of angelica.

7. The application according to claim 6, characterized in that: The Wumei Pill is made from the following raw materials in parts by weight: 30g of dried plum, 6g of asarum, 10g of dried ginger, 6g of aconite, 4g of Sichuan pepper, 6g of cinnamon twig, 16g of coptis, 6g of phellodendron bark, 6g of ginseng, and 4g of angelica.

8. The application according to claim 1 or 2, characterized in that: The dosage form of the Wumei Pill is an oral preparation.

9. The application according to claim 1 or 2, characterized in that: The drug includes pharmaceutically acceptable carriers and / or excipients for Wumei pills.

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