CN111617059B - Curcumadione application - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of curcuma zedoary diketone. The invention provides application of curdione, and experimental results show that curdione can effectively inhibit microglial activation and secretion of inflammatory factor TNF-alpha, control neuroinflammatory reaction, thereby relieving neuron injury caused by subsequent inflammatory factors, improving cognitive function, memory disorder and motor coordination ability of brain tissues, and can be used for Alzheimer's disease. The curcuma zedoary diketone is applied to preparing the medicine for preventing and/or treating the Alzheimer's disease, and has good application value and development prospect.

Description

Curcumadione application

technical field

The invention belongs to the technical field of medicine, and in particular relates to the application of curcumadione.

Background technique

Alzheimer's disease (AD) is a slowly degenerative disease of the central nervous system and is the most common form of senile dementia. The specific pathogenesis of Alzheimer's disease is still unclear. The current mainstream theory is that the abnormally deposited Aβ in the brain of patients directly or indirectly acts on neurons and microglia through a series of cascade reactions such as free radical reaction, mitochondrial oxidative damage, and inflammatory response, eventually leading to neuron death. At the same time, the activated microglia induces an inflammatory response and activation of astrocytes when cleaning up dead neurons, leading to more neuron death and forming a vicious cycle, eventually leading to Alzheimer's disease. According to a report, there were about 35.6 million AD patients in the world in 2010. At present, the number of Alzheimer's patients in the world has exceeded 47 million, and the number of Alzheimer's patients in my country has exceeded 10 million. It is estimated that by 2050, the number of AD patients will reach 115 million. The main clinical features of AD patients include progressive memory impairment, cognitive impairment, behavioral impairment, and decreased ability of daily living. Therefore, it is very important to detect early and delay or prevent the occurrence and development of such diseases.

The inflammatory response mediated by microglia and astrocytes is an important link in the pathological process of neuroinflammatory diseases. The stable maintenance of the physiological function and state of the brain is inseparable from the glial cells in the brain, and neuroinflammation mainly occurs in the inflammation of the brain tissue, which is caused by the excessive activation of the glial cells in the brain. The activation of glial cells may be the result of neuronal degeneration, and the activation of glial cells may exacerbate neuronal death. Excessive activation of glial cells in the brain in a resting state will mediate the release of a large number of pro-inflammatory factors, reactive oxygen/nitrogen, and prostaglandins to trigger an inflammatory cascade reaction, and inflammatory factors will further activate astrocytes and damage neurons, further expand the inflammatory response, and have a toxic effect on the brain. Inflammatory dysfunction of the nervous system can cause long-term learning and memory dysfunction, including the release of a large number of neuroinflammatory factors and the activation of immune system cells to participate in the regulation of neurogenesis, synaptic plasticity, neuron survival and other vicious circle processes, which affect the formation of learning and memory, and eventually lead to the formation of Alzheimer's disease.

Therefore, there is an urgent need to seek therapeutic and health care medicines for Alzheimer's disease.

Contents of the invention

In view of this, the present invention provides the application of curdione, which can inhibit the activation of microglial cells, relieve Alzheimer's disease symptoms, and improve memory impairment and motor coordination.

Concrete technical scheme of the present invention is as follows:

Application of curcumadione in the preparation of drugs for inhibiting microglia activation.

Preferably, the drug is used to reduce the expression of Iba-1 protein.

Preferably, the drug is used to reduce the expression of inflammatory factors.

Preferably, the inflammatory factor is TNF-α.

Experimental results show that curcumadione can reduce the expression level of TNF-α protein in hippocampus and cerebral cortex.

The present invention also provides the use of curcumadione in the preparation of drugs for preventing and/or treating Alzheimer's disease.

In the present invention, when curcumadione is used to prevent and/or treat Alzheimer's disease, the dosage is preferably 50 mg/kg/d-100 mg/kg/d.

Preferably, the medicament also includes pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the drug is oral preparation or injection.

In the present invention, the oral preparation is selected from capsules, microcapsules, pills, tablets, decoctions, granules, ointments, dispersible powders, dew oral preparations, dropping pills or liposomes;

The injection is powder injection or injection.

In summary, the present invention provides the application of curdione, and the experimental results show that curdione can effectively inhibit the activation of microglia and the secretion of the inflammatory factor TNF-α, control neuroinflammation, thereby alleviating the neuron damage caused by subsequent inflammatory factors, improving the cognitive function, memory impairment and motor coordination ability of brain tissue, and can be used for Alzheimer's disease. Curcumadione is applied to the preparation of drugs for the prevention and/or treatment of Alzheimer's disease, and has good application value and development prospect.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.

Fig. 1 is the mouse body weight change figure during the administration of embodiment 1;

Fig. 2 is the result figure of mouse water maze experiment of embodiment 1, (A) location and navigation experiment mouse trajectory figure (hidden platform), (B) location and navigation experiment mouse is in each quadrant escape latency (hidden platform), (C) space exploration experiment mouse spends the percentage of time in the original platform target quadrant (removal platform), (D) space exploration experiment mouse crosses the number of times of former platform area (removal platform), (E) space exploration experiment mouse movement track diagram (removal platform);

Fig. 3 is the mouse brain tissue analysis figure of embodiment 2, (A) the Nissl staining figure of the paraffin section of the mouse brain tissue of embodiment 2, wherein, the first horizontal row is the coronal section of the mouse brain tissue (magnification 40 times), the second horizontal row is the hippocampus area (magnification 400 times), the third horizontal row is the cerebral cortex region (magnification 400 times), (B) the quantitative analysis figure of the number of healthy neurons in the mouse brain tissue of embodiment 2;

Fig. 4 is the TNF-α protein expression diagram of the mouse brain tissue of embodiment 3, wherein, (A) the immunohistochemical diagram of the paraffin section of the mouse brain tissue of the embodiment 3, the first vertical row is the mouse brain tissue cross section, the second vertical row is the hippocampus region, and the third vertical row is the cerebral cortex region, (B) the quantitative map of TNF-α protein expression in the mouse brain tissue hippocampus region of Example 3, (C) the quantitative map of the TNF-α protein expression in the cerebral cortex region of the mouse brain tissue of embodiment 3;

5 is a map of Iba-1 protein expression in hippocampal microglial cells in the mouse brain tissue of Example 3, wherein, (A) immunofluorescence map, the first row is the Iba-1/DAPI fluorescence map, and the second row is the Iba-1 fluorescence map, and (B) the quantitative map of Iba-1 protein expression in the hippocampus of the mouse brain tissue in Example 3.

Detailed ways

The invention provides the application of curedione, which can inhibit the activation of microglial cells, alleviate the symptoms of Alzheimer's disease, and improve memory impairment and motor coordination ability.

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

In specific embodiments, the raw materials and reagents used can be purchased from the market. The experimental animals were SPF 8-9 week-old male C57 mice weighing 20-22 g, which were purchased from the Experimental Animal Center of Sun Yat-sen University, license number SCXK (Guangdong) 2016-0112. The mouse feed was purchased from the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine. The experimental animals were reared in a clean-grade laminar flow frame, the temperature of the feeding environment was controlled by air conditioning at 23±2°C, the relative humidity was 75±10%, and the lighting time was 12h/d (7:00-19:00).

Example 1 The water maze test was used to detect the improvement effect of curcumadione on the memory impairment of AD symptoms induced by LPS in mice.

In this embodiment, the experimental animals were randomly divided into 5 groups, namely: normal control group (Control), LPS group, positive drug group (LPS+TTP488, 5 mg/kg/day), low-dose curcumadione group (LPS+curcumadione, Cu-L, 50 mg/kg/day) and high-dose curcumadione group (LPS+curcumadione, Cu-H, 100 mg/kg/day), with 5 animals in each group. Each experimental group was administered 1 week before LPS injection, and administered continuously for 2 weeks, with daily administration of 250 μg/kg/d, and the body weight changes of the mice were recorded. Except for the normal group, LPS was injected intraperitoneally on the 7th day of administration in the other groups. After continuous injection for 1 week, the memory function of the mice was investigated by water maze test. The experimental data were expressed as Mean ± SD. All experimental data were statistically analyzed using SPSS software. The parameters between groups of mice in each group were tested by ANOVA, and P<0.05 was considered statistically significant. Please refer to Figure 1 for the weight changes of the mice during the administration period, and Figure 2 for the results of the water maze experiment.

Figure 1 is a graph showing changes in body weight of mice during administration of Example 1. Figure 1 shows that the weight of the mice in the Control group showed a trend of slowly increasing, and the weight of the mice decreased significantly in the first few days after the injection of LPS. LPS is a lipopolysaccharide produced by bacteria that can trigger inflammation in the body. After drug treatment, the body weight of the mice in the positive drug (TTP488) group and the curcumadione group recovered from the downward trend to normal faster (1-2 days earlier) than the LPS model group. In contrast, the body weight of the mice in the high-dose curcumadione group changed very little, indicating that curcumadione can better reduce the impact of LPS injection on body weight.

Long-term injection of LPS can cause inflammation of brain tissue, leading to damage to the nerves in the brain, thereby reducing the ability to learn and remember. Therefore, in this example, the water maze experiment was used to investigate whether curcumadione can improve the memory and cognitive impairment of mice induced by LPS. In the water maze navigation experiment, each mouse must start from quadrants 1, 2, 3, and 4 to find the hidden platform. Fig. 2 is the result figure of mouse water maze experiment of embodiment 1, (A) positioning and navigation experiment mouse movement track diagram (hidden platform), (B) location navigation experiment mouse is in each quadrant escape latency (hidden platform), (C) space exploration experiment mouse spends the percentage of time in the original platform target quadrant (removal platform), (D) space exploration experiment mouse crosses the number of times of the original platform area (removal platform), (E) space exploration experiment mouse movement trajectory diagram (removal platform). This embodiment records the latency time, and it can be seen that in the four quadrants of the positioning navigation experiment, compared with the Control group, intraperitoneal injection of LPS into the mice in the LPS model group significantly prolongs the latency of the mice in the water maze positioning navigation experiment. The memory function of mice in the experimental group given high-dose and low-dose curcumadione by continuous intragastric administration was significantly improved, and the latency in the four quadrants was shortened. In the space exploration experiment, especially the number and time ratio of mice entering the target platform in the high-dose curcumadione group were significantly increased, and the effect was comparable to that in the positive drug group. The number of times the mice in the low-dose curcumadione group entered the target platform was significantly increased (Figure 2C and 2D), indicating that curcumadione can slow down the effect of LPS-induced memory impairment in mice.

Example 2 Effect of Curcumadione on LPS-Induced Mouse Brain Neuron Damage

The experimental grouping and dosing regimen of this example are the same as in Example 1. After the administration of the mice, the mice were sacrificed under ether anesthesia, and the complete brain tissue was taken out to make paraffin sections. The main steps of making paraffin sections included washing the tissue and fixing it in 4% paraformaldehyde for 1 week → rinsing away excess fixative with tap water → performing gradient dehydration with alcohols with different concentration gradients (70%, 80%, 90%, 95% and 100%) → dehydration and then immersing in gradient xylene (50% and 100%) for transparency →Immerse the transparent sample in molten paraffin for 4 hours→embed (pour into the embedded paraffin until it solidifies)→section (slice with a thickness of 5μm)→expose the piece→baked the piece→do Nissl staining after dewaxing and rehydrating, and observe the pathological changes of neurons under an optical microscope. Please refer to Figure 3 for the results.

In order to further evaluate the protective effect of curcumadione on the nervous system, the present embodiment uses Nissl staining to detect the pathological changes of neurons. The experimental results are as shown in Figure 3A. Figure 3A is a Nissl staining figure of the paraffin section of the mouse brain tissue in Example 2. The first row is the coronal section of the mouse brain tissue (40 times magnification), the second row is the hippocampus (400 times magnification), and the third row is the cerebral cortex region (400 times magnification). Figure 3A shows that the neuropyramidal cells in the hippocampus and cortex of the mice in the normal control group contained a large number of Nissl bodies, which were darkly stained and the nuclei were clearly visible. However, the Nissl bodies in the neurons of the mice injected with LPS were significantly reduced, the coloring was lighter, and the nuclei were blurred. After curcumadione treatment, the number of Nissl bodies increased significantly, indicating that curcumadione can effectively relieve neuronal damage caused by LPS. Compared with the normal control group, through the quantitative analysis of the number of normal healthy neurons, please refer to Figure 3B for the quantitative analysis of the number of normal healthy neurons in the brain tissue of the mice in Example 2. Figure 3B shows that a large number of neurons in the hippocampus and cortex of the mice in the LPS group died, and the number of normal healthy neurons survived less, indicating that continuous intraperitoneal injection of LPS can damage neurons, and then induce chronic neuroinflammation, and curcumadione can effectively alleviate the neurons caused by LPS.

Example 3 Effect of Curcumadione on LPS-Induced Inflammatory Response in Mouse Brain Tissue

1) First, the experimental grouping and dosing regimen of this example are the same as those in Examples 1 and 2. After the administration, the mice were anesthetized with ether and sacrificed. The complete brain tissue was taken out to make paraffin sections, and the sections were subjected to immunohistochemistry or immunofluorescence. Please refer to Figure 4(A) and Figure 5(A) for the results, and then the expression levels of inflammatory factors TNF-α and Iba-1 in the hippocampus and cortex were determined by immunohistochemistry or immunofluorescence staining. The experimental data are expressed as Mean ± SD, and all experimental data are statistically analyzed using SPSS software, and the parameters between groups of mice in each group are tested by ANOVA, and P<0.05 is considered to be statistically different.

2) Immunohistochemical staining was performed on the tissue sections to detect the expression level of the inflammatory factor TNF-α. The process of making paraffin sections is the same as in Example 2. The cut paraffin sections are baked (after being baked in an oven at a temperature of 62-65°C for 1 hour, then dewaxed and hydrated in xylene alcohol solution) → antigen retrieval (tissue sections are placed in a restoration box containing EDTA antigen retrieval buffer pH 8.0, and antigen retrieval is performed in a microwave oven) → blocking endogenous peroxidase (use 3% hydrogen peroxide solution to incubate at room temperature in the dark for 25 minutes, then wash with PBS) → serum blocking (3% BSA incubation for 30 minutes) min) → primary antibody incubation at 4°C overnight (drop primary antibody TNF-α, 1:100, 1×PBS dilution) → primary antibody washing → secondary antibody incubation at room temperature for 50 min (drop the secondary antibody corresponding to the species of the primary antibody, 1:100, 1×PBS dilution) → secondary antibody washing → DAB color development (control the color development time under the microscope, the positive color is brown yellow, wash the section with tap water to stop color development) → hematoxylin counterstain the nuclei → ethanol dehydration and cover the slide (after washing 3 times on a shaker , dry, and seal with neutral gum) → xylene dehydration and transparency → microscope inspection and photographing (slices were observed under a microscope and images were collected), the results are shown in Figure 4 (A).

3) Immunofluorescence test was performed on the tissue sections to detect the inhibitory effect of curcumadione on the degree of activation of microglial cells induced by LPS. The process of making paraffin sections was the same as that in Example 2. The cut paraffin sections were baked (after being baked in an oven at a temperature of 62-65°C for 1 h, then dewaxed and hydrated in xylene ethanol solution) → antigen retrieval (tissue sections were placed in a restoration box filled with citric acid pH 6.0 antigen retrieval solution, and antigen retrieval was carried out in a microwave oven) → prevention of fluorescence quenching (after the sections were slightly dry, draw circles around the tissue with a histochemical pen) → serum blocking (incubate with 3% BSA for 30 min) → primary antibody incubation for 4 nights ( Add primary antibody Iba-1, 1:100, diluted in 1×PBS)→washing with primary antibody→incubate with secondary antibody for 50min at room temperature (drop the secondary antibody corresponding to the species of primary antibody, 1:100, diluted in 1×PBS)→wash with secondary antibody→counterstain cell nuclei with DAPI (add DAPI staining solution, incubate at room temperature for 10min in the dark)→wash and mount (after washing 3 times on a shaker, dry and seal with anti-fluorescence quenching mountant)→microscopic examination Observe and collect images under a fluorescence microscope, DAPI has an ultraviolet excitation wavelength of 330-380nm, an emission wavelength of 420nm, and emits blue light; CY3 has an excitation wavelength of 510-560nm, an emission wavelength of 590nm, and emits red light), and the results are shown in Figure 5(A).

Normally, microglia are in a quiescent state, producing anti-inflammatory factors and neurotrophic factors that protect brain tissue. In pathological conditions, microglia are widely activated, producing a large number of inflammatory factors to damage neurons, and even cause cognitive and metabolic disorders in brain tissue. 4 is a map of TNF-α protein expression in the mouse brain tissue of Example 3, wherein, (A) immunohistochemical map, the first vertical row is a cross-sectional view of the mouse brain tissue, the second vertical row is the hippocampus, and the third vertical row is the cerebral cortex area, (B) using Image-Proplus4. Figure 4 shows that combined with immunohistochemical section to characterize the expression level of TNF-α in the hippocampus and cortex (Figure 4A) and image analysis to quantify the expression level of TNF-α in the hippocampus and cortex (Figure 4B), after LPS injection, it caused the overexpression of the inflammatory factor TNF-α. After the administration of curedione, especially in the hippocampus, the concentrations of the inflammatory factor TNF-α in the Cu-L and Cu-H groups were significantly reduced. 5 is a map of Iba-1 protein expression in hippocampus microglial cells in the mouse brain tissue of Example 3, wherein, (A) immunofluorescence map, the first row is the Iba-1/DAPI fluorescence map, and the second row is the Iba-1 fluorescence map, (B) Image-Proplus4.1 software is used to perform quantitative analysis of the expression of Iba-1 protein fluorescence on the brain hippocampus image. Figure 5 shows that after LPS injection, it caused extensive activation of microglia. Compared with the normal control group, a large number of microglial cells were activated in the LPS group, and the red fluorescence increased significantly, and the injection of LPS caused neuroinflammation. After curcumadione was given again, the activation level of microglia was inhibited, and the red fluorescently labeled cells were significantly reduced. Compared with the LPS group, administration of high-dose curcumadione can significantly reduce the expression level of Iba-1 protein in the hippocampus, indicating that curcumadione can effectively inhibit the activation of microglia.

The above results show that curcumadione can effectively inhibit the activation of microglial cells and the secretion of the inflammatory factor TNF-α, control neuroinflammation, thereby alleviating subsequent neuronal damage, improving memory impairment and motor coordination, and can be used to prepare drugs for the prevention and/or treatment of Alzheimer's disease.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (6)

1. The use of curcumadione as the only active ingredient in the preparation of drugs for the prevention and/or treatment of Alzheimer's disease; The drug inhibits microglial activation. 2. The application according to claim 1, characterized in that the drug is used to reduce the expression of Iba-1 protein. 3. The application according to claim 1, characterized in that the drug is used to reduce the expression of inflammatory factors. 4. The application according to claim 3, characterized in that the inflammatory factor is TNF-α. 5. The application according to any one of claims 1 to 4, characterized in that the medicament further comprises pharmaceutically acceptable auxiliary materials. 6. The application according to any one of claims 1 to 4, characterized in that, the dosage form of the drug is oral preparation or injection.