Medicine for treating abnormal fat metabolism and related diseases caused by high fat diet

Abstract

The invention discloses a medicine for treating abnormal fat metabolism and related diseases caused by high fat diet. Experiments on a NDufa7 gene knockout mouse and a high-fat diet mouse find that inhibition of NDufa7 gene expression and high-fat diet can cause reduction of the activity ability of the mouse, weakening of the exploration ability and influence on the number of neuron Neun in the hippocampal region of the mouse. Inhibition of NDufa7 gene expression and high-fat diet may also cause myocardial hypertrophy of mice. Through overexpression of the Ndufa7 gene in cells, it is found that the survival rate of the cells under oleic acid stimulation can be increased, an oleic acid-induced cell model can be used for simulating the influence of high-fat diet, and the Ndufa7 gene expression promoter can be used for preparing drugs for treating fat metabolism disorder and related diseases caused by high-fat diet. The invention is beneficial to the development of drugs for treating cardiovascular diseases, neurodegenerative diseases and the like induced by high fat diet.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a therapeutic drug for abnormal fat metabolism and related diseases caused by a high-fat diet. Background Art

[0002] Abnormal fat metabolism refers to the abnormality of normal fat metabolism due to the combined effect of multiple factors, which can lead to the occurrence of various diseases. High fat diet (HFD) is closely related to abnormal fat metabolism. High fat diet can lead to abnormal fat metabolism, which in turn causes neurodegenerative diseases and cardiovascular diseases. For example, it has been reported that high-fat diet can cause neuronal damage and cerebrovascular dysfunction, leading to cognitive impairment and vascular cognitive impairment (VCI). It has also been reported that high-fat diet can promote cognitive impairment and chronic diseases such as Alzheimer's disease (AD) by affecting the intestinal microbiota. In addition, high-fat diet is also associated with the occurrence of neurodegenerative diseases such as Parkinson's disease (PD), Huntington's disease, and Lewy body dementia. In addition to neurodegenerative diseases, high-fat diet can also cause cardiovascular diseases such as atherosclerosis, coronary heart disease, myocardial infarction and heart failure.

[0003] A high-fat diet is difficult to avoid in modern society, so there is an urgent need to develop appropriate improvement measures for the harm caused by a high-fat diet. By studying metabolic pathways, it is highly feasible to find a solution that can reverse the negative effects of a high-fat diet. Summary of the invention

[0004] The present invention aims to provide gene targets that can reverse the negative effects caused by a high-fat diet, and to provide direction for the development of therapeutic drugs for abnormal fat metabolism and related diseases caused by a high-fat diet.

[0005] The first object of the present invention is to provide an application of an expression promoter of Ndufa7 gene in the preparation of a drug for preventing, improving and / or treating abnormal fat metabolism and related diseases caused by a high-fat diet.

[0006] The second object of the present invention is to provide the use of Ndufa7 gene as a target in the preparation of drugs for preventing, improving and / or treating abnormal fat metabolism and related diseases caused by a high-fat diet.

[0007] The third object of the present invention is to provide the use of Ndufa7 gene in the preparation of drugs for preventing, improving and / or treating abnormal fat metabolism and related diseases caused by a high-fat diet.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention has found through experiments such as mine field experiments on Ndufa7 knockout mice and mice on a high-fat diet that inhibiting Ndufa7 gene expression and a high-fat diet will lead to reduced activity and exploration ability of mice, and affect the number of neurons Neun in the hippocampus of mice. It was also found that inhibiting Ndufa7 gene expression and a high-fat diet will also lead to an increase in the weight of the left ventricular myocardium of mice, weakened cardiac contraction and pumping function, and may cause the occurrence of myocardial hypertrophy. By overexpressing the Ndufa7 gene in cells, it was found that the survival rate of cells under oleic acid stimulation can be increased, and the oleic acid-induced cell model can be used to simulate the effects of a high-fat diet, that is, the expression promoter of the Ndufa7 gene can be used to prepare drugs for treating abnormal fat metabolism and related diseases caused by a high-fat diet. The present invention fully proves that Ndufa7 is involved in the pathogenesis of abnormal fat metabolism and related diseases caused by a high-fat diet, including cardiovascular diseases, neurodegenerative diseases, etc., and can provide important support for the development of drugs for related diseases.

[0010] Therefore, the present invention claims protection for:

[0011] Use of an expression promoter of the Ndufa7 gene in preparing a drug for preventing, improving and / or treating abnormal fat metabolism and related diseases caused by a high-fat diet.

[0012] The use of Ndufa7 gene as a target in the preparation of drugs for preventing, improving and / or treating abnormal fat metabolism and related diseases caused by a high-fat diet.

[0013] Application of Ndufa7 gene in the preparation of drugs for preventing, improving and / or treating abnormal fat metabolism and related diseases caused by high-fat diet.

[0014] Specifically, the abnormal fat metabolism and related diseases caused by a high-fat diet are cardiovascular diseases caused by a high-fat diet.

[0015] Specifically, the cardiovascular disease includes atherosclerosis, coronary heart disease, myocardial infarction or heart failure.

[0016] More specifically, the cardiovascular disease is heart failure.

[0017] Specifically, the abnormal fat metabolism and related diseases caused by a high-fat diet are neurodegenerative diseases caused by a high-fat diet.

[0018] Specifically, the neurodegenerative disease includes vascular cognitive impairment, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lewy body dementia, primary tauopathy, frontotemporal dementia or amyotrophic lateral sclerosis.

[0019] Optionally, the Ndufa7 gene expression promoter includes an expression cassette of the Ndufa7 gene or an expression vector containing the Ndufa7 gene.

[0020] In a specific embodiment of the present invention, the Ndufa7 gene expression promoter is an overexpression vector containing the Ndufa7 gene.

[0021] In a specific embodiment of the present invention, the ID of the Ndufa7 gene in the NCBI database is 66416.

[0022] The present invention has the following beneficial effects:

[0023] The present invention conducts experiments on Ndufa7 knockout mice and mice on a high-fat diet and finds that inhibiting Ndufa7 gene expression and a high-fat diet will lead to reduced activity and exploration ability of mice, and affect the number of neurons Neun in the hippocampus of mice. Inhibiting Ndufa7 gene expression and a high-fat diet may also cause myocardial hypertrophy in mice. By overexpressing the Ndufa7 gene in cells, it was found that the survival rate of cells under oleic acid stimulation can be increased. The oleic acid-induced cell model can be used to simulate the effects of a high-fat diet, and the expression promoter of the Ndufa7 gene can be used to prepare drugs for treating abnormal fat metabolism and related diseases caused by a high-fat diet. The present invention is conducive to the development of therapeutic drugs for cardiovascular diseases, neurodegenerative diseases, etc. induced by a high-fat diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a trajectory diagram of the open field experiment of each model mouse (normal diet model (ND-WT) mouse, normal diet model (ND-KO) mouse, high-fat diet model (HFD-WT) mouse and high-fat diet model (HFD-KO) mouse).

[0025] Figure 2 The total movement distance, average movement speed, stationary time and center area residence time of each model mouse in the open field test, from left to right and from top to bottom are the total movement distance, average movement speed, stationary time and center area residence time of the mouse.

[0026] Figure 3 Figure 2 is a trajectory diagram of the elevated plus maze experiment for each model mouse.

[0027] Figure 4 It is the number of times each model mouse enters the open arm and the total number of times it enters the open arm and the closed arm in the elevated plus maze test.

[0028] Figure 5 The figures are the results of NeuN staining of neurons in the hippocampus of the brains of the model mice.

[0029] Figure 6 The results are the percentage of NeuN staining area of ​​neurons in the hippocampus of the brain of each model mouse.

[0030] Figure 7 Shown are the cardiac ultrasound results of each model mouse.

[0031] Figure 8 The heart-to-weight ratio calculation results of each model mouse.

[0032] Fig. 9 Shown are the heart staining results of each model mouse.

[0033] Fig.10 The results are statistical results of the degree of myocardial tissue fibrosis corresponding to Masson staining and Sirus red staining of the hearts of the mice in each model.

[0034] Fig.11 The transmission electron micrographs of the hearts of the model mice were magnified at 2.5K and 7.0K times.

[0035] Fig.12 The effect of overexpression of Ndufa7 gene on the survival rate of H9C2 cells stimulated by oleic acid.

[0036] Fig.13 The effect of overexpression of Ndufa7 gene on the survival rate of PC-12 cells stimulated by oleic acid.

[0037] In the figures, *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] The experimental mice used in the embodiments of the present invention are 5-month-old C57BL / 6 wild-type mice (WT mice) and 5-month-old Ndufa7 gene knockout mice (KO mice) constructed by CRISPR / Cas9 gene editing technology; the ID of the Ndufa7 gene in the NCBI database (https: / / www.ncbi.nlm.nih.gov) is 66416.

[0041] Example 1 Effects of knocking out the Ndufa7 gene and high-fat diet on the nervous system of mice

[0042] 1. Construction of mouse models with different diets

[0043] The present invention uses a normal diet (ND) and a high-fat diet (HFD) to feed WT mice and KO mice respectively, and constructs four model mice, namely, normal diet model (ND-WT) mice, normal diet model (ND-KO) mice, high-fat diet model (HFD-WT) mice and high-fat diet model (HFD-KO) mice. The nutritional composition of the normal diet and the high-fat diet are shown in Table 1 and Table 2, respectively.

[0044] Table 1 Nutrient composition of normal diet

[0045] Component (unit) gm(%) Kcal(%) protein 20 23.77 Fat 4.5 12.04 carbohydrate 54 64.19 total 100 Total energy (kcal / gm) 3.37

[0046] Table 2 Nutritional composition of high-fat diet

[0047]

[0048]

[0049] The high-fat model was established for 13 weeks. The mice were weighed and recorded at a fixed time every week, and the body weight changes of mice in each group were used to evaluate the establishment of the high-fat model.

[0050] 2. Analysis and evaluation of physiological functions of each model mouse

[0051] In order to explore whether the activity and exploratory abilities of mice are affected by Ndufa7 gene deletion and high-fat diet pressure, the present invention conducted open field experiments on the above four model mice, observed the spontaneous activities and exploratory behaviors of the mice, and then confirmed the results through elevated plus maze experiments.

[0052] The specific process is as follows:

[0053] (1) Open field test

[0054] Without interference from other animals, the mice were transferred from the mouse room to the special cage rack of the behavior laboratory two hours in advance. The open field box device consisted of a 50cm×50cm white plastic bottom plate and a 40cm black plastic high wall on four sides, made of medical ABS material. A monitoring camera dedicated to animal behavior experiments was installed above the instrument, which was connected to the SuperMaze animal behavior analysis software in the computer through an acquisition card. Before the experiment, all parameters were adjusted, the area of ​​the white plastic bottom plate was divided into 9 squares, the mice were placed in the central square, and the total distance, average speed, static time and residence time of the mice in the central square within 5 minutes were monitored by a video system. Before each mouse was placed in the experimental box, the white plastic bottom plate was wiped clean with alcohol to remove feces and urine to remove odor and prevent interference with the movement state of the next mouse. The entire experimental environment was carried out in a quiet environment with consistent light brightness at 25℃.

[0055] (2) Elevated plus maze test

[0056] The elevated cross maze consists of two open arms and two closed arms, which cross to form a central area, two open arms, and two closed arms. The arm width of the mouse experimental device is 5 cm, the arm length is 35 cm, the arm height is 15 cm, and the height of the maze is about 40 to 55 cm from the ground. A monitoring camera dedicated to animal behavior experiments is installed above the instrument, which is connected to the SuperMaze animal behavior analysis software in the computer through an acquisition card. After debugging the camera and various parameter values, it is divided into five areas: the central area, two open arms, and two closed arms. The mice are placed in the central area to monitor the number of times and retention time of the mice entering the open arms and closed arms within 5 minutes. Before each mouse is placed in the experimental box, the feces and urine left by the previous mouse are wiped clean with alcohol to prevent interference with the movement state of the next mouse. The entire experimental environment is kept at 25°C and quiet, with consistent light brightness.

[0057] The open field test trajectories of each model mouse are shown in the figure below. Figure 1 The total distance of movement, average speed, static time and time spent in the central area in the open field test are shown in Figure 2 As shown, Figure 2 From left to right and from top to bottom are the total distance traveled by the mouse, the average speed of movement, the static time, and the time spent in the center area. Figure 1 and 2 It can be seen that under normal dietary conditions, the total activity distance and total average speed of KO mice in the ND group were significantly lower than those of WT mice (P<0.001), and the exploration and residence time in the central area was also significantly reduced (P<0.05). At the same time, the resting and immobility time of KO mice during the monitoring period was significantly increased compared with WT mice (P<0.01). The poor motor ability and long immobility time of mice in behavioral experiments are manifestations of high anxiety, which indicates that the loss of the Ndufa7 gene will have adverse effects on the nervous system of mice. Compared with mice of the same WT type but fed with different diets, the total activity distance and total average speed of HFD mice were lower than those of ND mice (P<0.05), the resting and immobility time was higher than that of ND mice (P<0.05), and the exploration and residence time in the central area was reduced to a certain extent.

[0058] The elevated plus maze test uses the fear of animals to explore new and different environments, forming contradictory behaviors in animals, and has good test-retest reliability and good correlation. When faced with a new environment (open arms), mice will have a curiosity to explore. Due to the nature of mice approaching darkness (closed arms), an "exploration-avoidance" conflict arises between them, resulting in anxiety. The elevated plus maze test trajectory of each model mouse is shown in the figure below. Figure 3 The number of times it entered the open arm and the total number of times it entered the open arm and the closed arm in the elevated plus maze test are shown in Figure 4 (From top to bottom). Analysis of the number of times (OT) and total number (TNE) that mice entered the open arms within a specified time found that the KO mice in the ND group at 5 months old had lower time, number of times and total number of times entering the open arms than those in WT mice; compared with the same type of WT mice, the number of times that mice fed a high-fat diet entered the open arms was lower than that of mice fed a normal diet (P<0.05), and the total number was significantly lower than that of mice fed a normal diet (P<0.01), indicating that compared with WT mice of the same age, KO mice and WT mice fed a high-fat diet had reduced activity and weakened exploration ability in the open arm area in a high-suspended open state, indicating that the Ndufa7 gene and high-fat diet are associated with neurodegenerative diseases ( Figure 4 ). Inhibiting the expression of the Ndufa7 gene can alleviate the adverse effects of a high-fat diet on the activity and exploration abilities of mice to a certain extent.

[0059] 3. Histopathological analysis of each model mouse

[0060] In order to observe the effect of Ndufa7 gene deletion on the mouse nervous system from a pathological perspective, the present invention performed neuronal NeuN staining on the brains of each model mouse, and at the same time counted the area occupied by the hippocampal neurons NeuN (by statistical analysis using ImageJ software). The results of neuronal NeuN staining and area percentage in the hippocampal region of each model mouse brain are shown in Figure 2. Figure 5 and Figure 6 shown.

[0061] Depend on Figure 6 It can be seen that the proportion of Neun area in the hippocampus of the two groups of WT mice at 5 months old was slightly different, and the two groups of Ndufa7 gene-deficient mice at the same age had similar situations, indicating that changes in feeding conditions did not have a great effect on the area of ​​neurons in the hippocampus. Analysis of the proportion of Neun area of ​​neurons in different types of mice under the same feeding conditions found that the proportion of Neun area in the hippocampus of mice with Ndufa7 gene deficiency was lower than that of WT mice, regardless of whether it was a normal diet or a high-fat diet, indicating that the loss of Ndufa7 gene would damage the number of neurons Nuen in the hippocampus to a certain extent, thereby causing damage to the mouse nervous system, but there was no significant difference compared with mice without Ndufa7 gene knockout.

[0062] Example 2 Effects of knocking out the Ndufa7 gene and high-fat diet on the cardiovascular system of mice

[0063] 1. Mouse ultrasound experiment

[0064] Pathological myocardial hypertrophy is an important manifestation in the process of heart failure. In order to observe the effects of Ndufa7 deficiency and high-fat diet on the cardiac structure of mice, the present invention uses ultra-high resolution small animal echocardiography to evaluate the heart shape and function of each model mouse. This is a non-invasive, most effective and most commonly used method. Among them, B-ultrasound and M-ultrasound are used to monitor cardiac function, and M-ultrasound is analyzed to obtain cardiac function indicators. Stroke volume (SV) is one of the important indicators for measuring the heart's pumping function; changes in left ventricular myocardial weight (LV Mass) reflect the degree of left ventricular myocardial hypertrophy; ejection fraction (EF) and contraction fraction (FS) are important indicators for evaluating cardiac function, which reflect the contractile ability of the mouse heart and the degree of heart failure.

[0065] The cardiac ultrasound results of each model mouse are as follows Figure 7 As shown. Figure 7 The results show that compared with ND-WT mice, the stroke volume, ejection fraction and shortening fraction of ND-KO mice were significantly decreased (P<0.001). In addition, HFD induction affected the ejection fraction and contraction fraction of WT mice, which decreased to a certain extent (P<0.05). After Ndufa7 gene knockout, the left ventricular myocardial weight of KO mice increased significantly (P<0.01), showing left ventricular dilatation. HFD induction also had a statistically significant effect on the left ventricular myocardial weight of WT mice (P<0.05).

[0066] In summary, Ndufa7 knockout leads to changes in cardiac structure, with increased left ventricular myocardial weight and weakened cardiac contraction and pumping function at 5 months of age; in addition, HFD induction weakens the heart's contractile ability and aggravates the degree of heart failure.

[0067] 2. Organ coefficient analysis

[0068] The organ coefficient, i.e., organ mass ratio, is usually used as one of the indicators for evaluating the health status and functional status of mouse organs. The present invention evaluates the degree of myocardial hypertrophy by calculating the heart-to-weight ratio of mice. In order to observe the effects of Ndufa7 gene knockout and high-fat diet on the heart of mice, the hearts of mice were weighed and the heart-to-weight ratio was calculated. The calculation results of the heart-to-weight ratio of each model mouse are shown in Figure 2. Figure 8 As shown. Figure 8 The results show that there is a statistically significant difference in the heart-to-weight ratio between Ndufa7 knockout mice and WT mice (P<0.05), and Ndufa7 knockout affects the myocardial structure of mice to a certain extent; after HFD induction, there is a significant difference in the heart-to-weight ratio between mice of the same genotype (P<0.01).

[0069] In summary, Ndufa7 gene knockout or HFD induction affects the myocardial structure of mice.

[0070] 3. Histopathological analysis

[0071] Heart failure is the terminal manifestation of myocardial hypertrophy. Imaging analysis cannot prove that Ndufa7 gene deletion will lead to myocardial hypertrophy in mice. The ASCO guidelines state that pathological results can be used as the "gold standard" for disease diagnosis. To further prove our hypothesis, pathological section analysis of mouse heart tissue was performed.

[0072] In order to evaluate the changes in cardiac morphology of KO mice and mice fed a high-fat diet, and to observe the state, size changes and microscopic morphology of myocardial cells, the present invention uses 4% paraformaldehyde to fix the heart during the dissection process, and cross-sections and longitudinal sections are made on the largest cross-section of the fixed heart, and the sections are embedded and respectively stained with hematoxylin-eosin (HE), Masson staining and Sirius red staining, and the degree of myocardial tissue fibrosis is counted. Among them, the hematoxylin stain in the HE dye stains the chromatin and the cell nucleus into purple-blue, and the eosin stain stains the components in the cytoplasm and the extracellular matrix into red; after Masson staining, the muscle fibers are red, and the collagen fibers are green or blue; after Sirus red staining, the collagen fibers are stained red, and the muscle fibers are stained yellow.

[0073] The heart staining results of each model mouse are as follows Fig. 9 HE staining results showed that the myocardial cells of WT mice were closely arranged, with normal cell structure, clear nucleus and nucleolus structure, while the myocardial gaps of KO mice were significantly enlarged, and the cell boundaries were blurred ( Fig. 9 ).

[0074] Masson staining and Sirus red staining can evaluate the degree of myocardial fibrosis in pathological myocardial hypertrophy in mice. The statistical results of the degree of myocardial fibrosis corresponding to Masson staining and Sirus red staining of each model mouse are shown in the figure below. Fig.10 As shown. Fig.10 The results show that the area of ​​collagen fibers stained by Masson staining was significantly increased in KO mice under normal dietary conditions compared with WT mice; compared with isogenic mice, HFD induction increased the area of ​​collagen fibers, especially in KO mice, indicating that the induction of a high-fat diet would aggravate the degree of myocardial fibrosis caused by Ndufa7 gene knockout.

[0075] In summary, Ndufa7 gene knockout affects the myocardial structure of mice, leading to myocardial fibrosis in mice, and a high-fat diet will aggravate this result.

[0076] 4. Analysis of cardiac mitochondrial morphology

[0077] Transmission electron microscopy (TEM) can observe the morphological structure and changes of organelles in tissues at different magnifications, and can observe the effects of Ndufa7 gene deletion and HFD induction on the heart. Mitochondria are closed sac-like organelles surrounded by a double membrane, consisting of an outer membrane, an inner membrane, an outer cavity and an inner cavity. Between the inner and outer membranes of mitochondria is the outer chamber, and the inner membrane folds inward to form mitochondrial cristae. Between the cristae is the inner chamber, which is filled with matrix. Mitochondria are the energy supply sites in cells, and their morphology is constantly changing. Most of them are round or oval, and sometimes they appear long and thin. The transverse diameter is relatively consistent, but the long diameter varies greatly. One heart was randomly taken from each of the four groups of mice and fixed, and the morphological changes of mitochondria were observed under a transmission electron microscope.

[0078] Transmission electron microscopic images of the hearts of each model mouse at 2.5K and 7.0K times are shown in the figure below. Fig.11 As shown. The 2.5K-fold electron micrograph shows the general situation of the lipid metabolism capacity of the mouse heart. Different genotypes and different diets have different degrees of influence on the lipid metabolism of the heart. From the 7.0K-fold electron micrograph, under normal diet conditions, compared with WT mice, the outer membrane of some mitochondria of KO mice was damaged, the transverse diameter of mitochondria was inconsistent, and the inner chamber was swollen to a certain extent. Compared with WT mice, the structure of the heart mitochondria of HFD-WT mice was discontinuous and blurred. Under the condition of HFD and Ndufa7 gene knockout, the inner chamber of the heart mitochondria of HFD-KO mice was swollen, resulting in an increase in the volume of mitochondria and membrane coating. In addition, the mitochondrial cristae were significantly reduced or even disappeared.

[0079] In summary, Ndufa7 gene knockout affected the lipid metabolism and mitochondrial morphology of the mouse heart to a certain extent, leading to impaired mitochondrial function. In addition, HFD induction had a certain degree of impact on cardiac mitochondrial function.

[0080] Example 3 Effect of overexpression of Ndufa7 gene on cells

[0081] Oleic acid (OA) can produce a series of toxic effects on cells, leading to a series of pathological changes. Therefore, OA solutions are often used to treat cells to establish various cytotoxicity models. The constructed cell models can be used to simulate the effects of a high-fat diet on cells.

[0082] The present invention takes H9C2 cells (heart) and PC-12 cells (nerve) as examples, and explores the effect of overexpressing the Ndufa7 gene on the cells by detecting the survival rate of H9C2 cells and PC-12 cells overexpressing the Ndufa7 gene under oleic acid treatment conditions.

[0083] The specific process is as follows:

[0084] 1. Construction of recombinant overexpression vector

[0085] Based on the commercially available vector pEGFP-C1, the Ndufa7 gene (ID: 66416) was inserted between the multiple cloning sites Xho I and BamH I to construct the recombinant overexpression vector pEGFP-C1-Ndufa7.

[0086] 2. Cell culture

[0087] H9C2 cells and PC-12 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO. 2 Culture in an incubator, and when the cell confluence reaches 70% to 80%, digest the cells with 0.25% trypsin-EDTA and pass them at a ratio of 1:3. Take cells in the logarithmic growth phase for the experiment.

[0088] 3. Oleic acid treatment

[0089] 18 hours after cell plating, the culture medium was replaced with DMEM medium containing 5% fetal bovine serum and 0.5% fatty acid-free bovine serum albumin, and the cells were transfected with pEGFP-C1 empty vector and pEGFP-C1-Ndufa7, respectively; the cell lines were exposed to 500 μM oleic acid (OA) or BSA (as control) for 24 hours, and the cell viability was determined by CCK8. The experiment was repeated 3 times.

[0090] 4. CCK8 detection of cell viability

[0091] 10 μL of CCK8 working solution (the volume ratio of CCK8 reagent stock solution to complete culture medium was 1:9) was added to each well of the 96-well plate, incubated in a 37°C incubator for 45 min, and the absorbance at 450 nm was detected with an enzyme-labeled instrument to calculate the cell survival rate.

[0092] Cell survival rate / %=[(A experimental group-A blank group) / (A control group-A blank group)]×100%.

[0093] The effects of overexpression of Ndufa7 gene on the survival rates of H9C2 cells and PC-12 cells stimulated by oleic acid are shown in Figure 2 Fig.12 and Fig.13 As shown. Fig.12 and Fig.13 The results show that in the experimental group where Ndufa7 was not overexpressed in the cells, the survival rates of H9C2 and PC-12 cells in the OA stimulation group decreased after OA stimulation compared with the control group, indicating that OA stimulation caused toxicity to the cells and the cell survival rate decreased significantly. After the Ndufa7 gene was overexpressed in the cells, the decrease in cell survival rate was significantly improved under the same OA stimulation. Under the same OA stimulation, the survival rate of cells overexpressing the Ndufa7 gene was significantly higher than that of cells not overexpressing the Ndufa7 gene.

[0094] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of an expression promoter of the Ndufa7 gene in the preparation of a drug for preventing, improving and / or treating abnormal fat metabolism and related diseases caused by a high-fat diet.

2. Use of the Ndufa7 gene as a target in the preparation of drugs for preventing, improving and / or treating abnormal fat metabolism and related diseases caused by a high-fat diet.

3. Application of Ndufa7 gene in the preparation of drugs for preventing, improving and / or treating abnormal fat metabolism and related diseases caused by high-fat diet.

4. The use according to any one of claims 1 to 3, characterized in that: The abnormal fat metabolism and related diseases caused by a high-fat diet are cardiovascular diseases caused by a high-fat diet.

5. The use according to claim 4, characterized in that: The cardiovascular diseases include atherosclerosis, coronary heart disease, myocardial infarction or heart failure.

6. The use according to claim 5, characterized in that: The cardiovascular disease is heart failure.

7. The use according to any one of claims 1 to 3, characterized in that: The abnormal fat metabolism and related diseases caused by a high-fat diet are neurodegenerative diseases caused by a high-fat diet.

8. The use according to claim 7, characterized in that: The neurodegenerative disease includes vascular cognitive impairment, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lewy body dementia, primary tauopathy, frontotemporal dementia or amyotrophic lateral sclerosis.

9. The use according to claim 1, characterized in that: The Ndufa7 gene expression promoter includes an expression cassette of the Ndufa7 gene or an expression vector containing the Ndufa7 gene.

10. The use according to claim 9, characterized in that: The Ndufa7 gene expression promoter is an overexpression vector containing the Ndufa7 gene.

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