Protective effect of lithocarpus polystachyus on cardiomyopathy of diabetic patient

Abstract

The invention provides a protective effect of lithocarpus polystachyus on cardiomyopathy of diabetic patients, and belongs to the technical field of traditional Chinese medicinal materials. The lithocarpus polystachyus extract has the effect of protecting cardiomyopathy of diabetic patients; the health care product containing the lithocarpus polystachyus or the lithocarpus polystachyus extract is used for relieving cardiomyopathy of diabetic patients. The traditional Chinese medicinal materials containing lithocarpus polystachyus or lithocarpus polystachyus extract are used for relieving cardiomyopathy of diabetic patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and relates to the protective effect of Lithocarpus polystachyrus on cardiomyopathy in diabetic patients. Background Art

[0002] Lithocarpus polystachyrus Rehd, also known as Sweet tea, Lithocarpus polystachyrus, Wild gold firewood or Sweet tea, is an evergreen tree belonging to the genus Lithocarpus of the Fagaceae family. Its main effects include clearing heat and detoxifying, resolving phlegm, expelling wind, reducing blood pressure, etc., and it is mainly applied to diseases such as damp-heat diarrhea, lung-heat cough, carbuncles and sores, skin itching, etc. In 2017, it was approved as a new food raw material in China. Modern research has found that the extract of Lithocarpus polystachyrus can increase the intake of intracellular glucose, reduce the production of blood glucose and liver glycogen, and can enhance the expression of glucokinase, glucose transporter 2, insulin receptor and insulin receptor substrate in the liver tissue of type 2 diabetic mice. Although there is some research on its hypoglycemic effect, there is no literature on the protective effect of Lithocarpus polystachyrus on cardiomyopathy in diabetic patients. Diabetic cardiomyopathy is a heart disease related to diabetes mellitus (DM), which can cause significant changes in myocardial structure and function. It is a special form of cardiomyopathy that occurs independently of other cardiac risk factors and is promoted by long-term metabolic disturbances in diabetes, thereby having a direct toxic effect on the myocardium. Therefore, it is necessary for the present invention to further study the protective effect of Lithocarpus polystachyrus on diabetic cardiomyopathy.

[0003] Therefore, in order to further clarify the protective effect and mechanism of Lithocarpus polystachyrus on diabetic myocardium, the present invention takes the extract of Lithocarpus polystachyrus as the research object, and first clarifies the protective effect of the extract of Lithocarpus polystachyrus on diabetic cardiomyopathy through in vivo pharmacological experiments, which provides certain theoretical guidance for the development and utilization of Lithocarpus polystachyrus and the research on its pharmacodynamic substances. Summary of the Invention

[0004] The purpose of the present invention is to provide a protective effect of Lithocarpus polystachyrus on cardiomyopathy in diabetic patients in view of the above problems existing in the prior art. The technical problem to be solved by the present invention is to prove and clarify the protective effect of Lithocarpus polystachyrus on cardiomyopathy in diabetic patients.

[0005] Step 1: Preparation of the extract of Lithocarpus polystachyrus

[0006] After pulverizing Lithocarpus polystachyus leaves, sieve them through a 200-mesh sieve, put them into a round-bottom flask, add 20 times the volume of distilled water, let it stand for 1 hour, then perform two reflux extractions, each lasting 2 hours. Filter while it is hot and combine the filtrates. Next, perform alcohol precipitation and concentration steps: After concentrating the extract under reduced pressure, add high-concentration ethanol to make the ethanol concentration reach 65% (v / v%). Then let it stand at room temperature overnight. Finally, remove the turbid liquid at the bottom, take out the supernatant of the alcohol precipitation and concentrate it under reduced pressure, and store it for future use.

[0007] Step 2: Prepare diabetic rats by using a high-fat diet combined with streptozotocin, treat them with the extract of Lithocarpus polystachyus, and detect blood glucose, cardiac pathological observation, etc.

[0008] In Step 2, the preparation, grouping and administration of the animal model: Select 40 male SD rats weighing 200±20 g. The animals are kept at a room temperature of 20-24°C, a relative humidity of 50-70%, with free access to food and water, and adaptively fed for one week. Randomly select 8 healthy rats as the normal (Control) group, and the remaining rats are modeled. After the rats are fasted but allowed to drink water for 12 h, dissolve streptozotocin in 0.1 mol / L citric acid-sodium citrate buffer with a pH of 4.2-4.5, and prepare a solution with a mass concentration of 1% under light-proof conditions. Inject it intraperitoneally at a single dose of 35 mg / kg of streptozotocin. The normal control group is injected with the same volume of 0.1 mol / L citric acid-sodium citrate buffer with a pH of 4.2-4.5. After one week, collect blood from the tail vein continuously for three days to detect blood glucose. Diabetic rats with a fasting blood glucose ≥ 11.1 mmol / L are considered successfully modeled. The successfully modeled diabetic rats are randomly divided into a model (DM) group, a dapagliflozin (Dapa) group, a low-dose extract of Lithocarpus polystachyus (LAPE-L) group, and a high-dose extract of Lithocarpus polystachyus (LAPE-H) group, with 8 rats in each group. The extract groups are given the corresponding dose of the extract, and the blank control group and the model group are intragastrically administered the same volume of pure aqueous solution for 8 consecutive weeks.

[0009] In Step 2, the detection of blood glucose: Perform fasting blood glucose detection 8 weeks after administration.

[0010] In Step 2, cardiac pathological observation: Dissect the heart, wash it with normal saline to remove blood residues and dirt, then use filter paper to absorb the liquid and moisture on the tissue. Take a part of the heart, fix it with 4% paraformaldehyde, and perform histopathological observation.

[0011] Step 3: Use a small animal ultrasound instrument to detect the effects of Lithocarpus polystachyus extract on the cardiac function and morphology of diabetic rats. The specific detection indexes are as follows: Left ventricular systolic function: ejection fraction (EF), left ventricular shortening fraction (FS), left ventricular stroke volume (SV), cardiac output (CO); Left ventricular diastolic function: reflecting the blood flow pattern through the mitral valve orifice (E / A), reflecting the myocardial diastolic pattern of the mitral valve (E' / A'), the ratio of early diastolic mitral blood flow velocity to early diastolic mitral annulus velocity (E / E'), myocardial performance index (MPI); Left ventricular morphology of diabetic rats: left ventricular internal diameter at systole (LVID:s), left ventricular internal diameter at diastole (LVID:d), measuring the anterior wall thickness of the left ventricle at end-diastolic (LVAW:d), the anterior wall thickness of the left ventricle at end-systole (LVAW:s), the posterior wall thickness of the left ventricle at end-diastolic (LVPW:d), the posterior wall thickness of the left ventricle at end-systole (LVPW:s).

[0012] Step 4: Use Western Blot method to verify the targets and mechanisms of Lithocarpus polystachyus extract in protecting the hearts of diabetic rats.

[0013] In Step 4, Western Blot was used to detect the protein expressions of key proteins related to oxidative stress in cardiac tissues, namely Nuclear factor erythroid 2-related factor 2 (NRF2), Solute Carrier Family 7 Member 11 (SLC7A11), and Ferritin Heavy Chain 1 (FTH1).

[0014] The present invention uses small animal ultrasound combined with molecular analysis to study the protective effects and mechanisms of Lithocarpus polystachyus extract on cardiomyopathy in diabetic rats. Description of the Drawings

[0015] Figure 1 Effects of Lithocarpus polystachyus extract on fasting blood glucose and cardiac index in diabetic rats;

[0016] Figure 2 Lithocarpus polystachyus extract improves myocardial injury in diabetic rats;

[0017] Figure 3 Effects of Lithocarpus polystachyus extract on left ventricular systolic function;

[0018] Figure 4 Effects of Lithocarpus polystachyus extract on left ventricular diastolic function;

[0019] Figure 5 Effects of Lithocarpus polystachyus extract on left ventricular morphology;

[0020] Figure 6Effect of Lithocarpus polystachyus Extract on NRF2 / SLC7A11 / FTH1 Proteins; Specific Embodiments

[0021] The following are specific embodiments of the present invention and, in conjunction with the accompanying drawings, further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0022] The materials and reagents used in the embodiments can be obtained from commercial channels without special instructions.

[0023] Example 1: Diabetic rats were prepared by a high-fat diet combined with streptozotocin, treated with Lithocarpus polystachyus extract, and blood glucose was detected and cardiac pathology was observed.

[0024] Forty male SD rats weighing 200 ± 20 g were selected. The animals were fed freely at room temperature of 20 - 24°C, relative humidity of 50 - 70%, and adaptively fed for one week. Randomly, 8 healthy rats were selected as the normal (Control) group, and the remaining rats were modeled. After the rats were fasted but allowed to drink water for 12 h, streptozotocin was dissolved in 0.1 mol / L citric acid - sodium citrate buffer with a pH of 4.2 - 4.5, and a solution with a mass concentration of 1% was prepared under light - avoiding conditions. A single intraperitoneal injection was given at a dose of 35 mg / kg of streptozotocin. The normal control group was injected with the same volume of 0.1 mol / L citric acid - sodium citrate buffer with a pH of 4.2 - 4.5. After one week, blood was collected from the tail vein for three consecutive days to detect blood glucose. Diabetic rats with a fasting blood glucose ≥ 11.1 mmol / L were considered successfully modeled. The successfully modeled diabetic rats were randomly divided into a model (DM) group, a dapagliflozin (Dapa) group, a low - dose Lithocarpus polystachyus extract (LAPE - L) group, and a high - dose Lithocarpus polystachyus extract (LAPE - H) group, with 8 rats in each group. The Lithocarpus polystachyus extract groups were given the corresponding doses of the extract, and the blank control group and the model group were gavaged with the same volume of pure aqueous solution for eight consecutive weeks. A SanNuo blood glucose meter was used to detect the blood glucose of the rats after fasting for 12 h. Cardiac pathology observation: The heart was dissected, washed with normal saline to remove blood residues and dirt, and then the liquid and moisture on the tissue were absorbed with filter paper. A part of the heart was fixed with 4% paraformaldehyde for histopathological observation.

[0025] As Figure 1 shown, the Lithocarpus polystachyus extract can significantly reduce the fasting blood glucose and cardiac coefficient of diabetic rats.

[0026] Tissue fixation and HE staining. Tissue fixation: After grouping the tissues, place them in 4% paraformaldehyde for fixation; after fixing the tissue specimens in each group for at least 24 h, rinse them with tap water for 30 min; sequentially soak the tissues in 50% ethanol for 120 min, 70% ethanol for 120 min, 80% ethanol for 60 min, 95% ethanol (Ⅰ) for 60 min, 95% ethanol (Ⅱ) for 60 min, absolute ethanol (Ⅰ) for 45 min, and absolute ethanol (Ⅱ) for 45 min for dehydration treatment; continue to sequentially place the tissues in xylene (Ⅰ) for 16 min, xylene (Ⅱ) for 20 min, and xylene (Ⅲ) for 15 min for clearing treatment; then sequentially place the tissues in paraffin (Ⅰ) for 90 min and paraffin (Ⅱ) for 90 min for infiltration treatment; place the tissue blocks in a mold for embedding; use a microtome to cut the wax blocks into 4-μm-thick slices, then place them in water at 40 °C to spread, take out the tissue sections with an adhesive-coated glass slide, then place them in an incubator at 45 °C for drying, and finally store them at 4 °C. HE staining: Perform staining to observe the histopathological changes in rat tissues. Bake the cut tissue sections in an oven at 60 °C for 2 h; dewax the samples in xylene (Ⅰ) for 15 min and xylene (Ⅱ) for 15 min respectively; hydrate the sections by soaking them in absolute ethanol (Ⅰ) for 5 min, absolute ethanol (Ⅱ) for 5 min, 85% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and distilled water for 2 min respectively; control the hematoxylin staining time at 5 min, rinse with tap water for 1 min, then use 1% hydrochloric acid ethanol for differentiation treatment for 10 s, rinse with tap water for 1 min again, then perform eosin staining for 2 min, and finally rinse with tap water again; sequentially soak the sample slides in 95% ethanol (first time) for 5 min, 95% ethanol (second time) for 5 min, absolute ethanol (first time) for 5 min, and absolute ethanol (second time) for 5 min for dehydration treatment; sequentially place the sections in xylene (Ⅰ) for 5 min and xylene (Ⅱ) for 5 min for clearing treatment; seal the sections with neutral resin; observe and take pictures using an optical microscope.

[0027] Pathological observation of the heart found that in the normal group of rats, the heart tissue could show a normal morphology and regularity. As Figure 2 shown, the heart structure of the rats in the DM group became disordered, and at the same time, the presence of adipocytes could be seen. After treatment with Dapa and LAPE, there were obvious improvements compared with the DM group, and the cardiac lipid and vacuoles decreased.

[0028] Example 2: Use a small animal ultrasound instrument to detect the effects of Lithocarpus polystachyus extract on the cardiac function and morphology of diabetic rats;

[0029] Example 2 The specific implementation is as follows: Anesthetized rats are fixed in a supine position on an animal operation platform. The rats are mildly anesthetized with inhaled isoflurane (2 - 3%) and 1 L / min of oxygen, and the heart rate of the rats is maintained at 400 - 450 b / m for echocardiography (Vevo2100, FUJIFILM VisualSonics Inc, Toronto, Ontario, Canada). Echocardiography is performed using a 20 MHz linear transducer to obtain two-dimensional and two-dimensional-guided M-mode images. The following indicators are detected: Left ventricular systolic function: ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular stroke volume (SV), cardiac output (CO); Left ventricular diastolic function: reflecting the blood flow pattern through the mitral valve orifice (E / A), reflecting the myocardial diastolic pattern of the mitral valve (E' / A'), the ratio of early diastolic mitral blood flow velocity to early diastolic mitral annulus velocity (E / E'), myocardial performance index (MPI); Left ventricular morphology in diabetic rats: left ventricular internal diameter at systole (LVID:s), left ventricular internal diameter at diastole (LVID:d), measuring the anterior wall thickness of the left ventricle at end-diastolic (LVAW:d), the anterior wall thickness of the left ventricle at end-systole (LVAW:s), the posterior wall thickness of the left ventricle at end-diastolic (LVPW:d), the posterior wall thickness of the left ventricle at end-systole (LVPW:s).

[0030] In Example 2, in terms of the left ventricular systolic function indicators, as Figure 3 shown, compared with the Control group, the CO, EF, and FS of the rats in the DM group were significantly decreased (P < 0.05 or P < 0.01), indicating that the systolic function of the left ventricle in diabetic rats was impaired. Compared with the DM group, the CO, EF, FS, and SV of the rats in the Dapa and LAPE groups were significantly increased (P < 0.05 or P < 0.01); indicating that after administration of LAPE, the systolic function of the left ventricle in diabetic rats was somewhat restored.

[0031] In Example 2, in terms of the left ventricular diastolic function, as Figure 4 shown, compared with the Control group, E’ / A’ in the DM group was significantly decreased (P < 0.05), the MPI index was significantly increased (P < 0.01), and E / A and E / E’ increased to a certain extent, indicating that the diastolic function of the left ventricle in diabetic rats was impaired. Compared with the DM group, both Dapa and LAPE could significantly decrease E / A, E / E’, and the MPI index (P < 0.01), indicating that LAPE has the effect of alleviating the impairment of left ventricular diastolic function in diabetic rats.

[0032] In Example 2, in terms of the left ventricular morphology indicators, as Figure 5As shown, compared with the Control group, the LVID:d and LVID:s of diabetic rats were significantly increased (P<0.05 or P<0.01), and the LVPW:s was significantly decreased, indicating that diabetes led to an increase in the left ventricular volume of rats, and the internal and external diameter values of the left ventricle were significantly higher than those of normal rats. Compared with the DM group, Dapa and LAPE mainly improved the LVPW:s index (P<0.05). Combining with the previous evaluation of the heart coefficient, it shows that LAPE has a certain improvement in the change of left ventricular volume.

[0033] Example 3: Western Blot method was used to verify the targets and mechanisms of Lithocarpus polystachyus extract in protecting the hearts of diabetic rats.

[0034] In Example 3, Western Blot was used to detect the protein expressions of Nrf2, SLC7A11 and FTH1 in heart tissues.

[0035] According to Example 3, the method of Western blot is as follows: Extraction of total tissue protein: Take an appropriate amount of fresh tissue and place it in a sterile EP tube. At the same time, add tissue lysate to the EP tube (based on the size of the obtained tissue, this operation is carried out on ice), and finally add clean grinding beads. (The ratio of the tissue lysate is RIPA lysate: Phosphatase inhibitor A solution: Phosphatase inhibitor B solution: PMSF = 100:1:1:1). Homogenize the tissue at a frequency of 60 Hz and a temperature of 4°C for 5 - 10 minutes using a high-speed homogenizer. Centrifuge the homogenized liquid at 12,000 rpm at 4°C for 15 minutes, and aspirate the supernatant liquid and place it in a new sterile EP tube; Protein concentration determination (BCA): According to the instructions, mix BCA reagent A and BCA reagent B in a ratio of 50:1, and prepare the required volume of BCA working solution according to the total amount of the sample. Add 98 μL of BCA working solution to each well of the microplate. Subsequently, inject 2 μL of protein standard solution and the sample into each well in turn (each protein standard solution and the sample need to be injected 3 times repeatedly) (the concentrations of the protein standard solutions are 0, 2, 4, 6, 8 mg / mL). Place the microplate with the added liquid into a microplate reader, incubate it at 37°C for 30 minutes, shake it for 30 seconds, and then measure the absorbance at a wavelength of 562 nm, draw a standard curve, substitute the absorbance of the sample, calculate the sample concentration, and according to the concentration of the sample, add an appropriate amount of tissue lysate and 5× Loading buffer, and then place it in a metal bath at 98°C and shake it for 5 minutes, and let it cool naturally. Then carry out aliquoting and store it at -20°C; SDS-PAGE electrophoresis: Estimate the position where the protein molecular weight is to be determined and prepare separation gel and stacking gel with appropriate concentrations; After the electrophoresis equipment is installed, add 1× electrophoresis buffer to the electrophoresis tank. Then, remove the comb and load the samples in the order of marker, sample, and marker. The loading amount of total protein per well is 10 - 30 μg. Adjust the voltage to 80 V and electrophorese for about 30 minutes until the bromophenol blue band runs to the boundary between the stacking gel and the separation gel. Then adjust the voltage to a fixed voltage of 120 V and electrophorese for about 90 minutes. Stop electrophoresis when the bromophenol blue electrophoreses to the bottom of the separation gel, and remove the gel plate. Transfer membrane: Take out the gel from the glass plate and cut the gel according to the Marker position; Activate PVDF with methanol in advance and cut a membrane of appropriate size according to the size of the gel before use; Install the transfer membrane sandwich using the wet transfer method (according to the principle of "black gel and white membrane"), and pay attention to avoiding the generation of bubbles during membrane transfer; Place the sandwich into the transfer membrane tank, adjust it to a constant current of 250 mA, and set the membrane transfer time to 180 minutes; After the membrane transfer is completed, place the membrane in 1× TBST solution.Immunoblotting and development: Prepare 5% defatted milk powder, place the membrane in it, and shake and incubate at room temperature for 1 h; then wash the membrane 3 times with TBST for 5 min each time; cut the membrane according to the molecular weight of the target protein and the position of the Marker; place different bands in the corresponding primary antibody and incubate overnight at 4°C for immunoblotting; wash the membrane 3 times with TBST by shaking for 5 min each time; transfer different membrane bands to appropriate secondary antibody for incubation and hybridize by shaking at room temperature for 1 h; wash the membrane 3 times with TBST by shaking for 5 min each time; prepare the developing solution, completely immerse the membrane bands in the developing solution, and develop and photograph using the Bio-Rad imaging system; use ImageJ software to statistically analyze the gray value and use GraphPad Prism 9 software to process the data and plot the graph.

[0036] As Figure 6 , compared with the blank group, NRF2, SLC7A11 and FTH1 in the DM group were all significantly decreased (P<0.05 or P<0.01). After LAPE treatment, these indicators in the two dose groups increased to varying degrees. Among them, the LAPE-L group could significantly increase the protein expression of NRF2 and SLC7A11 in diabetic rats (P<0.05 or P<0.01). It shows that LAPE can reduce cardiac oxidative stress in diabetic rats.

[0037] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Those skilled in the art to which the present invention pertains can make various modifications or supplements to the described specific embodiments or use similar methods to replace them, but will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. The protective effect of Lithops multiflora on diabetic cardiomyopathy is characterized by: The extract of Lithospermum multiflorum has the effect of protecting diabetic patients from cardiomyopathy.

2. The protective effect of Lithops multiflora on diabetic cardiomyopathy according to claim 1, characterized in that: The polyspike Lithops includes health products containing polyspike Lithops or polyspike Lithops extract, which can alleviate cardiomyopathy in diabetic patients.

3. The protective effect of Lithops multiflora on diabetic cardiomyopathy according to claim 1, characterized in that: The polyspike Lithops includes Chinese medicinal materials containing polyspike Lithops or polyspike Lithops extract, which can alleviate cardiomyopathy in diabetic patients.

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