Application of gamma-linolenic acid in preparation of medicine for preventing or treating diabetic cardiomyopathy

Abstract

The invention provides application of gamma-linolenic acid in preparation of a medicine for preventing or treating diabetic cardiomyopathy. In-vivo and in-vitro experimental results show that the gamma-linolenic acid has a better effect in diabetic cardiomyopathy, which is mainly reflected in reducing oxidative stress (reducing lipid peroxidation), increasing expression of GSH and HO-1, reducing expression of MDA and Nrf2, increasing left ventricular ejection fraction (LVEF) and shortening fraction (FS), and repairing myocardial interstitial fibrosis. Therefore, the gamma-linolenic acid has the potential of being developed into the medicine for treating the diabetic cardiomyopathy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of gamma-linolenic acid in preparation of a drug for preventing or treating diabetic cardiomyopathy. BACKGROUND

[0002] Diabetic cardiomyopathy (DCM) refers to myocardial disease occurring in diabetic patients and cannot be explained by hypertensive heart disease, coronary atherosclerotic heart disease and other heart diseases. The pathological features are hypertrophy of myocardial cells, interstitial fibrosis, thickening of the basement membrane of coronary arterioles, and myocardial microvascular disease. Diabetic cardiomyopathy causes extensive focal necrosis of myocardium on the basis of metabolic disorders and microvascular disease, and subclinical cardiac dysfunction occurs, and eventually progresses to heart failure, arrhythmia and cardiogenic shock, and severe patients even die of sudden death.

[0003] With the continuous increase of the prevalence of diabetes, the incidence of diabetic cardiomyopathy is also increasing. However, there is currently no effective treatment for diabetic cardiomyopathy in clinical practice, and the treatment plan is still mainly to control blood sugar and improve insulin utilization, and there is a lack of treatment drugs for cardiac toxicity. The drug treatment targeting the control of blood sugar will bring the side effect of hypoglycemia. Therefore, it is very urgent to develop a new drug for treating diabetic cardiomyopathy. SUMMARY

[0004] In order to solve the problems in the prior art, the present application provides application of gamma-linolenic acid in preparation of a drug for preventing or treating diabetic cardiomyopathy.

[0005] The present application adopts the following technical solutions:

[0006] Application of gamma-linolenic acid in preparation of a drug for preventing or treating diabetic complications.

[0007] According to an embodiment of the present application, the diabetic complication is diabetic cardiomyopathy.

[0008] According to an embodiment of the present application, the application of gamma-linolenic acid in preparation of a drug for preventing or treating diabetic cardiomyopathy.

[0009] Gamma-linolenic acid (GLA), chemical name octadecatrienoic acid, molecular formula C 18 H 30 O2, CAS No. 463-40-1, is an essential unsaturated fatty acid for human body.

[0010] According to an embodiment of the present application, the application is to reduce oxidative stress. Further, the reduction of oxidative stress is to reduce lipid peroxidation.

[0011] According to an embodiment of the present application, the application is to increase GSH expression.

[0012] According to an embodiment of the present application, the application is to decrease MDA expression.

[0013] According to an embodiment of the present application, the application is to increase HO-1 expression and decrease Nrf2 expression.

[0014] According to an embodiment of the present application, the application is to increase left ventricular ejection fraction (LVEF).

[0015] According to an embodiment of the present application, the application is to increase fractional shortening (FS).

[0016] According to an embodiment of the present application, the application is to repair myocardial interstitial fibrosis.

[0017] Beneficial effects:

[0018] The present application provides the use of γ-linolenic acid in the preparation of a drug for preventing or treating diabetic cardiomyopathy. The results of in vivo and in vitro experiments show that γ-linolenic acid has good effects in diabetic cardiomyopathy, mainly in reducing oxidative stress (reducing lipid peroxidation), increasing GSH and HO-1 expression, decreasing MDA and Nrf2 expression, increasing left ventricular ejection fraction (LVEF) and fractional shortening (FS), and repairing myocardial interstitial fibrosis. Therefore, γ-linolenic acid has the potential to be developed into a drug for treating diabetic cardiomyopathy. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a flow chart of the treatment of diabetic cardiomyopathy in mice using γ-linolenic acid;

[0020] Figure 2 is a flow chart of the intervention of γ-linolenic acid in cell experiments;

[0021] Figure 3 is the cell survival rate of rat myocardial cells H9C2 under different glucose concentrations at different times;

[0022] Figure 4 is the cell survival rate of rat myocardial cells H9C2 under different palmitic acid concentrations at different times;

[0023] Figure 5 is the cell survival rate of rat myocardial cells H9C2 under different GLA concentrations at different times;

[0024] Figure 6 is the ROS detection result of rat myocardial cells H9C2;

[0025] Figure 7 is a graph of the statistical results of GSH and MDA in rat H9C2 myocardial cells;

[0026] Figure 8 Figure 3 is a Western Blot result chart of rat H9C2 myocardial cells HO-1 and Nrf2;

[0027] Figure 9 Figure 4 is a result chart of mouse echocardiogram;

[0028] Figure 10 Figure 5 is a masson and HE staining chart of myocardial cells of mice in each group. DETAILED DESCRIPTION

[0029] In order to further illustrate the present application and its advantages, the technical solutions of the present application will be further described below through specific embodiments, and it should be understood that these embodiments are only used to help understand the present application and should not be regarded as specific limitations to the present application.

[0030] Unless otherwise specified, the parts in the present application are weight parts, and the percentages are mass percentages.

[0031] Example 1

[0032] In the present study, the inventors studied the role of GLA in DCM and its influence on DCM through in vivo and in vitro experiments. The in vivo research process is shown in Figure 1 , and the in vitro research process is shown in Figure 2 .

[0033] In the in vivo experiment, the inventors established a DCM mouse model by feeding high-sugar high-fat feed combined with low-dose streptozotocin (STZ) injection. The protein regulation of heme oxygenase 1 (HMOX1) and nuclear factor erythroid-derived 2-like 2 (Nrf2) in oxidative stress, and the content of biochemical indicators of oxidative stress, malondialdehyde (MDA) and glutathione (GSH) were detected.

[0034] In the in vitro experiment, the inventors established an in vitro DCM model by culturing H9C2 cells with high-concentration glucose and palmitic acid intervention, which confirmed that GLA plays a crucial role in the diabetic microenvironment in vivo and in vitro.

[0035] Establishment of DCM animal model:

[0036] The newly purchased 3-week-old mice were first fed with ordinary feed for one week, and then randomly divided into two groups: control group (Control group) and experimental group (DCM group). From the age of 4 weeks, the DCM group mice were fed with high-fat feed containing 60% fat to induce insulin resistance; the Control group mice were fed with ordinary feed for 8 weeks until the mice were 12 weeks old.

[0037] Preparation of citric acid buffer: weigh 1.49 g of sodium citrate (MW: 294.10) and 1.05 g of citric acid (MW: 210.14), respectively, and dissolve in 50 mL of ddH2O. When used, mix them in equal proportions to adjust the pH to about 4.5. Weigh the STZ powder into a dry and sterile EP tube, wrap it with tin foil, and pre-cool it on ice together with the citric acid buffer, and take it to the animal room for use.

[0038] After the mice were fasted overnight, their body weight was measured; before use, 10 mg / mL of STZ injection was prepared with citric acid buffer. According to the fasting body weight of the mice, the DCM group mice were injected with 45 mg / kg of STZ injection intraperitoneally, and the control group mice were injected with the same amount of sodium citrate buffer, for 3 consecutive days. After the injection of STZ, the mice were given sufficient water and food, and the bedding was changed daily to keep the cages dry.

[0039] Detection of the effect of STZ-induced diabetic model: 7 days after STZ injection, the mice were fasted overnight, and the next morning at 8 o'clock, the fasting blood glucose of the mice was measured. Mice with FBG≥11.1 mmol / L were considered to have successfully modeled diabetes and were included in the subsequent experiments. Subsequently, the DCM group mice were continued to be fed with high-fat feed, and the Control group mice were fed with ordinary feed until the age of 25 weeks.

[0040] Echocardiography of mice:

[0041] (1) Use the small animal ultrasound imaging system VINNO 6 to detect the heart of the mouse, and the operation is performed by a technician who is unaware of the animal experiment grouping;

[0042] (2) Use 3% isoflurane to anesthetize the mouse, and after complete anesthesia, use depilatory cream to remove the hair on the chest to the upper abdomen of the mouse;

[0043] (3) Place the mouse on the ultrasound operation panel, fix the limbs of the mouse on the ultrasound electrode sheet with tape, and apply coupling agent on the skin of the limbs and the heart area to avoid air bubbles;

[0044] (4) The ultrasound probe notch is directed towards the head of the mouse, rotated counterclockwise by about 45°, and the probe is adjusted to fit the skin of the mouse;

[0045] (5) Turn on B-mode ultrasound mode, adjust the operation panel according to the image until the B-mode echocardiogram of the left ventricular long axis can be clearly observed; then turn on M-mode ultrasound mode, adjust the sampling line to the correct position and press M-mode ultrasound mode again to obtain the M-mode echocardiogram of the left ventricular long axis;

[0046] (6) Measure and record LVEF and FS by echocardiogram.

[0047] Western Blot:

[0048] (1) Preparation of related reagents (store at 4°C)

[0049] 1) 5× electrophoresis buffer: weigh SDS 5.0 g, Gly 72.0 g, Tris-base 15.15 g, dissolve with ddH2O, and make up to 1000 mL with water; dilute 5 times to get 1× electrophoresis buffer;

[0050] 2) 5× transfer buffer: weigh Gly 72.0 g, Tris-base 15.15 g, dissolve with ddH2O, and make up to 1000 mL with water; take 200 mL and add 200 mL of methanol, make up to 1000 mL with ddH2O and mix well to get 1× transfer buffer;

[0051] 3) TSBT buffer: weigh Tris-base 24.23 g, NaCl 80.06 g, dissolve with ddH2O, and make up to 1000 mL with water to get 10× TBS; take 100 mL of it + 900 mL of ddH2O + 1 mL of Tween 20 and mix well to get 1× TBST buffer;

[0052] 4) Protein lysis solution: take 5 mL of RIPA buffer and add 50 μL of protease inhibitor PMSF to get the required protein lysis solution (prepare fresh before use);

[0053] 5) 5% skimmed milk: dissolve 5.0 g of skimmed milk powder in 100 mL of TBST buffer.

[0054] (2) Extraction of heart tissue protein

[0055] 1) Weigh about 100 mg of mouse heart tissue and put it in a mortar, add 1 mL of protein lysis solution and liquid nitrogen, and grind the tissue quickly and thoroughly;

[0056] 2) Transfer the tissue homogenate to a 1.5 mL EP tube, stand for 10 min, then centrifuge at 12000 rpm for 15 min at 4°C;

[0057] 3) Take the supernatant, which is the total protein of the tissue required;

[0058] 4) Measure the protein concentration of each tube, calculate and add different amounts of RIPA lysis buffer to each protein sample to make the concentration uniform;

[0059] 5) Add 5x protein loading buffer at a volume ratio of 4:1, heat at 100°C for 10 min to make the protein boil and denature completely;

[0060] 6) Store the denatured protein at -20°C for subsequent use.

[0061] (3) Measure the protein concentration of the extracted tissue

[0062] 1) Use BCA method to measure;

[0063] 2) Prepare protein standard: prepare a protein standard solution with a concentration of 25 mg / mL according to the instructions; take 20 μL of it and add 980 μL of PBS to make a 0.5 mg / mL protein standard;

[0064] 3) Mix the standard 0, 1, 2, 4, 8, 12, 16, 20 μL with 20, 19, 18, 16, 12, 8, 4, 0 μL of PBS respectively and add them to the 96-well plate.

[0065] 4) Add 4 μL of different groups of samples + 16 μL of PBS to the sample wells of the 96-well plate, set 3 duplicate wells for each group;

[0066] 5) Mix A and B liquids at a ratio of 50:1 according to the instructions to prepare BCA detection working solution;

[0067] 6) Add 200 μL of working solution to each well and place it at 37°C for 0.5 h;

[0068] 7) Adjust the wavelength of the enzyme marker to 562 nm, measure the absorbance, and draw the standard curve;

[0069] 8) Calculate the protein concentration of the sample according to the average absorbance of the sample and the standard curve.

[0070] Cell protein extraction:

[0071] Trypsinize the cells and collect them in a centrifuge tube, discard the supernatant after centrifugation, add 1 mL of Ripa per 50 million cells, and sonicate the cells; centrifuge at 8000g at 4°C for 10 min, take the supernatant, and place it on ice for testing; the remaining steps are the same as above.

[0072] 9) Wash the glass plate with ddH2O the day before and dry it in the oven, install the glass plate in the gel preparation tank the next day, and prepare 10% SDS-PAGE gel according to the instructions;

[0073] 10) Fix the gel with the comb in the electrophoresis tank, add electrophoresis buffer to the upper part, then take out the comb vertically upward;

[0074] 11) Calculate the protein loading volume according to 20 μg protein per well, and add 5 μL marker on both sides as molecular weight marker;

[0075] 12) Electrophoresis: since the target protein molecular weight is small, first run at low voltage 30 V for 30 min, until all samples form a straight line; then continue running at 80 V, about 70 min, stop electrophoresis when the sample is in the middle of the glass plate;

[0076] 13) Electrotransfer: prepare in advance 15 min, cut the PVDF membrane, immerse in methanol for about 10 s; unfold the electrotransfer clamp, lay the sponge pad and filter paper on both sides, put the gel and membrane in the middle (be sure to "black glue white membrane", otherwise the protein will not be transferred to the membrane), clamp tightly, and use the roller to chase away small bubbles; 250 mA constant current for 100 min (note that heat is generated during electrotransfer, so the whole process is carried out in ice bath);

[0077] 14) Blocking: immerse the strip in 5% skimmed milk, shake on the shaker at room temperature for 2 h;

[0078] 15) Incubate the first antibody: immerse the strip in the first antibody prepared in milk, shake on the shaker at 4°C overnight (15-18 h);

[0079] 16) Wash the membrane: TBST buffer for 3 times, 10 min each time;

[0080] 17) Incubate the second antibody: room temperature shaker for 2 h;

[0081] 18) Wash the membrane: TBST buffer for 3 times, 10 min each time;

[0082] 19) Mix A and B luminescent solution in a ratio of 1:1 just before use, immerse in the PVDF membrane, and expose to imaging in the gel system.

[0083] CCK8 screening of optimal drug intervention concentration:

[0084] (1) H9C2 cells were seeded in a 96-well plate at a density of 103 cells per well, 100 μL of complete culture medium was added to each well, and the plate was incubated in an incubator;

[0085] (2) After 24 h, the original culture medium was removed, and complete culture medium containing 25, 33.3, 50, 60, 100 mM glucose and 0.1, 0.2, 0.4, 0.8 mM palmitic acid was added to the plate, 4 replicate wells were set for each group, and wells containing only DMEM were used as blank control group;

[0086] (3) Incubate each group of cells in the incubator for 24, 48 and 72h respectively;

[0087] (4) Add CCK-8 solution into the complete culture medium to make its concentration 10%, then add complete culture medium containing 10% CCK-8 solution into each well;

[0088] (5) Put the well plate into the cell culture incubator for 2h;

[0089] (6) Measure the absorbance of each well at 450nm by the microplate reader.

[0090] MDA content determination

[0091] (1) Sample preparation: prepare cell sample: trypsinize the cells and collect into centrifuge tube, discard the supernatant after centrifugation, add 1 mL extraction solution per 50 million cells, and break the cells by ultrasonic wave; centrifuge at 8000g at 4°C for 10min, take the supernatant, and place on ice for testing;

[0092] (2) Start the preheating microplate reader 30min in advance;

[0093] (3) Add sample according to the instruction;

[0094] (4) Heat the mixed solution at 100°C in a metal bath for 70min, a large amount of precipitate can be seen, and centrifuge at 10000g at room temperature for 10min;

[0095] (5) Take 200μL supernatant from each group and add into the 96-well plate, measure the absorbance of each well at 532nm and 600nm, and calculate the MDA content of the sample according to the formula in the instruction.

[0096] GSH content detection:

[0097] (1) Sample preparation: collect no less than 50 million cells, wash with PBS for 2 times, add 1 mL extraction solution to resuspend the cells, break the cells by ultrasonic wave; centrifuge at 8000g at 4°C for 10min, take the supernatant, and place on ice for testing;

[0098] (2) Add sample into the 96-well plate according to the instruction;

[0099] (3) Let the mixed solution stand at room temperature for 2min, measure the absorbance of each well at 412nm, and calculate the GSH content of the sample according to the formula in the instruction.

[0100] Cell MDA is as follows: the remaining steps are the same as those for tissues.

[0101] Cell reactive oxygen species (ROS) detection:

[0102] (1) Dilute DCFH-DA with DMEM at a ratio of 1:1000 to make its final concentration 10μmol / L;

[0103] (2) In situ loading of probes: remove the culture solution, add 1 mL of diluted DCFH-DA to each well of the twelve-well plate (for the positive control group, add an additional 1 μL of active oxygen positive control to each well);

[0104] (3) Place the well plate in a 37°C cell incubator and incubate for 20 min;

[0105] (4) Wash the cells with DMEM for 3 times, 2 min each time, to fully remove the DCFH-DA that does not enter the cells;

[0106] (5) Use the FITC channel, observe and take pictures under the fluorescence microscope.

[0107] Experimental results:

[0108] The inventors used the CCK-8 method to explore the effect of glucose and palmitic acid concentrations on the activity of cultured cells to obtain the optimal drug intervention concentration. The results showed that within 25-60 mmol / L, increasing the glucose concentration did not affect the cell activity, but 100 mmol / L glucose could significantly kill cells and reduce cell activity Figure 3 ). Finally, the inventors selected 60 mmol / L glucose as the intervention concentration. In addition, as the concentration of palmitic acid increased, the number of surviving cells decreased and the cell activity decreased significantly. Considering that too low a concentration of drugs is not enough to cause oxidation of cells, and too high a concentration of drugs causes large-area cell death, it is not enough to collect enough cells for subsequent experiments. Therefore, the inventors finally selected 0.1 mmol / L as the intervention concentration of palmitic acid Figure 4 ). The concentration of GLA showed an increasing trend within 0-100 umol / L, but it decreased sharply after exceeding 100 umol, so 100 umol was selected as the intervention concentration of the drug Figure 5 ).

[0109] High glucose and high fat combined intervention causes oxidative stress in H9C2 cells

[0110] In order to establish a DCM cell model in vitro, the inventors cultured H9C2 cells with 60 mmol / L glucose and 0.1 mmol / L palmitic acid for 48 h, and detected the occurrence of oxidative stress. The cell experiment was divided into the following four groups: control group (Control group), control plus drug group (control+GLA), high glucose and high fat group (High glucose+PA, DCM), and high glucose and high fat and GLA treatment group (DCM+GLA). The ROS detection results showed that high glucose and high fat increased the production of reactive oxygen species in H9C2 cells, and the addition of GLA reversed this trend. It is shown that lipid peroxidation increases when cells are stimulated by high glucose and high fat, and GLA can reduce lipid peroxidation Figure 6).

[0111] Biochemical kit detection results show that high glucose and high fat intervention increases the expression of MDA in cells, and reduces the expression of GSH, while adding GLA can reverse this trend. Figure 7 ).

[0112] Western Blot results show that high glucose and high fat intervention up-regulates the expression of HO-1 and Nrf2 proteins in H9C2 cells, and adding GLA can increase the expression of HO-1 and reduce the expression of Nrf2.

[0113] Mouse echocardiogram performance: diabetic mice showed reduced cardiac function, manifested as decreased left ventricular ejection fraction (LVEF) and fractional shortening (FS), while adding GLA can reverse this trend. Figure 9 ).

[0114] Pathological changes of diabetic cardiomyopathy mouse heart

[0115] Due to the long duration of diabetes, glycosylated collagen deposition can occur in myocardial interstitial fibrosis. In order to clarify the pathological changes of the heart of DCM mice, the inventors performed HE and Masson staining of heart tissue. HE staining showed that the myocardial cells of the Control group mice were arranged in a neat and dense manner, the cell structure was clear, and the extracellular matrix was less; while the myocardial fibers of the DCM group mice were disordered, the myocardial cells were hypertrophic and deformed, the intercellular space was increased, and the extracellular matrix around the blood vessels was increased, while adding GLA can reverse this trend. Masson trichrome staining showed that the interstitial and perivascular fibrosis of the heart of the DCM group mice was obvious, while adding GLA can reverse this trend. The above experimental results confirm that the cardiac function of the DCM group mice is impaired in the later stage, and fibrosis occurs in the myocardium and perivascular area, and diabetic cardiomyopathy occurs, while adding GLA can reverse this trend.

Claims

1. Use of γ-linolenic acid in the preparation of a medicament for the prevention or treatment of a diabetic complication; said γ-linolenic acid having the chemical name of octadecatrienoic acid, molecular formula C 18 H 30 O2, and said diabetic complication is diabetic cardiomyopathy.

2. Use according to claim 1, wherein The application is to reduce oxidative stress.

3. Use according to claim 2, wherein the compound is ###0002### The reduction of oxidative stress is to reduce lipid peroxidation.

4. Use according to any one of claims 1 to 3, wherein The application is to increase GSH expression.

5. The application as described in any one of claims 1-3, characterized in that, The application is to reduce MDA expression.

6. Use according to any one of claims 1 to 3, wherein The application is to increase HO-1 expression and reduce Nrf2 expression.

7. Use according to any one of claims 1 to 3, wherein the compound is ###0002### The application is to increase left ventricular ejection fraction (LVEF).

8. Use according to any one of claims 1 to 3, wherein The application is to increase fractional shortening (FS).

9. Use according to any one of claims 1 to 3, wherein the compound is ###0002### The application is to repair myocardial interstitial fibrosis.

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