Application of oral bear gall powder in preparation of medicine for treating myocardial infarction

Abstract

The invention mainly relates to application of oral bear gall powder in preparation of a medicine for treating myocardial infarction, and belongs to the technical field of biological medicines. The oral bear gall powder is used for treating myocardial infarction, can reduce the area of myocardial infarction, inhibit myocardial fibrosis and inflammatory response, recover cardiac functions, inhibit myocardial hypertrophy and apoptosis of myocardial cells, effectively treat myocardial infarction and improve prognosis conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of oral bear bile powder in preparing medicines for treating myocardial infarction. Background Art

[0002] Acute myocardial infarction (AMI) is an acute ischemic and hypoxic myocardial injury caused by coronary artery thrombosis or vasospasm, with clinical manifestations of chest pain, dyspnea, dizziness, nausea, sweating, etc. AMI is defined as a "four highs and one more" type of cardiovascular disease with high morbidity, high mortality, high disability rate, high recurrence rate and multiple cumulative organs, and has become the most important cardiovascular disease that endangers human life and health.

[0003] The local ischemia and hypoxia caused by AMI will lead to the death of a large number of myocardial cells, affecting the normal structure and function of the heart, and then the heart will enter the self-repair stage. First, a strong aseptic inflammatory response will be induced in the infarct and downstream areas to remove dead / damaged cardiac cells and extracellular matrix. This stage is called the acute inflammatory phase. After the inflammatory phase, the heart will enter the post-inflammatory repair phase. Fibroblasts proliferate and begin to synthesize collagen to form scar tissue. This scar tissue does not have the elasticity and contractile function of normal myocardium, but helps to maintain structural integrity. However, due to the non-renewability of myocardial cells, the heart repair process usually cannot completely restore the original myocardial structure and function. In addition, excessive activity, prolonged time or spatial expansion of the reactions involved in any of the above stages will lead to additional damage, including the loss of myocardial cells, inhibition of left ventricular contractile function, enhanced matrix degradation, etc., which will eventually lead to enlargement of the heart chamber, excessive tissue necrosis, destruction of the integrity of the ventricular wall, heart rupture, and even when scarring is formed, the degree of fibrosis will be aggravated far beyond the initial infarction lesion area, and the prognosis will be even worse. Therefore, there is an urgent need for a drug that can treat myocardial infarction and improve prognosis.

[0004] Bear bile powder (BBP) is the dried bile of the brown bear (Ursus arctos Linnaeus) or black bear (Selenarstos thibetanus G.Cuvier), which is of the Ursidae family. It has a glassy luster, is brittle and easy to absorb moisture, is cold in nature, has a fishy and bitter taste, and is in the liver, gallbladder, and heart meridians. The chemical composition of bear bile powder is relatively complex, mainly divided into: Ursodeoxycholic acid (UDCA), Chenodeoxycholic acid (CDCA), Tauroursideoxycholic acid (TUDCA), Taurochenodeoxycholic acid (TCDCA), cholesterol, bilirubin and other substances. It has the effects of clearing heat, calming the liver, and improving eyesight. It is used to treat symptoms such as eye diseases, convulsions, and sore throat caused by damp-heat accumulation in the liver meridian. Bear bile has been used as medicine for more than 2,000 years. It is known as the "gold of medicine" and ranks first among the four precious Chinese medicines. Ancient books have recorded many clinical applications of bear bile. The first record of bear bile as medicine was "Ming Yi Bie Lu" written in the late Han Dynasty. In the Song Dynasty, bear bile was used to treat diseases such as malnutrition, convulsions, hemorrhoids, and eye diseases. However, there are no reports on the oral use of bear bile powder to treat myocardial infarction. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an application of oral bear bile powder in the preparation of a drug for treating myocardial infarction, so as to effectively treat myocardial infarction and improve the prognosis.

[0006] The invention provides application of oral bear bile powder in preparing medicine for treating myocardial infarction.

[0007] Preferably, the oral bear bile powder consists of bear bile powder and sodium carboxymethyl cellulose.

[0008] Preferably, the sodium carboxymethyl cellulose is a sodium carboxymethyl cellulose solution with a mass volume fraction of 0.3% to 0.8%.

[0009] Preferably, the mass volume ratio of the bear bile powder to the sodium carboxymethyl cellulose solution is (70-80) mg: (5-15) mL.

[0010] Preferably, the oral bear bile powder can reduce the area of ​​myocardial infarction.

[0011] Preferably, the oral bear bile powder can restore heart function after myocardial infarction.

[0012] Preferably, the oral bear bile powder can inhibit myocardial hypertrophy.

[0013] Preferably, the oral bear bile powder can inhibit myocardial cell apoptosis.

[0014] Preferably, the oral bear bile powder can inhibit inflammatory response.

[0015] Preferably, the oral bear bile powder can inhibit myocardial fibrosis.

[0016] Beneficial effects of the present invention:

[0017] The present invention provides an application of oral bear bile powder in the preparation of a drug for treating myocardial infarction. The oral bear bile powder is used for treating myocardial infarction, can reduce the area of ​​myocardial infarction, inhibit myocardial fibrosis and inflammatory response, restore cardiac function, inhibit myocardial hypertrophy and apoptosis of myocardial cells, effectively treat myocardial infarction, and improve prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the ECG changes before and after modeling in mice; the left picture is a normal ECG before modeling; the right picture is an ECG with ST segment elevation after modeling.

[0019] Figure 2 The figure is the result of myocardial infarction area detection in mice; the left figure is the TTC staining image of the hearts of mice in each group; the right figure is the statistical graph of myocardial infarction area; among them, # indicates a significant difference compared with the Sham group, P < 0.05; * indicates a significant difference compared with the MI group, P < 0.05.

[0020] Figure 3 Figure 1 is the result of cardiac function test of mice; A is the echocardiogram of mice in each group (left ventricular short-axis papillary muscle slice); B is the short-axis shortening rate (FS) of mice in each group; C is the ejection fraction (EF) of mice in each group; D is the left ventricular wall thickness at end diastole (LVPWd) of mice in each group; E is the left ventricular wall thickness at end systole (LVPWs) of mice in each group; F is the left ventricular inner diameter at end systole (LVIDs) of mice in each group; G is the left ventricular inner diameter at end diastole (LVIDd) of mice in each group; H is the serum CK-MB content of mice in each group; # indicates significant difference compared with Sham group, P < 0.05; * indicates significant difference compared with MI group, P < 0.05.

[0021] Figure 4 The figures are the results of myocardial hypertrophy detection in mice; A is the observation diagram of heart size of mice in each group; B is the statistical diagram of heart weight / tibia length value of mice in each group; C is the H&E staining diagram of hearts of mice in each group; D is the WGA staining diagram of hearts of mice in each group, and E is the statistical diagram of cross-sectional area of ​​myocardial cells of mice in each group; wherein, # indicates significant difference compared with Sham group, P < 0.05; * indicates significant difference compared with MI group, P < 0.05.

[0022] Figure 5 Figure 1 is a graph showing the results of myocardial cell apoptosis detection in mice; A is a graph showing TUNEL staining in each group of mice; B is a graph showing the statistical rate of myocardial cell apoptosis in each group of mice; # indicates a significant difference compared with the Sham group, P < 0.05; * indicates a significant difference compared with the MI group, P < 0.05.

[0023] Figure 6 The figures are the results of myocardial inflammation detection in mice; A is the F4 / 80 immunohistochemical staining of mice in each group; B is the F4 / 80 level of myocardial tissue of mice in each group; C is the IL-10 level of serum of mice in each group; D is the IL-6 level of serum of mice in each group; E is the TNF-α level of serum of mice in each group; wherein, # indicates significant difference compared with the Sham group, P < 0.05; * indicates significant difference compared with the MI group, P < 0.05.

[0024] Figure 7 Figure 5 is the result of myocardial fibrosis detection in mice; A, from top to bottom, shows: Masson staining of the hearts of mice in each group, Sirius red staining of mice in each group, Coll-1 immunohistochemical staining of mice in each group, and α-SMA immunohistochemical staining of mice in each group; B is the area of ​​myocardial fibrosis in mice in each group statistically calculated by Masson staining; C is the area of ​​myocardial fibrosis in mice statistically calculated by Sirius red staining; D is the expression level of Coll-1 in each group of mice; E is the expression level of α-SMA in each group of mice; # indicates a significant difference compared with the Sham group, P < 0.05; * indicates a significant difference compared with the MI group, P < 0.05. DETAILED DESCRIPTION

[0025] The present invention provides the use of oral bear bile powder in preparing a medicine for treating myocardial infarction, wherein the myocardial infarction is preferably acute myocardial infarction.

[0026] In the present invention, the oral bear bile powder can preferably reduce the area of ​​myocardial infarction and restore heart function after myocardial infarction, and the restoration of heart function after myocardial infarction preferably includes restoring myocardial function and inhibiting the increase of serum creatine kinase content; the oral bear bile powder can preferably inhibit myocardial hypertrophy, and the inhibition of myocardial hypertrophy preferably includes inhibiting the degree of cardiac hypertrophy, reducing myocardial pathological damage and reducing the cross-sectional area of ​​myocardial cells; the oral bear bile powder can preferably also inhibit myocardial cell apoptosis and inhibit inflammatory response, and the inhibition of inflammatory response preferably includes inhibiting the infiltration of inflammatory factors in myocardial tissue and reducing the level of inflammatory factors in serum; the oral bear bile powder can also preferably inhibit myocardial fibrosis, and the inhibition of myocardial fibrosis preferably includes reducing the area of ​​myocardial fibrosis and inhibiting the expression of myocardial fibrosis-related proteins.

[0027] In the present invention, the oral bear bile powder is preferably composed of bear bile powder and sodium carboxymethyl cellulose. The sodium carboxymethyl cellulose is preferably a sodium carboxymethyl cellulose solution with a mass volume fraction of 0.3% to 0.8%. In one embodiment, the mass volume fraction of the sodium carboxymethyl cellulose solution can be 0.3%, 0.4%, 0.5%, 0.6%, 0.7% or 0.8%, more preferably 0.5%; then the suspension is prepared with sodium carboxymethyl cellulose, and the mass volume ratio of the bear bile powder to the sodium carboxymethyl cellulose solution is preferably (70-80) mg: (5-15) mL, more preferably (73-77) mg: (8-12) mL, and further preferably 75 mg: 10 mL.

[0028] In the present invention, the dosage form of the drug preferably includes powder, tablet, granule, capsule, solution or suspension, more preferably suspension.

[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] The bear bile powder used in the following examples is obtained by freeze-drying, bottling and sterilizing the bile, and is purchased from Yanbian Bear Farm with a product batch number of 20221011.

[0033] Example 1

[0034] An oral bear bile powder is obtained by mixing 10 mL of a 0.5% sodium carboxymethyl cellulose solution with 75 mg of bear bile powder, and is recorded as a bear bile powder suspension.

[0035] Example 2

[0036] An oral bear bile powder is obtained by mixing 15 mL of a 0.3% sodium carboxymethyl cellulose solution with 80 mg of bear bile powder, and is recorded as a bear bile powder suspension.

[0037] Example 3

[0038] An oral bear bile powder is obtained by mixing 5 mL of 0.8% sodium carboxymethyl cellulose solution with 70 mg of bear bile powder, and is recorded as a bear bile powder suspension.

[0039] Test Example 1

[0040] 1. Modeling, drug administration and sample processing

[0041] 1.1 Myocardial infarction model

[0042] The mouse chest and abdomen were depilated, and the isoflurane gas concentration in the anesthesia machine was adjusted to 2% to anesthetize the mouse. After anesthesia, surgery was performed, and the surgical procedure was as follows:

[0043] 1) Thoracotomy: Find the most obvious position of the mouse's apex beat, make a 1 cm incision on the left side of the sternum along the direction of the mouse's head, use hemostats and tweezers to bluntly separate the pectoralis major and minor muscles layer by layer, quickly open the chest cavity from the third and fourth intercostal spaces, rotate the hemostats about 45° to the left to open the intercostal space, and use the heartbeat rhythm to make the heart pop out automatically.

[0044] 2) Find the ligation position: Find the approximate position, gently scrape the heart with a 6-0 ligation needle, and you can clearly see the left coronary artery, which is different from the surrounding color. The specific ligation position is 1-2mm away from the root at the junction of the lower edge of the left atrial appendage and the pulmonary artery cone. Use 6-0 suture to quickly pass through and ligate. The ligation width and depth should be appropriate. The ligation is successful when the anterior wall of the left ventricle turns cyanotic or pale to the naked eye.

[0045] 3) Suture the chest: After ligation, check for bleeding, gently return the heart to the chest, and quickly squeeze out the air to avoid pneumothorax. Remove the anesthesia mask, wait for spontaneous breathing to resume, and suture the muscles and skin with 4-0 sutures.

[0046] 4) Awakening: After surgery, place the mouse on a heating blanket and wait for it to wake up. Return it to the cage after it regains mobility.

[0047] 5) Observe the changes in the electrocardiogram recorded before and after coronary artery ligation during the operation. Figure 1 As shown, before ligation of the left anterior descending coronary artery of mice, the electrocardiogram showed that the ST segment was not elevated; after ligation of the left anterior descending coronary artery of mice, the electrocardiogram showed that the ST segment was significantly elevated, and the absolute value of the elevation was ≥0.1mV.

[0048] 1.2 Oral administration

[0049] The sham group (sham operation group) was used as the control group, and the mice with successful surgical modeling were randomly divided into 4 groups: model group (MI), model group + low-dose bear bile powder group (MI+XL), model group + high-dose bear bile powder group (MI+XH), model group + positive drug group (MI+P) (positive drug: Tongxinluo Capsule, Shijiazhuang Yiling Pharmaceutical Co., Ltd., approval number: National Medicine Standard Z19980015, production batch number: A2212001). The MI+XL group and the MI+XH group were both intragastrically administered with the bear bile powder suspension prepared in Example 1. The low-dose bear bile powder dosage was 22.5 mg·kg -1 ·d -1 The high dose is 90 mg kg -1 ·d -1The positive drug dosage is 40 mg kg -1 ·d -1 The drugs were administered by gavage 2 hours after ligation and then every day for 28 days.

[0050] 1.3 Sampling

[0051] After the administration, ultrasound detection was performed first, and then blood was collected using a heparin sodium anticoagulation tube, centrifuged at 4°C and 3000 r / min for 20 minutes, and the supernatant was obtained to obtain a serum sample, which was stored at -80°C. After anesthesia, the mouse heart was taken, a part of which was fixed in 4% paraformaldehyde, and the rest was passed through liquid nitrogen and stored at -80°C for later use.

[0052] 1.4 Tissue sample processing

[0053] 1.4.1 Paraffin embedding

[0054] 1) Fix the heart portion taken out in 1.3 in 4% paraformaldehyde for more than 24 hours.

[0055] 2) Place the tissue in a labeled tissue embedding box and rinse under running water for 1 hour.

[0056] 3) Dehydration: Dehydrate with 75% ethanol (60 minutes), 85% ethanol (60 minutes), 95% ethanol (60 minutes), and 100% ethanol (5 times, 30 minutes each time, and replace with new 100% ethanol for the next treatment after each treatment).

[0057] 4) Deethanolization and transparency: Use xylene:ethanol (1:1, 30 minutes) and xylene (treat 3 times, 30 minutes each time, and replace new xylene after each treatment for the next treatment) to deethanol in sequence.

[0058] 5) Paraffin wax immersion: Soak in soft wax for 2 hours, then transfer to hard wax and soak for 2 hours.

[0059] 6) Embedding: Add a layer of melted hard wax to the bottom of the disposable embedding plate, clamp the heart tissue, place it with the cut surface facing down on the embedding plate, place it on the embedding table to allow it to solidify slightly, place the preheated embedding frame on the embedding plate, continue to add melted wax to the upper edge of the embedding frame, place it on a frozen ice plate, wait for it to completely solidify, gently peel off the embedding plate, and check for cracks.

[0060] 7) Storage: Store the embedded wax block (embedded block) at 4°C.

[0061] 1.4.2 Paraffin sections

[0062] 1) Fix the embedding block on the specimen table so that the bottom section of the embedding block is parallel to the section of the specimen holder. Push the knife table to a suitable position so that the slicer and the tissue section form an angle of about 5°, and the upper and lower edges of the embedding block are parallel to the knife edge.

[0063] 2) Trimming: Adjust the slice thickness on the micro-motion device. Start with 20 μm thickness and adjust the slice thickness to 4 μm when the tissue is cut.

[0064] 3) Slicing: Use your right hand to rotate the wheel, hold the brush in your left hand to pick up the slices under the blade, and drag the cut wax strip. When the wax strip contains the complete slices you need, stop rotating.

[0065] 4) Spreading: Use tweezers to gently pick up the cut wax tape and transfer it to room temperature water. Then use a glass slide to transfer the intact film to a spreader in a 45°C constant temperature water bath.

[0066] 5) Slice: After the tissue is fully unfolded, pick it up with a labeled slide, place it vertically on a slice rack, place it in an oven at 37°C overnight, and then collect the slices for later use.

[0067] 2. Detection of the effect of bear bile powder on myocardial infarction

[0068] 2.1 Effect of bear bile powder on myocardial infarction area

[0069] TTC staining was performed on the mouse hearts and the myocardial infarction area was calculated. The steps of TTC staining are as follows:

[0070] Preparation: 2% TTC solution (protected from light), preheat in a 37°C incubator for 30 minutes. Precool the blade in advance. Procedure: Cut 4-5 heart slices of 1 mm thickness from the apex to the ligation site, and dry them on filter paper. Pre-add 1 mL of TTC solution to each well of a six-well plate, place 2-3 heart slices symmetrically, and then add 1.5 mL of TTC solution. Observe the staining every 10 minutes and turn it over to ensure uniform staining. After the tissue turns dark red, pour out the TTC solution, add formalin fixative to immerse it, and stop staining. Store in a 4°C refrigerator protected from light.

[0071] The results are as follows Figure 2 As shown: TTC staining shows that the color of the mouse myocardium after surgery is divided into red and white, red is normal myocardium, and white is necrotic myocardium. 28 days after the left anterior descending branch of the mouse coronary artery was ligated, the color of the ligated part of the heart turned white. After calculation, the area of ​​myocardial infarction in mice was 57.75±4.99(%). After administration, high and low doses of bear bile powder can improve the area of ​​myocardial infarction in mice, which are 41.66±4.04(%) in the MI+XL group and 34.66±2.51(%) in the MI+XH group. The improvement effect of the high-dose group is better than that of the low-dose group.

[0072] 2.2 Effect of bear bile powder on cardiac function after myocardial infarction

[0073] 2.2.1 Ultrasonic testing

[0074] The cardiac ultrasound function of mice in each group was tested before surgery and on the 28th day after surgery. The cardiac function of mice was tested using a small animal ultrasound imager (VINNO). The specific operation is as follows:

[0075] The mouse chest and abdomen were depilated, and the isoflurane gas concentration in the anesthesia machine was adjusted to 2% to anesthetize the mouse. After applying the coupling agent, the probe was placed parallel to the midline of the mouse on its left chest, with the probe tip facing the mouse head. At this time, the probe tip was turned 45° to the left to make the probe parallel to the long axis of the mouse's left ventricle. The image obtained was the parasternal left ventricular long axis section. Based on the long axis, the probe tip was rotated about 90° to the right, and the probe was moved and tilted regularly to show the level of the papillary muscle. The image obtained was the left ventricular short axis papillary muscle section. Left ventricular function test: 8 points were selected, namely, the upper wall of the ventricular septum at end diastole, the lower wall of the ventricular septum at end diastole, the upper edge of the free wall at end diastole, the lower edge of the free wall at end diastole, the upper wall of the ventricular septum at end systole, the lower wall of the ventricular septum at end systole, the upper edge of the free wall at end systole, and the lower edge of the free wall at end systole for measurement to obtain the ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at end diastole (LVIDd), left ventricular internal diameter at end systole (LVIDs), left ventricular wall thickness at end diastole (LVPWd), and left ventricular wall thickness at end systole (LVPWs).

[0076] Short-axis left ventricular M-mode echocardiograms of mice in each group were shown in Figure 3 . The results showed that compared with the Sham group, the LVPWd, LVIDd, and LVIDs of the mice in the MI group were significantly increased, and the EF, FS, and LVPWs were significantly decreased (P<0.05); compared with the model group, the FS, LVPWd, LVIDd, and LVIDs of the mice in the MI+XL group were improved (P<0.05), and the EF, FS, LVPWs, and LVPWd of the mice in the MI+XH group were improved (P<0.05). This suggests that bear bile powder can improve the contractile function of the heart of mice after myocardial infarction, and it was found that the changes in the echocardiographic parameters of the mice treated with bear bile powder were reversed, indicating that bear bile powder has a repairing effect on myocardial dysfunction caused by myocardial infarction.

[0077] 2.2.2 Serum biochemical index detection

[0078] Take the serum sample in 1.3 and use a biochemical analyzer to detect the CK content in the serum

[0079] The results of CK content are as follows Figure 3As shown in H: Compared with the Sham group, the serum CK content of mice in the MI group was significantly increased (P<0.05). Compared with the MI group, the MI+XL group and the MI+XH group could reduce the serum CK content of mice (MI+XH group, P<0.05). This shows that bear bile powder can inhibit the increase of serum CK content in mice after myocardial infarction.

[0080] 2.3 Effect of bear bile powder on myocardial hypertrophy after myocardial infarction

[0081] 2.3.1 Observe the size of the mouse heart and calculate the cardiac hypertrophy index by heart weight / tibia length.

[0082] The results are as follows Figure 4 As shown in A and B in Figure 1, the heart of the MI group was significantly larger than that of the Sham group, and the cardiac hypertrophy index was significantly increased when the heart weight was compared with the tibia length (P<0.05). After drug treatment, the cardiac hypertrophy index of the MI+XL and MI+XH groups was significantly improved compared with the MI group (MI+XH group, P<0.05).

[0083] 2.3.2 H&E (Hematoxylin-Eosin) staining

[0084] 1) Slice drying: Place the cut paraffin slices (prepared in 1.4.2) into a slice rack and bake in an oven at 60°C for 2 hours.

[0085] 2) Dewaxing: Dewaxing in xylene twice, 10 minutes each time.

[0086] 3) Rehydration: In the order of 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol, each gradient ethanol treatment lasts for 3 minutes. Soak in distilled water for 2 minutes.

[0087] 4) Staining: Circle the stained tissue with an immunohistochemical pen, add hematoxylin stain solution, cover the tissue and stain the cell nucleus for 7 minutes, and wash away the floating color with distilled water; differentiate with differentiation solution for 1 minute, add tap water to soak twice, 5 minutes each time; stain with eosin stain solution for 2 minutes, and pour off the excess stain solution.

[0088] 5) Dehydration: Soak in 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 2 seconds each, then replace with new 100% ethanol and soak for 1 minute.

[0089] 6) Transparency: Soak in xylene twice, 1 minute each time.

[0090] 7) Sealing: After being transparentized with xylene, let the slices dry, add neutral gum, and seal with a clean cover glass. Allow to dry in a fume hood by self-heating and then observe under a microscope.

[0091] Results: H&E staining results of the hearts of mice in each group ( Figure 4C) shows that the myocardial cells in the Sham group are neatly arranged, the cytoplasm is uniform and rich, and the interstitium is normal. In the MI group, some myocardial cell nuclei are missing, the cells show vacuolar changes, the myocardial cells in the infarct area are disorderly arranged and the boundaries are unclear, and a large number of inflammatory cells are infiltrated. In contrast, the myocardial tissue damage in the MI+XL and MI+XH groups was significantly reduced, with only a small amount of inflammatory cells infiltrating. This shows that bear bile powder can effectively reduce the pathological damage caused by myocardial infarction in mice, and bear bile powder is more effective than positive drugs.

[0092] 2.3.3 WGA staining

[0093] WGA staining was used to assess the cross-sectional area (CSA) of cardiomyocytes as follows:

[0094] 1) Drying, dewaxing and rehydration are the same as steps 1)-3) under 2.3.2.

[0095] 2) Antigen repair: Place tissue sections in citrate repair solution (pH 6.0) and perform antigen repair in a pressure cooker. After the water boils, count for 5 minutes. Take out and naturally cool to room temperature, then wash twice with water, 5 minutes each time.

[0096] 3) Add WGA staining solution: Prepare WGA-AF488 staining solution (diluted with PBS 1:100), add 30 μL of WGA-AF488 staining solution to each tissue, and place in a wet box at 4°C overnight.

[0097] 4) Rewarming: Take out the slices on the next day and let them stand at room temperature or 37°C for 30 minutes to rewarm. Then discard the primary antibody and rinse with PBS three times, 5 minutes each time.

[0098] 5) DAPI nuclear staining: Add DAPI onto the tissue and let stand for 5-10 minutes.

[0099] 6) Add tissue autofluorescence quencher dropwise onto the tissue and let stand for 5-10 minutes.

[0100] 7) Sealing: Seal the slides with anti-fluorescence quenching sealing solution and take photos under a fluorescence microscope.

[0101] Results Figure 4 D and E in the figure showed that the CSA of the Sham group and MI+XH group was significantly lower than that of the MI group (P<0.05). Compared with the MI group, the MI+XL and MI+XH groups had an inhibitory effect on myocardial hypertrophy, indicating that bear bile powder can inhibit myocardial hypertrophy after myocardial infarction.

[0102] 2.4 Effect of bear bile powder on myocardial cell apoptosis after myocardial infarction

[0103] Cell apoptosis is the main factor affecting mortality and infarct size after myocardial infarction. TUNEL apoptosis staining was used to detect the effect of bear bile powder on myocardial cell apoptosis after myocardial infarction. The specific steps are as follows:

[0104] 1) Drying, dewaxing and rehydration are the same as steps 1)-3) under 2.3.2.

[0105] 2) Permeabilization: Add 20 μg / mL DNase-free proteinase K and allow to act at room temperature for 30 minutes to permeabilize the cell membrane and nuclear membrane to facilitate the TUNEL probe to enter the cell nucleus and ensure adequate staining.

[0106] 3) Washing: Wash 3 times with PBS, 5 minutes each time.

[0107] 4) Prepare TUNEL detection solution: TdT enzyme and fluorescent labeling solution are prepared in a ratio of 1:9. Each sample requires 50 μL. Prepare the detection solution according to the sample volume, mix thoroughly, and store in a dark place.

[0108] 5) Incubation and staining: Place each sample in a humidified chamber, add 50 μL of the prepared TUNEL detection solution to cover the tissue, and incubate at 37°C in the dark for 6 minutes.

[0109] 6) Washing: Wash 3 times with PBS, 5 minutes each time.

[0110] 7) Nuclear staining: Add appropriate amount of DAPI to each sample and stain for 10 minutes.

[0111] 8) Washing: Wash 3 times with PBS, 5 minutes each time.

[0112] 9) Sealing and observation: Seal the slides with anti-fluorescence quenching sealing solution and observe under a microscope.

[0113] Observation under fluorescence microscope showed Figure 5 Figure A shows that in the TUNEL colorimetric method, normal cardiomyocytes are stained blue, while apoptotic cardiomyocytes are stained red. After merging with DAPI, they become bright pink, which is used for statistical analysis to determine the apoptosis rate of cells. Figure 5 As shown in Figure B: Compared with the Sham group, the apoptosis rate of myocardial cells in the MI group was increased (P<0.05); compared with the MI group, the number of apoptotic myocardial cells in the MI+XL and MI+XH groups was reduced after intragastric administration (P<0.05), indicating that bear bile powder can significantly inhibit the apoptosis of myocardial cells in mice after myocardial infarction.

[0114] 2.5 Effect of bear bile powder on myocardial inflammation after myocardial infarction

[0115] 2.5.1 Immunohistochemical staining was used to detect the infiltration of macrophages in mouse myocardial tissue (F4 / 80). The specific steps are as follows:

[0116] 1) Drying, dewaxing and rehydration are the same as steps 1)-3) under 2.3.2.

[0117] 2) Antigen repair in a water bath at high temperature: Place a small container containing 0.01M sodium citrate buffer solution in a large container containing a certain amount of tap water, and heat to boil on an induction cooker. When the temperature in the small container reaches 92°C-98°C, place the slices in the sodium citrate buffer solution and time for 20 minutes. Then remove from the electric stove, cool naturally at room temperature for 30 minutes, and wash twice with PBS for 5 minutes each time.

[0118] 3) Blocking endogenous peroxidase: Place the sections in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Place the slides in PBS and wash them three times on a horizontal shaker, each time for 5 minutes.

[0119] 4) Blocking: Place the slices in a wet box, circle the tissue with an immunohistochemical pen, drop 5% BSA solution into the circle, and block in a 37°C incubator for 30 minutes.

[0120] 5) Incubate with primary antibody: Gently shake off the blocking solution, place the slices flat in a humidified box, add the primary antibody prepared in a certain ratio (F4 / 801:900), and place the humidified box in a 4°C refrigerator overnight.

[0121] 6) Rewarming: Place the wet box at room temperature for 1 hour, then wash 3 times with PBS, 5 minutes each time.

[0122] 7) Incubation with secondary antibody: After the sections are dried, add the prepared horseradish peroxidase-labeled secondary antibody staining solution in the circle and incubate at 37°C for 30 minutes, then wash with PBS three times, 5 minutes each time.

[0123] 8) DAB color development: Add the prepared DAB color development solution into the circle, observe the degree of staining under a microscope to control the color development time, the positive part is brown-yellow, and the color development is terminated by rinsing with tap water.

[0124] 9) Re-staining of cell nuclei: Add hematoxylin solution for 10 seconds and rinse with tap water.

[0125] 10) Dehydrate and seal the slides in the same manner as steps 5) to 7) under 2.3.2.

[0126] Immunohistochemistry results ( Figure 6 A and B in Figure 2 show that compared with the Sham group, the F4 / 80 level in the MI group was significantly increased (P<0.05). After administration, the F4 / 80 level in the myocardial tissue of mice in the MI+XH group and MI+P group was significantly decreased compared with the MI group (P<0.05). This result suggests that bear bile powder can inhibit the infiltration of inflammatory factors in myocardial tissue and has the effect of inhibiting inflammatory response.

[0127] 2.5.2ELISA detection of inflammatory factor expression levels

[0128] Take an appropriate amount of serum samples in 1.3, and use a microplate reader to detect the levels of IL-6, IL-10, and TNF-α in the serum of each group of mice according to the instructions of the IL-6 (Mouse IL-6 ELISA Kit Beyotime Catalog No.: PI326), IL-10 (Mouse Interleukin 10 (IL-10) ELISA Kit Bioswamp Catalog No.: MU30055), and TNF-α (Mouse TNF-α ELISA Kit Beyotime Catalog No.: PT512) ELISA kits.

[0129] ELISA results ( Figure 6 C, D, and E in Figure 2 showed that compared with the Sham group, the levels of IL-6, IL-10, and TNF-α in the MI group were significantly increased (all P<0.05). Compared with the MI group, the pro-inflammatory factors IL-6 and TNF-α in the serum of mice in the MI+XL group and the MI+XH group were significantly reduced (all P<0.05); the anti-inflammatory factor IL-10 in the MI+XH group was also significantly reduced (P<0.05). The results showed that bear bile powder inhibited the inflammatory response by regulating the levels of inflammatory factors in the serum of mice after MI.

[0130] 2.6 Effect of bear bile powder on myocardial fibrosis after myocardial infarction

[0131] 2.6.1 Detection of collagen fiber deposition

[0132] The proliferation of cardiac fibroblasts and the excessive deposition of extracellular matrix are characteristic changes of myocardial fibrosis after myocardial infarction. Masson staining and Sirius red staining are commonly used detection methods for evaluating myocardial fibrosis. Therefore, Masson staining and Sirius red staining were used to evaluate the deposition of collagen fibers after myocardial infarction in mice. The specific steps are as follows:

[0133] a.Masson's trichrome staining test

[0134] 1) The routine dewaxing and rehydration of the baking and slicing are the same as steps 1) to 3) under 2.3.2.

[0135] 2) Staining: Use the prepared Weigert iron hematoxylin stain (WeigertA and WeigertB mixed in equal amounts) to stain the nucleus for 5 minutes, and wash with distilled water; differentiate with acidic ethanol differentiation solution for 15 seconds, and wash with water; return to blue with Masson blueing solution for 5 minutes, and wash with distilled water for 1 minute; stain with Ponceau fuchsin stain for 10 minutes; add weak acid working solution for 1 minute to make the color clearer and brighter; differentiate with phosphomolybdic acid aqueous solution until the collagen fibers are light red and the fibers are red, usually 1-2 minutes; wash with weak acid working solution for 1 minute and then directly put into aniline blue stain for 20 seconds, and continue to wash with weak acid working solution for 1 minute.

[0136] 3) Dehydration and sealing are the same as steps 5)-7) under 2.3.2.

[0137] b. Sirius red staining

[0138] 1) The routine dewaxing and rehydration of the baking and slicing are the same as steps 1) to 3) under 2.3.2.

[0139] 2) Staining: Immerse the slices in Sirius Red Stain Solution A (Sirius Red Staining Kit, Sevier Biotechnology, Catalog No.: G1078-1) and place them in a 65°C oven for 30 minutes; wash the slices with water until the yellow color on the tissue fades, shake off the water, and soak in Sirius Red Stain Solution B (Sirius Red Staining Kit, Sevier Biotechnology, Catalog No.: G1078-2) for 2 minutes; wash with water, shake off the water, and soak in Sirius Red Stain Solution C (Sirius Red Staining Kit, Sevier Biotechnology, Catalog No.: G1078-3) for 30 minutes.

[0140] 3) Wash quickly with water for 1-2 seconds, and then put into three cylinders of anhydrous ethanol for 3 seconds each to quickly rinse and dehydrate.

[0141] 4) The transparent cover is the same as steps 6)-7) under 2.3.2.

[0142] Masson staining results showed that ( Figure 7 (A and B), compared with the Sham group, the normal muscle fiber tissue (red) in the MI group was significantly reduced, replaced by collagen fiber (blue) deposition, but the administration of bear bile powder can effectively inhibit the collagen fiber generation caused by myocardial infarction and improve myocardial fibrosis after myocardial infarction in mice. Similarly, Sirius red staining also reflects similar results to Masson staining ( Figure 7 A and C in the figure, that is, compared with the Sham group, the expression of collagen fibers (red) in the MI group was significantly increased, and the expression of collagen fiber content was significantly reduced after bear bile powder treatment, indicating that bear bile powder can effectively inhibit myocardial fibrosis after myocardial infarction in mice.

[0143] 2.6.2 Detection of myocardial fibrosis-related markers

[0144] The expression levels of myocardial fibrosis-related markers were evaluated by immunohistochemistry, and the method was the same as steps 1) to 10) under 2.5.1, with the only difference being that in step 5), the configuration ratio of the primary antibody was: Coll-1 (Collagen Ⅰ) 1:1000, α-SMA 1:500, and the rest was the same as steps 1) to 10) under 2.5.1.

[0145] The results show that Figure 7 A, D, and E): Compared with the Sham group, the expression levels of myocardial fibrosis-related proteins in the MI group were significantly increased, as shown by the increase in the expression levels of Collagen Ⅰ and α-SMA, while bear bile powder inhibited the increase in the expression of the above proteins.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of oral bear bile powder in the preparation of drugs for the treatment of myocardial infarction.

2. The use according to claim 1, characterized in that: The oral bear bile powder consists of bear bile powder and sodium carboxymethyl cellulose.

3. The use according to claim 2, characterized in that: The sodium carboxymethyl cellulose is a sodium carboxymethyl cellulose solution with a mass volume fraction of 0.3% to 0.8%.

4. The use according to claim 3, characterized in that: The mass volume ratio of the bear bile powder to the sodium carboxymethyl cellulose solution is (70-80) mg: (5-15) mL.

5. The use according to claim 1, characterized in that: The oral bear bile powder can reduce the area of ​​myocardial infarction.

6. The use according to claim 1, characterized in that: The oral bear bile powder can restore heart function after myocardial infarction.

7. The use according to claim 1, characterized in that: The oral bear bile powder can inhibit myocardial hypertrophy.

8. The use according to claim 1, characterized in that: The oral bear bile powder can inhibit myocardial cell apoptosis.

9. The use according to claim 1, characterized in that: The oral bear bile powder can inhibit inflammatory response.

10. The use according to claim 1, characterized in that: The oral bear bile powder can inhibit myocardial fibrosis.

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