Use of ganoderic acid a in prevention of acute kidney injury caused by renal ischemia-reperfusion
By using a drug form prepared from ganoderic acid A, apoptosis and inflammation of renal tubular epithelial cells are inhibited, thus solving the problem of acute kidney injury caused by renal ischemia-reperfusion and achieving a protective effect on the kidneys.
Patent Information
- Application Number
- CN202410793782.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-19
- Publication Date
- 2025-11-25
- Estimated Expiration
- 2044-06-19
AI Technical Summary
There is a lack of effective treatments in the current technology to alleviate or prevent acute kidney injury caused by renal ischemia-reperfusion, especially microcirculatory disturbances and tissue damage after organ transplantation, shock, trauma and surgery.
Using ganoderic acid A as the active ingredient, it is prepared into drug forms such as injections, tablets, pills, capsules, suspensions or emulsions. It provides protection against renal ischemia-reperfusion by inhibiting apoptosis and inflammation of renal tubular epithelial cells and reducing renal function indicators.
Ganoderma lucidum acid A significantly reduces apoptosis and inflammatory response of renal tubular epithelial cells at both the whole animal and cellular levels, lowers renal function indicators such as creatinine and blood urea nitrogen levels, provides protection against renal ischemia-reperfusion, and alleviates acute kidney injury.
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Figure CN118845794B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of pharmacotherapy, and particularly relates to application of ganoderic acid A in preparation of a medicine for preventing acute kidney injury caused by kidney ischemia-reperfusion. BACKGROUND
[0002] Microcirculation disorder and peripheral tissue injury caused by ischemia-reperfusion injury often occur after organ transplantation, shock, trauma, thrombosis and various operations, and can induce multiple organ injury, induce organ fibrosis and organ failure. As a high-perfusion organ of the human body, the kidney is very sensitive to ischemia and hypoxia, reperfusion; and when the body suffers from severe damage such as ischemia and hypoxia, shock, disseminated intravascular coagulation, multiple organ dysfunction syndrome and systemic inflammatory response syndrome, blood is redistributed to mainly supply important organs such as heart and brain, so the kidney is one of the most common organs of ischemia-reperfusion. Kidney ischemia-reperfusion (I / R) is an important cause of acute kidney injury, and has become a major obstacle to the long-term survival of transplant recipients / kidneys. Therefore, it is of great significance to the clinical treatment and scientific theory innovation to deeply understand the deep mechanism of kidney ischemia-reperfusion and explore the treatment strategy for preventing microcirculation disorder and tissue injury caused by ischemia-reperfusion.
[0003] So far, there is no effective treatment for acute kidney injury caused by kidney ischemia-reperfusion, therefore, it is of great significance to find a protective drug that can reduce microcirculation disorder and tissue injury. SUMMARY
[0004] To solve the above technical problems, the application first provides application of ganoderic acid A in preparation of a medicine for preventing acute kidney injury caused by kidney ischemia-reperfusion.
[0005] The technical solution adopted by the application is as follows:
[0006] The application of ganoderic acid A in preparation of a medicine for preventing acute kidney injury caused by kidney ischemia-reperfusion, wherein the ganoderic acid A has a structural formula as shown in Formula I:
[0007]
[0008] Preferably, the medicine is any one of an injection, a tablet, a pill, a capsule, a suspension or an emulsion.
[0009] The application further provides a medicine for preventing acute kidney injury caused by kidney ischemia-reperfusion, which contains a pharmaceutically effective dose of ganoderic acid A, wherein the ganoderic acid A has a structural formula as shown in Formula I:
[0010]
[0011] Preferably, the medicine further contains a pharmaceutically acceptable carrier.
[0012] Preferably, the pharmaceutically acceptable carrier comprises one or more of excipients, stabilizers, antioxidants, colorants, diluents, sustained-release agents, etc., such as starch, lipids, waxes, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc.
[0013] Preferably, the medicine is any one of an injection, a tablet, a pill, a capsule, a suspension or an emulsion.
[0014] The beneficial effects of the present application are:
[0015] Ganoderic acid A (GANODERIC ACID A, GAA) is a triterpene extracted from the fungus Ganoderma, which has the properties of anti-oxidative stress, anti-apoptosis and anti-inflammatory. Through the whole, cell and protein level research, the present application first proposes the protective effect of ganoderic acid A on acute kidney injury caused by renal ischemia-reperfusion and the possible mechanism, which provides experimental basis for preventing and reducing kidney ischemia-reperfusion and discovering new drug action targets.
[0016] The present application shows through pharmacodynamic results that, on the whole animal and cell levels, ganoderic acid A has a strong protective effect on the acute kidney injury model caused by renal ischemia-reperfusion. To explore the mechanism, it is confirmed on the whole animal and cell levels that ganoderic acid A can play a protective role on acute kidney injury caused by renal ischemia-reperfusion by inhibiting the apoptosis (PCD mediated by apoptotic bodies and executioner caspases), pyroptosis (inflammasome-dependent PCD executed by gasdermin family members) and necrosis (PCD mediated by RIPK3 and downstream effector MLKL) of renal tubular epithelial cells.
[0017] On the whole animal level, the protective effect of ganoderic acid A on renal ischemia-reperfusion is investigated, and through the pathological changes of the kidney, the expression changes of serum creatinine, urea nitrogen, Cleaved-caspase-3, GSDMD-N and pMLKL proteins, it is proved that ganoderic acid A has a protective effect on acute kidney injury caused by renal ischemia-reperfusion.
[0018] On the cell level, the renal tubular epithelial cells are subjected to hypoxia-reoxygenation, and at the same time, ganoderic acid A is given for treatment, and the CCK8 method and the detection of the expression changes of the renal tubular epithelial cell pan-apoptosis related markers Cleaved-caspase-3, GSDMD-N and pMLKL proteins are used. It is proved that ganoderic acid A can inhibit the pan-apoptosis of renal tubular epithelial cells and play a protective role on acute kidney injury caused by renal ischemia-reperfusion.
[0019] The experiment proves that ganoderic acid A can up-regulate the expression of EGR3, inhibit the EGR3 / Pstat3 / NLRP3 signal pathway, reduce the level of pan-apoptosis (Cleaved-caspase-3, GSDMD-N, pMLKL), and reduce the levels of creatinine and urea nitrogen, which are indicators of kidney function. The present application finds that ganoderic acid A has a protective effect on acute kidney injury caused by renal ischemia-reperfusion, and its mechanism is related to the inhibition of the activation and proliferation of hepatic stellate cells, thereby providing a new potential drug for the auxiliary treatment of kidney diseases by traditional Chinese medicine.
[0020] Ganoderic acid A has the prospect of developing into a drug for preventing acute kidney injury caused by renal ischemia-reperfusion. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 Figure 4 shows the effect of ganoderic acid A on the pathology of the kidney of mice with renal ischemia-reperfusion, and H&E staining (40x).
[0022] Figure 2 Figure 5 shows the effect of ganoderic acid A on serum BUN of mice with renal ischemia-reperfusion, and the effect of ganoderic acid A on serum CR of mice with renal ischemia-reperfusion.
[0023] Figure 3 、 Figure 4 Figure 6 shows the effect of ganoderic acid A on pan-apoptosis of mice with renal ischemia-reperfusion, wherein Figure 3 Figure 7 shows the results of Western blot detection, Figure 4 Figure 8 shows the results of immunohistochemistry of mouse kidney tissue.
[0024] Figures 5-7 Figure 9 shows the effect of ganoderic acid A on pan-apoptosis of renal tubular epithelial cells, wherein Figure 5 Figure 10 shows the results of CCK8 method for detecting the viability of renal tubular epithelial cells, Figure 6 Figure 11 shows the results of Western blot detection of Cleaved-caspase-3, GSDMD-N, and pMLKL of renal tubular epithelial cells, Figure 7 Figure 12 shows the results of immunofluorescence detection of Cleaved-caspase-3, GSDMD-N, and pMLK of hepatic stellate cells. DETAILED DESCRIPTION
[0025] Unless otherwise specified, the terms used herein have meanings commonly understood by those skilled in the art. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used herein can be purchased on the market or can be prepared by existing methods.
[0026] The technical solutions of the present application will be described in more detail below in conjunction with the examples:
[0027] Example 1
[0028] Effects of Ganoderic Acid A on Renal Pathology in Mice Undergoing Renal Ischemia-reperfusion Surgery
[0029] Experimental Design:
[0030] 60 male C57BL / 6J mice, aged 10-12 weeks, weighing 20±2 grams, were randomly divided into the following 7 groups (n=10): control group (Vehicle), control + Ganoderic Acid A (40 mg / kg) group, renal ischemia-reperfusion (I / R) model group, I / R + Ganoderic Acid A (10 mg / kg) group, I / R + Ganoderic Acid A (20 mg / kg) group, and I / R + Ganoderic Acid A (50 mg / kg) group, I / R + dexamethasone (3 mg / kg) group.
[0031] Ganoderic Acid A (10, 20 and 40 mg / kg) dissolved in sodium carboxymethylcellulose (CMC-Na) was administered intragastrically every day before the renal ischemia-reperfusion surgery, and intragastric administration was performed continuously for 7 days. The positive drug control group of mice was administered 0.1 mg / kg of dexamethasone intragastrically daily for 3 consecutive days. After the end of intragastric administration, the ischemia-reperfusion surgery was performed, and the mice were sacrificed 24 hours later under anesthesia.
[0032] The I / R experimental operation was as follows: after the mice were anesthetized, they were placed on a thermostatic plate to maintain a body temperature of 36.5°C. The bilateral renal pedicles were clamped with microaneurysm clips for 30 min. After ischemia, the clamps were released after 24 h of reperfusion, and all animals were sacrificed under anesthesia. The sham control group of animals received the same procedure without clamping the renal pedicles. The experiment was randomized. All animals received a 12-hour dark-light cycle, were fed standard pellet feed and had free access to water, and were kept in a temperature- and humidity-controlled room. After the modeling was completed, all mice were fasted and had no water for 24 h. The mice were sacrificed under anesthesia, and the mouse blood and kidney tissue were collected for subsequent studies.
[0033] The large lobe tissues of the kidneys of the mice in the different groups described above were fixed in 4% paraformaldehyde solution for 24-48 h, and the samples were dehydrated with alcohol and transparentized with xylene before being embedded with paraffin. After the embedded tissue blocks hardened, they were sectioned with a microtome. Before staining, the paraffin in the paraffin sections was removed in xylene, and after dehydration with ethanol from high concentration to low concentration, the final rinse with double distilled water was performed before staining. Staining was started after staining in hematoxylin staining solution for 8 min, and the excess staining solution was washed away with running water. After differentiation in 1% hydrochloric acid ethanol for 5 s, after water washing, an appropriate amount of 0.6% ammonia water was added until the color was slightly blue, and then the sample was rinsed for 10 min, and then the sample was stained with 0.5% eosin staining solution for 1-3 min. After washing with distilled water, the sample was dehydrated in ethanol solutions from low concentration to high concentration, and then the sample was transparentized in xylene, embedded with neutral resin, and observed under a microscope. After analyzing the results, the desired positions were selected for photography.
[0034] Results as shown in Figure 1 H&E staining showed that the kidney tissue structure of Ganoderic acid A (10, 20, 40 mg / kg) treated mice was significantly improved to varying degrees compared with the mice subjected to renal ischemia-reperfusion, and the renal tubular expansion, brush border and inflammatory cell infiltration model group were reduced to varying degrees.
[0035] Example 2
[0036] Effect of Ganoderic acid A on serum BUN and CR of mice subjected to renal ischemia-reperfusion operation
[0037] Creatinine (CR) and urea nitrogen (BUN) are the main indicators for checking kidney function, and the increase of creatinine, urea nitrogen represents the impairment of kidney function.
[0038] The blood serum of the mice obtained in Example 1 was taken as a sample, and the operation was carried out according to the instructions of the creatinine (CR) kit and the urea nitrogen (BUN) kit (microporous 96T microplate method) (Nanjing Jiancheng Biological Engineering Institute).
[0039] The detection results of serum CR and BUN are shown in Figure 2 Compared with the mice subjected to renal ischemia-reperfusion operation, Ganoderic acid A (10, 20, 40 mg / kg) can significantly reduce the serum CR and BUN levels of mice (p<0.01), suggesting that Ganoderic acid A has certain kidney protection effect on acute kidney injury caused by renal ischemia-reperfusion.
[0040] Example 3
[0041] Effect of Ganoderic acid A on pan-apoptosis of mice subjected to renal ischemia-reperfusion operation
[0042] Programmed cell death (PCD) is an evolutionarily conserved process that plays a central role in maintaining homeostasis. Three key PCD pathways have been studied in detail: pyroptosis (inflammasome-dependent PCD executed by gasdermin family members), apoptosis (PCD mediated by apoptosomes and executioner caspases), and necroptosis (PCD mediated by RIPK3 and downstream effector MLKL). The intersection of pyroptosis, apoptosis, and necroptosis can be subsumed into the concept of total cell death, or panoptosis. Among them, apoptosis (Cleaved-caspase-3) is a form of programmed cell death. A family of proteases called caspases can be activated by death receptors on the cell surface (extrinsic pathway) or by disturbances in the mitochondrial membrane (intrinsic pathway). The release of apoptotic factors can activate the caspase family, ultimately leading to apoptosis. Necrosis is a type of cell death that follows a signaling pathway closely related to apoptosis and is the most thoroughly studied form of regulated necrosis, executed by RIPK3 and its substrate, the mixed lineage kinase domain-like protein (MLKL). Pyroptosis (GSDMD-N) is involved in various kidney diseases, such as inflammatory caspase activation.
[0043] Take the appropriate amount of mouse kidney tissue from each group in Example 1, extract the total protein, and prepare the kidney tissue into a wax block and slice it. After routine deparaffinization and hydration, place the slices in an antigen repair box containing sufficient citrate buffer, and microwave heat at 100°C for 10 min. After taking out the box and allowing it to return to room temperature, slowly rinse the slices with distilled water and place them in a beaker containing 3% H2O2 at room temperature for 15 min. After rinsing with distilled water, draw a closed circle around the liver tissue on the slice with a histological pen, then add an appropriate amount of 3% bovine serum albumin to the circle with a pipette, and let it stand at room temperature for 30 min. Gently wipe off the blocking solution around the glass slide with absorbent paper, and add about 50 μl of primary antibody to the circle on each glass slide, incubate overnight at 4°C. After incubation, wash with PBS buffer and spin dry, add secondary antibody. Incubate in a 37°C incubator for 10 min. After incubation, wash with PBS buffer, add SABC, and incubate at 37°C for 30 min. Wash with PBS buffer, add DAB mixed staining solution, and observe under a microscope. Stop the color development immediately after DAB stains. Rinse with tap water for 5 min, stain with hematoxylin for 3-5 min, rinse with tap water for 5 min, and differentiate with 1% hydrochloric acid alcohol for 5 s, then rinse with tap water for 5 min. Dehydrate, transparentize, and mount the slide for observation. Perform Western blot and immunohistochemistry experiments according to the conventional method.
[0044] The results are as follows Figure 3 , Figure 4The expression levels of Cleaved-caspase-3, GSDMD-N, pMLKL in the model group were significantly higher than those in the normal group. The expression levels of Cleaved-caspase-3, GSDMD-N, pMLKL in the administration group were lower than those in the model group, indicating that ganoderic acid A can inhibit cell pan-apoptosis caused by renal ischemia-reperfusion, suggesting that ganoderic acid A can play a kidney protective role in renal ischemia-reperfusion mice by inhibiting the level of pan-apoptosis.
[0045] Example 4
[0046] Effect of ganoderic acid A on pan-apoptosis of renal tubular epithelial cells after hypoxia-reoxygenation
[0047] Renal tubular epithelial cells are sensitive to injury factors such as sepsis, poisoning and ischemia-hypoxia. Hypoxic injury is an important cause of various kidney diseases. The main pathological changes are apoptosis and necrosis of tubular epithelial cells. Therefore, inhibiting the necrosis and apoptosis of renal tubular epithelial cells has become a direction of treatment.
[0048] 96-well plates and six-well plates were used to culture cells, respectively. The cell H / R model was established: mTECs were cultured in a nutrient-free medium under hypoxic conditions (no sugar, no serum), 5% carbon dioxide) for 3 hours to induce hypoxic injury. Then, the culture medium was refreshed again, and the culture dish was moved to a normoxic cell incubator (5% carbon dioxide and 95% air) for 4 hours. The control cells were cultured in an incomplete medium and stimulated with ganoderic acid A (80 μM) for 48 hours in a conventional incubator (5% carbon dioxide and 95% air).
[0049] CCK8 was used to detect the cytotoxicity of renal tubular epithelial cells in 96-well plates, and the results are shown in Figure 5 It can be seen that ganoderic acid A has no toxicity to renal tubular epithelial cells at a concentration of 80 μM and below. The total protein and immunofluorescence of the cells in the six-well plate were extracted. The slices were blocked with 10% bovine serum albumin (BSA) solution to avoid non-specific staining. The slices were incubated with Cleaved-caspase-3, pMLKL, GSDMD-N antibodies. The slices were incubated at 4°C overnight, then incubated with goat anti-rabbit IgG and goat anti-mouse IgG antibodies, and stained with DAPI. The stained slices were observed under an inverted fluorescence microscope. (Western blot was used to detect the expression of Cleaved-caspase-3, GSDMD-N, pMLKL protein, and immunofluorescence was used to detect the fluorescence expression.
[0050] The results are shown in Figure 6 , Figure 7It can be seen that the expression levels of Cleaved-caspase-3, GSDMD-N and pMLKL in the renal tubular epithelial cells subjected to hypoxia-reoxygenation are obviously higher than those in the normal group, and the expression levels of Cleaved-caspase-3, GSDMD-N and pMLKL in the administration group are lower than those in the model group. It is indicated that ganoderic acid A can protect the renal tubular epithelial cells subjected to hypoxia-reoxygenation by inhibiting the level of pan-apoptosis.
[0051] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application; although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modification, equivalent replacement and improvement within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims
1. Use of ganoderic acid A in the preparation of a medicament for preventing acute kidney injury caused by renal ischemia-reperfusion, wherein the ganoderic acid A has a structural formula as shown in formula (I): ###0001### Formula (I) The medicament further comprises a pharmaceutically acceptable carrier. （Ⅰ）。 2. Use according to claim 1, wherein The pharmaceutically acceptable carrier comprises one or more of stabilizers, coloring agents, diluents, and sustained-release agents.
3. Use according to claim 2, wherein the compound is ###0002### The medicament is any one of an injection, a tablet, a pill, a capsule, a suspension, or an emulsion.
4. Use according to claim 1 or 3, wherein the compound is ###0002###