Application of isodan leaf emodin in preparation of medicine for preventing and treating acetaminophen-induced acute liver injury

Abstract

The invention provides an application of isodan leaf emodin in preparation of a medicine for preventing and treating acetaminophen-induced acute liver injury. The invention proves that the isodan-leaf emodin can obviously relieve the acute liver injury symptom induced by acetaminophen, the relieving effect is closely related to the fatty acid oxidation promoting capability of the isodan-leaf emodin, and the new application of the isodan-leaf emodin in pharmacology, new drug research and development and clinical treatment is expanded.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of isodanin in the preparation of a drug for preventing and treating acetaminophen-induced acute liver injury. Background Art

[0002] Acetaminophen (APAP) is the most commonly used anti-inflammatory, antipyretic and analgesic drug on the market. However, clinical studies have shown that overdose of APAP can lead to acute liver injury (AILI). Patients first experience symptoms such as abdominal pain and nausea, and then biochemical indicators such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood increase rapidly, causing liver cell death and large-scale necrosis of liver tissue. If AILI is not treated in time, it can further develop into acute liver failure, etc., and even lead to the death of patients, seriously endangering human public health. In the United States, AILI accounts for 46% of acute liver injury cases and has become the most common cause of acute liver failure in Western countries. At present, N-acetyl-L-cysteine ​​(NAC) is the only drug approved by the FDA for the treatment of AILI. However, the NAC treatment time window is narrow, and patients need to take it in time within 3-4 hours after the onset of liver toxicity to achieve a better treatment effect. Patients who are not treated in time may develop acute liver failure and need to be treated through expensive, long-term and extremely risky liver transplantation. In addition, there are no other effective means to treat AILI in clinical practice. Therefore, there is an urgent need to further explore new drugs to promote the clinical treatment of APAP acute liver injury.

[0003] APAP-induced acute liver injury involves multiple signaling pathways, including oxidative stress and inflammatory damage. Recent studies have shown that dysregulated fatty acid oxidation (FAO) plays an important role in the development of AILI. In a mouse model, activation of peroxisome proliferator-activated receptor α (PPAR-α), a key regulator of fatty acid oxidation, can alleviate APAP-induced acute liver injury. In addition, studies have found that Egr1 can play a protective role against APAP-induced acute liver injury by enhancing fatty acid oxidation. Therefore, targeting dysregulated fatty acid oxidation may be a potential intervention strategy for the treatment of AILI.

[0004] Isorhapontigenin (ISO) is a natural stilbene compound extracted from Chinese herbal medicine and grapes. 15 H 14 O4 has powerful pharmacological effects such as antioxidant, anti-inflammatory and lipid metabolism regulation. Studies have shown that isodanin has therapeutic effects on animal models of diseases such as diabetes, acute kidney injury and cerebral ischemia, but whether ISO has a therapeutic effect on AILI has not yet been clarified. Summary of the invention

[0005] The purpose of the present invention is to provide an application of isodanin in the preparation of a drug for preventing and treating acetaminophen-induced acute liver injury. The isodanin in the present invention can promote fatty acid oxidation, alleviate the acute liver injury caused by acetaminophen, solve the problem of shortage of existing therapeutic drugs, and provide a definite theoretical basis for the revelation of the pharmacodynamic mechanism of isodanin and its clinical application.

[0006] The structural formula of isodanin of the present invention is shown in (I):

[0007] Furthermore, the drug is a drug for improving liver function indicators, and the liver function indicators are alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase.

[0008] Furthermore, the drug is a drug for improving liver tissue structure.

[0009] Further, the drug is a drug for reducing inflammatory cell infiltration in the liver.

[0010] Furthermore, the drug is a drug that promotes fatty acid oxidation in the liver.

[0011] Furthermore, the drug is a drug that can enhance the effect of N-acetyl-L-cysteine ​​when administered in combination with N-acetyl-L-cysteine.

[0012] Furthermore, the medicine is any one of an injection, tablet, pill, capsule, suspension or emulsion.

[0013] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: The present invention discovers for the first time that isodan-emodin can promote fatty acid oxidation and significantly alleviate the symptoms of acute liver injury induced by acetaminophen. The mechanism is that isodan-emodin promotes fatty acid oxidation and reduces lipid accumulation in the liver of liver injury, thereby having a good improvement and therapeutic effect on acute liver injury caused by APAP. Moreover, the combined administration of isodan-emodin+NAC can enhance the therapeutic effect of NAC. The present invention expands the new uses of isodan-emodin in pharmacology, new drug development and clinical treatment, and has development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 .Figure 3. Test results of serum biochemical indices related to liver injury in the mouse model of acute liver injury induced by acetaminophen treated with isocyanate.

[0015] Figure 2 .Light microscopic results of H&E staining of liver pathology in the mouse model of acute liver injury induced by acetaminophen treated with isothiazolin.

[0016] Figure 3 .The results of Oil Red staining showing that isodanin promotes fatty acid oxidation in the liver of AILI mice.

[0017] Figure 4 .The results of protein expression level detection of the key fatty acid oxidation pathway PPAR-α / PGC-1α / CPT-1A.

[0018] Figure 5 .Results of serum biochemical index detection related to liver injury by inhibiting fatty acid oxidation and weakening the effect of isodanin in alleviating AILI.

[0019] Figure 6 .The results of liver pathological H&E staining light microscopy showed that inhibiting fatty acid oxidation weakened the effect of isotan-emodin in alleviating AILI.

[0020] Figure 7 .Figure 2. Results of serum biochemical index test on liver injury related to the enhancement of the effect of NAC by the combined administration of isodanin and NAC.

[0021] Figure 8 .The results of H&E staining light microscopy of liver pathology showed that the combined administration of isodanin and NAC enhanced the effect of NAC. DETAILED DESCRIPTION

[0022] The following drawings and embodiments are provided to further help those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0023] Example 1. Treatment of acute liver injury induced by acetaminophen in mice with isothiazolin

[0024] method: Male C57BL / 6 mice, weighing 18-22g, were randomly divided into 4 groups, 6 mice in each group. The 4 groups were normal control group, model group, low-dose isodanin group (25mg / kg) and high-dose isodanin group (50mg / kg). Except for the normal control group, the animals in the other groups were intraperitoneally injected with 350mg / kg APAP to construct AILI model. One hour after APAP administration, the low-dose and high-dose isodanin groups were gavaged at 25mg / kg and 50mg / kg, respectively, and the other groups were given the same volume of normal saline. After 24 hours of APAP action, the mice were eyeballed and blood was collected and killed. The mice were fixed on the dissection board and the liver was quickly removed. The collected blood samples were left to stand at room temperature for 30 minutes, centrifuged at 4°C, 6500rpm for 15 minutes, and the supernatant was pipetted into a new EP tube to measure the liver function indicators of mice, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). At the same time, liver tissue samples of mice in each group were collected, fixed with 4% formalin solution for 24 hours, and then rinsed with tap water to remove excess fixative. The tissues were dehydrated in 70%, 80%, 90%, 95%, and 100% ethanol in turn, with each step taking 20 minutes. After dehydration, the tissue was transparentized in xylene and then embedded in paraffin. The slices were sliced ​​into paraffin slices with a thickness of 5 μm. The paraffin slices were sequentially placed in xylene I for 10 minutes, xylene II for 10 minutes, and then placed in an alcohol concentration gradient of anhydrous alcohol, 95%, and 80% alcohol for 5 minutes each, then washed with distilled water for 10 minutes, and the slices were placed in hematoxylin stain for 2 minutes, rinsed with deionized water for 10 minutes, differentiated with differentiation solution, and rinsed with deionized water again for 10 minutes. Stained in eosin stain for 2 minutes and rinsed with deionized water for 10 minutes. The slices were placed in an alcohol solution with an alcohol concentration gradient of 80%, 95%, and anhydrous alcohol for 5 minutes each, and gradient dehydration was performed, and finally transparentized with xylene. After taking out the slices, an appropriate amount of neutral gum was quickly added before the xylene dried, and finally sealed with a cover glass, and the morphological changes of liver tissue were observed under an upright microscope.

[0025] result: The results of serum liver function indicators are as follows Figure 1 As shown in the figure, compared with the normal control group, the ALT, AST and LDH levels in the serum of the mice in the model group were significantly increased. Compared with the model group, the ALT, AST and LDH levels of the mice in the low and high dose groups of isodanin were significantly decreased.

[0026] H&E staining Figure 2As shown in the figure, the liver tissue of mice in the normal control group showed lobular structure, hepatocytes were arranged in cords, of uniform size, without atypia, and no inflammatory cell infiltration in the interstitium. In the model group, the hepatocytes were arranged in disordered cords, with nuclear condensation and cell punctate necrosis, and a large number of inflammatory cells infiltrated in the interstitium. The liver tissue of mice in the low- and high-dose isothiazin groups showed significant improvement compared with the model group, with neater hepatocyte cords, less hepatocyte necrosis and inflammatory cell infiltration.

[0027] The above results indicate that isotan-emodin can effectively alleviate APAP-induced liver cell damage and has a protective effect on the liver.

[0028] Example 2. Isodanin promotes fatty acid oxidation in the liver of AILI mice

[0029] method: Male C57BL / 6 mice, weighing 18-22g, were randomly divided into 4 groups, 4 mice in each group. They were normal control group, model group, low-dose isodanin group (25mg / kg) and high-dose isodanin group (50mg / kg). Except for the normal control group, the animals in the other groups were intraperitoneally injected with 350mg / kg APAP to establish AILI model. One hour after APAP administration, the low-dose and high-dose isodanin groups were gavaged at 25mg / kg and 50mg / kg, respectively, and the other groups were given the same volume of normal saline. After 24 hours of APAP, the mice were killed, fixed on the dissection board, and the fresh liver was quickly removed and trimmed with dissection scissors to make its shape and size suitable for subsequent processing. After trimming, the tissue blocks were placed in 4% PFA solution for fixation overnight, immersed in distilled water and placed on a shaker for washing for 20 seconds to remove excess fixative. The fixed tissue blocks were immersed in 30% sucrose solution and placed in a 4°C refrigerator overnight to dehydrate and prevent ice crystals from forming during freezing. After dehydration, the tissue block was directly placed in an appropriate amount of OCT embedding medium to ensure complete immersion and minimize bubbles. After embedding, it was placed in a -20℃ refrigerator for quick freezing. After freezing, the slices were sliced ​​using a cryostat with a thickness of 8μm. After slicing, they were stored in a -20℃ refrigerator. We used oil red staining to detect the lipid content in the liver. Take out the frozen sections that were pre-made and stored in a -20℃ refrigerator and put them in a slice rack to warm up for 5 minutes. Add 100μL of oil red staining wash solution to cover the sample for 20 seconds, remove the staining wash solution, add 100μL of oil red O staining working solution to cover the sample, and stain for 5 minutes. Remove the oil red O staining working solution, add 100μL of staining wash solution to the slice, let it stand for 30 seconds, remove the staining wash solution, immerse it in distilled water and wash it on a shaker for 20 seconds. The slices were immersed in hematoxylin staining solution to counterstain the nucleus for 2 minutes. After staining, the slices were sealed with aqueous sealing solution, observed under a microscope and photographed.

[0030] Male C57BL / 6 mice, weighing 18-22g, were randomly divided into 4 groups, 3 mice in each group. They were normal control group, model group, low-dose isodanin group (25mg / kg) and high-dose isodanin group (50mg / kg). Except for the normal control group, the animals in the other groups were intraperitoneally injected with 350mg / kg APAP to establish AILI model. One hour after APAP administration, the low-dose and high-dose isodanin groups were gavaged at 25mg / kg and 50mg / kg, respectively, and the other groups were given the same volume of normal saline. After 24 hours of APAP action, the mice were killed, fixed on the dissection board, and fresh livers were quickly removed. The PPAR-α / PGC-1α / CPT-1A pathway is a key pathway for fatty acid oxidation. PPAR-α controls the expression of genes encoding mitochondrial FAO enzymes. PGC-1α, as a coactivator of PPAR-α, controls the expression of CPT-1A, the rate-limiting enzyme for fatty acid oxidation, together with PPAR-α. To evaluate the level of fatty acid oxidation, we detected changes in the key fatty acid oxidation pathway PPAR-α / PGC-1α / CPT-1A. We took a small amount of tissue in an EP tube and added 200 μL of RIPA lysis buffer to it. After grinding the tissue, let it stand for 30 minutes. After centrifugation at 13500 rpm for 30 minutes, the supernatant was aspirated into a new EP tube and stored in a -80°C refrigerator. Prepare a protein standard solution with a concentration gradient for making a standard curve, as well as BCA working solution (A solution: B solution = 50:1). After diluting the sample protein supernatant, add the sample protein supernatant dilution, protein standard solution and BCA working solution (1:100) to the well plate, incubate at 37°C for 30 minutes, measure the absorbance value at a wavelength of 562nm, and calculate the sample protein concentration. Calculate according to the protein amount of 200ng, add appropriate protein supernatant, add 40μL of 5×Loading buffer and make up to 200μL with distilled water, boil at 95℃ for 15 minutes, and store in a -20℃ refrigerator. Wash the glass, make sure the bottom edges of the long and short glass are aligned, inject the separation gel into the two glass plates, then inject the concentrated gel into the two glass plates, insert the comb and wait for 20 minutes for the gel to solidify. After solidification, clamp the small glass of the gel plate toward the inside, fill the tank with 1×Running (10×Running is diluted with ultrapure water), unplug the comb, vortex the protein sample thoroughly, add 10μL of protein sample or 2.5μL of Marker to the lane, run the protein to the separation gel at a constant voltage of 80V, and then adjust the voltage to 150V constant voltage to continue electrophoresis. After running the gel, disconnect the power supply and place the PVDF membrane in anhydrous ethanol in advance to soak and activate. Pry off the small glass of the gel plate, gently scrape off the concentrated gel, and place the sponge, three-layer filter paper, gel, PVDF membrane, three-layer filter paper, and sponge in this order. Pour in 1× Trans buffer and anhydrous ethanol, and perform electrophoresis at a constant current of 330 mA for 60 minutes.After the transfer, dissolve skim milk powder in TPBS to prepare 5% blocking solution, take 5mL of blocking solution to cover the strips, block at room temperature for 60 minutes, wash three times with TPBS, 15 minutes for the first time, and 5 minutes for each of the next two times. Dilute the primary antibody with TPBS at a ratio of 1:1000, take 5mL to cover the strips, and incubate on a shaker at 4°C overnight. After the primary antibody incubation, wash three times with TPBS, 15 minutes for the first time, and 5 minutes for each of the next two times. Dilute the secondary antibody with blocking solution at a ratio of 1:5000, take 5mL to cover the strips, incubate on a shaker at room temperature for 1 hour, wash three times with TPBS, 15 minutes for the first time, and 5 minutes for each of the next two times. Prepare the ECL developer according to the instructions (A solution: B solution = 1:1), drop it evenly on the PVDF membrane, and place it in an exposure machine for development.

[0031] result: Oil red staining results Figure 3 As shown, the APAP model group showed a large number of red Oil Red O positive signals compared with the normal control group. Compared with the model group, the red Oil Red O positive signals were significantly reduced and weakened in the low and high doses of isothiazolin groups.

[0032] The protein expression level of the key fatty acid oxidation pathway PPAR-α / PGC-1α / CPT-1A was shown in Figure 4 As shown in the results, compared with the normal control group, the expression of PPAR-α / PGC-1α / CPT-1A in the liver tissue of the APAP model group was significantly downregulated, and isobutyric acid effectively inhibited the downregulation of PPAR-α / PGC-1α / CPT-1A caused by APAP. The experimental results suggest that isobutyric acid can promote fatty acid oxidation, thereby improving liver damage caused by APAP.

[0033] Example 3. Inhibition of fatty acid oxidation weakens the effect of isodanin in alleviating AILI

[0034] method: Etoxifen is an irreversible inhibitor of CPT-1A, the rate-limiting enzyme of fatty acid oxidation. We used male C57BL / 6 mice weighing 18-22g and randomly divided them into 4 groups, 5 mice in each group. The 4 groups were normal control group, model group, isodan-rhubarb administration group (50mg / kg) and isodan-rhubarb (50mg / kg) + etomoxifen combined administration group (5mg / kg). Except for the normal control group, the animals in the other groups were intraperitoneally injected with 350mg / kg APAP to construct AILI models. Etoxifen (5mg / kg) was intraperitoneally injected 1 hour before modeling and isodan-rhubarb (50mg / kg) was gavage. After 24 hours of APAP action, the mice were eyeballed and blood was collected and then killed. The mice were fixed on the dissection board and the liver was quickly removed. After the collected blood samples were allowed to stand at room temperature for 30 minutes, they were centrifuged at 4°C, 6500rpm for 15 minutes, and the supernatant was aspirated into a new EP tube to measure the liver function indicators of mice, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). At the same time, liver tissue samples of mice in each group were collected, fixed with 4% formalin solution for 24 hours, and then rinsed with tap water to remove excess fixative. The tissues were dehydrated in 70%, 80%, 90%, 95%, and 100% ethanol in turn, with each step taking 20 minutes. After dehydration, the tissue was transparentized in xylene and then embedded in paraffin. The slices were sliced ​​into paraffin slices with a thickness of 5 μm. The paraffin slices were sequentially placed in xylene I for 10 minutes, xylene II for 10 minutes, and then placed in an alcohol concentration gradient of anhydrous alcohol, 95%, and 80% alcohol for 5 minutes each, then washed with distilled water for 10 minutes, and the slices were placed in hematoxylin stain for 2 minutes, rinsed with deionized water for 10 minutes, differentiated with differentiation solution, and rinsed with deionized water again for 10 minutes. Stained in eosin stain for 2 minutes and rinsed with deionized water for 10 minutes. The slices were placed in an alcohol solution with an alcohol concentration gradient of 80%, 95%, and anhydrous alcohol for 5 minutes each, and gradient dehydration was performed, and finally transparentized with xylene. After taking out the slices, an appropriate amount of neutral gum was quickly added before the xylene dried, and finally sealed with a cover glass, and the morphological changes of liver tissue were observed under an upright microscope.

[0035] result: The results of serum liver function indicators are as follows Figure 5 As shown in the figure, compared with the normal control group, the ALT, AST and LDH levels in the serum of mice after APAP modeling were significantly increased. Compared with the model group, the administration of isodanin significantly inhibited the upregulation of ALT, AST and LDH levels caused by APAP. Compared with the isodanin-administered group, the levels of ALT, AST and LDH were significantly upregulated after the combined administration of isodanin + etomoxil, and returned to the level of the APAP model group.

[0036] H&E staining Figure 6As shown in the figure, the liver tissue structure of the APAP model group and the isodan-rhubarb + etomoxil co-administration group was severely damaged, manifested as liver necrosis and edema degeneration, accompanied by inflammatory cell infiltration. Compared with the model group, the liver tissue structure of the isodan-rhubarb group did not suffer obvious damage, and there was no obvious inflammatory infiltration. Compared with the model group, the liver damage of the isodan-rhubarb + etomoxil co-administration group did not decrease.

[0037] The above results indicate that isodanin can treat APAP-induced acute liver injury by promoting fatty acid oxidation.

[0038] Example 4. Combination of isothiazolin and NAC to enhance the effect of NAC

[0039] method: Male C57BL / 6 mice, weighing 18-22g, were randomly divided into 4 groups, 5 mice in each group. The 4 groups were normal control group, model group, isodanin-treated group (50mg / kg) and isodanin-treated group (50mg / kg) + NAC (300mg / kg). Except for the normal control group, the animals in the other groups were intraperitoneally injected with 350mg / kg APAP to construct AILI model. Isodanin (50mg / kg) and NAC (300mg / kg) were gavage 3 hours after APAP modeling. After 24 hours of action, the mice were eyeballed and blood was collected and then killed. The mice were fixed on the dissection board and the liver was quickly removed. After the collected blood samples were allowed to stand at room temperature for 30 minutes, they were centrifuged at 4°C, 6500rpm for 15 minutes, and the supernatant was aspirated into a new EP tube to measure the liver function indicators of mice, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). At the same time, liver tissue samples of mice in each group were collected, fixed with 4% formalin solution for 24 hours, and then rinsed with tap water to remove excess fixative. The tissues were dehydrated in 70%, 80%, 90%, 95%, and 100% ethanol in turn, with each step taking 20 minutes. After dehydration, the tissue was transparentized in xylene and then embedded in paraffin. The slices were sliced ​​into paraffin slices with a thickness of 5 μm. The paraffin slices were sequentially placed in xylene I for 10 minutes, xylene II for 10 minutes, and then placed in an alcohol concentration gradient of anhydrous alcohol, 95%, and 80% alcohol for 5 minutes each, then washed with distilled water, and the slices were placed in hematoxylin stain for 2 minutes, rinsed with deionized water for 10 minutes, differentiated with differentiation solution, and rinsed with deionized water again for 10 minutes. Stained in eosin stain for 5 minutes, rinsed with deionized water for 10 minutes. The slices were placed in an alcohol solution with an alcohol concentration gradient of 80%, 95%, and anhydrous alcohol for 5 minutes each, and gradient dehydration was performed, and finally transparentized with xylene. After taking out the slices, an appropriate amount of neutral gum was quickly added before the xylene dried, and finally sealed with a cover glass, and the morphological changes of liver tissue were observed under an upright microscope.

[0040] result: The results of serum liver function indicators are as follows Figure 7 As shown in the figure, compared with the normal control group, the ALT and AST levels in the serum of the model group mice were significantly increased. Compared with the model group, the administration of isodanin and NAC alone could reduce the ALT and AST caused by APAP, but there was no significant difference. The combined administration of isodanin + NAC could significantly reduce the ALT and AST caused by APAP.

[0041] H&E staining Figure 8 As shown in the figure, the liver tissue structure of mice in the normal control group was normal, and there was no inflammatory cell infiltration in the interstitium. The liver cell structure of the APAP model group was destroyed, with nuclear condensation and cell punctate necrosis, and a large number of inflammatory cells infiltrating the interstitium. The liver tissue damage of mice in the group treated with isodan-emodin and NAC alone was reduced compared with the model group, the hepatic cords were arranged more neatly, and the liver cell necrosis and inflammatory cell infiltration were reduced. However, the liver tissue damage of mice treated with isodan-emodin + NAC was significantly improved.

[0042] The above results prove that the combined administration of isocyanate+NAC can enhance the therapeutic effect of NAC.

Claims

1. Application of isodanin in the preparation of a drug for preventing and treating acetaminophen-induced acute liver injury, wherein the isodanin structural formula is shown in (I) 2. The use according to claim 1, It is characterized in that The medicine is a medicine for improving liver function indicators, and the liver function indicators are alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase.

3. The use according to claim 1, It is characterized in that The medicine is a medicine for improving liver tissue structure.

4. The use according to claim 1, It is characterized in that The drug is a drug for reducing inflammatory cell infiltration in the liver.

5. The use according to claim 1, It is characterized in that The drug is a drug that promotes liver fatty acid oxidation.

6. The use according to claim 1, It is characterized in that The drug is a drug which can be administered in combination with N-acetyl-L-cysteine ​​and can enhance the effect of N-acetyl-L-cysteine.

7. The use according to claim 1, It is characterized in that The medicine is any one of injection, tablet, pill, capsule, suspension or emulsion.

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