Use of phorbol in the preparation of a medicament for preventing and / or treating liver injury

Abstract

The application provides use of phorbol in preparation of a medicine for preventing and / or treating liver injury, and belongs to the technical field of medicines; in the application, the phorbol can prevent and / or treat chemical hepatocyte injury, can prevent and / or treat hepatocyte injury caused by high fat accumulation, can improve liver function, and can be developed into a new medicine for preventing and / or treating chemical liver injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of crotonin in the preparation of drugs for the prevention and / or treatment of liver injury. Background Technology

[0002] Chemical liver injury is liver damage caused by hepatotoxic chemicals. These chemicals include alcohol, food additives, and toxic organic solvents such as carbon tetrachloride. Alcoholic liver disease (ALD) is the most common chronic liver disease worldwide, also known as alcoholic liver injury or alcoholic liver disease. It is an alcoholic liver disease caused by long-term alcohol abuse, and the number of patients has increased significantly in recent years, showing a trend towards affecting younger people. Alcoholic liver disease can progress from alcoholic fatty liver (AFL) to alcoholic steatohepatitis (ASH), eventually leading to cirrhosis and, in some cases, hepatocellular carcinoma (HCC). Clinical diagnosis shows that alcohol damage to the liver manifests as hepatic edema, hepatic steatosis, and varying degrees of hepatic fibrosis. Biochemical changes show elevated alanine aminotransferase (AST) and aspartate aminotransferase (ALT). Its pathogenesis mainly includes acetaldehyde-induced toxicity, oxidative stress, hepatic steatosis, changes in intestinal permeability, and intestinal flora imbalance. The primary treatment for alcoholic liver disease is abstinence from alcohol to reverse early-stage alcoholic liver disease and reduce mortality. For patients with severe alcoholic liver disease, liver transplantation is the only fundamental treatment, but its clinical application is severely limited due to its complexity, high cost, and scarcity of donors. Meanwhile, with increased daily exposure to hepatotoxic substances such as carbon tetrachloride, the incidence of chemical liver injury has surged. The liver, as the central organ of metabolism, frequently produces substances that damage it; therefore, utilizing modern biotechnology to find treatments for chemical liver injury is crucial for preventing and mitigating the epidemic pressure of chemical liver injury.

[0003] In recent years, traditional Chinese medicine and its active ingredients have demonstrated significant advantages in the treatment of chemically induced liver injury. The active ingredients of single herbs and compound herbs in traditional Chinese medicine have good protective effects against liver damage, eliminating free radicals, lowering alanine aminotransferase (ALT) and aspartate aminotransferase (AST), reducing the production of inflammatory factors, and improving liver pathological tissue, thereby exerting their pharmacological effects. Croton glycoside is an important alkaloid component of croton, and its effects on antitumor, anti-inflammatory, and antiarrhythmic activity have been explored, but its therapeutic effects on liver diseases have not been reported. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides the use of crotonin in the preparation of drugs for the prevention and / or treatment of liver injury. In this invention, scientific experiments have shown that crotonin can prevent and / or treat hepatocellular damage, thereby effectively reducing lipid accumulation and inflammation in the liver, inhibiting hepatocellular apoptosis, and improving liver function. Therefore, it can be developed into a novel drug for the prevention and / or treatment of liver injury.

[0005] This invention first provides the use of crotonin in the preparation of medicaments for the prevention and / or treatment of liver injury, wherein the molecular formula of crotonin is C 10 H 13 N5O5 has the following structural formula:

[0006] .

[0007] The present invention also provides the use of crotonin in the preparation of medicaments for the prevention and / or treatment of fatty liver, which is caused by chemical liver damage or high lipid accumulation.

[0008] The present invention also provides the use of crotonin in the preparation of medicaments for the prevention and / or treatment of hepatitis caused by chemical liver injury.

[0009] The present invention also provides the use of crotonin in the preparation of medicaments for the prevention and / or treatment of liver fibrosis caused by chemical liver injury.

[0010] The present invention also provides the use of crotonin in the preparation of medicaments for the prevention and / or treatment of cirrhosis caused by chemical liver injury.

[0011] The present invention also provides the use of crotonin in the preparation of drugs for the prevention and / or treatment of lipid metabolism, inflammation and oxidative stress caused by liver injury.

[0012] The present invention also provides a medicament for the prevention and / or treatment of alcoholic liver injury, wherein the medicament uses crotonin as the active substance.

[0013] The drug is prepared into a pharmaceutically acceptable formulation by adding pharmaceutically acceptable excipients, wherein the acceptable formulation is granules, tablets, capsules, injections, pills, or oral liquids.

[0014] The drug exhibits effects as described in at least one of (a), (b), (c), and (d):

[0015] (a) Inhibits the increase in serum AST caused by liver injury;

[0016] (b) Inhibits the increase in serum ALT caused by liver injury;

[0017] (c) Inhibits the increase in serum triglyceride levels caused by liver injury;

[0018] (d) Inhibits the increase in triglyceride levels in liver tissue caused by liver injury.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] Croton seeds are pungent, hot, and highly toxic; they have the functions of purging cold accumulations, eliminating phlegm, promoting diuresis, killing parasites, and clearing the orifices. Furthermore, croton seeds treat cold accumulations and stagnation, abdominal distension and pain, blood stasis, phlegm accumulation, diarrhea, edema, sore throat, pharyngitis, and malignant sores and scabies. Croton glycosides are important alkaloids extracted and isolated from croton seeds, possessing strong purgative effects against cold accumulations, promoting diuresis and reducing swelling, and clearing phlegm and soothing the throat. Scientific experiments in this invention have shown that croton glycosides can significantly prevent and treat chemically induced hepatocellular damage caused by chemicals, including alcohol.

[0021] In this invention, crotonin is prepared as an active ingredient using conventional pharmaceutical formulation processes to create a pharmaceutical formulation suitable for clinical use. In a mouse normal hepatocyte (AML12) alcohol model, crotonin was found to significantly improve hepatocyte damage, reduce intracellular oxidative stress, and regulate the activity of alcohol metabolism-related enzymes, thereby exerting therapeutic effects against chemical liver damage such as ALD.

[0022] This invention elucidates the role of crotonin in ALD. In a mouse normal hepatocyte (AML12) alcohol model, crotonin reduced lipid accumulation and inflammation in the liver, inhibited hepatocyte apoptosis, and improved liver function, providing a potential treatment strategy for chemically induced liver damage such as ALD.

[0023] This invention also describes the use of crotonin in the prevention and / or treatment of hepatocellular damage caused by high lipid accumulation, such as fatty liver, and its excellent application in the preparation of related drugs. Attached Figure Description

[0024] Figure 1 This is a diagram showing the results of H&E staining in mouse livers.

[0025] Figure 2 The figures show the bacterial diversity statistics after alcohol modeling in mice, with Shannon index (a), Simpson index (b), evenness index (c), and richness index (d) showing the changes.

[0026] Figure 3 The figure shows the results of non-metric multidimensional scaling analysis of bacterial sequencing after alcohol modeling in mice.

[0027] Figure 4 The relative abundance changes of Prevotella (a) and Alternaria (b) after simulating alcoholic liver disease in mice.

[0028] Figure 5 Image showing the cell viability of normal mouse liver parenchymal cells (AML12) after 36 hours of crotonin treatment in an alcohol-induced model.

[0029] Figure 6 Image showing the cell viability of normal mouse hepatocytes (AML12) after 24 hours of treatment with crotonin, emodin, aloe-emodin, and rhein to induce an alcoholic model.

[0030] Figure 7 The image shows the cell viability after 24 hours of treatment with crotonin in a carbon tetrachloride-induced oxidative stress-induced hepatocyte injury model. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. To achieve the objectives of the present invention, the preventive and therapeutic effects of crotonin on alcoholic liver disease were demonstrated using normal liver parenchymal cells from alcohol-induced model mice. All materials used in the embodiments of the present invention are commercially available and can be purchased.

[0032] Example 1:

[0033] In this embodiment, an alcoholic liver disease mouse model induced by a high-fat diet was constructed to investigate the effect of crotonin on liver damage in the alcoholic liver disease model mice. The specific investigation method is as follows:

[0034] (1) Laboratory animals and grouping:

[0035] Twenty-four male C57BL / 6 mice (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) weighing 20-22g were randomly divided into four groups: blank group, high-fat control group, alcohol-high-fat model group, and crotonin group (25mg / kg), with six mice in each group.

[0036] The control group was fed with ordinary feed for 21 days.

[0037] The high-fat control group was fed dextrin-adapted diet for 1-5 days, and then fed a diet with 5% alcohol by volume starting on day 6, continuing until day 21.

[0038] The alcohol-high-fat model group was fed dextrin for 1-5 days, and then switched to a diet containing 5% alcohol and a high-fat diet for 6-21 days.

[0039] The crotonin group was fed the same way as the high-fat alcohol model group, and was administered the drug by gavage starting on day 1 and continuing until the day of dissection on day 21.

[0040] On day 20, except for the control group, all groups were administered 6 g / kg of 50% ethanol by gavage. The control group was administered isocaloric maltodextrin by gavage. After 9 hours, the animals were sacrificed, and their livers were harvested. The liver tissues obtained from each group were then soaked in 4% paraformaldehyde for later use.

[0041] (2) Liver pathological examination:

[0042] (1) Tissue dehydration:

[0043] Cut liver tissue of appropriate size that has been soaked in 4% paraformaldehyde, place it in a dehydration box, and put the dehydration box into a fully automatic dehydrator for dehydration, as follows:

[0044] The mixture was dehydrated 4 times with 70% ethanol (1 hour each time), 2 times with 80% ethanol (1 hour each time), 1 hour with 85% ethanol, 1 hour with 90% ethanol, 1 hour with 95% ethanol, 2 times with anhydrous ethanol (1 hour each time), 30 minutes with a mixed solution of anhydrous ethanol and xylene (1:1 volume ratio), 10 minutes with xylene, 30 minutes with a mixed solution of xylene and paraffin (1:1 volume ratio), and finally 1.5 hours with paraffin.

[0045] (2) Tissue embedding:

[0046] The dehydrated liver tissue was embedded in paraffin using a tissue embedding machine. The specific steps were as follows: the melted paraffin was placed into the embedding box, the liver tissue was then placed into the embedding box, the embedding box was placed in a -20℃ freezer, and after it was completely solidified, the paraffin block was removed and trimmed. The processed paraffin block was stored in a 4℃ freezer for later use.

[0047] (3) Slicing:

[0048] The paraffin block was placed on a tissue microtome for sectioning, with the section thickness controlled at 4 μm. The sections were then spread on warm water at 40°C and dried in an oven at 60°C for later use.

[0049] (4) Slicing and dehydration:

[0050] Immerse the sections in xylene I reagent for 15 min, then in xylene II reagent for 15 min, then in xylene III reagent for 15 min, then in anhydrous ethanol for 5 min. After immersion, replace with fresh anhydrous ethanol and continue immersion for 5 min. After immersion, replace with 95% ethanol for 5 min, then with 85% ethanol for 5 min. Finally, rinse with double-distilled water to obtain dehydrated sections for later use.

[0051] (5) Hematoxylin staining:

[0052] After dehydration in step (4), the slices were stained with hematoxylin for 2 min, rinsed with water, differentiated with differentiation solution, rinsed with double-distilled water, and then treated with blue solution. After treatment, the slices were rinsed with double-distilled water.

[0053] (6) Eosin staining: Stain the sections stained in step (5) with eosin staining solution for 3 min.

[0054] (7) Dehydration and mounting: Wash the stained sections three times in anhydrous ethanol for 5 minutes each time, then wash them with xylene for 5 minutes each time, and repeat the washing process twice until the sections are transparent. Finally, mount the sections with neutral resin.

[0055] (8) Observe under a microscope, take pictures, and collect images for analysis.

[0056] Figure 1 The image shows the results of H&E staining of mouse livers. Pathological results show that in the high-fat control group, the liver tissue structure of mice was intact, the hepatic lobules were clearly outlined, the hepatic cords were neatly arranged, and there was no obvious degeneration or necrosis of hepatocytes, nor any inflammatory cell infiltration or fibrosis. In the alcohol-induced high-fat model group, the liver tissue structure of mice showed significant changes, with swollen hepatocytes and fat vacuoles of varying sizes. In the crotonin group, the hepatocytes were round and plump, the hepatic plates were regularly and neatly arranged, there was no obvious dilation or compression of the hepatic sinusoids, and no obvious inflammatory cell infiltration was observed. Compared with the model group, the number of fat vacuoles was significantly reduced. This demonstrates that crotonin can improve the pathological condition of mice with alcoholic liver disease.

[0057] (3) Effects of crotonin on gut microbiota:

[0058] In this embodiment, the effects of crotonin on gut microbiota were also investigated using gut microbiota sequencing, as detailed below:

[0059] (1) Genomic DNA extraction: After genomic DNA extraction, the extracted genomic DNA was detected by 1% agarose gel electrophoresis.

[0060] (2) PCR amplification: Synthesize specific primers with barcodes according to the specified sequencing region. To ensure the accuracy and reliability of subsequent data analysis, two conditions must be met: 1) use the lowest possible cycle number for amplification; 2) ensure the same cycle number for each sample. Randomly select representative samples for preliminary experiments to ensure that the vast majority of samples can amplify products of appropriate concentrations within the lowest possible cycle number. All samples were processed according to the formal experimental conditions, with three replicates for each sample. The PCR products of the same sample were mixed and detected by 2% agarose gel electrophoresis. The PCR products were recovered by gel cutting using the AxyPrepDNA Gel Recovery Kit (AXYGEN), eluted with Tris-HCl, and detected by 2% agarose gel electrophoresis.

[0061] (3) Quantitative fluorescence: Based on the preliminary quantitative results of electrophoresis, the PCR products were detected and quantified using the QuantiFluor™-ST blue fluorescence quantitative system (Promega). Then, the products were mixed in the appropriate proportions according to the sequencing requirements of each sample.

[0062] (4) Building the PacBio library:

[0063] 1) Repair and level;

[0064] 2) The connecting joint forms a dumbbell-shaped structure;

[0065] 3) Exonuclease removes fragments without adapters.

[0066] (5) PacBio sequencing:

[0067] 1) Primer and template annealing;

[0068] 2) Fluorescently labeled dNTPs form a complex with the enzyme and DNA template, and bind briefly;

[0069] 3) Fluorescent dNTPs emit fluorescence when irradiated by a laser, and the fluorescence signal is collected;

[0070] 4) During the enzymatic reaction, the chain extends while the fluorescent group on the dNTPs is removed. The polymerization reaction continues, and sequencing continues simultaneously.

[0071] (6) Data Analysis

[0072] Figure 2 The figures show the bacterial diversity statistics after alcohol-induced bacterial modeling in mice, specifically the changes in Shannon index (a), Simpson index (b), evenness index (c), and richness index (d). Figure 3 A figure showing the results of nonmetric multidimensional scaling analysis of bacterial sequencing after alcohol modeling in mice. Figure 4 A graph showing the changes in the relative abundance of *Prevotella* and *Alternaria* species after inducing alcoholic liver disease in mice. (Combined with...) Figure 2 , 3 Figures 4 and 5 show significant differences in gut microbiota composition among the blank group, high-fat control group, model group, and crotonin group, demonstrating that crotonin improves the overall structure and diversity of the gut microbiota. The *Alloprevotella* spp. species increased in the model group compared to the control group, and the *Alloprevotella* spp. species in the crotonin group showed a significant difference compared to the control group. This study confirms that the bacterial composition of the gut microbiota changes in patients with liver disease, and that alcoholic liver disease patients exhibit gut bacterial overgrowth. Therefore, crotonin can improve alcoholic liver disease by improving gut microbiota, regulating the gut-liver axis, and thus improving gut microbiota.

[0073] In summary, crotonin significantly improves liver damage caused by alcoholic liver disease in vivo, and it can also effectively improve fatty liver induced by fat accumulation caused by intestinal bacterial dysbiosis due to high lipid intake.

[0074] Example 2:

[0075] In this embodiment, an alcoholic hepatocyte injury model was constructed by treating mouse liver parenchymal (AML12) cells with alcohol to investigate the effects of crotonin on alcohol-induced stem cell viability and liver injury.

[0076] AML12 cells were seeded in DMEM / F12 medium containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin, and cultured at 37°C and 5% CO2 until the logarithmic growth phase, ready for use.

[0077] Collect AML12 cells in the logarithmic growth phase at a ratio of 2 x 10⁻⁶ cells per well. -4 Cells were seeded into 96-well plates, with 100 μL of cell suspension added to each well, and cultured overnight. A blank control group, a model group, and a drug treatment group were set up. After 36 hours of treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for 2–4 hours. After incubation, the 96-well plates were shaken for 5 minutes, and the absorbance at 450 nm was measured using a microplate reader.

[0078] The blank control group consisted of cells cultured normally in complete culture medium; the model group consisted of cells with 100mM anhydrous ethanol and different concentrations of crotonin added to complete culture medium; the drug treatment group was modeled in the same way as the model group, and then different concentrations of crotonin were added and cultured for 36 hours.

[0079] Figure 5 The figures show the cell viability of normal mouse hepatocytes (AML12) treated with different concentrations of crotonin for 36 hours in an alcohol model. The figures clearly show a significant difference in relative cell viability between the drug-treated groups and the model group. Compared to previously reported hepatoprotective drugs, crotonin protects hepatocytes at nanomolar concentrations in vitro, demonstrating extremely high sensitivity to alcoholic liver injury models.

[0080] Example 3:

[0081] In this embodiment, an alcoholic hepatocellular injury model was constructed by treating AML12 cells with alcohol to investigate the therapeutic effect of crotonin on alcoholic hepatocellular injury.

[0082] AML12 cells were seeded in DMEM / F12 medium containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin, and cultured at 37°C and 5% CO2 until the logarithmic growth phase, ready for use.

[0083] Collect AML12 cells in the logarithmic growth phase at a ratio of 2 x 10⁻⁶ cells per well. -4 Cells were seeded into 96-well plates, with 100 μL of cell suspension added to each well, and cultured overnight. A blank control group, a model group, and a drug treatment group were set up. After 36 hours of treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for 2–4 hours. After incubation, the 96-well plates were shaken for 5 minutes, and the absorbance at 450 nm was measured using a microplate reader.

[0084] The blank control group and solvent control group were treated in the same manner as in Example 2. Different concentrations of crotonin, emodin, aloe-emodin, and rhein were added to the drug treatment groups for 24 hours.

[0085] Figure 6 The image shows the cell viability of normal mouse hepatocytes (AML12) after 24 hours of treatment with crotonin, emodin, aloe-emodin, and rhein to induce an alcoholic model. The image shows that crotonin improves alcohol-damaged cells at nanomolar concentrations, while aloe-emodin only works at micromolar concentrations. Therefore, crotonin has a good therapeutic effect.

[0086] Example 4:

[0087] In this embodiment, an oxidative stress-induced hepatocyte injury model was constructed by treating AML12 cells with carbon tetrachloride to investigate the therapeutic effect of crotonin on chemically induced hepatocyte injury.

[0088] AML12 cells were seeded in DMEM / F12 medium containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin and cultured at 37°C and 5% CO2 until the logarithmic growth phase, ready for use.

[0089] Collect AML12 cells in the logarithmic growth phase at a ratio of 2 x 10⁻⁶ cells per well. -4 Cells were seeded into 96-well plates, with 100 μL of cell suspension added to each well, and cultured overnight. A blank control group (without carbon tetrachloride and crotonin), a model group (with carbon tetrachloride only), and drug treatment groups (with carbon tetrachloride followed by different concentrations of crotonin) were set up. After 36 h of treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for 2–4 h. After incubation, the 96-well plates were shaken for 5 min, and the absorbance at 450 nm was measured using a microplate reader.

[0090] Figure 7 The graph shows the cell viability after 24 hours of treatment with different concentrations of crotonin in a carbon tetrachloride-induced hepatocyte injury model. As can be seen from the graph, crotonin has a therapeutic effect on hepatocyte injury caused by oxidative stress after carbon tetrachloride treatment.

[0091] In summary, crotonin can significantly improve hepatocellular damage and reduce intracellular oxidative stress levels, and can be used to prepare drugs for the prevention and / or treatment of liver damage.

[0092] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. The use of crotonin as the sole active ingredient in the preparation of a medicament for the prevention and / or treatment of chemically induced liver injury, wherein the molecular formula of crotonin is C2. 10 H 13 N5O5.

2. Use of crotonin as the sole active ingredient in the preparation of a medicament for the prevention and / or treatment of fatty liver, wherein the fatty liver is caused by chemical liver injury or hyperlipidemia, and the molecular formula of crotonin is C2. 10 H 13 N5O5.

Images