Application of cinobufagin in preparation of medicine for treating metabolic dysfunction related steatohepatitis

Abstract

The invention relates to the technical field of biological medicine, in particular to application of cinobufagin in preparation of a medicine for treating metabolic dysfunction related steatohepatitis. According to the application disclosed by the invention, an MASH mouse model is constructed by inducing methionine choline deficiency diet (MCD), and evaluation on hepatic histopathology, fibrosis degree and inflammatory cell infiltration condition of the MASH mouse model shows that ALT and AST levels of serum of mice in a low-dose and high-dose group of cinobufagin are remarkably reduced and are in a certain dose dependency relationship; after intervention of different doses of cinobufagin, the liver lipid degeneration, inflammatory cell infiltration and fibrosis degree of MASH mice are obviously improved. Through MASH mouse liver transcriptomics analysis, cinobufagin can significantly down-regulate lipid metabolism, inflammation, fibrosis and cell injury related gene expression. GSEA analysis results also show that cinobufagin is mainly used for negatively regulating lipid metabolism, inflammation, fibrosis and related signal channels of cell injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of cinobuccalin in the preparation of a drug for treating metabolic dysfunction-related fatty hepatitis. Background Art

[0002] Metabolic dysfunction-associated steatohepatitis (MASH), also known as non-alcoholic steatohepatitis, is the advanced stage of metabolic dysfunction-associated fatty liver disease. It is closely related to a variety of metabolic abnormalities such as type 2 diabetes, obesity, and cardiovascular disease. MASH is the main cause of liver-related death, mainly manifested as ballooning of hepatocytes, severe liver inflammation, and hepatocyte apoptosis, and will lead to liver fibrosis, cirrhosis, and eventually develop into liver cancer. Among them, liver fibrosis is an important feature of MASH. Studies have shown that whether the progression of liver fibrosis can be prevented is the main endpoint indicator for judging anti-MASH drugs. However, although the vast majority of existing drugs for treating MASH can improve the accumulation of fat in the liver, they rarely prevent the progression of liver fibrosis. Therefore, finding possible therapeutic drugs to slow down or even reverse the progression of liver fibrosis has become the key to treating MASH.

[0003] MASH belongs to the category of "liver obsession", "liver obstruction", "accumulation" and "phlegm turbidity" in traditional Chinese medicine. According to traditional Chinese medicine, fat comes from food, and is transformed into essence through the stomach's reception and the spleen's transportation and transformation. If the diet is not moderate and too much fat and sweet food is consumed, the spleen will be damaged. The spleen's transportation and transformation will not be sufficient, and fat cannot be transformed into essence, but accumulates to become phlegm, dampness and turbid fat. This is what "Xizhuo of the Origin and Development of Miscellaneous Diseases" said: "Excessive drinking and eating, and liking to eat oil, wheat and pig fat, will lead to poor spleen function and stagnation into phlegm". Turbid fat enters the liver along the pulse, accumulates in the liver, and is attached to the liver without being transformed, which is the basis for the onset of MASH. Turbid fat accumulates for a long time. On the one hand, it accumulates and turns into heat, leading to internal accumulation of dampness and heat; on the other hand, it accumulates for a long time, causing poor blood circulation, blood circulation delays and causes stasis. Therefore, the pathological factors of MASH are the mutual binding of "phlegm, dampness, stasis and heat", which blocks the liver pulse. Toad skin is the spleen of the Chinese giant toad or black-framed toad of the Toad family. It tastes bitter, is cool in nature, and is toxic. It enters the heart, lung, spleen, and large intestine meridians, and has the effects of clearing away heat and detoxifying, promoting diuresis and reducing swelling, promoting blood circulation and unblocking collaterals, and removing blood stasis and ulcers. The "Compendium of Materia Medica" says that it can "eliminate the accumulation of bad habits and break up hard masses and swellings", and the "Medical Collection" says that it "can disperse, move, penetrate, and soften", and the "Rihuazi Materia Medica" says that it can "break up masses and treat malnutrition". It is often used to treat malnutrition abdominal distension, swelling, toxicity, scrofula, and other diseases. It can be seen that the functions of toad skin in clearing heat and promoting diuresis, promoting blood circulation and removing blood stasis are consistent with the pathological factors of MASH "phlegm, dampness, stasis, and heat".

[0004] Cinobufagin is a refined aqueous extract of dried toad skin, a traditional Chinese medicine, which is commonly used in clinical treatment of diseases such as hepatitis B and liver cancer and has good safety. Modern pharmacological studies have shown that cinobufagin has anti-tumor activities against gastric cancer, pancreatic cancer, lung cancer, etc. However, there is no literature report on the application of cinobufagin in the treatment of MASH at present. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of cinobufagin in the preparation of a drug for treating metabolic dysfunction-related steatohepatitis.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] An application of cinobufagin in the preparation of a drug for treating metabolic dysfunction-related steatohepatitis. The present invention has confirmed through animal models that cinobufagin has significant curative effects in the treatment of MASH.

[0008] Preferably, the metabolic dysfunction-related steatohepatitis includes symptoms such as liver fibrosis, liver inflammation, and hepatocyte injury caused thereby.

[0009] Preferably, the drug is an oral preparation or an injection.

[0010] Preferably, the oral preparation is a powder, granule, capsule, powder, pill, tablet or oral liquid.

[0011] A drug for treating metabolic dysfunction-related steatohepatitis, wherein the active ingredient of the drug includes cinobufagin.

[0012] Preferably, the drug further includes pharmaceutically or physiologically acceptable excipients, and the excipients include one or more of a diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, solubilizer, preservative, pH regulator, osmotic pressure regulator, surfactant, coating material, antioxidant or buffer.

[0013] The beneficial effects of the present invention are:

[0014] In the present invention, a MASH mouse model was induced and constructed by a methionine-choline-deficient diet (MCD). The liver histopathology, fibrosis degree, and inflammatory cell infiltration were evaluated. The serum ALT and AST levels of the low- and high-dose cinobufagin groups of mice were significantly decreased, and showed a certain dose-dependent relationship; after intervention with different doses of cinobufagin, the liver lipid degeneration, inflammatory cell infiltration, and fibrosis degree of MASH mice were significantly improved. Through transcriptomics analysis of the livers of MASH mice, cinobufagin was able to significantly down-regulate the expression of genes related to lipid metabolism, inflammation, fibrosis, and cell injury. The results of GSEA analysis also showed that cinobufagin mainly negatively regulated the signaling pathways related to lipid metabolism, inflammation, fibrosis, and cell injury.

[0015] Therefore, cinobufacini of the present invention has the effect of treating MASH, which is mainly manifested in alleviating hepatic lipid degeneration, inhibiting liver inflammation and liver fibrosis, and has strong pharmacological effects, being safe and non-toxic. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Effects of cinobufacini on serum ALT and AST levels in MASH mice ( n = 8), where: NCD: normal diet group; MCD: methionine-choline deficient diet group; MCD + HCS-L: methionine-choline deficient diet + cinobufacini 30 mg / kg; MCD + HCS-H: methionine-choline deficient diet + cinobufacini 50 mg / kg;

[0017] Figure 2 Effects of cinobufacini on liver histopathology, inflammation and fibrosis in MASH mice, where: (A) H&E staining was used to observe the effects of cinobufacini on liver histopathological changes in MASH; (B) F4 / 80 immunohistochemical staining was used to observe the effects of cinobufacini on liver inflammatory cell infiltration in MASH; (C) Sirius red staining was used to observe the effects of cinobufacini on liver fibrosis in MASH, with the scale bar being 300 μm; (D) non-alcoholic fatty liver disease score (NAFLD activity score). NCD: normal diet group; MCD: methionine-choline deficient diet group; MCD + HCS-L: methionine-choline deficient diet + cinobufacini 30 mg / kg; MCD + HCS-H: methionine-choline deficient diet + cinobufacini 50 mg / kg;

[0018] Figure 3 Transcriptomic analysis of the livers of MASH mice, by transcriptomic analysis of the differential changes in liver gene expression between the MCD group and the MCD + HCS-H group of mice (n = 5, FC > 2, P < 0.05); where: (A) heat map of genes related to lipid metabolism, inflammation, fibrosis and cell damage with significant expression differences; (B) GSEA analysis of pathways related to liver lipid metabolism, inflammation, fibrosis and cell damage; MCD: methionine-choline deficient diet group; MCD + HCS-H: methionine-choline deficient diet + cinobufacini 50 mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0019] The technical solutions of the present invention will be further specifically described below through specific examples. It should be understood that the implementation of the present invention is not limited to the following examples, and any form of modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0020] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be obtained from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.

[0021] The reagents used in the following examples can be purchased from conventional biochemical reagent stores unless otherwise specified. The materials, reagents and experimental animals used in the following examples are as follows:

[0022] 1. Materials and Reagents

[0023] Cinobufagin was purchased from Shaanxi Dongtai Pharmaceutical Co., Ltd.; kits for detecting alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were purchased from Nanjing Jiancheng Bioengineering Institute; H&E staining kit and Sirius red staining kit were both purchased from Solarbio; TRIzol kit was purchased from Invitrogen, USA; NanoDrop ND-1000 was purchased from NanoDrop, USA; magnesium ion fragmentation kit was purchased from Magnesium RNA Fragmentation Module, USA; RNA reverse transcriptase was purchased from Invitrogen SuperScript TM II Reverse Transcriptase, USA; E.coli DNApolymerase I was purchased from NEB, USA; RNase H was purchased from NEB, USA.

[0024] 2. Experimental Animals

[0025] Thirty-two SPF-grade male C57BL / 6J mice, 6 - 8 weeks old, were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK(Zhe)2019 - 0001, and were housed in the Experimental Animal Center of Quzhou People's Hospital, use license number: SYXK(Zhe)2022 - 0042. All mice were housed in a specific pathogen-free environment at a temperature of (21 ± 1) °C and a humidity of 60% ± 5%, with a 12-h light-dark cycle. All animal experiments and research complied with all relevant ethical regulations. The animal experiments were approved by the Experimental Animal Welfare and Ethics Committee of Quzhou People's Hospital.

[0026] Examples

[0027] I. Methods

[0028] 1.1 Animal Grouping and Administration

[0029] The MASH mouse model was induced by methionine-choline-deficient diet (MCD). Thirty-two male C57BL / 6 mice (6 - 8 weeks old) were randomly divided into 4 groups according to the random number table method: NCD group, MCD group, MCD + HCS-L group, and MCD + HCS-H group, with 8 mice in each group. After one week of adaptive feeding for each group of mice, the NCD group continued to be fed with normal diet, while the MCD group, MCD + HCS-L group, and MCD + HCS-H group were fed with MCD diet for a total of 8 weeks. Meanwhile, starting from the 5th week of feeding, the mice in the NCD group and MCD group were given intragastric administration of 0.5% sodium carboxymethylcellulose solution, the MCD + HCS-L group was given intragastric administration of cinobufacini at 30 mg / kg / day, and the MCD + HCS-H group was given intragastric administration of cinobufacini at 50 mg / kg / day. The administration time was 4 weeks in total.

[0030] 1.2 Determination of serum biochemical indexes

[0031] Sixteen hours after the last administration, the serum was separated, and the serum AST and ALT levels of mice in each group were measured on an automatic biochemical analyzer.

[0032] 1.3 Evaluation of liver histopathology, fibrosis degree, and inflammatory cell infiltration

[0033] A part of the liver was taken, fixed with 10% formalin, and routinely embedded in paraffin for sectioning. H&E staining was used to observe the pathological morphology of the liver tissue of mice in each group; Sirius red staining was used to evaluate the liver fibrosis degree of mice in each group; F4 / 80 immunohistochemical staining was used to evaluate the inflammatory cell infiltration of the liver of mice in each group.

[0034] 1.4 Liver transcriptomics analysis

[0035] The total sample RNA was isolated and purified using a TRIzol kit according to the manufacturer's protocol. Then, the quantity and purity of the total RNA were quality controlled using a NanoDrop ND-1000. The integrity of the RNA was detected using a Bioanalyzer 2100 and verified using an agarose electrophoresis protocol. A concentration > 50 ng / μL, RIN value > 7.0, OD260 / 280 > 1.8, and total RNA > 1 μg were required to meet the downstream experiment requirements. Polyadenylated mRNA was specifically captured using oligo(dT) magnetic beads through two rounds of purification. The captured mRNA was fragmented using a magnesium ion fragmentation kit at high temperature, 94°C for 5 - 7 minutes. The fragmented RNA was reverse transcribed into cDNA using reverse transcriptase. Then, E. coli DNA polymerase I and RNase H were used for second-strand synthesis to convert the DNA-RNA hybrid double-strand into a DNA double-strand. At the same time, dUTP Solution was incorporated into the second strand to blunt the ends of the double-stranded DNA. An A base was added to each end to enable ligation to an adapter with a T base at the end. Magnetic beads were used to screen and purify the fragment sizes. The second strand was digested with UDG enzyme, and then PCR was performed - pre-denaturation at 95°C for 3 minutes, 98°C denaturation for a total of 8 cycles, 15 seconds each, annealing to 60°C for 15 seconds, extension at 72°C for 30 seconds, and finally extension at 72°C for 5 minutes to form a library with a fragment size of 300 bp ± 50 bp. Finally, the library was sequenced using an Illumina Novaseq TM 6000 (LC Bio Technology CO., Ltd. Hangzhou, China) according to the standard operation for paired-end sequencing, and the sequencing mode was PE150.

[0036] 1.5 Data processing

[0037] All data were processed using SPSS 17.0 statistical software, and the data were expressed as mean ± standard deviation and the inter-group differences were tested by analysis of variance. The least significant difference method was used for pairwise comparisons, and P < 0.05 was considered statistically significant.

[0038] II. Results

[0039] 1. Effect of cinobufacini on liver function in MASH mice

[0040] The results of the effect of cinobufacini on the serum ALT and AST levels in MASH mice are as follows Figure 1As shown, compared with the control group, the levels of liver function indexes ALT and AST in the MCD group of mice were significantly increased (P<0.01); compared with the MCD group, the levels of serum ALT and AST in the low- and high-dose cinobufacini groups of mice were significantly decreased (P<0.01), and showed a certain dose-dependent relationship.

[0041] 2. Effects of Cinobufacini on Liver Histopathology, Inflammation and Fibrosis in MASH Mice

[0042] The results of the effects of cinobufacini on liver histopathology, inflammation and fibrosis in MASH mice are as Figure 2 shown. In the NCD group of mice, the structure of liver hepatocytes was complete, arranged neatly, the hepatic lobules were clearly visible, and there was no obvious infiltration of inflammatory cells and fibrosis; in the MCD group of mice, the liver tissue structure was disordered, the boundaries of hepatic lobules were unclear, the volume of hepatocytes increased and swelled, a large number of macrovesicular vacuolar changes appeared in hepatocytes, and the degree of infiltration of inflammatory cells and fibrosis in the liver was obvious. After intervention with different doses of cinobufacini, the lipid degeneration, infiltration of inflammatory cells and fibrosis degree in the liver of MASH mice were significantly improved. In addition, low- and high-dose cinobufacini could significantly reduce the non-alcoholic fatty liver disease score (NAFLD activity score).

[0043] 3. Effects of Cinobufacini on Genes and Pathways Related to Liver Lipid Metabolism, Inflammation, Fibrosis and Apoptosis in MASH Mice

[0044] The results of transcriptomic analysis of the liver in MASH mice are as Figure 3 shown. By transcriptomic analysis, the differential changes in liver gene expression between the MCD group and the MCD+HCS-H group of mice were detected. Compared with the MCD group, a total of 408 differentially expressed genes were detected in the MCD+HCS-H group, including 93 up-regulated genes and 315 down-regulated genes (n=5, FC>2, P<0.05). The results showed that cinobufacini could significantly down-regulate the expression of genes related to lipid metabolism, inflammation, fibrosis and cell damage (P<0.05). The results of GSEA analysis also showed that cinobufacini mainly negatively regulated the signaling pathways related to lipid metabolism, inflammation, fibrosis and cell damage.

[0045] III. Conclusion

[0046] In this invention, MASH mice induced by methionine-choline-deficient diet (MCD) were used as the research object to observe the effects of cinobufacini on the liver function, liver histopathology, liver fibrosis and liver inflammation of MASH mice, and to observe the regulatory effects of cinobufacini on genes and pathways related to liver lipid metabolism, inflammation, fibrosis and cell damage through liver transcriptomic analysis. The results showed that cinobufacini could improve the liver function of MASH, inhibit liver inflammation and fibrosis, and systematically down-regulate the expression of genes related to liver lipid metabolism, inflammation, fibrosis and apoptosis.

[0047] In summary, the cinobufacini of the present invention has the effect of treating MASH, which is mainly manifested in relieving liver lipid degeneration, inhibiting liver inflammation and liver fibrosis, and has strong pharmacological effects, safety and no toxicity. Although the vast majority of existing drugs for treating MASH can improve the condition of liver fat accumulation, few can prevent the progression of liver fibrosis. It has been experimentally proven that in addition to improving the liver lipid accumulation in MASH mice, the cinobufacini drug of the present invention can also significantly inhibit liver fibrosis.

[0048] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple. For the relevant parts, reference can be made to the description in the method part.

[0049] The above has introduced in detail the application of the cinobufacini provided by the present invention in the preparation of drugs for treating metabolic dysfunction-related steatohepatitis. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A use of cinobucoids in the preparation of drugs for treating fatty liver disease associated with metabolic dysfunction.

2. The use according to claim 1, characterized in that: The metabolic dysfunction-related fatty hepatitis includes symptoms such as liver fibrosis, liver inflammation and liver cell damage caused by it.

3. The use according to claim 1, characterized in that: The medicine is an oral preparation or an injection.

4. The use according to claim 1, characterized in that: The oral preparation is a powder, granule, capsule, powder, pill, tablet or oral liquid.

5. A drug for treating metabolic dysfunction-related fatty liver disease, characterized in that: The active ingredient of the drug includes cinobucini.

6. The drug according to claim 5, characterized in that: The drug also includes pharmaceutically or physiologically acceptable excipients, which include one or more of diluents, adhesives, wetting agents, lubricants, disintegrants, solvents, emulsifiers, solubilizers, preservatives, pH regulators, osmotic pressure regulators, surfactants, coating materials, antioxidants or buffers.

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