Application of kaempferol in preparation of medicine for treating liver cancer related to non-alcoholic fatty liver

Abstract

The invention relates to application of kaempferol in preparation of drugs, food or health care products for preventing or treating liver cancer related to non-alcoholic fatty liver or non-alcoholic fatty liver. According to the kaempferol disclosed by the invention, the liver weight, the liver weight ratio, the tumor quantity and the maximum diameter of the tumor of a mouse with the non-alcoholic fatty liver-related liver cancer are obviously reduced; in histology, the kaempferol can obviously improve hepatic tissue fatty degeneration and liver lipid droplet density, and improve liver cell inflammation and fibrosis degree; kaempferol can obviously reduce the content of cholesterol serum and cholesterol in the liver of a mouse with non-alcoholic fatty liver-related liver cancer, and can reduce liver injury indexes ALT and AST; the kaempferol can obviously reduce the expression level of mRNA related to liver fibrosis of mice with liver cancer related to non-alcoholic fatty liver.

Description

Technical Field

[0002] This invention relates to the field of pharmaceutical biotechnology, and more specifically, to the application of kaempferol in the preparation of drugs for treating non-alcoholic fatty liver-related liver cancer. Background Technology

[0003] Non-alcoholic fatty liver disease (NAFLD) is a group of chronic liver diseases (CLDs) ranging from solitary hepatic steatosis and NASH to cirrhosis and / or hepatocellular carcinoma (HCC). The global prevalence of NAFLD is approximately 30%, meaning that 37% of adults worldwide may have NAFLD. HCC is one of the fourth leading causes of cancer death globally and the second leading cause of cancer-related life loss. According to the 2015 Global Burden of Disease Study, the incidence of liver cancer increased by 75% between 1990 and 2015. Currently, NAFLD-related HCC accounts for 1%–38% of the HCC burden in different countries. With the increasing prevalence of obesity, NAFLD-related HCC is likely to increase significantly.

[0004] However, there are currently no universally accepted drugs for the treatment of NAFLD in clinical practice. Therefore, the use of traditional Chinese medicine to treat NAFLD-related HCC has attracted much attention, and a deeper understanding of its pathogenesis and the search for effective therapeutic targets are of great significance.

[0005] Kaempferol (KAE) is a natural flavonoid with broad biological activities, including anti-inflammatory, antioxidant, cardiovascular protective, and anticancer effects. However, the therapeutic effect of KAE on NALFD-related hepatocellular carcinoma is currently unclear. This invention relates to the first application of kaempferol in the treatment of NALFD-related hepatocellular carcinoma.

[0006] Chinese patent document CN 111617092A discloses a drug for treating lung cancer and its preparation method. The drug contains etoposide and kaempferol, as well as a pharmaceutically acceptable carrier or conventional excipients. The combination of etoposide and kaempferol at specific dosage ratios has a synergistic effect in treating small cell lung cancer. The combined use of the two active pharmaceutical ingredients can reduce the individual dosage of etoposide and kaempferol in treating small cell lung cancer, thereby enhancing efficacy and reducing side effects. This phenomenon provides a scientific basis for the rational selection of clinical drugs, reducing drug toxicity, and improving the treatment effect of small cell lung cancer.

[0007] Chinese patent document CN111617092A discloses a pharmaceutical composition for treating liver cancer. This composition contains effective doses of icariin and kaempferol, with a mass ratio of icariin to kaempferol of 8-12:45-55. Experimental results show that, compared to KAE and ICA administered alone, the CMPLS group exhibited more necrotic tissue fragments in tumor cell clusters, moderate vascular dilation, congestion, and hemorrhage in the interstitial blood vessels, indicating a synergistic effect between KAE and ICA. Combined use of these drugs has a more significant killing effect on tumor cells, thereby inhibiting tumor cell proliferation. Therefore, this invention provides a new drug for the treatment of liver cancer.

[0008] Currently, there are no reports on the application of kaempferol in the preparation of drugs for the treatment of non-alcoholic fatty liver disease-related liver cancer. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing an application of a traditional Chinese medicine monomer in the preparation of a drug for non-alcoholic fatty liver disease-related hepatocellular carcinoma. The monomer, kaempferol (KAE), has the molecular formula C15H10O6 and its structural formula is shown below.

[0010]

[0011] To achieve the above objectives, the technical solution adopted by the present invention is: the application of a traditional Chinese medicine monomer, kaempferol, in the preparation of a drug for treating non-alcoholic fatty liver-related liver cancer.

[0012] In a first aspect, the present invention provides the use of kaempferol in the preparation of drugs, foods or health products for the prevention or treatment of non-alcoholic fatty liver disease-related liver cancer or non-alcoholic fatty liver disease.

[0013] As a preferred example, the non-alcoholic fatty liver disease manifests as one or more of the following: steatosis, lobular inflammation, hepatocyte ballooning degeneration, lipid droplet accumulation, and fibrosis.

[0014] As a preferred example, the kaempferol is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and a carrier, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

[0015] As a preferred example, kaempferol is dissolved in 0.5% sodium carboxymethyl cellulose.

[0016] As a preferred example, the dosage of kaempferol is 100 mg / kg / day, dissolved in 0.5% sodium carboxymethyl cellulose.

[0017] The advantages of this invention are as follows: Kaempferol (KAE) significantly reduces liver weight, liver weight ratio, tumor number, and maximum tumor diameter in mice with non-alcoholic fatty liver disease (NAFLD); histologically, Kaempferol (KAE) significantly improves steatosis and lipid droplet density in liver tissue, and improves hepatocyte inflammation and fibrosis; Kaempferol (KAE) significantly reduces serum and intrahepatic cholesterol levels in mice with NAFLD, and reduces liver damage indicators ALT and AST; Kaempferol (KAE) significantly reduces the expression level of liver fibrosis-related mRNAs in mice with NAFLD. Attached Figure Description

[0018] Figure 1 Macroscopic examination of the liver of mice with non-alcoholic fatty liver disease and liver cancer, including liver weight, liver weight ratio, number of tumors, and maximum tumor diameter.

[0019] Figure 2 Statistical graph showing changes in serum ALT, AST, TC, and liver TC in mice with non-alcoholic fatty liver disease-associated hepatocellular carcinoma.

[0020] Figure 3 HE staining results in mice with non-alcoholic fatty liver disease and liver lipid droplet accumulation, Oil Red O staining image and Sirius Red staining image.

[0021] Figure 4 Statistical graph showing changes in the expression levels of liver fibrosis-related mRNAs in mice with non-alcoholic fatty liver disease. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0023] Example 1: Animal Experiment

[0024] 1. Experimental Materials

[0025] 1.1 Experimental Animals and Traditional Chinese Medicine Materials: Male C57BL / 6 mice, weighing 20–25 g and 6 weeks old, were purchased from Jesstech and housed in an environment free from specific pathogen infection. The mice lived under a standard 12-hour light-dark cycle with free access to food and water. Kaempferol was purchased from Shidande.

[0026] 1.2 Establishment of a mouse model of non-alcoholic fatty liver disease-related hepatocellular carcinoma: Four-day-old mice were intraperitoneally injected with 200 μg / mouse of streptozotocin. Three weeks later, they were fed a high-fat diet (HFD) for 16 weeks, with fat in the HFD providing 60% of the total calories. Mice were randomly assigned to three groups: a normal diet (NCD) group (n=10), a streptozotocin (STZ) group (n=10), and a streptozotocin + kaempferol (KAE) group (n=10). After induced obesity by feeding the HFD diet for 12 weeks, the mice continued to be fed the HFD diet while simultaneously receiving the HFD diet via gavage for 4 weeks. Kaempferol was dissolved in 0.5% CMC-Na and administered to both the NCD and STZ groups via gavage with 0.5% CMC-Na. Mice were fasted overnight but allowed free access to water before sampling. On the day of sampling, mice were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital (50 μL / 10 g) and weighed. After the mice were fully anesthetized, blood was collected from their eyeballs. Whole blood was collected, left at room temperature for half an hour, and centrifuged at 3000 rpm for 15 min. The collected serum was stored at -80℃. After blood collection, the mice were euthanized by cervical dislocation. The chest wall was cut open layer by layer along the midline, and the left lobe of the liver was divided into two equal parts. Each part was fixed in 4% paraformaldehyde. The remaining liver was placed in EP tubes and frozen in liquid nitrogen for later use.

[0027] 1.3 Tissue H&E staining and Oil Red staining: (1) H&E staining: 1. Dewax the paraffin sections to water, and process the sections according to the following steps: soak in xylene for 30 min, 100% alcohol for 10 min, 95% alcohol for 10 min, ddH2O for 10 min, hematoxylin for 10 min, rinse with running water for 5 min, eosin for 90 s, rinse with running water for 10 min, 95% alcohol for 20 s, 100% alcohol for 20 s, xylene for 20 s, and mount with neutral resin. (2) Oil Red staining: Fixation of fresh frozen sections: Warm up and dry the frozen sections, fix them in fixative for 15 min, wash with tap water and air dry. Immerse the sections in oil red staining solution for 8-10 min (cover and protect from light). Background differentiation: Take out the sections, let them stand for 3 s, and then immerse them in two tanks of 60% isopropanol for differentiation, 3 s and 5 s respectively. Immerse the sections in two tanks of pure water for 10 s each. Hematoxylin staining: Remove the sections, let them sit for 3 seconds, then immerse them in hematoxylin for counterstaining for 3-5 minutes. Rinse with three tanks of pure water for 5 seconds, 10 seconds, and 30 seconds respectively. Differentiate with differentiation solution (60% alcohol as solvent) for 2-8 seconds, rinse with two tanks of distilled water for 10 seconds each, and then use blueing solution for 1 second. Gently immerse the sections in two tanks of tap water for 5 seconds and 10 seconds respectively, and examine the staining effect under a microscope. Mounting: Mount the sections with glycerol gelatin mounting medium.

[0028] 1.4 Biochemical Indicator Detection: Blood samples were collected from mice, and the supernatant was centrifuged at 3000 rpm for 10 minutes. Serum lipids and liver function-related indicators in mice were measured using a Hitachi fully automated biochemical analyzer.

[0029] 1.5 RT-qPCR

[0030] Total mRNA was extracted from mouse liver tissue or cell samples using a total RNA isolation reagent (Biosharp, BS258A) following the manufacturer's instructions. RNA concentration and the A260 / 280 ratio were determined using a Nanodrop 2000 spectrophotometer. Samples were considered suitable for subsequent quantitative analysis when this ratio was between 1.8 and 2.0. One microgram of mRNA was reverse transcribed into cDNA using ABScript III RT Master Mix for qPCR with gDNA Remover 202 (Abclonal, RK20429), following the manufacturer's instructions. Quantitative PCR amplification was performed using SYBR Green (Abclonal, RK21207), and expression levels of different genes were calculated using the 2^(-ΔΔCt) method.

[0031] 2. Experimental Results:

[0032] 2.1 Effects of kaempferol on liver tumors in mice with non-alcoholic fatty liver disease-associated hepatocellular carcinoma: (e.g.) Figure 1 As shown, kaempferol (KAE) significantly reduced liver weight, liver weight-to-weight ratio, tumor number, and maximum tumor diameter in mice with non-alcoholic fatty liver-associated hepatocellular carcinoma, indicating its therapeutic effect on fatty liver-associated hepatocellular carcinoma.

[0033] 2.2 Effects of kaempferol on serum liver function and total liver cholesterol in mice with non-alcoholic fatty liver disease-related hepatocellular carcinoma: (e.g.) Figure 2 As shown, kaempferol (KAE) significantly reduced serum and liver cholesterol levels in mice with non-alcoholic fatty liver-related hepatocellular carcinoma, and reduced liver damage indicators ALT and AST.

[0034] 2.3 Effects of kaempferol on liver morphology in mice with non-alcoholic fatty liver disease-related hepatocellular carcinoma: (e.g.) Figure 3 As shown, compared with the normal control group, the STZ model group mice exhibited significant hepatic steatosis, lobular inflammation, hepatocyte ballooning degeneration, lipid droplet accumulation, and fibrosis; compared with the STZ model group, the KAE group mice showed significantly reduced hepatic steatosis, lobular inflammation, hepatocyte ballooning degeneration, lipid droplet accumulation, and fibrosis. The results indicate that the kaempferol described in this invention can improve hepatic steatosis and fibrosis induced by a high-fat diet in mice.

[0035] 2.4 Effects of kaempferol on the expression level of liver fibrosis-related mRNAs in mice with non-alcoholic fatty liver disease-associated hepatocellular carcinoma: (e.g.) Figure 4 As shown, kaempferol (KAE) significantly reduced the expression levels of α-SMA, COL1A1, TGF-β, TIMP and FN mRNA in the livers of mice with non-alcoholic fatty liver-related hepatocellular carcinoma.

[0036] In summary, kaempferol is a drug that can be used to treat non-alcoholic fatty liver disease-associated hepatocellular carcinoma.

Claims

1. Use of kaempferol in the preparation of a medicine, food or health product for preventing or treating non-alcoholic fatty liver related hepatocarcinoma or non-alcoholic fatty liver.

2. Use according to claim 1, characterized in that, The non-alcoholic fatty liver shows one or more of the following: steatosis, lobular inflammation, hepatocyte ballooning, lipid droplet accumulation and fibrosis.

3. Use according to claim 1, characterized in that, The kaempferol is added with a pharmaceutically acceptable adjuvant and carrier to prepare a pharmaceutical preparation, and the adjuvant includes at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener and a colorant.

4. Use according to claim 1, characterized in that, Kaempferol is dissolved using 0.5% sodium hydroxymethyl cellulose.

5. Use according to claim 4, characterized in that, The dose of kaempferol is 100 mg / kg / d, and kaempferol is dissolved using 0.5% sodium hydroxymethyl cellulose.

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