Application of opuntia ficus-indica in preparation of medicine for treating non-alcoholic steatohepatitis

Abstract

The invention relates to application of opuntia ficus-indica in preparation of drugs, food or health-care products for preventing or treating non-alcoholic steatohepatitis. A non-alcoholic steatohepatitis mouse model fed with methionine and choline lacking high-fat feed finds that the opuntia molluscactus has an obvious effect of resisting the non-alcoholic steatohepatitis; compared with an MCD feed group, the opuntia ficus-indica can obviously reduce the liver weight ratio of mice with non-alcoholic steatohepatitis, compared with a low-dose group and a high-dose group of opuntia ficus-indica, the liver-to-body ratio of the mice is reduced, TC and TG levels in the liver are obviously reduced, and the opuntia ficus-indica can obviously reduce PAOA-induced mouse liver primary cell lipid accumulation and TG levels. In addition, the PAOA-induced mouse liver primary cell BODIPY dyeing fluorescence intensity is obviously reduced, and mouse liver bile acid substitution pathway related metabolic enzyme mRNA and protein expression can be up-regulated.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and more specifically, to the application of the Hoodia cactus in the preparation of a medicament for treating non-alcoholic steatohepatitis. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a chronic, progressive liver disease caused by overnutrition and insulin resistance (IR) in genetically susceptible individuals. Its spectrum includes non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and related fibrosis and cirrhosis. Diagnosis of NAFLD requires a liver fat accumulation of 5% or more, excluding other causes of liver disease such as viral hepatitis, autoimmune liver disease, hemochromatosis, Wilson's disease, drug-induced liver disease, and heavy alcohol consumption.

[0003] Bile acids (BAs) are synthesized from cholesterol in liver cells and are called primary bile acids. After being secreted into the intestinal lumen and modified by intestinal microorganisms, they become secondary bile acids. Bile acids can regulate their metabolism and transport through specialized nuclear and G protein-coupled receptors. Disruption of their transport and homeostasis can lead to the progression of cholestatic diseases and trigger various liver diseases, including non-alcoholic fatty liver disease, hepatocellular carcinoma, and cholangiocarcinoma.

[0004] The African cactus Hoodia gordonii is native to South Africa, Namibia, Botswana, and other regions, and was once used by local residents to suppress hunger and thirst.

[0005] There are currently no reports on the use of Hoodia gordonii (a type of cactus) in the preparation of drugs for the treatment of non-alcoholic steatohepatitis. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of the prior art by providing the application of *Opuntia ficus-indica* in the preparation of a medicament for treating non-alcoholic steatohepatitis.

[0007] In a first aspect, the present invention provides the use of the Hoodia cactus in the preparation of drugs, foods or health products for the prevention or treatment of non-alcoholic steatohepatitis.

[0008] As a preferred example, the non-alcoholic steatohepatitis manifests as one or more of the following: liver inflammation, hepatocyte structural disorder, and fatty degeneration.

[0009] As a preferred example, the non-alcoholic fatty liver disease is simple fatty liver disease, non-alcoholic steatohepatitis, and fatty liver infiltration.

[0010] As a preferred example, the *Opuntia ficus-indica* 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.

[0011] As a preferred example, the dosage of the *Opuntia ficus-indica* is 2.12 g / kg / d to 4.24 g / kg / d.

[0012] The advantages of this invention are as follows: Hoodia gordonii significantly reduces the liver weight ratio in mice with non-alcoholic steatohepatitis. Compared with the MCD diet group, the low-dose and high-dose Hoodia gordonii groups show a decrease in liver-to-body ratio and a significant decrease in intrahepatic TC and TG levels. Hoodia gordonii can significantly reduce PAOA-induced lipid accumulation and TG levels in mouse primary liver cells, and significantly reduce the intensity of BODIPY staining fluorescence in PAOA-induced mouse primary liver cells. It can also upregulate the mRNA and protein expression of bile acid replacement pathway-related metabolic enzymes in mouse liver. Attached Figure Description

[0013] Figure 1 Statistical chart of liver weight ratio and liver staining with HE, Oil Red, and Sirius Red in MCD mice.

[0014] Figure 2 Statistical graph of liver function and intrahepatic TC and TG levels in MCD mice.

[0015] Figure 3 Statistical graph showing changes in liver fibrosis and lipid-related mRNA expression levels in MCD mice.

[0016] Figure 4 Statistical graph of lipid accumulation and TG levels in primary mouse liver cells induced by PAOA.

[0017] Figure 5 Statistical graph of BODIPY fluorescence staining intensity in PAOA-induced primary mouse liver cells. Detailed Implementation

[0018] 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.

[0019] Example 1

[0020] 1. Experimental Materials

[0021] 1.1 Experimental Animals and Traditional Chinese Medicine: Male C57BL / 6 mice, weighing 20–25 g and 8 weeks old, were purchased from Jesstech. They were housed in an environment free from specific pathogens. The mice lived under a standard 12-hour light-dark cycle with free access to food and water. The NASH model was induced by feeding them a standard diet containing 10% fat and without methionine and choline supplements for 6 weeks. The African cactus *Hoodia gordonii*, commonly known as the Butterfly Cactus or South African Cactus, was provided by Professor Ma Xuesheng of the Department of Natural Sciences, University of the Western Cape.

[0022] 1.2 Establishment of a mouse model of non-alcoholic steatohepatitis (NASH): Mice were randomly assigned to a normal diet group, an MCD diet group, a low-dose Hoodia gordonii group, and a high-dose Hoodia gordonii group. After two weeks of MCD diet, mice were administered low-dose Hoodia gordonii (2.12 g / kg) or high-dose Hoodia gordonii (4.24 g / kg) by gavage for four weeks. After treatment, mice were deeply anesthetized with sodium pentobarbital (50 mg / kg). Blood was drawn directly from the inferior vena cava before liver extraction. Serum was collected and stored at -80°C. Liver tissue was rapidly removed, weighed, and then either fixed in a PBS solution of 4% paraformaldehyde and 2.5% glutaraldehyde or immediately frozen in liquid nitrogen and stored at -80°C for subsequent analysis.

[0023] 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.

[0024] 1.4 Biochemical index detection: Blood samples were collected from mice, and the supernatant was centrifuged at 3000 rpm for 10 minutes. Blood lipids and liver function-related indicators in mouse serum were measured using a Hitachi fully automated biochemical analyzer.

[0025] 1.5 RT-qPCR: Total mRNA was extracted from mouse liver tissue or cell samples using a total RNA isolation reagent (Biosharp, BS258A) according to 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.

[0026] 1.6 Cell culture and free fatty acid-induced fatty acid-induced steatosis model

[0027] Primary hepatocytes were obtained from fresh livers of C57BL / 6 mice and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. The culture conditions were 37°C and a humidified environment with 5% carbon dioxide. To induce hepatocyte steatosis in the PAOA model group, high-glucose DMEM medium containing 0.2 mM palmitic acid (PA) and 0.1 mM oleic acid (OA) was used. The control group was pretreated with an equal volume of bovine serum albumin for 24 hours. Lipid accumulation in each group was assessed by ORO or BODIPY staining.

[0028] 2. Experimental Results:

[0029] 2.1 Effects of Hoodia on liver pathology in mice with non-alcoholic steatohepatitis: such as Figure 1 As shown, Hoodia significantly reduced the liver weight ratio in mice with non-alcoholic steatohepatitis (NAH). HE staining showed that Hoodia improved liver inflammation, hepatocyte structural disorder, and steatosis in MCD mice. Oil Red staining showed that Hoodia reduced lipid accumulation in hepatocytes, and Sirius Red staining showed that Hoodia improved the degree of hepatocyte fibrosis. These phenotypic results demonstrate that Hoodia has a therapeutic effect on NHA.

[0030] 2.2 Effects of Hoodia on serum liver function and total cholesterol and triglycerides in mice with non-alcoholic steatohepatitis: such as Figure 2 As shown, both low and high doses of Hoodia significantly reduced cholesterol and triglyceride levels in the liver of mice with non-alcoholic steatohepatitis, and decreased liver damage indicators ALT and AST, with statistically significant differences.

[0031] 2.3 Effects of Hoodia on the expression levels of liver fibrosis and lipidogenesis-related mRNAs in mice with non-alcoholic steatohepatitis: such as Figure 3 As shown, both low-dose and high-dose Hoodia intervention significantly reduced the expression levels of fibrosis-related mRNAs such as α-SMA, COL1A1, TGF-β, TIMP, and FN in the liver of NAFLD mice, while also decreasing the expression levels of lipidogenesis-related mRNAs such as SCD1, SREBF1, and FASN. This indicates that Hoodia can improve liver fibrosis and affect lipidogenesis in NAFLD mice.

[0032] 2.4 Effects of Hoodia on lipid metabolism in mouse primary liver cells: such as Figure 4As shown, Oil Red staining of cells revealed that Hoodia intervention significantly reduced lipid accumulation and intracellular triglyceride levels in primary mouse liver cells induced by PAOA. Furthermore, the toxicity of Hoodia to primary liver cells was observed using the CCK8 cell viability assay. The results indicated that the Hoodia intervention dose concentration in this study had no cytotoxicity.

[0033] 2.5 Effect of Hoodia on BODIPY staining of mouse primary liver cells: such as Figure 5 As shown, compared with primary mouse liver cells not treated with PAOA, the model group cells had more positive BODIPY staining areas and stronger fluorescence, with statistically significant differences. This indicates that Hoodia can significantly reduce the fluorescence intensity and quantity of PAOA-induced BODIPY staining in primary mouse liver cells.

[0034] Hoodia gordonii improves the progression of non-alcoholic fatty liver disease by regulating bile acid metabolism homeostasis. In conclusion, Hoodia is a drug that can treat non-alcoholic steatohepatitis.

Claims

1. The use of butterfly subcereus in the preparation of a medicine, food or health product for preventing or treating non-alcoholic fatty hepatitis.

2. Use according to claim 1, characterized in that, The non-alcoholic fatty hepatitis is characterized by one or more of liver inflammation, liver cell structure disorder and fatty degeneration.

3. Use according to claim 1, characterized in that, The non-alcoholic fatty liver is simple fatty liver disease, non-alcoholic fatty hepatitis and fatty liver infiltration.

4. Use according to claim 1, characterized in that, The butterfly subcereus is added with pharmaceutically acceptable adjuvants and carriers, and is prepared into a pharmaceutical preparation, wherein the adjuvants include at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener and a colorant.

5. The use according to claim 1, characterized in that, The dosage of the butterfly subcereus is 2.12g / kg / d-4.24g / kg / d.

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