Application of 6-shogaol in preparation of medicine for preventing and treating hepatic fibrosis diseases

Abstract

The invention discloses application of 6-shogaol in preparation of a medicine for preventing and treating hepatic fibrosis diseases, and belongs to the field of natural medicines. According to the medicine, a glycolysis metabolic pathway of hepatic stellate cells is regulated and controlled by a targeted inhibition transcription factor HIF1-alpha; liver injury, collagen generation and extracellular matrix deposition are reversed; the invention discloses a medicine for improving liver index and liver function. The 6-shogaol provided by the invention can effectively relieve liver fibrosis and liver injury at a lower dosage; pathological injuries caused by CCl4-induced hepatic stellate cell activation and inflammatory cell infiltration are relieved, and the liver index and the liver function are improved; the raw material is from the natural product ginger, the active ingredients are convenient to collect, and the safety is high.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicines and liver fibrosis treatment, and particularly relates to the application of 6-shogaol in preparing medicines for preventing and treating liver fibrosis diseases. Background Art

[0002] Liver fibrosis is a core component of the progression of chronic liver disease and a major determinant of cirrhosis, liver cancer, and even liver failure. Liver fibrosis can be divided into two main types based on the mechanism of injury: hepatotoxic injury and cholestatic injury. Studies in patients with liver fibrosis and cirrhosis of varying etiologies, as well as in experimental rodent models of liver fibrosis, have revealed the core mechanisms of liver fibrosis progression. Specifically, initial hepatocyte injury triggers disruption of the epithelial / endothelial barrier, which in turn triggers an inflammatory cytokine cascade, ultimately driving the differentiation of hepatic stellate cells into myofibroblasts that secrete collagen I. This leads to abnormal deposition of this protein-based extracellular matrix and the formation of fibrotic scars. Although a variety of anti-fibrotic drugs have entered the basic research field, no clinically approved therapeutic agents exist due to complex etiologies, insufficient drug targeting, and significant side effects. Notably, natural products, with their multi-component synergistic effects and excellent biosafety, offer unique advantages in modulating complex pathological mechanisms and have become a key research direction for overcoming current treatment challenges.

[0003] Ginger (Zingiber officinale Roscoe) is a pungent, aromatic spice that has been used in food and medicinal recipes worldwide for thousands of years. Numerous studies have shown that ginger contains a rich array of chemical compounds, including volatile oils, gingerol analogs, diarylheptanes, phenylalanoids, sulfonates, steroids, and monoterpenoid glycosides. These compounds play important roles in protecting liver and kidney function, the gastrointestinal system, and preventing diabetes. 6-Shogaol, in particular, exhibits multifaceted bioactivity on the liver. On the one hand, 6-Shogaol can reduce hepatic lipid accumulation by inhibiting the expression of lipid biosynthesis genes; on the other hand, it can optimize glucose metabolism by promoting hepatic glycogen synthesis. This demonstrates the therapeutic advantage of 6-Shogaol as a natural hepatoprotective agent, embodying its multi-pathway synergistic effects.

[0004] However, there are no patent literature reports on the activity and mechanism of 6-gingerol in protecting liver damage caused by CCl4 and bile duct ligation (BDL) by targeting HIF1-α to regulate glycometabolism reprogramming.

[0005] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the Invention

[0006] The purpose of the present invention is to solve the problem of how to find a safe and effective drug for preventing or treating liver fibrosis and liver damage from natural products, and provides the use of 6-shogaol in the preparation of drugs for preventing and treating liver fibrosis.

[0007] In order to achieve the above object, the present invention discloses the use of 6-shogaol in preparing a medicine for preventing and treating liver fibrosis.

[0008] The pathological process of liver fibrosis involves chronic liver damage caused by various etiologies and excessive accumulation of the extracellular matrix (ECM). In particular, the continuous progression of liver fibrosis has been recognized as the core pathological process of cirrhosis and end-stage liver failure. Although several candidate drugs have been included in clinical trials, there is currently no specific drug available for the clinical treatment of liver fibrosis. In this context, highly effective and low-toxic active ingredients of traditional Chinese medicine are becoming an emerging strategic direction for the development of anti-liver fibrosis drugs due to their multi-target regulation advantages. Notably, 6-shogaol, an active ingredient extracted from ginger, has shown unique potential in intervening in liver fibrosis due to its multiple pharmacological activities, including antioxidant, anti-inflammatory and anti-cancer properties, but the more specific molecular mechanisms still need to be further studied.

[0009] The present invention has found through a large number of studies that increased expression of glycolysis-related proteins in hepatic stellate cells will aggravate the development of liver fibrosis.

[0010] Based on extensive previous research, the present invention discovered that 6-singerol can be used as a single agent and has a very significant therapeutic effect on the treatment of liver fibrosis. The inventors' research has shown that 6-singerol can reduce the levels of liver injury markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL), and reduce the expression of liver fibrosis markers such as type I collagen (Collagen I), α-smooth muscle actin (α-SMA), and vimentin. Therefore, 6-singerol is used to prepare a drug for the treatment of liver fibrosis.

[0011] It is understood that the actual dosage of 6-shogaol administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, organism type or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0012] Furthermore, the drug uses 6-shogaol as the only active ingredient.

[0013] The drug for treating liver fibrosis of the present invention uses 6-gingerol as an active ingredient, and can inhibit liver fibrosis by targeting HIF1-α to inhibit the reprogramming of glycolytic metabolism in hepatic stellate cells.

[0014] Furthermore, the 6-gingerol is used to prepare drugs for inhibiting the expression of liver fibrosis marker molecules type I collagen (Collagen I), α-smooth muscle actin (α-SMA) and vimentin (Vimentin), thereby inhibiting liver fibrosis.

[0015] Furthermore, the 6-singerol is used to prepare a drug for reducing the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) and improving liver tissue pathological damage.

[0016] Furthermore, the 6-gingerol is used to prepare a drug for reducing collagen deposition in liver tissue and improving liver fibrosis damage.

[0017] Furthermore, the 6-gingerol is used to prepare a drug that reduces the levels of glycolytic metabolites lactate and pyruvate, inhibits the reprogramming of glycolytic metabolism in hepatic stellate cells, and inhibits liver fibrosis.

[0018] Furthermore, the 6-gingerol is used to inhibit the expression of hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), and glucose transporter 1 (GLUT1), and to prepare a drug that inhibits the reprogramming of glycolytic metabolism in hepatic stellate cells and inhibits liver fibrosis.

[0019] Furthermore, the 6-gingerol is used to prepare drugs that target and inhibit HIF1-α protein expression, inhibit hepatic stellate cell glycometabolism reprogramming, and inhibit liver fibrosis.

[0020] Furthermore, the 6-gingerol is used to prepare a lead drug for inhibiting CCl4-induced liver fibrosis mouse model.

[0021] Furthermore, the 6-shogaol is used to prepare a lead drug for inhibiting a bile duct ligation (BDL)-induced liver fibrosis mouse model.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The present invention develops a new use for 6-shogaol, using it in the preparation of a drug for treating liver fibrosis. Studies have shown that 6-shogaol, as an active ingredient in drugs for treating liver fibrosis, can target and inhibit the expression of HIF1-α protein, inhibit the reprogramming of glycolytic metabolism in hepatic stellate cells, reduce the degree of structural disorder in liver tissue of mice with liver fibrosis, reduce the levels of ALT, AST, and TBIL, reduce the expression of liver fibrosis markers Collagen I, α-SMA, and Vimentin, reduce the expression of glycolysis-related proteins HK2, PKM2, and GLUT1, reduce the levels of glycolysis metabolites lactate and pyruvate, inhibit hepatic stellate cell activation, and reduce collagen deposition in liver tissue. This invention confirms that 6-shogaol can be used to treat liver fibrosis, providing a new theoretical basis and experimental foundation for the clinical development of new drugs to prevent or reverse the progression of liver fibrosis, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The liver tissue sections obtained from the blank control group and CCl4 model group were stained with hematoxylin-eosin (HE).

[0025] Figure 2 The liver tissue sections obtained from the blank control group and BDL model group were stained with hematoxylin-eosin (HE).

[0026] Figure 3 The liver tissue sections obtained from the blank control group and CCl4 model group were stained with α-SMA and Collagen I respectively;

[0027] Figure 4 The liver tissue sections obtained from the blank control group and BDL model group were stained with α-SMA and Collagen I respectively;

[0028] Figure 5 The liver tissue sections of the blank control group and CCl4 model group were stained with Sirius Red.

[0029] Figure 6 The liver tissue sections obtained from the blank control group and BDL model group were stained with Sirius Red.

[0030] Figure 7 The liver tissue sections obtained from the blank control group and CCl4 model group were stained for HK2, PKM2, and GLUT1 respectively;

[0031] Figure 8 The liver tissue sections obtained from the blank control group and BDL model group were stained for HK2, PKM2, and GLUT1 respectively;

[0032] Figure 9 HIF1-α staining was performed on the liver tissue sections obtained from the blank control group and the model group;

[0033] Figure 10 The liver tissue sections of the blank control group, CCl4 model group, and 6-gingerol treatment group were stained with hematoxylin-eosin (HE).

[0034] Figure 11 The liver tissue sections of the blank control group, BDL model group, and 6-gingerol treatment group were stained with hematoxylin-eosin (HE).

[0035] Figure 12 The liver tissue sections obtained from the blank control group, CCl4 model group, and 6-gingerol treatment group were stained with α-SMA and Collagen I respectively;

[0036] Figure 13 The liver tissue sections obtained from the blank control group, BDL model group, and 6-gingerol treatment group were stained for α-SMA and type I collagen.

[0037] Figure 14 The liver tissue sections of the blank control group, CCl4 model group, and 6-gingerol treatment group were stained with Sirius Red.

[0038] Figure 15 The liver tissue sections of the blank control group, BDL model group, and 6-gingerol treatment group were stained with Sirius Red.

[0039] Figure 16 RT-qPCR was performed on the liver tissue sections obtained from the blank control group, CCl4 model group, and 6-gingerol treatment group;

[0040] Figure 17 RT-qPCR was performed on the liver tissue sections obtained from the blank control group, BDL model group, and 6-gingerol treatment group;

[0041] Figure 18 The liver tissue sections obtained from the blank control group, CCl4 model group, and 6-gingerol treatment group were stained for HK2, PKM2, and GLUT1 respectively;

[0042] Figure 19 Liver tissue sections from the blank control group, BDL model group, and 6-gingerol treatment group were stained for HK2, PKM2, and GLUT1;

[0043] Figure 20 Western blot was performed on the liver tissue sections of the blank control group, CCl4 model group, and 6-gingerol treatment group.

[0044] Figure 21 Western blot was performed on the liver tissue sections of the blank control group, BDL model group, and 6-gingerol treatment group.

[0045] Figure 22 The liver tissue sections obtained from the blank control group, CCl4 model group, and 6-gingerol treatment group were stained for α-SMA, Collagen I, HK2, PKM2, and GLUT1 after HIF1-α overexpression and knockout respectively;

[0046] Figure 23 The liver tissue sections obtained from the blank control group, CCl4 model group, and 6-gingerol treatment group were stained for α-SMA, Collagen I, HK2, PKM2, and GLUT1 after HIF1-α overexpression and knockout. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with embodiments of the present invention. Obviously, the described embodiments are part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific techniques or conditions are not indicated in the embodiments, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not indicate manufacturers are conventional products that can be obtained commercially.

[0048] As used herein, the term "treating" refers in some embodiments to ameliorating a disease or condition (i.e., slowing, arresting, or alleviating the development of a disease or condition or at least one of its clinical symptoms). In other embodiments, "treating" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treating" refers to regulating a disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both. In other embodiments, "treating" refers to preventing or delaying the onset, occurrence, or worsening of a disease or condition.

[0049] The application of 6-shogaol provided by the present invention in preparing a drug for treating liver fibrosis is described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0050] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.

[0051] Example 1

[0052] Construction of CCl4 and BDL-induced liver fibrosis mouse model:

[0053] 1. Experimental Materials

[0054] Male C57BL / 6J mice were purchased from the Experimental Animal Research Center of Anhui Medical University.

[0055] 2. Construction of CCl4-induced liver fibrosis mouse model

[0056] Ten 6-week-old male C57BL / 6J mice were randomly divided into two groups: a blank control group (n=5) and a model group (injected with carbon tetrachloride (CCl4) alone, n=5). The model group was induced to develop liver fibrosis by intraperitoneal injection of CCl4 for 4 weeks. CCl4 was diluted 1:5 with olive oil (Product No. O108686, Aladdin) and injected at 1 μl / g twice weekly. The blank control group received no treatment.

[0057] All animal experimental procedures were approved by the Animal Ethics Committee of Anhui Qing Medical University (No. 2023-019).

[0058] 3. Construction of a BDL-induced liver fibrosis mouse model

[0059] Ten male C57BL / 6J mice, 6 weeks old, were randomly divided into two groups: a blank control group (n=5) and a model group (n=5). The model group underwent bile duct ligation to induce liver fibrosis for 3 weeks. The blank control group received no treatment.

[0060] IV. Obtaining Mouse Liver Tissue and Preparing Liver Tissue Sections

[0061] After modeling, mice were killed by cervical dislocation, and liver tissues were removed and prepared into liver tissue slices. The specific steps are as follows:

[0062] 1. Fixation: Fix the liver tissue with 4% paraformaldehyde fixative for 24 hours to obtain fixed tissue;

[0063] 2. Dehydration: Dehydrate the fixed tissue using graded alcohol concentrations, sequentially immersing it in 70%, 80%, 90%, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II. Soak in 70% ethanol for 30 minutes, and in anhydrous ethanol I and anhydrous ethanol II for 1 hour.

[0064] 3. Transparency: Soak the dehydrated tissue in xylene I for 1 hour and xylene II for 1 hour;

[0065] 4. Wax immersion: After the tissue is transparent, place it in low-melting-point paraffin for 1 hour and high-melting-point paraffin for 1 hour;

[0066] 5. Embedding: Melt the high melting point wax in an automatic embedding machine in advance and keep it in a molten state. Adjust the wax-soaked tissue to the state required for sectioning and place it in the embedding machine. Pour the high melting point wax and place it on ice to cool and solidify.

[0067] 6. Sectioning: Set the water temperature of the slide spreading pool to 42°C, the temperature of the drying area to 65°C, and pre-cool the wax block in the refrigerator. After trimming the embedded tissue to the required cross-section, adjust the thickness to 3 μm and start slicing to obtain liver tissue sections. Place the obtained liver tissue sections into the slide spreading pool to remove wrinkles. After spreading, use a slide pre-coated with APES to pick up the liver tissue sections. Place the liver tissue sections in the drying area and bake for 2 hours before storing at 4°C.

[0068] The above method was used to obtain liver tissue and liver tissue sections of mice in the blank control group and model group, respectively.

[0069] 5. Hematoxylin-eosin (HE) staining

[0070] The liver tissue sections of the blank control group and the model group were stained with hematoxylin-eosin (HE). Figure 1 and Figure 2 shown.

[0071] Depend on Figure 1 and Figure 2 It can be seen that compared with the blank control group, the liver tissue sections of the model group had disordered structure, hepatocyte edema and degeneration, and the degree of inflammatory cell infiltration was significantly increased.

[0072] 6. α-SMA and Collagen I Staining

[0073] The liver tissue sections of the blank control group and the model group were stained with α-SMA and Collagen I respectively to detect the activation of hepatic stellate cells in the liver tissue of mice. Figure 3 and Figure 4 shown.

[0074] Depend on Figure 3 and Figure 4 It can be seen that compared with the blank control group, the expressions of hepatic stellate cell activation markers α-SMA and Collagen I in the model group were significantly increased.

[0075] 7. Sirius Red staining

[0076] The liver tissue sections of the blank control group and the model group were stained with Sirius Red to detect the collagen deposition in the liver tissue of mice. The results are as follows: Figure 5 and Figure 6 shown.

[0077] Depend on Figure 5 and Figure 6 It can be seen that compared with the blank control group, the collagen deposition in the liver tissue of mice in the model group was significantly increased.

[0078] 8. Glycolysis Metabolism Analysis in Mouse Liver Tissue

[0079] HK2, PKM2, and GLUT1 staining were performed on the liver tissue sections of the blank control group and the model group, respectively. The results are as follows: Figure 7 and Figure 8 shown.

[0080] Depend on Figure 7 and Figure 8 It can be seen that compared with the blank control group, the expression of HK2, PKM2, and GLUT1 in the liver tissue of mice in the model group increased, indicating that the level of glycolysis increased in the process of liver fibrosis.

[0081] IX. Analysis of HIF1-α Expression in Mouse Liver Tissue

[0082] Immunohistochemistry (IHC) was performed on the liver tissue sections of the blank control group and the model group, and the results were as follows: Figure 9 shown.

[0083] Depend on Figure 9 It can be seen that compared with the blank control group, the expression of HIF1-α in the liver tissue of mice in the model group was increased.

[0084] The above results show that CCl4-induced and BDL-induced liver fibrosis mouse models were successfully constructed, and the glycolysis metabolism level and HIF1-α protein expression level increased in the liver tissue of liver fibrosis mice.

[0085] Example 2

[0086] Effects of 6-shogaol on mice with hepatic fibrosis

[0087] 1. Experimental Materials

[0088] 6-Shogaol (CAS No.: 1801724-76-4, purchased from MedChemExpress).

[0089] Male C57BL / 6 mice were purchased from the Experimental Animal Research Center of Anhui Medical University.

[0090] 2. Construction of CCl4 Liver Fibrosis Mouse Model

[0091] Thirty six-week-old male C57BL / 6J mice were randomly divided into six groups: a blank control group (n=5), a model group (injected with carbon tetrachloride (CCl4) alone, n=5), a low-dose 6-shogaol group (10 mg / kg, n=5), a medium-dose 6-shogaol group (20 mg / kg, n=5), and a high-dose 6-shogaol group (40 mg / kg, n=5). The mice were administered with low (10 mg / kg body weight), medium (20 mg / kg body weight), and high (40 mg / kg body weight) doses of 6-shogaol for 7 days. The control group, CCl4-treated group, and colchicine-treated group mice were gavaged with the corresponding solvent. The CCl4-treated group, colchicine-treated group, and 6-shogaol-treated group mice were intraperitoneally injected with 10 mg / kg CCl4 solution twice a week. The mice were dissected after 4 weeks.

[0092] All animal experimental procedures were approved by the Animal Ethics Committee of Anhui Medical University (No. 2023-019).

[0093] 3. Construction of BDL liver fibrosis mouse model

[0094] Thirty six-week-old male C57BL / 6J mice were randomly divided into six groups: a blank control group (n=5), a model group (bile duct ligation (BDL) alone, n=5), a low-dose 6-shogaol group (10 mg / kg, n=5), a medium-dose 6-shogaol group (20 mg / kg, n=5), and a high-dose 6-shogaol group (40 mg / kg, n=5). Mice were administered low (10 mg / kg body weight), medium (20 mg / kg body weight), and high (40 mg / kg body weight) doses of 6-shogaol for 7 days. Mice in the control, BDL, and colchicine groups were gavaged with the corresponding solvents and dissected three weeks later.

[0095] All animal experimental procedures were approved by the Animal Ethics Committee of Anhui Medical University (No. 2023-019).

[0096] IV. Construction of HIF1-α Knockout Mouse Model

[0097] Fifteen 6-week-old male HIF1-α conditional knockout mice (C57BL / 6J background, genotype: HIF1-α ^flox / flox^; Alb-Cre ^+^, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into three groups: a blank control group (n=5), a model group (n=5), and a 6-shogaol-treated group (n=5). The blank control group, which did not undergo HIF1-α gene knockout, was injected with saline alone; the model group, which was HIF1-α knockout mice, was treated with carbon tetrachloride (CCl4) intraperitoneally to induce liver fibrosis; and the 6-shogaol-treated group, which was HIF1-α knockout mice, was given 6-shogaol (40 mg / kg body weight) daily orally, in addition to CCl4-induced liver fibrosis. Dissections were performed 4 weeks later.

[0098] All animal experimental procedures were approved by the Animal Ethics Committee of Anhui Medical University (No. 2023-019).

[0099] 5. Obtaining Mouse Liver Tissue and Preparing Liver Tissue Sections

[0100] After the modeling was completed, the mice were killed by cervical dislocation, and the liver tissue was removed and prepared into liver tissue slices. The specific steps were as follows:

[0101] 1. Fixation: Fix the liver tissue with 4% paraformaldehyde fixative for 24 hours to obtain fixed tissue;

[0102] 2. Dehydration: Dehydrate the fixed tissue using graded alcohol concentrations, sequentially immersing it in 70%, 80%, 90%, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II. Soak in 70% ethanol for 30 minutes, and in anhydrous ethanol I and anhydrous ethanol II for 1 hour.

[0103] 3. Transparency: Soak the dehydrated tissue in xylene I for 1 hour and xylene II for 1 hour;

[0104] 4. Wax immersion: After the tissue is transparent, place it in low-melting-point paraffin for 1 hour and high-melting-point paraffin for 1 hour;

[0105] 5. Embedding: Melt the high melting point wax in an automatic embedding machine in advance and keep it in a molten state. Adjust the wax-soaked tissue to the state required for sectioning and place it in the embedding machine. Pour the high melting point wax and place it on ice to cool and solidify.

[0106] 6. Sectioning: Set the water temperature of the slide spreading pool to 42°C, the temperature of the drying area to 65°C, and pre-cool the wax block in the refrigerator. After trimming the embedded tissue to the required cross-section, adjust the thickness to 3 μm and start slicing to obtain liver tissue sections. Place the obtained liver tissue sections into the slide spreading pool to remove wrinkles. After spreading, use a slide pre-coated with APES to pick up the liver tissue sections. Place the liver tissue sections in the drying area and bake for 2 hours before storing at 4°C.

[0107] The above method was used to obtain liver tissues and liver tissue sections of mice in the blank control group, model group, and 6-gingerol treatment group.

[0108] 6. Hematoxylin-eosin (HE) staining

[0109] The liver tissue sections of the blank control group, model group, and 6-gingerol treatment group were stained with hematoxylin-eosin (HE). Figure 10 and Figure 11 shown.

[0110] Depend on Figure 10 and Figure 11 As shown, compared with the blank control group, hepatocyte degeneration was observed in the model group and the 6-shogaol treatment group. The model group showed liver tissue disorder and inflammatory cell infiltration, while the degree of liver tissue disorder and inflammatory cell infiltration in the 6-shogaol treatment group was significantly less than that in the model group, indicating that 6-shogaol can improve liver fibrosis and inflammation.

[0111] 7. α-SMA and Collagen I Staining

[0112] The liver tissue sections of the blank control group, model group, and 6-gingerol treatment group were stained with α-SMA and Collagen I to detect the activation of hepatic stellate cells in the mouse liver tissue. The results are as follows: Figure 12 and Figure 13 shown.

[0113] Depend on Figure 12 and Figure 13 It can be seen that compared with the blank control group, the expression of α-SMA, a marker molecule of hepatic stellate cell activation, was significantly increased in the model group, and the expression of α-SMA, a marker molecule of hepatic stellate cell activation, in the liver tissue of mice in the 6-gingerol treatment group was significantly decreased, indicating that 6-gingerol significantly inhibited the activation of hepatic stellate cells.

[0114] 8. Sirius Red staining

[0115] The liver tissue sections of the blank control group, model group, and 6-gingerol treatment group obtained above were stained with Sirius Red to detect the collagen deposition in the mouse liver tissue. The results are as follows: Figure 14 and Figure 15 shown.

[0116] Depend on Figure 14 and Figure 15 It can be seen that compared with the blank control group, the collagen deposition in the liver tissue of the model group mice increased significantly, while the collagen deposition in the liver tissue of the 6-shogaol treatment group mice decreased significantly, indicating that 6-shogaol significantly inhibited the formation of collagen.

[0117] 9. Real-time quantitative PCR (qRT-PCR) detection

[0118] qRT-PCR was performed on the liver tissues of the blank control group, model group, and 6-gingerol treatment group to detect the expression of liver fibrosis markers Collagen I, α-SMA, and Vimentin in the liver tissues of each group of mice. The specific steps were to extract RNA from the mouse liver tissue according to the manufacturer's instructions for the qRT-PCR kit (9108, Takara, Japan), and analyze the qRT-PCR results. The results are shown in Figure 2. Figure 16 and Figure 17 shown.

[0119] Depend on Figure 16 and Figure 17 It can be seen that compared with the model group, the mRNA expression of liver fibrosis markers Collagen I, α-SMA and Vimentin in the liver tissue of mice in the 6-shogaol treatment group was significantly reduced, indicating that 6-shogaol significantly inhibited liver fibrosis.

[0120] 10. Glycolysis Metabolism Analysis in Mouse Liver Tissue

[0121] HK2, PKM2, and GLUT1 staining were performed on the liver tissue sections of the blank control group, model group, and 6-gingerol treatment group, respectively. The results are as follows: Figure 18 and Figure 19 shown.

[0122] Depend on Figure 18 and Figure 19 It can be seen that compared with the model group, the expression of HK2, PKM2, and GLUT1 in the liver tissue of mice in the 6-shogaol treatment group was significantly reduced, indicating that 6-shogaol significantly inhibited the increased level of glycolysis metabolism in the process of liver fibrosis.

[0123] 11. Protein immunoblotting (WB) detection

[0124] Western blot analysis was performed on the liver tissue sections of the blank control group, model group, and 6-gingerol treatment group to detect the expression of liver fibrosis markers CollagenI, α-SMA, and Vimentin and glycolysis-related proteins HK2, PKM2, and GLUT1 in the liver tissue of mice. The results are as follows: Figure 20 and Figure 21 shown.

[0125] Depend on Figure 20 and Figure 21 It can be seen that compared with the model group, the expression levels of liver fibrosis marker molecules and glycolysis-related proteins in the liver tissue of mice in the 6-shogaol treatment group were significantly reduced, indicating that 6-shogaol significantly inhibited the progression of liver fibrosis and the increase in glycolysis metabolism levels.

[0126] XI. Analysis of HIF1-α Expression in Mouse Liver Tissue

[0127] The liver tissue sections of the blank control group, CCl4 model group, and 6-gingerol treatment group of HIF1-α overexpression and knockout mice were stained for α-SMA, CollagenI, HK2, PKM2, and GLUT1, respectively. The results are as follows: Figure 22 and Figure 23 shown.

[0128] Depend on Figure 22 and Figure 23 It can be seen that 6-singerol can improve liver damage and liver fibrosis. However, after HIF1-α conditional gene knockout mice were treated with 6-singerol, pathological damage and liver fibrosis were not improved, indicating that 6-singerol lost its anti-fibrotic effect when treating HIF1-α conditional gene knockout mice.

[0129] All these results confirmed that the anti-fibrotic effect of 6-shogaol was associated with its inhibitory effect on HIF1-α, which regulates glycolytic metabolism during liver fibrosis.

[0130] The above description is merely a preferred embodiment of the present invention and is intended to be illustrative rather than restrictive of the present invention. Those skilled in the art will appreciate that many changes, modifications, and even equivalents may be made to the present invention within the spirit and scope of the claims, all of which fall within the scope of protection of the present invention.

Claims

Application of 1.6-gingerol in the preparation of drugs for preventing and treating liver fibrosis.

2. The use of 6-shogaol according to claim 1 in preparing a drug for preventing and treating liver fibrosis, wherein: The medicine uses 6-shogaol as the only active ingredient.

3. The use of 6-shogaol according to claim 1 in preparing a drug for preventing and treating liver fibrosis, wherein: The 6-gingerol is used to prepare a drug for inhibiting the expression of liver fibrosis marker molecules type I collagen, α-smooth muscle actin and vimentin, thereby inhibiting liver fibrosis.

4. The use of 6-shogaol according to claim 1 in preparing a drug for preventing and treating liver fibrosis, wherein: The 6-gingerol is used to prepare a drug for reducing the levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin, improving liver tissue pathological damage and inhibiting liver fibrosis.

5. The use of 6-shogaol according to claim 1 in preparing a drug for preventing and treating liver fibrosis, wherein: The 6-gingerol is used to prepare medicine for reducing liver tissue collagen deposition and improving liver fibrosis damage.

6. The use of 6-shogaol according to claim 1 in preparing a drug for preventing and treating liver fibrosis, wherein: The 6-gingerol is used to prepare a drug that reduces the levels of glycolytic metabolites lactic acid and pyruvic acid, inhibits glycolytic metabolic reprogramming of hepatic stellate cells, and improves liver fibrosis.

7. The use of 6-shogaol according to claim 1 in preparing a drug for preventing and treating liver fibrosis, wherein: The 6-gingerol is used to prepare a drug that inhibits the expression of hexokinase 2, pyruvate kinase M2 and glucose transporter 1, inhibits the glycolysis metabolic reprogramming of hepatic stellate cells, and improves liver fibrosis.

8. Use of 6-shogaol according to claim 1 in the preparation of a drug for preventing and treating liver fibrosis, wherein the 6-shogaol is used to prepare a drug for targeted inhibition of HIF1-α protein expression, inhibition of hepatic stellate cell glycometabolism reprogramming, and inhibition of liver fibrosis.

9. Use of 6-shogaol according to claim 1 in preparing a drug for preventing and treating liver fibrosis, wherein the 6-shogaol is used to prepare a lead drug for inhibiting CCl4-induced liver fibrosis in a mouse model.

10. Use of 6-shogaol according to claim 1 in preparing a drug for preventing and treating liver fibrosis, wherein the 6-shogaol is used to prepare a lead drug for inhibiting liver fibrosis in a mouse model induced by bile duct ligation.

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