Application of Physagulin C in the preparation of drugs for treating liver fibrosis

Abstract

The present invention belongs to the field of pharmaceutical technology, specifically to the use of physagulin C in the preparation of a drug for treating liver fibrosis. Using human hepatic stellate cells (LX‑2) as a model cell line, the present invention experimentally demonstrates that physagulin C can significantly reduce the expression of liver fibrosis markers, such as collagen, in LX‑2 hepatic stellate cells, while effectively inhibiting their proliferation and inducing apoptosis. This indicates that physagulin C has the potential to inhibit or reverse liver fibrosis in a dose-dependent manner. This indicates that physagulin C has promising application prospects in the preparation of drugs for treating liver fibrosis, and is of great significance for overcoming the challenges in treating liver fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the application of physagulin C in preparing a medicine for treating liver fibrosis. Background Art

[0002] Liver fibrosis is a compensatory response to liver damage caused by the sustained effects of multiple factors on hepatocytes, primarily leading to liver inflammation or injury. It primarily refers to an imbalance in the synthesis and degradation of the extracellular matrix (ECM) in liver tissue. Studies have shown that liver fibrosis is a common pathological process in various chronic liver diseases. If liver fibrosis is not promptly prevented or reversed, it can lead to cirrhosis and various complications, severely impacting the patient's health. Therefore, the search for effective drugs to prevent or reverse liver fibrosis is an urgent issue. Hepatic stellate cells (HSCs) are non-parenchymal cells in the liver and are the primary source of liver ECM. Activation of HSCs is a key step in the development and progression of liver fibrosis. When HSCs transition from a quiescent state to an activated state, their gene expression and phenotype change, transforming them into myofibroblasts with the ability to proliferate, fibroblastize, and contract. Activated HSCs synthesize and secrete large amounts of ECM components, such as type I collagen, type III collagen, and fibronectin, which accumulate in damaged liver areas, leading to the development of liver fibrosis. Therefore, finding drugs that can inhibit the activation and proliferation of HSCs and promote the apoptosis of HSCs has become an important approach and research direction to slow down and reverse liver fibrosis.

[0003] Physagulin C is a typical ergostane compound from the genus Physalis. Its side chain has a δ-lactone unit at carbon positions 22 and 26. Physagulin C was originally isolated from Physalis sangulata L. (Shingu K, et al. Chemical Pharmaceutical Bulletin, 1991, 39(6):1591-1593). It exhibits anti-tumor, HIV-inhibiting, and anti-inflammatory activities.

[0004] Patent publication number CN111000852 A discloses the use of a withanolide extract from physalin B in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease. The extract contains a mixture of any two or more of physalin B, physalin D, physalin F, physalin I, and physalin H, and does not involve the physagulin C mentioned in this application. Furthermore, physalins and physagulins are structurally distinct compounds. Currently, no relevant research literature has been found reporting the use of physagulin C in the preparation of drugs for treating liver fibrosis. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides the use of physagulin C in preparing a drug for treating liver fibrosis.

[0006] This is achieved specifically through the following technical solutions:

[0007] Application of Physagulin C in the preparation of drugs for treating liver fibrosis.

[0008] Furthermore, the chemical name of physagulin C is (20R, 22R)-15α-acetoxy-5β(6), 16β(17)-diepoxy-4β, 14β-dihydroxy-1-oxo-witha-2,24-dienolide; the molecular formula is C 30 H 38 O9; molecular weight is 542.63; specific structural formula is as follows:

[0009]

[0010] The physagulin C described in the present application can be directly isolated and extracted from natural products or prepared by chemical synthesis. As long as their structures are the same, they all have corresponding effects.

[0011] Furthermore, the drug for treating liver fibrosis is a drug that inhibits the proliferation or activation of hepatic stellate cells.

[0012] Furthermore, the drug for treating liver fibrosis is a drug that inhibits the expression of hepatic stellate cell fibrosis marker proteins. Specifically, the proteins inhibiting hepatic stellate cell fibrosis marker proteins are α-SMA and Collagen I proteins. This can achieve an anti-liver fibrosis effect by inhibiting the expression of hepatic stellate cell fibrosis marker proteins.

[0013] Furthermore, the drug for treating liver fibrosis is a drug that promotes apoptosis of hepatic stellate cells.

[0014] Furthermore, the drug for treating liver fibrosis includes an oral preparation or an injectable preparation, which is usually prepared with physagulin C as the active ingredient and other pharmaceutical excipients or carriers. The oral preparation can be any one or more of granules, capsules, tablets, powders, pills, and sustained-release preparations.

[0015] Furthermore, in the drug for treating liver fibrosis, the concentration of physagulin C is 0.5 to 2 μM.

[0016] In summary, the beneficial effects of the present invention are as follows: the present invention provides for the first time the use of physagulin C in the preparation of drugs for the treatment of liver fibrosis. By using human hepatic stellate cells LX-2 as model cells, experiments have shown that physagulin C can significantly reduce the expression levels of liver fibrosis markers such as collagen in hepatic stellate cells LX-2, while effectively inhibiting the proliferation ability of hepatic stellate cells LX-2 and inducing their apoptosis. This shows that physagulin C has the effect of inhibiting or reversing liver fibrosis, and its effect is dose-dependent, and has good application prospects in the preparation of drugs for the treatment of liver fibrosis. The new application of physagulin C provided by the present invention in the preparation of drugs for the treatment of liver fibrosis is of great significance for overcoming the difficulties in the treatment of liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the effects of different concentrations of physagulin C on the proliferation activity of LX-2 cells in this application.

[0018] Figure 2 This is the protein expression bar diagram of α-SMA / collagenⅠ with different concentrations of physagulin C in this application.

[0019] Figure 3 This is a statistical graph showing the effects of different concentrations of physagulin C on the protein expression level of α-SMA in this application.

[0020] Figure 4 This is a statistical graph showing the effects of different concentrations of physagulin C on the protein expression level of collagen I in this application.

[0021] Figure 5 The inverted fluorescence microscope images of LX-2 cells treated with different concentrations of physagulin C in this application were used to observe the apoptosis of the cells by AO / EB dual fluorescence staining; wherein, AO-stained green fluorescence indicates living cells, and EB-stained cells indicate apoptotic cells or dead cells. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are further described in detail below, but the present invention is not limited to these embodiments. Any improvement or replacement based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0023] Example 1

[0024] MTT assay to detect the effect of physagulin C on the proliferation activity of LX-2 cells

[0025] LX-2 cells were cultured in a high-glucose medium (DMEM) containing 10% fetal bovine serum, 100 kU / L penicillin, and 100 mg / L streptomycin at 37°C in a 5% CO2 incubator. LX-2 cells in the logarithmic growth phase were obtained, digested with 0.25% trypsin, and then adjusted to a cell density of 5 × 10 cells / ml using culture medium (10% FBS + 1% double-antibody). 4 Cells were plated in 96-well plates and divided into control, TGF-β1 (10 ng / mL), physagulin C (0.5 μM), physagulin C (1 μM), and physagulin C (2 μM) groups. Six replicate wells were plated per group, with 100 μL of cell suspension per well. A blank well was also set up for zeroing. 200 μL of sterile PBS was added around the wells and the cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 24 hours. Subsequently, 20 μL of MTT was added to each well and the cells were incubated under the same conditions for another 3 hours. Subsequently, the culture medium was aspirated, 100 μL of DMSO was added, and the cells were shaken for 10 minutes. The absorbance (A) of each well at 490 nm was measured using a microplate reader to calculate the cell proliferation rate. The cell proliferation rate was calculated as follows: (A experimental group - A zeroing group) / (A control group - A zeroing group) × 100%.

[0026] from Figure 1 It can be concluded that physagulin C can significantly reduce the proliferation of LX-2 cells in a concentration-dependent manner.

[0027] Example 2

[0028] Effects of Physagulin C on Liver Fibrosis Markers in Activated LX-2 Cells

[0029] After treating LX-2 cells with physagulin C, 50 μL of lysis buffer was added to each well of a 6-well plate to fully lyse the cells. The cells were centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA assay kit. After adjusting the protein concentration, the cells were mixed with 4× sample buffer and heated at 100°C for 10 minutes to completely denature the proteins. The extracted protein samples were stored at -80°C until further use. The target bands were transferred to a PVDF membrane using a 10% or 8% (by weight) SDS-PAGE gel electrophoresis. The membranes were blocked in 5% (by weight) skim milk for 2 hours at room temperature. The membranes were incubated with the primary antibody overnight at 4°C. After incubation, the membranes were washed three times with TBST for 10 minutes each. The secondary antibody was incubated at room temperature for 2 hours, followed by three washes with TBST for 10 minutes each. The membranes were immersed in developer solution, and the target protein bands were visualized using a chemiluminescence imaging system.

[0030] from Figures 2 to 4 It can be seen that compared with the control group, the expression levels of α-SMA and Collagen I proteins in the TGF-β1 group were increased, and compared with the TGF-β1 group, the expression levels of α-SMA and Collagen I proteins in the physagulin C group were decreased.

[0031] Example 3

[0032] Effect of Physagulin C on apoptosis of LX-2 cells

[0033] Take LX-2 cells in the logarithmic growth phase and adjust the cell density to 1×10 per ml. 5 1 mL / well of physagulin C was added to a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. The next day, LX-2 cells were treated with different concentrations of physagulin C. After 24 hours of treatment, the culture medium was removed and the cells were washed three times with PBS. 10 μL of AO / EB staining solution was added to each well. After incubation at 37°C in the dark for 1 hour, the cells were washed again three times with PBS. Fluorescence intensity changes were observed under an inverted fluorescence microscope and photographed.

[0034] from Figure 5 It can be seen that with the increase of physagulin C dose, the number of apoptotic cells increased.

[0035] It can be seen from this that physagulin C can significantly inhibit the proliferation of hepatic stellate cells LX-2, reduce the protein expression of major markers of liver fibrosis in hepatic stellate cells LX-2, induce apoptosis of hepatic stellate cells LX-2, and has the effect of inhibiting or reversing liver fibrosis. It has good application prospects in the preparation of drugs for the treatment of liver fibrosis.

[0036] In summary, the present invention provides for the first time the use of physagulin C in the preparation of a drug for treating liver fibrosis. The novel use of physagulin C in the preparation of a drug for treating liver fibrosis provided by the present invention is of great significance for breaking through the bottleneck of liver fibrosis treatment.

Claims

1. Application of Physagulin C in the preparation of drugs for the treatment of liver fibrosis.

2. The use of physagulin C in the preparation of a drug for treating liver fibrosis according to claim 1, wherein The chemical name of physagulin C is (20R, 22R)-15α-acetoxy-5β(6), 16β(17)-diepoxy-4β, 14β-dihydroxy-1-oxo-witha-2, 24-dienolide; its molecular formula is C 30 H 38 O9; molecular weight is 542.63; specific structural formula is as follows:

3. The use of physagulin C in the preparation of a drug for treating liver fibrosis according to claim 1, wherein The drug for treating liver fibrosis is a drug that inhibits the proliferation or activation of hepatic stellate cells.

4. The use of physagulin C in the preparation of a drug for treating liver fibrosis according to claim 1, wherein The drug for treating liver fibrosis is a drug that inhibits the expression of hepatic stellate cell fibrosis marker protein.

5. The use of physagulin C in the preparation of a drug for treating liver fibrosis according to claim 4, characterized in that: The proteins that inhibit hepatic stellate cell fibrosis markers are α-SMA and Collagen I protein.

6. The use of physagulin C in the preparation of a drug for treating liver fibrosis according to claim 1, wherein: The drug for treating liver fibrosis is a drug that promotes apoptosis of hepatic stellate cells.

7. The use of physagulin C in the preparation of a medicament for treating liver fibrosis according to claim 1, wherein: The medicine for treating liver fibrosis includes oral preparations or injection preparations.

8. The use of physagulin C in preparing a drug for treating liver fibrosis according to claim 7, wherein: The oral preparation is any one or more of granules, capsules, tablets, powders, pills, and sustained-release preparations.

9. The use of physagulin C in preparing a drug for treating liver fibrosis according to claim 1, wherein In the drug for treating liver fibrosis, the concentration of physagulin C is 0.5-2 μM.

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