Application of gypenoside A in preparation of medicine for treating diseases caused by iron metabolism disorder

Abstract

The invention provides novel application of gypenoside A. Verification shows that the gypenoside A can improve problems caused by cell iron ion accumulation by improving the level of cell iron metabolism related protein, and can be used for preparing medicines for treating diseases caused by iron metabolism disorder, such as neurodegenerative diseases, thalassemia and hemochromia.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of Gynostemma pentaphyllum saponin A in the preparation of therapeutic drugs for diseases caused by iron metabolism disorders. Background Technology

[0002] Gynostemma pentaphyllum saponins are the dried aerial parts of *Gynostemma pentaphyllum*, a plant in the Cucurbitaceae family, also known as Seven-Leaf Gall, Sweet Tea Vine, Small Bitter Medicine, and Gongluoguodi. It has opposite, foot-like compound leaves, and small, pale greenish-white, unisexual, dioecious flowers. The whole plant is used medicinally for clearing heat and detoxifying, relieving cough and expectorating phlegm. It can be found on hillsides, forest edges, ditches, and in thickets. It possesses the functions of clearing heat and detoxifying, relieving cough and treating ailments.

[0003] There are approximately 140 species of total saponins belonging to Gynostemma pentaphyllum, named Gynostemma pentaphyllum saponins I to LXXVIII. Among them, some are ginsenosides Rb1, Rb3, and Rd, as well as ginsenoside diol, 2α-hydroxyginsenoside diol, 2α,19-dihydroxy-12-deoxyginsenoside diol, etc.

[0004] Gynostemma pentaphyllum saponin A, belonging to the dammarane-type triterpenoid saponin, is an important active ingredient in Gynostemma pentaphyllum. It possesses various biological activities, including anti-tumor, anti-diabetic, cardiovascular protective, and anti-aging effects. Furthermore, Gynostemma pentaphyllum saponin A also exhibits antioxidant and anti-inflammatory properties. Currently, there are no literature reports on the application of Gynostemma pentaphyllum saponin A in diseases caused by iron metabolism disorders. This invention investigates its association with diseases caused by iron metabolism disorders by treating a cell model with Gynostemma pentaphyllum saponin A. Summary of the Invention

[0005] This invention aims to provide a new use for Gynostemma pentaphyllum saponin A, which has a good regulatory effect on the expression of iron metabolism-related proteins, effectively alleviates iron overload and cell damage caused by iron overload, and can be used to prepare therapeutic drugs for diseases caused by iron metabolism disorders; and for the treatment of neurodegenerative diseases, hemochromatosis, and thalassemia.

[0006] To achieve the above objectives, the present invention provides the application of Gynostemma pentaphyllum saponin A in the preparation of a therapeutic drug for diseases caused by iron metabolism disorders.

[0007] Furthermore, diseases caused by iron metabolism disorders include neurodegenerative diseases, thalassemia, and hemochromatosis.

[0008] Furthermore, neurodegenerative diseases include Alzheimer's disease and Parkinson's disease.

[0009] To achieve the above objectives, the present invention provides the application of Gynostemma pentaphyllum saponin A in the preparation of a therapeutic drug for diseases caused by iron metabolism disorders.

[0010] Furthermore, diseases caused by iron metabolism disorders include neurodegenerative diseases, thalassemia, and hemochromatosis.

[0011] Furthermore, neurodegenerative diseases include Alzheimer's disease and Parkinson's disease.

[0012] Neurodegenerative diseases are a group of conditions affecting the function of the nervous system, including Alzheimer's disease and Parkinson's disease. Recent studies have found that iron overload is associated with the occurrence and development of these diseases. Specifically, there is a clear link between iron overload and Alzheimer's disease. Iron overload can promote the production of β-amyloid protein (Aβ), a key pathological feature of Alzheimer's disease. In addition, iron overload can induce oxidative stress and inflammatory responses, thereby exacerbating neuronal damage.

[0013] Patients with thalassemia typically have low ferritin levels due to abnormal red blood cell development. However, in cases requiring repeated blood transfusions, ferritin levels may increase, leading to iron overload and consequently increasing the risk of oxidative stress. Iron overload can catalyze the production of reactive oxygen species (ROS), which can damage cell membranes, proteins, and DNA, resulting in cellular dysfunction and tissue damage. Because of the increased iron load in their bodies due to long-term or repeated blood transfusions, thalassemia patients are more prone to iron overload, thus increasing their risk of oxidative stress.

[0014] This invention verifies that Gynostemma pentaphyllum saponin A in HT22 cells can improve cell damage caused by iron metabolism homeostasis, thereby achieving the treatment of neurodegenerative diseases.

[0015] Furthermore, the concentration of Gynostemma pentaphyllum saponin A at the cellular level is 10 μM-20 μM.

[0016] Meanwhile, the present invention provides a pharmaceutical composition for treating diseases caused by iron metabolism disorders, comprising an effective amount of Gynostemma pentaphyllum saponin A and its pharmaceutically acceptable salt and one or more pharmaceutically acceptable excipients.

[0017] Compared with the prior art, the present invention has the following significant advantages:

[0018] This study provides a new use for Gynostemma pentaphyllum saponin A, which has been validated to improve problems caused by cellular iron accumulation by improving the levels of cellular iron metabolism-related proteins. It can be used to prepare treatments for diseases caused by iron metabolism disorders, such as neurodegenerative diseases, thalassemia, and hemochromatosis. Attached Figure Description

[0019] Figure 1 The results of cell viability analysis obtained by CCK8 assay after treatment of HT22 cells with Gynostemma pentaphyllum saponin A are presented.

[0020] Figure 2 The figure shows the effect of Gynostemma pentaphyllum saponin A on the expression of iron metabolism-related proteins.

[0021] Figure 3 The figure shows the effect of Gynostemma pentaphyllum saponin A on the expression of iron metabolism-related proteins.

[0022] Figure 4 The effects of Gynostemma pentaphyllum saponin A on iron levels and the expression of ASCL4 and MDA in a FAC (ferric amine chelator) induced HT22 cell model were investigated. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] (1) CCK8 experiment

[0026] HT22 cells were digested to prepare a cell suspension, and the cell concentration was adjusted to 5 × 10⁻⁶. 4 / ml. The dissolved Gynostemma pentaphyllum saponin A was diluted with culture medium to different concentrations and used to incubate HT22 cells seeded in 96 wells for 24 hours. The CCK8 assay was performed according to the manufacturer's manual (Cat#C0037, Beyotime, Nanjing, China). Cell viability was calculated as follows: Cell viability (%) = (Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells) × 100%.

[0027] (2) Cell culture and treatment

[0028] HT22 cells were used for cell experiments. Cells were incubated in 50% Dulbecco's modified Eagle's medium (DMEM) (Gibico, USA), supplemented with 50% Opti-MEM, 5% fetal bovine serum, 0.5% penicillin, and streptomycin. Cells were cultured at 37°C and 5% CO2. After near-confluence, cells were treated with / without 200 μM ferric ammonium citrate in DMEM (1% fetal bovine serum) for 24 hours, with / without gypenosides saponin A (10 μM, 20 μM), before subsequent experiments.

[0029] (3) Measurement of cellular iron ion levels

[0030] Cell culture was performed in the same manner as in step (2), and the level of ferrous ions was measured using the ferrous ion fluorescence assay kit (Elabscience, E-BC-F101).

[0031] (4) MDA level test

[0032] Cell culture was performed in the same manner as in step (2), and the level of ferrous ions was determined using the malondialdehyde (MDA) colorimetric assay kit (Elabscience, E-BC-K028-M).

[0033] (5) Protein extraction and Western blotting

[0034] Prepare lysis buffers for protein lysis using lysis buffers (including chilled RIPA lysis buffer and protease and phosphatase inhibitors) in the specified proportions, following the instructions for each reagent. Determine protein concentration using the BCA Protein Assay Kit (Cat#23235, Thermo Scientific, USA) according to the manufacturer's instructions. Select an appropriate SDS-PAGE gel for electrophoresis based on the molecular weight of the target protein. Load 40-60 μg per well. Then, transfer the protein strips to polyvinylidene fluoride (PVDF, Cat#ISEQ00010, Millipore, USA) with charged charge. After transfer, remove the PVDF membrane, soak it in 1X TBST, and then transfer it to a box containing Beyotime blocking buffer. Shake on a decolorizing shaker at room temperature for 0.5-1 h or block overnight at 4°C. Then remove the PVDF membrane from the blocking solution, place it in primary antibody dilution buffer, and incubate overnight at 4°C. The next day, the primary antibody diluent was recovered. The PVDF membrane incubated with the primary antibody was placed in 1X TBST and washed three times at room temperature using a decolorizing shaker, 10-15 minutes each time. A secondary antibody diluent (Cat#ab288151, Abcam, USA) was prepared in the same manner and brought into contact with the PVDF membrane. After incubation at room temperature for 1-2 hours, the secondary antibody diluent was discarded. The PVDF membrane incubated with the secondary antibody was then placed in 1X TBST and washed three times at room temperature using a decolorizing shaker, 10-15 minutes each time. After the above steps, the PVDF membrane was uniformly immersed in the developing solution and developed using a Tanon-5200 chemiluminescence developing system.

[0035] The developed images were adjusted to the optimal contrast using ImageJ or QuantityOne analysis software, and the gray values ​​of protein bands and fluorescence images were analyzed. The data were analyzed using GraphpadPrism 8 software. One-way ANOVA was performed, and p < 0.05 was considered statistically significant.

[0036] Cell CCK8 assays revealed that the survival rate of HT22 cells treated with 60 μM Gynostemma pentaphyllum saponin A was lower than that of the control group. Therefore, we subsequently used 10 μM and 20 μM Gynostemma pentaphyllum saponin A for further experiments.

[0037] Iron ions, especially ferrous ions, cause toxic damage to cells. In the HT22 cell line, after 24 hours of induction with ferric ammonium citrate (FAC), we used gypenosides A (10 μM). First, using a ferrous ion level assay kit, we found that gypenosides A effectively promoted the downregulation of iron ion levels in HT22 cells in the presence of FAC. Further, Western blot analysis revealed that gypenosides A downregulated the upregulation of ASCL4 caused by iron accumulation in the presence of iron. Additionally, we found that 20 μM gypenosides A downregulated the excessive MDA caused by FAC treatment. These preliminary experiments suggest that gypenosides A can improve iron accumulation and the oxidative damage it causes.

[0038] In further experiments, we measured the levels of iron metabolism-related proteins in HT22 cells treated with FAC (ferric amine chelator). The results showed a significant decrease in the level of the iron export protein FPN (ferroportin), while a significant upregulation of the level of the transferrin receptor TFR (transferrin receptor). These results indicate that Gynostemma pentaphyllum saponin A has a physiological effect of promoting iron ion excretion and inhibiting iron ion uptake, thereby affecting intracellular iron homeostasis.

[0039] Furthermore, we observed upregulation of the levels of iron regulatory elements IRP1 (iron response element-binding protein 1) and IRP2 (iron response element-binding protein 2). This suggests that Gynostemma pentaphyllum saponin A may regulate intracellular iron metabolism by affecting the expression of these iron regulatory proteins.

[0040] Finally, the experimental results also showed that the level of ferritin FPN (ferritin, an iron storage protein) was upregulated. This increase suggests that gypenosin A may also play a role in iron storage, further emphasizing its importance in the regulation of iron metabolism.

[0041] In summary, iron overload can promote the production of β-amyloid protein (Aβ), induce oxidative stress and inflammatory responses, thereby exacerbating neuronal damage; it also catalyzes the production of reactive oxygen species, leading to cellular dysfunction and tissue damage. The experiments of this invention demonstrate that Gynostemma pentaphyllum saponin A improves cellular iron ion accumulation by enhancing the levels of cellular iron metabolism-related proteins, thereby achieving the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, thalassemia, and hemochromatosis.

[0042] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of gypenoside A in the preparation of a medicament for treating a disease caused by iron metabolism disorder, characterized in that, The diseases caused by iron metabolism disorder are neurodegenerative diseases, thalassemia, and hemochromatosis.

2. Use according to claim 1, characterized in that The neurodegenerative diseases include Parkinson's disease and Alzheimer's disease.

3. Use according to claim 1, characterized in that At the cellular level, the effective concentration range of gypenoside A is 10-20 μM.

4. A pharmaceutical composition for treating diseases caused by iron metabolism disorder, which is composed of an effective amount of gypenoside A and its pharmaceutically acceptable salts and one or more pharmaceutically acceptable adjuvants.

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