Application of Liao's Huafeng Dan in the preparation of drugs for preventing and treating multiple myeloma

Abstract

This invention discloses the application of the Miao medicine Liao's Huafengdan in the preparation of drugs for the prevention and treatment of multiple myeloma, belonging to the field of pharmaceutical application technology. In vitro detection using RPMI8226 and MM1.S multiple myeloma cells revealed that Liao's Huafengdan has a significant inhibitory effect on multiple myeloma. Flow cytometry analysis further showed its effect on cell apoptosis, indicating that Liao's Huafengdan significantly affects apoptosis in multiple myeloma cells, with higher drug concentrations leading to more pronounced apoptosis. Therefore, it can be used as a preparation for the prevention and treatment of multiple myeloma, and can be formulated into pharmaceutically permissible oral liquids, injections, tablets, capsules, and drop pills, providing a new application for Liao's Huafengdan. Moreover, Liao's Huafengdan is already a marketed drug and can be directly applied to humans without further clinical safety assessments, which can rapidly increase the market share of this product.

Description

Technical Field

[0001] This invention belongs to the fields of biology and medicine, specifically relating to the application of a Miao medicine, Liao's Huafeng Dan, in the preparation of drugs for the prevention and treatment of multiple myeloma. Background Technology

[0002] Liao's Wind-Dispelling Pill, a Miao medicine, is manufactured by Guizhou Wansheng Pharmaceutical Co., Ltd. It is a pill form with the following functions: calming wind and relieving spasms, clearing phlegm and opening the orifices. It is used for wind-phlegm obstruction, stroke hemiplegia, epilepsy, facial nerve palsy, and facial paralysis. The main ingredients include more than 20 kinds of Chinese medicinal herbs such as Gastrodia elata, scorpion, silkworm, Aconitum carmichaelii, Atractylodes lancea, Arisaema heterophyllum, and musk. The pills are vermilion in color, with a brownish-yellow cross-section; they have a strong aroma and a pungent taste.

[0003] Currently, treatment options for multiple myeloma include chemotherapy, hematopoietic stem cell transplantation, chimeric antigen receptor T cells (CAR-T), proteasome inhibitors, immunomodulators, histone deacetylase inhibitors, and monoclonal antibodies, among other supportive and symptomatic therapies. These drugs and techniques have significantly advanced the treatment of multiple myeloma and markedly improved patient prognosis; however, multiple myeloma remains incurable. Therefore, finding effective treatments for multiple myeloma is currently a top priority. Summary of the Invention

[0004] The purpose of this invention is to provide a new application for the Miao medicine Liao's Huafeng Dan, and to solve the problem of difficult treatment of multiple myeloma.

[0005] To achieve the above objectives, this invention provides the application of Miao medicine Liao's Huafeng Dan in the preparation of drugs for the prevention and treatment of multiple myeloma, which has a significant therapeutic effect on multiple myeloma.

[0006] This invention, using in vitro detection of multiple myeloma cells RPMI8226 and MM1.S, revealed that Liao's Huafengdan has a significant inhibitory effect on multiple myeloma cells. Flow cytometry analysis further investigated its effect on apoptosis, showing that Liao's Huafengdan significantly affects apoptosis in multiple myeloma cells, with higher drug concentrations leading to more pronounced apoptosis. Western blotting experiments indicated that Liao's Huafengdan overactivates caspase 9 and caspase 3 proteins, causing cleavage of both proteins and inducing apoptosis. Therefore, it can be used as a drug for the prevention and treatment of multiple myeloma.

[0007] This invention directly prepares Liao's Huafeng Dan into a preparation for the prevention and treatment of multiple myeloma, or combines it with other drugs for the prevention and treatment of multiple myeloma to prepare a compound preparation for the prevention and treatment of multiple myeloma. The preparation can be any one of the pharmaceutically permissible forms such as oral liquid, injection, tablet, capsule, or drop pill.

[0008] The beneficial effects of this invention are as follows:

[0009] Compared to existing uses of Liao's Huafeng Dan, the inventors discovered that Liao's Huafeng Dan has a good in vitro inhibitory effect on multiple myeloma, revealing a new use for this drug and solving the problem of difficult treatment of multiple myeloma. Furthermore, Liao's Huafeng Dan is a marketed drug that can be directly applied to humans without further clinical safety assessments, showing great application potential and the ability to rapidly increase its market share. Attached Figure Description

[0010] Figure 1 The inhibition rate of different concentrations of Liao's Huafengdan on the proliferation of human multiple myeloma cells RPMI8226 is shown.

[0011] Figure 2 The inhibition rate of different concentrations of Liao's Huafengdan on the proliferation of human multiple myeloma cells MM1.S is shown.

[0012] Figure 3 The effect of different concentrations of Liao's Huafengdan on apoptosis of human multiple myeloma cells RPMI8226.

[0013] Figure 4 The effect of different concentrations of Liao's Huafengdan on apoptosis proteins in human multiple myeloma cells RPMI8226. Detailed Implementation

[0014] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0015] Example 1

[0016] In vitro activity test of Miao medicine Liao's Huafengdan against multiple myeloma cells

[0017] 1. Experimental materials

[0018] Preparation of sample solutions: Liao's Huafeng Dan was prepared into sample solutions with concentrations of 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL and 3.125 μg / mL using DMSO.

[0019] Preparation of 5% MTT solution: Weigh 0.5g of MTT powder, add 100mL of sterile PBS, dissolve at 60℃, filter sterilize through a 0.22μm filter membrane, and store at 4℃.

[0020] Cell lines: Human multiple myeloma cells RPMI8226 and MM1.S were both preserved by the Guizhou Provincial Natural Products Research Center. Human multiple myeloma cells RPMI8226 and MM1.S were cultured in DMEM medium containing 5% FBS in a 37℃ 5% CO2 incubator.

[0021] DMEM culture medium and trypsin were purchased from Hycone, fetal bovine serum (FBS) was purchased from Shanghai Unimicron Biotechnology Co., Ltd., and tetramethyl azolidinyl ether salt (MTT) and dimethyl sulfoxide (DMSO) were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0022] 2. Experimental Methods

[0023] (1) The MTT assay was used to determine the inhibitory effect of different concentrations of Liao's Huafengdan on the proliferation of multiple myeloma cells.

[0024] With 5×10 3 To determine the well density, human multiple myeloma cells RPMI8226 and MM1.S in the exponential proliferation phase were seeded into 96-well plates with 190 μL of DMEM medium containing 5% FBS per well. After 24 h of culture, 10 μL of sample solution containing different concentrations of Liao's Huafengdan was added to each well, with 5 replicates for each concentration. Cells were cultured at 37°C for another 72 h, followed by the addition of 20 μL of 5% MTT solution to each well. After another 4 h of culture, the cells were centrifuged at 1200 rpm for 10 min, the supernatant was discarded, and 160 μL of LDMSO was added to each well. The mixture was thoroughly mixed using a microplate reader at 570 nm as the test wavelength and 630 nm as the reference wavelength. The absorbance of each well was measured, and the inhibition rate was calculated. The results are shown below. Figure 1 and Figure 2 .

[0025] Figure 1 and Figure 2 The results showed that Liao's Huafengdan significantly inhibited the growth of human multiple myeloma cells RPMI8226 and MM1.S, and the inhibition of RPMI8226 and MM1.S cells was significantly concentration-dependent. At a drug concentration of 100 μg / mL, the inhibition rate of human multiple myeloma cells RPMI8226 was greater than 80%. The IC50 was calculated based on the processed data. 50 The value was 31.23 ± 2.67 μg / mL. At a drug concentration of 50 μg / mL, the inhibition rate of human multiple myeloma cells MM1.S was greater than 90%. The IC50 was calculated based on the processed data. 50 The value was 25.93 ± 6.75 μg / mL.

[0026] (2) Flow cytometry analysis of changes in cell apoptosis

[0027] Human multiple myeloma cells RPMI8226, growing in the logarithmic growth phase, were seeded into 60 mm round culture dishes and incubated at 37°C for 6 h. Then, sample solutions containing different concentrations of Liao's Huafengdan (a traditional Chinese medicine) were added, with final concentrations of 12.5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively. The control group received an equal volume of DMSO. The cells were incubated at 37°C with 5% CO2 for 24 h. The drug-treated cells were collected in 15 mL centrifuge tubes, centrifuged at 1000 g at 4°C for 5 min, the supernatant was discarded, and the cells were resuspended and washed with PBS. The cells were then centrifuged again at 1000 g at 4°C for 5 min, the supernatant was discarded, and the cells were resuspended in 1×binding buffer. 2.5 μL of FITC and propidium iodide (PI) staining solution was added, mixed well, and stained at room temperature in the dark for 15 min. The stain was removed by centrifugation, and the cells were washed with 100 μL of 1×binding buffer and resuspended. The cells were then analyzed by flow cytometry. The results are shown in the figure below. Figure 3 .

[0028] Figure 3 Flow cytometry results showed that Liao's Huafengdan significantly affected the apoptosis of human multiple myeloma cells RPMI8226, and the higher the drug concentration, the more obvious the apoptosis.

[0029] (3) Detection of changes in apoptosis proteins by Western blotting

[0030] Human multiple myeloma cells RPMI8226 in the logarithmic growth phase were seeded in 60 mm round culture dishes and incubated at 37°C for 6 h. Then, sample solutions containing different concentrations of Liao's Huafengdan (a traditional Chinese medicine) were added, with final concentrations of 12.5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively. An equal volume of DMSO was added to the control group. The cells were incubated at 37°C with 5% CO2 for 24 h. Cells were collected, washed twice with PBS, and collected in 15 mL centrifuge tubes. The cells were centrifuged at 1000 g at 4°C for 5 min, the supernatant was discarded, and protein lysis buffer was added. The cells were lysed on ice for 30 min, then centrifuged at 12000 g at 4°C for 15 min, and the supernatant was collected. Protein concentration was determined using a BCA protein concentration kit. Based on a protein volume: 5×loading buffer volume ratio of 4:1, the corresponding volume of 5×loading buffer was added to the extracted protein sample and vortexed until homogeneous. The sample was then placed on a preheated 100°C heating module for 5 min to deform, and stored at -20°C. Western blotting was used to detect the expression of Caspase 3, cleave-caspase 3, Caspase 9, and cleave-caspase 9 after treatment with different concentrations of cyclophosphamide. The results are shown in [Figure number missing]. Figure 4 .

[0031] Figure 4The results showed that Liao's Huafengdan can induce apoptosis by activating caspase 9 and caspase 3 proteins, causing the cleavage of caspase 9 and caspase 3 proteins.

[0032] Based on the above results, it can be concluded that Liao's Huafengdan has an inhibitory effect on human multiple myeloma, indicating that Liao's Huafengdan has potential value in the preparation of drugs for the prevention and treatment of multiple myeloma or as an inhibitor of multiple myeloma cell proliferation.

Claims

1. The application of Liao's Huafeng Dan in the preparation of a medicine for preventing and treating multiple myeloma.

2. The use of the seed medicine Liao's Huafeng Dan according to claim 1 in the preparation of a drug for preventing and treating multiple myeloma, characterized in that: The dosage form of the medicine is any one of oral liquid, injection, tablet, capsule, and dripping pill.

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