Medicinal drug and food product produced from small-spored ganoderma lucidum-derived gmi

Abstract

To provide a medicinal drug or a food product produced from small-spored Ganoderma lucidum-derived GMI.SOLUTION: Provided is a medicinal drug or a food product produced from small-spored Ganoderma lucidum-derived GMI (Ganoderma immunoregulatory protein) . The medicinal drug or the food product is produced by using small-spored Ganoderma lucidum-derived GMI (Ganoderma immunoregulatory protein) fermentatively produced by genetically modified Pichia pastoris Ey72 strain, GMI has an amino acid sequence shown by SEQ ID NO:1, and is used for treatment, prevention or amelioration of pulmonary fibrosis caused by a factor other than a virus.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a Ganoderma immunomodulatory protein (GMI) derived from microspore reishi, and more specifically to a medicine or food product using a Ganoderma immunomodulatory protein (GMI) derived from microspore reishi. [Background technology]

[0002] Idiopathic pulmonary fibrosis (IPF) is a lung inflammatory disease characterized by progressive fibrosis, leading to irreversible decline in lung function. Its exact cause remains unknown. In recent years, the incidence of IPF has rapidly increased worldwide due to the COVID-19 pandemic.

[0003] Recent studies have shown that transforming growth factor β1 (TGF-β1) is an important promoter of fibrosis in IPF. Current IPF treatments include pirfenidone (PFD) and nintedanib, of which PFD is known to inhibit the action of TGF-β1.

[0004] However, taking PFD or nintedanib can cause many side effects, including photosensitivity, loss of appetite, stomach discomfort, nausea, vomiting, abdominal pain, rash, diarrhea, liver dysfunction, headache, and hypertension, significantly reducing patients' quality of life (QOL) and not leading to a clear improvement in survival rates.

[0005] For this reason, the biopharmaceutical field continues to develop more effective treatments for IPF with fewer side effects, and treatment techniques using the endogenous protein TXNDC5, mulberry yellow extract, and human umbilical cord-derived mesenchymal stem cells (HUMSCs) are being researched.

[0006] On the other hand, microspore reishi contains a protein called GMI (Ganoderma immunomodulatory protein) that has immunomodulatory effects. GMI (Ganoderma immunomodulatory protein) is known to promote cytokine secretion from immune cells and induce immune cell activation, thereby contributing to the reduction of bacterial infections and the maintenance of normal immune responses.

[0007] In February 2022, Taiwan's Ministry of Health and Welfare's Food and Drug Administration (TFDA) approved a globulin concentrate derived from microspores of Ganoderma lucidum, produced by fermentation using the genetically modified yeast Pichia pastoris Ey72 strain, as a food ingredient. Currently, several studies are underway regarding the use of GMI (Ganoderma immunomodulatory protein) to treat various diseases. For example, Taiwan Patent Publication No. I670066 discloses a therapeutic drug for oral submucous fibrosis (OSF) using GMI (Ganoderma immunomodulatory protein).

[0008] However, the patent does not provide specific data on the therapeutic effect of GMI on fibrosis in other organs, and there is a lack of evidence supporting the claim that GMI has a general therapeutic effect on fibrosis, which has different pathological mechanisms depending on the organ.

[0009] Through research into IPF and microspore Ganoderma lucidum-derived GMI, the present inventors have found that GMI has therapeutic, preventive, and / or ameliorative effects on TGF-β-induced IPF fibrosis. To date, no technology has been known to use GMI to treat, prevent, or ameliorate TGF-β-induced IPF fibrosis. Summary of the Invention [Problem to be solved by the invention]

[0010] The main object of the present invention is to demonstrate that GMI derived from microspore Ganoderma lucidum is effective in treating, preventing and / or ameliorating TGF-β-induced idiopathic pulmonary fibrosis (IPF), and to provide pharmaceuticals or foods produced from said GMI. [Means for solving the problem]

[0011] To achieve this objective, the present invention provides a medicine or food product produced from GMI derived from microspore Ganoderma lucidum. The medicine or food is manufactured from GMI derived from microspore Ganoderma lucidum, which is fermented using the genetically modified Pichia yeast strain Ey72. GMI has the amino acid sequence set forth in SEQ ID NO:1 and is used to treat, prevent or ameliorate pulmonary fibrosis caused by factors other than viruses, particularly idiopathic pulmonary fibrosis.

[0012] In the method of the reference embodiment, pulmonary fibrosis is treated, prevented, or ameliorated by administering to an individual microspore reishi-like globular protein GMI produced by fermentation using recombinant Pichia pastoris Ey72 strain. [Brief explanation of the drawings]

[0013] [Figure 1] Statistical graph of changes in viability of LL29 cells due to GMI. [Figure 2A] Microscopic images and quantitative graphs of the effect of GMI on TGF-β1-induced LL29 cell migration. [Figure 2B] Microscopic images and quantitative graphs of the effect of GMI on TGF-β1-induced LL29 cell migration. [Figure 3A] Western blot analysis and quantitative graph showing the anti-fibrotic activity of GMI in TGF-β1-induced fibrotic LL29 cells. [Figure 3B] Western blot analysis and quantitative graph showing the anti-fibrotic activity of GMI in TGF-β1-induced fibrotic LL29 cells. [Figure 4A] Graph of weight change and weight gain / loss rate in a bleomycin (BLM)-induced mouse model administered GMI. [Figure 4B] Graph of weight change and weight gain / loss rate in a bleomycin (BLM)-induced mouse model administered GMI. [Figure 5]Graph showing changes in survival rate of BLM-induced mice by GMI treatment. [Figure 6A] Graph of changes in lung weight and lung weight change rate in mice after GMI treatment. [Figure 6B] Graph of changes in lung weight and lung weight change rate in mice after GMI treatment. [Figure 7] Images of the appearance of mouse lungs in the normal group (NT) and each control group (GMI, BLM, PFD). DETAILED DESCRIPTION OF THE INVENTION

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0015] As used herein, "treatment" refers to a means of obtaining a beneficial or desired effect, including a therapeutic effect or a prophylactic effect. A therapeutic effect includes the elimination or improvement of a target disease.

[0016] "Prevention" and "amelioration" are used interchangeably herein to refer to a reduction in a measured parameter in a cell, tissue, or individual after treatment compared to an untreated cell, tissue, or individual. Comparisons can be made before and after treatment in the same cell, tissue, or individual.

[0017] "Individual" includes human and non-human animals.

[0018] In one embodiment, the present invention provides a use of GMI derived from microspore reishi mushroom for the preparation of a medicine for the treatment, prevention, or amelioration of pulmonary fibrosis, which is prepared using microspore reishi-like globular protein produced by fermentation using a recombinant Pichia pastoris Ey72 strain.

[0019] In another embodiment, the present invention provides a use of GMI derived from microspore reishi in the production of food, which is a microspore reishi-like globular protein produced by fermentation using the Ey72 strain, for the purpose of preventing or ameliorating pulmonary fibrosis.

[0020] In some embodiments, Microspore Ganoderma lucidum GMI exerts its effect by suppressing the activity of human lung fibroblasts (LL29 cells) involved in the formation of pulmonary fibrosis.

[0021] Furthermore, GMI suppresses the expression of three fibrotic marker proteins in LL29 cells: fibronectin, COL1A1 gene, and α-smooth muscle actin (α-SMA).

[0022] In one embodiment, the concentration of the GMI is less than 0.4 μM.

[0023] Furthermore, in another embodiment, the present invention provides a method for treating, preventing, or ameliorating pulmonary fibrosis by administering the GMI to an individual as a globular protein derived from microspores of Ganoderma lucidum produced by fermentation using a genetically modified Pichia yeast strain Ey72.

[0024] The GMI of the present invention can be administered parenterally, such as intravenously, intraperitoneally, subcutaneously, or intramuscularly, or orally, and can be administered in the form of tablets, pills, capsules, liquids, gels, syrups, emulsions, suspensions, etc., after mixing with a carrier or excipient.

[0025] Needless to say, the technology according to the present invention can be easily implemented by those skilled in the art based on the present specification, and the following examples are merely for illustrative purposes and do not limit the technical scope of the present invention.

[0026] (Example)

[0027] The present study aimed to investigate and verify the anti-fibrotic properties of GMI derived from microspore Ganoderma lucidum to combat TGF-β-induced fibrosis using an in vitro model. The therapeutic activity of GMI was evaluated using an in vitro model using an idiopathic pulmonary fibrosis (IPF) cell line.

[0028] (1) Experiments using human-derived in vitro cell models: The effect of GMI on pulmonary fibrosis was analyzed using LL29 human lung fibroblasts derived from IPF patients. Cells were induced into a fibrotic state by combined TGF-β1 (5 ng / mL) and then pretreated with GMI (2 concentrations) or the control drug pirfenidone (PFD) in serum-reduced medium for 2 hours. After treatment, cells were exposed to TGF-β1 for 48 hours.

[0029] Microspore Ganoderma GMI: GMI was a concentrate produced by Mycomagic Biotechnology Co., Ltd., Taipei, Taiwan. GMI has the amino acid sequence set forth in SEQ ID NO:1. MSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRPSYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWN(SEQ ID NO:l)

[0030] Cell acquisition and culture: LL29 cells are human lung fibroblasts isolated from the lung tissue of an IPF patient and were obtained from ATCC (Registered Trademark). The cells were cultured in Gibco's Dulbecco's Modified Eagle's Medium (containing 10% FBS) at 37°C, 5% CO2, and >95% humidity, and were passaged every 2–3 days.

[0031] Cytotoxicity test: The cytotoxicity of GMI was evaluated by MTT assay using LL29 cells. LL29 cells were trypsinized and seeded at 3,000 cells per well in a 96-well plate. After overnight incubation, the medium was replaced with GMI-supplemented medium. After 48 hours of treatment, the medium was replaced with MTT medium (final concentration 0.5 mg / mL) and incubated at 37°C for 4 hours. The dissolution solution was then added and the plate was left overnight. After confirming complete dissolution of the purple formazan crystals, the absorbance was measured using an ELISA reader at wavelengths of 550–600 nm, with a reference wavelength of 650 nm or higher.

[0032] Wound healing assay: A gap of 500±100 μm was formed between the cells using a double insert, and microscopic images were taken at 0 and 24 hours. The image analysis was performed using the Robust Quantitative Scratch Assay (RQSA) algorithm. The wound healing rate was calculated using the following formula: Healing(%)=((Areat-Area0) / Area0)×100 where Area0 is the initial wound area and Areat is the open area at each time.

[0033] Western blot analysis: Cell lysates were prepared using RIPA buffer, separated via SDS-PAGE, and transferred to a PVDF membrane. Anti-COL1A1, fibronectin, and α-SMA antibodies were used from GeneTex, Taiwan, and HRP-labeled secondary antibodies were used from Jackson ImmunoResearch Laboratories, USA. Proteins were detected using WesternBright® ECL (Advansta), and quantified using Fiji software.

[0034] Statistical analysis: Statistical analysis and graphing were performed using GraphPad Prism 7.03 (GraphPad Software, USA). Data are presented as mean ± standard error of the mean (SEM). Comparisons between two groups were performed using Student's t-test, and comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test. Significance levels were indicated as *p<0.05, **p<0.01, and ***p<0.001.

[0035] (Effect of GMI on LL29 cell viability) LL29 cells are lung fibroblasts isolated from IPF patients, and the cytotoxicity of GMI was evaluated by MTT assay. GMI concentrations ranged from 0 to 0.4 μM, and the viability after 48 hours of treatment is shown in Figure 1. As long as the GMI concentration was below 0.4 μM, no significant cytotoxicity was observed (see Figure 1). Values ​​are shown as mean ± SEM, and statistical significance was analyzed by one-way ANOVA and Dunnett's test (*p<0.05; **p<0.01; ***p<0.001).

[0036] (GMI inhibits TGF-β1-induced LL29 cell migration) TGF-β1 (5 ng / mL) induces the differentiation of fibroblasts into myofibroblasts. In this study, we performed a 48-hour wound healing assay using co-treatment with GMI (0.1 and 0.2 μM) and TGF-β1. Pirfenidone (PFD) was used as a positive control.

[0037] Figure 2A shows a microscopic image (40x magnification), and Figure 2B shows the quantification of the area of ​​the healing zone. Significant wound closure was observed in the TGF-β1 alone group, but a dose-dependent inhibitory effect was observed in the GMI-treated group. In particular, GMI demonstrated a stronger inhibitory effect than PFD. The suppression of cell migration (crawling) ability suggests that GMI contributes to the suppression of fibrosis progression.

[0038] (GMI suppresses the expression of fibrosis-related proteins) The expression of fibronectin, COL1A1, and α-SMA under TGF-β1 induction is an indicator of fibrosis progression. In the GMI-treated group, the expression levels of these proteins were significantly reduced, confirming the anti-fibrotic effect (see Figure 3). Figure 3A shows the results of Western blot analysis, and Figure 3B shows the relative expression levels of each protein relative to α-tubulin. GMI showed a clear inhibitory effect even at a concentration of 0.1 μM, while PFD did not show a similar effect. These results indicate that GMI has the effect of suppressing the progression of IPF by intervening in the TGF-β1 pathway and suppressing the formation of myofibroblasts and the excessive production of extracellular matrix.

[0039] (2) Pulmonary fibrosis model experiments in mice: In this study, male C57BL / 6J mice (6-8 weeks old) were divided into five groups, with at least six mice per group (see Table 1). Four of the groups were administered a single dose of 2.5 U / kg of bleomycin (BLM) via the tail vein on the first day of the experiment to induce pulmonary fibrosis, and the remaining group served as a normal control. The BLM-administered group was treated as follows: Group 1: BLM only (negative control group) Group 2: GMI (1 mg / kg) was administered after BLM administration. Group 3: GMI (2 mg / kg) was administered after BLM administration. Group 4: After BLM administration, 200 mg / kg of clinically used drug pirfenidone (PFD) was administered. On the 22nd day, all surviving mice were euthanized and various evaluations were performed.

[0040] [Table 1]

[0041] (Observation of changes in mouse weight) As shown in Figures 4A and 4B, mice administered BLM showed significant weight loss compared to the control group. On the other hand, the BLM-induced weight loss was suppressed in the GMI-administered group, demonstrating a weight-maintenance effect.

[0042] (Observation of mouse survival rate) As shown in Figure 5, the survival rate in the BLM-only group was 40%, whereas the PFD-administered group was 50%, the GMI 1 mg / kg-administered group was 70%, and the GMI 2 mg / kg-administered group showed a 100% survival rate at day 14, demonstrating a superior therapeutic effect to PFD.

[0043] (Observation of mouse lung weight) As shown in Figures 6A and 6B, BLM-induced lung weights in mice tended to increase, due to the progression of inflammation and fibrosis. In the GMI-treated group, lung weights remained close to the normal range, and the rate of change in lung weight was significantly lower than in the BLM-only group.

[0044] (Observation of the appearance of mouse lungs) As shown in Figure 7, the lungs of BLM-induced mice were redder in appearance compared to the control group. On the other hand, the lungs of the GMI and PFD-treated groups had a color similar to that of the control group, suggesting a therapeutic effect even with the naked eye.

[0045] These results demonstrate that the microspore Ganoderma lucidum-derived GMI of the present invention has the effect of suppressing the progression of pulmonary fibrosis in both TGF-β1-induced LL29 cells and BLM-induced mouse models. GMI has been confirmed to have multiple mechanisms of action, including suppressing myofibroblast activation, reducing cell migration, and suppressing the expression of extracellular matrix (ECM) components and fibrosis marker proteins.

[0046] In particular, GMI exhibits stronger anti-fibrotic effects than the existing clinical drug pirfenidone, and is expected to have high applicability as a future IPF treatment.

[0047] The application of the microspore Ganoderma lucidum GMI of the present invention to pharmaceuticals or foods has been scientifically demonstrated to have therapeutic, preventive and ameliorative effects on pulmonary fibrosis through experiments using human cell models and mouse models. Therefore, the present invention is industrially applicable and satisfies the requirements for invention as stipulated in the Patent Act.

[0048] The embodiments described in this specification are merely preferred examples of the present invention and do not limit the technical scope of the present invention. Those skilled in the art can make various changes, modifications, and substitutions without departing from the spirit and scope of the present invention. Therefore, the technical scope of the present invention is to be determined by the following claims.

Claims

1. It is produced from GMI (Ganoderma immunomodulatory protein) derived from microspores of Ganoderma lucidum, which are fermented and produced by genetically modified Pichia pastoris Ey72 strain. The GMI has the amino acid sequence shown in SEQ ID NO: 1, and is used for the treatment, prevention, or amelioration of pulmonary fibrosis caused by factors other than viruses, particularly idiopathic pulmonary fibrosis (IPF). A medicine or food product produced from the GMI derived from microspore Ganoderma lucidum.

2. 2. A medicine or food prepared from the GMI derived from microspore Ganoderma lucidum according to claim 1, wherein the GMI inhibits the formation of pulmonary fibrosis by inhibiting the activity of LL29 cells, which are human lung fibroblasts.

3. The GMI of claim 2 is characterized in that it suppresses the expression of the following three types of fibrosis-related proteins in LL29 cells: fibronectin, COL1A1 gene, and α-smooth muscle actin (α-SMA).

4. The medicine or food produced from the GMI derived from microspore Ganoderma lucidum according to claim 1, wherein the concentration of the GMI is less than 0.4 μM.

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