Application of GMI of Ganoderma microspore to preparation of medicines and foods

Abstract

The invention provides an application of GMI of Ganoderma microspore in preparation of drugs and food, and the drugs and food are prepared from Ganoderma microspore globulin produced by fermentation of a genetically modified pichia pastoris Ey72 strain and used for treating, preventing or improving pulmonary fibrosis.

Description

Technical Field

[0001] This invention relates to GMI of microspore Ganoderma lucidum, and more specifically to the use of a GMI of microspore Ganoderma lucidum in the preparation of pharmaceuticals and food. Background Technology

[0002] Note: Idiopathic pulmonary fibrosis (IPF) is an inflammatory condition that causes progressive fibrosis in the lungs, leading to a chronic and irreversible decline in lung function. The recent COVID-19 pandemic has further exacerbated the rapid increase in the global incidence of IPF.

[0003] Current research on the fibrotic process of IPF (intrauterine fibrosis) has found that transforming growth factor-β1 (TGF-β1) promotes the fibrotic process. Among the currently used clinical medications for IPF patients, Pirfenidone (PFD) and nintedanib, PFD has an inhibitory effect on TGF-β1. However, taking PFD and nintedanib can cause various side effects, such as photosensitivity, decreased appetite, stomach discomfort, nausea, vomiting, abdominal pain, skin rashes, diarrhea, elevated liver enzymes, headache, and hypertension. These symptoms not only affect the quality of life of IPF patients but also do not significantly improve survival rates. Therefore, researchers in the biomedical field continue to study drugs and technologies for treating IPF fibrosis, such as endoplasmic reticulum protein TXNDC5, Phellinus linteus extract, and human umbilical cord mesenchymal stem cell (HUMSC) therapy.

[0004] Furthermore, Ganoderma lucidum contains immunomodulatory proteins (GMIs), which can promote the secretion of cytokines by immune cells and activate immune cells, thereby reducing bacterial infections and allowing the immune response to function normally. In February 2022, Taiwan announced that a concentrated extract of Ganoderma lucidum globulin produced by fermentation with the genetically modified Pichia pastoris strain Ey72 can be used as a food ingredient. Currently, many companies are conducting research on using GMIs to combat various diseases. For example, Taiwan Patent No. I670066 discloses a drug using Ganoderma lucidum GMIs to treat oral submucosal fibrosis (OSF).

[0005] Although the aforementioned patent discloses the concept of using Ganoderma lucidum microspore GMI to treat fibrosis in different organs, fibrosis in different organs has different pathological mechanisms. The patent only records relevant efficacy data of GMI for oral submucosal fibrosis and cannot prove that GMI also has therapeutic effects on fibrosis in other organs.

[0006] Through research on IPF and Ganoderma lucidum microspore GMI, the inventors discovered that GMI has the effect of treating, preventing and / or improving TGF-β-induced IPF fibrosis, while there is currently no technology to use GMI to treat, prevent and / or improve TGF-β-induced IPF fibrosis. Summary of the Invention

[0007] The main objective of this invention is to provide a use of GMI from Ganoderma lucidum microspores for the preparation of pharmaceuticals and food products, which confirms that GMI does indeed have the effect of treating, preventing and / or improving TGF-β-induced IPF fibrosis.

[0008] Therefore, in order to achieve the aforementioned objectives, the present invention provides the use of GMI of Ganoderma lucidum microspores for the preparation of pharmaceuticals and food, wherein the aforementioned pharmaceuticals are prepared from Ganoderma lucidum microspore globulins produced by fermentation of yeast strains, and are used to treat, prevent or improve pulmonary fibrosis.

[0009] Furthermore, GMI from Ganoderma lucidum microspores inhibits the formation of pulmonary fibrosis by suppressing the activity of LL29 cells in the lungs.

[0010] Furthermore, GMI from Ganoderma lucidum microspores can inhibit the three pulmonary fibrosis protein indices in LL29 cells. These three pulmonary fibrosis proteins include fibronectin, COL1A1 gene, and alpha smooth muscle actin (α-SMA).

[0011] Furthermore, the present invention provides the use of GMI from Ganoderma lucidum microspores for the preparation of food, wherein the aforementioned drug is prepared from Ganoderma lucidum microspore globulin produced by fermentation of genetically modified Pichia pastoris strain Ey72, and is used to prevent or improve pulmonary fibrosis.

[0012] Furthermore, the aforementioned drug is prepared using microspore Ganoderma lucidum globulin produced by fermentation of genetically modified Pichia pastoris strain Ey72, and pulmonary fibrosis refers to idiopathic pulmonary fibrosis (IPF).

[0013] Furthermore, the concentration of GMI in Ganoderma lucidum microspores was less than 0.4 μM.

[0014] Additionally, the present invention also provides a method for treating, preventing or improving pulmonary fibrosis by administering to an individual microspore Ganoderma globulin GMI produced by fermentation of genetically modified Pichia pastoris strain Ey72.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention provides the use of GMI from Ganoderma lucidum microspores in the preparation of pharmaceuticals and food, and confirms that GMI does indeed have the effect of treating, preventing and / or improving TGF-β-induced IPF fibrosis. Attached Figure Description

[0017] Figure 1 This is a statistical graph showing the effect of GMI on the survival rate of LL29 cells.

[0018] Figure 2A This is a microscopic image of LL29 cell migration induced by GMI and TGF-β1.

[0019] Figure 2B for Figure 2A Quantitative statistical charts.

[0020] Figure 3A Western ink dot analysis of the antifibrotic activity of GMI in TGF-β1-induced fibrotic LL29 cells.

[0021] Figure 3B , Figure 3C , Figure 3D The graphs show the quantitative statistical analysis of the antifibrotic activities of GMI in α-SMA, COL1A1, and fibronectin in TGF-β1-induced fibrotic LL29 cells.

[0022] Figure 4A , Figure 4B The figures show the changes in body weight and the rate of change in body weight in mice treated with GMI to induce BLM-induced antifibrosis.

[0023] Figure 5 The graph shows the changes in survival rate in mice treated with GMI to induce BLM-induced antifibrosis.

[0024] Figure 6A , Figure 6B The figures show the changes in lung weight and the rate of change in lung weight in mice treated with GMI to induce BLM-induced antifibrosis.

[0025] Figure 7 The images show the color of the lungs of mice in the normal group and each control group. Detailed Implementation

[0026] The following are some preferred embodiments of the present invention, along with further detailed descriptions in conjunction with the accompanying drawings:

[0027] As used in this specification, “treatment” means a method of obtaining a favorable or desired outcome, including but not limited to therapeutic and / or preventive benefits, where the therapeutic benefit relates to the eradication or improvement of the underlying condition being treated.

[0028] As used in this specification, the terms "preventing" or "ameliorating" are used interchangeably, referring to the reduction in measurable parameters of a cell (tissue or individual) after treatment compared to an untreated cell (tissue or individual). Such comparisons may also be made between the same cells, tissues, or individuals before and after treatment.

[0029] As used in this specification, "individual" refers to a human or a non-human animal.

[0030] In one embodiment, the present invention provides the use of GMI from *Ganoderma lucidum* for the preparation of a pharmaceutical product, wherein the pharmaceutical product is prepared from *Ganoderma lucidum* globulin produced by fermentation of a yeast strain, and is used to treat, prevent, or improve pulmonary fibrosis. The aforementioned yeast strain is, for example, the genetically modified *Pichia pastoris* strain Ey72.

[0031] In addition, in one embodiment, the present invention provides the use of GMI of Ganoderma lucidum microspores for the preparation of food, wherein the aforementioned drug is prepared from Ganoderma lucidum microspore globulin produced by fermentation of genetically modified Pichia pastoris Ey72 strain, and is used to prevent or improve pulmonary fibrosis.

[0032] In some embodiments, GMI from Microspore Ganoderma lucidum inhibits the formation of pulmonary fibrosis by suppressing the activity of LL29 cells (human pulmonary fibroblasts) in the lungs.

[0033] In some embodiments, GMI from Ganoderma lucidum microspores can inhibit the three pulmonary fibrosis protein indices of LL29 cells. These three pulmonary fibrosis proteins include fibronectin, the COL1A1 gene, and alpha smooth muscle actin (α-SMA).

[0034] In some embodiments, the aforementioned drug is prepared from microspore Ganoderma lucidum globulin produced by fermentation of genetically modified Pichia pastoris strain Ey72, and pulmonary fibrosis refers to idiopathic pulmonary fibrosis (IPF).

[0035] In some embodiments, the concentration of Ganoderma lucidum microspore GMI is less than 0.4 μM.

[0036] Furthermore, in one embodiment, the present invention also provides a method for treating, preventing, or improving pulmonary fibrosis by administering to an individual microspore Ganoderma globulin GMI produced by fermentation of a genetically modified Pichia pastoris strain Ey72.

[0037] The microspore Ganoderma lucidum GMI of the present invention can be administered without intestinal administration, such as by intravenous injection or infusion, intraperitoneal injection, subcutaneous injection, or intramuscular injection. The microspore Ganoderma lucidum GMI can also be administered orally or by formulation with a carrier and / or excipients to form tablets, pills, capsules, liquids, gels, syrups, liquids, suspensions, and the like.

[0038] Of course, no further detailed description is necessary; those skilled in the art can utilize this invention to the fullest extent possible based on existing techniques. Therefore, the following examples should be understood as illustrative only and in no way limit the scope of the invention.

[0039] Example:

[0040] The purpose of this invention is to study and verify the anti-fibrotic properties of GMI using an in vitro model to combat TGF-β-induced fibrosis. The therapeutic activity of GMI will be determined in in vitro model (IPF cell line) experiments.

[0041] 1. Human in vitro cell experiments: Studying the effects of GMI on pulmonary fibrosis using a human IPF cell model:

[0042] In in vitro experiments, cells were pretreated with drugs and stimulated with recombinant TGF-β1 to induce fibrosis. In in vitro studies, TGF-β1 plays a crucial role in inducing epithelial-mesenchymal transition (EMT), a driving force behind myofibroblast transformation. LL29 cells were derived from lung fibroblasts of IPF patients. After reaching 70% confluence in culture dishes, cells were pretreated for 2 hours in low-serum medium with selected drugs (two concentrations each). Subsequently, cells were exposed to rTGF-β at a concentration of 5 ng / ml (R&D Systems, Minnesota, USA) for 48 hours.

[0043] Microspore Ganoderma GMI:

[0044] This is a concentrated extract of Ganoderma lucidum microspores manufactured by Mycomagic Biotechnology Co., Ltd.

[0045] Cell acquisition and culture:

[0046] LL29 cells are human lung fibroblasts / pulmonary fibroblasts isolated from IPF lung tissue. LL29 cells were purchased from [unclear - possibly a company name]. Cells were cultured in Dulbecco's modified Eagle's medium (Gibco, USA) with 10% FBS (Gibco, USA) at 37°C, >95% humidity and 5% CO2, and passaged every 2-3 days.

[0047] Cytotoxicity test:

[0048] LL29 cells were used to evaluate the toxicity of the GMI reagent. The toxicity assay was a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Prior to the cytotoxicity test, LL29 cells were trypsinized and seeded into 96-well plates at 3000 cells per well. After overnight attachment, the cell culture medium was replaced with medium containing the GMI reagent. After 48 hours of treatment, the test medium was replaced with MTT-labeled medium (final MTT concentration 0.5 mg / ml). After further incubation at 37°C for 4 hours, 100 μl of dissolving solution was added, and the plates were allowed to stand overnight in an incubator. Complete dissolution of the purple azo crystals was checked, and the absorbance of the sample was measured using a microplate (ELISA) reader. The absorbance of the azo product was measured at wavelengths between 550 and 600 nm, depending on the filter available on the ELISA reader. A reference wavelength greater than 650 nm was recommended.

[0049] Wound healing test:

[0050] A 500 ± 100 μm cell gap was created using a two-well culture insert. Microscopic images were captured at the same location in each group at 0 and 24 hours. The cell gap area in the micrographs was quantified using the Robust Quantitative Scratch Assay (RQSA) algorithm. The wound healing percentage was calculated as ((Area0 - Area0)) / (Area0 × 100%), where Area0 is the area of ​​the injured region immediately after the scratch in each microscopic image, and Area0 is the area of ​​the open region after the specified time.

[0051] Western ink dot analysis:

[0052] Cell lysates were prepared in RIPA buffer. For Western ink dot analysis, the same amount of lysate sample was separated by SDS-PAGE and then transferred to a PVDF membrane. Antibodies against COL1A1, fibronectin, and α-SMA were purchased from GeneTex (Taiwan). HRP-labeled secondary antibodies were obtained from Jackson Immuno Research Laboratories, Inc. (USA). Protein visualization using the ECL suite (Advansta Inc., USA). Protein expression levels were quantified using Fiji.

[0053] Statistical analysis:

[0054] Data (N=3) were analyzed and plotted using GraphPad Prism 7.03 (GraphPad, USA). Numerical values ​​are expressed as mean ± standard error mean (SEM). t-tests were performed to determine statistical significance between two groups. Dunnett's multiple comparison test was used after one-way analysis of variance to determine statistical significance between the control group and two or more treatment groups. Statistical significance is indicated by an asterisk or topic label (e.g., *, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0055] The effect of GMI on LL29 cell survival:

[0056] To investigate the effect of GMI on the pulmonary fibrosis process, this invention used the LL29 cell line, which is a lung fibroblast isolated from IPF patients. First, this invention evaluated the cytotoxicity of GMI in the LL29 cell line using an MTT assay. The LL29 cell line was treated for 48 hours with GMI concentrations ranging from 0 to 0.4 μM. When the GMI concentration was less than 0.4 μM, no significant cytotoxicity was observed in the LL29 cell line. Figure 1 As shown, Figure 1 In this study, the cell viability of LL29 cells treated with different concentrations of GMI was determined by the MTT assay. Data are presented as mean ± standard error. A Dunnett test was performed after one-way variance analysis to determine statistical significance between the control group and other groups.

[0057] GMI inhibits TGF-β1-induced LL29 cell migration:

[0058] Transformation of fibroblasts into myofibroblasts was induced using TGF-β1 (transforming growth factor beta 1) at 5 ng / mL. This invention performed a co-treatment experiment with TGF-β1 (5 ng / mL) and GMI (0.1 and 0.2 μM) in LL29 cells. This invention also includes a positive control group treated with the clinically used drug Pirfenidone (PFD). A wound healing assay was performed for 48 hours, with observations at different time points of 0, 24, and 48 hours after co-treatment. Figure 2A and Figure 2B As shown, compared with the NT group, TGF-β1 treatment significantly increased cell wound healing. Conversely, when TGF-β1 was combined with GMI to treat LL29 cells, the wound healing effect showed a dose-dependent inhibitory effect. Notably, the inhibitory effect of GMI on wound healing was stronger than that observed in the PFD group. Figure 2ATo capture microscopic images of the wound healing experiment at 40x magnification, the leading edge of the transition is indicated by a dashed line. Figure 2B To quantify the percentage of wound healing (area in the middle of the dashed line) in each microscopic image and compare it with the 0-hour control group at different time points, data were presented as mean ± standard error. A Dunnett's test following one-way variable analysis was performed to determine statistical significance between the control group and other groups (*p<0.05; **p<0.01; ***p<0.001).

[0059] Secondly, by Figure 2A and Figure 2B It is known that TGF-β1 induces pulmonary fibrosis in LL29 cells. During the fibrosis process, the crawling ability of LL29 cells increases, leading to accelerated closure of the two dashed lines in various microscopic images; the faster the closure, the more severe the fibrosis. Conversely, GMI can reduce the closure speed of the two dashed lines in various microscopic images. GMI is effective at a concentration of 0.1 μM, effectively reducing the crawling (tissue proliferation) and closure of LL29 cells, thus reducing the condition of pulmonary fibrosis tangles. Of course, GMI at concentrations of 0.1 μM and 0.2 μM can effectively inhibit the crawling ability of LL29 cells and the formation of pulmonary fibrosis.

[0060] GMI improves the pathological mechanism of TGF-β1-induced fibrotic LL29 cells:

[0061] This invention determined the levels of fibronectin, COL1A1, and α-SMA, as these proteins are associated with myofibroblasts and the extracellular matrix (ECM). Figures 3A to 3D As shown, compared with the control group, the levels of COL1A1, α-SMA, and fibronectin were increased in the TGF-β1 group, while GMI treatment decreased the expression of these proteins. The results indicate that GMI can reduce TGF-β1-induced fibrosis in LL29 cells, producing an anti-fibrotic effect, while PFD cannot reduce the increased expression levels of these proteins induced by TGF-β1. Figure 3A Western ink dot analysis to visualize fibronectin, COL1A1, α-SMA, and α-tubulin. Figures 3B to 3D To be Figure 3A Protein expression was quantified, showing fold changes in Fibronectin / α-tubulin, COL1A1 / α-tubulin, and α-SMA / α-tubulin (NT group for the standard group). Data were presented as mean ± standard error. Dunnett's test was performed after one-way variable analysis to determine statistical significance between the control group and other groups (*p<0.05; **p<0.01; ***p<0.001).

[0062] Increased expression and cell migration of α-smooth muscle actin are closely associated with acute and chronic fibrotic states. Furthermore, TGF-β1 is known to promote fibrotic processes, including the differentiation of fibroblasts into myofibroblasts. Therefore, this invention utilizes the LL29 cell migration model to test and validate the efficacy of GMI in the treatment of pulmonary fibrosis.

[0063] 2. Mouse pulmonary fibrosis experiment:

[0064] As shown in Table 1, five groups of C57BL / 6J mice aged 6 to 8 weeks were fed, with at least 6 mice in each group. On day 1, four control groups were injected with BLM (Bleomycin) 2.5 U / kg to induce pulmonary fibrosis, and one group was a normal group. Among them, one control group was injected with BLM only, two control groups were injected with different doses of GMI (1 mg / kg and 2 mg / kg) respectively, and another control group was injected with the clinical drug PFD 200 mg / kg. All mice that were still alive on day 22 were sacrificed.

[0065] Table 1:

[0066]

[0067] Mouse body weight observation:

[0068] like Figure 4A , Figure 4B As shown, the body weight of mice induced by BLM was significantly lower than that of normal mice, and the use of GMI can effectively prevent the weight loss of mice induced by BLM.

[0069] Mouse survival rate observation:

[0070] Secondly, such as Figure 5 As shown, the survival rate of mice induced by BLM was 40%, the survival rate of mice treated with the clinical drug PFD was 50%, the survival rate of mice treated with GMI 1 mg / kg was 70%, and the survival rate of mice treated with GMI 2 mg / kg was as high as 100% (day 14), which is better than the clinical drug PFD.

[0071] Lung weight observation:

[0072] like Figure 6A , Figure 6B As shown, BLM-induced pulmonary fibrosis in mice increases lung weight (inflammation and fibrosis increase lung weight), while GMI treatment effectively maintains normal lung weight in BLM-induced mice. Furthermore, BLM-induced fibrosis results in a high rate of change in lung weight in mice, while GMI treatment reduces this rate of change.

[0073] Observation of lung color:

[0074] like Figure 7 As shown, the lung color of mice in the normal group and each control group was observed. The lungs of mice induced by BLM appeared redder, while the lungs of mice treated with GMI and PFD were closer to the lung color of mice in the normal group.

[0075] Accordingly, this invention demonstrates the therapeutic potential of GMI in alleviating TGF-β1-induced intracellular pulmonary fibrosis in LL29 cells, primarily through inhibiting myofibroblast activation. Specifically, GMI exhibits unique functions in suppressing migration activity and reducing ECM components and fibrotic factor protein levels. Notably, the inhibitory effect of GMI on myofibroblast activation is even more pronounced than that in the PFD group, indicating greater promise for clinical treatment of IPF.

[0076] In summary, the use of GMI from Ganoderma lucidum microspores provided by this invention for the preparation of medicines and food has been demonstrated through in vitro human cell experiments and mouse experiments that GMI does indeed have the effect of treating, preventing or improving pulmonary fibrosis.

[0077] The above description is only a few preferred embodiments of the present invention and is not intended to limit the present invention. Any person skilled in the art can make some modifications, alterations and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. The use of a GMI of Ganoderma lucidum microspores in the preparation of a drug, characterized in that, This drug is prepared from microspore Ganoderma lucidum globulin produced by fermentation of yeast strains and is used to treat, prevent and / or improve pulmonary fibrosis.

2. The use as described in claim 1, characterized in that, GMI from microspore Ganoderma lucidum inhibits the formation of pulmonary fibrosis by suppressing the activity of LL29 cells in the lungs.

3. The use as described in claim 2, characterized in that, GMI from microspore Ganoderma lucidum can inhibit three pulmonary fibrosis protein indices in LL29 cells, including fibronectin, COL1A1 gene and α smooth muscle actin.

4. The use as described in claim 1, characterized in that, This drug is prepared using microspore Ganoderma lucidum-like globulin produced by fermentation of genetically modified Pichia pastoris strain Ey72. Pulmonary fibrosis refers to idiopathic pulmonary fibrosis.

5. The use as described in claim 1, characterized in that, The concentration of GMI in microspore Ganoderma lucidum is less than 0.4 μM.

6. The use of a GMI of microspore Ganoderma lucidum in the preparation of food, characterized in that, This drug is prepared using microspore Ganoderma lucidum globulin produced by fermentation of genetically modified Pichia pastoris strain Ey72, and is used to prevent and / or improve pulmonary fibrosis.

7. The use as described in claim 6, characterized in that, GMI from microspore Ganoderma lucidum inhibits the formation of pulmonary fibrosis by suppressing the activity of LL29 cells in the lungs.

8. The use as described in claim 7, characterized in that, GMI from microspore Ganoderma lucidum can inhibit the indices of three pulmonary fibrosis proteins in LL29 cells. These three pulmonary fibrosis proteins include reticulin, COL1A1 gene and α smooth muscle actin.

9. The use as described in claim 6, characterized in that, Pulmonary fibrosis refers to idiopathic pulmonary fibrosis.

10. The use as described in claim 6, characterized in that, The concentration of GMI in microspore Ganoderma lucidum is less than 0.4 μM.

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