Application of Pleurotus ostreatus extract in the preparation of programmed necrosis inhibitors

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of styrax extract in the preparation of programmed necrosis inhibitors. In vitro experiments demonstrate that styrax extract can inhibit TSZ-induced programmed necrosis in HT29 cells (human colorectal adenocarcinoma cells). Therefore, styrax extract can be used to construct an anti-programmed necrosis colorectal adenocarcinoma cell model. This model can be used to screen or develop targeted drugs specifically targeting the anti-programmed necrosis mechanism of tumor cells, overcoming the treatment resistance problem caused by the anti-programmed necrosis characteristics of tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of styrax extract in the preparation of programmed necrosis inhibitors. Background Technology

[0002] Necroptosis is a newly discovered form of programmed cell death mediated by death receptors and independent of caspase proteolytic enzymes. It is characterized by organelle swelling, loss of cell membrane integrity, and release of cytoplasmic contents and is one of the most important programmed cell death pathways in ulcerative colitis ectopic cells (IECs).

[0003] As a pleiotropic pro-inflammatory cytokine, tumor necrosis factor (TNF) is considered a major driver of intestinal epithelial cell death and is currently the most studied pathway inducing programmed necrosis. Upon TNF stimulation, TNFR1 typically recruits the TNF receptor-associated death domain, TNF receptor-associated factor 2, apoptosis inhibitor protein 1 / 2, and receptor-interacting protein kinase 1 (RIPK1) to form complex I on the plasma membrane, thereby mediating NF-κB signaling. When Caspase-8 activity is inhibited, RIPK1 and RIPK3 interact through their RIP homology interaction domains to form the RIPK1-RIPK3 necrosome, activating the mixed lineage kinase domain-like protein (MLKL). MLKL oligomerizes and translocates to the cell membrane, leading to cell membrane rupture and the occurrence of programmed necrosis. After the cell membrane ruptures, a large amount of cellular contents are released, and damage-associated molecular patterns (DAMPs) activate pattern recognition receptors, inducing a severe inflammatory response in the gut.

[0004] Programmed necrosis plays a crucial role in physiological and pathological processes such as growth and development, cell proliferation, immunity, and inflammation. It primarily affects inflammatory diseases, neurological diseases, immune disorders, and ischemia-reperfusion disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Alzheimer's disease, inflammatory bowel disease, and rheumatoid arthritis. Therefore, the discovery of programmed necrosis inhibitors is of great significance for the treatment of cell death-related diseases. Summary of the Invention

[0005] The purpose of this invention is to further expand the applications of sclerotium truncatum and provide its use in the preparation of inhibitors of programmed necrosis.

[0006] To achieve the above objectives, the present invention first provides the application of styrax extract in the preparation of programmed necrosis inhibitors.

[0007] This invention demonstrates through in vitro experiments that styrax extract can inhibit TSZ-induced programmed cell death in HT29 cells (human colorectal adenocarcinoma cells).

[0008] Therefore, the programmed necrosis inhibitor is further used to construct an anti-programmed necrosis cell model.

[0009] Anti-programmed necrosis tumors refer to tumors that resist or inhibit the programmed necrosis process in tumor cells. Tumor cells may evade programmed necrosis through certain mechanisms, thus continuing to survive, proliferate, and develop into tumors. For example, some anti-necrosis proteins, such as FLIP and PARP-1, can inhibit programmed necrosis in cancer cells, which is one manifestation of tumor cells' resistance to programmed necrosis.

[0010] Studying tumors resistant to programmed necrosis (CNN) helps to deepen our understanding of drug resistance mechanisms in tumor cells. When tumor cells become resistant to programmed necrosis, they may develop resistance to existing cancer treatments based on inducing CNN (such as certain chemotherapy drugs, radiotherapy, and targeted therapies, which induce CNN in cancer cells). Investigating these resistance mechanisms can provide insights into developing new cancer treatment strategies to overcome tumor cell resistance and improve the effectiveness of cancer treatment.

[0011] Furthermore, the programmed necrosis inhibitor is used to prepare a drug for treating programmed necrosis-related diseases.

[0012] Furthermore, the drug uses styrax as its sole active ingredient, and is formulated from styrax and pharmaceutically acceptable excipients selected from carriers or excipients. In preparing drugs for treating diseases related to programmed necrosis, styrax is typically mixed with excipients, diluted with excipients, or encapsulated in a carrier that can be in capsule or pouch form. When the excipient acts as a diluent, it can be a solid, semi-solid, or liquid material serving as the medium for the excipient, carrier, or active ingredient. Therefore, the drug can be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions, etc. Examples of suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, etc. The drug may also include: wetting agents, emulsifiers, preservatives (such as methylparaben and propylparaben), sweeteners, etc.

[0013] Furthermore, the programmed necrosis-related diseases include ulcerative colitis.

[0014] Secondly, this invention provides a method for constructing a colorectal adenocarcinoma cell model resistant to programmed necrosis, using the aforementioned styraxein. The method includes: pretreating colorectal adenocarcinoma cells with 9.5–10.5 nM SM-164 and 19.5–20.5 μM caspase inhibitor z-VAD-fmk for 28–32 min, adding 19.5–20.5 ng / mL h-TNF-α and 0.15–8 μM of the aforementioned styraxein, and incubating together for 10–12 h, using h-TNF-α to induce programmed necrosis of colorectal adenocarcinoma cells, and using styraxein to inhibit the necrosis of colorectal adenocarcinoma cells.

[0015] Furthermore, the colorectal adenocarcinoma cells are HT-29 cells.

[0016] Furthermore, this invention provides a colorectal adenocarcinoma cell model resistant to programmed necrosis, which is constructed using the above-described method.

[0017] Finally, the present invention provides any of the following applications of the aforementioned resistant colorectal adenocarcinoma cell model:

[0018] 1) To study the pathogenesis of colorectal adenocarcinoma resistant to programmed necrosis;

[0019] 2) Screening for drugs to treat colorectal adenocarcinoma with anti-programmed necrosis.

[0020] The present invention has the following beneficial effects:

[0021] This invention is the first to discover that phenomenin can act as a programmed necrosis inhibitor to suppress programmed necrosis in colorectal adenocarcinoma cells. Pphenomenin can be used to construct a programmed necrosis-resistant colorectal adenocarcinoma cell model. This model can be used to screen or develop targeted drugs that specifically target the programmed necrosis-resistant mechanism of tumor cells, thereby overcoming the treatment resistance problem caused by the programmed necrosis-resistant properties of tumor cells. Attached Figure Description

[0022] Figure 1 The anti-programmed necrosis activity of sclerotin includes:

[0023] A represents the chemical structural formula of scleroderma.

[0024] B shows representative images of HT-29 cells stained with Calcein-AM / PI 24 hours after receiving different treatments.

[0025] C represents the statistical graph of cell viability obtained by chemiluminescence assay after treating HT-29 cells with different concentrations of styraxin and then stimulating the cells with TSZ for 12 hours.

[0026] D represents the EC50 curve calculated based on cell viability.

[0027] *P<0.05 and **P<0.01 compared with the TSZ group.

[0028] Figure 2 The inhibitory effect of different doses and durations of treatment with styraxein on programmed necrosis.

[0029] A shows the results of Western Blot analysis of HT-29 cells treated with TSZ and different concentrations (10 μM, 5 μM and 2.5 μM) of styraxin for 6 hours.

[0030] B represents the Western Blot results of HT-29 cells treated with TSZ and 10 μM for 0, 2, 4, and 6 hours.

[0031] Figure 3 The protective effect of sclerotium twig extract against DSS-induced ulcerative colitis in mice, wherein:

[0032] A shows the DSS-induced mouse ulcerative colitis model and its drug administration flowchart.

[0033] B represents the change in the mouse's body weight during the experiment.

[0034] C is a representative image of a mouse colon.

[0035] D represents the DAI score in the experiment.

[0036] E represents the length of the colon on day 12.

[0037] F is a representative image of HE staining.

[0038] *P<0.05 and **P<0.01 compared with the DSS group.

[0039] Figure 4 The effect of sclerotin on the mucus barrier in UC mice, wherein:

[0040] A is a PAS-stained image of a representative colon tissue section.

[0041] Image B is an immunohistochemical image of MUC-2 staining in representative colon tissue.

[0042] C represents the quantitative result of PAS.

[0043] D represents the quantitative result after Muc2 standardization.

[0044] Compared with the DSS group, *P<0.05 and **P<0.01.

[0045] Figure 5 The effect of scleroderma on the intestinal mechanical barrier of UC mice, wherein:

[0046] A represents the effect of immunohistochemical detection on the distribution and expression of occupiedin in DSS-induced UC mice.

[0047] B represents the effect of immunohistochemical detection on the distribution and expression of ZO-1 in DSS-induced UC mice.

[0048] Quantitative results after standardization of Occludin (C) and ZO-1 (D).

[0049] *P<0.05 and **P<0.01 compared with the DSS group.

[0050] Figure 6 The effect of styrax extract on programmed necrosis of intestinal epithelial cells in UC mice, wherein:

[0051] A represents the distribution and expression of Tunel in colon tissue determined by immunofluorescence.

[0052] B represents the immunofluorescence assay for the distribution and expression of p-RIPK3 in colon tissue.

[0053] C represents the distribution and expression of p-MLKL in colon tissue determined by immunofluorescence.

[0054] Figure 7 The effects of styrax extract on the pathological characteristics of the heart, liver, spleen, lungs, and kidneys in UC mice. Detailed Implementation

[0055] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0056] Example 1: Flat-skinned vine extract has anti-programmed necrosis activity.

[0057] 1. Test Methods

[0058] Evaluation of in vitro anti-programmed necrosis activity: HT-29 cells were seeded in 96-well plates at 10,000 cells per well. The culture medium was McCoy's 5A (Procell, CM-0118) supplemented with 10% fetal bovine serum (Procell, 164210-50) and 1% penicillin antibody (Gibco, 15140-122).

[0059] Cells were divided into four groups: a DMSO group, a TSZ group, a DMSO+pristimerin group, and a TSZ+pristimerin group. The DMSO+pristimerin and TSZ+pristimerin groups each contained different concentrations of pristimerin. Pristimerin was sequentially diluted in DMSO to concentrations of 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15 μM, and 0.075 μM. After 12 h, cells in the TSZ and TSZ+pristimerin groups were pretreated with 10 nM SM-164 and 20 μM caspase inhibitor z-VAD-fmk for 30 min, followed by stimulation with 20 ng / mL h-TNF-α for 12 h. After 12 h, CellTiter-Lumi was used... TM Cell viability was determined using a luminescence assay kit, and luminescence readings were recorded using a SpectraMax M5 microplate reader. The effect of phenomenin on improving TSZ-induced programmed cell death in HT-29 cells was calculated using Prism software. 50 Values. Double staining of live / dead cells: HT-29 cells were seeded at a density of 1×10,000 cells / well in 96-well plates and cultured for 12 hours. A programmed cell necrosis model was induced using TSZ, and after intervention with sclerotin, the culture medium was discarded, and Calcein-AM / PI working solution was added. The plates were incubated at 37°C in the dark for 30 minutes. Cell viability was observed under a microscope, with red representing dead cells and green representing live cells.

[0060] 2. Test Results

[0061] like Figure 1 As shown in the results, the assay results indicated that styraxin significantly enhanced cell viability in a dose-dependent manner, with an EC50 value of 8.289 μM calculated based on cell viability. Cell death was improved after styraxin treatment, and an increase in the number of viable cells was clearly observed after staining with a live / dead cell kit.

[0062] Example 2: The inhibitory effect of styrax extract on programmed necrosis in a dose- and time-dependent manner.

[0063] 1. Test Methods

[0064] A TSZ-induced programmed necrosis model was used in HT-29 cells. Different concentrations of styraxin were incubated for 0-6 hours. Western blotting was used to determine the effects of styraxin on the key proteins p-RIPK1, p-RIPK3, p-MLKL, and MLKL in programmed necrosis. The specific procedure is as follows:

[0065] (1) Protein sample preparation

[0066] Discard the supernatant from the processed cells and place them on ice. Wash twice with pre-cooled PBS, add an appropriate amount of cell lysis buffer, and incubate on ice for 10 min. Collect the cells using a cell scraper into a 1.5 mL EP tube and centrifuge at 12000 RPM for 5 min at 4°C. Take the supernatant, add 5× protein electrophoresis buffer, and boil for 7 min.

[0067] (2) Protein concentration determination

[0068] Set up the standard curve according to the BCA protein concentration assay kit instructions, add the test samples, and set up one auxiliary well for each group. Add 200 μL of BCA working solution to each well, incubate in a 55℃ shaking incubator for 15 min, and measure the absorbance at 562 nm wavelength on a microplate reader. Calculate the protein concentration based on the standard curve and the absorbance values ​​of each group, and calculate the loading volume of each group based on the equal amount of protein loaded.

[0069] (3) Gel preparation

[0070] Place the cleaned and dried glass plate on the gel preparation rack, check for leaks, and prepare the lower layer gel (8%-12%) according to the SDS-PAGE separating gel composition table. Add 200μL of isopropanol to remove air bubbles and flatten the gel surface. After the lower layer gel solidifies, blot the isopropanol with absorbent paper, prepare the upper layer gel, insert the comb, and wait for the gel to solidify before use.

[0071] (4) Electrophoresis

[0072] Load the sample according to the sample volume calculated based on the protein concentration. First, use 80V constant voltage electrophoresis until the protein markers are clear. After all groups of samples have reached the same level, adjust the voltage to 120V.

[0073] (5) Transfer membrane

[0074] Protein molecules from the SDS-PAGE gel were transferred to a solid support, a nitrocellulose filter membrane (NC membrane), which was matched to the size of the gel. Filter paper and a sponge were then placed on the NC membrane before it was placed in the transfer tank. The black electroporation tank, with the negative end of the gel on one side, was filled with ice. Electroporation was performed at a constant current of 250 mA for 60-90 minutes, depending on the molecular weight.

[0075] (6) Closed

[0076] Remove the NC membrane after electroporation termination, quickly place it in Ponceau S staining solution for staining, rinse with ultrapure water, and then seal it in 5% skim milk for 30 minutes.

[0077] (7) Incubation of primary antibody

[0078] Wash the membrane with TBST solution on a shaker for 5 min × 4 times. Dilute the primary antibody (anti-p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL antibody) in 1% skim milk, place it in the PVDF membrane with the corresponding molecular weight, and incubate overnight at 4°C with gentle shaking.

[0079] (8) Incubation of secondary antibody and membrane scanning

[0080] After incubating with enzyme-labeled secondary antibody for 1 hour, the membrane was scanned using an imaging system (Bio-Rad, Hercules, USA), and the optical density of the bands was analyzed using Image-J software.

[0081] 2. Test Results

[0082] like Figure 2 The results show that styraxin can inhibit programmed necrosis in a dose- and time-dependent manner.

[0083] Example 3: Protective effect of sclerotium tsao-ko on DSS-induced mouse UC model

[0084] 1. Test Methods

[0085] To confirm the protective effect of prismine on ulcerative colitis, a mouse model of ulcerative colitis induced by primidone saline (DSS) was used. After one week of acclimatization, mice were randomly divided into a control group (Con) and a model group (DSS). The control group received normal diet and water, while the other groups received 2.5% DSS for 7 days to induce ulcerative colitis (UC). After 7 days, DSS was removed, and the model group was randomly divided into four groups: DSS treatment group (DSS), high-dose primidone saline (PH, 3 mg / kg), low-dose primidone saline (PL, 1 mg / kg), mesalazine (100 mg / kg), and Nec-1 (5 mg / kg), with six mice in each group. The DSS group received normal diet and water. All treatment groups began administration after model establishment, once daily, with primidone and mesalazine administered by gavage and Nec-1 by intraperitoneal injection for 5 consecutive days. Blood and colon tissue samples were collected from all animals after the last administration on day 12. The length of the mouse colon was measured, and the colon tissue was used to prepare paraffin tissue sections, which were stained with hematoxylin and eosin (HE). Pathological changes were observed and photographed under an optical microscope.

[0086] 2. Test Results

[0087] like Figure 3The results showed that the phenomenol significantly improved the symptoms of ulcerative colitis caused by DSS. Compared with the model group, the mice recovered their body weight significantly, their DAI scores decreased, their stool bleeding and stool quality improved, their colon length increased, their intestinal permeability decreased, their submucosal edema in the colon was reduced, their loss of mucosal epithelial cells and goblet cells decreased, their inflammatory infiltration improved, and the effect of PH was comparable to that of the Nec-1 group.

[0088] Example 4: Flat-leaved vine extract can protect the intestinal barrier in DSS-induced UC mice

[0089] 1. Test Methods

[0090] Colon histopathology: Colon tissue of about 1 cm was taken from the severely ulcerated area of ​​the mouse, fixed in 4% paraformaldehyde, routinely embedded in paraffin, sectioned, and subjected to PAS staining and immunohistochemistry. Pathological changes were observed and photographed under an optical microscope.

[0091] 2. Test Results

[0092] like Figure 4 The results showed that in the DSS model, goblet cells were significantly reduced due to inflammatory damage, leading to a decrease in PAS-positive areas. The number of goblet cells in the treated mice was significantly improved, suggesting that Pristimerin can repair DSS-induced colonic tissue damage in UC mice. Immunohistochemistry revealed decreased Muc2 expression in the model group, while Pristimerin significantly promoted Muc2 expression in a dose-dependent manner, with the high-dose group showing the same effect as the positive control drug Nec-1.

[0093] like Figure 5 The results showed that, compared with the control group, the expression of Occludin and ZO-1 proteins in the colon tissue of mice in the DSS group was decreased, indicating that the integrity of the intestinal mucosal barrier of mice was damaged to varying degrees; after administration, treatment with pristimerin, mesalazine, and Nec-1 could significantly upregulate the expression of Occludin and ZO-1 proteins. Figure 5 The above results indicate that phenoxylate can promote the repair of the intestinal mucosal barrier and maintain the integrity of the intestinal barrier by upregulating the expression of tight junction protein in the intestine of UC mice.

[0094] Example 5: Flat-skinned vine extract alleviates programmed necrosis of intestinal epithelial cells in UC mice

[0095] 1. Test Methods

[0096] Using a DSS-induced mouse ulcerative colitis model, the distribution and expression of Tunel, p-RIPK3, and p-MLKL were determined by immunofluorescence.

[0097] 2. Test Results

[0098] like Figure 6 The results showed that, compared with the control group, the number of Tunel-positive cells in the intestinal epithelial cells of the model group mice was significantly increased, and the expression of pristimerin in both groups was reduced, with the high-dose effect consistent with that of the positive control drug. Figure 6 A). The results showed that pristimerin could improve apoptosis of intestinal epithelial cells. The phosphorylation levels of RIPK3 and MLKL were increased in the colon tissue of DSS mice. After administration of pristimerin, the phosphorylation levels of RIPK3 and MLKL were inhibited in a dose-dependent manner, and its high-dose effect was comparable to that of the Nec-1 group and the mesalazine group.

[0099] Example 6: Oral administration of *Platycodon grandiflorus* extract without side effects such as liver and kidney damage.

[0100] 1. Test Methods

[0101] Mouse heart, lung, liver, spleen, and kidney tissues were fixed with 4% paraformaldehyde, embedded, and prepared into paraffin sections. The sections were dewaxed to water, reheated, and fixed. The sections were then stained with hematoxylin for 3-5 minutes, washed, and then blued again. The sections were dehydrated in 95% alcohol for 1 minute, stained with eosin for 15 seconds, dehydrated, mounted, and examined under a microscope to collect images for analysis.

[0102] 2. Test Results

[0103] like Figure 6 The results showed that the herbal extract had no significant effect on the function of the heart, liver, spleen, lungs, and kidneys.

[0104] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0105] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0106] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for constructing a model of anti-programmed necrosis of colorectal adenocarcinoma cells, characterized by, The method is constructed by adopting the flat platicylate, and the method comprises the following steps: pre-treating colorectal adenocarcinoma cells by 9.5-10.5 nM SM-164 and 19.5-20.5 muM caspase inhibitor z-VAD-fmk for 28-32 min, adding 19.5-20.5 ng / mL h-TNF-alpha and 0.15-8 muM of the flat platicylate, and incubating for 10 h-12 h, wherein h-TNF-alpha is used to induce programmed necrosis of the colorectal adenocarcinoma cells, and the flat platicylate is used to inhibit necrosis of the colorectal adenocarcinoma cells, and the colorectal adenocarcinoma cells are HT-29 cells.

2. A model of anti-programmed necrosis of colorectal adenocarcinoma cells, characterized by, The model is constructed by the construction method in claim 1.

3. The anti-programmed necrosis colorectal adenocarcinoma cell model in claim 2 is applied to screening drugs for treating anti-programmed necrosis colorectal adenocarcinoma.

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