Application of flat plastic cane element in preparation of programmed necrosis inhibitor

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of flat plastic cane in preparation of a programmed necrosis inhibitor. In-vitro experiments prove that the flat plastic rattans can inhibit TSZ-induced programmed cell necrosis on HT29 cells (human colorectal adenocarcinoma cells). Therefore, a colorectal adenocarcinoma cell model capable of resisting programmed necrosis can be constructed by utilizing the flat plastic rattans, and a targeted drug specially aiming at a tumor cell anti-programmed necrosis mechanism can be screened or researched and developed by utilizing the model, so that the problem of treatment drug resistance caused by the characteristic of the tumor cell anti-programmed necrosis is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to the application of pyrrolizidine in the preparation of programmed necrosis inhibitors. Background Art

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

[0003] As a pleiotropic proinflammatory cytokine, tumor necrosis factor (TNF) is considered to be the main driver of intestinal epithelial cell death and the most studied pathway for inducing programmed necrosis. Under the stimulation of TNF, TNFR1 usually recruits tumor necrosis factor receptor-associated death domain, TNF receptor-associated factor 2, inhibitor of apoptosis 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 the RIP homology interaction domain to form RIPK1-RIPK3 necrosomes and activate mixed lineage kinase domain like protein (MLKL), which oligomerizes and translocates to the cell membrane and causes cell membrane rupture, leading to 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 to induce severe inflammatory response in the intestine.

[0004] Programmed necrosis plays an important role in physiological and pathological processes such as growth and development, cell proliferation, immunity and inflammation, mainly including inflammatory diseases, neurological diseases, immune diseases, ischemia-reperfusion, including amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, inflammatory bowel disease, rheumatoid arthritis, etc. 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 the present invention is to further expand the use of pyrrolidone and provide the use of pyrrolidone in the preparation of programmed necrosis inhibitors.

[0006] To achieve the above object, the present invention first provides the use of pyrrolizidine in the preparation of programmed necrosis inhibitors.

[0007] The invention proves through in vitro experiments that pyrrolidone can inhibit the programmed cell necrosis induced by TSZ method on HT29 cells (human colorectal adenocarcinoma cells).

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

[0009] Necrotic-resistant tumors refer to tumors that resist or inhibit the necrosis process of tumor cells. Tumor cells may escape necrosis through certain mechanisms, thereby continuing to survive, proliferate, and develop into tumors. For example, some anti-necrosis proteins such as FLIP and PARP-1 can inhibit cancer cell necrosis, which is a manifestation of tumor cell resistance to necrosis.

[0010] Studying necrosis-resistant tumors helps us gain a deeper understanding of the drug resistance mechanisms of tumor cells. When tumor cells are able to resist programmed necrosis, they may develop resistance to existing cancer treatments based on the induction of programmed necrosis (such as certain chemotherapy drugs, radiotherapy, and targeted therapy drugs, because these can induce programmed necrosis in cancer cells). By studying this resistance mechanism, we can provide ideas for the development of new cancer treatment strategies, overcome the drug resistance of tumor cells, and improve the effectiveness of cancer treatment.

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

[0012] Furthermore, the drug has pyrrolidone as the only active ingredient, and the drug is compounded with pyrrolidone and a pharmaceutically acceptable excipient, and the excipient is selected from one of a carrier or an excipient. When preparing a drug for treating a disease related to programmed necrosis, pyrrolidone is usually mixed with an excipient, or diluted with an excipient, or encapsulated in a carrier that can be in the form of a capsule or a sachet. When the excipient acts as a diluent, it can be a solid, semisolid or liquid material as an excipient, a carrier or a medium for the active ingredient. Therefore, the drug can be a tablet, a pill, a powder, a solution, a syrup, a sterile injection solution, etc. Examples of suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, etc. The drug may also include: a wetting agent, an emulsifier, a preservative (such as methyl and propyl hydroxybenzoate), a sweetener, etc.

[0013] Furthermore, the necroptosis-related disease includes ulcerative colitis.

[0014] Secondly, the present invention provides a method for constructing a colorectal adenocarcinoma cell model resistant to programmed necrosis, which is constructed using the above-mentioned pyrrolidone. The method comprises: pretreating colorectal adenocarcinoma cells with 9.5-10.5nM SM-164 and 19.5-20.5μM caspase inhibitor z-VAD-fmk for 28-32min, adding 19.5-20.5ng / mL of h-TNF-α and 0.15-8μM of the above-mentioned pyrrolidone, and incubating them together for 10h-12h, inducing programmed necrosis of colorectal adenocarcinoma cells using h-TNF-α, and inhibiting necrosis of colorectal adenocarcinoma cells using pyrrolidone.

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

[0016] Again, the present invention provides a colorectal adenocarcinoma cell model resistant to programmed necrosis, which is constructed by the above-mentioned construction method.

[0017] Finally, the present invention provides any of the following uses of the necrosis-resistant colorectal adenocarcinoma cell model:

[0018] 1) To study the pathogenesis of necroptosis-resistant colorectal adenocarcinoma;

[0019] 2) Screening for drugs to treat necroptosis-resistant colorectal adenocarcinoma.

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

[0021] The present invention discovers for the first time that pyrrolidone can be used as a programmed necrosis inhibitor to inhibit the programmed necrosis of colorectal adenocarcinoma cells. Pyrrolidone can be used to construct a colorectal adenocarcinoma cell model that is resistant to programmed necrosis. This model can be used to screen or develop targeted drugs specifically targeting the anti-programmed necrosis mechanism of tumor cells, so as to overcome the problem of treatment resistance caused by the anti-programmed necrosis characteristics of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The anti-necrosis activity of pyrrolidone, wherein:

[0023] A is the chemical structural formula of pyrrolizidine.

[0024] B is a representative image of Calcein-AM / PI staining of HT-29 cells after receiving different treatments for 24 h.

[0025] C is a statistical graph of cell activity obtained by chemiluminescence detection after HT-29 cells were treated with different concentrations of scutellaria baicalensis and then stimulated with TSZ for 12 hours.

[0026] D is the EC50 curve calculated based on cell activity.

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

[0028] Figure 2 The inhibition of programmed necrosis by treatment with different doses and durations of pyrrolizidine.

[0029] A is the Western Blot detection result of HT-29 cells treated with TSZ and different concentrations (10μM, 5μM and 2.5μM) of pyrrolizidine for 6 hours.

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

[0031] Figure 3 The protective effect of pyrrolidone on DSS-induced ulcerative colitis in mice:

[0032] A is the DSS-induced mouse ulcerative colitis model and drug administration flow chart.

[0033] B is the changes in mouse body weight during the experiment.

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

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

[0036] E is the colon length on the 12th day.

[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 pyrrolidone on the mucus barrier of UC mice:

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

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

[0042] C is the PAS quantitative result.

[0043] D is the quantitative result after Muc2 normalization.

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

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

[0046] A is the immunohistochemical detection of the effect of scutellaria baicalensis on the distribution and expression of occludin in DSS-induced UC mice.

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

[0048] Quantitative results after normalization 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 pyrrolidone on programmed necrosis of intestinal epithelial cells in UC mice, including:

[0051] A: Immunofluorescence determination of the distribution and expression of Tunel in colon tissue.

[0052] B: Immunofluorescence determination of the distribution and expression of p-RIPK3 in colon tissue.

[0053] C: Immunofluorescence analysis of the distribution and expression of p-MLKL in colon tissue.

[0054] Figure 7 The effects of pyrrolizidine on the heart, liver, spleen, lung and kidney pathologies of UC mice. DETAILED DESCRIPTION

[0055] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0056] Example 1: Psoralen has anti-necrosis activity

[0057] 1. Test methods

[0058] In vitro evaluation of anti-necroptosis activity: HT-29 cells were seeded in 96-well plates, with 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% double antibody (Gibco, 15140-122).

[0059] The DMSO group, TSZ group, DMSO+administration group (DMSO+pristimerin) and TSZ+administration group (TSZ+pristimerin) were set up, where the DMSO+administration group and the TSZ+administration group both included different concentrations of pristimerin. Pristimerin was diluted in DMSO to concentrations of 5μM, 2.5μM, 1.25μM, 0.625μM, 0.3125μM, 0.15μM, and 0.075μM. After 12 hours, the cells in the TSZ group and the TSZ+administration group were pretreated with 10nM SM-164 and 20μM caspase inhibitor z-VAD-fmk for 30 minutes, and then the cells were stimulated with 20ng / mL h-TNF-α for 12 hours. After 12 hours, the cells were treated with CellTiter-Lumi TM Cell viability was determined using a luminescence detection kit, and luminescence readings were recorded using a SpectraMax M5 microplate reader. The EC value of pyrrolidone to improve TSZ-induced necrosis in HT-29 cells was calculated using Prism software. 50 Live / dead cell double staining, HT-29 cells were plated in 96-well plates at a density of 1×10000 / well and cultured for 12 hours. TSZ-induced programmed necrosis model, and after intervention with plastolide, the solution was discarded, Calcein-AM / PI working solution was added, and the cells were incubated at 37℃ in the dark for 30 minutes. The survival of cells in each group was observed under a microscope, where red represents dead cells and green represents live cells.

[0060] 2. Test results

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

[0062] Example 2: The inhibition of programmed necrosis by pyrrolidone is dose- and time-dependent

[0063] 1. Test methods

[0064] The HT-29 cell programmed necrosis model was induced by TSZ, and different concentrations of pyralidin were incubated for 0-6 hours. The WB method was used to determine the effects of pyralidin on the key proteins of programmed necrosis, p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL. The specific process is as follows:

[0065] (1) Protein sample preparation

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

[0067] (2) Protein concentration determination

[0068] According to the instructions of the BCA protein concentration determination kit, set up the standard curve, add the sample to be tested, and set up one secondary well for each group. Add 200μL of BCA working solution to each well, incubate in a 55℃ shaking incubator for 15min, measure the absorbance at a wavelength of 562nm on an ELISA reader, and obtain the protein concentration based on the standard curve and the absorbance of each group. Calculate the loading volume of each group based on the equal amount of protein loading.

[0069] (3) Gel preparation

[0070] Place the washed and dried glass plate on the gel preparation rack, check for leaks, prepare the lower gel (8%-12%) according to the SDS-PAGE separation gel composition table, add 200μL of isopropanol to remove bubbles and flatten the gel surface. After the lower gel solidifies, absorb the isopropanol with absorbent paper, prepare the upper gel, and then insert the comb. After the gel solidifies, set aside.

[0071] (4) Electrophoresis

[0072] Load the sample according to the sample volume calculated according to the protein concentration, and first use 80V constant voltage electrophoresis until the protein marker runs away. After each group of samples runs to the same horizontal line, adjust the voltage to 120V.

[0073] (5) Transfer

[0074] Transfer the protein molecules on the SDS-PAGE gel to a solid phase carrier nitrocellulose filter membrane (NC membrane) that matches the gel size, cover the NC membrane with filter paper and sponge in turn, and then put it into the transfer tank. Add ice cubes to the black electrotransfer tank at the negative end of the gel side, and electrotransfer at a constant current of 250mA for 60-90min according to the molecular weight.

[0075] (6) Closed

[0076] The NC membrane after electrotransfer was terminated was taken out, and was quickly placed in Ponceau red dye solution for staining, and then rinsed with ultrapure water and placed in 5% skim milk for blocking for 30 minutes.

[0077] (7) Incubation with primary antibody

[0078] The membrane was washed with TBST solution on a shaker for 5 min × 4 times, and the primary antibody (anti-p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL antibody) was diluted in 1% skim milk, placed on a PVDF membrane corresponding to the molecular weight, and incubated overnight in a 4°C refrigerator with slow shaking.

[0079] (8) Incubation with secondary antibody and scanning

[0080] After incubation with enzyme-labeled secondary antibody for 1 h, 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 showed that pyrrolizidine could inhibit programmed necrosis in a dose- and time-dependent manner.

[0083] Example 3: Protective effect of pyrrolizidine on DSS-induced UC model in mice

[0084] 1. Test methods

[0085] To confirm the protective effect of Pristimerin on ulcerative colitis, a DSS-induced mouse ulcerative colitis model was used. After one week of adaptive feeding, the mice were randomly divided into a control group (Con) and a model group (DSS). The control group received normal diet and water, and the other groups were given 2.5% DSS for 7 days to induce UC. After 7 days, DSS was withdrawn, and the model group was randomly divided into a DSS-treated group (DSS), a high-dose Pristimerin (PH, Pristimerin 3mg / kg), a low-dose Pristimerin (PL, Pristimerin 1mg / kg), Mesalazine (Mesalazine, 100mg / kg) and Nec-1 (5mg / kg), with six mice in each group. The DSS group received normal diet and water, and each drug-treated group began to receive drugs after the model was established, once a day, Pristimerin and Mesalazine were gavaged, and Nec-1 was intraperitoneally injected, for 5 consecutive days. After the last drug administration on the 12th day, blood and colon tissue samples were collected from all animals. The length of the mouse colon was measured, and the colon tissue was used to make paraffin tissue sections, which were stained with HE. The pathological changes were observed under an optical microscope and photographed.

[0086] 2. Test results

[0087] like Figure 3The results showed that plastolide can significantly improve the symptoms of ulcerative colitis caused by DSS. Compared with the model group, the mice recovered significantly, the DAI score was reduced, the blood in stool and stool consistency were improved, the colon length was increased, the intestinal permeability was reduced, the submucosal edema of the colon of mice was alleviated, the loss of mucosal epithelial cells and goblet cells was reduced, the inflammatory infiltration was improved, and the PH effect was equivalent to that of the Nec-1 group.

[0088] Example 4: Psoralen can protect the intestinal barrier of DSS-induced UC mice

[0089] 1. Test methods

[0090] Colon tissue pathology: Colon tissue of mice with more serious ulcers, about 1 cm, was obtained and fixed with 4% paraformaldehyde, embedded in conventional paraffin, sectioned, and subjected to PAS staining and immunohistochemistry. Pathological changes were observed under an optical microscope and photographed.

[0091] 2. Test results

[0092] like Figure 4 The results showed that in the DSS model, due to inflammatory damage, goblet cells were significantly reduced, resulting in a decrease in PAS-positive areas. The number of goblet cells in the drug-treated group was significantly improved, suggesting that Pristimerin can repair DSS-induced colon tissue damage in UC mice. Immunohistochemistry showed that the expression of Muc2 in the model group decreased, while Pristimerin could significantly promote the expression of Muc2 in a dose-dependent manner, and the effect of the high-dose group was the same as that of the positive 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 decreased, indicating that the integrity of the intestinal mucosal barrier of mice was damaged to varying degrees; after administration of pristimerin, mesalazine, and Nec-1 treatments could significantly upregulate the expression of Occludin and ZO-1 proteins ( Figure 5 The above results indicate that pyrrolizidine can promote the repair of intestinal mucosal barrier and maintain the integrity of intestinal barrier by upregulating the expression of intestinal tight junction proteins in UC mice.

[0094] Example 5: Psoralen reduces programmed necrosis of intestinal epithelial cells in UC mice

[0095] 1. Test methods

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

[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 increased significantly, and the expression of pristimerin-treated groups decreased, and the high-dose effect was consistent with that of the positive drug ( Figure 6 A). The results showed that pristimerin can improve the apoptosis of intestinal epithelial cells. The phosphorylation levels of RIPK3 and MLKL in the colon tissue of DSS mice increased. After administration of pristimerin, the phosphorylation levels of RIPK3 and MLKL were inhibited in a dose-dependent manner. The high-dose effect was comparable to that of the Nec-1 group and the mesalazine group.

[0099] Example 6: No side effects such as liver and kidney damage after oral administration of pyrrolidone

[0100] 1. Test methods

[0101] The heart, lung, liver, spleen and kidney tissues of mice were fixed with 4% paraformaldehyde, embedded, made into paraffin sections, dewaxed to water, rewarmed and fixed, and the sections were dyed in hematoxylin solution for 3-5 minutes, washed and blued, dehydrated in 95% alcohol for 1 minute, dyed in eosin solution for 15 seconds, dehydrated, sealed and examined under a microscope for image analysis.

[0102] 2. Test results

[0103] like Figure 6 The results showed that pyrrolizidine had no significant effect on the heart, liver, spleen, lung and kidney functions.

[0104] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0105] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0106] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of physostigmine in the preparation of programmed necrosis inhibitors.

2. The use of pyrrolizidine in the preparation of a programmed necrosis inhibitor according to claim 1, characterized in that: The programmed necrosis inhibitor is used to construct a programmed necrosis-resistant tumor cell model.

3. The use of pyrrolizidine in the preparation of a programmed necrosis inhibitor according to claim 2, characterized in that: The tumor cells include colorectal adenocarcinoma cells.

4. The use of pyrrolizidine in the preparation of a programmed necrosis inhibitor according to claim 1, characterized in that: The programmed necrosis inhibitor is used for preparing medicines for treating diseases related to programmed necrosis.

5. The use of pyrrolizidine in the preparation of a programmed necrosis inhibitor according to claim 1, characterized in that: The medicine has pyrrolidone as the only effective ingredient and is prepared by compounding pyrrolidone and pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials are selected from one of carriers or excipients.

6. The use of pyrrolizidine in the preparation of a programmed necrosis inhibitor according to claim 5, characterized in that: The necroptosis-related diseases include ulcerative colitis.

7. A method for constructing a colorectal adenocarcinoma cell model resistant to programmed necrosis, characterized in that: The method is constructed using the planifolia described in claim 1, and the method comprises: pretreating colorectal adenocarcinoma cells with 9.5-10.5nM SM-164 and 19.5-20.5μM caspase inhibitor z-VAD-fmk for 28-32min, adding 19.5-20.5ng / mL h-TNF-α and 0.15-8μM planifolia, incubating together for 10h-12h, inducing programmed necrosis of colorectal adenocarcinoma cells using h-TNF-α, and inhibiting necrosis of colorectal adenocarcinoma cells using planifolia.

8. The method for constructing a colorectal adenocarcinoma cell model resistant to programmed necrosis according to claim 7, characterized in that: The colorectal adenocarcinoma cells are HT-29 cells.

9. A colorectal adenocarcinoma cell model resistant to programmed necrosis, characterized in that: The method according to claim 8 is used to construct the present invention.

10. Any of the following uses of the necroptosis-resistant colorectal adenocarcinoma cell model according to claim 9: 1) To study the pathogenesis of necroptosis-resistant colorectal adenocarcinoma; 2) Screening for drugs to treat necroptosis-resistant colorectal adenocarcinoma.

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