CN114470015A

CN114470015A - Application of total coumarins of degummed croton herb in serving as and/or preparing cell iron death inducer

Info

Publication number: CN114470015A
Application number: CN202210191776.0A
Authority: CN
Inventors: 黎骊; 刘瑛; 陆国寿; 王丽; 吕纪华; 胡筱希; 黄建猷
Original assignee: Guangxi Institute Of Chinese Medicine & Pharmaceutical Science
Current assignee: Guangxi Institute Of Chinese Medicine & Pharmaceutical Science
Priority date: 2022-03-01
Filing date: 2022-03-01
Publication date: 2022-05-13

Abstract

The invention discloses application of total coumarins of degummed rattan in serving as and/or preparing a cell iron death inducer, belonging to the technical field of medicines. The experimental research shows that: the total coumarins of the tumor-removing rattan can induce cell iron death by improving the intracellular ROS and lipid peroxidation level and through oxidative stress and iron metabolism pathways; the total coumarins of the degummed rattan can reduce the expression level of iron death marker proteins GPX4, xCT, NRF2 and FTH1 in cells and can up-regulate the expression level of NCOA4, DMT1 and KEAP 1. The invention discloses the effect of total coumarins of tumor-eliminating rattan on inducing cell iron death for the first time, and can be used as or for preparing a cell iron death inducer.

Description

Application of total coumarins of degummed croton herb in serving as and/or preparing cell iron death inducer

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of total coumarins of degummed rattan as and/or in preparation of a cell iron death inducer.

Background

Dixon in 2012 reported that iron death (ferroptosis) was an iron ion-dependent non-apoptotic cell necrosis. The iron death is the regulated cell necrosis caused by lipid peroxidation induced by iron ions and active oxygen, and is obviously different from other forms of regulated cell necrosis such as apoptosis, necrosis, autophagy and the like on the morphological, biological and gene levels, and has become a hot point of research in recent years. The essence of iron death is metabolic disorder of lipid oxide in cells, and then abnormal metabolism is performed under the catalysis of iron ions, so that a large amount of lipid is produced, oxidation-reduction balance in the cells is destroyed, biomacromolecules are attacked, and cell death is triggered. Iron death is characterized by disappearance of the mitochondrial cristae and increased membrane density, as well as by increased cytoplasmic and lipid reactive oxygen radicals.

Iron death is mainly caused by iron-dependent oxidative damage, involving a complex series of biochemical reactions, gene expression and signaling. Iron death is closely related to diseases such as nervous system diseases, tumors, ischemia-reperfusion injury, kidney injury, atherosclerosis, diabetes, heart disease and the like. A large number of research reports show that iron death plays a key negative regulation and control role in the occurrence and development processes of tumors, and researches find that the iron death is involved in the occurrence and development processes of various tumors such as liver cancer, kidney cancer, pancreatic cancer and the like, and the iron death can effectively kill tumor cells and prevent the growth of the tumors. There is increasing evidence that induction of iron death can serve as an important mechanism for anti-tumor therapy, and therefore, induction of iron death in tumor cells is a novel anti-tumor therapeutic strategy.

The currently discovered iron death inducers mainly include the following two classes: one class is systemic Xc-inhibitors, such as Erastin; another class is GPx4 inhibitors, such as RSL 3. The natural products which can induce iron death are mainly artemisinin and dangshen ketone derivatives, and the natural products have more practical values due to the advantages of rich medicine sources, small side effect, multi-link integral treatment and the like. Because few iron death inducers are on the market at present, and reports of traditional Chinese medicines or traditional Chinese medicine extracts with the iron death inducers are few, the research and development of new iron death inducers are of great significance for developing anti-tumor medicines.

The Marsdenia tenacissima (Roxb.) wight of Marsdenia of hydrangeaceaePileostegia tomentella Hand. MazzThe whole herb of the formula belongs to Guangxi traditional Yao medicinal materials, and is a main drug for treating various tumors in the oncology department of Guangxi JinxiYao nationality Yao medical hospitals. Earlier researches found that the Total Coumarins (TCPT) in the tumor-eliminating rattan is the main active site and has a certain anti-tumor effect, but the deep action mechanism of the TCPT is not clarified. However, no relevant report is found on the application of the total coumarins of the tumor-eliminating rattan in the aspect of being used as and/or preparing the cell iron death inducer at present.

Disclosure of Invention

The invention aims to provide application of total coumarins of degummed croton herb in serving as and/or preparing a cell iron death inducer, and provides a new natural, efficient and safe medicine source for the cell iron death inducer.

The technical scheme adopted by the invention is as follows:

application of total coumarins of Kadsura heteroclita (Roxb.) Craib in preparing cell iron death inducer is provided. The tumor-eliminating vine is neocanopy planisporus of Cymbopogon of SparassaceaePileostegia tomentella Hand. MazzThe total coumarins of the degummed rattan are extracted from the degummed rattan.

The total coumarins of the degummed rattan can induce cell iron death by improving the intracellular ROS and lipid peroxidation level.

The total coumarins of the tumor-eliminating rattan can induce cell iron death through oxidative stress and an iron metabolism path.

Furthermore, the total coumarins of the degummed rattan can down-regulate the expression level of iron death marker proteins GPX4 and xCT in cells and up-regulate the expression level of NCOA4 and DMT 1.

The total coumarins of the degummed rattan can reduce the expression level of iron death marker proteins GPX4, NRF2 and FTH1 in cells and can increase the expression level of KEAP 1.

Research experiments show that:

(1) the total coumarins of the degummed rattan can induce the death of HT-29 iron of colorectal cancer cells by increasing the Reactive Oxygen Species (ROS) and lipid peroxidation levels in the cells. The invention adopts total coumarins (total coumarins) from which the tumor can be eliminatedpileostegia tomentellaTCPT) treated human colorectal cancer cells HT-29, cell viability decreased significantly with increasing concentrations of TCPT. The transmission electron microscope detection shows that the cells of the TCPT treated group have typical iron death morphological characteristics. The detection of the probe shows that the intracellular ROS and lipid peroxide levels are obviously increased and are dose-dependent. The expression of iron death marker proteins GPX4 (glutathione peroxidase 4) and xCT (cystine-glutamic acid antiporter subunit) in HT-29 cells is down-regulated, while the expression level of NCOA4 (nuclear receptor co-activator 4) and DMT1 (divalent metal ion transporter) is up-regulated, and the difference has statistical significance (theP＜0.05，P＜0.01）。

(2) The total coumarins of the degummed rattan can obviously inhibit the proliferation of H1688 cells of small cell lung cancer, and the mechanism of the total coumarins of the degummed rattan can finally induce cell iron death by increasing the intracellular ROS and lipid peroxidation level. The invention adopts total coumarins (total coumarins) from which the tumor can be eliminatedpileostegia tomentellaTCPT) has obvious inhibition effect on cell proliferation after intervention of small cell lung cancer H1688 cellsP＜0.05，P< 0.01). The morphological change of cell iron death caused by TCPT can be seen under a transmission electron microscope. TCPT is effective in increasing intracellular ROS and lipid peroxide levels. The expression of the iron death marker proteins GPX4 (glutathione peroxidase 4), NRF2 (nuclear factor E2-related factor 2) and FTH1 (ferritin heavy chain 1) in H1688 cells is down-regulated, while the expression of KEAP1 (Kelch-like epoxy chloropropylamine-related protein 1) is up-regulated, and the difference has statistical significance (1)P＜0.05，P＜0.01）。

The invention has the beneficial effects that:

the invention discloses application of total coumarins of degummed rattan in serving as and/or preparing a cell iron death inducer. The total coumarins of the Kadsura heteroclita can induce cell iron death by improving the intracellular ROS and lipid peroxidation level and through oxidative stress and iron metabolism. The total coumarins of Kadsura japonica can reduce the expression level of iron death marker proteins GPX4, xCT, NRF2 and FTH1 in cells and can up-regulate the expression level of NCOA4, DMT1 and KEAP 1.

The invention provides a new natural, efficient and safe medicine source for the iron death inducer. The invention discloses the effect of total coumarins of tumor-eliminating rattan on inducing cell iron death for the first time, which can be used as or for preparing a cell iron death inducer to further inhibit the proliferation of cells, especially tumor cells, and can be further used for preventing and/or treating tumors (such as small cell lung cancer), thereby providing a new idea for treating tumor diseases.

Drawings

FIG. 1 is a graph of the results of flow cytometry for the detection of HT-29 cell activity in different groups;

FIG. 2 Transmission electron microscopy of the changes in mitochondria of HT-29 cells (magnification 30000 ×);

FIG. 3 DCFH-DA assay of ROS levels in HT-29 cells in different groups;

FIG. 4 flow cytometry detected lipid peroxide levels in different groups of HT-29 cells;

FIG. 5 Western blotting to detect the expression level of the iron death marker protein in HT-29 cells;

FIG. 6 is a diagram showing the inhibition rate of TCPT on the proliferation of H1688 cells measured by the CCK-8 method;

FIG. 7 Transmission electron microscopy of changes in morphology of H1688 cells (magnification 30000 ×);

FIG. 8 DCFH-DA assay of ROS levels in H1688 cells of different groupings;

FIG. 9 flow cytometry detected lipid peroxide levels in different groupings of H1688 cells;

FIG. 10 Western blotting detection of the expression level of the iron death marker protein in H1688 cells.

Detailed Description

The invention is further described with reference to specific examples, without limiting the scope and application of the invention; the reagents, methods and apparatus employed in the present examples are conventional in the art, unless otherwise indicated.

Example 1 Effect of Total coumarin of Oncorhynchus Marsdenia on the Induction of iron death in colorectal cancer cells

1. Material

1.1 preparation of TCPT tumor-eliminating rattan is collected from Guangxi Jinxiu county, and identified as whole herb of Pileostegia tomentosa Hand, Mazz, belonging to genus Cyperus of family hydrangeaceae, by the assistant investigator of Huangyunfeng, a Chinese medicine institute of Guangxi Chinese medicine. 250 g of the medicinal material of the tumor-eliminating rattan is taken and crushed into coarse powder, 8 times of 95 percent ethanol is refluxed and extracted for 5 times and 2 hours/time, the filtering is carried out, the extracting solutions are combined, and the solvent is recovered to obtain 46 g of extract. Suspending the extract in water, sequentially extracting with petroleum ether (60-90 ℃), extracting with ethyl acetate, taking an ethyl acetate extraction part, separating by using a silica gel chromatographic column, performing gradient elution with ethyl acetate-methanol (100: 0-90: 0-80: 20-50: 50), collecting fractions, and combining coumarin components by using thin-layer chromatography to finally obtain 6.28 g of Total Coumarins (TCPT) of the Kadsura longipedunculata.

1.2 cell lines human colorectal cancer HT-29 cell lines were purchased from Guangzhou Suyao Biotech limited (STR identified by).

1.3 apoptosis detection kit (A211-02, Nanjing Vazyme); dichlorofluorescein (DCFH-DA) dye (S0033S-1, Shanghai Biyuntian); rosu (S0033S-2, Shanghai Biyun day); BODIPY 581/589C 11 probe dye (0622937, Cayman, usa); rabbit anti-human GPX4 monoclonal antibody (52455T-10, U.S. CST); rabbit anti-human NCOA4 monoclonal antibody (66849T-2, usa CST); a rabbit anti-human xCT monoclonal antibody (12691T-5, U.S. CST); rabbit anti-human DMT1 monoclonal antibody (15083T-1, U.S. CST); rabbit anti-human GAPDH monoclonal antibody (D110016-0100, Shanghai Production); horse radish peroxidase-labeled goat anti-rabbit IgG (GR 3307268-2, Abcame, USA).

1.4 major instruments biosafety cabinet (ESCO AC2-4S1, Singapore); CO 2₂Cell culture incubator (MCO-18 AIC, Japan Song Fang); transmission electron microscope (H-7650, Hitachi, Japan); microcapillary cytoflow analysis platform (Guava easyCyte HT, Merck Millipore, usa); inverted fluorescence microscope (DMi 8, come card, germany); vertical electrophoresis and transferSystem (Mini-PROTEAN Tetra/Mini Trans-Blot Module, Bio-Rad, USA); multifunctional imaging analysis system (Protein simple fluorochem R, U.S. Protein simple).

2. Method of producing a composite material

2.1 cell culture of human colorectal cancer HT-29 cells DMEM high-sugar Medium containing 10% fetal bovine serum, 37 ℃, 5% CO₂Culturing under the condition, and digesting and passaging by using 0.25 percent trypsin when the cell fusion rate reaches 80-90 percent.

2.2 flow cytometry detection of cellular Activity

Taking HT-29 cells in logarithmic growth phase at 5 × 10⁵Individual cells/well were seeded in 6-well plates and grown adherent for 24 h. TCPT treatment groups were added with different concentrations of TCPT (50. mu.g/mL, 100. mu.g/mL), and blank control groups were added with an equal volume of medium and incubated for an additional 24 h. The cell and supernatant from each group were collected by digestion, centrifuged, washed, and the cells resuspended in 100ul loading buffer. Adding 5 ul annexin V-FITC and 5 ul PI staining solution, mixing, incubating for 15 min in the dark, adding 200 ul loading buffer, mixing, sieving, and detecting cell activity by microcapillary cell flow analysis platform.

2.3 Transmission Electron microscopy for observing characteristic ultrastructure of cell

Taking HT-29 cells in logarithmic growth phase, and performing cytotoxicity experiment IC according to TCPT₅₀As a result, a blank control group and a TCPT-treated group (100. mu.g/mL) were set, and an equal volume of the medium was added to the blank control group, and the incubation was continued for 24 hours. After the intervention, the cells were digested and collected in groups of 2X 10 cells⁶Individual cells, washed with PBS, double fixed with 2.5% glutaraldehyde and 1% osmate, routinely dehydrated embedded, sectioned, stained with uranyl acetate-lead citrate, and observed for characteristic changes in the mitochondria of the cells by transmission electron microscopy.

2.4 detection of intracellular ROS by DCFH-DA Probe assay

Taking HT-29 cells in logarithmic growth phase, and culturing at 3 × 10³Individual cells/well were seeded in 96-well plates and grown adherent for 24 h. TCPT treatment groups were added with different concentrations of TCPT (50. mu.g/mL, 100. mu.g/mL), ROS positive control group was added with Rosu (50. mu.g/mL), and blank control group was added with an equal volume of medium and incubation was continued for 2 h. After the intervention of each group of cells according to the grouping setting is finished, abandoningRemoving original culture medium, diluting DCFH-DA probe dye with serum-free culture medium at a ratio of 1:1000, adding into a well plate at a ratio of 0.1 mL/well, incubating in an incubator for 30min, removing the culture medium containing the probe, washing for 3 times with the serum-free culture medium, and observing the fluorescence intensity of cells under a fluorescence microscope.

2.5 intracellular lipid peroxide content determination

Taking HT-29 cells in logarithmic growth phase at a ratio of 1 × 10⁶Individual cells/well were seeded in 6-well plates and grown adherent for 24 h. TCPT treatment groups were added with different concentrations of TCPT (50. mu.g/mL, 100. mu.g/mL), and blank control groups were added with an equal volume of medium and incubated for an additional 6 h. After the set of cell groups in groups is finished, replacing a fresh culture medium, adding stock solution (1.5 mmol/L) of BODIPY 581/589C 11 probe dye according to the proportion of 1:1000, uniformly mixing, and placing in an incubator for incubation for 30 min. Removing the dye, washing with PBS, collecting cells, suspending the cells in PBS, sieving, and detecting and analyzing by a microcapillary cell flow analysis platform.

2.6 Western blot detection of the expression level of proteins associated with iron death

Taking HT-29 cells in logarithmic growth phase at a ratio of 1 × 10⁶Individual cells/well were seeded in 6-well plates and grown adherent for 24 h. TCPT treatment groups were added with different concentrations of TCPT (50. mu.g/mL, 100. mu.g/mL), and blank control groups were added with an equal volume of medium and incubated for an additional 24 h. After the intervention of each group of cells according to grouping setting is finished, extracting total cell protein by RIPA lysate, and determining the protein concentration by a BCA protein kit. 10 mu g of total protein was electrophoresed by SDS-PAGE using 12% separation gel and 5% concentrated gel, transferred onto PVDF membrane, blocked with PBST containing 5% skim milk powder for 1 h, incubated with rabbit anti-human GPX4 (1: 1000 dilution), rabbit anti-human NCOA4 (1: 1000 dilution), rabbit anti-human CD98 (1: 1000 dilution), rabbit anti-human xCT (1: 1000 dilution), rabbit anti-human GAPDH (1:2000 dilution) overnight at 4 ℃, PBST washed 3 times, HRP-labeled goat anti-rabbit IgG (1:2000 dilution) incubated for 2h at room temperature, PBST washed 3 times, ECL luminescence substrate soaked for 1 min, and pictures were collected using ProteinSimple multifunctional imaging analysis system.

2.7 statistical treatment

Data are averaged. + -. standard deviation

Showing that SPSS22.0 software is used for statistical analysis, t test is adopted for comparison between two groups, one-factor analysis of variance (ANOVA) is adopted for comparison between multiple groups, the total variance p is larger than 0.05, LSD-t test is adopted, and when the normality condition is not met, data are properly converted.

3. Results

3.1 Effect of TCPT on HT-29 cellular Activity

Cell Annexin V-FITC double staining by flow cytometry, and detection results show that the death number of HT-29 cells is remarkably increased along with the increase of TCPT concentration, and the difference among groups has statistical significance (thePLess than 0.01), suggesting that TCPT can effectively reduce the activity of HT-29 cells and induce cell death. See fig. 1.

3.2 Effect of TCPT on the mitochondria of HT-29 cells

Observed under a transmission electron microscope, the blank control group HT-29 cells have normal mitochondria size, continuous outer membranes and uniform mitochondrial cristae arrangement. After TCPT treatment of cells, the cells have characteristic morphological changes of iron death, which are represented by aggregation of mitochondrial membrane matrixes, increase of density, reduction of mitochondrial cristae, swelling of partial mitochondria, shallow matrixes, vacuolated structures and no obvious change of nuclear morphology. See fig. 2.

3.3 Effect of TCPT on the intracellular ROS content of HT-29

The DCFH-DA probe is not fluorescent, and can be oxidized by ROS in cells after being combined with the DCFH-DA probe to generate a green fluorescent substance. The detection result shows that the blank control group HT-29 cells have weak green fluorescence, which indicates that the blank control group HT-29 cells have certain ROS generating capacity, and the number of positive staining cells and the fluorescence intensity in the cells are obviously increased after TCPT treatment (the detection result shows that the blank control group HT-29 cells have certain ROS generating capacityP＜0.05，P< 0.01), the fluorescence is distributed throughout the cell, suggesting that TCPT is effective in increasing the level of ROS in HT-29 cells. See fig. 3.

3.4 Effect of TCPT on HT-29 intracellular lipid peroxide levels

The cells were incubated with the specific fluorescent probe BODIPY 581/589C 11, and the flow cytometry detection results showed that the blank controlGroup HT-29 intracellular lipid peroxide levels were low and after TCPT treatment, intracellular lipid peroxide levels were significantly increased, with statistical differences between groups: (P＜0.05，P< 0.01), suggesting that TCPT is effective in increasing the level of lipid peroxides in HT-29 cells. See fig. 4.

3.5 Effect of TCPT on the expression level of iron death marker protein in HT-29 cells

GPX4, xCT, NCOA4 and DMT1 are key molecular markers related to iron death, and Western blotting detection results show that, compared with a blank control group, after TCPT treatment is carried out, along with the increase of TCPT concentration, the expression levels of GPX4 and xCT proteins in HT-29 cells are obviously reduced, the expression levels of NCOA4 and DMT1 proteins are obviously increased, and the differences have statistical significance (theP＜0.05，P< 0.01), suggesting that TCPT induces HT-29 cell iron death through oxidative stress and iron metabolic pathways, as shown in FIG. 5.

Example 2 inhibition of proliferation of small cell lung carcinoma H1688 cells by inducing iron death by Total coumarin of Oncomelania

1. Material

1.1 preparation of TCPT example 1

1.2 cell line Small cell Lung cancer H1688 cell line was purchased from Guangzhou Sugao Biotech Co.

1.3 Cell proliferation assay Kit (Cell Counting Kit-8, CCK-8, lot No. KN658, Japan Dojindo); dichlorofluorescein (DCFH-DA) dye (S0033S-1, Shanghai Biyuntian); rosu (S0033S-2, Shanghai Biyun day); BODIPY 581/589C 11 probe dye (0622937, Cayman, usa); rabbit anti-human GPX4 monoclonal antibody (52455T-10, U.S. CST); rabbit anti-human KEAP1 monoclonal antibody (8047T-3, usa CST); rabbit anti-human NRF2 monoclonal antibody (12721T-10, usa CST); rabbit anti-human FTH1 monoclonal antibody (4393T-4, CST, USA); rabbit anti-human GAPDH monoclonal antibody (D110016-0100, Shanghai Production); horse radish peroxidase-labeled goat anti-rabbit IgG (GR 3307268-2, Abcam, USA).

1.4 Main Instrument CO₂Cell culture incubator (MCO-18 AIC, Japan Song Fang); biological safety cabinet (ESCO AC2-4S1, newly-addedA slope); multifunctional detectors (Synergy H1, Biotek, usa); transmission electron microscope (H-7650, Hitachi, Japan); inverted fluorescence microscope (DMi 8, come card, germany); microcapillary cytoflow analysis platform (Guava easyCyte HT, Merck Millipore, usa); vertical electrophoresis and transfer systems (Mini-PROTEAN Tetra/Mini Trans-Blot Module, Bio-Rad, USA); multifunctional imaging analysis system (Protein simple fluorochem R, U.S. Protein simple).

2. Method of producing a composite material

2.1 cell culture Small cell Lung cancer H1688 cells in RPMI-1640 medium (37 ℃, 5% CO) containing 10% fetal bovine serum₂) When the cells grew to log phase, the cells were digested with 0.25% trypsin for grouping experiments.

2.2 CCK-8 method for detecting cell proliferation ability H1688 cells in logarithmic growth phase were taken, digested and counted, and then counted according to 5X 10⁴One/well was seeded in 96-well plates and grown adherent for 24 h. Adding TCPT (12.5, 25, 50, 100 and 200 mu g/mL) with gradient concentration, setting 4 multiple wells for each concentration, taking RPMI-1640 culture medium without medicine as blank control, acting for 24, 48 and 72 hours, adding 10 mu L CCK-8 for each well, continuing to incubate for 2 hours, detecting absorbance (OD value) of each well by a microplate reader at a wavelength of 450 nm, calculating the cell proliferation inhibition rate, and drawing a growth curve. Inhibition (%) =1- (experimental OD value/control OD value) × 100%.

2.3 Transmission Electron microscopy to observe changes in cell morphology

Taking H1688 cells in logarithmic growth phase, and performing cell proliferation experiment IC₅₀Results A blank control group and a TCPT treated group (50. mu.g/mL TCPT) were set, and the blank control group was added with the same volume of RPMI-1640 medium. After 48h, each group of cells was collected and counted at 2X 10⁶The individual cells were washed with PBS, double-fixed with 2.5% glutaraldehyde and 1% osmate, dehydrated and then embedded in sections, stained with uranyl acetate-lead citrate, and the morphological changes of the mitochondria of the cells were observed under a transmission electron microscope.

2.4 detection of intracellular ROS content by DCFH-DA dye

Taking H1688 cells in logarithmic growth phase, digesting and counting according to 3X 10³Inoculating each cell/well in 96-well plate, and growing adherentThe length is 24 h. TCPT treated group and blank control group were set, TCPT treated group was added with different concentrations of TCPT (25. mu.g/mL, 50. mu.g/mL), ROS positive control group was added with Rosu (50. mu.g/mL), blank control group was added with equal volume of RPMI-1640 medium, and culture was continued for 2 h. After the intervention of each group of cells according to grouping setting is finished, the original culture medium is discarded, the DCFH-DA probe dye is diluted by serum-free culture medium according to the proportion of 1:1000, 0.1 mL/hole is added into a pore plate, the pore plate is placed in an incubator for incubation for 30min, the culture medium containing the probe is removed, the serum-free culture medium is washed for 3 times, and the fluorescence intensity of the cells is observed under a fluorescence microscope.

2.5 intracellular lipid peroxide content determination

Taking H1688 cells in logarithmic growth phase, digesting, counting, and counting according to 1 × 10⁶Individual cells/well were seeded in 6-well plates and grown adherent for 24 h. TCPT treated group and blank control group were set, and TCPT treated group was added with different concentrations of TCPT (25. mu.g/mL, 50. mu.g/mL), and blank control group was added with the same volume of RPMI-1640 medium, and culture was continued for 48 h. After the intervention of each group of cells according to the grouping setting is finished, the fresh culture medium is replaced, stock solution (1.5 mmol/L) of BODIPY 581/589C 11 probe dye is added according to the proportion of 1:1000, and the cells are incubated for 30min in an incubator. Dye was removed, washed with PBS, cells were resuspended in PBS, sieved, and analyzed by microcapillary cytoflow assay platform assay.

2.6 expression of iron death marker protein by Western blot detection

Taking H1688 cells in logarithmic growth phase, digesting, counting, and counting according to 1 × 10⁶Individual cells/well were seeded in 6-well plates and grown adherent for 24 h. TCPT treated group and blank control group were set, and TCPT treated group was added with different concentrations of TCPT (25. mu.g/mL, 50. mu.g/mL), and blank control group was added with the same volume of RPMI-1640 medium, and culture was continued for 48 h. The total cell protein of each group was extracted by RIPA lysate and the protein concentration was measured by BCA method. Taking 10 mu g of total protein, carrying out SDS-PAGE electrophoresis, transferring the membrane, blocking with 5% skimmed milk powder for 1 h, respectively incubating with rabbit anti-human GPX4 (1: 1000 dilution), rabbit anti-human KEAP1 (1: 1000 dilution), rabbit anti-human NRF2 (1: 1000 dilution), rabbit anti-human FTH1 (1: 1000 dilution), rabbit anti-human GAPDH (1:2000 dilution) at 4 ℃ overnight, PBST washing, HRP-labeled goatAnti-rabbit IgG and goat anti-mouse IgG (diluted 1: 2000) were incubated at room temperature for 2h, PBST washed, ECL illuminated and then pictures were taken with a ProteinSimple multifunctional imaging analysis system.

2.7 statistical treatment

Statistical analysis was performed using SPSS22.0, data mean. + -. standard deviation

The results show that the comparison between groups adopts one-way ANOVA analysis, the total variance p is more than 0.05, LSD-t test is adopted, and when the variances are not uniform, the data are properly converted.

3. Results

3.1 Effect of TCPT on H1688 cell proliferation

CCK-8 detection results show that the proliferation inhibition rate of the small cell lung cancer H1688 cells is increased along with the increase of the concentration of TCPT, and the TCPT has time dependence, and the IC of the TCPT after the TCPT acts on the H1688 cells for 24H, 48H and 72H₅₀The values were 174.4, 53.61, 35.91. mu.g/mL, respectively, as shown in FIG. 6.

3.2 Effect of TCPT on H1688 cell morphology

The ultrastructure under an electron microscope shows that the H1688 cell of the blank control group has normal size of mitochondria and uniformly arranged mitochondrial cristae. After TCPT intervenes in cells, intracellular mitochondria become small and shrink, vacuoles can be seen in part of mitochondrial cytoplasm, the membrane density is increased, ridges are reduced, and the nuclear morphology is normal. See fig. 7.

3.3 Effect of TCPT on the production of ROS in H1688 cells

DCFH-DA dye detection results showed that the positive control group H1688 cells significantly enhanced green fluorescence compared to the blank control group (PLess than 0.01), the number of positive staining cells and the fluorescence intensity in the cells are obviously increased after TCPT intervenes the cells (P< 0.01). See fig. 8.

3.4 Effect of TCPT on H1688 cell lipid peroxide levels

Flow cytometry results showed that the blank control group H1688 had a lower level of intracellular lipid peroxide, and after TCPT treatment, the intracellular lipid peroxide level was significantly increased, and differences among groups had statistical significance (P＜0.05，P< 0.01). See the figure9。

3.5 Effect of TCPT on iron death marker protein expression levels

Western blotting detection results show that, compared with a blank control group, after TCPT treatment, the expression levels of GPX4, NRF2 and FTH1 proteins in H1688 cells are obviously reduced, the expression level of KEAP1 protein is obviously increased, and the difference has statistical significance (A)P＜0.05，P< 0.01), see FIG. 10.

Through the research experiments of the embodiment 1 and the embodiment 2, the technical scheme of the invention is realized:

The total coumarins of the degummed rattan can induce cell iron death by improving the intracellular ROS and lipid peroxidation level. The total coumarins of the tumor-eliminating rattan can induce cell iron death through oxidative stress and an iron metabolism path.

Furthermore, the total coumarins of the degummed rattan can down-regulate the expression level of iron death marker proteins GPX4 and xCT in cells and up-regulate the expression level of NCOA4 and DMT 1. The total coumarins of the degummed rattan can reduce the expression level of iron death marker proteins GPX4, NRF2 and FTH1 in cells and can increase the expression level of KEAP 1.

Claims

1. Application of total coumarins of Kadsura heteroclita (Roxb.) Craib in preparing cell iron death inducer is provided.

2. Use according to claim 1, characterized in that: the total coumarins of the degummed rattan can induce cell iron death by improving the intracellular ROS and lipid peroxidation level.

3. Use according to claim 1, characterized in that: the total coumarins of the tumor-eliminating rattan can induce cell iron death through oxidative stress and an iron metabolism path.

4. Use according to claim 1, characterized in that: the total coumarins of the degummed rattan can reduce the expression level of iron death marker proteins GPX4 and xCT in cells and can up-regulate the expression level of NCOA4 and DMT 1.

5. Use according to claim 1, characterized in that: the total coumarins of the degummed rattan can reduce the expression level of iron death marker proteins GPX4, NRF2 and FTH1 in cells and can increase the expression level of KEAP 1.