Application of combination of radix tetrastigme polysaccharide and anthracycline compound in preparation of medicine for treating triple-negative breast cancer

Abstract

The invention relates to the field of biological medicines, and discloses application of combination of radix tetrastigme polysaccharide and anthracycline compounds in preparation of a medicine for treating triple-negative breast cancer. Firstly, it is found that the radix tetrastigme polysaccharide is a cell ferroptosis inducer for the first time; secondly, the combination of the radix tetrastigme polysaccharide and the anthracycline compound is found for the first time, the radix tetrastigme polysaccharide can play a role in enhancing the effect and reducing toxicity in the treatment process of the triple negative breast cancer, and the toxic and side effects of the anthracycline compound are greatly reduced, so that the prognosis and life quality of patients with the triple negative breast cancer are expected to be improved, and the survival rate of the patients is increased.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of trifoliate green polysaccharide combined with anthracycline compounds in the preparation of a drug for treating triple-negative breast cancer. Background Art

[0002] Triple-negative breast cancer, as the most malignant breast cancer subtype, is characterized by the lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor 2 receptor. Therefore, traditional endocrine therapy and targeted anti-HER2 drugs are ineffective in treating it, which leads to poor clinical prognosis and greatly limits the choice of treatment strategies. In addition, compared with other breast cancer subtypes, triple-negative breast cancer has a higher risk of distant metastasis, recurrence and death.

[0003] Since triple-negative breast cancer is rich in iron and lipids, this property makes ferroptosis inducers ideal for its treatment. In recent years, rapid progress has been made in the understanding of the mechanism of ferroptosis, and a variety of oncoproteins, tumor suppressor genes, and oncogenic signaling pathways have been found to regulate ferroptosis. In general, ferroptosis is a form of regulated necrosis that is strictly controlled at multiple levels. Ferroptosis can occur through three major pathways, namely the glutathione pathway and the iron autophagy pathway (such as SLC7A11, GPX4, NCOA4, and FTH1) and the lipid metabolism pathway (such as ACSL4, ALOXs, GPX4, POR, GCH1, NOS, and AIFM2)

[0004] However, at present, there is no consensus on the best treatment for triple-negative breast cancer. Anthracyclines (doxorubicin, daunomycin, epirubicin, aclarubicin, idarubicin, valrubicin, etc.) combined with other chemotherapy drugs such as paclitaxel are still the main treatment for triple-negative breast cancer. However, this chemotherapy regimen has strong heterogeneity and limited efficacy. More complicated is that residual lesions, gene mutations, amplification and immune escape after chemotherapy can lead to the development of chemotherapy resistance, further aggravating the poor prognosis of patients with triple-negative breast cancer. In addition, although anthracyclines have achieved certain results in anti-tumor treatment, they are also faced with some challenges and limitations in practical applications. Among them, the biggest problem in clinical treatment is its limited efficacy and significant toxic side effects. Since doxorubicin is currently one of the most effective anti-tumor drugs among anthracyclines, it can inhibit the growth of a variety of solid and hematological malignancies. Therefore, the present invention takes doxorubicin as an example to propose solutions to the problems existing in the treatment of anthracycline drugs against triple-negative breast cancer.

[0005] We believe that the combination of ferroptosis inducers and anthracyclines may achieve unexpected results in the treatment of triple-negative breast cancer. However, the ferroptosis inducers discovered so far are very limited, and there are even fewer studies on the application and therapeutic effects of ferroptosis inducers. How to effectively develop new ferroptosis inducers and solve the problems of current anthracyclines to effectively treat triple-negative breast cancer has become a severe challenge that needs to be solved urgently.

[0006] Sanyeqing belongs to the genus of the genus Tricholoma in the grape family. It is a precious medicinal material unique to my country and was originally used for folk cancer treatment. Now it is not only a traditional Chinese medicine, but also a functional food. Sanyeqing contains a variety of phytochemicals, such as flavonoids, phenolic acids, polysaccharides and phytosterols. Modern pharmacological studies have shown that Sanyeqing and its active ingredients have multiple pharmacological activities such as anti-inflammatory, antioxidant, antiviral, antipyretic, anti-liver damage, immunomodulatory, and antibacterial. After long-term research, our preliminary work has confirmed that Sanyeqing polysaccharide is the main medicinal substance of Sanyeqing. Sanyeqing polysaccharide has multiple pharmacological functions, such as anti-inflammatory, antioxidant and immunomodulatory functions. The applicant's prior patent CN105816430B (Chinese patent application number: 201610149782.4, published on April 9, 2019) discloses a method for extracting Sanyeqing polysaccharide. Based on this, the applicant further explored the synergistic and toxicity-reducing effects of the extracted Tripterygium wilfordii polysaccharide on anthracycline drugs represented by doxorubicin in the treatment of triple-negative breast cancer. Summary of the invention

[0007] In order to solve the problems of poor efficacy, strong heterogeneity and significant toxic side effects of anthracycline compounds in clinical applications, the present invention provides the use of Tripterygium wilfordii polysaccharide combined with anthracycline compounds in the preparation of triple-negative breast cancer treatment drugs. First, the present invention first discovered that Tripterygium wilfordii polysaccharide is a ferroptosis inducer; secondly, the present invention first discovered that Tripterygium wilfordii polysaccharide combined with anthracycline compounds can play a role in enhancing efficacy and reducing toxicity in the treatment of triple-negative breast cancer, greatly reducing the toxic side effects of anthracycline compounds, thereby expected to improve the prognosis and quality of life of patients with triple-negative breast cancer and improve the survival rate of patients.

[0008] The specific technical scheme of the present invention is: In a first aspect, the present invention provides the use of Tripterygium wilfordii polysaccharide in the preparation of a cell ferroptosis inducer.

[0009] The present invention discovers for the first time through experiments that Tripterygium wilfordii polysaccharide is a natural cell ferroptosis inducer, which can promote the ferroptosis of cancer cells.

[0010] Preferably, the cells are triple-negative breast cancer cells.

[0011] In a second aspect, the present invention provides the use of a cell ferroptosis inducer in combination with an anthracycline compound in the preparation of a drug for treating triple-negative breast cancer.

[0012] In our previous studies, we found that the combination of ferroptosis inducers and anthracyclines can achieve unexpected effects in the treatment of triple-negative breast cancer.

[0013] Preferably, the cell ferroptosis inducer is Tripterygium wilfordii polysaccharide.

[0014] The present invention discovers for the first time that the combination of Tripterygium wilfordii polysaccharide and anthracycline drugs can significantly reverse the negative immune regulation of anthracycline drugs and further promote ferroptosis of triple-negative breast cancer.

[0015] In a third aspect, the present invention provides the use of Tripterygium wilfordii polysaccharide in combination with anthracycline compounds in the preparation of a drug for the treatment of triple-negative breast cancer.

[0016] The present invention first discovered that after the combination of Tripterygium wilfordii polysaccharide and anthracycline compounds, in the process of treating triple-negative breast cancer, Tripterygium wilfordii polysaccharide has a significant synergistic and toxicity-reducing effect on anthracycline compounds, which is specifically embodied in: (1) When combined with anthracycline drugs, trifoliate green polysaccharide mainly regulates the xCT / GSH / GPX4 and Nrf2 / NCOA4 / FTH1 pathways, thereby improving the therapeutic effect on triple-negative breast cancer; (2) The present invention found through flow cytometry that after administration of anthracyclines, the number of Treg cells increased, CD8 + The number of T cells decreased, showing negative immune regulation. When combined with Tripterygium wilfordii polysaccharide, it can significantly improve the tumor microenvironment, as shown by a decrease in the proportion of Treg cells and a decrease in CD4 + and CD8 + The proportion of T cells increased; (3) Tripterygium wilfordii polysaccharide is a natural inducer of cell ferroptosis. When combined with anthracyclines, it can further promote ferroptosis in triple-negative breast cancer, thereby improving the therapeutic effect on triple-negative breast cancer. (4) The present invention detected biochemical indicators (CK-MB) reflecting cardiac function after multiple groups of drug administration, and detected cardiac morphology. The results showed that anthracyclines have significant toxic side effects, which were significantly alleviated after combined use with Tripterygium wilfordii polysaccharide; the present invention further detected biochemical indicators reflecting liver (ALT, AST), kidney (ALB) and bone marrow hematopoietic function (WBC, PLT) after multiple groups of drug administration. The results showed that anthracyclines have significant toxic side effects, which were significantly alleviated after combined use with Tripterygium wilfordii polysaccharide. Therefore, Tripterygium wilfordii polysaccharide can significantly alleviate the toxic side effects of anthracyclines, including: heart, liver, kidney and bone marrow suppression toxicity and other aspects.

[0017] Preferably, the anthracycline compound is selected from doxorubicin, daunomycin, epirubicin, aclarubicin, idarubicin and valrubicin.

[0018] Preferably, the mass ratio of the trifoliate green polysaccharide to the anthracycline compound is 15-60:0.25.

[0019] The present invention found through in vitro CCK8 test that the drug concentration range of the combination of Tripterygium wilfordii polysaccharide and anthracycline compounds (such as doxorubicin) with synergistic anti-triple-negative breast cancer effect is: doxorubicin (0.25 μg / ml) is combined with Tripterygium wilfordii polysaccharide (15, 30, 60 μg / ml) respectively.

[0020] Preferably, the drug comprises: anthracycline compounds as main active ingredients, Tripterygium wilfordii polysaccharide as auxiliary ingredient for enhancing efficacy and reducing toxicity, and pharmaceutically acceptable excipients.

[0021] Furthermore, the toxicity reduction is to reduce the toxic effects of anthracycline compounds on the heart, liver, kidneys and bone marrow.

[0022] Preferably, the drug is in the form of an oral preparation.

[0023] In a sixth aspect, the present invention provides a method for promoting cell ferroptosis in vitro: adding Tripterygium wilfordii polysaccharide and anthracycline compounds to a cell suspension to promote cell ferroptosis.

[0024] Compared with the prior art, the beneficial effects of the present invention are: First, the present invention discovers for the first time that Tripterygium wilfordii polysaccharide is an inducer of cell ferroptosis; second, the present invention discovers for the first time that Tripterygium wilfordii polysaccharide combined with anthracycline compounds can play a synergistic and toxic reduction role in the treatment of triple-negative breast cancer, significantly reducing the toxic and side effects of anthracycline compounds, thereby hopefully improving the prognosis and quality of life of patients with triple-negative breast cancer and increasing the survival rate of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1It is that Tripterygium wilfordii polysaccharide inhibits the proliferation of triple-negative breast cancer cells, and when combined with doxorubicin, it enhances the anti-triple-negative breast cancer effect of doxorubicin. Among them: (AB) The effect of doxorubicin on MDA-MB-231 and 4T1 cells after 24, 48 and 72 hours. (CD) The effect of Tripterygium wilfordii polysaccharide on MDA-MB-231 and 4T1 cells after 24, 48 and 72 hours. (EF) The effect of 0.25μg / ml doxorubicin combined with 15, 30, 60, 120μg / ml Tripterygium wilfordii polysaccharide on the viability of MDA-MB-231 and 4T1 cells after 24 and 48 hours. (GH) The effect of 0.5μg / ml doxorubicin combined with 15, 30, 60, 120μg / ml Tripterygium wilfordii polysaccharide on the viability of MDA-MB-231 and 4T1 cells after 24 and 48 hours. (IJ) Based on the CCK8 results of MDA-MB-231 cells, the HSA method was used to perform synergistic analysis of cell viability using Combefit software, and the average synergistic score was reported. A score greater than 10 indicates synergy for each dose combination, and a score less than 10 indicates antagonism. (KL) Based on the CCK8 results of 4T1 cells, the HSA method was used to perform synergistic analysis of cell viability using Combefit software, and the average synergistic score was reported. A score greater than 10 indicates synergy for each dose combination, and a score less than 10 indicates antagonism. * P <0.05,** P <0.01,*** P <0.001 vs. Control group.

[0026] Figure 2 It is the trifoliate green polysaccharide and the combination of the two drugs that induce ferroptosis in triple-negative breast cancer cells. Among them: (A) GSEA pathway enrichment showed that the glutathione metabolic signal was downregulated in the medium-dose trifoliate green polysaccharide group (THP-M) compared with the control group. (B) Electron microscopy images of MDA-MB-231 cells treated with the control group, doxorubicin group, medium-dose trifoliate green polysaccharide group and two-drug combination group (THP-M+DOX) for 24 hours, with 4 fields of view selected for each group; scale bar: 0.5μm. (CD) GSH levels of each drug group in MDA-MB-231 and 4T1 cells. (EF) Fe levels of each drug group in MDA-MB-231 and 4T1 cells 2+ Level. (GH) MDA levels in each drug-treated group in MDA-MB-231 and 4T1 cells. * P <0.05,** P <0.01,*** P <0.001 vs. Controlgroup.

[0027] Figure 3It is the combination of Tripterygium wilfordii polysaccharide and the two drugs that induce ferroptosis through the glutathione pathway. Among them: (A) GSEA analysis showed that compared with the doxorubicin group, the ferroptosis signal in the combination group was enhanced. (B) The heat map shows the differentially expressed genes in the combination group compared with the doxorubicin group in MDA-MB-231 cells after 24 hours of treatment. (C) The protein changes of p53, xCT, GPX4, Nrf2, NCOA4 and FTH1 in MDA-MB-231 and 4T1 cells after 24 hours of treatment with low, medium and high doses of Tripterygium wilfordii polysaccharide. (D) The protein changes of p53, xCT, GPX4, Nrf2, NCOA4 and FTH1 in the control group, doxorubicin group and combination group after 24 hours of treatment with MDA-MB-231 and 4T1 cells.

[0028] Figure 4 In vivo, Tripterygium wilfordii polysaccharide can enhance the anti-triple-negative breast cancer effect of doxorubicin. Among them: (A) Tumor images of model group (MOD), low-dose doxorubicin group (DOX-L), Tripterygium wilfordii polysaccharide group, low-dose doxorubicin combined with Tripterygium wilfordii group (DOX-L+THP), low-dose doxorubicin combined with dexamethasone group (DOX-L+DEX), high-dose doxorubicin group (DOX-H), high-dose doxorubicin combined with Tripterygium wilfordii group (DOX-H+THP), and high-dose doxorubicin combined with dexamethasone group (DOX-H+DEX). (B) Statistical results of tumor volume after treatment with DOX-L, THP, DOX-L+THP, DOX-L+DEX, DOX-H, DOX-H+THP, and DOX-H+DEX compared with the model group. (C) Comparison of mouse body weight after treatment with DOX-L, THP, DOX-L+THP, DOX-L+DEX, DOX-H, DOX-H+THP, and DOX-H+DEX with the model group. (D) Comparison of tumor weight after treatment with DOX-L, THP, DOX-L+THP, DOX-L+DEX, DOX-H, DOX-H+THP, and DOX-H+DEX with the model group. (E) HE staining analysis of tumor tissue morphology. * P <0.05,** P <0.01,*** P <0.001 vs. MOD group.

[0029] Figure 5 In vivo, Tripterygium wilfordii polysaccharide enhances the anti-triple-negative breast cancer effect of doxorubicin by inducing ferroptosis and ferroptosis. 2+Levels; (B) GSH levels in tumors of MOD, DOX-L, THP and DOX-L+THP groups; (C) MDA levels in tumors of MOD, DOX-L, THP and DOX-L+THP groups; (D) Western blot detection of p53, xCT, GPX4, Nrf2, NCOA4 and FTH1 expression levels in tumors after DOX-L, THP and DOX-L+THP treatment; (E) Immunohistochemistry detection of GPX4 and NCOA4 expression in tumor tissues. * P <0.05,** P <0.01,*** P <0.001 vs. MOD group.

[0030] Figure 6 Tripterygium wilfordii polysaccharide can improve the tumor microenvironment and reverse the negative immune regulation of doxorubicin. Among them: (A) After THP-M treatment of MDA-MB-231 cells for 24 hours, KEGG analysis showed the enrichment pathways of differentially expressed genes compared with the control group; the larger the bubble, the more differential genes there are between the two groups, the darker the color, and the more significant the difference. (BD) Treg and CD8 in tumor tissues of mice in the DOX-L, THP, DOX-L+THP and MOD groups + T cell levels; (E) Treg, CD4 + T and CD8 + T cell levels.* P <0.05,** P <0.01,*** P <0.001 vs. MOD group.

[0031] Figure 7 Tripterygium wilfordii polysaccharide can reduce the cardiotoxicity caused by doxorubicin. Among them: (AB) Heart organ coefficient of each group (organ coefficient is calculated by dividing organ weight by body weight). (C) CK-MB (U / L) level of each group. (D) Electrocardiogram of mice in each group. (E) HE staining analysis of cardiac histological morphology of each group. * P <0.05,** P <0.01,*** P <0.001 vs. CONgroup. # P <0.05, ## P <0.01, ### P <0.001 vs. DOX-H group.

[0032] Figure 8 Tripterygium wilfordii polysaccharide can alleviate the liver, kidney and bone marrow suppression toxicity caused by doxorubicin. Among them: (A) Organ coefficient of each organ in mice; organ coefficient is calculated by dividing organ weight by body weight. (BC) Creatinine (CREA) and lactate dehydrogenase (LDH) levels of mice in each group. (DF) Normal reference range of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB) in mice and the average values ​​of ALT, AST and ALB in each group of mice. (GI) ALT, AST and ALB values ​​in each group of mice. (JL) Normal reference range of neutrophil ratio (NEUT%), white blood cells (WBC) and platelets (PLT) in mice and the average values ​​of NEUT%, WBC and PLT in each group. (MO) NEUT%, WBC and PLT values ​​in each group of mice. For Figures (DF) and (JL), in the discussion of biochemical indicators, we believe that due to individual differences in mice, biochemical indicators should have a normal range. We determined the maximum and minimum values ​​of each indicator. Compare the mean and range of each metric. * P <0.05,** P <0.01,*** P <0.001 vs. MODgroup. # P <0.05, ## P <0.01, ### P <0.001 vs. DOX-H group. DETAILED DESCRIPTION

[0033] In order to make the purpose of the present application, the technical scheme and the benefits clearer, the technical scheme of the present invention is described in detail below in conjunction with specific experiments and data processing. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, the changes and optimizations that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention. Unless otherwise described, the implementation of the present invention will adopt conventional techniques such as cell biology, molecular biology, and genetic engineering technology, which are all known to those skilled in the art.

[0034] Overall embodiment In a first aspect, the present invention provides the use of Tripterygium wilfordii polysaccharide in the preparation of a cell ferroptosis inducer; further, the cells are triple-negative breast cancer cells.

[0035] In a second aspect, the present invention provides the use of a cell ferroptosis inducer combined with an anthracycline compound in the preparation of a drug for the treatment of triple-negative breast cancer; further, the cell ferroptosis inducer is trifoliate green polysaccharide.

[0036] In a third aspect, the present invention provides the use of Tripterygium wilfordii polysaccharide in combination with anthracycline compounds in the preparation of a drug for the treatment of triple-negative breast cancer.

[0037] Furthermore, the anthracycline compound is selected from doxorubicin, daunomycin, epirubicin, aclarubicin, idarubicin and valrubicin.

[0038] Furthermore, the mass ratio of the trifoliate green polysaccharide to the anthracycline compound is 15-60:0.25.

[0039] Furthermore, the drug includes: anthracycline compounds as main active ingredients, Tripterygium wilfordii polysaccharide as auxiliary ingredient for enhancing synergy and reducing toxicity, and pharmaceutically acceptable excipients; wherein the toxicity reduction is to reduce the toxic effects of anthracycline compounds on the heart, liver, kidneys, and bone marrow.

[0040] Furthermore, the drug is in the form of an oral preparation.

[0041] In a sixth aspect, a method for promoting cell ferroptosis in vitro: adding trifoliate green polysaccharide and anthracycline compounds to a cell suspension to promote cell ferroptosis.

[0042] Example 1: Preparation of Tripterygium wilfordii polysaccharide The required experimental materials, instruments and specific experimental methods are as described in patent CN105816430B (Chinese patent application number: 201610149782.4). The process is briefly described as follows: Take the above-ground part of Tripterygium wilfordii and dry it in an oven at 40 °C, dry it, and crush it. Put it in a round-bottom flask, and add double distilled water to reflux and extract it three times at a solid-liquid ratio of 1:15 (g / ml), each time for 30 minutes. After filtering, combine the three extracts, concentrate them by vacuum distillation, add 95% ethanol with a mass of 3 times the concentrate, extract them with water and precipitate them with alcohol three times, let them stand overnight at 4 °C, and centrifuge them at high speed for 15 minutes at 4200rpm to obtain the precipitate. Wash it twice with anhydrous ethanol and obtain Tripterygium wilfordii polysaccharide powder by vacuum freeze drying.

[0043] Example 2: The combination of Tripterygium wilfordii polysaccharide and anthracycline drugs has a significant synergistic effect against triple-negative breast cancer 1. Experimental materials, reagents and instruments Breast cancer cell lines MDA-MB-231 and 4T1 were purchased from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. K1018CCK-8 reagent (APExBio, USA), fetal bovine serum (BI, USA), trypsin (Gibco, USA), DOX (doxorubicin, Shanghai Biotech), DEX (dexamethasone, Shyuanye, B25793), cell culture flask (25 cm 3 , 75cm3 ) (Wuxi Nice Biotechnology Co., Ltd.), cell culture plates (96-well, 24-well, 6-well) (Wuxi Nice Biotechnology Co., Ltd.), PBS (Jiangsu KeyGen Biotech Co., Ltd.), Leibbovitz's L-15 medium (Shanghai Yuanpei Biotechnology Co., Ltd.), microplate reader (Rayto, USA), cell culture incubator (Thermo Fisher Scientific, USA).

[0044] 2. Experimental methods The polysaccharide extract of the trifoliate green ginseng obtained in Example 1 was used in combination with doxorubicin to synergistically induce triple-negative breast cancer cell death. Specifically, the following steps were included: (1) MDA-MB-231 and 4T1 cells in the exponential proliferation phase were digested with trypsin and blown into a single uniform cell suspension. A portion of the suspension was taken with a 20µl pipette tip and counted with a cell counting plate. After counting, the cells were mixed and 100µl (2000 cells / well) was added to a 96-well plate. (2) The 96-well cell culture plate was placed in a cell culture incubator and cultured for 12 hours, and then DOX and THP were added in a concentration gradient and incubated for 24, 48, and 72 hours respectively; (3) The culture medium was aspirated without touching the bottom attached cells, and 100μl of culture medium containing 10% CCK-8 solution was added to the 96-well plate; (4) The plate was incubated in a 37°C incubator for 3.5 hours, and the absorbance at 480nm was measured.

[0045] Healthy female BALB / C mice were obtained from the Experimental Animal Center of Zhejiang Chinese Medical University (License No.: SYXK (Zhe) 2021-0012, Animal Model Research Ethics Approval No.: IACUC-20220627-30). All animals were housed under standard conditions (25 ± 3 °C, 60 ± 5% humidity, 12 h light-dark cycle) with free access to complete pelleted feed and water. All animals were acclimated for 7 days before the experiment. 4T1 cells were injected into the mammary fat pad of BALB / C mice, and drug treatment was started 5 days later.

[0046] Mice were grouped as follows: 4T1 tumor-bearing mice were randomly divided into 9 groups (8 mice in each group), MOD (saline injection) group; DOX-L (3 mg / kg) group; THP (150 mg / kg) group; DEX (5 mg / kg) group; DOX-L + THP (3 mg / kg + 150 mg / kg) group; DOX-L + DEX (3 mg / kg + 5 mg / kg) group; DOX-H (5 mg / kg) group; DOX-H + THP (5 mg / kg + 150 mg / kg) group; DOX-H + DEX (5 mg / kg + 5 mg / kg) group. All compounds were diluted in saline.

[0047] Weigh the body every other day, and use a vernier caliper to measure the length and width of the tumor. Calculate the tumor volume: tumor volume = (tumor length) × (tumor width) 2 / 2. At the end of the experiment, tumors and major organs (heart, liver, spleen, and kidney) were separated from the mouse corpses. Organ index was calculated as follows: organ index (%) = organ weight (g) / mouse body weight (g) × 100%.

[0048] 3. Data Statistics All measurement data were expressed as mean ± SD, unpaired t-test was used for comparison between two groups, analysis of variance was used for comparison between multiple groups, and one-way ANOVA was used for comparison between multiple groups. P< 0.05 was used to judge statistical significance, * P <0.05,** P <0.01,*** P <0.001; Graphpad Prism 9.0 was used for statistics, and Combenefit software was used for synergy scoring.

[0049] (IV) Experimental results The results are as follows Figure 1 As shown. Using CCK8 detection, we established a concentration gradient of DOX and THP (0.25-4μg / ml and 15-240μg / ml, respectively) ( Figure 1 AD). Then we combined four THP concentrations (15, 30 and 60, 120 μg / ml) with 0.25 μg / ml DOX-L and 0.5 μg / ml DOX-H to test the combined effect. Based on the CCK8 results and the combined score, we determined that the optimal THP concentrations for the combination of the two drugs were low (THP-L, 15 μg / ml), medium (THP-M, 30 μg / ml) and high (THP-H, 60 μg / ml), and combined them with DOX-L ( Figure 1 EH). Then, according to the CCK8 results, the HSA method was used to perform synergistic analysis of cell viability using Combenefit software, and the average synergistic score was reported. The results showed that 0.25 μg / ml doxorubicin combined with 15, 30, 60, and 120 μg / ml of Tripterygium wilfordii polysaccharide had a significant synergistic effect. The synergistic scores were all greater than 10 ( Figure 1 IL).

[0050] To evaluate the in vivo anti-TNBC effects of THP and THP+DOX, we constructed a mouse subcutaneous transplant tumor model using TNBC cells. Dexrazoxane (DEX) is the only effective drug approved by the FDA to prevent anthracycline cardiotoxicity. However, studies have reported that it affects the anti-tumor effect of anthracyclines and bone marrow suppression. Therefore, we used 5 mg / kg DEX as a positive control to comprehensively compare the synergistic and toxic effects of THP on DOX. In the preliminary experimental exploration, we established three THP concentrations (100, 150, and 200 mg / kg) to explore its optimal dosage. The results showed that the anti-TNBC effect of THP was concentration-dependent within a certain range, and the best effect was achieved at about 150 mg / kg. When the concentration exceeded this range, its anti-TNBC effect did not show a dose-dependent enhancement. The in vivo regulation of DOX was consistent with our in vitro experimental results, and its anti-TNBC effect was dose-dependent; low concentration of DOX (DOX-L, 3 mg / kg) had a poor anti-TNBC effect, while high concentration of DOX (DOX-H, 5 mg / kg) had a better anti-TNBC effect, but was accompanied by severe weight loss and extremely serious toxic side effects.

[0051] Specific results such as Figure 4 As shown, the combination of THP and DOX-L has a significant anti-TNBC effect, and its anti-TNBC effect is basically the same as that of the DOX-H group ( Figure 7 AD). Hematoxylin and eosin (HE) staining of tumor tissues confirmed that the combined use of DOX-L+THP and DOX-H+THP groups had the best anti-TNBC effect, with relatively regular cell morphology, reduced tumor cell counts and nuclear divisions, which was better than the DEX positive control group ( Figure 7 E).

[0052] Example 3: Tripterygium wilfordii polysaccharide induces ferroptosis in triple-negative breast cancer cells, and ferroptosis is further enhanced after combination with anthracyclines.

[0053] 1. Experimental materials and instruments HBSS (Gibco, USA), BODIPY 581 / 591 C11 (Invitrogen, USA), Hoechst (Invitrogen, USA), GSH / GSSG-glo detection kit (Beyotime, China), PVDF membrane (Millipore, USA), analytical grade anhydrous ethanol (Wuxi Yasheng Chemical Co., Ltd.), analytical grade methanol (Shanghai Lingfeng Chemical Reagent Co., Ltd.), P0010 BCA protein concentration determination kit (Shanghai Biotech Co., Ltd.), SDS-PAGE gel rapid preparation kit (Shanghai Biotech Co., Ltd.), SDS-PAGE electrophoresis buffer (Shanghai Biotech Co., Ltd.), SDS-PAGE protein loading buffer (5×) (Shanghai Biotech Co., Ltd.), RIPA lysis buffer (Shanghai Biotech Co., Ltd.), P0010 BCA protein concentration determination kit (Shanghai Biotech Co., Ltd.), electrophoresis instrument (bio-rad, USA), vertical electrophoresis tank (bio-rad, USA), transfer equipment (Beijing Wex Technology Co., Ltd.), GAPDHMouse McAb 60004 (Proteintech, USA), D8340 skim milk powder (Beijing Solebow Technology Co., Ltd.), Goat anti-Rabbit IgG (White Shark Biotechnology Co., Ltd.), Goat anti-Mouse IgG (White Shark Biotechnology Co., Ltd.), 20×TBST (Beijing Solebow Technology Co., Ltd.), PVDF membrane (Millipore, USA) 2. Experimental methods The extract of the polysaccharide of the trifoliate green ginseng obtained in Example 1 was used for transcriptomic sequencing, and it was found that the polysaccharide of the trifoliate green ginseng had a significant regulatory effect on the glutathione pathway. The mitochondrial morphology was observed by transmission electron microscopy, and the specific method was as follows: (1) Sample collection: After the cell suspension was centrifuged, the cell pellet was collected and fixed and stored in 2.5% glutaraldehyde at 4°C. (2) Sample fixation: The sample was fixed to the transmission electron microscope sample holder. Then the sample was glued to a copper mesh and placed in a container containing a fixative for fixation. (3) Dehydration treatment: The cell pellet was rinsed with phosphate buffer and 1% osmium acid, and then the sample was gradient dehydrated with acetone. (4) Embedding: The immersed sample was wrapped with an embedding agent and cut into ultrathin sections; (5) Sectioning: The embedded sample was sectioned to obtain ultrathin sections. The thickness of the section was between 50-100nm. (6) Staining: The section was stained so that the structure of the sample could be better distinguished when observed under a transmission electron microscope. (7) Loading the sample: Load the prepared sample onto the sample stage of the transmission electron microscope. This step requires the use of a special sample fixture to fix the sample on the sample stage and adjust the position and angle of the sample to make it suitable for electron beam irradiation. (8) Loading the sample: Load the prepared sample onto the sample stage of the transmission electron microscope. This step requires the use of a special sample fixture to fix the sample on the sample stage and adjust the position and angle of the sample to make it suitable for electron beam irradiation. (9) Equipment debugging: After the sample is loaded, the transmission electron microscope needs to be debugged to ensure that the equipment is in the best working condition. The debugging process includes adjusting the brightness, focus, magnification and other parameters of the electron gun, as well as calibrating the operating voltage and current of the instrument. (10) Transmission electron microscope observation: After the equipment debugging is completed, transmission electron microscope observation begins. After the electron beam passes through the sample, the microscopic structure inside the sample will be revealed by the information carried by the electron beam. By adjusting the magnification and focal length of the transmission electron microscope, clear images at different scales can be obtained. (11) Image acquisition and analysis: Ultra-thin sections were imaged and photographed under a H7500 transmission electron microscope to observe mitochondrial morphology.

[0054] Detection of GSH content: (1) Preparation of GSSG stock solution: Dissolve 5 mg GSSG in the kit in 816 μl ultrapure water to obtain a stock solution with a concentration of 10 mM. (2) Preparation of DTNB stock solution: Dissolve 4.5 mg DTNB in ​​1.5 ml DMSO provided in the kit and mix well to obtain DTNB stock solution. (3) Preparation of protein removal reagent M solution: Weigh 0.2 g protein removal reagent M and mix it with 4 ml total glutathione detection buffer to prepare a 5% aqueous solution. (4) Preparation of NADPH stock solution: Dissolve 4 mg NADPH in the kit in 100 μl ultrapure water to obtain a high concentration stock solution. (5) Dilution of glutathione reductase: Take 50 μl glutathione reductase and mix it with 200 μl total glutathione detection buffer to obtain diluted reductase. (6) Preparation of total glutathione detection working solution: According to the number of samples to be tested, mix the three reagents according to the proportions in the instructions to prepare a working solution. (7) Prepare 0.5 mg / ml NADPH solution: Take an appropriate amount from the NADPH stock solution and add total glutathione detection buffer to dilute to the required concentration. (8) Preparation of GSH scavenging auxiliary solution and working solution: Mix the GSH scavenging auxiliary solution with ultrapure water to obtain a diluent, which must be freshly prepared each time it is used. (9) Preparation of standard: Use protein removal reagent M solution to dilute the GSSG stock solution and prepare a series of GSSG solutions of different concentrations as standards. (10) Determination of GSSG content in samples: Add the diluted GSH scavenging auxiliary solution and GSH scavenging reagent working solution to the standard. After a certain reaction time, it can be used for subsequent detection. (11) Preparation of cell samples: Wash the cells with PBS, collect them by centrifugation, remove the supernatant, add protein removal reagent M solution for freeze-thaw treatment, and centrifuge to obtain the supernatant for subsequent determination. (12) Determination of total glutathione: Add diluted GSH scavenging auxiliary solution and GSH scavenging reagent working solution to the sample to be tested. After the reaction, add the sample or standard to the 96-well plate, then add the total glutathione detection working solution and NADPH solution, and measure the absorbance after incubation. (13) Centrifuge the sample at 10,000g for 10 minutes at 4°C to separate the supernatant, which will be used for the subsequent total glutathione determination. (14) Take a portion of the prepared sample to be tested, add the diluted GSH scavenging auxiliary solution according to the established ratio (add 20μl per 100μl sample), and then immediately mix it evenly by vortex. (15) Subsequently, add the GSH scavenging reagent working solution to the sample according to another ratio (add 4μl per 100μl sample), also immediately mix it evenly by vortex, and react at 25°C for 60 minutes. (16) When measuring samples and standards, use a 96-well plate, add samples or standards in sequence, and mix thoroughly.Then, add 150 μl of total glutathione detection working solution to each well, mix well, and incubate at 25°C or room temperature for 5 minutes. (17) Next, add 50 μl of 0.5 mg / ml NADPH solution to each well and mix well. (18) After the NADPH solution is mixed well, wait 25 minutes and then use a microplate reader to measure the absorbance of each well at A412. These steps will accurately determine the glutathione content in the sample.

[0055] Fe 2+ Content detection: (1) After cell plating and culture for 12 hours, add drugs for 24 hours. (2) Trypsin digestion, centrifugation to collect cells, and dilute the sample. (3) Add 50μl of sample to the sample well of the 96-well plate and adjust the volume to 100μl / well with AssayBuffer. (4) Add 5μl of iron assay buffer to each sample. Use a pipette to pipette evenly, incubate the reaction at 25℃ for 30 minutes, and protect the culture dish from light during the incubation period. (5) Add 100μl of iron probe to each well, each well contains standard, use a pipette to pipette evenly, and react at 25℃ for 60 minutes. Protect the culture dish from light during the incubation period. (6) Measure the absorbance at 593nm.

[0056] MDA content detection: (1) Preparation of TBA storage solution: Weigh an appropriate amount of TBA as required, and use a specific TBA preparation solution to dissolve it to a concentration of 0.37%. (2) Preparation of MDA detection solution: According to the number of samples to be tested (including the control group), refer to the instructions to prepare an appropriate amount of MDA detection solution immediately before the test. (3) Dilution of standard: Use distilled water to dilute the standard to a concentration gradient of 1, 2, 5, 10, 20, and 50 μM for subsequent construction of the standard curve. If the MDA concentration in the sample is high, 100, 150, and 200 μM concentration points can be added as appropriate. (4) Sample measurement steps: In an appropriate container, add 0.1 ml of homogenate, lysate or PBS as a blank control, and add the same volume of different concentrations of standard to construct the standard curve, and then add 0.1 ml of the sample to be tested. Subsequently, add 0.2 ml of MDA detection solution. (5) After mixing evenly, place it at 100°C or in boiling water and heat for 15 minutes. During the heating process, it is necessary to prevent the liquid from boiling violently and splashing. If a heating block is used, it is recommended to press the centrifuge tube cover with a heavy object to ensure safety. (6) After the solution cools to room temperature, centrifuge it at room temperature for 10 minutes at a centrifugal force of 1000g. Take 200μl of the supernatant, add it to a 96-well plate, and measure the absorbance at a wavelength of 532nm using an enzyme reader. (7) Calculation of MDA content: Based on the measured absorbance value, calculate the MDA content in the sample solution. Subsequently, convert it by unit weight of protein content or tissue weight to obtain the MDA content in the initial sample, expressed as μmol / mg protein.

[0057] In addition, the polysaccharide extract of the trifoliate green ginseng obtained in Example 1 was used in combination with doxorubicin. By transcriptomic sequencing, it was found that ferroptosis was significantly upregulated after the combination compared with doxorubicin alone. Western Blot was further used to detect the cell signaling pathway, and the specific steps were as follows: (1) Collect cells; lyse cells on ice (RIPA: PMSF = 99:1), centrifuge at 4°C, 10000rpm for 10 minutes, and take the supernatant. (2) Add 0, 1, 2, 4, 8, 12, 16, 20μl of protein standard solution to the wells in turn, and add 1μl of the protein sample to be tested to each duplicate well of the sample to be tested. (3) Add 200μl of BCA working solution (A solution: B solution = 50:1) to each well and let it stand at room temperature for 2 minutes or at 37°C for 1 hour. (4) Use a 562nm microplate reader to detect the OD value. First, make a standard curve based on the OD value of the standard well, then insert the average OD value of the sample to be tested into the curve, and multiply the value by 20 to get the actual protein concentration (μg / μl). (5) Use cell lysis buffer to dilute the protein sample with a measured concentration to a concentration of 2μg / μl. Add 20μl of 5× protein loading buffer to 80μl of the diluted sample and mix thoroughly. Place it in a 100℃ metal bath and heat for 5min to fully denature the protein. (6) Prepare SDS-PAGE gel. (7) Connect the power supply and set the working voltage to 80V constant voltage for 30min. (8) After the bromophenol blue indicator in the sample migrates to the separation gel, change to 120V constant voltage for 1h until the blue band approaches the bottom edge of the glass gel plate and stop electrophoresis. (9) Transfer, prepare transfer solution, the ratio is: glycine 14.4g, tris(hydroxymethyl)aminomethane 3g, methanol 200ml, ultrapure water 800ml. Shake the mixture evenly until there are no obvious solid particles, and place it in a 4℃ refrigerator for later use. (10) Soak the PVDF membrane in methanol for 3-5min and transfer it. (11) Generally, proteins below 30kDa are transferred at 100V constant voltage for 1h, proteins between 30-80kDa are transferred at 250mA constant current for 1h, and proteins above 80kDa are transferred at 300mA constant current for 2h. (12) Membrane blocking and antibody incubation, slowly shake on a shaker at room temperature for 1-5h; (13) Discard the milk powder and rinse the PVDF membrane twice with 1×TBST. (14) Add 4 ml of primary antibody diluent and appropriate amount of antibody (antibody dilution concentration: GAPDH1:10000, xCT1:2000, Nrf21:2000, GPX41:5000, p531:2000, NCOA41:2000, FTH11:2000), incubate at 4°C, shake slowly on a shaker for 12-16 hours. (15) Recover the primary antibody and store at 4°C, pour 1×TBST into the incubation box, and wash the PVDF membrane by shaking rapidly on a shaker for 10 minutes each time, for a total of 3 times.(16) Add an appropriate amount of 5% skimmed milk powder to the incubation box, add the corresponding species' secondary antibodies at a ratio of 1:10000, and incubate on a shaker for 1 hour. (17) Discard the secondary antibody and wash the PVDF membrane for 10 minutes each time, for a total of 3 times. (18) Use ImageJ software to measure the gray value of the band and perform quantitative analysis.

[0058] At the in vivo level, immunohistochemistry was used to detect the expression of NCOA4 and GPX4 in tumor tissues: (1) Specimen collection and fixation: Tissues were collected from the samples to be tested, and then the collected samples were fixed on slides or slices to maintain their structural and morphological integrity. (2) Paraffin dewaxing: The fixed tissue slices were placed in xylene for dewaxing for 10 minutes to remove the paraffin. Then they were placed in anhydrous ethanol, 90% alcohol, 80% alcohol, and 70% alcohol in turn, each step for 2 minutes for dehydration. (3) Permeabilization: The tissues were permeabilized using hydrochloric acid alcohol to expose the antigens. (4) Antigen heat repair: The slices were placed in antigen heat repair buffer and heated to 100°C in a microwave oven for 4 minutes to expose the antigens. After cooling to room temperature, the tissue slices were rinsed with washing solution. (5) Elimination of nonspecific antibody binding: The tissues were incubated with normal serum to eliminate nonspecific antibody binding. After incubation, the tissue slices were rinsed with washing solution. (6) Primary antibody incubation: Use the primary antibody diluent to prepare the target antibody (GPX4 1:100; NCOA4 1:50), add it to the tissue section, and incubate it in a dark box refrigerator overnight. (7) Secondary antibody incubation: Take out the dark box from the refrigerator and place it at room temperature for a certain period of time to rewarm. Then add the corresponding secondary antibody and incubate. (8) Add horseradish enzyme-labeled streptavidin working solution: After incubation at room temperature for a certain period of time, wash three times with PBS. (9) DAB color development: Prepare DAB color development solution, absorb an appropriate amount of DAB color development solution and add it to the tissue. Under the background of A4 paper, observe the color development every 20 seconds. When the tissue turns yellow, immediately use tap water to wash off the DAB color development solution. (10) Hematoxylin nuclear staining: Add the prepared hematoxylin solution to the tissue and observe the stained cell nuclei under a microscope. (11) Dehydration and sealing: Dehydrate in sequence and then seal the slides. (12) After completing the above steps, observe under a microscope and take photos to record the results.

[0059] 3. Data Statistics All measurement data were expressed as mean ± SD. Unpaired t-test was used for comparison between two groups. ANOVA was used for comparison between multiple groups. One-way ANOVA was used for comparison between multiple groups. P< 0.05 was used to judge statistical significance, * P <0.05,** P <0.01,***P <0.001; statistics were performed using Graphpad Prism 9.0.

[0060] (IV) Experimental results To further investigate the mechanism of THP's anti-TNBC effect, we performed RNA sequencing analysis. Gene set enrichment analysis (GSEA) results showed that GSH signaling was downregulated in the THP-M group compared with the negative control group (NC). Figure 2 A). Subsequently, we used electron microscopy to observe the morphological characteristics of cells in each group. Compared with the NC and DOX groups, the THP-M and THP-M+DOX groups showed a decrease in the number of mitochondrial cristae, mitochondrial distortion, and mitochondrial folding ( Figure 2 B). In addition, we examined other indicators of ferroptosis and found that THP decreased GSH levels and increased Fe 2+ and MDA levels ( Figure 3 CH). After combined administration, DOX not only did not inhibit THP-induced ferroptosis; on the contrary, it led to a further decrease in GSH levels and Fe 2+ And MDA levels further increased.

[0061] In addition, GSEA showed that DOX+THP-M significantly promoted ferroptosis in MDA-MB-231 cells compared with the DOX group ( Figure 3 A). Next, using RNA sequencing data, we identified differentially expressed genes (DEGs) related to ferroptosis and ferroptosis pathways in the DOX and DOX+THP-M groups ( Figure 3 B) The DOX+THP-M group significantly regulated the three regulatory pathways of ferroptosis: lipid metabolism, glutathione pathway, and ferroptosis pathway. In conclusion, our results confirm that THP, but not DOX, can induce ferroptosis.

[0062] To verify the RNA sequencing results, we detected the key proteins involved in these pathways by Western blot. The results showed that THP treatment reduced the levels of xCT and GPX4 and increased the level of p53 in a dose-dependent manner, indicating that THP activated the p53 / xCT / GSH / GPX4 pathway and promoted ferroptosis in TNBC cells. In addition, THP treatment caused a concentration-dependent increase in NCOA4 expression and a concentration-dependent decrease in Nrf2 and FTH1 expression, indicating that THP can also regulate iron autophagy through the Nrf2 / NCOA4 / FTH1 pathway, induce ferroptosis, and inhibit TNBC cell proliferation ( Figure 3 C). Similar results were obtained when DOX was used in combination with THP ( Figure 3D). In conclusion, these results once again confirmed that THP has the effect of inducing ferroptosis and ferroptosis in TNBC.

[0063] In summary, THP alone or in combination with DOX can induce ferroptosis and ferroptosis in vitro, exerting an anti-TNBC effect. Consistent with these findings, the in vivo experimental results showed that Fe 2+ and MDA levels were significantly higher than those in the other groups. In addition, compared with the MOD group (saline treatment), the GSH level in the DOX group was significantly increased, while the GSH level in the THP+DOX group was significantly decreased ( Figure 5 AC). Western blotting further confirmed that the THP and THP+DOX groups reduced the levels of xCT and GPX4, increased the level of p53, and inhibited the malignant development of TNBC by participating in the xc-system pathway of ferroptosis. The level of NCOA4 increased; while the levels of Nrf2 and FTH1 decreased in tumor tissues ( Figure 5 D). Immunohistochemical staining confirmed that GPX4 expression was downregulated and NCOA4 expression was upregulated in tumor tissues of mice treated with THP and THP+DOX ( Figure 5 E). These results indicate that THP can promote ferroptosis and ferroptosis in vivo, and can further increase ferroptosis when combined with DOX, thereby effectively enhancing its inhibitory effect on TNBC.

[0064] Example 4: Tripterygium wilfordii polysaccharide reverses the negative immune regulation of anthracyclines, and the combination of the two drugs significantly improves the tumor microenvironment Example: 1. Experimental materials, reagents and instruments Accuri™ C6 flow cytometer (BD, USA) 2. Experimental methods The polysaccharide extract of the trifoliate green ginseng obtained in Example 1 was used in combination with doxorubicin to detect changes in the proportion of immune cells in the tumor microenvironment. The specific method is as follows: (1) Sampling: After the tumor block is removed, it is placed in a 15ml centrifuge tube containing 5ml PBS and temporarily stored on ice. (2) Pre-shredding: The tumor block and about 2ml of PBS are poured out from the centrifuge tube and placed on the bottom of a 6cm culture dish, and the remaining about 3ml PBS is placed on the lid of the culture dish. Use surgical scissors to cut the tumor block into a mud-like shape, and transfer it back to the 15ml centrifuge tube with a 1ml pipette with the tip cut off; rinse the bottom of the culture dish with PBS on the lid of the culture dish, and transfer it back to the centrifuge tube. Store on ice temporarily. (3) Digestion: Centrifuge at 1000rpm for 5min at room temperature and discard the supernatant. Add 4 ml collagenase (1 mg / ml collagenase IV, 0.1 mg / ml DNase, 2% FBS, RMPI1640) to resuspend, place in a 37°C water bath for 1 hour, and shake every 5 minutes to resuspend. (4) Grinding: Pour the digested tumor tissue and liquid in the 15 ml centrifuge tube directly into the bottom of a 6 cm culture dish, and rinse the centrifuge tube twice with 1 ml PBS and pour it in; transfer an appropriate amount of liquid to the lid of the culture dish so that the remaining liquid is suitable for grinding. Clamp the sterilized stainless steel mesh to the bottom of the culture dish, fix it with straight tweezers, and grind the tumor tissue on the stainless steel mesh with curved tweezers. Clamp the nylon filter (200 mesh) to the mouth of the 15 ml centrifuge tube, and use a 1 ml pipette with the tip cut off to transfer the ground tumor tissue back to the centrifuge tube through the nylon filter; rinse the bottom of the culture dish with the liquid on the lid of the culture dish, and transfer it back to the centrifuge tube. (5) Washing: Centrifuge at 1000 rpm for 5 minutes at room temperature and resuspend with 5 ml PBS. Store on ice. (6) Dead cell staining: Centrifuge at 1000 rpm for 5 min at room temperature, add 200 μl LIVE / DEADFixableDeadCellStains diluent to each tube (50 μl Component B is added to 1 tube of Component A to make a master solution, and dilute it with PBS 1:1000 when used), vortex to mix, and incubate at room temperature and away from light for 30 min. Add 1 ml PBS and centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, and resuspend with 1 ml PBS. (7) Fixation: Centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant. Resuspend the tumor suspension in 1 ml fixative (eBioscience Foxp3 / Transcription Factor Staining Buffer Set, Cat: 00-5523-00, Concentrate: Diluent ratio 1:3, ready to use) and fix (overnight, 4°C, away from light). (8) Storage: Centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant. Wash twice with 1 ml of Buffer, resuspend and store at 4°C in the dark.(9) Counting and staining: Dilute the sample to 1 million cells per tube with buffer, wash once, add 50 μl blocking solution (anti-CD16 / 32) and incubate at 4°C for 15 min, then add 50 ul of antibody diluted to the corresponding concentration and incubate at 4°C for 30 min, wash twice with 1 ml PBS and resuspend, store or flow cytometry. (10) Use FCAP Array v3 software to calculate the percentage of T lymphocytes and CD4. + / CD8 + ratio.

[0065] 3. Data Statistics All measurement data were expressed as mean ± SD. Unpaired t-test was used for comparison between two groups. ANOVA was used for comparison between multiple groups. One-way ANOVA was used for comparison between multiple groups. P< 0.05 was used to judge statistical significance, * P <0.05,** P <0.01,*** P <0.001; statistics were performed using Graphpad Prism 9.0.

[0066] (IV) Experimental results like Figure 6 As shown in the above, we detected immune cells in tumor tissues and found that DOX-L treatment led to an increase in the number of Treg cells and CD8 + In contrast, THP treatment alone reduced the number of Tregs and increased CD8 + Compared with the use of either drug alone, the combination of the two drugs significantly improved the tumor microenvironment, reduced the proportion of Treg cells, and increased the number of CD4 + and CD8 + The proportion of T cells increased.

[0067] Example 5: Example of how Tripterygium wilfordii polysaccharide significantly alleviates the cardiotoxicity caused by anthracyclines 1. Experimental materials, reagents and instruments Neutral gum (Shanghai Bio-Tech Biotechnology Co., Ltd.), paraformaldehyde (Shanghai Bio-Tech Biotechnology Co., Ltd.), hematoxylin (Shanghai Bio-Tech Biotechnology Co., Ltd.), xylene (Shanghai Lingfeng Chemical Reagent Co., Ltd.), film viewing tool (produced by Jiangsu Suhong Medical Instrument Co., Ltd.).

[0068] 2. Experimental methods (1) Tissue fixation: Collect tissue from the sample to be tested and fix it with 4% paraformaldehyde to maintain its structural and morphological integrity. (2) Tissue dehydration: Use alcohol with a gradient concentration to dehydrate and remove water from the tissue. (3) Tissue embedding: Embed the dehydrated tissue in paraffin for easy sectioning. (4) Tissue sectioning: Slice the embedded tissue block and make slices of appropriate thickness. (5) Dewaxing: Dewax the slices in xylene for 10 minutes, then place them in anhydrous ethanol, 90% alcohol, 80% alcohol, and 70% alcohol in turn, each step for 2 minutes. (6) Staining: Stain the slices in hematoxylin staining solution for 10 minutes, then wash away the hematoxylin and floating color in water for about 2 minutes. Then place the slices in differentiation solution for a few seconds to 30 seconds to make the slices fade to light blue-red. Then wash them in running water for 30 minutes to make the tissue bright blue or sky blue. Finally, place the slices in eosin solution for staining for 3 minutes. (7) Dehydration: Place the slices in 75% alcohol, 95% alcohol, and anhydrous ethanol in turn, 30 seconds for each step. (8) Transparency: Place the slices in xylene for 1 minute. (9) Sealing: Take the slices out of xylene and seal them with neutral gum. After staining, the cell nucleus appears blue, and the cytoplasm, muscle, connective tissue, red blood cells, and eosinophilic granules appear pink to varying degrees.

[0069] 3. Data Statistics All measurement data were expressed as mean ± SD. Unpaired t-test was used for comparison between two groups. Analysis of variance was used for comparison between multiple groups. One-way ANOVA was used for comparison between multiple groups. P< 0.05 was used to judge statistical significance, * P <0.05,** P <0.01,*** P <0.001; statistics were performed using Graphpad Prism 9.0.

[0070] (IV) Experimental results like Figure 7 As shown in the experiment, we observed that both low and high concentrations of DOX led to a decrease in heart mass in BALB / C mice ( Figure 7 AB). High concentrations of DOX were more significant, so we analyzed the various indicators of mice in the DOX-H group. The CK-MB results showed that DOX-H was significantly toxic, while THP was non-toxic. After combined use, the CK-MB level was significantly reduced, and the toxicity was effectively alleviated ( Figure 7 C). The electrocardiogram of the THP group was very similar to that of the CON group, and the heart rate and rhythm were basically the same ( Figure 7D). In the DOX-H group, the heart rate slowed down, the P wave disappeared, and the PR interval was prolonged. However, these phenomena can be alleviated when used in combination with THP. This was also confirmed by histological examination of cardiomyocytes. The cardiomyocytes in the CON, MOD, and THP groups were transverse, arranged non-randomly, and were short cylindrical. The cell nuclei were branched and located in the center of the myocardium. The branches of the outer wall of the myocardium were interconnected to form a myofibril network. In the DOX-H group, the myocardial interstitium was widened, the blood vessels were dilated and congested, the horizontal lines of the sarcoplasm were disordered, the nuclear staining became darker, and the cytoplasm was vacuolated. When used in combination with THP, the morphology of cardiomyocytes treated with DOX-H was significantly improved ( Figure 7 E).

[0071] Example 6: An example of how Tripterygium wilfordii polysaccharide significantly alleviates the liver, kidney and bone marrow suppression toxicity caused by anthracyclines.

[0072] 1. Experimental materials and instruments 5427R high-speed refrigerated centrifuge (Eppendorf, Germany), fully automatic biochemical analyzer 2. Experimental methods Detection of mouse biochemical indicators (1) Layer the mouse blood samples, centrifuge at 4℃ for 10 minutes, and separate the supernatant. (2) Use a fully automatic biochemical analyzer to detect mouse serum biochemical indicators. (3) Analyze and plot the results based on the normal range of mouse biochemical indicators.

[0073] 3. Data Statistics All measurement data were expressed as mean ± SD. Unpaired t-test was used for comparison between two groups. Analysis of variance was used for comparison between multiple groups. One-way ANOVA was used for comparison between multiple groups. P< 0.05 was used to judge statistical significance, * P <0.05,** P <0.01,*** P <0.001; statistics were performed using Graphpad Prism 9.0.

[0074] (IV) Experimental results like Figure 8 However, we observed a significant decrease in the weight of the liver, kidney, and spleen after DOX treatment during the experiment, especially in the DOX-H group ( Figure 8 A). Therefore, we further tested some biochemical indicators of liver and kidney function, and found that DOX was significantly toxic, and each indicator showed different degrees of changes. Figure 8As shown in BC, the levels of creatinine (CREA) and lactate dehydrogenase (LDH) in the DOX-H+THP group were significantly reduced compared with those in the DOX-H group. According to the clinical pathological data of BALB / C mice from Charles River Laboratories Inc. from 2008 to 2012, the 95% interval was used as the normal range for reference of physiological indicators of BALB / C mice. The ALT level in the DOX-H group exceeded the maximum limit. Although the AST level was within this range, it was significantly higher than that in the other groups. In addition, the albumin (ALB) level in the DOX-H group was significantly reduced; however, these indicators recovered after the administration of THP ( Figure 8 DF and GI). These results indicate that DOX-H caused severe renal injury and hepatotoxicity, which could be significantly improved by co-administration with THP. In addition, we observed that the neutrophil ratio (NEUT%) and white blood cell (WBC) count in the MOD group exceeded the highest standard, indicating that this group of mice had a higher level of inflammation ( Figure 8 J, K, M, and N). After treatment, except for the DOX-H+DEX group, in which NEUT% decreased, the NEUT% and WBC counts in the other groups were within the normal range. In addition, the NEUT% and WBC counts in the DOX-H group were higher than those in the other groups, indicating the presence of infection-related inflammation. In summary, our results suggest that DOX-H treatment is accompanied by bone marrow suppression, leading to adverse toxic reactions, and effective relief can be achieved when DOX-H is combined with THP.

[0075] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the experimental methods described herein are conventional methods unless otherwise specified; the reagents, biological materials and equipment described herein are commercially available unless otherwise specified.

[0076] The above embodiments are preferred implementations of the present invention, and the present invention is not limited to the above embodiments. It is particularly pointed out that, for ordinary technicians in this field, several modifications and embellishments can be made without departing from the principles of the present invention, and these improvements and embellishments should also be considered as the protection scope of the present invention.

Claims

1. Application of Tripterygium wilfordii polysaccharide in the preparation of cell ferroptosis inducer.

2. The use according to claim 1, characterized in that: The cells are triple-negative breast cancer cells.

3. Application of cell ferroptosis inducers combined with anthracycline compounds in the preparation of drugs for the treatment of triple-negative breast cancer.

4. The use according to claim 3, characterized in that: The cell ferroptosis inducer is Tripterygium wilfordii polysaccharide.

5. Application of Tripterygium wilfordii polysaccharide combined with anthracycline compounds in the preparation of drugs for the treatment of triple-negative breast cancer.

6. The use according to claim 5, characterized in that: The anthracycline compound is selected from doxorubicin, daunomycin, epirubicin, aclarubicin, idarubicin and valrubicin.

7. The use according to claim 6, characterized in that: The mass ratio of the trifoliate green polysaccharide to the anthracycline compound is 15-60:0.

25.

8. The use according to any one of claims 5 to 7, characterized in that: The medicine comprises: anthracycline compounds as main medicinal ingredients, trifoliate green polysaccharide as auxiliary ingredient for enhancing efficacy and reducing toxicity, and pharmaceutically acceptable excipients.

9. The use according to claim 8, characterized in that: The toxicity reduction is to reduce the toxic effects of anthracycline compounds on the heart, liver, kidneys and bone marrow.

10. The use according to claim 8, characterized in that: The preparation form of the drug is an oral preparation.

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