Application of polysaccharide from tetrastigma hemsleyanum combined with anthracycline in preparation of triple-negative breast cancer treatment drug

Abstract

The present application relates to the field of biological medicine, and discloses application of polysaccharide of smilax sieboldi and anthracycline compound in preparation of triple-negative breast cancer treatment drugs. First, the present application first discovers that the polysaccharide of smilax sieboldi is a cell ferroptosis inducer. Secondly, the present application first discovers that the polysaccharide of smilax sieboldi and the anthracycline compound can play a synergistic and attenuating role in the treatment of triple-negative breast cancer, greatly reduce the toxic and side effects of the anthracycline compound, so as to improve the prognosis and survival quality of the triple-negative breast cancer patients, and improve the survival rate of the patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to the application of a combination of Radix Tetrastigmae Hemsleyanum Polysaccharide and anthracycline compounds in preparing a triple-negative breast cancer treatment drug. BACKGROUND

[0002] Triple-negative breast cancer, as the subtype of breast cancer with the highest malignancy, 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 have poor therapeutic effect, which leads to poor clinical prognosis and greatly limits the choice of treatment strategies. In addition, compared with other subtypes of breast cancer, triple-negative breast cancer has a higher risk of distant metastasis, recurrence and death.

[0003] Due to the high iron and lipid content of triple-negative breast cancer, this feature makes iron death inducers an ideal choice for its treatment. In recent years, rapid progress has been made in understanding the mechanism of iron death, and it has been found that various cancer proteins, tumor suppressor genes and oncogenic signaling pathways can regulate iron death. Generally speaking, iron death is a form of regulated necrosis, which is strictly controlled at multiple levels. Iron death can occur through three main 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 plan for triple-negative breast cancer, and anthracycline drugs (doxorubicin, daunorubicin, epirubicin, aclarubicin, idarubicin, valrubicin, etc.) combined with other chemotherapy drugs such as paclitaxel are still the main treatment method for triple-negative breast cancer. However, this chemotherapy regimen is highly heterogeneous and has limited efficacy. More complexly, residual lesions after chemotherapy, gene mutations, amplification and immune escape can lead to the development of chemotherapy resistance, further aggravating the poor prognosis of triple-negative breast cancer patients. In addition, although anthracycline drugs have achieved certain success in anti-tumor treatment, they also face some challenges and limitations in practical application, of which the biggest problem in clinical treatment is their limited efficacy accompanied by significant side effects. Since doxorubicin is one of the most effective anthracycline drugs in inhibiting the growth of various solid and hematological malignancies, the present application takes doxorubicin as an example to propose a solution to the problems existing in the treatment of triple-negative breast cancer with anthracycline drugs.

[0005] We think that the combination of ferroptosis inducer and anthracycline may have an unexpected effect on the treatment of triple-negative breast cancer. However, the number of ferroptosis inducers is very limited, and the exploration of the application and therapeutic effect of ferroptosis inducers is even less. How to effectively develop new ferroptosis inducers and solve the current problems of anthracyclines, so as to effectively treat triple-negative breast cancer has become a severe challenge that needs to be solved urgently.

[0006] Ampelopsis brevifolia is a rare medicinal material in China, which was originally used to treat cancer in folk. Now it is not only a traditional Chinese medicine, but also a functional food. Ampelopsis brevifolia contains various phytochemicals, such as flavonoids, phenolic acids, polysaccharides and phytosterols. Modern pharmacological studies have shown that Ampelopsis brevifolia and its active ingredients have anti-inflammatory, antioxidant, antiviral, antipyretic, anti-liver injury, immunomodulatory, antibacterial and other pharmacological activities. Through long-term research, our previous work has confirmed that Ampelopsis brevifolia polysaccharide is the main medicinal material of Ampelopsis brevifolia. Ampelopsis brevifolia polysaccharide has various pharmacological functions, such as anti-inflammatory, antioxidant and immunomodulatory functions. The applicant's prior patent CN105816430B (Chinese patent application number: 201610149782.4, publication date April 9, 2019) discloses an extraction method of Ampelopsis brevifolia polysaccharide. Based on this, the applicant further explores the synergistic and detoxification effects of the extracted Ampelopsis brevifolia polysaccharide on anthracyclines represented by doxorubicin in the treatment of triple-negative breast cancer. SUMMARY

[0007] In order to solve the problems of poor efficacy, strong heterogeneity and significant toxic side effects of anthracyclines in clinical application, the application provides the use of Ampelopsis brevifolia polysaccharide combined with anthracyclines in the preparation of triple-negative breast cancer treatment drugs. First of all, the application first discovers that Ampelopsis brevifolia polysaccharide is a ferroptosis inducer; secondly, the application first discovers that Ampelopsis brevifolia polysaccharide combined with anthracyclines can play a synergistic and detoxification role in the treatment of triple-negative breast cancer, greatly reducing the toxic side effects of anthracyclines, thereby hopefully improving the prognosis and quality of life of triple-negative breast cancer patients and improving the survival rate of patients.

[0008] The specific technical scheme of the application is as follows:

[0009] In the first aspect, the application provides the use of Ampelopsis brevifolia polysaccharide in the preparation of a cell ferroptosis inducer.

[0010] The application first discovers through experiments that Ampelopsis brevifolia polysaccharide is a natural cell ferroptosis inducer, which can promote cancer cell ferroptosis.

[0011] As a preferred, the cell is a triple-negative breast cancer cell.

[0012] In a second aspect, the present application provides a use of a cell ferroptosis inducer in combination with an anthracycline compound in the preparation of a drug for treating triple-negative breast cancer.

[0013] In our previous studies, it was found that the combination of a cell ferroptosis inducer and an anthracycline drug can achieve unexpected effects on the treatment of triple-negative breast cancer.

[0014] Preferably, the cell ferroptosis inducer is a Radix Tetrastigmae Polysaccharide.

[0015] The present application first found that the Radix Tetrastigmae Polysaccharide in combination with an anthracycline drug can significantly reverse the negative immune regulation of the anthracycline drug, and further promote the ferroptosis of triple-negative breast cancer.

[0016] In a third aspect, the present application provides a use of a Radix Tetrastigmae Polysaccharide in combination with an anthracycline compound in the preparation of a drug for treating triple-negative breast cancer.

[0017] The present application first found that the Radix Tetrastigmae Polysaccharide in combination with an anthracycline compound can significantly enhance the efficacy and reduce the toxicity of the anthracycline compound in the treatment of triple-negative breast cancer, which is embodied in:

[0018] (1) The Radix Tetrastigmae Polysaccharide in combination with an anthracycline drug mainly regulates the xCT / GSH / GPX4 and Nrf2 / NCOA4 / FTH1 pathways, thereby improving the treatment effect on triple-negative breast cancer;

[0019] (2) The present application found through flow detection that the administration of an anthracycline drug can increase the number of Treg cells, reduce the number of CD8 + T cells, and present negative immune regulation. The combination with the Radix Tetrastigmae Polysaccharide can significantly improve the tumor microenvironment, which is manifested by a decrease in the proportion of Treg cells and an increase in the proportion of CD4 + and CD8 + T cells;

[0020] (3) The Radix Tetrastigmae Polysaccharide is a natural cell ferroptosis inducer, which can further promote the ferroptosis of triple-negative breast cancer when combined with an anthracycline drug, thereby improving the treatment effect on triple-negative breast cancer;

[0021] (4) The present application detects multiple groups of biochemical indicators (CK-MB) reflecting heart function after administration, and detects the morphology of the heart. The results show that anthracyclines have significant toxic side effects, which are significantly alleviated when used with Radix Tetrastigme Polysaccharide; The present application further detects multiple groups of biochemical indicators reflecting liver (ALT, AST), kidney (ALB) and bone marrow hematopoietic function (WBC, PLT) after administration. The results show that anthracyclines have significant toxic side effects, which are significantly alleviated when used with Radix Tetrastigme Polysaccharide. Therefore, Radix Tetrastigme Polysaccharide can significantly alleviate the toxic side effects of anthracyclines, including heart, liver, kidney and bone marrow suppression toxicity and other aspects.

[0022] As a preferred, the anthracycline compound is selected from doxorubicin, daunorubicin, epirubicin, aclarubicin, idarubicin and valrubicin, etc.

[0023] As a preferred, the mass ratio of Radix Tetrastigme Polysaccharide to anthracycline compound is 15-60:0.25.

[0024] The present application finds through in vitro CCK8 test that the drug concentration range of Radix Tetrastigme Polysaccharide combined with anthracycline compound (such as doxorubicin) for synergistic anti-triple negative breast cancer effect is: doxorubicin (0.25 μg / ml) combined with Radix Tetrastigme Polysaccharide (15, 30, 60 μg / ml) respectively.

[0025] As a preferred, the drug includes: anthracycline compound as the main pharmaceutical ingredient, Radix Tetrastigme Polysaccharide as the synergistic and attenuated efficacy auxiliary ingredient, and pharmaceutically acceptable excipients.

[0026] Further, the attenuation is to reduce the toxic effects of anthracyclines on heart, liver, kidney and bone marrow.

[0027] As a preferred, the preparation form of the drug is oral preparation.

[0028] In a sixth aspect, the present application provides a method for promoting in vitro cell ferroptosis: adding Radix Tetrastigme Polysaccharide and anthracycline compound to a cell suspension to promote cell ferroptosis.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] Firstly, the present application first finds that Radix Tetrastigme Polysaccharide is a cell ferroptosis inducer; secondly, the present application first finds that Radix Tetrastigme Polysaccharide combined with anthracycline compound can play a synergistic and attenuated effect in the treatment of triple negative breast cancer, significantly reducing the toxic side effects of anthracyclines, thereby improving the prognosis and quality of life of triple negative breast cancer patients and improving the survival rate of patients. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the proliferation of triple-negative breast cancer cells inhibited by Tripterygium wilfordii Hook f. polysaccharides, and the anti-triple-negative breast cancer effect of doxorubicin is enhanced after combined with Tripterygium wilfordii Hook f. polysaccharides. Among them: (A-B) the effect of doxorubicin on MDA-MB-231 and 4T1 cells after 24, 48 and 72h of action. (C-D) The effect of Tripterygium wilfordii Hook f. polysaccharides on MDA-MB-231 and 4T1 cells after 24, 48 and 72h of action. (E-F) The effect of 0.25μg / ml of doxorubicin combined with 15, 30, 60, 120μg / ml of Tripterygium wilfordii Hook f. polysaccharides on MDA-MB-231 and 4T1 cell viability after 24, 48h of action. (G-H) The effect of 0.5μg / ml of doxorubicin combined with 15, 30, 60, 120μg / ml of Tripterygium wilfordii Hook f. polysaccharides on MDA-MB-231 and 4T1 cell viability after 24, 48h of action. (I-J) According to the CCK8 results of MDA-MB-231 cells, the cell viability was analyzed by Combenefit software using HSA method, and the average synergistic score was reported. A score greater than 10 indicates synergistic effect of each dose combination, and a score less than 10 indicates antagonistic effect. (K-L) According to the CCK8 results of 4T1 cells, the cell viability was analyzed by Combenefit software using HSA method, and the average synergistic score was reported. A score greater than 10 indicates synergistic effect of each dose combination, and a score less than 10 indicates antagonistic effect. P <0.05,** P <0.01,*** P <0.001 vs. Control group.

[0032] Figure 2 is the induction of ferroptosis in triple-negative breast cancer cells by Tripterygium wilfordii Hook f. polysaccharides and the combination of the two drugs. Among them: (A) GSEA pathway enrichment shows that the glutathione metabolism signal is down-regulated in the medium dose group of Tripterygium wilfordii Hook f. polysaccharides (THP-M) compared with the control group. (B) Electron microscope images of each group after treating MDA-MB-231 cells with the control group, doxorubicin administration group, medium dose Tripterygium wilfordii Hook f. polysaccharide group and two drug combination group (THP-M+DOX) for 24h, 4 fields of view were selected from each group; scale bar: 0.5μm. (C-D) GSH levels in MDA-MB-231 and 4T1 cells in each administration group. (E-F) Fe 2+ levels in MDA-MB-231 and 4T1 cells in each administration group. P <0.05,** P <0.01,*** P <0.001 vs. Control group.

[0033] Figure 3 (A) GSEA analysis showed that the iron death signal was enhanced in the combination group compared with the doxorubicin group. (B) Heat map showing the differentially expressed genes in MDA-MB-231 cells after 24 h of treatment with the combination group compared with the doxorubicin group. (C) Changes in the proteins of p53, xCT, GPX4, Nrf2, NCOA4, and FTH1 after 24 h of treatment of MDA-MB-231 and 4T1 cells with low, medium, and high doses of tri-leaf green polysaccharide. (D) Changes in the proteins of p53, xCT, GPX4, Nrf2, NCOA4, and FTH1 after 24 h of treatment of MDA-MB-231 and 4T1 cells with the control group, the doxorubicin group, and the combination group.

[0034] Figure 4 Tri-leaf green polysaccharide can enhance the anti-triple-negative breast cancer effect of doxorubicin at the in vivo level. Among them: (A) Tumor images of the model group (MOD), the low-dose doxorubicin group (DOX-L), the tri-leaf green polysaccharide group, the low-dose doxorubicin and tri-leaf green polysaccharide combination group (DOX-L+THP), the low-dose doxorubicin and dexamethasone combination group (DOX-L+DEX), the high-dose doxorubicin group (DOX-H), the high-dose doxorubicin and tri-leaf green polysaccharide combination group (DOX-H+THP), and the high-dose doxorubicin and dexamethasone combination 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) Mice body weight 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. (D) Tumor weight 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. (E) HE staining analysis of tumor tissue morphology. P <0.05,** P <0.01,*** P <0.001 vs. MOD group.

[0035] Figure 5 Tri-leaf green polysaccharide can enhance the anti-triple-negative breast cancer effect of doxorubicin at the in vivo level by inducing iron death and iron autophagy. Among them: (A) Fe 2+(a) Levels of GSH in tumors treated with MOD, DOX-L, THP, and DOX-L+THP; (b) Levels of MDA in tumors treated with MOD, DOX-L, THP, and DOX-L+THP; (c) Levels of MDA in tumors treated with MOD, DOX-L, THP, and DOX-L+THP; (d) Western blot analysis of the expression levels of p53, xCT, GPX4, Nrf2, NCOA4, and FTH1 in tumors after DOX-L, THP, and DOX-L+THP treatment; (e) Immunohistochemical analysis of GPX4 and NCOA4 expression levels in tumor tissues. P <0.05,** P <0.01, *** P <0.001 vs. MOD group.

[0036] Figure 6 *Trifolium repens* polysaccharide can improve the tumor microenvironment and reverse the negative immune regulation of doxorubicin. Specifically: (A) KEGG analysis of MDA-MB-231 cells treated with THP-M for 24 h showed differentially expressed gene enrichment pathways compared to the control group; the larger the bubble, the more differentially expressed genes between the two groups, and the darker the color, the more significant the difference. (BD) Treg and CD8 expression levels in tumor tissues of mice in the DOX-L, THP, DOX-L+THP, and MOD groups... + T cell levels; (E) Treg and CD4 levels in tumor tissues of mice in the DOX-L, THP, DOX-L+THP and MOD groups + T and CD8 + T cell levels. P <0.05,** P <0.01, *** P <0.001 vs. MOD group.

[0037] Figure 7 *Trifolium repens polysaccharide can alleviate doxorubicin-induced cardiotoxicity. The results included: (AB) cardiac organ coefficients (calculated by dividing organ weight by body weight); (C) CK-MB (U / L) levels in each group; (D) electrocardiogram findings in each group of mice; and (E) HE staining analysis of cardiac histological morphology in 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.

[0038] Figure 8 The Trientalis Davidiana polysaccharide can reduce the liver, kidney and bone marrow suppression toxicity caused by doxorubicin. Among them: (A) the organ coefficient of each organ of the mouse; the organ coefficient is calculated by dividing the organ weight by the body weight. (B-C) The creatinine (CREA) and lactate dehydrogenase (LDH) levels of each group of mice. (D-F) The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) normal value reference range of the mouse and the average value of ALT, AST, and ALB of each group of mice. (G-I) The ALT, AST, and ALB values of each group of mice. (J-L) The normal value reference range of the neutrophil ratio (NEUT%), white blood cells (WBC), and platelets (PLT) of the mouse and the average value of NEUT%, WBC, and PLT of each group. (M-O) The NEUT%, WBC, and PLT values of each group of mice. For Figures (D-F) and (J-L), in the discussion of biochemical indicators, we believe that due to the individual differences of mice, biochemical indicators should have a normal range. We determined the maximum and minimum values of each indicator. Compare the average value of each indicator with the interval. P <0.05, P <0.01, P <0.001 vs. MOD group. # P <0.05, ## P <0.01, ### P <0.001 vs. DOX-H group. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and benefits of the present application clearer, the technical scheme of the present application will be described in detail below in combination with specific experiments and data processing. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. Changes and optimizations that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and the appended claims and any equivalents thereof are the protection scope of the present application. Unless otherwise described, the implementation of the present application will employ conventional techniques of cell biology, molecular biology, genetic engineering technology, etc., which are known to those skilled in the art.

[0040] General examples

[0041] In a first aspect, the present application provides the use of Trientalis Davidiana polysaccharide in the preparation of a cell ferroptosis inducer; further, the cell is a triple-negative breast cancer cell.

[0042] In a second aspect, the present application provides a use of a cell ferroptosis inducer in combination with an anthracycline compound in the preparation of a drug for treating triple-negative breast cancer.

[0043] In a third aspect, the present application provides a use of a Radix Tetrastigme polysaccharide in combination with an anthracycline compound in the preparation of a drug for treating triple-negative breast cancer.

[0044] Further, the anthracycline compound is selected from the group consisting of doxorubicin, daunorubicin, epirubicin, aclarubicin, idarubicin, and valrubicin.

[0045] Further, the mass ratio of the Radix Tetrastigme polysaccharide to the anthracycline compound is 15-60:0.25.

[0046] Further, the drug comprises: the anthracycline compound as a main pharmaceutical active ingredient, the Radix Tetrastigme polysaccharide as a synergistic and attenuated efficacy auxiliary ingredient, and a pharmaceutically acceptable excipient; wherein the attenuation is to reduce the toxic effects of the anthracycline compound on the heart, liver, kidney, and bone marrow.

[0047] Further, the preparation form of the drug is an oral preparation.

[0048] In a sixth aspect, a method for promoting cell ferroptosis in vitro: adding a Radix Tetrastigme polysaccharide and an anthracycline compound to a cell suspension to promote cell ferroptosis.

[0049] Example 1: Preparation of Radix Tetrastigme polysaccharide

[0050] The required experimental materials, instruments, and specific experimental methods are described in patent CN105816430B (Chinese patent application number: 201610149782.4). The process is briefly described as follows: The above-ground parts of Radix Tetrastigme are dried in a 40℃ oven, dried, and crushed. They are placed in a round-bottom flask and extracted with double-distilled water by reflux for 3 times, 30 min each time, at a solid-liquid ratio of 1:15 (g / ml). After filtration, the 3 times of extraction solutions are combined, concentrated under reduced pressure, and 3 times of 95% ethanol is added, which is 3 times the mass of the concentrated solution. The water extraction and alcohol precipitation are performed 3 times, and after standing overnight at 4℃, high-speed centrifugation is performed at 4200 rpm for 15 min to obtain the precipitate. The precipitate is washed with anhydrous ethanol 2 times, and vacuum freeze-drying is performed to obtain Radix Tetrastigme polysaccharide powder.

[0051] Example 2: Significant synergistic anti-triple-negative breast cancer effect of Radix Tetrastigme polysaccharide in combination with an anthracycline drug

[0052] (I) Experimental materials, reagents, and instruments

[0053] MDA-MB-231 and 4T1 cell lines were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. CCK-8 reagent (APExBio, USA), fetal bovine serum (BI, USA), trypsin (Gibco, USA), DOX (Doxorubicin, Shanghai Shenguo), DEX (Dexamethasone, Shyuanye, B25793), cell culture bottles (25 cm 3 , 75 cm 3 ) (Wuxi Naisi Biological Technology Co., Ltd.), cell culture plates (96-well, 24-well, 6-well) (Wuxi Naisi Biological Technology Co., Ltd.), PBS (Jiangsu Kaikai Biological Technology Co., Ltd.), leibbovitz's L-15 medium (Shanghai Yuancheng Biological Technology Co., Ltd.), microplate reader (Rayto, USA), cell incubator (Thermo Fisher Scientific, USA).

[0054] (II) Experimental method

[0055] The three-leaf clover polysaccharide extract obtained in Example 1 was combined with doxorubicin to synergistically induce triple-negative breast cancer cell death. The specific steps include the following: (1) MDA-MB-231 and 4T1 cells in the exponential growth phase were digested with trypsin, blown into a single uniform cell suspension, and a portion was taken with a 20 μl gun head. The cell counting plate was counted, and after counting, the cells were mixed, and 100 μl (2000 cells / well) was added to the 96-well plate. (2) The 96-well cell culture plate was placed in the cell incubator for 12 h, then DOX and THP were added at different concentrations, and incubated for 24, 48, and 72 h, respectively; (3) the culture medium was aspirated, without touching the bottom adherent cells, 100 μl of culture medium containing 10% CCK-8 solution was added to the 96-well plate; (4) incubate in a 37°C incubator for 3.5 h, and measure the absorbance at 480 nm.

[0056] Healthy female BALB / C mice were obtained from Zhejiang Chinese Medical University Experimental Animal Center (license number: SYXK (Zhejiang) 2021-0012, animal model research ethics approval number: IACUC-20220627-30). All animals were raised under standard conditions (25±3℃, 60±5% humidity, 12h light-dark cycle) and had free access to whole grain feed and water. All animals were adapted for 7 days before the experiment. 4T1 cells were injected into the mammary fat pad of BALB / C mice, and drug treatment began 5 days later.

[0057] The mice were grouped as follows: 4T1 tumor-bearing mice were randomly divided into 9 groups (8 mice per group), MOD (injection of normal saline) 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 normal saline.

[0058] The body weight was measured every other day, and the length and width of the tumor were detected using a vernier caliper. The tumor volume was calculated: tumor volume = (tumor length) x (tumor width) 2 / 2. At the end of the experiment, the tumors and major organs (heart, liver, spleen, kidney) were isolated from the mouse cadavers. The organ index was calculated: organ index (%) = organ weight (g) / mouse weight (g) x 100%.

[0059] (Three) Data statistics

[0060] The measurement data were expressed as mean ± SD. The unpaired t-test was used for comparison between two groups, and the variance analysis test was used for comparison of differences among multiple groups. The one-way ANOVA was used for pairwise comparison among multiple groups. The statistical significance was judged at P< 0.05, P <0.05, P <0.01, P <0.001; the statistics were performed using Graphpad Prism 9.0, and the synergistic score was performed using Combenefit software.

[0061] (Four) Experimental results

[0062] The results are shown in Figure 1 . Through CCK8 detection, we established the concentration gradient of DOX and THP (0.25-4 μg / ml and 15-240 μg / ml, respectively) Figure 1A-D). Then we combined four concentrations of THP (15, 30 and 60, 120 pg / ml) with 0.25 pg / ml of DOX-L and 0.5 pg / ml of DOX-H, respectively, to detect the combination effect. According to the CCK8 results and combination scores, we determined the optimal concentrations of THP for combination with DOX as low (THP-L, 15 pg / ml), medium (THP-M, 30 pg / ml) and high (THP-H, 60 pg / ml), and combined with DOX-L (0.25 pg / ml) respectively. Figure 1 E-H). Subsequently, according to the CCK8 results, we used the HSA method to analyze the synergy of cell viability using Combenefit software and reported the average synergy score. The results showed that 0.25 pg / ml of doxorubicin combined with 15, 30, 60, 120 pg / ml of THP had a significant synergistic effect. The synergy scores were all greater than 10 ( Figure 1 I-L).

[0063] To evaluate the anti-TNBC effect of THP and THP+DOX in vivo, we constructed a mouse subcutaneous tumor model with TNBC cells. Dexrazoxane (DEX) is the only effective drug approved by FDA to prevent anthracycline cardiotoxicity. However, studies have reported that it can affect 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 efficacy and toxicity of THP on DOX. In the pre-experiment exploration, we established three concentrations of THP (100, 150 and 200 mg / kg) to explore the optimal dosage, and the results showed that the anti-TNBC effect of THP was concentration-dependent within a certain range, and reached the best effect at about 150 mg / kg. When the concentration exceeds this range, its anti-TNBC effect does not show a dose-dependent enhancement. The in vivo regulation of DOX is consistent with the results of our in vitro experiments, and its anti-TNBC effect shows a dose-dependent effect; low concentration DOX (DOX-L, 3 mg / kg) has poor anti-TNBC effect, and high concentration DOX (DOX-H, 5 mg / kg) has good anti-TNBC effect, but with severe weight loss and extremely serious side effects.

[0064] The specific results are shown in Figure 4 Fig. 6, and 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 4 A-D). Hematoxylin and eosin (HE) staining of tumor tissue confirmed that the combination of DOX-L+THP and DOX-H+THP had the best anti-TNBC effect, the cell morphology was relatively regular, the tumor cell count and nuclear division were reduced, and it was better than the DEX positive control group ( Figure 4 E).

[0065] Example 3: Radix tetrastigmae polysaccharide induces ferroptosis in triple-negative breast cancer cells, and ferroptosis is further enhanced after combination with anthracycline drugs.

[0066] (I) Experimental materials and instruments

[0067] HBSS (Gibco, USA), BODIPY 581 / 591 C11 (Invitrogen, USA), Hoechst (Invitrogen, USA), GSH / GSSG-glo assay kit (Beyotime, China), PVDF membrane (Millipore, USA), analytical grade anhydrous ethanol (Yasheng Chemical Industry Co., Ltd. Wuxi), analytical grade methanol (Shanghai Lingfeng Chemical Reagent Co., Ltd.), P0010 BCA protein concentration determination kit (Biuntian Biotechnology Co., Ltd., Shanghai), SDS-PAGE gel rapid preparation kit (Biuntian Biotechnology Co., Ltd., Shanghai), SDS-PAGE electrophoresis liquid (Biuntian Biotechnology Co., Ltd., Shanghai), SDS-PAGE protein loading buffer (5x) (Biuntian Biotechnology Co., Ltd., Shanghai), RIPA lysis buffer (Biuntian Biotechnology Co., Ltd., Shanghai), P0010 BCA protein concentration determination kit (Biuntian Biotechnology Co., Ltd., Shanghai), electrophoresis instrument (bio-rad, USA), vertical electrophoresis tank (bio-rad, USA), transfer device (produced by Beijing Weikesi Technology Co., Ltd.), GAPDH Mouse McAb 60004 (Proteintech, USA), D8340 skimmed milk powder (Solabio Technology Co., Ltd., Beijing), Goat anti-Rabbit IgG (Baishabiological Technology Co., Ltd.), Goat anti-Mouse IgG (Baishabiological Technology Co., Ltd.), 20x TBST (Solabio Technology Co., Ltd., Beijing), PVDF membrane (Millipore, USA)

[0068] (II) Experimental methods

[0069] The three-leafed greenbrier polysaccharide extract obtained in Example 1 was used to find that the three-leafed greenbrier polysaccharide has a significant regulatory effect on the glutathione pathway through transcriptome sequencing. The transmission electron microscope was used to observe the morphology of mitochondria, and the specific method was as follows: (1) sample collection: after centrifugation of the cell suspension, the cell precipitate was collected and stored at 4°C in 2.5% glutaraldehyde. (2) sample fixation: the sample was fixed on the transmission electron microscope sample holder. Then the sample was stuck on the copper mesh and placed in a container containing the fixing solution for fixation. (3) dehydration treatment: the cell precipitate was washed with phosphate buffer and 1% osmium acid, and then the sample was gradient dehydrated with acetone. (4) embedding: the immersed sample was wrapped with embedding agent and cut into ultrathin sections; (5) sectioning: the embedded sample was sectioned to obtain ultrathin sections. The section thickness was between 50-100 nm. (6) staining: the section was stained to better distinguish the structure of the sample under the transmission electron microscope. (7) sample loading: the prepared sample was loaded onto the sample stage of the transmission electron microscope. This step requires the use of a special sample clamp 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) sample loading: the prepared sample was loaded onto the sample stage of the transmission electron microscope. This step requires the use of a special sample clamp 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, focusing, magnification and other parameters of the electron gun, and calibrating the working voltage and current of the instrument, etc. (10) transmission electron microscope observation: after the equipment debugging is completed, the transmission electron microscope observation is started. After the electron beam passes through the sample, the microstructure 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 are obtained. (11) picture taking and analysis: the morphology of mitochondria was observed and imaged under the H7500 transmission electron microscope, and the pictures were taken.

[0070] GSH content detection: (1) GSSG stock solution preparation: Dissolve 5 mg GSSG in 816 μl ultrapure water to obtain a 10 mM stock solution. (2) DTNB stock solution preparation: Dissolve 4.5 mg DTNB in 1.5 ml DMSO provided by the kit, and mix well to obtain the DTNB stock solution. (3) Preparation of protein removal reagent M solution: Weigh 0.2 g of protein removal reagent M, and mix with 4 ml total glutathione detection buffer to prepare a 5% aqueous solution. (4) NADPH stock solution preparation: Dissolve 4 mg NADPH in 100 μl ultrapure water to obtain a high-concentration stock solution. (5) Glutathione reductase dilution: Mix 50 μl glutathione reductase with 200 μl total glutathione detection buffer to obtain the diluted reductase. (6) Preparation of total glutathione detection working solution: According to the number of samples to be tested, mix the three reagents in the proportion specified in the instructions to prepare the working solution. (7) Preparation of 0.5 mg / ml NADPH solution: Take an appropriate amount of NADPH stock solution, and dilute to the desired concentration with total glutathione detection buffer. (8) Preparation of GSH removal auxiliary solution and working solution: Mix the GSH removal auxiliary solution with ultrapure water to obtain a dilution solution, which needs to be freshly prepared each time. (9) Standard preparation: Dilute the GSSG stock solution with the protein removal reagent M solution to prepare a series of GSSG solutions with different concentrations as standards. (10) Determination of GSSG content in samples: Add the diluted GSH removal auxiliary solution and GSH removal reagent working solution to the standards, and react for a certain period of time before subsequent detection. (11) Cell sample preparation: Wash the cells with PBS, centrifuge to collect, remove the supernatant, add the protein removal reagent M solution for freeze-thaw treatment, centrifuge to take the supernatant for subsequent determination. (12) Total glutathione determination: Add the diluted GSH removal auxiliary solution and GSH removal reagent working solution to the sample to be tested, react, then add the sample or standard to the 96-well plate, followed by the total glutathione detection working solution and NADPH solution, incubate, and then measure the absorbance. (13) Centrifuge the sample at 10,000 g at 4°C for 10 min to separate the supernatant, which will be used for subsequent total glutathione determination. (14) Take a portion of the prepared sample to be tested, add the diluted GSH removal auxiliary solution according to the specified proportion (20 μl per 100 μl sample), and then immediately mix well by vortex. (15) Then, add the GSH removal reagent working solution to the sample according to another proportion (4 μl per 100 μl sample), mix well by vortex immediately, and react at 25°C for 60 min. (16) When measuring the sample and standard, use a 96-well plate, add the sample or standard in order, and mix well.Then, 150 μl of total glutathione detection working solution was added to each well, mixed well, and incubated at 25°C or room temperature for 5 min. (17) Next, 50 μl of 0.5 mg / ml NADPH solution was added to each well and mixed well. (18) After the NADPH solution was mixed well, 25 min was waited, and then the absorbance of each well at A412 was measured using an enzyme marker. These steps will accurately determine the glutathione content in the sample.

[0071] Fe 2+ Content detection: (1) After 12 h of cell plating culture, drug treatment was performed for 24 h. (2) Trpsin digestion treatment, centrifugal collection of cells, and dilution of samples. (3) 50 μl of sample was added to the sample well of a 96-well plate, and the volume was adjusted to 100 μl / well with Assay Buffer. (4) 5 μl of iron assay buffer was added to each sample. Mix well with a pipette, incubate the reaction at 25°C for 30 min, and protect the culture dish from light during incubation. (5) Add 100 μl of iron probe to each well, and each well contains a standard sample. Mix well with a pipette, react at 25°C for 60 min, and protect the culture dish from light during incubation. (6) Measure the absorbance at 593 nm.

[0072] MDA content detection: (1) Preparation of TBA stock solution: The required amount of TBA was weighed, and a specific TBA preparation solution was used 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 and prepare the appropriate amount of MDA detection solution immediately before testing. (3) Dilution of standard samples: Use distilled water to dilute the standard samples to concentration gradients of 1, 2, 5, 10, 20, and 50 μM for subsequent construction of standard curves. If the MDA concentration in the sample is high, 100, 150, and 200 μM concentration points can be added as needed. (4) Sample determination steps: In a suitable container, add 0.1 ml of homogenate, lysate, or PBS as a blank control, and add the same volume of different concentrations of standard samples for constructing a standard curve, and then add 0.1 ml of the sample to be tested. Then, add 0.2 ml of MDA detection solution. (5) After mixing well, place in a 100°C or boiling water bath for 15 min. During the heating process, prevent the liquid from splashing out, and if a heating block is used, it is recommended to press the centrifuge tube cap with a weight to ensure safety. (6) After the solution cools to room temperature, centrifuge at 1000 g for 10 min at room temperature. Take 200 μl of supernatant and add it to a 96-well plate, and measure the absorbance at a wavelength of 532 nm using an enzyme marker. (7) MDA content calculation: According to the measured absorbance value, calculate the MDA content in the sample solution. Then, convert it to the original MDA content in the sample by unit weight of protein content or tissue weight, etc., and express it as μmol / mg protein.

[0073] Further, the three-leaf extract obtained in Example 1 was combined with doxorubicin, and through transcriptome sequencing, it was found that ferroptosis was significantly up-regulated after combination compared with doxorubicin alone. Further, Western Blot was used to detect the cell signal pathway, and the specific steps were as follows: (1) collect cells; lyse cells on ice (RIPA: PMSF = 99:1), 4°C, 10000 rpm centrifugation for 10 min, take the supernatant. (2) Add 0, 1, 2, 4, 8, 12, 16, 20 μl of protein standard solution to the hole in turn, and add 1 μl of the measured protein sample to each duplicate hole of the measured sample. (3) Add 200 μl of BCA working solution (A liquid: B liquid = 50:1) to each hole, and stand at room temperature for 2 min or at 37°C for 1 h. (4) Detect the OD value with an enzyme marker with a wavelength of 562 nm. First, make a standard curve according to the OD value of the standard hole, then bring the average OD value of the measured sample into the curve, and the obtained value multiplied by 20 times is the actual concentration of protein (μg / μl). (5) Dilute the protein sample with a measured concentration to 2 μg / μl with a cell lysis buffer, add 20 μl of 5x protein loading buffer to 80 μl of the diluted sample, and mix well by blowing, then heat at 100°C metal bath for 5 min to denature the protein. (6) Prepare SDS-PAGE gel. (7) Connect the power supply and set the working voltage to 80V constant voltage for 30 min. (8) After the bromophenol blue indicator in the sample migrates to the separation gel, change to 120V constant voltage for 1 h, until the blue band approaches the lower edge of the glass gel plate, stop electrophoresis. (9) Transfer the membrane, prepare the transfer solution, and the ratio is: glycine 14.4 g, tris-hydroxymethyl aminomethane 3 g, methanol 200 ml, ultrapure water 800 ml. Shake evenly until there are no obvious solid particles, and store in a 4°C refrigerator for standby. (10) Soak the PVDF membrane in methanol for 3-5 min for membrane transfer. (11) Generally, proteins below 30 kDa are transferred at 100V constant voltage for 1 h, proteins between 30-80 kDa are transferred at 250mA constant current for 1 h, and proteins above 80 kDa are transferred at 300mA constant current for 2 h. (12) Membrane blocking and antibody incubation: slowly shake on a shaker at room temperature for 1-5 h; (13) Discard the milk powder and rinse the PVDF membrane with 1xTBST twice. (14) Add 4 ml of primary antibody diluent and an appropriate amount of antibody (antibody dilution concentration: GAPDH 1:10000, xCT 1:2000, Nrf2 1:2000, GPX4 1:5000, p53 1:2000, NCOA4 1:2000, FTH1 1:2000), 4°C, slowly shake on a shaker for 12-16 h. (15) Recover the primary antibody and store it at 4°C. Pour 1xTBST into the incubation box, and quickly shake the PVDF membrane on a shaker for 10 min each time, for a total of 3 times.(16) Add an appropriate amount of 5% skimmed milk powder to the incubation box, and add the corresponding secondary antibody of the species at a ratio of 1:10000, and slowly shake the incubator for 1 h. (17) Discard the secondary antibody, wash the PVDF membrane for 10 min each time, a total of 3 times. (18) The band is developed by measuring the gray value of the band with ImageJ software and quantitatively analyzing it.

[0074] At the in vivo level, the expression of NCOA4 and GPX4 in tumor tissues was detected by immunohistochemistry: (1) Specimen collection and fixation: collect tissues from the samples to be detected, and then fix the collected samples on glass slides or sections to maintain the integrity of their structure and morphology. (2) Paraffin deparaffinization: place the fixed tissue sections in xylene for 10 min to remove paraffin. Then place them in anhydrous ethanol, 90% alcohol, 80% alcohol, and 70% alcohol, respectively, for 2 min each time, for dehydration. (3) Permeation: use hydrochloric acid alcohol to permeate the tissue to expose the antigen. (4) Antigen heat repair: place the sections in the antigen heat repair buffer and heat them to 100°C in a microwave oven for 4 min to expose the antigen. After cooling to room temperature, rinse the tissue sections with washing solution. (5) Non-specific antibody binding elimination: use normal serum to incubate the tissue to eliminate non-specific antibody binding. After incubation, rinse the tissue sections with washing solution. (6) Primary antibody incubation: use primary antibody diluent to prepare the target antibody (GPX4 1:100; NCOA4 1:50), and drop it on the tissue sections, and incubate in a dark box refrigerator overnight. (7) Secondary antibody incubation: take the dark box out of the refrigerator, and let it warm up at room temperature for a certain period of time. Then add the corresponding secondary antibody, and incubate. (8) Drop horseradish enzyme-labeled streptavidin working solution: after incubation at room temperature for a certain period of time, wash with PBS for three times. (9) DAB color development: prepare DAB color developing solution, and drop an appropriate amount of DAB color developing solution on the tissue, and observe the color development every 20 s under an A4 paper background. When the tissue turns yellow, immediately wash away the DAB color developing solution with tap water. (10) Hematoxylin nuclear staining: drop the prepared hematoxylin solution on the tissue, and observe the cell nucleus under a microscope. (11) Dehydration and mounting: dehydrate in order, and then mount. (12) After the above steps are completed, observe and take pictures under a microscope to record the results.

[0075] (Three) Data statistics

[0076] The measurement data were expressed as mean ± SD, and the comparison between two groups was performed using unpaired t-test, and the difference between multiple groups was analyzed using one-way ANOVA, and the difference between two groups was analyzed using LSD-t test, and P< 0.05 was considered statistically significant. P <0.05, **P <0.01, *** P <0.001; statistics were performed using Graphpad Prism 9.0.

[0077] (iv) Experimental Results

[0078] To further investigate the mechanism of THP's anti-TNBC effect, we performed RNA sequencing analysis. Gene set enrichment analysis (GSEA) results showed that, compared with the negative control group (NC), GSH signaling was downregulated in the THP-M group ( Figure 2 A). Subsequently, we observed the morphological characteristics of the cells in each group using electron microscopy. 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 tortuosity, and mitochondrial folding. Figure 2 B). Furthermore, we examined other indicators of ferroptosis and found that THP reduced GSH levels in MDA-MB-231 and 4T1 cells and increased Fe... 2+ and MDA level ( Figure 2 CH). Following combined administration, DOX not only failed to inhibit THP-induced ferroptosis; on the contrary, it led to a further decrease in GSH levels and Fe... 2+ And MDA levels further increased.

[0079] Furthermore, GSEA showed that, compared with the DOX group, DOX+THP-M significantly promoted ferroptosis of MDA-MB-231 (…). Figure 3 A). Next, using RNA sequencing data, we identified differentially expressed genes (DEGs) associated with ferroptosis and ferrophagy pathways in the DOX and DOX+THP-M groups ( Figure 3 (B) The DOX+THP-M group significantly regulated all three pathways of ferroptosis: lipid metabolism, the glutathione pathway, and ferrophagy. In conclusion, our results confirm that THP, but not DOX, can induce ferroptosis.

[0080] To validate the RNA sequencing results, we used Western blot to detect key proteins involved in these pathways. The results showed that THP treatment decreased xCT and GPX4 levels and increased p53 levels in a dose-dependent manner, indicating that THP activates the p53 / xCT / GSH / GPX4 pathway and promotes ferroptosis in TNBC cells. Furthermore, THP treatment caused a concentration-dependent increase in NCOA4 expression and a concentration-dependent decrease in Nrf2 and FTH1 expression, suggesting that THP can also regulate ferroptosis through the Nrf2 / NCOA4 / FTH1 pathway, inducing ferroptosis and inhibiting TNBC cell proliferation. Figure 3 C). Similar results were also obtained when DOX was used in combination with THP.Figure 3 D). In summary, these results again verified that THP has the effect of inducing iron death and iron autophagy of TBC.

[0081] In summary, in vitro, THP alone or in combination with DOX can induce iron death and iron autophagy, and play an anti-TNBC effect. Consistent with these findings, the results of in vivo experiments showed that Fe 2+ and MDA levels were significantly higher than those in 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 (P<0.05) Figure 5 A-C). Western blotting further confirmed that the THP and THP+DOX groups reduced the levels of xCT and GPX4 and increased the level of p53, thereby inhibiting the malignant development of TBC through the xc-system pathway involved in iron death. The level of NCOA4 was increased; while the levels of Nrf2 and FTH1 were decreased in tumor tissues (P<0.05) Figure 5 D). Immunohistochemical staining confirmed that in the tumor tissues of mice treated with THP and THP+DOX, GPX4 expression was down-regulated and NCOA4 expression was up-regulated (P<0.05) Figure 5 E). These results showed that THP can promote iron death and iron autophagy in vivo, and when combined with DOX, it can further increase iron death, thereby effectively enhancing its inhibitory effect on TBC.

[0082] Example 4: Reversal of anthracycline negative immunomodulation by Tetrastigma hemsleyanum polysaccharide, and significant improvement in tumor microenvironment when the two drugs are combined

[0083] (I) Experimental materials, reagents and instruments

[0084] Accuri™ C6 flow cytometer (BD Company, USA)

[0085] (II) Experimental method

[0086] The three-leafed greenbrier polysaccharide extract obtained in Example 1 was combined with doxorubicin to detect the change in the proportion of immune cells in the tumor microenvironment, and the specific method was as follows: (1) sample collection: after the tumor mass was taken out, it was placed in a 15 ml centrifuge tube containing 5 ml PBS and stored on ice. (2) pre-cutting: the tumor mass and about 2 ml of PBS were poured out from the centrifuge tube and placed on the bottom of a 6 cm culture dish, and the remaining about 3 ml of PBS was placed on the culture dish cover. The tumor mass was cut into a paste with surgical scissors, and a 1 ml pipette without a suction head was used to transfer it back into a 15 ml centrifuge tube; and the bottom of the culture dish was washed with PBS on the culture dish cover and transferred back into the centrifuge tube. Store on ice. (3) digestion: centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant. Add 4 ml of collagenase (1 mg / ml collagenase IV, 0.1 mg / ml DNAase, 2% FBS, RMPI1640) to resuspend, and place in a 37°C water bath for 1 h, resuspend every 5 min. (4) grinding: pour the digested tumor tissue and liquid in the 15 ml centrifuge tube directly into the 6 cm culture dish bottom, and pour twice with 1 ml PBS into the centrifuge tube and pour into the culture dish cover; transfer an appropriate amount of liquid to the culture dish cover, and adjust the remaining liquid volume for grinding. Sterilize the stainless steel clamp and place it on the culture dish bottom, fix it with a straight forceps, and grind the tumor tissue on the stainless steel mesh with a curved forceps. Clamp the nylon filter (200 mesh) to the mouth of the 15 ml centrifuge tube, and use a 1 ml pipette without a suction head to transfer the ground tumor tissue back into the centrifuge tube; and wash the culture dish bottom with the liquid on the culture dish cover and transfer it back into the centrifuge tube. (5) washing: centrifuge at 1000 rpm for 5 min at room temperature, 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 of LIVE / DEAD Fixable Dead Cell Stains diluent (50 μl Component B added to 1 tube Component A to make a mother solution, and use PBS 1:1000 dilution when needed) to each tube, vortex to mix, and incubate at room temperature for 30 min. After adding 1 ml of PBS, centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, and resuspend with 1 ml of PBS. (7) fixation: centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant. Resuspend the tumor suspension with 1 ml of fixing solution (eBioscience Foxp3 / Transcription Factor Staining Buffer Set, Cat: 00-5523-00, Concentrate: Diluent ratio 1:3 freshly prepared) and fix (overnight, 4°C, avoid light). (8) preservation: centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant. Wash twice with 1 ml of Buffer and resuspend, store at 4°C in the dark.(9) Count and Stain: Dilute the sample to 1 million cells per tube with Buffer, wash once, add 50 μl blocking solution (anti-CD16 / 32) 4°C for 15 min, then add 50ul of the corresponding concentration of antibody 4°C for 30 min, 1ml PBS wash 2 times, resuspended, save or flow. + / CD8 + Ratio.

[0087] (Three) Data Statistics

[0088] The measurement data were expressed as mean ± SD. The unpaired t-test was used for comparison between two groups, and the variance analysis test was used for comparison among multiple groups. The one-way ANOVA was used for multiple comparisons. P < 0.05 was considered statistically significant. P< 0.05, * P <0.05, ** P <0.01, *** P <0.001; statistical analysis was performed using Graphpad Prism 9.0.

[0089] (Four) Experimental Results

[0090] As shown in Figure 6 , we found that DOX-L treatment led to an increase in the number of Treg cells and a decrease in the number of CD8 + T cells in tumor tissue immune cell detection. In contrast, THP alone can reduce the number of Treg cells and increase the number of CD8 + T cells. Compared with any single drug treatment, the combination of the two drugs can significantly improve the tumor microenvironment, reduce the proportion of Treg cells, and increase the proportion of CD4 + and CD8 + T cells.

[0091] Example 5: Sanjiaqing polysaccharide significantly alleviates the cardiotoxicity caused by anthracyclines

[0092] (I) Experimental materials, reagents and instruments

[0093] Neutral gum (Shanghai Biyun Tian Biological Co., Ltd.), paraformaldehyde (Shanghai Biyun Tian Biological Co., Ltd.), hematoxylin (Shanghai Biyun Tian Biological Co., Ltd.), xylene (Shanghai Lingfeng Chemical Reagent Co., Ltd.), film viewing tools (produced by Jiangsu Su Hong Medical Instrument Co., Ltd.).

[0094] (II) Experimental methods

[0095] (1) Tissue fixation: Collect tissue from the sample to be tested and fix it with 4% paraformaldehyde to maintain the integrity of its structure and morphology. (2) Tissue dehydration: Dehydrate the tissue using alcohol of varying concentrations to remove water. (3) Tissue embedding: Embed the dehydrated tissue in paraffin for easy sectioning. (4) Tissue sectioning: Section the embedded tissue block to make sections of appropriate thickness. (5) Dewaxing: Dewax the sections in xylene for 10 min, then sequentially in anhydrous ethanol, 90% alcohol, 80% alcohol, and 70% alcohol for 2 min each. (6) Staining: Stain the sections in hematoxylin solution for 10 min, then wash away the hematoxylin and excess dye in water for about 2 min. After that, immerse the sections in differentiation solution for a few seconds to 30 s to decolorize the sections to a pale blue-red. Then wash in running water for 30 min to make the tissue bright blue or sky blue. Finally, stain the sections in eosin solution for 3 min. (7) Dehydration: Place the sections in 75% alcohol, 95% alcohol, and anhydrous ethanol sequentially, for 30 seconds each. (8) Clearing: Clear the sections in xylene for 1 minute. (9) Mounting: Remove the sections from the xylene and mount them with neutral resin. After staining, the cell nuclei are blue, while the cytoplasm, muscle, connective tissue, erythrocytes, and eosinophilic granules are pink to varying degrees.

[0096] (III) Data Statistics

[0097] Quantitative data are expressed as mean ± standard deviation (mean ± SD). Unpaired t-tests were used for comparisons between two groups, ANOVA was used for comparisons among multiple groups, and one-way ANOVA was used for pairwise comparisons among multiple groups. P< 0.05 is used to determine statistical significance, * P <0.05,** P <0.01, *** P <0.001; statistics were performed using Graphpad Prism 9.0.

[0098] (iv) Experimental Results

[0099] 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 toxic, so we analyzed various indicators in the DOX-H group mice. CK-MB results showed that DOX-H was significantly toxic, while THP was non-toxic. After combined use, CK-MB levels were significantly reduced, and the toxicity was effectively alleviated. Figure 7 C). The electrocardiograms of the THP group were very similar to those of the CON group, with essentially the same heart rate and rhythm.Figure 7 D). DOX-H group showed decreased heart rate, P-wave disappeared, and prolonged PR interval. However, the use of THP can alleviate these phenomena. Histological examination of cardiomyocytes also confirmed this. In the CON, MOD, and THP groups, cardiomyocytes were in a horizontal type, arranged non-randomly, short cylindrical, with a branched nucleus located in the center of the myocardium, and the branches of the outer wall of the myocardium were connected to form a myofibril network. In the DOX-H group, the myocardial interstitium was widened, the blood vessels were dilated and congested, the sarcoplasmic horizontal arrangement was disordered, the nuclear staining was deepened, 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).

[0100] Example 6: Tripterygium wilfordii polysaccharides significantly alleviate the liver, kidney, and bone marrow suppression toxicity caused by anthracyclines

[0101] (I) Experimental materials and instruments

[0102] 5427R high-speed refrigerated centrifuge (Eppendorf, Germany), automatic biochemical analyzer

[0103] (II) Experimental methods

[0104] Detection of biochemical indicators in mice (1) The mouse blood samples were layered and centrifuged at 4°C for 10 min to separate the supernatant. (2) The automatic biochemical analyzer was used to detect the serum biochemical indicators of mice. (3) According to the normal range of mouse biochemical indicators, the results were analyzed and plotted.

[0105] (III) Data statistics

[0106] The measurement data were expressed as mean ± SD, and the comparison between two groups used unpaired t-test, the difference between multiple groups was analyzed by variance analysis, and the multiple comparisons between groups used one-way ANOVA, P<0.05 was considered statistically significant, P< <0.05,** P <0.01,*** P <0.001; statistical analysis was performed using Graphpad Prism 9.0. P

[0107] (IV) Experimental results

[0108] As Figure 8 shown, but we observed a significant decrease in the weight of the liver, kidney, and spleen after DOX treatment, especially in the DOX-H group ( Figure 8 A). Therefore, we further detected some biochemical indicators of liver and kidney function, and found that DOX had significant toxicity, and each indicator showed varying degrees of change. For example,​Figure 8 B-C, the levels of creatinine (CREA) and lactate dehydrogenase (LDH) were significantly reduced in the DOX-H+THP group compared to the DOX-H group. According to the Charles River Laboratories Inc. 2008-2012 BALB / C mouse clinical pathology data, the 95% interval was used as the normal range of BALB / C mouse physiological indicators. The ALT level in the DOX-H group exceeded the maximum limit. The AST level was within the range, but was significantly higher than that of the other groups. In addition, the albumin (ALB) level in the DOX-H group was significantly reduced; however, these indicators were restored after the administration of THP Figure 8 D-F and G-I). These results indicate that DOX-H causes severe kidney injury and liver toxicity, which can be significantly improved by the combination 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 the mice in this group had a higher level of inflammation Figure 8 J, K, M, and N). After treatment, the NEUT% and WBC count in all groups were within the normal range, except for the DOX-H+DEX group, which had a decreased NEUT%. In addition, the NEUT% and WBC count in the DOX-H group were higher than those in the other groups, indicating the presence of infection-related inflammation. In summary, our research results indicate that DOX-H treatment is associated with myelosuppression, leading to adverse toxic reactions, which can be effectively alleviated when DOX-H is used in combination with THP.

[0109] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Moreover, the experimental methods described herein, unless otherwise specified, are conventional methods; and the reagents, biological materials, and equipment described herein, unless otherwise specified, are commercially available.

[0110] The above examples are preferred embodiments of the present application, and the present application is not limited by the above examples. It is particularly pointed out that, for those skilled in the art, a number of modifications and refinements can be made without departing from the principles described herein, and these modifications and refinements should also be considered within the scope of protection of the present application.

Claims

1. The application of *Trifolium repens* polysaccharide combined with doxorubicin in the preparation of a therapeutic drug for triple-negative breast cancer, characterized in that: The mass ratio of *Trifolium repens* polysaccharide to doxorubicin is 15-60:0.25; The preparation of *Trifolium repens* polysaccharide includes: drying the aerial parts of *Trifolium repens* in an oven at 40℃ and pulverizing them; placing them in a round-bottom flask, adding double-distilled water at a material-to-liquid ratio of 1:15 (g / ml) and refluxing for extraction 3 times, 30 min each time; combining the 3 extracts after filtration, concentrating by vacuum distillation, adding 3 times the mass of 95% ethanol of the concentrate, water extraction and alcohol precipitation 3 times, standing overnight at 4℃, centrifuging at 4200 rpm for 15 min to obtain the precipitate; washing twice with anhydrous ethanol, and freeze-drying under vacuum to obtain *Trifolium repens* polysaccharide powder.

2. The application according to claim 1, characterized in that: The drug comprises: doxorubicin as the main active ingredient, *Trifolium repens* polysaccharide as an adjuvant ingredient for enhancing efficacy and reducing toxicity, and pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that: The attenuation refers to reducing the toxic effects of doxorubicin on the heart, liver, kidneys, and bone marrow.

4. The application according to claim 2, characterized in that: The drug is in the form of an oral formulation.

5. The application according to claim 1, characterized in that: The combination of Tripterygium wilfordii polysaccharide and doxorubicin improves the treatment efficacy for triple-negative breast cancer by regulating the xCT / GSH / GPX4 and Nrf2 / NCOA4 / FTH1 pathways.

6. The application according to claim 1, characterized in that: The combined use of *Trifolium repens* polysaccharide and doxorubicin improved the tumor microenvironment, manifested as a decrease in the proportion of Treg cells and CD4+. + and CD8 + The proportion of T cells increased.

7. The application according to claim 1, characterized in that: The combination of Tripterygium wilfordii polysaccharide and doxorubicin improves the treatment efficacy of triple-negative breast cancer by promoting ferroptosis in triple-negative breast cancer.

8. The application according to claim 1, characterized in that: The combined use of Tripterygium wilfordii polysaccharide and doxorubicin alleviated the toxic side effects of doxorubicin.

9. The application according to claim 8, characterized in that: The toxic side effects include those affecting the heart, liver, kidneys, and bone marrow.

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