A Chinese medicine composition for treating castration-resistant prostate cancer and its application

Abstract

The present invention provides a Chinese medicine composition for treating castration-resistant prostate cancer and its application. The Chinese medicine composition is prepared by including the following raw materials in parts by weight: 10-30 parts of Astragalus, 3-15 parts of Curcuma, 3-6 parts of Piper longum, and 3-6 parts of Apis cerevisiae. The Chinese medicine composition has a rigorous formula, concise medicinal taste, and clear efficacy. The Chinese medicine composition can increase TNF-α content, reduce Ki67 expression, IL-10 content, triglyceride content, GPX4 mRNA expression, SLC7A11 mRNA expression and protein expression, etc., and can activate ferroptosis and inhibit tumor growth by inducing the SLC7A11 / GPX4 pathway, thereby achieving the purpose of treating castration-resistant prostate cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and specifically comprises a traditional Chinese medicine composition for treating castration-resistant prostate cancer and application thereof. Background Art

[0002] Prostate cancer refers to an epithelial malignant tumor that occurs in the prostate. According to the latest data released by the World Health Organization's International Agency for Research on Cancer (IARC), the incidence and mortality of prostate cancer rank sixth and seventh respectively among male cancer patients in China. Early prostate cancer is a hormone-dependent tumor. After endocrine therapy, most of them turn into castration-resistant prostate cancer, and the median survival period is less than two years. The treatment of castration-resistant prostate cancer is a globally recognized difficulty and hot spot. In the treatment of castration-resistant prostate cancer, second-generation hormone therapy, second-generation anti-androgen drugs (apalutamide, enzalutamide, darolutamide) and androgen metabolism inhibitor abiraterone are used in first-line endocrine therapy. During the treatment process, patients will develop drug resistance again. At present, radiotherapy or drug treatment of CRPC is not effective and has low specificity. Therefore, exploring drugs that can selectively induce CRPC cell death has a far-reaching impact on the treatment of prostate cancer.

[0003] Current research shows that Chinese medicine can help patients recover after surgery, improve their symptoms and quality of life, and delay the progression of the disease by combining Chinese medicine with chemotherapy and radiotherapy to enhance efficacy and reduce toxicity. However, the Chinese medicines used in the past have many flavors and are mostly empirical formulas, lacking scientific evidence, and there is relatively little research on the mechanism of action of Chinese medicine compositions. Based on this, it is urgent to develop a Chinese medicine composition with simple formulation, scientific compatibility, and significant efficacy for the clinical treatment of prostate cancer. Summary of the invention

[0004] In view of the above problems existing in the prior art, the first object of the present invention is to provide a Chinese medicine composition for treating castration-resistant prostate cancer.

[0005] The second object of the present invention is to provide a drug for treating castration-resistant prostate cancer.

[0006] The third object of the present invention is to provide an application of the above-mentioned traditional Chinese medicine composition in the preparation of a drug for treating castration-resistant prostate cancer.

[0007] To achieve the above first object, the technical solution adopted by the present invention includes:

[0008] The present invention discloses a traditional Chinese medicine composition for treating castration-resistant prostate cancer. The traditional Chinese medicine composition comprises the following raw materials in parts by weight:

[0009] 10-30 parts of Astragalus, 3-15 parts of Curcuma, 3-6 parts of Piper longum, and 3-6 parts of Apium graveolens.

[0010] The present invention takes the traditional Chinese medicine treatment of castration-resistant prostate cancer as the starting point, and combines many years of clinical experience to create a traditional Chinese medicine composition for the treatment of castration-resistant prostate cancer (the traditional Chinese medicine composition is also known as: Yiqi Huoxue Fang), which hits the key pathogenesis of castration-resistant prostate cancer in the form of invigorating qi and activating blood circulation, strengthening the spleen and removing dampness, and replenishing deficiency and detoxifying. At the same time, a C57BL / 6 male mouse CRPC subcutaneous transplant tumor model is established, and its mechanism of action in inhibiting the growth of castration-resistant prostate cancer cells is studied.

[0011] The sources and functions of the medicinal materials in the above Chinese medicine prescriptions are as follows:

[0012] Astragalus: A plant of the genus Astragalus in the leguminous family, it tastes sweet and is slightly warm in nature. It has the effects of tonifying Qi and strengthening the exterior, promoting diuresis, strengthening the heart, lowering blood pressure, antibacterial, detoxifying, draining pus, promoting tissue regeneration, strengthening capillary resistance, stopping sweating and having sex hormone-like effects. It can treat spontaneous sweating due to deficiency of the exterior, internal injuries due to deficiency of Qi, diarrhea due to deficiency of the spleen, edema and carbuncle, etc.

[0013] Curcuma: A perennial herb with pungent, bitter and warm nature and flavor. It enters the liver and spleen meridians. It is mainly used to treat stagnation of qi and blood, distension and pain in the heart and abdomen, masses, accumulation, indigestion, blood stasis and amenorrhea in women, and pain caused by falls. The tuber is called "green silk turmeric" and has the functions of promoting qi and relieving depression, breaking up blood stasis and relieving pain.

[0014] Piper longum: Piperaceae, Piper genus, pungent and hot in nature, enters the spleen meridian, stomach meridian, large intestine meridian, lung meridian, bladder meridian, liver meridian, kidney meridian, warms the middle and dispels cold, relieves gas and relieves pain, used for cold pain in the abdomen, vomiting, diarrhea, migraine, sinusitis, external treatment of toothache, coronary heart disease, angina pectoris. Treats cold pain in the heart and abdomen, vomiting and acid regurgitation, intestinal rumbling and diarrhea, cold dysentery, hernia, headache, sinusitis, toothache.

[0015] Beehive: The nest of the fruit wasp Polistes olivaceous (DeGeer), the Japanese long-legged wasp Polistes japonicus Saussure or the heterogastric wasp Parapolybia varia Fabricius. It has the effects of attacking poison and killing insects, dispelling wind and relieving pain. It tastes sweet and is neutral in nature. It enters the stomach meridian and kidney meridian. It is often used for sores, swellings, mastitis, scrofula, stubborn skin ringworm, goose foot wind, toothache, rheumatism and pain.

[0016] The solution of this prescription is as follows:

[0017] Through literature review, Chinese medicine believes that the location of castration-resistant prostate cancer is in the kidney and prostate. Castration-resistant prostate cancer is generally characterized by deficiency of the body's vital energy, with excess of pathogenic factors as the symptom, and deficiency of the spleen and kidney as the root cause. Diet, emotions, and exogenous pathogenic factors can all lead to spleen deficiency. Spleen deficiency can produce dampness, which blocks the Qi mechanism, causing Qi and blood to stagnate and turn dampness into phlegm. When cancer invades the human body, deficiency, dampness, blood stasis, phlegm, and toxins are intertwined in the body. The formula for invigorating qi and activating blood circulation is composed of Astragalus, Curcuma, Piper longum, and Beehive. The "Shennong Bencao Jing" says about Astragalus: "It tastes sweet and slightly warm. It is used to treat carbuncles, long-term wounds, discharge pus, relieve pain, leprosy, five types of hemorrhoids, fistula, tonic, and all kinds of children's diseases." The "New Edition of Materia Medica" says about Curcuma: "Curcuma is bitter and pungent in taste, warm in nature, and non-toxic. It enters the liver and spleen meridians and is an important medicine for the blood. It is specifically used to break up the blood in the qi, remove bad habits, stop heartache, regulate menstruation, eliminate blood stasis, treat cholera, drain accumulation, and regulate the qi in the middle. It is a drug for attacking tough targets and can be used as an adjuvant, but it should not be used for a long time. It specifically enters the qi to break up blood, and although it breaks up blood, it does not hurt the qi." The "Compendium of Materia Medica" says that Piper longum is "pungent and warm in nature, non-toxic, and mainly used to treat cold phlegm." The "Shennong's Materia Medica" says that "Phoenix bee house is used to treat epilepsy, convulsions, cold and heat evil spirits, scrofula, ghost spirits, and intestinal hemorrhoids." Astragalus invigorates the spleen and warms the middle to replenish deficiency, Curcuma zedoaria promotes blood circulation and removes blood stasis to eliminate blood stasis, Piper longum warms the middle to invigorate the spleen to eliminate phlegm and dampness, and Bee house reduces swelling and detoxifies to treat poison. The whole formula uses Astragalus as the main ingredient and Curcuma zedoaria as the auxiliary ingredient, which invigorates Qi and promotes blood circulation, strengthens the spleen and removes dampness, replenishes deficiency and detoxifies, and hits the key to the pathogenesis of castration-resistant prostate cancer. Piper longum and Bee house are both adjuvants, and the combination of these medicines can achieve the effects of invigorating Qi, promoting blood circulation and removing blood stasis, invigorating the spleen and removing dampness, replenishing deficiency and detoxifying.

[0018] Furthermore, the Chinese medicine composition is prepared by including the following raw materials in parts by weight:

[0019] 10-15 parts of Astragalus, 3-9 parts of Curcuma, 3-5 parts of Piper longum, and 3-5 parts of Apium graveolens.

[0020] Furthermore, the Chinese medicine composition is prepared by including the following raw materials in parts by weight:

[0021] 15 parts of Astragalus, 6 parts of Curcuma, 3 parts of Piper longum, and 3 parts of Nigella ovata.

[0022] To achieve the above second purpose, the technical solution adopted by the present invention includes:

[0023] The invention discloses a medicine for treating castration-resistant prostate cancer, wherein the medicine uses the above-mentioned traditional Chinese medicine composition as an active ingredient.

[0024] Furthermore, the drug also includes pharmaceutically acceptable excipients, including but not limited to fillers, disintegrants, lubricants, suspending agents, adhesives, sweeteners, flavoring agents, preservatives, and the like.

[0025] Furthermore, the dosage form of the drug includes but is not limited to decoction, powder, granule, pill, granule, drop pill, tablet, capsule, oral liquid, paste or decoction.

[0026] To achieve the third objective above, the technical solution adopted by the present invention includes:

[0027] The present invention discloses an application of the above-mentioned traditional Chinese medicine composition in preparing a medicine for treating castration-resistant prostate cancer.

[0028] Furthermore, the drug is used for one or more of the following effects:

[0029] 1) Increase TNF-α content;

[0030] 2) Reduce Ki67 expression;

[0031] 3) Reduce the level of IL-10;

[0032] 4) Reduce triglyceride levels;

[0033] 5) Reduce the mRNA expression of GPX4;

[0034] 6) Reduce the mRNA expression of SLC7A11;

[0035] 7) Reduce the protein expression of SLC7A11.

[0036] Furthermore, the drug activates ferroptosis and inhibits tumor growth by inducing the SLC7A11 / GPX4 pathway.

[0037] Beneficial effects of the present invention:

[0038] The traditional Chinese medicine composition provided by the invention has a rigorous formula, concise medicinal taste and clear therapeutic effect.

[0039] Based on the test results in the specific implementation, the Yiqi Huoxue recipe can reduce tumor mass, reduce the expression of Ki67 in tumor tissue, reduce the content of triglycerides in mice, increase liver and spleen mass, and stimulate the immune ability of mice, so as to achieve the purpose of strengthening the body and fighting cancer. And by establishing a C57 BL / 6 male mouse CRPC subcutaneous transplant tumor model, as well as HE staining, Elisa detection, RT-PCR, Westen Blot and other methods, it was preliminarily verified from the tissue-transcription-translation level, from the perspective of immunity, ferroptosis, and lipid metabolism that the Yiqi Huoxue recipe can inhibit the growth of castration-resistant prostate cancer.

[0040] Furthermore, the inventors used the CCK8 method, observed cell morphology, detected intracellular reactive oxygen species (ROS) content, GSH content, lipid peroxide ratio, Fe ion content, and RT-PCR to detect the mRNA expression of GPX4 and SLC7A11, verifying that the Yiqi Huoxue recipe can activate the ferroptosis pathway through the SLC7A11 / GPX4 pathway and inhibit the growth of RM-1 cells, thereby establishing a clear mechanism of action for researchers. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The effect of Yiqi Huoxue Fang (YQHXF) on the body weight, liver and spleen weight of mice with C57 subcutaneous transplanted tumors is shown;

[0042] (A): Mouse body weight change diagram (n=59, `X±S); (B): Mouse liver weight diagram (n=59, `X±S, one-way ANOVA); (C): Mouse spleen weight diagram (n=59, `X±S, one-way ANOVA); Note: Compared with the normal group, *p<0.05, **p<0.001, compared with the model group, #p<0.05.

[0043] Figure 2 The effect of Yiqi Huoxue Fang (YQHXF) on tumor mass and volume in mice with subcutaneous transplanted C57 tumors is shown;

[0044] (A): Entity image of tumor in C57 mice (n=30); (B): Mass image of tumor in C57 mice (n=57, `X±S, one-way ANOVA); (C): Volume image of tumor in C57 mice (n=57, `X±S, one-way ANOVA); Note: Compared with the model group, *p<0.05, **p<0.001.

[0045] Figure 3 The results of Yiqi Huoxue Fang (YQHXF) on Ki67 immunohistochemistry in tumor tissues of mice with subcutaneous transplanted C57 tumors are shown (n=3); Note: The gray part in the figure shows the expression of Ki67; In the table: compared with the model group, *p<0.05.

[0046] Figure 4 The effect of Yiqi Huoxue Fang (YQHXF) on the levels of TNF-α and IL-10 in mice with C57 subcutaneous tumor transplantation was shown;

[0047] (A): TNF-α content in serum of C57 mice (n=57, X±S, one-way ANOVA); (B): IL-10 content in serum of C57 mice (n=57, X±S, one-way ANOVA); Note: Compared with the normal group, *p<0.05; Compared with the model group, #p<0.05.

[0048] Figure 5 The effects of Yiqi Huoxue Fang (YQHXF) on four blood lipids in mice with C57 subcutaneous transplanted tumors are shown;

[0049] (A): Serum triglyceride content in C57 mice (n=57, `X±S, one-way ANOVA); (B): Serum total cholesterol content in C57 mice (n=57, `X±S, one-way ANOVA); (C): Serum low-density lipoprotein cholesterol content in C57 mice (n=57, `X±S, one-way ANOVA); (D): Serum high-density lipoprotein cholesterol content in C57 mice (n=57, `X±S, one-way ANOVA); Note: Compared with the normal group, *p<0.05, **p<0.001; Compared with the model group, # p<0.05.

[0050] Figure 6 The results show the effect of Yiqi Huoxue Fang (YQHXF) on the mRNA expression of SLC7A11 and GPX4 in tumor tissues of mice with subcutaneous transplanted C57 tumors.

[0051] (A): SLC7A11 mRNA expression in mouse tumor tissue (n=3, X±S, one-way ANOVA); (B): GPX4 mRNA expression in mouse tumor tissue (n=3, X±S, one-way ANOVA); Note: Compared with the model group, *p<0.05, **p<0.01; Compared with the cyclophosphamide group, # p<0.05, ## p<0.01.

[0052] Figure 7 The results show the effect of Yiqi Huoxue Fang (YQHXF) on the expression of GPX4 and SLC7A11 proteins in tumor tissues of mice with subcutaneous transplanted C57 tumors.

[0053] (A): Wseten Blot of mouse tumor tissue; (B): SLC7A11 protein expression in mouse tumor tissue (n=3, X±S, one-way ANOVA); (C): GPX4 protein expression in mouse tumor tissue (n=3, X±S, one-way ANOVA); Note: Compared with the model group, *p<0.05, **p<0.01; Compared with the cyclophosphamide group, # p<0.05, ## p<0.0.

[0054] Figure 8 Shown are the cell survival curves of RM-1 cells treated with YQHXF drug-containing serum, RSL3, and Fer-1.

[0055] Fig. 9 The effect of YQHXF-containing serum on the cell morphology of RM-1 is shown (10x).

[0056] Fig.10 The effect of YQHXF-containing serum on the cell morphology of RM-1 is shown (40x).

[0057] Fig.11 The figure shows the effect of YQHXF-containing serum on the ROS content in RM-1 cells.

[0058] Fig.12 The effect of YQHXF-containing serum on GSH content and lipid peroxides in RM-1 cells is shown;

[0059] (A): GSH content in RM-1 cells (n=3, X±S, one-way ANOVA); (B): Lipid peroxide ratio in RM-1 cells (n=3, X±S, one-way ANOVA); Note: Compared with the model group, *p<0.05, **p<0.01; Compared with the YQHXF group, #p<0.05; Compared with the Fer-1 group, ^p<0.05.

[0060] Fig.13 Shown is the effect of YQHXF-containing serum on Fe content in RM-1 cells;

[0061] (A): Fe 2+ Content (n=3, X±S, one-way ANOVA); (B): Total Fe ion content in RM-1 cells (n=3, X±S, one-way ANOVA); Note: Compared with the model group, *p<0.05; compared with the RSL3 group, #p<0.05; compared with the Fer-1 group, ^p<0.05.

[0062] Fig.14 The effect of YQHXF-containing serum on the expression of GPX4 and SLC7A11 in RM-1 cells is shown;

[0063] (A): mRNA expression of SLC7A11 in RM-1 cells (n=3, X±S, one-way ANOVA); (B): mRNA expression of GPX4 in RM-1 cells (n=3, X±S, one-way ANOVA); Note: Compared with the model group, *p<0.05; Compared with the RSL3 group, # p<0.05; compared with Fer-1 group, ^p<0.05. DETAILED DESCRIPTION

[0064] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and drawings. It should be clear that the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0065] Example 1

[0066] The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 15g of Astragalus, 6g of Curcuma, 3g of Piper longum and 3g of Nymphaeaceae.

[0067] The effect of the Yiqi Huoxue prescription (abbreviated as YQHXF) of the present invention on the growth of castration-resistant prostate cancer cells and its mechanism of action were verified by the following experiments.

[0068] Part I

[0069] 1 Experimental Materials

[0070] 1.1 Experimental animals

[0071] 60 C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center, license number SCXK (Beijing) 2019-0008, ordinary laboratory animal environment. Male mice, 4-5 weeks old, weight 18-20g, caged. During the experiment, they were raised in an ordinary environment, with a constant temperature of 22-25°C and a constant humidity of 50%-65%. Drinking water, feed and experimental supplies were sterilized and disinfected, and the experimental operation strictly followed the principle of aseptic operation. Mice were purchased one week before the experiment to adapt them to the new environment.

[0072] 1.2 Experimental cells

[0073] Cell line preparation: Mouse cell line RM-1 was provided by the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in F-12K medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. When the color of the medium gradually changed from red to yellow, fresh medium was replaced. After about 3 days, the cells covered the bottom of the bottle, and then digested with trypsin and subcultured at a ratio of 1:3. Cells in good cell condition were used for experiments.

[0074] 1.3 Experimental drugs

[0075] The Yiqi Huoxue Fang granules consisted of 15g of Astragalus, 6g of Curcuma, 3g of Piper longum, and 3g of Nigella ovata, and were prepared by the pharmacy of Guang'anmen Chinese Medicine Hospital. The granules and quality control were provided by Sichuan Xinlu Pharmaceutical Technology Development Co., Ltd.

[0076] Cyclophosphamide powder: 25 mg, (Cat. No. C12147599), purchased from Maclean.

[0077] 2 Experimental methods

[0078] 2.1 Establishment of CRPC subcutaneous transplant tumor model in C57 BL / 6 male mice

[0079] The RM-1 cells cultured in vitro were collected. The trypan blue staining experiment showed that the viable cells were >95%. 0.25 ml of tumor cell suspension was taken. The total number of cells was 1×10 7 The cells were inoculated in the left groin of each mouse (to avoid spillage of the cell suspension during transplantation).

[0080] Weigh the mice every day after inoculation, adjust the dosage in time, and observe and record the general conditions of the mice, such as food intake, activity level, coat color, etc.

[0081] 2.2 Experimental groups and drug administration methods

[0082] After subcutaneous transplantation, the mice were divided into balanced groups according to their body weight, with 10 mice in each group.

[0083] Group setting and processing:

[0084] ① Normal group: No special treatment, pure water gavage

[0085] ②Model group (implanted RM-1 cells): pure water was administered intragastrically.

[0086] ③Cyclophosphamide group: After the model grouping, cyclophosphamide was intraperitoneally injected once a week, with an injection dose of 25 mg / kg.

[0087] ④YQHXF low-dose group: After the model grouping, the mice were gavaged once a day.

[0088] ⑤YQHXF medium-dose group: After the model was grouped, the mice were gavaged once a day.

[0089] ⑥YQHXF high-dose group: After the model grouping, gavage was performed once a day.

[0090] Each group of mice was gavaged with the corresponding drugs. The dosage of Chinese medicine was calculated according to the conversion formula between mice and humans. The YQHXF low-dose group, YQHXF medium-dose group, and YQHXF high-dose group contained crude drugs of 0.9g / kg, 2.7g / kg, and 8.1g / kg, respectively, which were 1, 3, and 9 times the conventional human dose, respectively. The gavage volume was 0.1mL / 10g per day. The drug was administered for a total of 3 weeks.

[0091] 2.3 Sample collection

[0092] Samples were collected at the end of week 3. The mice were fasted for 12 hours and then weighed.

[0093] ① Remove the eyeball and draw blood:

[0094] Use the thumb, index finger and middle finger of your left hand to grab the scalp of the mouse's neck, and fix the tail with the little finger and ring finger. Gently press the eye skin that needs to be removed to make the eyeball congested and protruding. Use ophthalmic scissors to cut the mouse's whiskers to prevent blood from flowing from the whiskers and causing hemolysis. Use ophthalmic curved forceps to clamp the eyeball and remove it quickly, and let the blood flow from the eye socket into a 0.5ml EP tube. After the mouse is bled, break the neck and kill it. Take about 500-600μL of blood. Let it stand for 4h, centrifuge at 4℃, 3000r / min for 20min. Use a pipette to aspirate the upper layer of serum, use centrifuge tubes to divide it, and transfer it to a -80℃ refrigerator for storage.

[0095] ②Remove the mouse tumor:

[0096] Use a marker to roughly mark the range of the mouse tumor, use ophthalmic scissors to gently cut the mouse skin, use ophthalmic curved forceps to clamp the mouse skin, and continue to use ophthalmic scissors to remove excess hair until the complete mouse tumor is removed. Weigh the mass of the mouse tumor and soak it in 4% paraformaldehyde according to the experimental requirements, or put it into a 1.5ml EP tube and freeze it in dry ice and transfer it to a -80℃ refrigerator on the same day for storage.

[0097] ③Remove the liver and spleen of the mouse and weigh their mass.

[0098] 2.4 Paraffin sections & immunohistochemistry

[0099] 2.4.1 Paraffin sections

[0100] ① Fixation: The tumor tissue was fixed in 4% paraformaldehyde solution;

[0101] ② Dehydration: Place the fixed tissue in 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol I and anhydrous ethanol II for 1 hour each;

[0102] ③ Transparency: Place the dehydrated tissue blocks in equal volumes of anhydrous ethanol and xylene mixed solution, xylene I and xylene II for 1 hour each;

[0103] ④ Wax immersion: Place the tissue in a mixed solution of equal volumes of paraffin and xylene, and then transfer it to the melted paraffin I and paraffin II for 1 hour each;

[0104] ⑤Embedding: Place the tissue into a paraffin mold and place it in a paraffin embedding machine to cool;

[0105] ⑥ Sectioning: Fix the embedded wax block on the microtome, cut thin slices of about 3μm, transfer the slices to warm water with a brush for spreading, then pick up the slices with a glass slide and put them in an oven to dry at 45℃.

[0106] 2.4.2 HE staining

[0107] ① Dewaxing: Place the paraffin sections in xylene I and xylene II for 15 min each;

[0108] ② Hydration: Place the slices in absolute ethanol I, absolute ethanol II, 95% ethanol, 75% ethanol for 1 min each, and distilled water for 2 min;

[0109] ③ Staining: immerse the tissue in hematoxylin staining solution for 5 minutes, rinse with running water for 5 minutes, differentiate with 1% hydrochloric acid alcohol for 5 seconds, rinse with running water for 5 minutes, reverse blue with bluing solution for 10 seconds, rinse with running water for 5 minutes, eosin staining solution for 3 minutes, rinse with running water, and distilled water for 2 minutes;

[0110] ④ Dehydration and transparency: 85% ethanol, 95% ethanol, anhydrous ethanol II, xylene I, xylene II for 5 minutes each, and seal with neutral resin to avoid bubbles;

[0111] ⑤Microscopic examination: Observe under an optical microscope. The cytoplasm of the tissue cells appears pink and the nuclei appear blue.

[0112] 2.4.3 Immunohistochemistry

[0113] ① Dewax and rehydrate, wash twice with PBS, 5 minutes each time.

[0114] ② Antigen repair: Prepare fresh 3% H2O2 with distilled water or PBS, seal for 5-10 minutes at room temperature, wash 3 times with distilled water and 5 minutes with PBS.

[0115] ③ Block, add normal goat serum blocking solution, store at room temperature for 20 minutes, and remove excess liquid.

[0116] ④ Incubate with primary antibody: incubate antibody I at room temperature for 1 hour, at 4°C overnight, or at 37°C for 1 hour (reheat at 37°C for 45 minutes after overnight at 4°C).

[0117] ⑤ Wash three times with PBS, 2 minutes each time. Add biotinylated secondary antibody at 20℃~37℃ for 20 minutes. Wash three times with PBC, 2 minutes each time.

[0118] ⑥ Add reagent SABC at 20℃~37℃ for 20 minutes. Wash with PBS 4 times, 5 minutes each time.

[0119] ⑦DAB color development: DAB color development kit or homemade color development reagent (control the degree of color development under the microscope).

[0120] ⑧ Wash with distilled water. Stain with hematoxylin for 2 minutes and differentiate with hydrochloric acid and alcohol.

[0121] ⑨ Dehydration, transparency, sealing and microscopic examination.

[0122] Results: Five high-power microscopic fields (400 times) were selected for each slice, and the image analysis system was used for quantitative grayscale scanning and analysis.

[0123] 2.5ELISA test

[0124] Enzyme-linked immunosorbent assay was used to detect the levels of interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) in the serum of each group of rats. According to the instructions of the kit, the operation process of the IL-10-ELISA detection kit is taken as an example and briefly summarized as follows:

[0125] ① Prepare all the necessary reagents and working concentration standards. Centrifuge at 6000-10000 rpm for 30s. Then dilute with standard diluent to 8 different concentrations, namely: 4000pg / mL, 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 0pg / mL;

[0126] ②Remove unnecessary slats, put them back into the aluminum foil bag containing desiccant, and reseal it.

[0127] ③ Soak ELISA plate: add 300 μl 1× washing solution and soak for 30 seconds.

[0128] ④ Spike addition: Add 100 μl of 2-dilution standard to the standard wells. Add 100 μl of standard diluent (serum / plasma sample) to the blank.

[0129] ⑤ Add sample: Serum / plasma: Add 100 μl of pre-diluted sample to the sample pool. Cell culture supernatant: Add 100 μl of cell culture supernatant to the sample well.

[0130] Ensure that steps 4 and 5 are loaded continuously without interruption. The loading process is completed within 15 minutes.

[0131] ⑥ Incubation: Seal the plate with a sealing film, shake at 300 rpm, and incubate at room temperature for 1.5 hours.

[0132] ⑦ Washing: Discard the liquid, add 300μl washing solution to each well, and wash the plate 6 times. After each wash, pat the board dry with absorbent paper. In order to obtain ideal experimental performance, the residual liquid must be completely removed.

[0133] ⑧ Add detection antibody: Add 100 μl of diluted detection antibody (1:100 dilution) to each well. Seal the plate with sealing film. Shake at 300 rpm and incubate at room temperature for 45 minutes.

[0134] ⑨Washing: Repeat step 7.

[0135] ⑩Incubate with enzyme: Add 100 μl diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well. Seal the board with a new sealing film. Oscillate at 300 rpm and incubate at room temperature for 30 minutes.

[0136] Wash: Repeat step 7.

[0137] Add chromogenic substrate: Add 100 μl chromogenic substrate TMB to each well, protect from light, and incubate at room temperature for 5-30 minutes.

[0138] Add stop solution: add 100 μl stop solution to each well. Measure the OD value of the optical density at 450 nm wavelength with a microplate within 3 minutes after mixing. Use ELISAcalc software to draw a standard curve regression equation with the standard concentration as the ordinate and the OD value as the abscissa. Substitute the sample OD value into the equation, calculate the sample concentration, take the average value, and perform statistical analysis.

[0139] 2.6 RT-PCR detection of tumor tissue mRNA

[0140] RNA extraction:

[0141] ① Homogenization treatment:

[0142] Add 300 μl of lysis buffer RL for every 10-20 mg of tissue (please check whether β-mercaptoethanol has been added before use), and grind the tissue thoroughly with a pestle (if it is difficult to grind the tissue thoroughly, use an electric or glass homogenizer); then add 590 μl of RNase-Free ddH2O and 10 μl of pproteinasek to the homogenate and treat at 56°C for 10-20 min.

[0143] Note: The amount of tissue should not exceed 20 mg, otherwise the yield and quality of RNA will be reduced.

[0144] ②12000rpm (~13000), continue for 2-5min. Take the supernatant and perform the following operations.

[0145] ③ Slowly add 0.5 times the volume of supernatant ethanol, mix well (precipitation may occur at this time), transfer the resulting solution and precipitate to the adsorption column CR3 (the adsorption column is placed in the collection tube), centrifuge at 12000rpm (~13400) for 30-60 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0146] ④ Add 350 μl of deproteinized solution RW1 to the adsorption column CR3 at 12000 rpm (~13400), centrifuge for 30-60 seconds, discard the waste liquid, and put the adsorption column back into the collection tube.

[0147] ⑤ Preparation of DNase working solution: Place 10 μl of DNase stock solution into a new RNase-free centrifuge tube, add 70 μl of DD buffer, and mix gently.

[0148] ⑥ Add 80 μl of DNA working solution to the center of adsorption column CR3 and leave at room temperature for 15 minutes.

[0149] ⑦ Add 350 μl of deproteinized solution RW1 to adsorption column CR3 at 12000 rpm (~13400), centrifuge for 30-60 seconds, discard the waste liquid, and put the adsorption column back into the collection tube.

[0150] ⑧ Add 500 μl of washing solution RW to the adsorption column CR3 (please check whether ethanol has been added before use), let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm (~13400) for 30-60 seconds, discard the waste liquid, and put the adsorption column CR3 back into the collection tube.

[0151] ⑨Repeat step 8.

[0152] ⑩ Centrifuge at 12000 rpm (~13400) for 2 minutes and discard the waste liquid. Place the adsorption column CR3 at room temperature for a few minutes to completely dry the residual washing liquid in the adsorption material.

[0153] Transfer the adsorption column CR3 to a new RNase-free centrifuge tube, add 30-100 μl RNase-Free ddH2O in the middle of the adsorption membrane, let stand at room temperature for 2 minutes, and centrifuge at 12000 rpm (~13400) for 2 minutes to obtain RNA solution.

[0154] Check the nucleic acid concentration with a nanodropper and determine whether the nucleic acid extraction is successful according to the ratio of 260 / 280. Determine the amount of RNA solution to be added to the next reaction system based on the standard amount of 1ug.

[0155] Synthesis of cDNA

[0156] 50n-2μg total RNA can be used to establish a 20μl reaction system.

[0157] ① Thaw the template RNA 5×gDNABuffer, FQ-RTPrimerMix, 10×KingRTBuffer,

[0158] Thaw RNase-Free ddH2O at room temperature (15-25°C) and quickly place on ice. Before use, mix each solution and centrifuge briefly to collect the liquid remaining on the tube wall. Please do the following steps on ice. To ensure the accuracy of the reaction solution preparation, it should be prepared before each reaction. Make a mixture and then dispense it into each reaction tube.

[0159] ② Prepare a mixed solution according to the genomic DNA removal system in Table 1 and mix thoroughly. Centrifuge briefly and incubate at 42℃ for 3 minutes. Then place on ice.

[0160] Table 1

[0161]

[0162] ③ Prepare the mixed solution according to the reverse transcription reaction system in Table 2.

[0163] Table 2

[0164]

[0165] ④ Add the Mix in the reverse transcription reaction to the reaction solution in the gDNA removal step and mix thoroughly.

[0166] ⑤Incubate at 42℃ for 15 minutes.

[0167] ⑥Incubate at 95℃ for 3 minutes and place on ice to obtain cDNA.

[0168] ⑦ Primer design

[0169] Primers were designed using PrimerPremier 6 software and synthesized by Beijing Bomade Co., Ltd. (see Table 3).

[0170] Table 3

[0171]

[0172] ⑧Configure the reaction system:

[0173] Prepare the PCR reaction system according to Table 4 (Note: When preparing multiple reaction wells, please reserve 10% of the remaining volume for each component to avoid pipetting loss.)

[0174] Table 4

[0175]

[0176] ⑨After the reaction system is prepared, cover the reaction cap, vortex mix thoroughly, and centrifuge.

[0177] ⑩ Dispense the reaction solution into each reaction well, seal with film, and centrifuge to avoid bubbles.

[0178] Place the reaction plate on the fluorescence quantitative PCR instrument, select the fast or standard PCR reaction program as needed, and set the reaction parameters according to Table 5.

[0179] Table 5

[0180]

[0181] Using StepOnePlus TM Real-TimePCRSystem fluorescent quantitative PCR instrument was used for amplification. After the reaction was completed, the amplification curve and melting curve were confirmed and the data were output. -ΔΔCt Calculation method: ΔΔCt = (Ct target gene - Ct internal reference gene) experimental group - (Ct target gene - Ct internal reference gene) control group.

[0182] 2.7 Western Blot detection of tumor tissue protein expression

[0183] Extraction of total protein from tissues

[0184] 1. Place a small amount of tissue chunks into a 1-2 ml homogenizer and use clean scissors to cut off as much tissue chunks as possible.

[0185] 2. Add 400 μl RIPA, lyse the liquid (including PMSF) and homogenize in a homogenizer. Then put it on ice.

[0186] 3. After a few minutes, grind for a while and place on ice. Repeat the grinding several times to crush as much tissue as possible.

[0187] 4. After 30 minutes of lysis, the lysate can be transferred to a 1.5 ml centrifuge tube using a pipette, and then centrifuged at 12,000 rpm for 5 minutes at 4°C. The supernatant can be dispensed into 0.5 ml centrifuge tubes and stored at -20°C.

[0188] Determination of protein content

[0189] 1. Make a standard curve

[0190] (1) Take out 1 mg / ml BSA from -20°C and melt it at room temperature for later use.

[0191] (2) Take 18 1.5 ml centrifuge tubes, three in a group, and label them as 0 mg, 2.5 mg, 5.0 mg, 10.0 mg, 20.0 mg, and 40.0 mg respectively.

[0192] (3) Add various reagents to each test tube according to the table below.

[0193] (4) After mixing, the mixture was placed at room temperature for 2 minutes and analyzed by biospectrophotometer.

[0194] 2. Detect the protein content of the sample

[0195] (1) Take enough 1.5 ml centrifuge tubes and add 1 ml of Coomassie Brilliant Blue solution stored at 4°C to each tube. After standing at room temperature for 30 minutes, it can be used for protein detection.

[0196] (2) Take a tube of Coomassie Brilliant Blue and 100 ml of 0.15 mol / L NaCl solution, mix well and let stand for 2 minutes to serve as a blank sample. Pour the blank into a cuvette and measure the blank sample according to the blank under the standard curve program.

[0197] (3) Discard the blank sample, wash the test tube twice with anhydrous ethanol (0.5 ml each time), and then wash again with sterile water.

[0198] (4) Take a tube of Coomassie Brilliant Blue, add 95 ml of 0.15 mol / L NaCl solution and 5 ml of the protein sample to be tested, mix well, let stand for 2 min, pour into the drained cuvette, press the sample button, and test the sample.

[0199] Protein gel electrophoresis

[0200] 1. Run the glue

[0201] (1) Clean the glass panel thoroughly and install it according to the manufacturer's instructions;

[0202] (2) Prepare a separation gel with a concentration of 12% and mix well;

[0203] (3) Quickly pour the separation gel into the glass dish. Use a pipette to carefully add a layer of isopropanol to seal it;

[0204] (4) Wait for about 30 minutes until the separation gel is completely polymerized, and then gently pour out the covering isopropanol;

[0205] (5) Prepare 5% concentrated gel;

[0206] (6) Pour 5% concentrated gel into a glass dish, and then insert a 15-hole sampling comb into the concentrated gel solution;

[0207] (7) After the concentrated gel solidifies, carefully remove the comb teeth. Place the glass plate with gel into the electrophoresis tank and fill the electrophoresis box with 1× electrophoresis buffer;

[0208] (8) Sample loading: Gently pull out the gel comb and add the protein marker and the prepared protein sample into the sample loading well in sequence;

[0209] (9) Electrophoresis: ① Constant voltage 80 V, current 250 mA, electrophoresis for about 30 minutes; ② Constant voltage 120 V, current 250 mA (after the sample enters the separation gel), until the bromophenol blue dye just electrophoreses out of the gel.

[0210] 2. Transfer

[0211] (1) Prepare 1 L of 1× film transfer solution and precool it in a 4°C refrigerator;

[0212] (2) Complete the assembly according to the following steps: membrane transfer plate negative electrode - sponge - filter paper - separation gel - PVDF membrane (methanol activated) - filter paper - sponge - membrane transfer plate positive electrode, ensuring that there are no bubbles;

[0213] (7) Immediately insert the transfer splint into the transfer tank, add 1× transfer solution, place the ice box into the transfer tank, transfer the membrane in a cold room at 4°C, set the voltage to 130 V, the constant current to 250 mA, and transfer for 2.5 hours; hours;

[0214] (8) Disconnect the power supply and remove the conveyor from top to bottom.

[0215] 3. Staining

[0216] (1) Immerse the polyvinylidene fluoride membrane in the poncho dye solution; refer to the molecular weight of the protein marker, cut off the PVDF membrane where the protein to be tested is located, and cut off the upper left corner as a marker;

[0217] 2) Rinse with 1×TBST for 3 min.

[0218] 4. Closure

[0219] (1) Sealing: Use the prepared sealing liquid (5% skimmed milk powder) to seal on a rotary shaker at room temperature for 1-2 h;

[0220] (2) Rinse: 1‰ TBST × 3 times, 10 min each time;

[0221] (3) Primary antibody incubation: The primary antibody was diluted with 5% skim milk powder. The final concentration of SV40LT was generally 1-5 μg / ml; the final concentration of Cas9-Flag was generally 1:2000, and that of GAPDH was 1:2000. Shake well at 4°C overnight.

[0222] (4) Rinse: 1‰ TBST × 3 times, 10 min each time;

[0223] (5) Incubation with the secondary antibody: dilute the secondary antibody according to the dilution ratio specified in the antibody instructions and shake at room temperature for 1-1.5 hours;

[0224] (6) Rinse: 1‰ TBST × 3 times, 10 min each time.

[0225] 5. Exposure and Development

[0226] (1) Prepare the ECL working solution according to the instructions;

[0227] (2) Use flat tweezers to take the membrane out of the antibody incubation box, absorb the excess liquid with absorbent paper, soak the PVDF membrane thoroughly with the working solution, and then place it in the black box of the automatic colorimeter;

[0228] (3) Exposure, development, and fixing: Exposure and development are performed according to the operation of the image analysis system. The exposure time can be adjusted according to the results, and finally a photo with the appropriate exposure time is saved.

[0229] 6. Result analysis.

[0230] 3. Data Analysis

[0231] Excel was used to analyze and organize the data, and SPSS 27 software was used for statistical analysis. The data were expressed as mean ± standard deviation (Mean ± SD). The comparison between two independent groups used the T test (normal distribution data) and Wilcoxon rank sum test (non-normal distribution data). The comparison between multiple independent groups used one-way analysis of variance (normal distribution data) and Kruskal-Wallis rank sum test (non-normal distribution data). All statistical analyses were bilateral tests, and P < 0.05 was considered statistically significant. All experiments were repeated 3 times.

[0232] 4. Experimental results

[0233] 4.1 Effects of Yiqi Huoxue Fang (YQHXF) on body weight changes and liver and spleen weights in C57 subcutaneous transplanted tumor mice. Figure 1 In terms of body weight of C57 mice, there was a significant difference between the low-dose YQHXF group and the model group (p<0.05), among which the body weight of the high-dose YQHXF group was closer to that of the normal group; in terms of liver weight of C57 mice, compared with the normal group and the model group, the medium-dose YQHXF group had increased liver weight (p<0.05); in terms of spleen weight of C57 mice, compared with the normal group, the model group, cyclophosphamide group, low-dose YQHXF group, medium-dose YQHXF group and high-dose YQHXF group had increased spleen weight (p<0.05), and compared with the model group, the medium-dose YQHXF group had significantly increased spleen weight (p<0.05).

[0234] 4.2 Effect of Yiqi Huoxue Fang (YQHXF) on tumor mass and volume in mice with C57 subcutaneous tumor transplantation

[0235] Depend on Figure 2 It was found that three weeks after oral administration, there was no significant difference in body weight among the C57 mice groups. In terms of tumor weight, compared with the model group, the YQHXF medium-dose group and the YQHXF high-dose group could reduce tumor weight (p<0.05), and the cyclophosphamide chemotherapy group could significantly reduce tumor weight (p<0.001). In terms of tumor volume, compared with the model group, the cyclophosphamide chemotherapy group could significantly reduce tumor volume (p<0.001).

[0236] 4.3 Effect of Yiqi Huoxue Fang (YQHXF) on Ki67 expression in tumor tissue of C57 subcutaneous transplanted tumor mice

[0237] See also Figure 3 After immunohistochemical treatment of the tumor tissue of C57 mice, Ki67 was strongly expressed in the model group. Compared with the model group, the Ki67 expression in the cyclophosphamide group and the YQHXF high-dose group, YQHXF medium-dose group, and YQHXF low-dose group was significantly decreased, and the tumor invasion degree of the model group was high.

[0238] 4.4 Effect of Yiqi Huoxue Fang (YQHXF) on TNF-α and IL-10 levels in mice with C57 subcutaneous tumor transplantation

[0239] See also Figure 4 , the content of TNF-α in each treatment group of C57 mice was lower than that in the normal group (p<0.05); compared with the model group, the content of TNF-α in the cyclophosphamide group and the YQHXF low-dose group was significantly higher than that in the model group (p<0.05). The content of IL-10 in each treatment group of C57 mice was higher than that in the normal group (p<0.05); compared with the model group, the content of IL-10 in the YQHXF low-dose group and the YQHXF medium-dose group was significantly lower (p<0.05).

[0240] 4.5 Effects of Yiqi Huoxue Fang (YQHXF) on four blood lipid parameters in mice with C57 subcutaneous transplanted tumors

[0241] Detection of 4 blood lipids in C57 subcutaneous transplanted tumor mice, see Figure 5 Compared with the normal group, the triglyceride content of each treatment group of C57 mice was significantly increased in the model group (p<0.05), and the triglyceride content of the YQHXF low-dose group, YQHXF medium-dose group and YQHXF high-dose group was significantly decreased compared with the model group (p<0.05); while in serum total cholesterol and low-density lipoprotein LDL-C, the model group and other treatment groups were higher than the normal group, and compared with the model group, there were no significant differences in TC and LDL-C content in the cyclophosphamide group, YQHXF low-dose group, YQHXF medium-dose group and YQHXF high-dose group; in C57 subcutaneous transplanted tumor small high-density lipoprotein HDL-C, there were no significant differences between each treatment group and the normal group.

[0242] 4.6 Effect of Yiqi Huoxue Fang (YQHXF) on the mRNA expression of GPX4 and SLC7A11 in tumor tissues of C57 subcutaneous transplanted mice

[0243] See also Figure 6In the mRNA expression of GPX4 in each treatment group of C57 mice, compared with the model group, the mRNA expression of YQHXF low-dose group increased (p<0.01), and the mRNA expression of GPX4 in YQHXF medium-dose group and YQHXF high-dose group decreased (p<0.01). Compared with the cyclophosphamide group, the mRNA expression of YQHXF low-dose group increased (p<0.01), and the mRNA expression of GPX4 in YQHXF medium-dose group and YQHXF high-dose group was significantly decreased. (p<0.01); in the mRNA expression of SLC7A11, compared with the model group, the mRNA expression of SLC7A11 in the cyclophosphamide group was significantly increased (p<0.01), the mRNA expression of SLC7A11 in the YQHXF high-dose group was significantly decreased (p<0.05), and compared with the cyclophosphamide group, the mRNA expression of SLC7A11 in the YQHXF low-dose group, YQHXF medium-dose group and YQHXF high-dose group was significantly decreased (p<0.05).

[0244] 4.7 Effect of Yiqi Huoxue Fang (YQHXF) on GPX4 and SLC7A11 protein expression in tumor tissues of C57 subcutaneous transplanted tumor mice

[0245] Western Blot results showed ( Figure 7 ), GPX4 protein expression in tumor tissue of C57 mice, compared with the model group, GPX4 protein expression in the YQHXF high-dose group was lower (p<0.05), and SLC7A11 expression in tumor tissue, compared with the model group, SLC7A11 protein in the YQHXF medium-dose group and the YQHXF high-dose group was significantly decreased (p<0.01).

[0246] 5. Conclusion

[0247] By establishing a CRPC subcutaneous transplant tumor model in C57 BL / 6 male mice, HE staining, Elisa detection, RT-PCR, Western Blot and other methods were used to preliminarily verify that the Yiqi Huoxue recipe (YQHXF) can inhibit the growth of castration-resistant prostate cancer from the perspectives of tissue-transcription-translation level, immunity, ferroptosis, and lipid metabolism.

[0248] The experimental results showed that compared with the model group, Yiqi Huoxue Fang (YQHXF) could reduce tumor mass and inhibit tumor growth; in terms of ki67 expression, compared with the model group, Yiqi Huoxue Fang (YQHXF) could reduce the expression of KI67 and reduce the malignancy of tumor invasion; Elisa test showed that Yiqi Huoxue Fang (YQHXF) could increase the content of TNF-α, reduce the content of IL-10, and improve the inflammatory response in the body. At the same time, YQHXF could reduce the content of triglycerides in mice and improve the survival status of mice. RT-PCR and Western Blot results showed that the medium and high dose groups of YQHXF could significantly reduce the mRNA expression of GPX4, and the low, medium and high dose groups of YQHXF could significantly reduce the mRNA expression of SLC7A11. Western Blot results showed that the high dose group of YQHXF could reduce the protein expression of GPX4, which was inconsistent with the RT-PCR results. The low, medium and high dose groups of YQHXF could significantly reduce the protein expression of SLC7A11, which was consistent with the RT-PCR results.

[0249] Yiqi Huoxue recipe (YQHXF) increased the content of TNF-α in C57 mice and reduced the expression of AR receptors. It also reduced the content of IL-10 in C57 mice, thereby regulating the immune response and inhibiting tumor growth. Yiqi Huoxue recipe (YQHXF) increased the spleen mass of C57 mice, which may be related to stimulating the immune function of the spleen. It may also be due to the fact that beehive in traditional Chinese medicine is slightly toxic. Future research should focus on the toxicological effects of Yiqi Huoxue recipe.

[0250] YQHXF increases the TNF-α content in C57 mice, promotes the enzymatic hydrolysis of AR protein, induces the production of oxygen free radicals, and promotes the "oxidative burst" of neutrophils; YQHXF reduces the content of interleukin-10, enhances the body's immune response, and reversely increases the production of mitochondrial ROS; YQHXF reduces the content of triglycerides in the body, reduces the impact of lipids on the immune function of regulatory T cells and DC cells, and jointly inhibits tumor growth.

[0251] The Yiqi Huoxue recipe reduced the mRNA and protein expression of SLC7A11 in the tumor, and also reduced the mRNA and protein expression of GPX4. It is speculated that the Yiqi Huoxue recipe may induce ferroptosis through the SLC7A11 / GPX4 pathway and thus inhibit the growth of the tumor. To further confirm this hypothesis, the inventors conducted a second part of the experiment.

[0252] Part 2

[0253] 1 Experimental Materials

[0254] 1.1 Cell culture and treatment

[0255] The cell sources are the same as those in Part 1.2 Experimental cells.

[0256] Recovery of frozen cells: Take out the frozen cells from liquid nitrogen and immediately put them into a 42℃ water bath to fully thaw, then wipe off the water stains; enter the cell space and centrifuge at 1000rpm for 3min; remove the supernatant, add cell culture medium to resuspend the cells, add the cell suspension to a T25 flask, add cell culture medium to 5-6mL, and mark it; place the T25 flask in a 37℃, 5% CO2 incubator for culture.

[0257] Cell passaging: Use 1640 medium to prepare the medium according to 10% FBS + 1% ITS + 1% NEAA, suspend the cells, and culture them in a 37°C, 5% CO2 incubator; when the cells reach about 80% confluence, add 1-2mL of trypsin to digest; observe under a microscope, when the cells become round, discard the trypsin, add 1-2mL of fresh medium, gently blow the cells, and then add fresh medium according to the cell type, density, growth status and needs, and continue to culture.

[0258] Cryopreservation of cells: Observe the cells under a microscope. When the cells grow to the logarithmic growth phase, add 3-4mL trypsin to gently wash the cells, then add 1mL trypsin and place in a 37℃ incubator to digest the cells. When the cell morphology becomes round, discard the trypsin, add cell culture medium and blow to collect the cells; centrifuge at 1 000rpm for 3min, remove the supernatant; add cell freezing solution to blow the cells apart; transfer to a 2mL cryopreservation tube with a pipette, and mark the cryopreservation tube with the cell name, passage number and cryopreservation date. Wrap the cryopreservation tube with cotton, then wrap it with rubber gloves and mark it, put it in a -80℃ refrigerator overnight, and put it in a liquid nitrogen tank for long-term storage the next day.

[0259] 1.2 Preparation of serum containing Yiqi Huoxue prescription

[0260] Animal preparation: 20 SD rats were purchased from the National Rodent Laboratory Animal Seed Center and bred in a normal-grade laboratory animal environment. Male rats, 6-7 weeks old, weighing 240±20g, were raised in separate cages. During the experiment, they were raised in a normal environment with a constant temperature of 22-25°C and a constant humidity of 50%-65%. Drinking water, feed, and experimental supplies were sterilized and disinfected, and the experimental operation strictly followed the principle of aseptic operation. The rats were purchased 2 days before the experiment to adapt them to the new environment.

[0261] Drug preparation: Yiqi Huoxue Fang granules are composed of 15g of Astragalus, 6g of Curcuma, 3g of Piper longum, and 3g of Nigella ovata. Granules and quality control are provided by Sichuan Xinlu Pharmaceutical Technology Development Co., Ltd. The dosage of Chinese medicine is calculated according to the conversion formula between rats and humans, and the dosage is 3 times the effective dosage of human body, that is, 0.945g / kg, and the dosage is 1ml / 100g.

[0262] Blood collection from abdominal aorta: After 3 days of intragastric gavage, fast for 12 hours, prepare 10% chloral hydrate at 350 mg / kg for intraperitoneal anesthesia, and collect blood from abdominal aorta of rats, about 0.5-1 ml of blood from each rat.

[0263] Serum treatment: After the collected blood is left to rest for 2 hours, it is placed in a 45°C water bath for 10 minutes and centrifuged at 3000r / min for 5 minutes. The upper serum is aspirated and filtered 1-2 times with a 0.22μm syringe filter in a cell clean bench. The serum is packaged according to experimental requirements, frozen at -20°C overnight, and then stored in a -80°C refrigerator.

[0264] 1.3 Experimental drugs

[0265] Ferropoptosis inhibitor (Ferrostatin-1): 25 mg, powder, (Cat. No. TQ009), purchased from MCE. Prepared into 10 mM solution with DMSO solution and stored at -20°C.

[0266] Ferroptosis activator RSL3: 10 mg, powder, (Cat. No. TQ008), purchased from MCE. Prepared into 10 mM solution with DMSO solution and stored at -20°C.

[0267] 1.4 Main Reagents

[0268] Table 6

[0269]

[0270] 2 Experimental methods

[0271] 2.1 CCK8 assay

[0272] Relevant operations were performed according to the CCK8 kit, including preparation of CCK8 standard curve, detection of cell activity, detection of cell proliferation-toxicity, and calculation of cell survival rate according to the formula.

[0273] According to the calculation formula, cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0274] As: optical density of experimental wells (containing cells, culture medium, CCK-8 solution and drug solution);

[0275] Ac: optical density of control wells (containing cells, culture medium, CCK-8 solution, but no drug);

[0276] Ab: optical density of blank wells (containing culture medium and CCK-8 solution, but not cells or drugs).

[0277] 2.2 ROS Reactive Oxygen Species Detection Kit

[0278] 1. Loading ROS probe

[0279] 1) Cell preparation: Use 6-well plates for cell plating, with a cell number of approximately 2×10 6 / ml.

[0280] 2) Drug induction: Remove the cell culture medium, add the drug diluted in serum-free medium (the specific method of adding drugs is shown in Table 7), and incubate in a cell culture incubator at 37°C in the dark for 3 h.

[0281] Table 7

[0282]

[0283] 3) Positive control: dilute the positive control (Rosup, 100mM) to the commonly used working concentration of 100μM with serum-free medium, add to the cells, and incubate at 37℃ in the dark for 0.5-4h to increase the level of reactive oxygen species, which varies for different cell types. For example, HeLa cells need to be incubated for 30-60min, while MRC5 human embryonic fibroblasts need to be incubated for 90min.

[0284] 4) Preparation of ROS probe: Before probe loading, dilute DCFH-DA with serum-free culture medium at 1:1000 to a final concentration of 10 μM. Pipette 6 μL of diluted DCFH-DA, add 6 ml of serum-free culture medium, and mix thoroughly.

[0285] 5) Loading ROS probe: Remove the treatment drug and add an appropriate volume of diluted DCFH-DA working solution. The added volume should be sufficient to cover the cells. (6-well plate is usually not less than 1000μL). Wrap the culture plate with tin foil and incubate in a 37℃ cell culture incubator away from light for 30 minutes.

[0286] 6) Cell washing: Wash the cells 1 to 2 times with serum-free culture medium to fully remove DCFH-DA that has not entered the cells.

[0287] 2. Fluorescence microscopy operation method

[0288] 1) Adherent cells or living tissues can be directly observed under a fluorescence microscope;

[0289] 2) Under a fluorescence microscope, use a FITC filter to observe fluorescence and remove the background to observe changes in fluorescence.

[0290] 2.3C11BODIPY581 / 591 lipid peroxide probe and GSH kit

[0291] 1) Dissolve 1 mg of this product in 198.2 μL DMSO to prepare a 10 mM C11BODIPY581 / 591 stock solution.

[0292] 2) Culture the target cells to an appropriate density and, after appropriate treatment, add an appropriate volume of lipid peroxide probe storage solution to the proposed cell model and incubate for 1 hour.

[0293] 3) The cells were washed twice with PBS to remove excess dye, then digested with trypsin, resuspended in PBS containing 5% FBS, and the cells were collected in a 1.5 ml EP tube.

[0294] 4) Using a flow cytometer, using 499nm and 565nm laser excitation, measure the signal corresponding to 505-550nm in the FL1 channel, and measure the signal greater than 580nm in the FL2 channel.

[0295] 5) After this dye is excited with a blue laser (488nm), the wavelength of emission light emitted by the dye bound to normal cells is 595nm at maximum. Once the cell lipids bound to the dye are peroxidized, the polyunsaturated butadiene part of C11BODIPY581 / 591 is oxidized, causing its maximum emission fluorescence to migrate from 590nm to around 510nm. It is proportional to the production of lipid reactive oxygen species. The arithmetic mean ± SEM is calculated based on the FL1 and FL2 channel signals of the flow cytometer to quantify the proportion of lipid peroxidized cells.

[0296] GSH content determination: After RM-1 was divided into groups for treatment, the treated cells were collected and the GSH content was determined according to the operation of the kit.

[0297] 2.4 Iron ion detection

[0298] 1) Dilute 10 μL of iron standard with 990 μL ddH2O to prepare a 1 mM standard.

[0299] 2) Referring to Table 8, prepare a standard curve using 1 mM standard in a microplate or microcentrifuge tube.

[0300] Dilution:

[0301] Table 8

[0302]

[0303] 3) Add samples according to Table 9. The amount of standard in each dilution should be sufficient for two replicates (2×100 μL). Standard wells = 100 μL standard dilution. Sample wells = 2-50 μL sample (adjust volume to 100 μL / well with iron assay buffer). Mix standards and samples and incubate at 37°C for 30 minutes.

[0304] Add 100 μL of iron probe to the iron standard well and test sample well, mix well and incubate at 37°C in the dark for 60 minutes. Immediately place on a colorimetric microplate reader to measure the absorbance (OD593nm).

[0305] Table 9

[0306]

[0307] 4) Take the average of the readings of the two replicate wells for each standard and sample. Subtract the average absorbance value of the blank sample (standard #1) from the readings of all standards and samples to obtain the corrected absorbance.

[0308] 5) Plot the calibration values ​​for each standard as a function of the final iron concentration. Using these scattered points, create a smooth curve that is ideal for the standard curve. Most microplate reader software or Excel can plot these values ​​and fit the curve. The trend line equation can be calculated from the standard curve data (using the equation that produces the most accurate fit to the data).

[0309] 6) Obtain the iron (II) and total iron (II+III) content in the test sample directly from the standard curve according to the following calculation formula. Calculate the iron (III) content in the test sample: Iron (III) = Total Iron (II+III) - Iron (II)

[0310] 2.5RT-QPCR detection

[0311] After trypsin digestion, the cells were collected, and cDNA was extracted according to the above-mentioned qPCR operation, and the expression levels of GPX4 and SLC7A11 mRNA were calculated.

[0312] 3 Data analysis

[0313] Same as data analysis in Part 1.3.

[0314] 4 Experimental results

[0315] 4.1RM-1 cell growth curve

[0316] Results Figure 8 As shown in Table 10, the number of RM-1 cells is linearly related to the absorbance of the cck8 solution. The IC50 values ​​of YQHXF-containing serum, RSL3, and Fer-1 for treating RM-1 cells were 57%, 1.87μM, and 13.5μM, respectively. According to specific experimental observations and convenient experimental operations, the drug concentrations for treating cells were determined to be 40% YQHXF-containing serum, and the dosing concentrations of RSL3 and Fer-1 were 1μM and 10μM, respectively.

[0317] Table 10 IC50 values ​​of RM-1 cells after treatment with YQHX-containing serum, RSL3, and Fer-1

[0318]

[0319]

[0320] 4.2 Effect of YQHXF-containing serum on RM-1 cell morphology

[0321] The growth morphology of RM-1 cells is as follows Fig. 9 : After 6 hours of treatment, the RM-1 cells in the model group were spindle-shaped, colony-like, with clear colony growth boundaries, and epithelial cell proliferation; the RM-1 cells in the RSL3 group became oval in color, and under a high-power microscope (40X) (such as Fig.10 ), it can be seen that the cell membrane of the marked cells has ruptured, the growth of other cells has slowed down, and the cells in the Fer-1 group have colony growth, which is not significantly different from the model group. In the treatment groups with Chinese medicine, the RM-1 cells have different morphologies, with spindle cells and small round cells scattered, and no obvious colony phenomenon under low power microscope (10X).

[0322] Effect of 4.3YQHXF-containing serum on ROS content in RM-1 cells

[0323] Immunofluorescence results showed that ( Fig.11 ): The expression of ROS after 12 hours of treatment with different methods. Compared with the control group, the RSL3 group, YQHXF+RSL3 group, and YQHXF+Fer-1 group all showed different degrees of fluorescence, while no fluorescence was collected in the FER-1 group.

[0324] Effects of 4.4 YQHXF-containing serum on GSH content and lipid peroxides in RM-1 cells

[0325] The results show that Fig.12 ), after various treatments, the GSH content in the YQHXF group and the Fer-1 group increased compared with the model group, and the GSH content in the YQHXF+Fer-1 group was significantly reduced compared with the Fer-1 group (p<0.05); compared with the YQHXF group, the GSH content in the YQHXF+RSL3 group was reduced (p<0.05). In terms of the lipid peroxide ratio in RM-1 cells, compared with the model group, the lipid peroxide ratios in the YQHXF group, the RSL3 group, the YQHXF+RSL3 group, and the YQHXF+Fer-1 group were higher than those in the model group (p<0.05); compared with the YQHXF group, the lipid peroxide ratio in the YQHXF+RSL3 group was reduced (p<0.05), and the lipid peroxide ratio in the YQHXF+Fer-1 group was increased (p<0.05).

[0326] Effect of 4.5YQHXF-containing serum on Fe ion content in RM-1 cells

[0327] The occurrence of ferroptosis is related to the accumulation of iron ions in the body. The content of Fe ions in each treatment group of RM-1 was detected, and the results showed that ( Fig.13 ): Compared with the model group, the total iron ions (Fe 2+ , Fe 3+ ) content was significantly higher than that in the model group (p<0.05). Compared with the RSL3 group, the content in the YQHXF+RSL3 group was significantly higher than that in the RSL3 group (p<0.05). Compared with the Fer-1 group, the content in the YQHXF+Fer-1 group was significantly higher than that in the Fer-1 group (p<0.05). 2+ The total iron ion (Fe) content in the YQHXF group, YQHXF+RSL3 group and YQHXF+Fer-1 group 2+ , Fe 3+ ) content was significantly increased compared with the model group (p<0.05), the content in the YQHXF+RSL3 group was significantly increased compared with the RSL3 group (p<0.05), and the content in the YQHXF+Fer-1 group was significantly increased compared with the Fer-1 group (p<0.05).

[0328] Effect of 4.6YQHXF-containing serum on the expression of GPX4 and SLC7A11 in RM-1 cells

[0329] RT-PCR results showed that in the expression of SLC7A11, compared with the model group, the SLC7A11 in the YQHXF+Fer-1 group was increased (p<0.05); compared with the RSL3 group, the SLC7A11 expression in the YQHXF+RSL3 group was decreased (p<0.05); compared with the YQHXF group, the SLC7A11 expression in the YQHXF+RSL3 group was decreased (p<0.05), and the SLC7A11 in the YQHXF+Fer-1 group was increased (p<0.05). In terms of GPX4 expression, compared with the model group, the GPX4 expression in the YQHXF group and YQHXF+RSL3 group was significantly decreased (p<0.05), and the GPX4 expression in the Fer-1 group and YQHXF+Fer-1 group was significantly increased (p<0.05). Compared with the YQHXF group, the SLC7A11 expression in the YQHXF+RSL3 group was decreased (p<0.05), and the SLC7A11 in the YQHXF+Fer-1 group was increased (p<0.05).

[0330] 5. Conclusion

[0331] In the in vitro experiment, the cck8 method, observation of cell morphology, detection of intracellular reactive oxygen species (ROS) content, GSH content, lipid peroxide ratio, Fe ion content and RT-PCR detection of GPX4 and SLC7A11 mRNA expression were used to verify whether the Yiqi Huoxue prescription (YQHXF) activates the ferroptosis pathway through the SLC7A11 / GPX4 pathway and inhibits the growth of RM-1 cells.

[0332] The effect of Yiqi Huoxue Fang on cell morphology was not drastic, but rather moderate. After RM-1 was treated for 6 hours in each experimental group, the morphology of RM-1 cells in the RSL3 group changed to a small round shape, and cell membrane rupture was found under a high-power microscope. In the three treatment groups with Yiqi Huoxue Fang, the morphology of RM-1 cells did not change significantly, suggesting that the effect of Yiqi Huoxue Fang on RM-1 cells was milder than that of the ferroptosis activator RSL3.

[0333] The Yiqi Huoxue recipe increases the reactive oxygen content in RM-1 cells and promotes ferroptosis. ROS can be detected under the microscope in the normal group of RM-1 cells. During cell growth, a certain amount of ROS is produced, which is caused by the growth and development of tumor cells. In the RSL3 group and the Yiqi Huoxue recipe + RSL3 group, an increase in ROS levels was observed under the microscope. The inhibitor Fer-1 is a ROS scavenger, so the generation of ROS cannot be observed under the microscope. After adding the Yiqi Huoxue recipe to Fer-1, the increase in ROS can be cleared.

[0334] The Yiqi Huoxue recipe promoted the accumulation of lipid peroxides through other pathways and promoted ferroptosis in RM-1 cells. The experiment showed that although the Yiqi Huoxue recipe temporarily increased the content of GSH in RM-1 cells, it did not affect the proportion of lipid peroxides in the cells. The three treatment groups that added the Yiqi Huoxue recipe all increased the proportion of lipid peroxides to varying degrees.

[0335] Yiqi Huoxue Fang increases intracellular Fe 2+ The experiment showed that both total Fe ions and Fe 2+ In the group containing the Yiqi Huoxue recipe, the content of Fe ions was significantly higher than that in the model group and the control inhibitor and activator groups. The Yiqi Huoxue recipe may increase Fe ions, destroy iron homeostasis, activate the Fenton reaction in cells, produce a large amount of ROS, and induce ferroptosis in RM-1 cells.

[0336] RT-PCR results showed that compared with the model group, the Yiqi Huoxue group did not reduce the expression of GPX4 mRNA. Compared with the activator RSL3, the Yiqi Huoxue group combined with RSL3 reduced the expression of GPX4. Compared with the inhibitor Fer-1, the Yiqi Huoxue group combined with Fer-1 enhanced the expression of GPX4. In terms of the mRNA expression of SLC7A11, RSL3 directional regulated the expression of GPX4 and could not affect the expression of the upstream factor SLC7A11. However, the Yiqi Huoxue group combined with RSL3 could reduce the expression of SLC7A11. Compared with the inhibitor Fer-1, the Yiqi Huoxue group combined with Fer-1 could enhance the expression of SLC7A11.

[0337] In summary, the Yiqi Huoxue recipe increases the Fe ion content, lipid peroxide level, and ROS content in RM-1 cells, promotes ferroptosis in combination with the ferroptosis activator RSL3, and weakens the inhibitory effect on ferroptosis in combination with the ferroptosis inhibitor Fer-1, thereby regulating the SLC7A11 / GPX4 pathway to induce ferroptosis in RM-1 cells.

[0338] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A Chinese medicine composition for treating castration-resistant prostate cancer, characterized in that: The Chinese medicine composition is prepared from the following raw materials in parts by weight: 10-30 parts of Astragalus, 3-15 parts of Curcuma, 3-6 parts of Piper longum, and 3-6 parts of Apium graveolens.

2. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is prepared from the following raw materials in parts by weight: 10-15 parts of Astragalus, 3-9 parts of Curcuma, 3-5 parts of Piper longum, and 3-5 parts of Apium graveolens.

3. The Chinese medicine composition according to claim 1, characterized in that The Chinese medicine composition is prepared from the following raw materials in parts by weight: 15 parts of Astragalus, 6 parts of Curcuma, 3 parts of Piper longum, and 3 parts of Nigella ovata.

4. A drug for treating castration-resistant prostate cancer, characterized in that: The medicine uses the Chinese medicine composition according to any one of claims 1 to 3 as an active ingredient.

5. The drug according to claim 4, characterized in that The drug is also added with pharmaceutically acceptable excipients.

6. The drug according to claim 4, characterized in that The dosage form of the medicine is decoction, powder, pill, granule, tablet, capsule, oral liquid or ointment.

7. Use of the traditional Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a drug for treating castration-resistant prostate cancer.

8. The use according to claim 7, characterized in that: The drug is used for one or more of the following effects: 1) Increase TNF-α content; 2) Reduce Ki67 expression; 3) Reduce the content of IL-10; 4) Reduce triglyceride levels; 5) Reduce GPX4 mRNA expression; 6) Reduce the mRNA expression of SLC7A11; 7) Reduce the protein expression of SLC7A11.

9. The use according to claim 7, characterized in that: The drug activates ferroptosis and inhibits tumor growth by inducing the SLC7A11 / GPX4 pathway.

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