WO2023051088A1 - Use of dihydrotanshinone i in preparation of nrf2 inhibitor - Google Patents

Use of dihydrotanshinone i in preparation of nrf2 inhibitor Download PDF

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WO2023051088A1
WO2023051088A1 PCT/CN2022/113764 CN2022113764W WO2023051088A1 WO 2023051088 A1 WO2023051088 A1 WO 2023051088A1 CN 2022113764 W CN2022113764 W CN 2022113764W WO 2023051088 A1 WO2023051088 A1 WO 2023051088A1
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dht
cells
nrf2
cell
dihydrotanshinone
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French (fr)
Chinese (zh)
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开国银
韩冰
孙承韬
王瑶
翟宇飞
赵欢
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浙江中医药大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the invention belongs to the technical field of medicine, specifically relates to the function of dihydrotanshinone I in inhibiting Nrf2 activity, and is also related to the application of dihydrotanshinone I in the treatment of ovarian cancer and the active ingredients of anti-ovarian cancer drugs.
  • Salvia miltiorrhiza is a plant of the genus Salvia in the dicotyledonous Lamiaceae. It has a long history and was first recorded in "Shen Nong Baicao Jing". The medicinal parts are dry roots and rhizomes. Tanshinone compounds have been widely studied because of their high content in Danshen and their significant antitumor effects. Tanshinone IIA (Tanshinone IIA, TAN IIA), Tanshinone I (Tanshinone I, TAN I), Cryptotanshinone (Cryptotanshinone, CT) and dihydrotanshinone I (15,16-Dihydrotanshinone I, DHT) are fat-soluble Active ingredients of phenanthrene quinones.
  • DHT can play an anti-tumor effect by inhibiting tumor cell proliferation, promoting apoptosis and blocking cell cycle.
  • DHT can induce the apoptosis of human osteosarcoma 143B cells through the Caspase pathway, and down-regulate the adhesion molecules VCAM-1 and ICAM-1 to weaken the migration of osteosarcoma cells (Anti-Cancer Agent Me, 2017; 1234-1242).
  • DHT can activate JNK/p38MAPK signaling pathway to induce apoptosis of gastric cancer cells SGC7901 and MGC803 (Pharm Biol, 2016; 3019-3025).
  • DHT can also induce the production of reactive oxygen species and inhibit the growth of breast cancer stem cells through the ROS/Stat3 signaling pathway (Oxid Med Cell Longev, 2019).
  • DHT can regulate the MAPK/Akt/mTOR pathway to induce liver cancer SK-HEP-1 cells to arrest in the G0/G1 phase (Journal of cancer prevention, 2018; 63-69).
  • MAPK/Akt/mTOR pathway to induce liver cancer SK-HEP-1 cells to arrest in the G0/G1 phase
  • Nuclear factor E2-related factor 2 (Nuclear factor E2-related factor 2, Nrf2) is an endogenous regulator of antioxidant stress, regulated by cytoplasmic adapter protein (Kelch-like ECH-associated protein 1, Keap1 ) negative regulation. Keap1 is able to accelerate the ubiquitination of lysine residues in the Nrf2Neh2 domain, leading to Nrf2 degradation. Nrf2 is normally anchored in the cytoplasm by Keap1 and regulated by the Keap1-Cul3E3 ubiquitin ligase.
  • Keap1 When oxidative stress and electrophile stimulation occur, the conformation of Keap1 changes, Nrf2 dissociates from Keap1 and enters the nucleus to bind with the Antioxidant response element (ARE), activating the downstream related transcription factor heme oxidative synthase (HMOX) -1), ferritin heavy chain polypeptide (FTH1), NADPH quinone oxidoreductase 1 (NQO1), glutamic acid cysteine ligase (GCLM) and other expressions, so as to resist the damage of oxidative stress to the body (Free Radic Biol Med, 2014; 36-44).
  • ARE Antioxidant response element
  • HMOX downstream related transcription factor heme oxidative synthase
  • FTH1 ferritin heavy chain polypeptide
  • NQO1 NADPH quinone oxidoreductase 1
  • GCLM glutamic acid cysteine ligase
  • p62 also known as SQSTM1
  • SQSTM1 is a ubiquitin-binding protein involved in cell signaling, oxidative stress, and autophagy.
  • p62 can bind to the DC pocket of Keap1 to inhibit its binding to Nrf2, block the regulation of Nrf2 by Keap1, further inhibit the ubiquitination of Nrf2, stabilize Nrf2 and promote the downstream genes. Expression (Redox Biol, 2018; 246-258). This pathway can lead to the abnormal accumulation of Nrf2 in tumor cells, thereby obtaining protection against apoptosis, promoting tumorigenesis, and making tumor cells resistant to chemotherapy (Curr Cancer Drug Targets, 2019; 468-478).
  • Nrf2 is one of the effective ways to treat ovarian cancer and its drug resistance.
  • the small molecule inhibitor ML385 specifically binds to Neh1, the DNA-binding site of Nrf2 in lung cancer cells, interferes with the binding of MAFG-Nrf2 protein complexes to DNA, thereby inhibiting the expression of downstream genes, and is a selection for Keap1-mutated non-small cell lung cancer Stronger and more specific (ACS Chem Biol, 2016; 3214-3225).
  • DHT as an Nrf2 inhibitor, there is no corresponding technical plan for the treatment of ovarian cancer with abnormally activated Nrf2.
  • the object of the present invention is to provide a new application of DHT, that is, the application for preparing Nrf2 inhibitors and anticancer drugs related to Nrf2 inhibition, so as to solve one or more of the above problems.
  • DHT in the preparation of Nrf2 inhibitors
  • structural formula of DHT is as follows:
  • the medicament consists of DHT and a pharmaceutically acceptable excipient.
  • the drug is administered by intraperitoneal injection.
  • the disease is cancer.
  • the cancer is lung cancer, ovarian cancer, and the like.
  • DHT can down-regulate the expression level of Nrf2 protein and reduce the anti-oxidative stress ability of human ovarian cancer cells HO8910PM and SKOV3.
  • DHT and cisplatin for preparing anticancer drugs is provided.
  • the medicament consists of the DHT and cisplatin and pharmaceutically acceptable excipients.
  • the combination of DHT and cisplatin is administered by intraperitoneal injection.
  • anti-cancer specifically refers to anti-ovarian cancer.
  • the present invention is based on the traditional Chinese medicine Salvia miltiorrhiza, from which DHT is separated, extracted, purified and researched. It is found through experiments that the inhibitory effect of DHT alone on human ovarian cancer cell lines HO8910PM and SKOV3 subcutaneous transplanted tumors is equivalent to that of DDP, and comparable to that of Shun
  • the combined use of platinum has a stronger effect on inhibiting the growth of ovarian cancer cells, thus providing a new use of DHT, which can down-regulate the expression level of Nrf2 protein, reduce the anti-oxidative stress ability of human ovarian cancer cells HO8910PM and SKOV3, and improve the effect of chemotherapy drugs.
  • the proliferation inhibitory effect of cisplatin on HO8910PM and SKOV3 can be used as an Nrf2 inhibitory drug, and as an anti-cancer drug, especially an anti-ovarian cancer drug; when DHT is used as a drug, animal experiments have shown that DHT has an important effect on the main organs (heart) of animals. , liver, spleen, lung, kidney) without obvious toxicity.
  • the present invention verifies the inhibitory effect of DHT on Nrf2, and proves that DHT can play an anti-ovarian cancer effect by inhibiting NRF2.
  • the object of the present invention is to provide an Nrf2 inhibitor and a drug for clinically treating ovarian cancer, providing a new application of DHT.
  • the present invention proves that DHT can be used as an inhibitor of Nrf2 protein, and plays an anti-cancer effect by inhibiting Nrf2 protein, providing a new idea for the treatment of cancer.
  • the DHT in the present invention has low toxicity, high efficiency and abundant sources, which can greatly reduce the toxic and side effects of drugs on the human body in the process of cancer treatment, and significantly reduce the treatment cost.
  • Figure 1 is a structural diagram of DHT, CT, TAN I and TAN II A;
  • Figure 2 is a graph showing the effects of different concentrations of DHT, CT, TAN I and TAN II A on the cell viability of ovarian cancer cells HO8910PM, SKOV3 and A2780 for 24 hours and 48 hours;
  • Fig. 3 is a diagram showing the effect of different concentrations of cisplatin, doxorubicin and 5-fluorouracil on the viability of ovarian cancer cells HO8910PM, SKOV3 and A2780 after 24 hours of treatment.
  • Figure 4 is a schematic diagram of the cell morphology changes of HO8910PM (top), SKOV3 (middle) and A2780 (bottom) before and after DHT intervention;
  • Figure 5 is the results and statistics of HO8910PM cell apoptosis after administration of different concentrations (A: 0 ⁇ M, D: 1.6 ⁇ M, E: 3.2 ⁇ M, F: 6.4 ⁇ M) of DHT and DDP (B: 20.0 ⁇ M) (C) ;
  • Figure 6 is the results and statistics of SKOV3 cell apoptosis after administration of different concentrations (A: 0 ⁇ M, D: 4.0 ⁇ M, E: 8.0 ⁇ M, F: 16.0 ⁇ M) of DHT and DDP (B: 20.0 ⁇ M) (C) ;
  • Fig. 7 is different concentrations (A: 0 ⁇ M, B: 1.6 ⁇ M, C: 3.2 ⁇ M, D: 6.4 ⁇ M) after DHT administration, HO8910PM cell S, G1, G2 phase result chart and statistical chart (E); Show that S, G1 Phase cell arrest;
  • Fig. 8 is different concentration (A: 0 ⁇ M, B: 4.0 ⁇ M, C: 8.0 ⁇ M, D: 16.0 ⁇ M) SKOV3 cell S, G1, G2 phase result chart and statistical chart (E) after DHT administration; Show that S, G1 Phase cell arrest;
  • Figure 9 is the result graph (A, C) and histogram (B, D) of the increase of ROS levels in HO8910PM and SKOV3 cells caused by administration of different concentrations of DHT;
  • Fig. 10 is the graph and statistical graph (E) of mitochondrial depolarization of HO8910PM cells induced by different concentrations (A: 0 ⁇ M, B: 1.6 ⁇ M, C: 3.2 ⁇ M, D: 6.4 ⁇ M) of DHT;
  • Fig. 11 is the graph and statistical graph (E) of mitochondrial depolarization of SKOV3 cells induced by different concentrations (A: 0 ⁇ M, B: 4.0 ⁇ M, C: 8.0 ⁇ M, D: 16.0 ⁇ M) of DHT;
  • Figure 12 is the effect of DHT combined with ROS inhibitor NAC on the viability of HO8910PM and SKOV3 cells after administration of different treatment groups (A), and the statistics of ROS expression levels (B, C);
  • Figure 13 is the results of mitochondrial depolarization of HO8910PM cells and statistical charts (E) after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT);
  • Figure 14 is the results and statistics of mitochondrial depolarization in SKOV3 cells after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT) (E) ;
  • Figure 15 is the results and statistics of HO8910PM cell apoptosis after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT);
  • Figure 16 is the results and statistics of SKOV3 cell apoptosis after administration of different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT) combined with ROS inhibitor NAC (E);
  • Figure 17 is the results and statistics of HO8910PM cells in S, G1, and G2 phases after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT) (E ); indicating cell arrest in S and G1 phases;
  • Figure 18 is the results and statistics of SKOV3 cells in S, G1, and G2 phases after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT) (E ); indicating cell arrest in S and G1 phases;
  • Figure 19 is the result of expression of related proteins in HO8910PM cells after administration of different concentrations of DHT (A), the expression statistics of apoptosis-related proteins Bcl-2, Bax, Caspase-3 and cycle proteins CyclinB1 and Cdc2 (B);
  • Figure 20 is the result of expression of related proteins in SKOV3 cells after administration of different concentrations of DHT (A), the expression statistics of apoptosis-related proteins Bcl-2, Bax, Caspase-3 and cycle proteins CyclinB1 and Cdc2 (B);
  • Figure 21 is the results of p62/Keap1/Nrf2 protein expression in HO8910PM cells after administration of different concentrations of DHT (A) statistical chart (B);
  • Fig. 22 is a graph (A) statistical graph (B) of p62/Keap1/Nrf2 protein expression results in SKOV3 cells after administration of different concentrations of DHT;
  • Figure 23 is a graph showing the mRNA level of Nrf2 protein in HO8910PM (A), SKOV3 (B) cells after DHT administration;
  • Figure 24 is the results of HO8910PM, SKOV3Nrf2 protein expression results (A) and statistical charts (B, C) after administration of different treatment groups of DHT combined with proteasome inhibitor MG132;
  • Figure 25 is the results of HO8910PM, SKOV3Nrf2 protein expression results (A) and statistical charts (B, C) after administration of different treatment groups of DHT combined with Nrf2 inhibitor tBHQ;
  • Fig. 26 is the body weight (A), tumor volume (B) of mice in different treatment groups changing with time result figure, isolated tumor photo (C), isolated tumor volume statistical figure (D), serum tumor marker CA125 (E ), HE4(F) horizontal result graph;
  • Fig. 27 is HE pathological slices (200 ⁇ ) (A) and visceral index (B) of hearts, livers, spleens, lungs, and kidneys of mice in different treatment groups.
  • Main reagent materials RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin double antibody, phosphate buffer (Gibco); tanshinone II A, MTT powder, dimethyl sulfoxide solution, formaldehyde (Sigma); Giemsa staining solution (Shanghai Merck Chemical Technology Co., Ltd.); Apoptosis detection kit, cell cycle detection kit (Becton Dickinson and Company); RIPA protein lysate (ThermoFisher Scientific company); protease inhibitor, phosphatase inhibition Reagent (Bimake Company); SDS-PAGE gel, ECL chemiluminescence solution (Shanghai Yazyme Biotechnology Co., Ltd.); Western blot antibody (Abcam Company).
  • mice Human ovarian cancer cell lines HO8910PM, SKOV3 and A2780 were purchased from Wuhan Punuosai Biotechnology Co., Ltd. and frozen in the cell storage liquid nitrogen tank in our laboratory. The mice required for the experiment were purchased from the Shanghai Slack Animal Experiment Center, SPF grade healthy and mature 6-week-old female NOD/SCID mice.
  • MTT Tetramethylazolazolium blue, the full name is 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide, which is a kind of reagent that can indirectly detect the number of living cells yellow dye.
  • concentration of MTT used in this cell viability detection experiment is 5 mg/mL: Weigh 0.1 g of MTT and dissolve it in 20 mL of sterile phosphate buffered saline (PBS), filter and sterilize with a 0.22 ⁇ m filter membrane and a sterile syringe, and dispense to 1 mL Store in a sterile brown centrifuge tube at 4°C in the dark for later use.
  • PBS sterile phosphate buffered saline
  • Cell counting First, gently blow the cell suspension evenly with a pipette gun, and then perform trypan blue cell counting. Mix trypan blue and cell suspension 1:1, pipette 20 ⁇ L of the mixture onto a cell counting plate, and then use a cell counter to count the concentration of cells per ml.
  • DHT dihydrotanshinone
  • CT cryptotanshinone
  • TAN I tanshinone I
  • TAN II A tanshinone II A
  • IC50 of comparative chemotherapy drugs cisplatin (DDP): 17.80 ⁇ M, 115.57 ⁇ M, 519.5 ⁇ M; doxorubicin (DOX): 2.29 ⁇ M, 46.29 ⁇ M, 4.4 ⁇ M; 5-fluorouracil (5-FU): 395.29 ⁇ M, - -, 985.86 ⁇ M) ( Figure 3), indicating that the inhibitory effect of DHT on ovarian cancer cells is equivalent to that of doxorubicin, and better than that of cisplatin and 5-fluorouracil.
  • Cell treatment collect HO8910PM and SKOV3 cells in good growth state, count the cells, adjust the cell concentration, take 2 ⁇ 10 5 /mL cells and plant them in a six-well plate, add different concentrations of DHT and 20.0 ⁇ M DDP to treat for 24 hours .
  • Flow cytometry detection Filter the treated cell suspension into a flow tube with a 200-mesh filter, and finally analyze the distribution of the cell cycle by flow cytometry within 1 hour.
  • the washed cells were resuspended by adding 500 ⁇ L of DCFH-DA probe with a final concentration of 10 ⁇ M, and then placed in a constant temperature incubator at 37°C in the dark for 20 minutes to detect the level of intracellular reactive oxygen species by flow cytometry.
  • ROS reactive oxygen species
  • Flow cytometry was used to analyze the intracellular reactive oxygen species levels in different groups. As shown in Fig. 12B-D, ROS increased, mitochondrial membrane potential decreased, apoptosis and cycle arrest (Figure 12-18). It was further confirmed that DHT induces apoptosis and cycle arrest in ovarian cancer cells by activating oxidative stress.
  • FIG. 21-22 shows the results of Keap1/Nrf2 protein expression analysis.
  • the Keap1/Nrf2 signaling pathway is an important oxidative stress regulation system in cells.
  • Keap1 negatively regulates Nrf2 expression through ubiquitination and degradation.
  • the results in the figure show that DHT increases the expression of Keap1, down-regulates the expression of Nrf2, and inhibits the entry of Nrf2 into the nucleus.
  • qRT-PCR analysis was used to analyze the mRNA level of Nrf2 protein. qRT-PCR analysis found that DHT reduced the target genes of Nrf2 such as HMOX1, GCLM, and FTH1 at the mRNA level, and increased KEAP1 and NFE2L2 in a dose-dependent manner. Transcript levels ( Figures 23A and 23B). However, DHT inhibits the expression of Nrf2 at the protein level, indicating that DHT regulates the expression of Nrf2 through the post-transcriptional level.
  • DHT combined with proteasome inhibitor MG132 treatment analysis: the control group, MG132 group (10 ⁇ M), DHT group (6.4 ⁇ M), MG132 (10 ⁇ M) + DHT group (6.4 ⁇ M) were set up to treat human ovarian cancer cells HO8910PM, SKOV3 After 24 hours, Western blot analysis was used to test the expression of Nrf2 protein in different groups of ovarian cancer cells. Further analysis found that the co-incubation of DHT and MG132 could reverse the inhibitory effect of DHT on Nrf2 ( Figure 25), indicating that DHT inhibited Nrf2 by promoting the degradation of Nrf2 protein. its expression.
  • mice Balb/c female mice, 4-6 weeks old, with an initial body weight between 18 grams and 22 grams. Collect HO8910PM cells in good growth state, adjust the cell concentration after counting, take HO8910PPM cells (1 ⁇ 107 cells/100 ⁇ L), use a 1mL sterile syringe to subcutaneously inoculate the cell suspension into Balb/c small The left side of the mouse armpit.
  • mice were treated in groups: 50vol% PEG 400 + 35vol% DMSO + 15vol% physiological saline were used, mixed evenly as a solvent for dissolving DHT.
  • mice were intraperitoneally injected about 100 ⁇ L of 50vol% PEG 400+35vol% DMSO+15vol% normal saline (blank control group TC), 10 mg/kg/d of DHT (DHT low-dose group DHT- L), DHT at 20 mg/kg/d (DHT-H in DHT high-dose group), cisplatin injection at 2 mg/kg/d (DDP in cisplatin group), once every other day, for a total of 14 days.
  • blade control group TC 10 mg/kg/d of DHT
  • DHT- L DHT at 20 mg/kg/d
  • DHT-H in DHT high-dose group DHT-H in DHT high-dose group
  • cisplatin injection at 2 mg/kg/d (DDP in cisplatin group), once every other day, for a total of 14 days.
  • the levels of HE4 and CA125 in mouse serum were detected by ELISA, and the extraction method was as follows: (1) Coating detection plate: Dilute the antigen used to 1 ⁇ g/ ⁇ L with coating diluent, add 100 ⁇ L of antigen to each well, 37 After incubation at °C for 4 hours, the liquid in the wells was discarded.
  • enzyme-labeled antibody according to the reference working dilution provided by the enzyme conjugate provider: add 100 ⁇ L of enzyme-labeled antibody to each well, incubate at 37°C for 60 minutes, wash with PBS 3 times after incubation, 3 minutes each time.
  • Termination reaction 50 ⁇ L of stop solution was added to each well to terminate the reaction, and the experimental results were measured at a wavelength of 450 nm using an enzyme-linked immunosorbent reaction detector within 20 minutes.
  • DHT can reduce the weight of isolated tumors ( Figure 26C and 26D) and the levels of serum tumor markers CA125 and HE4 (Figure 26E and 26F). died (Fig. 26G).
  • DHT administration had no significant effect on the pathology of major organs (Fig. 27A) and organ index (Fig. 27B) in mice, indicating that DHT treatment can delay the growth of tumors in mice, and has Without potential toxic effects, DHT is relatively safe for mice in vivo.

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Abstract

Disclosed in the present invention is the use of dihydrotanshinone I in the preparation of an Nrf2 inhibitor. Provided in the present invention is the new use of dihydrotanshinone, wherein dihydrotanshinone can down-regulate the protein expression level of Nrf2 (nuclear factor E2-related factor 2), and reduce the oxidative stress resistance of human ovarian cancer cells. Therefore, dihydrotanshinone I can be used as a drug for inhibiting Nrf2, and can be used for treating ovarian cancer and used as an active ingredient of an anti-ovarian cancer drug. When dihydrotanshinone I is used as a drug, animal experiments show that dihydrotanshinone I can significantly inhibit the occurrence and development of tumors and has no effect on major organs such as the liver and the kidneys of an animal.

Description

二氢丹参酮Ⅰ在制备Nrf2抑制剂中的应用Application of Dihydrotanshinone Ⅰ in the Preparation of Nrf2 Inhibitors 技术领域technical field
本发明属于医药技术领域，具体涉及二氢丹参酮Ⅰ抑制Nrf2活性的功能，同时也与二氢丹参酮Ⅰ应用于治疗卵巢癌和抗卵巢癌药物的活性成分有关。The invention belongs to the technical field of medicine, specifically relates to the function of dihydrotanshinone I in inhibiting Nrf2 activity, and is also related to the application of dihydrotanshinone I in the treatment of ovarian cancer and the active ingredients of anti-ovarian cancer drugs.
背景技术Background technique
丹参为双子叶唇形科鼠尾草属植物，来源历史悠久，最先记载于《神农百草经》，药用部位为干燥根及根茎。丹参酮类化合物因在丹参中含量较高，且具有显著的抗肿瘤作用而得到广泛的研究。丹参酮IIA(Tanshinone IIA，TAN IIA)，丹参酮I(Tanshinone I，TAN I)，隐丹参酮(Cryptotanshinone，CT)和二氢丹参酮Ⅰ(15,16-Dihydrotanshinone I，DHT)是从丹参中提取的脂溶性菲醌类有效成分。其中，DHT可以通过抑制肿瘤细胞增殖、促进凋亡以及阻滞细胞周期从而发挥抗肿瘤作用。DHT能够通过Caspase途径诱导人骨肉瘤143B细胞的凋亡，下调黏附分子VCAM-1和ICAM-1来减弱骨肉瘤细胞的迁移(Anti-Cancer Agent Me,2017；1234-1242)。DHT能够激活JNK/p38MAPK信号通路诱导胃癌细胞SGC7901和MGC803凋亡(Pharm Biol,2016；3019-3025)。DHT还能够通过ROS/Stat3信号途径，诱导活性氧的生产，抑制乳腺癌干细胞的生长(Oxid Med Cell Longev,2019)。此外，DHT能够调控MAPK/Akt/mTOR通路诱导肝癌SK-HEP-1细胞在G0/G1期发生阻滞(Journal of cancer prevention,2018；63-69)。然而，目前尚无DHT抗卵巢癌的体内药效研究报道，同时，DHT的抗卵巢癌机制尚未明确。Salvia miltiorrhiza is a plant of the genus Salvia in the dicotyledonous Lamiaceae. It has a long history and was first recorded in "Shen Nong Baicao Jing". The medicinal parts are dry roots and rhizomes. Tanshinone compounds have been widely studied because of their high content in Danshen and their significant antitumor effects. Tanshinone IIA (Tanshinone IIA, TAN IIA), Tanshinone I (Tanshinone I, TAN I), Cryptotanshinone (Cryptotanshinone, CT) and dihydrotanshinone I (15,16-Dihydrotanshinone I, DHT) are fat-soluble Active ingredients of phenanthrene quinones. Among them, DHT can play an anti-tumor effect by inhibiting tumor cell proliferation, promoting apoptosis and blocking cell cycle. DHT can induce the apoptosis of human osteosarcoma 143B cells through the Caspase pathway, and down-regulate the adhesion molecules VCAM-1 and ICAM-1 to weaken the migration of osteosarcoma cells (Anti-Cancer Agent Me, 2017; 1234-1242). DHT can activate JNK/p38MAPK signaling pathway to induce apoptosis of gastric cancer cells SGC7901 and MGC803 (Pharm Biol, 2016; 3019-3025). DHT can also induce the production of reactive oxygen species and inhibit the growth of breast cancer stem cells through the ROS/Stat3 signaling pathway (Oxid Med Cell Longev, 2019). In addition, DHT can regulate the MAPK/Akt/mTOR pathway to induce liver cancer SK-HEP-1 cells to arrest in the G0/G1 phase (Journal of cancer prevention, 2018; 63-69). However, there is no report on the in vivo efficacy of DHT against ovarian cancer, and the mechanism of DHT's resistance to ovarian cancer has not yet been clarified.
核转录因子NF-E2相关因子2(Nuclear factor E2-related factor 2，Nrf2)是一种内源性的抗氧化应激调节因子，受胞质接头蛋白(Kelch-like ECH-associated protein 1，Keap1)负调控。Keap1能够加速Nrf2Neh2结构域中赖氨 酸残基的泛素化，导致Nrf2降解。正常情况下Nrf2被Keap1锚定在细胞质中，并受Keap1-Cul3E3泛素连接酶调控。当产生氧化应激和亲电子试剂刺激时，Keap1构象发生改变，Nrf2与Keap1解离并进入细胞核与抗氧化反应元件(Antioxidant response element，ARE)结合，激活下游相关转录因子血红素氧化合酶(HMOX-1)、铁蛋白重链多肽(FTH1)、NADPH醌氧化还原酶1(NQO1)、谷氨酸半胱氨酸连接酶(GCLM)等表达，从而抵抗氧化应激对机体的损伤(Free Radic Biol Med,2014；36-44)。同时，有研究表明，与Keap1相互作用蛋白的积累，可阻止Nrf2与Keap1结合，导致细胞内Nrf2的积累。p62，也称作SQSTM1，是一种与细胞信号转导、氧化应激和自噬有关的泛素结合蛋白。p62作为Keap1-Nrf2的一个重要调控因子，能够与Keap1的DC口袋结合从而抑制其对Nrf2的结合，阻断Keap1对Nrf2的调控，进一步抑制Nrf2的泛素化，使Nrf2稳定并促进下游基因的表达(Redox Biol,2018；246-258)。该途径可导致肿瘤细胞内Nrf2的异常积累，由此获得对凋亡的保护，促进肿瘤的发生，使肿瘤细胞对化疗产生一定的耐药性(Curr Cancer Drug Targets,2019；468-478)。Nuclear factor E2-related factor 2 (Nuclear factor E2-related factor 2, Nrf2) is an endogenous regulator of antioxidant stress, regulated by cytoplasmic adapter protein (Kelch-like ECH-associated protein 1, Keap1 ) negative regulation. Keap1 is able to accelerate the ubiquitination of lysine residues in the Nrf2Neh2 domain, leading to Nrf2 degradation. Nrf2 is normally anchored in the cytoplasm by Keap1 and regulated by the Keap1-Cul3E3 ubiquitin ligase. When oxidative stress and electrophile stimulation occur, the conformation of Keap1 changes, Nrf2 dissociates from Keap1 and enters the nucleus to bind with the Antioxidant response element (ARE), activating the downstream related transcription factor heme oxidative synthase (HMOX) -1), ferritin heavy chain polypeptide (FTH1), NADPH quinone oxidoreductase 1 (NQO1), glutamic acid cysteine ligase (GCLM) and other expressions, so as to resist the damage of oxidative stress to the body (Free Radic Biol Med, 2014; 36-44). At the same time, studies have shown that the accumulation of proteins interacting with Keap1 can prevent the combination of Nrf2 and Keap1, resulting in the accumulation of Nrf2 in cells. p62, also known as SQSTM1, is a ubiquitin-binding protein involved in cell signaling, oxidative stress, and autophagy. As an important regulator of Keap1-Nrf2, p62 can bind to the DC pocket of Keap1 to inhibit its binding to Nrf2, block the regulation of Nrf2 by Keap1, further inhibit the ubiquitination of Nrf2, stabilize Nrf2 and promote the downstream genes. Expression (Redox Biol, 2018; 246-258). This pathway can lead to the abnormal accumulation of Nrf2 in tumor cells, thereby obtaining protection against apoptosis, promoting tumorigenesis, and making tumor cells resistant to chemotherapy (Curr Cancer Drug Targets, 2019; 468-478).
现有研究表明，在各种人类癌症中，p62和Keap1/Nrf2系统经常失调，能够导致Nrf2的异常激活(Pharmacol Rep,2017；393-402)。肿瘤中Nrf2的积累与患者预后不良密切相关，Nrf2高表达卵巢癌患者无病生存期(HR：2.084；95％CI：1.229-3.536)和总生存期(HR：2.487；95％CI：1.443-4.286)降低(Cancer Cell Int,2021；116)。因此，靶向Nrf2是治疗卵巢癌及其耐药性的有效途径之一。有报道称，植物鸦胆子的活性成分鸦胆苦醇可作为Nrf2抑制剂，通过增强Nrf2泛素化选择性降低其蛋白表达，抑制肺癌荷瘤小鼠的肿瘤生长(Proc Natl Acad Sci,2011；1433-1438)。小分子抑制剂ML385特异性与肺癌细胞中Nrf2的DNA结合位点Neh1结合，干扰MAFG-Nrf2蛋白复合物与DNA的结合，从而抑制下游基因的表达，并且对Keap1突变的非小细胞肺癌的选择性和 特异性更强(ACS Chem Biol,2016；3214-3225)。然而基于Nrf2抑制对卵巢癌的治疗作用鲜有报道。基于DHT作为Nrf2抑制剂，针对Nrf2异常激活的卵巢癌进行治疗并未有相应技术方案的提出。Existing studies have shown that in various human cancers, the p62 and Keap1/Nrf2 systems are often dysregulated, which can lead to abnormal activation of Nrf2 (Pharmacol Rep, 2017; 393-402). The accumulation of Nrf2 in the tumor is closely related to the poor prognosis of patients. The disease-free survival (HR: 2.084; 95% CI: 1.229-3.536) and overall survival (HR: 2.487; 95% CI: 1.443- 4.286) reduction (Cancer Cell Int, 2021; 116). Therefore, targeting Nrf2 is one of the effective ways to treat ovarian cancer and its drug resistance. It has been reported that the active ingredient Brucea javanica can act as an Nrf2 inhibitor, selectively reduce its protein expression by enhancing Nrf2 ubiquitination, and inhibit tumor growth in lung cancer tumor-bearing mice (Proc Natl Acad Sci, 2011; 1433-1438). The small molecule inhibitor ML385 specifically binds to Neh1, the DNA-binding site of Nrf2 in lung cancer cells, interferes with the binding of MAFG-Nrf2 protein complexes to DNA, thereby inhibiting the expression of downstream genes, and is a selection for Keap1-mutated non-small cell lung cancer Stronger and more specific (ACS Chem Biol, 2016; 3214-3225). However, there are few reports on the therapeutic effect of Nrf2 inhibition on ovarian cancer. Based on DHT as an Nrf2 inhibitor, there is no corresponding technical plan for the treatment of ovarian cancer with abnormally activated Nrf2.
发明内容Contents of the invention
本发明的目的在于提供DHT的一种新用途，即：用于制备Nrf2抑制剂、与Nrf2抑制有关的抗癌药物的应用，以解决上述问题中的一个或几个。The object of the present invention is to provide a new application of DHT, that is, the application for preparing Nrf2 inhibitors and anticancer drugs related to Nrf2 inhibition, so as to solve one or more of the above problems.
根据本发明的一个方面，提供了DHT在制备Nrf2抑制剂中的应用，其中，DHT结构式如下：According to one aspect of the present invention, the application of DHT in the preparation of Nrf2 inhibitors is provided, wherein the structural formula of DHT is as follows:
Figure PCTCN2022113764-appb-000001
Figure PCTCN2022113764-appb-000001
实验表明，不同浓度的DHT均能够抑制卵巢癌细胞中Nrf2的活性，且抑制能力随DHT剂量的增加而上升。Experiments have shown that different concentrations of DHT can inhibit the activity of Nrf2 in ovarian cancer cells, and the inhibitory ability increases with the increase of DHT dose.
根据本发明的另一个方面，提供了DHT在制备与Nrf2抑制有关的疾病的治疗药物中的应用。According to another aspect of the present invention, there is provided the use of DHT in the preparation of therapeutic drugs for diseases related to Nrf2 inhibition.
在一些实施方式中，药物由DHT和药学上可接受的赋形剂组成。In some embodiments, the medicament consists of DHT and a pharmaceutically acceptable excipient.
在一些实施方式中，药物的给药方式为腹腔注射。In some embodiments, the drug is administered by intraperitoneal injection.
在一些实施方式中，疾病为癌症。具体地，癌症为肺癌、卵巢癌等。实验表明，DHT可下调Nrf2蛋白表达水平，降低人卵巢癌细胞HO8910PM和SKOV3的抗氧化应激能力。In some embodiments, the disease is cancer. Specifically, the cancer is lung cancer, ovarian cancer, and the like. Experiments have shown that DHT can down-regulate the expression level of Nrf2 protein and reduce the anti-oxidative stress ability of human ovarian cancer cells HO8910PM and SKOV3.
根据本发明的另一个方面，提供了DHT和顺铂用于制备抗癌药物的用途。According to another aspect of the present invention, the use of DHT and cisplatin for preparing anticancer drugs is provided.
在一些实施方式中，药物由所述DHT和顺铂以及药学上可接受的赋形剂 组成。In some embodiments, the medicament consists of the DHT and cisplatin and pharmaceutically acceptable excipients.
在一些实施方式中，DHT和顺铂药物组合的给药方式为腹腔注射。In some embodiments, the combination of DHT and cisplatin is administered by intraperitoneal injection.
在一些实施方式中，抗癌具体指抗卵巢癌。In some embodiments, anti-cancer specifically refers to anti-ovarian cancer.
本发明是基于中药丹参，从中分离、提取、纯化得到DHT并对其进行研究而完成，通过试验发现DHT单独使用对人卵巢癌细胞株HO8910PM和SKOV3皮下移植瘤的抑制作用与DDP相当，与顺铂联合使用后抑制卵巢癌细胞生长的效果更强，因而提供了DHT的一种新用途，其可下调Nrf2蛋白表达水平，降低人卵巢癌细胞HO8910PM和SKOV3的抗氧化应激能力，提高化疗药物顺铂对HO8910PM和SKOV3的增殖抑制作用，可作为Nrf2抑制药物使用，以及作为抗癌尤其是抗卵巢癌药物使用；当DHT作为药物使用时，动物实验表明，DHT对动物的主要脏器(心、肝、脾、肺、肾)无明显毒性。The present invention is based on the traditional Chinese medicine Salvia miltiorrhiza, from which DHT is separated, extracted, purified and researched. It is found through experiments that the inhibitory effect of DHT alone on human ovarian cancer cell lines HO8910PM and SKOV3 subcutaneous transplanted tumors is equivalent to that of DDP, and comparable to that of Shun The combined use of platinum has a stronger effect on inhibiting the growth of ovarian cancer cells, thus providing a new use of DHT, which can down-regulate the expression level of Nrf2 protein, reduce the anti-oxidative stress ability of human ovarian cancer cells HO8910PM and SKOV3, and improve the effect of chemotherapy drugs. The proliferation inhibitory effect of cisplatin on HO8910PM and SKOV3 can be used as an Nrf2 inhibitory drug, and as an anti-cancer drug, especially an anti-ovarian cancer drug; when DHT is used as a drug, animal experiments have shown that DHT has an important effect on the main organs (heart) of animals. , liver, spleen, lung, kidney) without obvious toxicity.
本发明是验证了DHT对于Nrf2的抑制作用，并证实DHT能够通过抑制NRF2起到抗卵巢癌的作用。本发明的目的在于提供一种Nrf2抑制剂，以及临床上治疗卵巢癌的药物，提供了DHT的新用途。The present invention verifies the inhibitory effect of DHT on Nrf2, and proves that DHT can play an anti-ovarian cancer effect by inhibiting NRF2. The object of the present invention is to provide an Nrf2 inhibitor and a drug for clinically treating ovarian cancer, providing a new application of DHT.
本发明的有益效果是：The beneficial effects of the present invention are:
(1)本发明证实了DHT可用于Nrf2蛋白的抑制剂，并且通过抑制Nrf2蛋白起到抗癌作用，为癌症的治疗提供了一种新的思路。(1) The present invention proves that DHT can be used as an inhibitor of Nrf2 protein, and plays an anti-cancer effect by inhibiting Nrf2 protein, providing a new idea for the treatment of cancer.
(2)本发明中的DHT低毒高效且来源丰富，能够大大减少癌治疗过程中药物对人体的毒副作用，并显著降低治疗成本。(2) The DHT in the present invention has low toxicity, high efficiency and abundant sources, which can greatly reduce the toxic and side effects of drugs on the human body in the process of cancer treatment, and significantly reduce the treatment cost.
附图说明Description of drawings
图1是DHT、CT、TAN Ⅰ和TAN Ⅱ A结构图；Figure 1 is a structural diagram of DHT, CT, TAN Ⅰ and TAN Ⅱ A;
图2是不同浓度DHT、CT、TAN Ⅰ和TAN Ⅱ A处理卵巢癌细胞HO8910PM、SKOV3和A2780 24h和48h对细胞活力的影响结果图；Figure 2 is a graph showing the effects of different concentrations of DHT, CT, TAN Ⅰ and TAN Ⅱ A on the cell viability of ovarian cancer cells HO8910PM, SKOV3 and A2780 for 24 hours and 48 hours;
图3是不同浓度顺铂、阿霉素和5-氟尿嘧啶对卵巢癌细胞HO8910PM、 SKOV3和A2780给药处理24h后对细胞活力的影响结果图。Fig. 3 is a diagram showing the effect of different concentrations of cisplatin, doxorubicin and 5-fluorouracil on the viability of ovarian cancer cells HO8910PM, SKOV3 and A2780 after 24 hours of treatment.
图4是DHT干预前后HO8910PM(上)、SKOV3(中)和A2780(下)的细胞形态变化示意图；Figure 4 is a schematic diagram of the cell morphology changes of HO8910PM (top), SKOV3 (middle) and A2780 (bottom) before and after DHT intervention;
图5是不同浓度(A:0μM、D:1.6μM、E:3.2μM、F:6.4μM)DHT和DDP(B:20.0μM)给药后引起HO8910PM细胞凋亡结果图和统计图(C)；Figure 5 is the results and statistics of HO8910PM cell apoptosis after administration of different concentrations (A: 0 μM, D: 1.6 μM, E: 3.2 μM, F: 6.4 μM) of DHT and DDP (B: 20.0 μM) (C) ;
图6是不同浓度(A:0μM、D:4.0μM、E:8.0μM、F:16.0μM)DHT和DDP(B:20.0μM)给药后引起SKOV3细胞凋亡结果图和统计图(C)；Figure 6 is the results and statistics of SKOV3 cell apoptosis after administration of different concentrations (A: 0 μM, D: 4.0 μM, E: 8.0 μM, F: 16.0 μM) of DHT and DDP (B: 20.0 μM) (C) ;
图7是不同浓度(A:0μM、B:1.6μM、C:3.2μM、D:6.4μM)DHT给药后HO8910PM细胞S、G1、G2期结果图和统计图(E)；表明S、G1期细胞阻滞；Fig. 7 is different concentrations (A: 0 μ M, B: 1.6 μ M, C: 3.2 μ M, D: 6.4 μ M) after DHT administration, HO8910PM cell S, G1, G2 phase result chart and statistical chart (E); Show that S, G1 Phase cell arrest;
图8是不同浓度(A:0μM、B:4.0μM、C:8.0μM、D:16.0μM)DHT给药后SKOV3细胞S、G1、G2期结果图和统计图(E)；表明S、G1期细胞阻滞；Fig. 8 is different concentration (A: 0 μ M, B: 4.0 μ M, C: 8.0 μ M, D: 16.0 μ M) SKOV3 cell S, G1, G2 phase result chart and statistical chart (E) after DHT administration; Show that S, G1 Phase cell arrest;
图9是不同浓度DHT给药后引起HO8910PM、SKOV3细胞内ROS水平升高结果图(A、C)和柱状图(B、D)；Figure 9 is the result graph (A, C) and histogram (B, D) of the increase of ROS levels in HO8910PM and SKOV3 cells caused by administration of different concentrations of DHT;
图10是不同浓度(A:0μM、B:1.6μM、C:3.2μM、D:6.4μM)DHT给药后引起HO8910PM细胞线粒体去极化结果图和统计图(E)；Fig. 10 is the graph and statistical graph (E) of mitochondrial depolarization of HO8910PM cells induced by different concentrations (A: 0 μM, B: 1.6 μM, C: 3.2 μM, D: 6.4 μM) of DHT;
图11是不同浓度(A:0μM、B:4.0μM、C:8.0μM、D:16.0μM)DHT给药后引起SKOV3细胞线粒体去极化结果图和统计图(E)；Fig. 11 is the graph and statistical graph (E) of mitochondrial depolarization of SKOV3 cells induced by different concentrations (A: 0 μM, B: 4.0 μM, C: 8.0 μM, D: 16.0 μM) of DHT;
图12是DHT联合ROS抑制剂NAC不同处理组给药后HO8910PM、SKOV3细胞活力的影响结果图(A)、ROS表达水平统计图(B、C)；Figure 12 is the effect of DHT combined with ROS inhibitor NAC on the viability of HO8910PM and SKOV3 cells after administration of different treatment groups (A), and the statistics of ROS expression levels (B, C);
图13是DHT联合ROS抑制剂NAC不同处理组A:Ctrl.、B:NAC、C:DHT、D:NAC+DHT)给药后引起HO8910PM细胞线粒体去极化结果图和统计图(E)；Figure 13 is the results of mitochondrial depolarization of HO8910PM cells and statistical charts (E) after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT);
图14是DHT联合ROS抑制剂NAC不同处理组(A:Ctrl.、B:NAC、C:DHT、D:NAC+DHT)给药后引起SKOV3细胞线粒体去极化结果图和统计图(E)；Figure 14 is the results and statistics of mitochondrial depolarization in SKOV3 cells after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT) (E) ;
图15是DHT联合ROS抑制剂NAC不同处理组(A:Ctrl.、B:NAC、C:DHT、D:NAC+DHT)给药后引起HO8910PM细胞凋亡结果图和统计图(E)；Figure 15 is the results and statistics of HO8910PM cell apoptosis after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT);
图16是DHT联合ROS抑制剂NAC不同处理组(A:Ctrl.、B:NAC、C:DHT、D:NAC+DHT)给药后引起SKOV3细胞凋亡结果图和统计图(E)；Figure 16 is the results and statistics of SKOV3 cell apoptosis after administration of different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT) combined with ROS inhibitor NAC (E);
图17是DHT联合ROS抑制剂NAC不同处理组(A:Ctrl.、B:NAC、C:DHT、D:NAC+DHT)给药后HO8910PM细胞S、G1、G2期结果图和统计图(E)；表明S、G1期细胞阻滞；Figure 17 is the results and statistics of HO8910PM cells in S, G1, and G2 phases after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT) (E ); indicating cell arrest in S and G1 phases;
图18是DHT联合ROS抑制剂NAC不同处理组(A:Ctrl.、B:NAC、C:DHT、D:NAC+DHT)给药后SKOV3细胞S、G1、G2期结果图和统计图(E)；表明S、G1期细胞阻滞；Figure 18 is the results and statistics of SKOV3 cells in S, G1, and G2 phases after administration of DHT combined with ROS inhibitor NAC in different treatment groups (A: Ctrl., B: NAC, C: DHT, D: NAC+DHT) (E ); indicating cell arrest in S and G1 phases;
图19是不同浓度DHT给药后HO8910PM细胞内相关蛋白表达结果图(A)、凋亡相关蛋白Bcl-2、Bax、Caspase-3和周期蛋白CyclinB1、Cdc2的表达统计图(B)；Figure 19 is the result of expression of related proteins in HO8910PM cells after administration of different concentrations of DHT (A), the expression statistics of apoptosis-related proteins Bcl-2, Bax, Caspase-3 and cycle proteins CyclinB1 and Cdc2 (B);
图20是不同浓度DHT给药后SKOV3细胞内相关蛋白表达结果图(A)、凋亡相关蛋白Bcl-2、Bax、Caspase-3和周期蛋白CyclinB1、Cdc2的表达统计图(B)；Figure 20 is the result of expression of related proteins in SKOV3 cells after administration of different concentrations of DHT (A), the expression statistics of apoptosis-related proteins Bcl-2, Bax, Caspase-3 and cycle proteins CyclinB1 and Cdc2 (B);
图21是不同浓度DHT给药后HO8910PM细胞内p62/Keap1/Nrf2蛋白表达结果图(A)统计图(B)；Figure 21 is the results of p62/Keap1/Nrf2 protein expression in HO8910PM cells after administration of different concentrations of DHT (A) statistical chart (B);
图22是不同浓度DHT给药后SKOV3细胞内p62/Keap1/Nrf2蛋白表达结果图(A)统计图(B)；Fig. 22 is a graph (A) statistical graph (B) of p62/Keap1/Nrf2 protein expression results in SKOV3 cells after administration of different concentrations of DHT;
图23是DHT给药后HO8910PM(A)、SKOV3(B)细胞内Nrf2蛋白的mRNA水平图；Figure 23 is a graph showing the mRNA level of Nrf2 protein in HO8910PM (A), SKOV3 (B) cells after DHT administration;
图24是DHT联合蛋白酶体抑制剂MG132不同处理组给药后HO8910PM、SKOV3Nrf2蛋白表达结果图(A)和统计图(B、C)；Figure 24 is the results of HO8910PM, SKOV3Nrf2 protein expression results (A) and statistical charts (B, C) after administration of different treatment groups of DHT combined with proteasome inhibitor MG132;
图25是DHT联合Nrf2抑制剂tBHQ不同处理组给药后HO8910PM、SKOV3Nrf2蛋白表达结果图(A)和统计图(B、C)；Figure 25 is the results of HO8910PM, SKOV3Nrf2 protein expression results (A) and statistical charts (B, C) after administration of different treatment groups of DHT combined with Nrf2 inhibitor tBHQ;
图26是不同处理组小鼠的体重(A)、肿瘤体积(B)随时间变化结果图、离体肿瘤照片(C)、离体肿瘤体积统计图(D)、血清肿瘤标志物CA125(E)、HE4(F)水平结果图；Fig. 26 is the body weight (A), tumor volume (B) of mice in different treatment groups changing with time result figure, isolated tumor photo (C), isolated tumor volume statistical figure (D), serum tumor marker CA125 (E ), HE4(F) horizontal result graph;
图27是不同处理组小鼠心、肝、脾、肺、肾的HE病理切片图(200×)(A)和脏器指数(B)。Fig. 27 is HE pathological slices (200×) (A) and visceral index (B) of hearts, livers, spleens, lungs, and kidneys of mice in different treatment groups.
具体实施方式Detailed ways
下面结合附图以及具体实施例对本发明所提供的DHT用于NRF2抑制效果、治疗与NRF2抑制有关疾病作进一步详细的说明。The application of DHT provided by the present invention in NRF2 inhibition effect and treatment of diseases related to NRF2 inhibition will be further described in detail below in conjunction with the accompanying drawings and specific examples.
以下实施例不对本发明做任何形式的限定。本发明具体实施方式中使用的原料、设备均为已知产品，除非特别说明，以下实施例所用试剂和材料均通过购买市售产品获得。The following examples do not limit the present invention in any form. The raw materials and equipment used in the specific embodiments of the present invention are all known products. Unless otherwise specified, the reagents and materials used in the following examples are obtained by purchasing commercially available products.
主要仪器设备：超纯水机(Millipore公司)；细胞计数仪(Couter Star公司)；涡旋仪(Midwest公司)；CO 2恒温细胞培养箱(ThermoFisher Scientific公司)；冰箱(Haier公司)；光学显微镜(Olympus公司)；金属加热仪(杰瑞尔电器有限公司)；低温离心机(Beckman公司)；-80℃冰箱(ThermoFisher Scientific公司)；荧光显微镜(Olympus公司)；流式细胞仪(Beckman公司)；超净工作台(上海沪净医疗器械有限公司)；酶联免疫反应检测仪(Biotek Epoch公司)；电泳仪、转膜仪和WB显影装置(上海天能科技有限公司) Main equipment: ultrapure water machine (Millipore Company); cell counter (Couter Star Company); vortex instrument (Midwest Company); CO 2 constant temperature cell incubator (ThermoFisher Scientific Company); refrigerator (Haier Company); optical microscope (Olympus Company); metal heater (Jerrell Electric Co., Ltd.); cryogenic centrifuge (Beckman Company); -80 ℃ refrigerator (ThermoFisher Scientific Company); fluorescence microscope (Olympus Company); flow cytometer (Beckman Company) ;Ultra-clean workbench (Shanghai Hujing Medical Instrument Co., Ltd.); ELISA detector (Biotek Epoch Company); electrophoresis instrument, membrane transfer instrument and WB developing device (Shanghai Tianneng Technology Co., Ltd.)
主要试剂材料：RPMI-1640培养基、胎牛血清、青霉素-链霉素双抗、磷酸盐缓冲液(Gibco公司)；丹参酮ⅡA、MTT粉末、二甲基亚砜溶液、甲醛(Sigma公司)；吉姆萨染液(上海默克化工技术有限公司)；细胞凋亡检测试剂盒、细胞周期检测试剂盒(Becton Dickinson and Company)；RIPA蛋白裂解液(ThermoFisher Scientific公司)；蛋白酶抑制剂、磷酸酶抑制剂(Bimake公司)；SDS-PAGE凝胶、ECL化学发光液(上海雅酶生物科技有限公司)；Western blot抗体(Abcam公司)。Main reagent materials: RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin double antibody, phosphate buffer (Gibco); tanshinone Ⅱ A, MTT powder, dimethyl sulfoxide solution, formaldehyde (Sigma); Giemsa staining solution (Shanghai Merck Chemical Technology Co., Ltd.); Apoptosis detection kit, cell cycle detection kit (Becton Dickinson and Company); RIPA protein lysate (ThermoFisher Scientific company); protease inhibitor, phosphatase inhibition Reagent (Bimake Company); SDS-PAGE gel, ECL chemiluminescence solution (Shanghai Yazyme Biotechnology Co., Ltd.); Western blot antibody (Abcam Company).
实验细胞株及动物：人卵巢癌细胞株HO8910PM，SKOV3和A2780，购自于武汉普诺赛生物科技有限公司，冻存于本实验室的细胞存储液氮罐中。从上海斯莱克动物实验中心购买实验所需小鼠，SPF级健康成熟的6周龄的雌性NOD/SCID小鼠。Experimental cell lines and animals: Human ovarian cancer cell lines HO8910PM, SKOV3 and A2780 were purchased from Wuhan Punuosai Biotechnology Co., Ltd. and frozen in the cell storage liquid nitrogen tank in our laboratory. The mice required for the experiment were purchased from the Shanghai Slack Animal Experiment Center, SPF grade healthy and mature 6-week-old female NOD/SCID mice.
所有统计分析均采用Graphpad 5软件进行。多组比较采用单因素方差分析(ANOVA)检验。数据以独立实验次数的平均数±标准差(SD)表示。流式图使用Flowjo 7.6软件进行圈门和细胞百分比的统计。P值＜0.5为有统计学差异。All statistical analyzes were performed using Graphpad 5 software. Multiple groups were compared using one-way analysis of variance (ANOVA) test. Data are expressed as mean ± standard deviation (SD) of independent experiments. The flow diagram uses Flowjo 7.6 software for the statistics of circle gate and cell percentage. P value<0.5 was considered to be statistically different.
实施例1 DHT对卵巢癌细胞的体外增殖影响的研究Example 1 Study on the Influence of DHT on the In vitro Proliferation of Ovarian Cancer Cells
1.1细胞培养1.1 Cell culture
(1)细胞复苏：从细胞存储液氮罐中取出人卵巢癌细胞株HO8910PM，SKOV3和A2780，分别置于37℃水浴锅中,轻轻晃动使冻存的细胞完全融化，然后用3mL的RPMI-1640完全培养基(包含10vol％的胎牛血清+1vol％青霉素-链霉素双抗)洗去细胞冻存液中的二甲基亚砜,1800rpm/min离心5分钟，将上清弃去，加入1mL RPMI-1640完全培养基重悬细胞，然后将细胞悬液转移至培养皿中，补加7mL的培养基,轻轻晃动细胞培养皿并置于37℃的CO 2细胞培养箱中培养。 (1) Cell recovery: Take out the human ovarian cancer cell lines HO8910PM, SKOV3 and A2780 from the cell storage liquid nitrogen tank, place them in a 37°C water bath, shake gently to completely thaw the frozen cells, and then use 3mL of RPMI -1640 complete medium (contains 10vol% fetal bovine serum + 1vol% penicillin-streptomycin double antibody) to wash away the dimethyl sulfoxide in the cell freezing solution, centrifuge at 1800rpm/min for 5 minutes, and discard the supernatant , add 1mL RPMI-1640 complete medium to resuspend the cells, then transfer the cell suspension to a culture dish, add 7mL of medium, gently shake the cell culture dish and place it in a CO 2 cell culture incubator at 37°C .
(2)细胞传代：待人卵巢癌细胞株呈圆形透明状，生长状态良好，且细胞生长到培养皿的八九成满时进行细胞传代。首先用移液枪将细胞悬液轻轻吹匀，然后吸取一半细胞悬液转移至一个新的细胞培养皿中,两个培养皿中均补加4mL的新培养基，置于细胞培养箱中进行细胞培养。(2) Cell passage: When the human ovarian cancer cell line is round and transparent, the growth state is good, and the cells grow to 80-90% of the culture dish, the cell passage is carried out. First, blow the cell suspension gently with a pipette gun, then transfer half of the cell suspension to a new cell culture dish, add 4mL of new medium to both culture dishes, and place them in the cell culture incubator Perform cell culture.
1.2 MTT法分析DHT对卵巢癌细胞增殖的影响1.2 MTT method to analyze the effect of DHT on the proliferation of ovarian cancer cells
MTT：四甲基偶氮唑蓝，全称为3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐，是一种可以间接检测活细胞数量的黄色染料。本次细胞活性检测实验所用MTT浓度为5mg/mL：称取MTT 0.1g溶于20mL无菌磷酸盐缓冲液(PBS)中，用0.22μm滤膜和无菌注射器过滤除菌，分装至1mL无菌棕色离心管中，置于4℃避光保存，备用。MTT: Tetramethylazolazolium blue, the full name is 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide, which is a kind of reagent that can indirectly detect the number of living cells yellow dye. The concentration of MTT used in this cell viability detection experiment is 5 mg/mL: Weigh 0.1 g of MTT and dissolve it in 20 mL of sterile phosphate buffered saline (PBS), filter and sterilize with a 0.22 μm filter membrane and a sterile syringe, and dispense to 1 mL Store in a sterile brown centrifuge tube at 4°C in the dark for later use.
(1)细胞计数：首先用移液枪将细胞悬液轻轻吹匀，然后进行台盼蓝细胞计数。台盼蓝和细胞悬液1:1混合，吸取20μL混合液于细胞计数板上，然后用细胞计数仪计数每毫升所含细胞浓度。(1) Cell counting: First, gently blow the cell suspension evenly with a pipette gun, and then perform trypan blue cell counting. Mix trypan blue and cell suspension 1:1, pipette 20 μL of the mixture onto a cell counting plate, and then use a cell counter to count the concentration of cells per ml.
(2)细胞铺板：取对数生长期的人卵巢癌细胞系(HO8910PM，SKOV3，A2780)接种于96孔板中，加入不同浓度的DHT，CT，TAN IA，TAN IIA(化学结构如图1所示)，顺铂，阿霉素和5-氟尿嘧啶(0μM,1μM,2μM,4μM,8μM,16μM,32μM,64μM)处理细胞24或48小时。(2) Cell plating: the human ovarian cancer cell line (HO8910PM, SKOV3, A2780) in the logarithmic growth phase was inoculated in a 96-well plate, and different concentrations of DHT, CT, TAN IA, TAN IIA were added (the chemical structure is shown in Figure 1 indicated), cisplatin, doxorubicin and 5-fluorouracil (0 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM) treated cells for 24 or 48 hours.
(3)酶联免疫反应检测仪检测：给药结束后每孔避光加入10μL的MTT溶液，37℃培养箱中继续培养4小时。最后加入100μL的DMSO溶解甲瓒结晶体。在酶联免疫吸附测定仪中490nm处检测细胞的光密度值。根据以下公式进行细胞活性的计算：细胞活性＝(给药组细胞平均吸光度值-空白组细胞平均吸光度值)/(对照组细胞平均吸光度值-空白组细胞平均吸光度值)×100％。Graphpad 5软件进行数据处理，计算不同药物对细胞的IC50值。(3) Detection by enzyme-linked immunosorbent reaction detector: After the administration, 10 μL of MTT solution was added to each well in the dark, and cultured in a 37° C. incubator for 4 hours. Finally, 100 μL of DMSO was added to dissolve the formazan crystals. The optical density of the cells was detected at 490 nm in an enzyme-linked immunosorbent assay instrument. Carry out the calculation of cell activity according to the following formula: cell activity=(the average absorbance value of the cells in the administration group-the average absorbance value of the cells in the blank group)/(the average absorbance value of the cells in the control group-the average absorbance value of the cells in the blank group)×100%. Graphpad 5 software was used for data processing to calculate the IC50 values of different drugs on the cells.
二氢丹参酮(DHT)、隐丹参酮(CT)、丹参酮Ⅰ(TAN Ⅰ)和丹参酮Ⅱ A (TAN Ⅱ A)对三种不同卵巢癌细胞增殖的作用MTT法检测结果如图2所示，其中DHT的效果最好，可显著抑制卵巢癌细胞增殖，呈剂量和时间依赖性。处理24h后，DHT对三种卵巢癌细胞HO8910PM、SKOV3和A2780的IC50分别为3.26μM，8.03μM和4.05μM。对比化疗药物的IC50(顺铂(DDP)：17.80μM，115.57μM，519.5μM；阿霉素(DOX)：2.29μM，46.29μM，4.4μM；5-氟尿嘧啶(5-FU)：395.29μM，--，985.86μM)(图3)，说明DHT对卵巢癌细胞的抑制效果与阿霉素相当，优于顺铂和5-氟尿嘧啶。The MTT assay results of dihydrotanshinone (DHT), cryptotanshinone (CT), tanshinone Ⅰ (TAN Ⅰ) and tanshinone Ⅱ A (TAN Ⅱ A) on the proliferation of three different ovarian cancer cells are shown in Figure 2, in which DHT The effect is the best, and can significantly inhibit the proliferation of ovarian cancer cells in a dose- and time-dependent manner. After 24 hours of treatment, the IC50 of DHT on three ovarian cancer cells HO8910PM, SKOV3 and A2780 were 3.26μM, 8.03μM and 4.05μM, respectively. IC50 of comparative chemotherapy drugs (cisplatin (DDP): 17.80 μM, 115.57 μM, 519.5 μM; doxorubicin (DOX): 2.29 μM, 46.29 μM, 4.4 μM; 5-fluorouracil (5-FU): 395.29 μM, - -, 985.86μM) (Figure 3), indicating that the inhibitory effect of DHT on ovarian cancer cells is equivalent to that of doxorubicin, and better than that of cisplatin and 5-fluorouracil.
1.3细胞形态学观察(结晶紫染色)1.3 Cell morphology observation (crystal violet staining)
(1)细胞处理：将生长状态良好的HO8910PM，SKOV3和A2780细胞计数后，取2×10 5/mL细胞种植于六孔板中，分别加入不同浓度DHT处理细胞24小时。 (1) Cell treatment: After counting HO8910PM, SKOV3 and A2780 cells in good growth state, 2×10 5 /mL cells were planted in a six-well plate, and different concentrations of DHT were added to treat the cells for 24 hours.
(2)结晶紫染色：给药结束后，每孔分别加入磷酸盐缓冲液(PBS)溶液1mL，润洗2遍，之后每孔分别加入4％多聚甲醛溶液1mL，固定细胞15分钟。固定结束后，再次加入PBS溶液1mL润洗2遍。最后，每孔加入0.1％结晶紫染色液1mL染色15分钟。染色结束后取六孔板于低速流水下轻轻冲洗，晾干，在倒置显微镜下观察细胞形态并拍照。结果如图4所示，随着DHT给药浓度的增加，细胞数量减少，且伴随着细胞破裂、核固缩等现象，表明DHT能够抑制卵巢癌细胞的增殖并导致细胞损伤。(2) Crystal violet staining: After administration, 1 mL of phosphate buffered saline (PBS) solution was added to each well, rinsed twice, and then 1 mL of 4% paraformaldehyde solution was added to each well, and the cells were fixed for 15 minutes. After the fixation, 1 mL of PBS solution was added to rinse twice. Finally, 1 mL of 0.1% crystal violet staining solution was added to each well for 15 minutes. After staining, the six-well plate was gently rinsed under low-speed running water, dried, and the cell morphology was observed and photographed under an inverted microscope. The results are shown in Figure 4. With the increase of DHT concentration, the number of cells decreased, accompanied by cell rupture and nuclear pyknosis, indicating that DHT can inhibit the proliferation of ovarian cancer cells and cause cell damage.
1.4流式细胞术分析细胞周期1.4 Analysis of cell cycle by flow cytometry
(1)细胞处理：收集生长状态良好的HO8910PM，SKOV3细胞计数后，调整细胞浓度，取2×10 5/mL细胞种植于六孔板中，分别加入不同浓度的DHT和20.0μM DDP处理24小时。 (1) Cell treatment: collect HO8910PM and SKOV3 cells in good growth state, count the cells, adjust the cell concentration, take 2×10 5 /mL cells and plant them in a six-well plate, add different concentrations of DHT and 20.0 μM DDP to treat for 24 hours .
(2)细胞固定：分别收集不同处理组别的HO8910PM，SKOV3和A2780细胞于离心管中，使用PBS溶液于1800rpm/min下离心5分钟洗涤两次，弃去 上清。加入250μL预冷的PBS轻轻重悬细胞，之后在细胞涡旋仪上一边涡旋一边逐滴加入750μL预冷的乙醇固定细胞。细胞放置在4℃过夜孵育。(2) Cell fixation: HO8910PM, SKOV3 and A2780 cells of different treatment groups were collected in centrifuge tubes, washed twice by centrifugation at 1800rpm/min for 5 minutes with PBS solution, and the supernatant was discarded. Add 250 μL pre-cooled PBS to gently resuspend the cells, then add 750 μL pre-cooled ethanol drop by drop while vortexing on the cell vortex to fix the cells. Cells were incubated overnight at 4°C.
(3)细胞染色：取出固定好的细胞，3000rpm/min离心5分钟，将细胞中的乙醇除去，使用PBS溶液于1800rpm/min下离心5分钟洗涤一次，弃上清。之后加入200μL的PBS重悬细胞，然后加入核糖核酸酶(RNase A)和50μg/mL的碘化丙啶(PI)，将细胞混合均匀，置于室温避光孵育30分钟使细胞着色。(3) Cell staining: Take out the fixed cells, centrifuge at 3000rpm/min for 5 minutes, remove the ethanol in the cells, wash once with PBS solution at 1800rpm/min for 5 minutes, and discard the supernatant. Then add 200 μL of PBS to resuspend the cells, then add ribonuclease (RNase A) and 50 μg/mL propidium iodide (PI), mix the cells evenly, and incubate at room temperature in the dark for 30 minutes to color the cells.
(4)流式细胞仪检测：用200目滤网将处理好的细胞悬液过滤于流式管中，最后于1小时之内通过流式细胞术分析细胞周期的分布情况。(4) Flow cytometry detection: Filter the treated cell suspension into a flow tube with a 200-mesh filter, and finally analyze the distribution of the cell cycle by flow cytometry within 1 hour.
结果表明，DHT可剂量依赖性降低G1期细胞数量，升高S期和G2期数量，从而诱导细胞周期S、G2期阻滞(图7-8)。The results showed that DHT could dose-dependently reduce the number of cells in G1 phase and increase the number of cells in S phase and G2 phase, thereby inducing cell cycle arrest in S and G2 phases (Figure 7-8).
1.5流式细胞术分析细胞凋亡1.5 Analysis of cell apoptosis by flow cytometry
(1)细胞处理：收集生长状态良好的HO8910PM，SKOV3和A2780细胞计数后，调整细胞浓度，取2×10 5/mL细胞种植于六孔板中，分别加入不同浓度的DHT处理24小时。 (1) Cell treatment: HO8910PM, SKOV3 and A2780 cells in good growth state were collected and counted, the cell concentration was adjusted, 2×10 5 /mL cells were planted in six-well plates, and different concentrations of DHT were added for 24 hours.
(2)细胞收集：分别收集不同组别的HO8910PM，SKOV3和A2780细胞于离心管中，使用PBS溶液于1800rpm/min下离心5分钟洗涤两次，弃去上清。(2) Cell collection: HO8910PM, SKOV3 and A2780 cells of different groups were collected in centrifuge tubes, washed twice by centrifugation at 1800 rpm/min for 5 minutes with PBS solution, and the supernatant was discarded.
(3)细胞染色：首先将细胞凋亡试剂盒中的10×结合缓冲液用去离子水稀释成1×结合缓冲液。接着加入400μL的1×结合缓冲液重悬细胞，使最终浓度为5×10 6个细胞/mL。每个样品取100μL的细胞悬液，分别加入5μL的膜联蛋白V-FITC和5μL的碘化丙啶(PI)，将细胞混匀，在室温避光条件下培养30分钟使细胞着色。 (3) Cell staining: firstly, the 10× binding buffer in the cell apoptosis kit was diluted into 1× binding buffer with deionized water. Then add 400 μL of 1× binding buffer to resuspend the cells to make a final concentration of 5×10 6 cells/mL. Take 100 μL of cell suspension for each sample, add 5 μL of annexin V-FITC and 5 μL of propidium iodide (PI), mix the cells, and incubate at room temperature for 30 minutes in the dark to stain the cells.
(4)流式细胞仪检测：每个样本中补加300μL的1×结合缓冲液，将细胞 轻轻混匀。200目滤网将细胞悬液过滤于流式管中，最后于1小时之内通过流式细胞术分析细胞周期的分布情况。(4) Flow cytometry detection: 300 μL of 1× binding buffer was added to each sample, and the cells were mixed gently. A 200-mesh filter was used to filter the cell suspension into the flow tube, and finally the distribution of the cell cycle was analyzed by flow cytometry within 1 hour.
通过流式细胞检测发现，DHT剂量依赖性诱导卵巢癌HO8910PM和SKOV3细胞凋亡，处理24h后HO8910PM凋亡率为45.90％(6.4μM)，SKOV3凋亡率为56.60％(16.0μM)(图5-6)。Through flow cytometry, it was found that DHT dose-dependently induced the apoptosis of ovarian cancer HO8910PM and SKOV3 cells. After 24 hours of treatment, the apoptosis rate of HO8910PM was 45.90% (6.4 μM), and the apoptosis rate of SKOV3 was 56.60% (16.0 μM) (Figure 5 -6).
1.6流式细胞术分析细胞内活性氧水平和线粒体膜电位DHT通过激活卵巢癌细胞氧化应激诱导细胞凋亡和周期阻滞1.6 Flow cytometry analysis of intracellular reactive oxygen species levels and mitochondrial membrane potential DHT induces apoptosis and cycle arrest by activating oxidative stress in ovarian cancer cells
(1)细胞处理：收集生长状态良好的HO8910PM，SKOV3细胞计数后，调整细胞浓度，取2×10 5/mL细胞种植于六孔板中，分别加入不同浓度的DHT处理24小时。 (1) Cell treatment: HO8910PM and SKOV3 cells in good growth state were collected and counted, and the cell concentration was adjusted. 2×10 5 /mL cells were planted in a six-well plate, and treated with different concentrations of DHT for 24 hours.
(2)细胞染色：分别收集不同组别的HO8910PM，SKOV3细胞于离心管中，使用PBS溶液于1800rpm/min下离心5分钟洗涤两次，弃去上清。取洗涤后的细胞加入1mL的RPMI-1640培养基重悬。随后，加入浓度为2mM的罗丹明123染液2μL，置于37℃恒温培养箱中避光孵育15分钟，使细胞着色用于流式细胞仪检测线粒体膜电位。取洗涤后的细胞加入500μL终浓度为10μM的DCFH-DA探针重悬，随后置于37℃恒温培养箱中避光孵育20分钟用于流式细胞仪检测细胞内活性氧水平。(2) Cell staining: HO8910PM and SKOV3 cells of different groups were collected in centrifuge tubes, washed twice by centrifugation at 1800 rpm/min for 5 minutes with PBS solution, and the supernatant was discarded. Take the washed cells and add 1 mL of RPMI-1640 medium to resuspend. Subsequently, 2 μL of rhodamine 123 staining solution with a concentration of 2 mM was added, and placed in a constant temperature incubator at 37° C. for 15 minutes in the dark to incubate the cells to stain the cells for flow cytometry to detect mitochondrial membrane potential. The washed cells were resuspended by adding 500 μL of DCFH-DA probe with a final concentration of 10 μM, and then placed in a constant temperature incubator at 37°C in the dark for 20 minutes to detect the level of intracellular reactive oxygen species by flow cytometry.
(3)流式细胞仪检测：细胞着色完成后，用RPMI-1640培养基1800rpm/min离心5分钟洗涤2次，弃去上清。紧接着再加入400μL的RPMI-1640培养基重悬细胞，于37℃恒温培养箱中避光孵育1小时。用200目滤网将处理好的细胞悬液过滤于流式管中，在1小时之内使用流式细胞仪对样品进行线粒体膜电位检测。(3) Flow cytometry detection: After the coloring of cells is completed, centrifuge with RPMI-1640 medium at 1800 rpm/min for 5 minutes and wash twice, and discard the supernatant. Immediately afterwards, 400 μL of RPMI-1640 medium was added to resuspend the cells, and incubated in a 37°C constant temperature incubator in the dark for 1 hour. Filter the treated cell suspension into the flow tube with a 200-mesh filter, and use the flow cytometer to detect the mitochondrial membrane potential of the sample within 1 hour.
细胞内活性氧水平(ROS)结果如图9所示，发现DHT处理后，卵巢癌HO8910PM和SKOV3细胞ROS水平显著升高，分别升高6.69倍(6.4μM)和 15.16倍(16.0μM)，且呈剂量依赖性。The results of intracellular reactive oxygen species (ROS) levels are shown in Figure 9. It was found that after DHT treatment, the ROS levels of ovarian cancer HO8910PM and SKOV3 cells increased significantly, increasing by 6.69 times (6.4 μM) and 15.16 times (16.0 μM), respectively, and It is dose-dependent.
ROS升高伴随线粒体膜电位的变化，DHT剂量依赖性降低细胞中线粒体膜电位水平，分别降低92.5％(6.4μM)和98.7％(16.0μM)，导致线粒体去极化(图10-11)。The increase of ROS was accompanied by the change of mitochondrial membrane potential, and DHT dose-dependently reduced the level of mitochondrial membrane potential in cells, respectively by 92.5% (6.4 μM) and 98.7% (16.0 μM), resulting in mitochondrial depolarization (Figure 10-11).
表明DHT升高肿瘤细胞中ROS水平可激活细胞氧化应激，从而杀伤肿瘤细胞。It shows that the increase of ROS level in tumor cells by DHT can activate cellular oxidative stress, thereby killing tumor cells.
(4)DHT联合ROS抑制剂NAC处理分析：为了进一步印证DHT对SKOV3和HO8910PM的细胞毒性是否与诱导氧化应激有关，使用ROS抑制剂NAC进行干预，分别设置对照组ctrl、NAC组(10mM)、DHT组(6.4μM)、NAC(10mM)+DHT组(6.4μM)处理人卵巢癌细胞HO8910PM，SKOV3 24小时，并用MTT法统计不同组别卵巢癌细胞增殖结果，发现预孵育NAC(10mM)可以完全逆转DHT诱导的增殖抑制(图12A)，采用流式细胞术分析不同组别细胞内活性氧水平结果如图12B-D所示，ROS上升，线粒体膜电位下降，凋亡和周期阻滞(图12-18)。进一步证实了DHT通过激活氧化应激诱导卵巢癌细胞凋亡和周期阻滞。(4) Analysis of DHT combined with ROS inhibitor NAC treatment: In order to further confirm whether the cytotoxicity of DHT on SKOV3 and HO8910PM is related to the induction of oxidative stress, the ROS inhibitor NAC was used to intervene, and the control group ctrl and NAC group (10mM) were respectively set , DHT group (6.4μM), NAC (10mM)+DHT group (6.4μM) treated human ovarian cancer cells HO8910PM, SKOV3 for 24 hours, and used the MTT method to count the proliferation results of ovarian cancer cells in different groups, and found that pre-incubated NAC (10mM) DHT-induced proliferation inhibition can be completely reversed (Fig. 12A). Flow cytometry was used to analyze the intracellular reactive oxygen species levels in different groups. As shown in Fig. 12B-D, ROS increased, mitochondrial membrane potential decreased, apoptosis and cycle arrest (Figure 12-18). It was further confirmed that DHT induces apoptosis and cycle arrest in ovarian cancer cells by activating oxidative stress.
1.7蛋白免疫印迹分析DHT通过促进Nrf2蛋白降解从而抑制其表达1.7 Western blot analysis DHT inhibits the expression of Nrf2 protein by promoting its degradation
(1)细胞处理：收集生长状态良好的HO8910PM，SKOV3细胞计数后，调整细胞浓度，取2×10 5/mL细胞种植于六孔板中，分别加入不同浓度的DHT处理24小时。 (1) Cell treatment: HO8910PM and SKOV3 cells in good growth state were collected and counted, and the cell concentration was adjusted. 2×10 5 /mL cells were planted in a six-well plate, and treated with different concentrations of DHT for 24 hours.
(2)蛋白提取：分别收集不同组别的HO8910PM，SKOV3细胞于离心管中，使用PBS溶液于1800rpm/min下离心5分钟洗涤两次，弃去上清。加入含有1％蛋白酶抑制剂和磷酸酶抑制剂的高强度细胞裂解缓冲液裂解细胞的总蛋白。裂解期间每隔5分钟超声一次样品，裂解20min。然后按照4：1的比例加入5×的蛋白上样缓冲液，将蛋白样品在金属加热器中100℃下煮沸10分 钟。提取好的蛋白样品置于-20℃保存备用。(2) Protein extraction: HO8910PM and SKOV3 cells of different groups were collected in centrifuge tubes, washed twice by centrifugation at 1800 rpm/min for 5 minutes with PBS solution, and the supernatant was discarded. The total protein of the cells was lysed by adding high-strength cell lysis buffer containing 1% protease inhibitors and phosphatase inhibitors. During lysis, the samples were sonicated every 5 minutes for 20 minutes. Then add 5× protein loading buffer at a ratio of 4:1, and boil the protein sample in a metal heater at 100°C for 10 minutes. The extracted protein samples were stored at -20°C for future use.
(3)Western blot分析：将等量的细胞总蛋白加到12％的十二烷基硫酸盐聚丙烯酰胺凝胶(SDS-PAGE)上进行电泳(浓缩胶部分80V，分离胶部分为120V)，分离相对分子质量不同的蛋白质。电泳结束后用湿转(恒压100V转膜60min)的方法将凝胶上的蛋白转移到聚偏二氟乙烯(PVDF)膜上。PVDF膜与5％的脱脂牛奶于室温下，在摇床上孵育1小时，封闭PVDF膜上的非特异性蛋白结合位点。封闭结束后，用TBST摇床上洗三次，每次8分钟。然后在4℃摇床上孵育各种主要的兔抗抗体：β-actin，Keap1，Nrf2，Caspase-3，Bax，Bcl-2，CyclinB1，cdc-2，抗体孵育过夜。次日，回收一抗于-20℃保存，将PVDF膜用TBST摇床上洗三次，每次8分钟。然后将孵育过一抗的PVDF膜与辣根过氧化物酶结合的抗兔免疫球蛋白G(IgG)抗体于室温摇床上孵育1小时。之后用TBST摇床上洗三次，每次8分钟。最后采用增强型化学发光检测试剂盒(ECL)检测抗体的结合。β-actin蛋白表达用于内部参照。(3) Western blot analysis: add an equal amount of total cell protein to 12% dodecyl sulfate polyacrylamide gel (SDS-PAGE) for electrophoresis (80V for the stacking gel part, 120V for the separating gel part) , to separate proteins with different relative molecular masses. After electrophoresis, the protein on the gel was transferred to a polyvinylidene fluoride (PVDF) membrane by wet transfer (constant voltage 100V transfer membrane for 60 min). The PVDF membrane was incubated with 5% skimmed milk at room temperature on a shaker for 1 hour to block the non-specific protein binding sites on the PVDF membrane. After the end of blocking, wash three times on a TBST shaker, 8 minutes each time. Then various major rabbit anti-antibodies: β-actin, Keap1, Nrf2, Caspase-3, Bax, Bcl-2, CyclinB1, cdc-2 were incubated on a shaker at 4°C overnight. The next day, the primary antibody was recovered and stored at -20°C, and the PVDF membrane was washed three times on a TBST shaker for 8 minutes each time. Then the PVDF membrane incubated with the primary antibody was incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody on a shaker at room temperature for 1 hour. Then wash three times on a TBST shaker, 8 minutes each time. Finally, the enhanced chemiluminescence detection kit (ECL) was used to detect the binding of the antibody. β-actin protein expression was used for internal reference.
结果如图19-20所示，发现DHT剂量依赖性下调凋亡相关蛋白Bcl-2、Caspase-3表达，下调周期蛋白CyclinB1、Cdc2表达，升高SKOV3细胞中Bax表达，对HO8910PM细胞中Bax表达无影响，进一步说明DHT抑制卵巢癌细胞增殖的作用与诱导细胞凋亡和周期阻滞有关。The results are shown in Figures 19-20. It was found that DHT dose-dependently down-regulated the expressions of apoptosis-related proteins Bcl-2 and Caspase-3, down-regulated the expressions of cyclins CyclinB1 and Cdc2, increased the expression of Bax in SKOV3 cells, and affected the expression of Bax in HO8910PM cells. It has no effect, which further shows that the effect of DHT on inhibiting the proliferation of ovarian cancer cells is related to the induction of apoptosis and cycle arrest.
图21-22是Keap1/Nrf2蛋白表达分析结果，Keap1/Nrf2信号通路是细胞中重要的氧化应激调节系统，Keap1通过泛素化降解负调控Nrf2表达。图中结果表明，DHT升高Keap1表达，下调Nrf2表达，并且抑制Nrf2进入细胞核。Figure 21-22 shows the results of Keap1/Nrf2 protein expression analysis. The Keap1/Nrf2 signaling pathway is an important oxidative stress regulation system in cells. Keap1 negatively regulates Nrf2 expression through ubiquitination and degradation. The results in the figure show that DHT increases the expression of Keap1, down-regulates the expression of Nrf2, and inhibits the entry of Nrf2 into the nucleus.
(4)qRT-PCR分析：利用qRT-PCR分析Nrf2蛋白的mRNA水平，qRT-PCR分析发现在mRNA水平上DHT降低Nrf2的靶基因如HMOX1、GCLM、FTH1，剂量依赖性升高KEAP1和NFE2L2的转录水平(图23A和23B)。而在蛋白水平DHT抑制Nrf2的表达，说明DHT是通过转录后水平对Nrf2表达 进行调控。(4) qRT-PCR analysis: qRT-PCR was used to analyze the mRNA level of Nrf2 protein. qRT-PCR analysis found that DHT reduced the target genes of Nrf2 such as HMOX1, GCLM, and FTH1 at the mRNA level, and increased KEAP1 and NFE2L2 in a dose-dependent manner. Transcript levels (Figures 23A and 23B). However, DHT inhibits the expression of Nrf2 at the protein level, indicating that DHT regulates the expression of Nrf2 through the post-transcriptional level.
(5)DHT联合Nrf2激活剂tBHQ处理分析：分别设置对照组、tBHQ组(10μM)、DHT组(6.4μM)、tBHQ(10μM)+DHT组(6.4μM)处理人卵巢癌细胞HO8910PM，SKOV3 24小时，并用Western blot分析法测试不同组别卵巢癌细胞Nrf2蛋白表达，进一步分析发现DHT与tBHQ共孵育可逆转DHT对Nrf2的抑制作用(图24)，证明DHT的作用靶点为Nrf2。(5) Analysis of DHT combined with Nrf2 activator tBHQ: control group, tBHQ group (10 μM), DHT group (6.4 μM), tBHQ (10 μM)+DHT group (6.4 μM) were set up to treat human ovarian cancer cells HO8910PM, SKOV3 24 After 1 hour, the expression of Nrf2 protein in different groups of ovarian cancer cells was tested by Western blot analysis. Further analysis found that the co-incubation of DHT and tBHQ could reverse the inhibitory effect of DHT on Nrf2 (Figure 24), proving that the target of DHT is Nrf2.
(6)DHT联合蛋白酶体抑制剂MG132处理分析：分别设置对照组、MG132组(10μM)、DHT组(6.4μM)、MG132(10μM)+DHT组(6.4μM)处理人卵巢癌细胞HO8910PM，SKOV3 24小时，并用Western blot分析法测试不同组别卵巢癌细胞Nrf2蛋白表达，进一步分析发现DHT与MG132共孵育可逆转DHT对Nrf2的抑制作用(图25)，说明DHT是通过促进Nrf2蛋白降解从而抑制其表达。(6) DHT combined with proteasome inhibitor MG132 treatment analysis: the control group, MG132 group (10 μM), DHT group (6.4 μM), MG132 (10 μM) + DHT group (6.4 μM) were set up to treat human ovarian cancer cells HO8910PM, SKOV3 After 24 hours, Western blot analysis was used to test the expression of Nrf2 protein in different groups of ovarian cancer cells. Further analysis found that the co-incubation of DHT and MG132 could reverse the inhibitory effect of DHT on Nrf2 (Figure 25), indicating that DHT inhibited Nrf2 by promoting the degradation of Nrf2 protein. its expression.
实施例二 DHT在小鼠体内抗肿瘤疗效的研究Example 2 Study on the Anti-tumor Curative Effect of DHT in Mice
小鼠皮下移植：Balb/c雌性小鼠，4-6周龄，初始体重在18克到22克之间。收集生长状态良好的HO8910PM细胞，计数后调整细胞浓度，取HO8910PPM细胞(1×10 7个细胞/100μL)，使用1mL无菌注射器在超净工作台上将细胞悬液皮下接种到Balb/c小鼠腋窝左侧。 Subcutaneous transplantation of mice: Balb/c female mice, 4-6 weeks old, with an initial body weight between 18 grams and 22 grams. Collect HO8910PM cells in good growth state, adjust the cell concentration after counting, take HO8910PPM cells (1× 107 cells/100μL), use a 1mL sterile syringe to subcutaneously inoculate the cell suspension into Balb/c small The left side of the mouse armpit.
小鼠分组给药治疗：使用50vol％PEG 400+35vol％DMSO+15vol％生理盐水，混合均匀后作为溶解DHT的溶剂。Mice were treated in groups: 50vol% PEG 400 + 35vol% DMSO + 15vol% physiological saline were used, mixed evenly as a solvent for dissolving DHT.
当肿瘤发展到约100mm 3时，小鼠腹腔注射约100μL的50vol％PEG 400+35vol％DMSO+15vol％生理盐水(空白对照组TC)，10mg/kg/d的DHT(DHT低剂量组DHT-L)，20mg/kg/d的DHT(DHT高剂量组DHT-H)，2mg/kg/d的顺铂注射液(顺铂组DDP)，隔天一次，共给药14天。期间隔天测量和计算一次小鼠的体重和肿瘤体积(用游标卡尺测量肿瘤的最长直径(a)和最 短直径(b)，按照V＝1/2ab 2计算肿瘤体积并记录)。给药结束后利用ELISA法检测小鼠血清中HE4和CA125水平，提取方法如下：(1)包被检测板：将所用抗原用包被稀释液稀释至1μg/μL，每孔抗原加入100μL，37℃孵育4小时后弃去孔中液体。 When the tumor had grown to about 100 mm 3 , mice were intraperitoneally injected about 100 μL of 50vol% PEG 400+35vol% DMSO+15vol% normal saline (blank control group TC), 10 mg/kg/d of DHT (DHT low-dose group DHT- L), DHT at 20 mg/kg/d (DHT-H in DHT high-dose group), cisplatin injection at 2 mg/kg/d (DDP in cisplatin group), once every other day, for a total of 14 days. The body weight and tumor volume of the mice were measured and calculated every other day during this period (the longest diameter (a) and shortest diameter (b) of the tumor were measured with a vernier caliper, and the tumor volume was calculated and recorded according to V=1/2ab 2 ). After administration, the levels of HE4 and CA125 in mouse serum were detected by ELISA, and the extraction method was as follows: (1) Coating detection plate: Dilute the antigen used to 1 μg/μL with coating diluent, add 100 μL of antigen to each well, 37 After incubation at °C for 4 hours, the liquid in the wells was discarded.
(2)封闭酶标反应孔：5％胎牛血清于37℃下封闭40分钟，封闭时将封闭液加满各反应孔，并去除各孔中的气泡，封闭结束后用PBS洗涤3遍，每遍3分钟。(2) Seal the enzyme-labeled reaction wells: 5% fetal bovine serum was sealed at 37°C for 40 minutes. When sealing, the blocking solution was filled to the top of each reaction well, and the air bubbles in each well were removed. After the sealing was completed, it was washed 3 times with PBS. 3 minutes each time.
(3)加入待检测样品：吸取50μL不同组别Balb/c小鼠血清加入酶标反应孔中，每个样品做3个复孔,每孔100μL，置于37℃孵育60分钟，孵育结束后用PBS洗涤3遍，每遍3分钟。(3) Add the sample to be tested: pipette 50 μL of Balb/c mouse serum from different groups and add it to the enzyme-labeled reaction well. Make 3 replicate wells for each sample, 100 μL per well, and incubate at 37°C for 60 minutes. After the incubation Wash 3 times with PBS, 3 minutes each time.
(4)加入酶标抗体：酶标抗体：根据酶结合物提供商提供的参考工作稀释度进行：每孔加入酶标抗体100μL，37℃下孵育60分钟，孵育结束后用PBS洗涤3遍，每遍3分钟。(4) Add enzyme-labeled antibody: enzyme-labeled antibody: according to the reference working dilution provided by the enzyme conjugate provider: add 100 μL of enzyme-labeled antibody to each well, incubate at 37°C for 60 minutes, wash with PBS 3 times after incubation, 3 minutes each time.
(5)加入底物液：每孔加入100μL底物液，置37℃避光放置5分钟，之后加入终止液显色。(5) Add substrate solution: add 100 μL substrate solution to each well, place at 37°C in the dark for 5 minutes, then add stop solution to develop color.
(6)终止反应：每孔加入终止液50μL终止反应，20min内使用酶联免疫反应检测仪于450nm波长下测定实验结果。(6) Termination reaction: 50 μL of stop solution was added to each well to terminate the reaction, and the experimental results were measured at a wavelength of 450 nm using an enzyme-linked immunosorbent reaction detector within 20 minutes.
结果如图26所示，DHT(20mg/kg)腹腔注射对小鼠体重无显著性影响(图26A)，能显著抑制卵巢癌荷瘤小鼠肿瘤生长(图26B)。实验结束后处死小鼠，测量肿瘤组织和主要脏器组织(肝脏、脾脏)重量，并进行病理组织学染色，具体为：用石蜡切片机进行石蜡样本的切片，厚度3.5μm，连续切片，染色程序为烤片干燥20分钟，二甲苯2次×10分钟，无水乙醇2次×2分钟，95％乙醇1分钟，80％乙醇1分钟，70％乙醇1分钟，水洗1分钟，苏木素8分钟，水洗2次×1分钟，0.5％盐酸酒精10秒，水洗10分钟，伊红2分 钟，水洗1分钟，80％乙醇5秒，85％乙醇5秒，90％乙醇5秒，95％乙醇1分钟，无水乙醇2次×2分钟，无水乙醇3分钟，二甲苯2次×2分钟，结束染色后直接用中性树胶封片。The results are shown in Figure 26, DHT (20 mg/kg) intraperitoneal injection had no significant effect on the body weight of the mice (Figure 26A), but could significantly inhibit tumor growth in ovarian cancer-bearing mice (Figure 26B). After the experiment, the mice were sacrificed, the weight of the tumor tissue and major organ tissues (liver, spleen) was measured, and histopathological staining was carried out, specifically: paraffin samples were sliced with a paraffin microtome, with a thickness of 3.5 μm, serially sectioned, and stained The program is to dry the slices for 20 minutes, xylene 2 times x 10 minutes, absolute ethanol 2 times x 2 minutes, 95% ethanol for 1 minute, 80% ethanol for 1 minute, 70% ethanol for 1 minute, wash with water for 1 minute, hematoxylin for 8 minutes , wash 2 times with water for 1 minute, 0.5% hydrochloric acid alcohol for 10 seconds, wash with water for 10 minutes, eosin for 2 minutes, wash with water for 1 minute, 80% ethanol for 5 seconds, 85% ethanol for 5 seconds, 90% ethanol for 5 seconds, 95% ethanol for 1 Minutes, absolute ethanol 2 times × 2 minutes, absolute ethanol 3 minutes, xylene 2 times × 2 minutes, directly seal the slide with neutral gum after staining.
研究发现，DHT能够降低离体肿瘤重量(图26C和26D)和血清肿瘤标志物CA125、HE4水平(图26E和26F)，肿瘤组织HE病理切片表明DHT能够导致荷瘤中卵巢癌细胞坏死及凋亡(图26G)。此外，与模型组相比，DHT给药后对小鼠主要脏器病理(图27A)和脏器指数(图27B)无显著性影响，说明DHT治疗可延缓小鼠肿瘤生长，且对小鼠无潜在毒性作用，DHT体内对小鼠相对较安全。Studies have found that DHT can reduce the weight of isolated tumors (Figure 26C and 26D) and the levels of serum tumor markers CA125 and HE4 (Figure 26E and 26F). died (Fig. 26G). In addition, compared with the model group, DHT administration had no significant effect on the pathology of major organs (Fig. 27A) and organ index (Fig. 27B) in mice, indicating that DHT treatment can delay the growth of tumors in mice, and has Without potential toxic effects, DHT is relatively safe for mice in vivo.
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其他不同形式的变化或变动。这里无需也无法把所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围。Apparently, the above-mentioned embodiments are only examples for clear description, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all implementation modes here. However, the obvious changes or variations derived therefrom still fall within the protection scope of the present invention.

Claims (6)

  1. 一种二氢丹参酮Ⅰ在制备Nrf2抑制剂中的应用。An application of dihydrotanshinone I in the preparation of Nrf2 inhibitors.
  2. 根据权利要求1所述的应用，其特征在于，所述二氢丹参酮Ⅰ作为药理学成分抑制Nrf2活性，作为药物应用于Nrf2抑制有关的疾病。The application according to claim 1, characterized in that the dihydrotanshinone I is used as a pharmacological component to inhibit Nrf2 activity, and is used as a drug for diseases related to Nrf2 inhibition.
  3. 根据权利要求2所述的应用，其特征在于，所述Nrf2抑制剂由所述二氢丹参酮Ⅰ和药学上可接受的赋形剂组成。The use according to claim 2, characterized in that said Nrf2 inhibitor consists of said dihydrotanshinone I and pharmaceutically acceptable excipients.
  4. 根据权利要求2所述的应用，其特征在于，所述Nrf2抑制剂为胶囊剂、颗粒剂、片剂、口服液或注射剂。The application according to claim 2, characterized in that the Nrf2 inhibitor is capsule, granule, tablet, oral liquid or injection.
  5. 根据权利要求2～4任一项所述的应用，其特征在于，所述疾病为癌症。The use according to any one of claims 2-4, characterized in that the disease is cancer.
  6. 根据权利要求5所述的应用，其特征在于，所述癌症为卵巢癌。The use according to claim 5, characterized in that the cancer is ovarian cancer.
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