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WO2024169348A1 - Use of alnustone in inhibiting and treating gastric cancer - Google Patents

Use of alnustone in inhibiting and treating gastric cancer Download PDF

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Publication number
WO2024169348A1
WO2024169348A1 PCT/CN2023/136957 CN2023136957W WO2024169348A1 WO 2024169348 A1 WO2024169348 A1 WO 2024169348A1 CN 2023136957 W CN2023136957 W CN 2023136957W WO 2024169348 A1 WO2024169348 A1 WO 2024169348A1
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Prior art keywords
gastric cancer
ketone
alnus
protein
cancer cells
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French (fr)
Chinese (zh)
Inventor
全娟花
王洋
高菲菲
杜海燕
潘昭彬
谭治明
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Affiliated Hospital of Guangdong Medical University
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Affiliated Hospital of Guangdong Medical University
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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
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/20—Unsaturated compounds containing keto groups bound to acyclic carbon atoms
    • C07C49/213—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing six-membered aromatic rings
    • C07C49/217—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing six-membered aromatic rings having unsaturation outside the aromatic rings
    • C07C49/223—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing six-membered aromatic rings having unsaturation outside the aromatic rings polycyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/20—Unsaturated compounds containing keto groups bound to acyclic carbon atoms
    • C07C49/225—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing six-membered aromatic rings and other rings

Definitions

  • the invention relates to the field of medical technology, and in particular to use of alnus ketone in inhibiting and treating gastric cancer, and use of alnus ketone in preparing a drug for treating gastric cancer.
  • Gastric cancer is a malignant tumor originating from the gastric epithelium, and most of them are adenocarcinomas.
  • the Lauren classification system there are four main subtypes of gastric cancer: well-differentiated (non-cardia/intestinal), poorly differentiated (cardia/diffuse), mixed, and a newly added subtype - solid gastric cancer; according to the WHO classification system, there are four main subtypes: papillary, tubular, signet ring cell, and mucinous, and other subtypes: adenosquamous carcinoma, squamous cell carcinoma, Paneth cell carcinoma, undifferentiated carcinoma, etc.
  • the cause and pathogenesis of gastric cancer have not yet been elucidated. Studies have shown that the occurrence of gastric cancer is the result of the combined action of multiple factors.
  • Hp Helicobacter pylori
  • Epstein-Barr virus infection is the main risk factor for gastric cancer, with an attributable risk of 75% to 88%. This is because Helicobacter pylori has important virulence and oncogene types, such as cytotoxin-associated gene A (CagA) and vacuolating toxin A (VacA).
  • CagA protein is one of the most intensively studied proteins of Helicobacter pylori.
  • This pathogenic protein affects the development of peptic ulcers and is almost 100% expressed by Helicobacter pylori strains infected in Asian patients.
  • Helicobacter pylori can also secrete peptidoglycan into host cells, leading to the upregulation of various proinflammatory cytokines, such as IL-8 and COX, leading to chronic inflammation and the development of cancer.
  • WHO has classified Helicobacter pylori as a Class I carcinogen. Under the influence of multiple factors such as Hp infection, adverse environment, unhealthy diet and lifestyle, the formation of gastric cancer goes through a series of histopathological stages: normal mucosa-chronic gastritis-atrophic gastritis-intestinal metaplasia-dysplasia-gastric cancer.
  • Gastric cancer is currently the fifth most common cancer and the fourth leading cause of cancer death worldwide. In 2020, it is estimated that nearly 10 million cancer deaths occurred, of which gastric cancer accounted for 7.7%. The median overall survival of advanced gastric cancer does not exceed 12 months. Studies have shown that undifferentiated gastric cancer is more malignant than differentiated gastric cancer, has a higher rate of lymph node metastasis, and lacks targeted treatments.
  • the purpose of the present invention is to avoid the shortcomings of the prior art and provide a use of alnus ketone in preparing a drug for treating gastric cancer.
  • the use of alnus ketone in preparing a drug for treating gastric cancer has a significant apoptosis-promoting effect on gastric cancer cells.
  • alnus ketone in preparing a medicine for treating gastric cancer.
  • the alnus ketone of the present invention inhibits the proliferation of undifferentiated gastric cancer cells HGC-27.
  • the alnus ketone of the present invention induces apoptosis of gastric cancer cells by down-regulating the expression of anti-apoptotic protein, wherein the anti-apoptotic protein is Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.
  • the alnus ketone of the present invention induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, wherein the pro-apoptotic protein is Cleaved PARP protein.
  • the alnus ketone of the invention induces the occurrence of gastric cancer cell apoptosis by up-regulating the expression of p-JNK protein or Bak protein.
  • the target of the alnus ketone of the present invention is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway and the JNK/Bcl-2 signaling pathway.
  • the alnus ketone of the invention is dissolved in dimethyl sulfoxide, and the concentration is 10 ⁇ g/ml to 50 ⁇ g/ml.
  • the above concentration is 15 ⁇ g/ml to 40 ⁇ g/ml.
  • the invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer.
  • Alnus ketone has an obvious apoptosis-promoting effect on gastric cancer cells.
  • Figure 1 is a growth inhibition curve of undifferentiated gastric cancer cells HGC-27 treated with different concentrations of alderone for 24h, 48h and 72h.
  • Figure 2 shows the expression levels of apoptosis-related proteins in undifferentiated gastric cancer cells HGC-27 after treatment with different concentrations of alderone for 48 hours.
  • Figure 3 shows the expression levels of p-JNK protein, JNK protein and Bak protein in undifferentiated gastric cancer cells HGC-27 after treatment with different concentrations of alderone for 48 hours.
  • Figure 4 shows the apoptosis rate of undifferentiated gastric cancer cells HGC-27 after being treated with different concentrations of alderone for 48 hours.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the raw materials, reagents, etc. used in the following examples can be purchased from conventional biochemical reagent stores or pharmaceutical companies unless otherwise specified.
  • the cells used in the invention are undifferentiated gastric cancer cells HGC-27 purchased from the Chinese Academy of Sciences.
  • RPMI Medium 1640 basic was purchased from Gibco, USA, with the product number C11875500BT.
  • 1X phosphate buffered saline (1XPBS buffer) was purchased from Solarbio, China, with the catalog number P1020.
  • Fetal bovine serum of French origin was purchased from ZETA Company of the United States with the item number Z7186FBS-500.
  • Trypsin-EDTA (0.25%) containing phenol red (0.25wt% Trypsin-EDTA) was purchased from Gibco, USA, with the product number 25200-072.
  • CCK-8 kit France Origin CCK-8 Cell Counting Kit was purchased from China Novozymes Co., Ltd. with the catalog number A311-01.
  • BCA protein concentration assay kit (BCA Protein Assay Kit) was purchased from China Kangrun Company with the product number E162-01.
  • the cell apoptosis detection kit (Annexin V, FITC Apoptosis Detection Kit) was purchased from DOJINDO Company of Japan with the catalog number AD10.
  • the substrate color development reagent (Immobilon Forte Western HRP substrate) was purchased from Milipore, USA, with the catalog number WBLUF0500.
  • Anti-PARP antibody Anti-PARP(46D11)antibody(9532S)
  • anti-Bcl-2 antibody Anti-Bcl-2(D55G8)antibody(4223S)
  • anti-Bcl-xL antibody Anti-Bcl-xL(54H6)antibody(2764S)
  • anti-Mcl-1 antibody Anti-Mcl-1(D35A5)antibody(5453S)
  • anti-Bak antibody Anti-Bak (D4E4) antibody (12105S)
  • anti-p-JNK antibody Anti-Phospho-SAPK/JNK (Thr183/Tyr185) antibody (9251S)
  • anti-JNK antibody Anti-SAPK/JNK antibody (9252S)
  • anti-GAPDH antibody Anti-GAPDH antibody (2118S) were all purchased from CST, USA.
  • the invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.
  • Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.
  • Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.
  • alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.
  • the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.
  • alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.
  • alnus ketone has a significant pro-apoptotic effect on gastric cancer cells and can be used as a drug for the treatment of gastric cancer.
  • the invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.
  • Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.
  • Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.
  • alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.
  • the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.
  • alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.
  • Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when alnus ketone was dissolved in dimethyl sulfoxide and the concentration was in the range of 15 ⁇ g/ml to 40 ⁇ g/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.
  • the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.
  • the invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.
  • Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.
  • Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.
  • alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.
  • the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.
  • alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.
  • Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 10 ⁇ g/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.
  • the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.
  • the invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.
  • Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.
  • Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.
  • alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein, Bak protein or JNK protein.
  • the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.
  • alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.
  • Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 15 ⁇ g/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.
  • the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.
  • the invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.
  • Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.
  • Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.
  • alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.
  • the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.
  • alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.
  • Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 20 ⁇ g/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.
  • the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.
  • the invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.
  • Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.
  • Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.
  • alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.
  • the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.
  • alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.
  • Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 30 ⁇ g/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.
  • the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.
  • the invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.
  • Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.
  • Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.
  • alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein, Bak protein or JNK protein.
  • the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.
  • alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.
  • Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 40 ⁇ g/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.
  • the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells, and can be used to prepare drugs for treating gastric cancer.
  • the alnus ketone at the concentration of this example has the best effect of promoting apoptosis of gastric cancer cells, and the inhibition rate of the growth of undifferentiated gastric cancer cells HGC-27 is high, with a 72h inhibition rate greater than 80%, a 48h inhibition rate greater than 60%, and a 24h inhibition rate greater than 40%, and can be used to prepare drugs for treating gastric cancer.
  • the invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.
  • Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.
  • Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.
  • alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein, Bak protein or JNK protein.
  • the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.
  • alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.
  • Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 50 ⁇ g/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.
  • the concentration of alnus ketone in this example has the highest apoptosis-promoting effect on gastric cancer cells and the highest inhibition rate on the growth of undifferentiated gastric cancer cells HGC-27, with the inhibition rates at 72h and 48h both greater than 80%, and the inhibition rate at 24h greater than 60%, and can be used to prepare drugs for treating gastric cancer.
  • Differentiated HGC-27 cells fall off into single cell suspension, and undifferentiated HGC-27 gastric cancer cells are transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 3min. Take out the centrifuge tube, aspirate the supernatant, add an appropriate amount of culture medium, and fully suspend the undifferentiated HGC-27 gastric cancer cells.
  • the undifferentiated HGC-27 gastric cancer cells can be subcultured and divided into new culture dishes or culture bottles at a ratio of 1:2 to 1:3 or after counting the undifferentiated HGC-27 gastric cancer cells, and cultured in a cell culture incubator at 37°C and 5% v/v CO2.
  • the undifferentiated gastric cancer cells HGC-27 in the logarithmic growth phase were resuspended in complete culture medium, and the cells were counted using a cell counting plate to prepare a cell suspension of 5 ⁇ 103 cells/100 ⁇ L.
  • the cell suspension was inoculated in a 96-well plate, with 100 ⁇ L of cell suspension in each well, and then placed in a 37°C, 5% v/v CO2 saturated humidity incubator for overnight culture.
  • the undifferentiated gastric cancer cells HGC-27 were treated with different concentrations of alderone (such as 0 ⁇ g/ml, 15 ⁇ g/ml, 20 ⁇ g/ml, 30 ⁇ g/ml, 40 ⁇ g/ml, 50 ⁇ g/ml), and 3 replicates were set for each concentration.
  • the cell activity was detected by CCK-8 kit at 24h, 48h and 72h, respectively, as shown in Figure 1.
  • Gastric cancer undifferentiated cells HGC-27 in the logarithmic growth phase were inoculated in a culture dish. After the cells adhered to the wall, the gastric cancer undifferentiated cells HGC-27 were treated with different concentrations of alderone (such as 0 ⁇ g/ml, 15 ⁇ g/ml, 20 ⁇ g/ml, 30 ⁇ g/ml, 40 ⁇ g/ml). After 48 hours, the supernatant and adherent cells were collected, and the cell proteins were extracted and prepared by lysis with RIPA Buffer protein lysis buffer and ultrasonic cell disruptor. SDS-PAGE gel electrophoresis was performed and the proteins were transferred to PVDF membranes.
  • alderone such as 0 ⁇ g/ml, 15 ⁇ g/ml, 20 ⁇ g/ml, 30 ⁇ g/ml, 40 ⁇ g/ml.
  • GAPDH is the internal reference
  • Cleaved PARP is a pro-apoptotic protein
  • Bcl-2, Bcl-xL, and Mcl-1 are anti-apoptotic proteins.
  • the expression of pro-apoptotic proteins increased gradually, while the expression of anti-apoptotic proteins decreased significantly gradually, which shows that alder ketone has a concentration-dependent promoting effect on cell apoptosis.
  • GAPDH is the internal reference.
  • the c-Jun N-terminal kinase (JNK) signaling pathway is an important pathway involved in the initiation of cell apoptosis. After being activated by extracellular stimuli, it exerts biological effects, mainly mediating physiological functions such as differentiation, proliferation, and apoptosis.
  • Bak protein is a major family member of Bcl2 pro-apoptotic proteins and an essential molecule for apoptotic cell death.
  • alder ketone can induce apoptosis of human gastric cancer cell HGC-27 cells by activating the JNK signaling pathway, and regulate the mitochondrial apoptosis of gastric cancer cells through the JNK/Bak signaling pathway and the JNK/Bcl-2 signaling pathway.
  • the inhibitory effect of alderone on the proliferation of undifferentiated gastric cancer cells HGC-27 was time-concentration dependent within a certain concentration range.
  • Alderone induced apoptosis of undifferentiated gastric cancer cells HGC-27 by upregulating the expression of related pro-apoptotic proteins and downregulating the expression of related anti-apoptotic proteins, or upregulating the expression of p-JNK and Bak proteins.
  • the apoptosis rate of gastric cancer cells increased with increasing concentration.
  • Alderone had a significant pro-apoptotic effect on gastric cancer cells.
  • alnus ketone has a significant apoptosis-promoting effect on gastric cancer cells. Therefore, alnus ketone is used as a raw material to prepare a drug for treating gastric cancer, which also has the same inhibitory effect on gastric cancer cells.

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Abstract

A use of alnustone in preparing a drug for treating gastric cancer. Alnustone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27. Alnustone induces the occurrence of apoptosis of gastric cancer cells by down-regulating the expression of an anti-apoptotic protein and/or up-regulating the expression of a pro-apoptotic protein, and/or up-regulating the expression of a p-JNK protein or a Bak protein. Alnustone has a time-concentration dependence on the proliferation inhibition effect on the gastric cancer undifferentiated cells HGC-27 within a certain concentration range, and the apoptosis rate of the gastric cancer cells is increased along with the increase of the concentration.

Description

桤木酮在抑制和治疗胃癌中的用途Application of alderone in inhibiting and treating gastric cancer 技术领域Technical Field

本发明涉及医药技术领域，特别涉及桤木酮在抑制和治疗胃癌的用途，以及桤木酮在制备治疗胃癌药物中的用途。The invention relates to the field of medical technology, and in particular to use of alnus ketone in inhibiting and treating gastric cancer, and use of alnus ketone in preparing a drug for treating gastric cancer.

背景技术Background Art

胃癌是起源于胃上皮的恶性肿瘤，绝大多是腺癌。根据Lauren分类系统，胃癌有四种主要亚型：高分化(非贲门/肠道)、低分化(贲门/弥漫性)、混合型及新增添的亚型—实体性胃癌；根据WHO分类系统，主要有四种亚型：乳头型、管状型、印戒细胞型和粘液型，及其它亚型：腺鳞癌、鳞状细胞癌、潘氏细胞癌、未分化癌等。胃癌的病因和发病机制尚未阐明，研究表明胃癌的发生是多因素综合作用的结果。Gastric cancer is a malignant tumor originating from the gastric epithelium, and most of them are adenocarcinomas. According to the Lauren classification system, there are four main subtypes of gastric cancer: well-differentiated (non-cardia/intestinal), poorly differentiated (cardia/diffuse), mixed, and a newly added subtype - solid gastric cancer; according to the WHO classification system, there are four main subtypes: papillary, tubular, signet ring cell, and mucinous, and other subtypes: adenosquamous carcinoma, squamous cell carcinoma, Paneth cell carcinoma, undifferentiated carcinoma, etc. The cause and pathogenesis of gastric cancer have not yet been elucidated. Studies have shown that the occurrence of gastric cancer is the result of the combined action of multiple factors.

目前认为胃癌发生的常见因素包括男性、年龄较大、家族史、饮食、饮酒、吸烟、社会经济地位低、既往有胃手术史、恶性贫血、幽门螺旋杆菌(Hp)和EB病毒感染。其中幽门螺旋杆菌感染是胃癌的主要危险因素，其归因风险为75％至88％。这是由于幽门螺旋杆菌具有重要的毒力和致癌基因类型，如细胞毒素相关基因A(CagA)和空泡毒素A(VacA)。其中CagA蛋白是幽门螺杆菌被研究最深入的蛋白之一，这种致病蛋白影响消化性溃疡的发展，亚洲患者感染的幽门螺杆菌菌株几乎100％表达。此外幽门螺旋杆菌还可以分泌肽聚糖进入宿主细胞，导致各种促炎细胞因子的上调，如IL-8和COX，从而导致慢性炎症和癌症的发展。WHO已将幽门螺旋杆菌列为I类致癌物。在Hp感染、不良环境、不健康饮食生活习惯等多种因素作用下，胃癌的形成经过了一系列组织病理学阶：正常黏膜-慢性胃炎-萎缩性胃炎-肠化生-异型增生-胃癌。在此过程中，胃黏膜细胞增殖和凋亡之间的正常动态平衡被打破。许多与胃癌发生相关的分子事件已经被广泛研究，包括HER2表达模块、p53基因突变、抑癌基因缺失或因高甲基化而失活、某些癌基因(c-met、EGFR)扩增、调节细胞凋亡的因素、细胞周期调节因素、影响细胞膜特性的因素、多药耐药蛋白和微卫星不稳定性等。It is currently believed that common factors for the occurrence of gastric cancer include male sex, older age, family history, diet, drinking, smoking, low socioeconomic status, previous history of gastric surgery, pernicious anemia, Helicobacter pylori (Hp) and Epstein-Barr virus infection. Among them, Helicobacter pylori infection is the main risk factor for gastric cancer, with an attributable risk of 75% to 88%. This is because Helicobacter pylori has important virulence and oncogene types, such as cytotoxin-associated gene A (CagA) and vacuolating toxin A (VacA). Among them, CagA protein is one of the most intensively studied proteins of Helicobacter pylori. This pathogenic protein affects the development of peptic ulcers and is almost 100% expressed by Helicobacter pylori strains infected in Asian patients. In addition, Helicobacter pylori can also secrete peptidoglycan into host cells, leading to the upregulation of various proinflammatory cytokines, such as IL-8 and COX, leading to chronic inflammation and the development of cancer. WHO has classified Helicobacter pylori as a Class I carcinogen. Under the influence of multiple factors such as Hp infection, adverse environment, unhealthy diet and lifestyle, the formation of gastric cancer goes through a series of histopathological stages: normal mucosa-chronic gastritis-atrophic gastritis-intestinal metaplasia-dysplasia-gastric cancer. In this process, the normal dynamic balance between proliferation and apoptosis of gastric mucosal cells is broken. Many molecular events related to gastric cancer have been widely studied, including HER2 expression module, p53 gene mutation, tumor suppressor gene deletion or inactivation due to hypermethylation, amplification of certain oncogenes (c-met, EGFR), factors regulating cell apoptosis, cell cycle regulatory factors, factors affecting cell membrane properties, multidrug resistance proteins and microsatellite instability.

目前胃癌是全球第五大常见癌症和第四大癌症死亡原因，2020年估计发生了近1000万例癌症死亡，其中胃癌占7.7％。晚期胃癌的中位总生存期不超过12个月。研究表明，未分化胃癌比分化型胃癌恶性程度更高，淋巴结转移率更高，且缺乏针对性的治疗方法。Gastric cancer is currently the fifth most common cancer and the fourth leading cause of cancer death worldwide. In 2020, it is estimated that nearly 10 million cancer deaths occurred, of which gastric cancer accounted for 7.7%. The median overall survival of advanced gastric cancer does not exceed 12 months. Studies have shown that undifferentiated gastric cancer is more malignant than differentiated gastric cancer, has a higher rate of lymph node metastasis, and lacks targeted treatments.

因此，针对现有技术不足，提供一种桤木酮在抑制和治疗胃癌中的用途，以及桤木酮在制备治疗胃癌药物中的用途以解决现有技术不足甚为必要。Therefore, in view of the shortcomings of the prior art, it is necessary to provide a use of alnus ketone in inhibiting and treating gastric cancer, and a use of alnus ketone in preparing a drug for treating gastric cancer to solve the shortcomings of the prior art.

发明内容 Summary of the invention

本发明的目的在于避免现有技术的不足之处而提供一种桤木酮在制备治疗胃癌药物中的用途。该桤木酮在制备治疗胃癌药物中的用途，桤木酮对胃癌细胞有明显的促凋亡作用。The purpose of the present invention is to avoid the shortcomings of the prior art and provide a use of alnus ketone in preparing a drug for treating gastric cancer. The use of alnus ketone in preparing a drug for treating gastric cancer has a significant apoptosis-promoting effect on gastric cancer cells.

本发明的上述目的通过以下技术措施实现：The above-mentioned purpose of the present invention is achieved by the following technical measures:

提供桤木酮在制备治疗胃癌药物中的用途。Provided is the use of alnus ketone in preparing a medicine for treating gastric cancer.

本发明桤木酮抑制胃癌未分化细胞HGC-27的增殖。The alnus ketone of the present invention inhibits the proliferation of undifferentiated gastric cancer cells HGC-27.

本发明桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生。其中抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。The alnus ketone of the present invention induces apoptosis of gastric cancer cells by down-regulating the expression of anti-apoptotic protein, wherein the anti-apoptotic protein is Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.

本发明桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生。其中促凋亡蛋白为Cleaved PARP蛋白。The alnus ketone of the present invention induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, wherein the pro-apoptotic protein is Cleaved PARP protein.

本发明桤木酮通过上调p-JNK蛋白或者Bak蛋白的表达，诱导胃癌细胞凋亡的发生。The alnus ketone of the invention induces the occurrence of gastric cancer cell apoptosis by up-regulating the expression of p-JNK protein or Bak protein.

本发明桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。The target of the alnus ketone of the present invention is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway and the JNK/Bcl-2 signaling pathway.

本发明桤木酮溶于二甲基亚砜，且浓度为10μg/ml～50μg/ml。The alnus ketone of the invention is dissolved in dimethyl sulfoxide, and the concentration is 10 μg/ml to 50 μg/ml.

进一步优选的，上述浓度为15μg/ml～40μg/ml。More preferably, the above concentration is 15 μg/ml to 40 μg/ml.

本发明的一种桤木酮在制备治疗胃癌药物中的用途，桤木酮对胃癌细胞有明显的促凋亡作用。The invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer. Alnus ketone has an obvious apoptosis-promoting effect on gastric cancer cells.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

利用附图对本发明作进一步的说明，但附图中的内容不构成对本发明的任何限制。The present invention is further described with reference to the accompanying drawings, but the contents in the accompanying drawings do not constitute any limitation to the present invention.

图1为不同浓度桤木酮处理胃癌未分化细胞HGC-2724h、48h和72h生长抑制率曲线。Figure 1 is a growth inhibition curve of undifferentiated gastric cancer cells HGC-27 treated with different concentrations of alderone for 24h, 48h and 72h.

图2为不同浓度桤木酮处理胃癌未分化细胞HGC-2748h后凋亡相关蛋白表达水平。Figure 2 shows the expression levels of apoptosis-related proteins in undifferentiated gastric cancer cells HGC-27 after treatment with different concentrations of alderone for 48 hours.

图3为不同浓度桤木酮处理胃癌未分化细胞HGC-2748h后p-JNK蛋白、JNK蛋白和Bak蛋白表达水平。Figure 3 shows the expression levels of p-JNK protein, JNK protein and Bak protein in undifferentiated gastric cancer cells HGC-27 after treatment with different concentrations of alderone for 48 hours.

图4为不同浓度桤木酮处理胃癌未分化细胞HGC-2748h后细胞凋亡率。Figure 4 shows the apoptosis rate of undifferentiated gastric cancer cells HGC-27 after being treated with different concentrations of alderone for 48 hours.

具体实施方式DETAILED DESCRIPTION

结合以下实施例对本发明的技术方案作进一步说明。The technical solution of the present invention is further described in conjunction with the following embodiments.

下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均可自常规生化试剂商店或药品经营企业购买得到。The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples can be purchased from conventional biochemical reagent stores or pharmaceutical companies unless otherwise specified.

其中发明所用的细胞为胃癌未分化细胞HGC-27购于中国科学院。 The cells used in the invention are undifferentiated gastric cancer cells HGC-27 purchased from the Chinese Academy of Sciences.

RPMI Medium 1640培养基(RPMI Medium 1640 basic)购自美国Gibco公司，货号为C11875500BT。RPMI Medium 1640 basic was purchased from Gibco, USA, with the product number C11875500BT.

1X磷酸盐缓冲液(1XPBS缓冲液)购自中国Solarbio公司，货号为P1020。1X phosphate buffered saline (1XPBS buffer) was purchased from Solarbio, China, with the catalog number P1020.

法国原产胎牛血清(France Origin)购自美国ZETA公司，货号为Z7186FBS-500。Fetal bovine serum of French origin (France Origin) was purchased from ZETA Company of the United States with the item number Z7186FBS-500.

胰蛋白酶-EDTA(0.25％)，含酚红(0.25wt％Trypsin-EDTA)购自美国Gibco公司，货号为25200-072。Trypsin-EDTA (0.25%) containing phenol red (0.25wt% Trypsin-EDTA) was purchased from Gibco, USA, with the product number 25200-072.

CCK-8试剂盒(France Origin CCK-8 Cell Counting Kit)购自中国诺唯赞公司，货号为A311-01。CCK-8 kit (France Origin CCK-8 Cell Counting Kit) was purchased from China Novozymes Co., Ltd. with the catalog number A311-01.

BCA蛋白浓度测定试剂盒(BCA Protein Assay Kit)购自中国康润公司，货号为E162-01。BCA protein concentration assay kit (BCA Protein Assay Kit) was purchased from China Kangrun Company with the product number E162-01.

细胞凋亡检测试剂盒(Annexin V,FITC Apoptosis Detection Kit)购自日本DOJINDO公司，货号为AD10。The cell apoptosis detection kit (Annexin V, FITC Apoptosis Detection Kit) was purchased from DOJINDO Company of Japan with the catalog number AD10.

底物显色试剂(Immobilon Forte Western HRP底物)购自美国Milipore公司，货号为WBLUF0500。The substrate color development reagent (Immobilon Forte Western HRP substrate) was purchased from Milipore, USA, with the catalog number WBLUF0500.

抗PARP抗体(Anti-PARP(46D11)antibody(9532S))、抗Bcl-2抗体(Anti-Bcl-2(D55G8)antibody(4223S))、抗Bcl-xL抗体(Anti-Bcl-xL(54H6)antibody(2764S))、抗Mcl-1抗体(Anti-Mcl-1(D35A5)antibody(5453S))、抗Bak抗体(Anti-Bak(D4E4)antibody(12105S))、抗p-JNK抗体(Anti-Phospho-SAPK/JNK(Thr183/Tyr185)antibody(9251S))、抗JNK抗体(Anti-SAPK/JNK antibody(9252S))、抗GAPDH抗体(Anti-GAPDHantibody(2118S))均购自美国CST公司。Anti-PARP antibody (Anti-PARP(46D11)antibody(9532S)), anti-Bcl-2 antibody (Anti-Bcl-2(D55G8)antibody(4223S)), anti-Bcl-xL antibody (Anti-Bcl-xL(54H6)antibody(2764S)), anti-Mcl-1 antibody (Anti-Mcl-1(D35A5)antibody(5453S)), anti-Bak antibody ( Anti-Bak (D4E4) antibody (12105S)), anti-p-JNK antibody (Anti-Phospho-SAPK/JNK (Thr183/Tyr185) antibody (9251S)), anti-JNK antibody (Anti-SAPK/JNK antibody (9252S)), and anti-GAPDH antibody (Anti-GAPDH antibody (2118S)) were all purchased from CST, USA.

实施例1Example 1

一种桤木酮在制备治疗胃癌药物中的用途，具体是桤木酮抑制胃癌未分化细胞HGC-27的增殖。The invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.

桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中促凋亡蛋白为Cleaved PARP蛋白。Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.

桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.

具体是桤木酮通过上调p-JNK蛋白或者Bak蛋白的表达，诱导胃癌细胞凋亡的发生。Specifically, alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.

申请人进一步研究发现桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。 The applicant further discovered that the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.

具体是桤木酮通过激活JNK信号通路诱导胃癌细胞凋亡的发生。通过调控JNK/Bak通路或者JNK/Bcl-2通路，使胃癌细胞发生线粒体凋亡。Specifically, alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.

通过不同浓度的桤木酮对胃癌细胞进行处理，观察桤木酮对胃癌细胞增殖的抑制作用，实验发现，当桤木酮溶于二甲基亚砜，且浓度10μg/ml～50μg/ml范围内时，对胃癌细胞增殖的抑制作用明显。Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. The experiment found that when alnus ketone was dissolved in dimethyl sulfoxide and the concentration was in the range of 10μg/ml to 50μg/ml, it had a significant inhibitory effect on the proliferation of gastric cancer cells.

综上所述，桤木酮对胃癌细胞有明显的促凋亡作用，可作为治疗胃癌的药物用途。In summary, alnus ketone has a significant pro-apoptotic effect on gastric cancer cells and can be used as a drug for the treatment of gastric cancer.

实施例2Example 2

一种桤木酮在制备治疗胃癌药物中的用途，具体是桤木酮抑制胃癌未分化细胞HGC-27的增殖。The invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.

桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中促凋亡蛋白为Cleaved PARP蛋白。Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.

桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.

具体是桤木酮通过上调p-JNK蛋白或者Bak蛋白的表达，诱导胃癌细胞凋亡的发生。Specifically, alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.

申请人进一步研究发现桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。The applicant further discovered that the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.

具体是桤木酮通过激活JNK信号通路诱导胃癌细胞凋亡的发生。通过调控JNK/Bak通路或者JNK/Bcl-2通路，使胃癌细胞发生线粒体凋亡。Specifically, alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.

通过不同浓度的桤木酮对胃癌细胞进行处理，观察桤木酮对胃癌细胞增殖的抑制作用，发现，当桤木酮浓度溶于二甲基亚砜，且浓度15μg/ml～40μg/ml范围内对胃癌细胞增殖的抑制作用明显。Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when alnus ketone was dissolved in dimethyl sulfoxide and the concentration was in the range of 15μg/ml to 40μg/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

与实施例1相比，本实施例桤木酮对胃癌细胞促凋亡作用较优，可用于制备治疗胃癌药物。Compared with Example 1, the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.

实施例3Example 3

一种桤木酮在制备治疗胃癌药物中的用途，具体是桤木酮抑制胃癌未分化细胞HGC-27的增殖。The invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.

桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中促凋亡蛋白为Cleaved PARP蛋白。Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.

桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。 Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.

具体是桤木酮通过上调p-JNK蛋白或者Bak蛋白的表达，诱导胃癌细胞凋亡的发生。Specifically, alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.

申请人进一步研究发现桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。The applicant further discovered that the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.

具体是桤木酮通过激活JNK信号通路诱导胃癌细胞凋亡的发生。通过调控JNK/Bak通路或者JNK/Bcl-2通路，使胃癌细胞发生线粒体凋亡。Specifically, alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.

通过不同浓度的桤木酮对胃癌细胞进行处理，观察桤木酮对胃癌细胞增殖的抑制作用，发现，其中当桤木酮浓度的二甲基亚砜溶液浓度为10μg/ml对胃癌细胞增殖的抑制作用明显。Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 10 μg/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

与实施例1相比，本实施例桤木酮对胃癌细胞促凋亡作用较优，可用于制备治疗胃癌药物。Compared with Example 1, the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.

实施例4Example 4

一种桤木酮在制备治疗胃癌药物中的用途，具体是桤木酮抑制胃癌未分化细胞HGC-27的增殖。The invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.

桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中促凋亡蛋白为Cleaved PARP蛋白。Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.

桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.

具体是桤木酮通过上调p-JNK蛋白、Bak蛋白或者JNK蛋白的表达，诱导胃癌细胞凋亡的发生。Specifically, alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein, Bak protein or JNK protein.

申请人进一步研究发现桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。The applicant further discovered that the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.

具体是桤木酮通过激活JNK信号通路诱导胃癌细胞凋亡的发生。通过调控JNK/Bak通路或者JNK/Bcl-2通路，使胃癌细胞发生线粒体凋亡。Specifically, alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.

通过不同浓度的桤木酮对胃癌细胞进行处理，观察桤木酮对胃癌细胞增殖的抑制作用，发现，其中当桤木酮的二甲基亚砜溶液浓度为15μg/ml对胃癌细胞增殖的抑制作用明显。Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 15 μg/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

与实施例1相比，本实施例桤木酮对胃癌细胞促凋亡作用较优，可用于制备治疗胃癌药物。Compared with Example 1, the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.

实施例5Example 5

一种桤木酮在制备治疗胃癌药物中的用途，具体是桤木酮抑制胃癌未分化细胞HGC-27的增殖。 The invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.

桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中促凋亡蛋白为Cleaved PARP蛋白。Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.

桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.

具体是桤木酮通过上调p-JNK蛋白或者Bak蛋白的表达，诱导胃癌细胞凋亡的发生。Specifically, alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.

申请人进一步研究发现桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。The applicant further discovered that the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.

具体是桤木酮通过激活JNK信号通路诱导胃癌细胞凋亡的发生。通过调控JNK/Bak通路或者JNK/Bcl-2通路，使胃癌细胞发生线粒体凋亡。Specifically, alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.

通过不同浓度的桤木酮对胃癌细胞进行处理，观察桤木酮对胃癌细胞增殖的抑制作用，发现，当桤木酮的二甲基亚砜溶液浓度为20μg/ml对胃癌细胞增殖的抑制作用明显。Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 20 μg/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

与实施例1相比，本实施例桤木酮对胃癌细胞促凋亡作用较优，可用于制备治疗胃癌药物。Compared with Example 1, the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.

实施例6Example 6

一种桤木酮在制备治疗胃癌药物中的用途，具体是桤木酮抑制胃癌未分化细胞HGC-27的增殖。The invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.

桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中促凋亡蛋白为Cleaved PARP蛋白。Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.

桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.

具体是桤木酮通过上调p-JNK蛋白或者Bak蛋白的表达，诱导胃癌细胞凋亡的发生。Specifically, alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein.

申请人进一步研究发现桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。The applicant further discovered that the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.

具体是桤木酮通过激活JNK信号通路诱导胃癌细胞凋亡的发生。通过调控JNK/Bak通路或者JNK/Bcl-2通路，使胃癌细胞发生线粒体凋亡。Specifically, alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.

通过不同浓度的桤木酮对胃癌细胞进行处理，观察桤木酮对胃癌细胞增殖的抑制作用，发现，当桤木酮的二甲基亚砜溶液浓度为30μg/ml对胃癌细胞增殖的抑制作用明显。 Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 30 μg/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

与实施例1相比，本实施例桤木酮对胃癌细胞促凋亡作用较优，可用于制备治疗胃癌药物。Compared with Example 1, the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.

实施例7Example 7

一种桤木酮在制备治疗胃癌药物中的用途，具体是桤木酮抑制胃癌未分化细胞HGC-27的增殖。The invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.

桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中促凋亡蛋白为Cleaved PARP蛋白。Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.

桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.

具体是桤木酮通过上调p-JNK蛋白、Bak蛋白或者JNK蛋白的表达，诱导胃癌细胞凋亡的发生。Specifically, alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein, Bak protein or JNK protein.

申请人进一步研究发现桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。The applicant further discovered that the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.

具体是桤木酮通过激活JNK信号通路诱导胃癌细胞凋亡的发生。通过调控JNK/Bak通路或者JNK/Bcl-2通路，使胃癌细胞发生线粒体凋亡。Specifically, alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.

通过不同浓度的桤木酮对胃癌细胞进行处理，观察桤木酮对胃癌细胞增殖的抑制作用，发现，当桤木酮的二甲基亚砜溶液浓度为40μg/ml对胃癌细胞增殖的抑制作用明显。Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 40 μg/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

与实施例1相比，本实施例桤木酮对胃癌细胞促凋亡作用较优，可用于制备治疗胃癌药物。与实施例1至7相比，在本实施例的浓度下的桤木酮对胃癌细胞促凋亡作用是最优，对胃癌未分化细胞HGC-27生长的抑制率均较高，72h抑制率大于80％，48h抑制率大于60％，24h抑制率大于40％，可用于制备治疗胃癌药物。Compared with Example 1, the alnus ketone in this example has a better effect of promoting apoptosis of gastric cancer cells, and can be used to prepare drugs for treating gastric cancer. Compared with Examples 1 to 7, the alnus ketone at the concentration of this example has the best effect of promoting apoptosis of gastric cancer cells, and the inhibition rate of the growth of undifferentiated gastric cancer cells HGC-27 is high, with a 72h inhibition rate greater than 80%, a 48h inhibition rate greater than 60%, and a 24h inhibition rate greater than 40%, and can be used to prepare drugs for treating gastric cancer.

实施例8Example 8

一种桤木酮在制备治疗胃癌药物中的用途，具体是桤木酮抑制胃癌未分化细胞HGC-27的增殖。The invention discloses a use of alnus ketone in preparing a drug for treating gastric cancer, specifically alnus ketone inhibits the proliferation of gastric cancer undifferentiated cells HGC-27.

桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中促凋亡蛋白为Cleaved PARP蛋白。Alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins, among which the pro-apoptotic protein is Cleaved PARP protein.

桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生，其中抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。Alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins, where the anti-apoptotic proteins are Bcl-2 protein, Bcl-xL protein or Mcl-1 protein.

具体是桤木酮通过上调p-JNK蛋白或者Bak蛋白或者JNK蛋白的表达，诱导胃癌细胞凋亡的发生。 Specifically, alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein, Bak protein or JNK protein.

申请人进一步研究发现桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。The applicant further discovered that the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway.

具体是桤木酮通过激活JNK信号通路诱导胃癌细胞凋亡的发生。通过调控JNK/Bak通路或者JNK/Bcl-2通路，使胃癌细胞发生线粒体凋亡。Specifically, alder ketone induces apoptosis of gastric cancer cells by activating the JNK signaling pathway and by regulating the JNK/Bak pathway or the JNK/Bcl-2 pathway, causing mitochondrial apoptosis of gastric cancer cells.

通过不同浓度的桤木酮对胃癌细胞进行处理，观察桤木酮对胃癌细胞增殖的抑制作用，发现，当桤木酮的二甲基亚砜溶液浓度为50μg/ml对胃癌细胞增殖的抑制作用明显。Gastric cancer cells were treated with different concentrations of alnus ketone to observe the inhibitory effect of alnus ketone on the proliferation of gastric cancer cells. It was found that when the concentration of alnus ketone in dimethyl sulfoxide solution was 50 μg/ml, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

与实施例1至7相比，在本实施例的浓度下的桤木酮对胃癌细胞促凋亡作用是最高，对胃癌未分化细胞HGC-27生长的抑制率最高，72h和48h的抑制率均大于80％，24h的抑制率大于60％，可用于制备治疗胃癌药物。Compared with Examples 1 to 7, the concentration of alnus ketone in this example has the highest apoptosis-promoting effect on gastric cancer cells and the highest inhibition rate on the growth of undifferentiated gastric cancer cells HGC-27, with the inhibition rates at 72h and 48h both greater than 80%, and the inhibition rate at 24h greater than 60%, and can be used to prepare drugs for treating gastric cancer.

实施例9Example 9

为了阐明桤木酮对胃癌的抑制机理，进行了一系列的实验研究，具体如下：In order to clarify the inhibitory mechanism of alder ketone on gastric cancer, a series of experimental studies were conducted, as follows:

1、胃癌未分化细胞HGC-27培养处理1. Culture and treatment of undifferentiated gastric cancer cells HGC-27

(1)细胞传代：待胃癌未分化细胞HGC-27增长至密度70％～80％之间，吸弃原培养液，用1X磷酸盐缓冲液(洗涤细胞2次。加入1mL胰蛋白酶-EDTA，常温下放置30s～1min，相差显微镜下见胃癌未分化细胞HGC-27收缩变圆后加入3mL含10wt％法国原产胎牛血清的RPMI Medium1640培养基细胞培养液终止消化。吹打胃癌未分化细胞HGC-27脱落成单细胞悬液，将胃癌未分化细胞HGC-27转移至15mL离心管中，1000rpm离心3min。取出离心管，吸掉上清液，加入适量培养液，使胃癌未分化细胞HGC-27充分混悬，可按1：2～1：3或进行胃癌未分化细胞HGC-27计数后传代分装到新的培养皿或培养瓶中，置于细胞培养箱中37℃，5％v/v CO2条件下培养。(1) Cell passaging: When the HGC-27 gastric cancer undifferentiated cells grow to a density of 70% to 80%, the original culture medium is discarded and the cells are washed twice with 1X phosphate buffer. 1 mL of trypsin-EDTA is added and the cells are placed at room temperature for 30 s to 1 min. Under a phase contrast microscope, the HGC-27 gastric cancer undifferentiated cells shrink and become round. Then 3 mL of RPMI Medium 1640 culture medium containing 10 wt% French fetal bovine serum is added to terminate the digestion. The HGC-27 gastric cancer undifferentiated cells are blown to remove the original culture medium. Differentiated HGC-27 cells fall off into single cell suspension, and undifferentiated HGC-27 gastric cancer cells are transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 3min. Take out the centrifuge tube, aspirate the supernatant, add an appropriate amount of culture medium, and fully suspend the undifferentiated HGC-27 gastric cancer cells. The undifferentiated HGC-27 gastric cancer cells can be subcultured and divided into new culture dishes or culture bottles at a ratio of 1:2 to 1:3 or after counting the undifferentiated HGC-27 gastric cancer cells, and cultured in a cell culture incubator at 37℃ and 5% v/v CO2.

(2)细胞计数：待胃癌未分化细胞HGC-27生长至密度70％～80％之间，弃原培养液，用1X磷酸盐缓冲液(洗涤细胞2次)。加入1mL含0.25wt％胰蛋白酶，常温下放置30s～1min，相差显微镜下见胃癌未分化细胞HGC-27收缩变圆后加入3mL完全培养液终止消化。吹打使胃癌未分化细胞HGC-27落成单细胞悬液，将胃癌未分化细胞HGC-27转移至15mL离心管中，1000rpm离心3min。弃上清液，加入培养基吹打混匀细胞，将胃癌未分化细胞HGC-27制成单细胞悬液。使用细胞计数板计数，遵循计上不计下，计左不计右的原则，二次重复计数误差不应超过±5％，算出所得胃癌未分化细胞HGC-27悬液浓度。(2) Cell counting: When the undifferentiated gastric cancer cells HGC-27 grow to a density of 70% to 80%, discard the original culture medium and use 1X phosphate buffer (wash the cells twice). Add 1mL of 0.25wt% trypsin and place at room temperature for 30s to 1min. After the undifferentiated gastric cancer cells HGC-27 shrink and become round under a phase contrast microscope, add 3mL of complete culture medium to terminate digestion. Puff to make the undifferentiated gastric cancer cells HGC-27 fall into a single cell suspension, transfer the undifferentiated gastric cancer cells HGC-27 to a 15mL centrifuge tube, and centrifuge at 1000rpm for 3min. Discard the supernatant, add culture medium and blow to mix the cells, and make the undifferentiated gastric cancer cells HGC-27 into a single cell suspension. Use a cell counting plate to count, follow the principle of counting the top but not the bottom, and counting the left but not the right, and the error of the second repeated counting should not exceed ±5%, and calculate the concentration of the obtained undifferentiated gastric cancer cell HGC-27 suspension.

(3)细胞铺板：铺一定量的细胞数及培养基体积将胃癌未分化细胞HGC-27接种到各型号培养板或培养皿上，胃癌未分化细胞HGC-27长至约为70％～80％时进行后续实验。 (3) Cell plating: A certain amount of cells and culture medium volume are plated to inoculate undifferentiated gastric cancer HGC-27 cells onto various types of culture plates or dishes. Subsequent experiments are performed when the undifferentiated gastric cancer HGC-27 cells grow to about 70% to 80%.

2、桤木酮对胃癌未分化细胞HGC-27增殖的抑制作用2. Inhibitory effect of alderone on proliferation of undifferentiated gastric cancer cells HGC-27

将处于对数生长期的胃癌未分化细胞HGC-27在胰酶消化后，用完全培养液重悬，用细胞计数板进行细胞计数后配成5×103个细胞/100μL的细胞悬液，将细胞悬液接种于96孔板中，每孔100μL的细胞悬液，然后将其置于37℃、5％v/vCO2饱和湿度培养箱中进行培养过夜。用不同浓度的桤木酮(如0μg/ml、15μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml)处理胃癌未分化细胞HGC-27，每个浓度设置3个复孔，分别于24h、48h和72h用CCK-8试剂盒检测细胞活性，如图1。After trypsin digestion, the undifferentiated gastric cancer cells HGC-27 in the logarithmic growth phase were resuspended in complete culture medium, and the cells were counted using a cell counting plate to prepare a cell suspension of 5×103 cells/100 μL. The cell suspension was inoculated in a 96-well plate, with 100 μL of cell suspension in each well, and then placed in a 37°C, 5% v/v CO2 saturated humidity incubator for overnight culture. The undifferentiated gastric cancer cells HGC-27 were treated with different concentrations of alderone (such as 0 μg/ml, 15 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml), and 3 replicates were set for each concentration. The cell activity was detected by CCK-8 kit at 24h, 48h and 72h, respectively, as shown in Figure 1.

在图1中，使用浓度为0μg/ml、15μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml桤木酮作用胃癌未分化细胞HGC-27后，然后再使用细胞毒性检测试剂盒检测不同浓度桤木酮及在不同时间点对细胞的毒性作用，结果显示桤木酮对胃癌未分化细胞HGC-27增殖有明显抑制作用并呈时间及浓度依赖性。当桤木酮的浓度从40μg/ml升至50μg/ml时，细胞生长抑制率增加放缓，因此桤木酮的最佳浓度为40μg/ml。In Figure 1, after using alnus ketone at concentrations of 0μg/ml, 15μg/ml, 20μg/ml, 30μg/ml, 40μg/ml, and 50μg/ml to act on gastric cancer undifferentiated cells HGC-27, a cytotoxicity detection kit was used to detect the toxic effects of alnus ketone at different concentrations and at different time points on the cells. The results showed that alnus ketone had a significant inhibitory effect on the proliferation of gastric cancer undifferentiated cells HGC-27 and was time- and concentration-dependent. When the concentration of alnus ketone increased from 40μg/ml to 50μg/ml, the cell growth inhibition rate increased slowly, so the optimal concentration of alnus ketone was 40μg/ml.

3、桤木酮对HGC-27细胞凋亡诱导作用3. Effect of Alnus ketone on apoptosis in HGC-27 cells

3.1、蛋白印迹或免疫印迹(WB)检测相关凋亡蛋白3.1. Detection of related apoptosis proteins by Western blotting or immunoblotting (WB)

取对数生长期的胃癌未分化细胞HGC-27接种于培养皿中，细胞贴壁后，用不同浓度的桤木酮(如0μg/ml、15μg/ml、20μg/ml、30μg/ml、40μg/ml)处理胃癌未分化细胞HGC-27在48h后，收集上清液和贴壁细胞，用RIPA Buffer蛋白裂解液和超声细胞破碎仪裂解提取细胞蛋白并制备蛋白样品，进行SDS-PAGE凝胶电泳，将蛋白电转至PVDF膜。5％脱脂奶粉封闭液孵育1h，一抗(如抗PARP抗体、抗Bcl-2抗体、抗Mcl-1抗体、抗Bcl-xL抗体、抗p-JNK抗体、抗Bak抗体和抗JNK抗体)孵育过夜，二抗室温孵育1h～2h。用Azure BiosystemsC500近红外成像系统图像采集，如图2和图3。Gastric cancer undifferentiated cells HGC-27 in the logarithmic growth phase were inoculated in a culture dish. After the cells adhered to the wall, the gastric cancer undifferentiated cells HGC-27 were treated with different concentrations of alderone (such as 0μg/ml, 15μg/ml, 20μg/ml, 30μg/ml, 40μg/ml). After 48 hours, the supernatant and adherent cells were collected, and the cell proteins were extracted and prepared by lysis with RIPA Buffer protein lysis buffer and ultrasonic cell disruptor. SDS-PAGE gel electrophoresis was performed and the proteins were transferred to PVDF membranes. Incubate with 5% skim milk powder blocking solution for 1 hour, incubate with primary antibodies (such as anti-PARP antibody, anti-Bcl-2 antibody, anti-Mcl-1 antibody, anti-Bcl-xL antibody, anti-p-JNK antibody, anti-Bak antibody and anti-JNK antibody) overnight, and incubate with secondary antibodies at room temperature for 1 hour to 2 hours. Images were collected using the Azure Biosystems C500 near-infrared imaging system, as shown in Figures 2 and 3.

在图2中，GAPDH为内参，Cleaved PARP是促凋亡蛋白，Bcl-2、Bcl-xL和Mcl-1是抑凋亡蛋白。在图2中显示，随着桤木酮的浓度升高，促凋亡蛋白表达呈梯度上调，而抑凋亡蛋白呈梯度显著下调，由此可见桤木酮对细胞凋亡具有浓度依赖性的促进作用。In Figure 2, GAPDH is the internal reference, Cleaved PARP is a pro-apoptotic protein, and Bcl-2, Bcl-xL, and Mcl-1 are anti-apoptotic proteins. As shown in Figure 2, with the increase of the concentration of alder ketone, the expression of pro-apoptotic proteins increased gradually, while the expression of anti-apoptotic proteins decreased significantly gradually, which shows that alder ketone has a concentration-dependent promoting effect on cell apoptosis.

在图3中，GAPDH为内参，随着桤木酮的浓度升高，p-JNK蛋白和Bak蛋白表达显著上调，而Bcl-2蛋白和Bcl-xL蛋白表达下调。c-Jun氮末端激酶(JNK)信号通路是参与启动细胞凋亡重要通路，被细胞外刺激激活后发挥生物学效应，主要是介导分化、增殖、凋亡等生理功能。Bak蛋白是Bcl2促凋亡蛋白的主要家族成员，也是凋亡细胞死亡的必需分子。由此可见，桤木酮可通过激活JNK信号通路诱导人胃癌细胞HGC-27细胞凋亡，并通过JNK/Bak信号通路、JNK/Bcl-2信号通路调控胃癌细胞的线粒体凋亡。In Figure 3, GAPDH is the internal reference. As the concentration of alder ketone increases, the expression of p-JNK protein and Bak protein is significantly upregulated, while the expression of Bcl-2 protein and Bcl-xL protein is downregulated. The c-Jun N-terminal kinase (JNK) signaling pathway is an important pathway involved in the initiation of cell apoptosis. After being activated by extracellular stimuli, it exerts biological effects, mainly mediating physiological functions such as differentiation, proliferation, and apoptosis. Bak protein is a major family member of Bcl2 pro-apoptotic proteins and an essential molecule for apoptotic cell death. It can be seen that alder ketone can induce apoptosis of human gastric cancer cell HGC-27 cells by activating the JNK signaling pathway, and regulate the mitochondrial apoptosis of gastric cancer cells through the JNK/Bak signaling pathway and the JNK/Bcl-2 signaling pathway.

3.2、流式细胞技术仪检测细胞凋亡率 3.2. Detection of cell apoptosis rate by flow cytometry

按实验要求处理胃癌未分化细胞HGC-27在48h后，收集上清细胞及消化后的细胞，1×PBS缓冲液洗涤2次；用500μL1×Binding Buffer缓冲液重悬细胞，取100μL细胞溶液转移到流式管中；加入5μLFITC Annexin V和5μL碘化丙啶(PI)染色液，混匀室温避光孵育15min，加入400μL 1×Binding Buffer缓冲液，混匀，进行流式细胞仪检测，结果如图4。After 48 hours of treatment of undifferentiated gastric cancer cells HGC-27 according to the experimental requirements, the supernatant cells and digested cells were collected and washed twice with 1×PBS buffer; the cells were resuspended with 500μL 1×Binding Buffer, and 100μL of the cell solution was transferred to the flow tube; 5μL FITC Annexin V and 5μL propidium iodide (PI) staining solution were added, mixed and incubated at room temperature in the dark for 15 minutes, 400μL 1×Binding Buffer was added, mixed, and detected by flow cytometry. The results are shown in Figure 4.

在图4中，用AnnexinV-FITC及PI双染法检测细胞凋亡率，结果显示随着桤木酮浓度增加，细胞早期凋亡率(Q3)及晚期凋亡率(Q2)也逐渐升高，这表明桤木酮对细胞凋亡的诱导作用呈浓度依赖性。In Figure 4, the cell apoptosis rate was detected by AnnexinV-FITC and PI double staining. The results showed that with the increase of alnus ketone concentration, the early apoptosis rate (Q3) and late apoptosis rate (Q2) of cells also gradually increased, indicating that the induction effect of alnus ketone on cell apoptosis was concentration-dependent.

综上所述，桤木酮在一定浓度范围内对胃癌未分化细胞HGC-27增殖抑制作用呈时间-浓度依赖性，其中桤木酮通过上调相关促凋亡蛋白和下调相关抑凋亡蛋白的表达，或者上调p-JNK、Bak蛋白的表达从而诱导胃癌未分化细胞HGC-27凋亡的发生，并随着浓度增加胃癌细胞凋亡率也随之增加，桤木酮对胃癌细胞有明显的促凋亡作用。In summary, the inhibitory effect of alderone on the proliferation of undifferentiated gastric cancer cells HGC-27 was time-concentration dependent within a certain concentration range. Alderone induced apoptosis of undifferentiated gastric cancer cells HGC-27 by upregulating the expression of related pro-apoptotic proteins and downregulating the expression of related anti-apoptotic proteins, or upregulating the expression of p-JNK and Bak proteins. The apoptosis rate of gastric cancer cells increased with increasing concentration. Alderone had a significant pro-apoptotic effect on gastric cancer cells.

实施例10Example 10

桤木酮在治疗胃癌药物中的应用，在实施例9的验证桤木酮对胃癌细胞有明显的促凋亡作用，因此将桤木酮作为原料制备成治疗胃癌药物，对胃癌细胞也具有相同的抑制作用。The application of alnus ketone in the drug for treating gastric cancer. In Example 9, it was verified that alnus ketone has a significant apoptosis-promoting effect on gastric cancer cells. Therefore, alnus ketone is used as a raw material to prepare a drug for treating gastric cancer, which also has the same inhibitory effect on gastric cancer cells.

最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。 Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims (10)

桤木酮在制备治疗胃癌药物中的用途。Use of alnus ketone in preparing medicine for treating gastric cancer. 根据权利要求1所述的桤木酮在制备治疗胃癌药物中的用途，其特征在于：桤木酮抑制胃癌未分化细胞HGC-27的增殖。The use of alnus ketone in the preparation of a drug for treating gastric cancer according to claim 1, characterized in that alnus ketone inhibits the proliferation of undifferentiated gastric cancer cells HGC-27. 根据权利要求1所述的桤木酮在制备治疗胃癌药物中的用途，其特征在于：桤木酮通过下调抑凋亡蛋白的表达，诱导胃癌细胞凋亡的发生。The use of alnus ketone in the preparation of a drug for treating gastric cancer according to claim 1, characterized in that alnus ketone induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic proteins. 根据权利要求1所述的桤木酮在制备治疗胃癌药物中的用途，其特征在于：桤木酮通过上调促凋亡蛋白的表达，诱导胃癌细胞凋亡的发生。The use of alnus ketone in the preparation of a drug for treating gastric cancer according to claim 1, characterized in that alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of pro-apoptotic proteins. 根据权利要求3所述的桤木酮在制备治疗胃癌药物中的用途，其特征在于：所述抑凋亡蛋白为Bcl-2蛋白、Bcl-xL蛋白或者Mcl-1蛋白。The use of alnus ketone in the preparation of a drug for treating gastric cancer according to claim 3, characterized in that the anti-apoptotic protein is Bcl-2 protein, Bcl-xL protein or Mcl-1 protein. 根据权利要求4所述的桤木酮在制备治疗胃癌药物中的用途，其特征在于：所述促凋亡蛋白为Cleaved PARP蛋白。The use of alnus ketone in the preparation of a drug for treating gastric cancer according to claim 4, characterized in that the pro-apoptotic protein is Cleaved PARP protein. 根据权利要求1所述的桤木酮在制备治疗胃癌药物中的用途，其特征在于：桤木酮通过上调p-JNK蛋白或者Bak蛋白的表达，诱导胃癌细胞凋亡的发生。The use of alnus ketone in the preparation of a drug for treating gastric cancer according to claim 1, characterized in that alnus ketone induces apoptosis of gastric cancer cells by upregulating the expression of p-JNK protein or Bak protein. 根据权利要求1所述的桤木酮在制备治疗胃癌药物中的用途，其特征在于：桤木酮的作用靶点为JNK信号通路、JNK/Bak信号通路、JNK/Bcl-2信号通路中至少一种。The use of alnus ketone in the preparation of a drug for treating gastric cancer according to claim 1, characterized in that the target of alnus ketone is at least one of the JNK signaling pathway, the JNK/Bak signaling pathway, and the JNK/Bcl-2 signaling pathway. 根据权利要求1所述的桤木酮在制备治疗胃癌药物中的用途，其特征在于：桤木酮溶于二甲基亚砜，且浓度为10μg/ml～50μg/ml。The use of alnus ketone in the preparation of a drug for treating gastric cancer according to claim 1, characterized in that alnus ketone is dissolved in dimethyl sulfoxide and the concentration is 10 μg/ml to 50 μg/ml. 根据权利要求9所述的桤木酮在制备治疗胃癌药物中的用途，其特征在于：所述浓度为15μg/ml～40μg/ml。 The use of alnus ketone in the preparation of a drug for treating gastric cancer according to claim 9, characterized in that the concentration is 15 μg/ml to 40 μg/ml.
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