The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells is characterized in that the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on liver cancer HepG-2 cells based on a p53 target spot, and comprises the following steps:

the following detection steps are not in sequence;

1. detecting the influence of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell proliferation;

1.1 determination of MTT in vitro anti-tumor activity, detecting the proliferation and survival condition of HepG-2 cells;

1.2IncuCyte living cell imaging system, detecting the cell fusion rate;

2. detecting the influence of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell migration;

3. detecting the influence of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell apoptosis;

3.1 staining Hoechst33258 to detect apoptosis;

3.2Annexin V/PI double staining to detect apoptosis;

4. detecting the influence of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the HepG-2 cell cycle;

5. detecting the change condition of related proteins in vivo after 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HepG-2 cells;

5.1 Western blot experiment;

5.2 Small interfering RNA cell transfection experiment.

2. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells according to claim 1, wherein the step 1.1 determination of the in vitro anti-tumor activity of MTT specifically comprises:

(1) plate preparation: push buttonAccording to 4X 10⁴Taking HepG-2 cells at the density of each ml, inoculating the HepG-2 cells into a 96-well plate, and culturing the cells;

(2) treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: when the cells grow to 50% -60%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given; the concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is 1.56. mu.M, 3.125. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M;

(3) MTT detection: adding MTT stock solution in a dark place, mixing uniformly, culturing in an incubator, discarding old culture medium, adding DMSO to dissolve formazan crystal, shaking, and detecting OD value at 490nm wavelength;

(4) calculating the survival rate of the cells:

wherein the experimental group is a treatment group with different concentrations, and the control group is a group added with DMSO;

(5) compound IC₅₀The measurement of (1).

3. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells according to claim 1, wherein the step 1.2IncuCyte live cell imaging system is used for detecting the cell fusion rate, and comprises the following specific steps:

(1) plate preparation: according to 4X 10⁴Taking HepG-2 cells at a cell density of each ml, and plating the HepG-2 cells in a 96-well plate to divide the HepG-2 cells into a control group and a 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group;

(2) treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: after the cells are attached to the wall, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is given for treatment, and the method is the same as the step 1 (2);

(3) data processing: and (3) placing the 96-well plate in an IncuCyte living cell imaging system, collecting cell image dynamics at regular time, and calculating the cell fusion rate under different treatment conditions.

4. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells according to claim 1, wherein the step 2 is to detect the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell migration, and comprises the following specific steps:

(1) plate preparation: according to 4X 10⁴Taking HepG-2 cells at the cell density of each ml, and laying the HepG-2 cells in a 96-well plate for cell culture;

(2) scratching and administration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: after the cells are full, abandoning the culture medium, scratching, washing with a buffer solution, and treating with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene at a concentration of 0.4 μ M;

(3) data processing: in the IncuCyte live cell imaging system, photographs were taken at different time points and the mobility of the cells was analyzed at each time point.

5. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells according to claim 1, wherein the step 3.1Hoechst 33258 staining detects apoptosis, and comprises the following specific steps:

(1) plate preparation: according to the formula of 7.5X 10⁴Taking HepG-2 cells with the cell density of each ml, and laying the HepG-2 cells in a six-hole plate for culture;

(2) treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given; the concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is 1.56 mu M and 3.125 mu M;

(3) fixing: after HepG-2 cells are treated by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48 hours, 4 percent of paraformaldehyde is added, and the mixture is fixed at normal temperature in a dark place;

(4) dyeing: discarding the stationary liquid, adding Hoechst33258 staining solution, and incubating at 37 ℃ in a dark place;

(5) and (3) observation by a fluorescence microscope: the cells were washed with buffer, observed under a fluorescent microscope for morphology, and photographed.

6. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells according to claim 1, wherein the step 3.2Annexin V/PI double staining is used for detecting apoptosis, and comprises the following specific steps:

(1) plate preparation: according to 7.5X 10⁴Taking HepG-2 cells with the cell density of each ml, inoculating the HepG-2 cells into a six-hole plate, and culturing;

(2) treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given; the concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is 1.56 mu M, 3.125 mu M and 6.25 mu M;

(3) collecting cells: treating 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h, and collecting cells;

(4) grouping: respectively adding 1 × Annexin V binding Solution into a control group and a 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group; the control component is a negative control group, an Annexin V-FITC staining group, a PI staining group, an Annexin V-FITC and PI double staining group; the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene component is Annexin V-FITC and PI double staining group;

(5) dyeing: adding Annexin V-FITC conjugate into each group in the step (4), uniformly mixing, adding PI Solution, uniformly mixing, and culturing at room temperature in a dark place;

(6) flow detection: adding 1 × Annexin V Binding Solution, and performing machine detection within 1 h.

7. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells according to claim 1, wherein the step 4 is to detect the cell cycle by adopting PI single staining in combination with a flow cytometer, and comprises the following specific steps:

(1) plate preparation: according to 7.5X 10⁴Taking HepG-2 cells with the cell density of each ml, inoculating the HepG-2 cells into a six-hole plate, and culturing;

(2) treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given; the concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is 3.125 mu M;

(3) collecting cells: collecting cells after 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts for 36 hours;

(4) ethanol fixation: adding 70% ethanol, and fixing at 4 deg.C;

(5) propidium iodide staining: after the ethanol is fixed, adding propidium iodide staining solution into each tube of cell sample, and carrying out light-shielding warm bath at 37 ℃;

(6) flow detection: cell cycle assays were performed using a flow cytometer over 1 h.

8. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells according to claim 1, wherein the step 5.1 is performed by Western blotting, and comprises the following steps:

(1) extraction of total cellular protein:

plate preparation: according to 7.5X 10⁴Taking HepG-2 cells with the cell density of each ml, inoculating the HepG-2 cells into a six-hole plate, and culturing;

treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given; the concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is 1.56 mu M, 3.125 mu M and 6.25 mu M;

collecting cells: after the intervention of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h, collecting cells;

cell lysis: adding cracking mixed solution, cracking for 30min, then performing ultrasonic treatment, and centrifuging; the cracking mixed solution is a mixed solution with the volume ratio of RIPA to PMSF to phosphatase inhibitor being 100:1: 1;

(2) BCA assay protein concentration

Diluting a standard product: diluting 0.5mg/ml protein standard;

dilution of the protein to be tested: diluting by 40 times;

loading a 96-well plate: adding a protein standard diluent and a protein diluent to be detected into a 96-well plate, and then adding a BCA working solution;

and (3) incubation: incubating at 37 ℃ in the dark;

measuring absorbance by an enzyme-linked immunosorbent assay (ELIAS): measuring the absorbance of each hole under A562nm, and drawing a standard curve by taking the concentration as an abscissa and the absorbance as an ordinate;

(3) denaturation of proteins

Adding 5 Xloading buffer solution into protein sample, and performing constant temperature denaturation at 100 ℃;

(4) glue preparation

And (3) leak detection: adding deionized water, and detecting whether glue leaks or not;

preparing glue: respectively preparing separation gel and concentrated gel, adding the separation gel into the plate, adding the concentrated gel after solidification, inserting a 9-hole comb, and standing for gel polymerization of the concentrated gel;

(5) electrophoretic preparation and sampling

The installation device comprises: adding 1X electrophoresis liquid into an electrophoresis tank, pulling a comb to remove bubbles, and designing a loading pore channel;

protein sample and Marker loading: loading 15ul-20ul of protein denaturation sample on each pore channel, and adding a Marker with the same volume as the protein;

electrophoresis: keeping the voltage at 80V, adjusting the voltage to 120V after the bromophenol blue runs through the concentrated gel, and stopping electrophoresis when the bromophenol blue runs to the bottom of the concentrated gel and does not run out;

(6) film transfer: removing concentrated gel without protein by using a wane, respectively spreading a layer of sponge on black and white of a transfer membrane clamp, respectively adding 4 layers of filter paper, soaking, spreading, placing the gel on the black transfer membrane clamp, activating a PVDF membrane in methanol, transferring to the gel, closing the transfer membrane clamp, and transferring to a membrane;

(7) sealing milk: transferring the PVDF membrane into milk sealing liquid, and sealing at room temperature;

(8) primary antibody incubation: adding primary antibody according to the size of the membrane, and incubating at 4 ℃;

(9) and (3) secondary antibody incubation: adding a secondary antibody according to the size of the membrane, and incubating at room temperature;

(10) and (3) developing: mixing ECl hypersensitive luminescent solution A and solution B in equal volume, covering the mixture on the surface of a membrane, and performing machine loading and development;

(11) and (4) analyzing results: images were collected and analyzed using an Image lab gel analysis system.

9. The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells according to claim 1, wherein the step 5.2 is a small interfering RNA cell transfection experiment, and comprises the following specific steps:

(1) plate preparation: according to 7.5X 10⁴Taking HepG-2 cells with the cell density of/ml, inoculating the HepG-2 cells into a six-hole plate, and culturing;

(2) transfection: adding a transfection reagent after the cell density reaches 30% -50%, and performing transfection; the Transfection reagent is siRNA control, p53 siRNA #1, p53 siRNA #2, p53 siRNA #3, jetPRIME Buffer, jetPRIME Transfection reagent;

(3) after transfection for 16h, collecting transfected cells, counting again, inoculating the cells to a 96-well plate, culturing for 10h, and treating with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene; the concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is 1.56 mu M and 3.125 mu M;

(4) MTT assay and analytical data: and after 48h, MTT detection is carried out to calculate the survival rate, and images are collected in a fixed time mode in an IncuCyte zooming live cell imaging system and used for analyzing the survival condition of the cells.

10. The application of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene in the aspect of resisting liver cancer is characterized in that the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene inhibits migration of HepG-2 cells, induces apoptosis of the HepG-2 cells, induces G0/G1 phase block of the HepG-2 cells, and is applied to preparation of a medicine for treating and/or preventing liver cancer.

Background

Liver cancer refers to malignant tumor derived from liver cells and hepatobiliary cells, and is classified into primary and secondary malignant tumors. Common symptoms of early liver cancer are not specific, the symptoms of middle and late liver cancer are more, and common clinical manifestations include liver region pain, abdominal distension, anorexia, hypodynamia, emaciation, progressive hepatomegaly or epigastric mass and the like; some patients have low fever, jaundice, diarrhea, upper gastrointestinal hemorrhage, etc. The treatment of primary liver cancer comprises systemic chemotherapy, targeted therapy and biological therapy, the treatment of secondary liver cancer patients comprises surgical treatment, and conservative treatment can be adopted when the surgical treatment is ineffective.

The choice of treatment modality depends on the location of the tumor, the degree of malignancy, the degree of progression, and the physical state of the patient. The treatment of liver cancer, whether chemotherapy, surgery or radiotherapy, is a great burden on the body, and after malignant metastasis, it is difficult to cure the cancer completely in any way. Oncogene, oncogene suppressor gene discovery, and the theory of apoptosis have been developed, and their understanding has also been carried out from the cellular level to the molecular level. Cancer is related to changes of a plurality of oncogenes and cancer suppressor genes, information transmission and protease activity related to apoptosis are researched, and a new means for treating cancer is hopefully provided.

In recent years, some progress is made in the research of the cancer suppressor gene p53, and the target is widely concerned as a target drug for researching and treating cancer. The MDM2 protein is the most important negative regulator of p53, and is involved in regulating the stability and activity of p53 protein, inhibiting cell growth, inducing apoptosis and regulating cell cycle function. However, the application of the p53 target and the MDM2-p53 protein and protein action pathway in the preparation of antitumor drugs for treating and/or preventing human liver cancer is only reported.

Disclosure of Invention

The invention aims to provide a method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on liver cancer HepG-2 cells based on a p53 target spot, and also provides application of the method in the aspect of resisting liver cancer.

In order to realize the purpose, the following technical scheme is adopted:

apoptosis is one of the programmed forms of cell death and is an important form of cell homeostasis, whether endogenous or exogenous, induced by the target gene p 53. Combining with a flow cytometer, Hoechst33258, the method proves that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can induce HepG-2 cells to generate apoptosis in a concentration-dependent manner, and the cell cycle is blocked at the G0/G1 stage. This is consistent with the expression that activation of the p53 pathway inhibits cell cycle, promotes damaged cellular DNA repair, and promotes apoptosis when normal cells are damaged. In order to further discuss the action mechanism of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for inducing HepG-2 cells to generate apoptosis, Western Blot is used for detecting the influence on the expression of proteins such as a p53 channel, a Bcl-2 family (Bax, Bcl-2 and Mcl-1) and the like. The results showed that in HepG-2 cells, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene increased the expression level of p53 protein, increased the Bax/Bcl-2 ratio, and found that the expression level of MDM2, a downstream target gene of p53, was decreased. As is known, p53 and MDM2 have negative feedback regulation, and the expression of MDM2 protein is reduced, so that p53 protein is aggregated in vivo, the expression level is increased, and the normal biological function is exerted. Therefore, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene was shown to act on the p53-MDM2 pathway, blocking the inverse regulation of p53 by MDM 2. In order to further prove whether the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is dependent on p53 for exerting the anti-tumor activity, a cell p53 siRNA knockdown experiment is carried out on the HepG-2 cell, the result shows that the survival rate of the p53 siRNA + Drug group cell is obviously higher than that of the Control siRNA + Drug group, and p53 participates in the anti-proliferation activity of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the HepG-2 cell. Based on the research, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene has potential to develop anti-liver cancer therapeutic drugs.

In conclusion, the invention proves that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene inhibits HepG-2 cell proliferation in the G0/G1 phase through cell cycle arrest, plays an anti-tumor role through inducing apoptosis, and realizes an inhibition effect through activating P53, and the specific mechanism is based on a P53 target and MDM2-P53 protein and protein action pathways, as shown in figure 1.

The method for detecting the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on liver cancer HepG-2 cells based on a p53 target point specifically comprises the following steps:

the following tests are not in order.

1. The influence of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell proliferation was examined.

1.1 assay of MTT in vitro antitumor Activity to detect proliferation and survival of HepG-2 cells.

MTT is known as 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide. The detection principle is that exogenous MTT can be reduced into water-insoluble blue-purple crystalline Formazan (Formazan) by succinate dehydrogenase in mitochondria of living cells and deposited in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value is measured by an enzyme-labeling instrument at 490nm wavelength, and the MTT crystal formation amount is in direct proportion to the number of cells in a certain cell number range. The number of living cells was judged from the measured absorbance value (OD value), and the larger the OD value, the stronger the cell activity (in this experiment, the lower the drug toxicity).

The method comprises the following specific steps:

(1) plate preparation: according to 4X 10⁴HepG-2 cells were harvested at a density of one cell per ml, seeded into a 96-well plate, and cultured.

(2) Treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: when the cells grow to 50% -60%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given; the concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is 1.56. mu.M, 3.125. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M.

(3) MTT detection: adding MTT stock solution in dark place, mixing, culturing in an incubator, discarding old culture medium, adding DMSO to dissolve formazan crystal, shaking, and detecting OD at 490nm wavelength.

(4) Calculating the survival rate of the cells:

wherein, the experimental groups are treatment groups with different concentrations. The control group was a group to which DMSO was added.

(5) Compound IC₅₀The measurement of (1).

1.2IncuCyte live cell imaging system, and detecting the cell fusion rate.

The method comprises the following specific steps:

(1) plate preparation: according to 4X 10⁴At a cell density of one/ml, HepG-2 cells were plated in a 96-well plate and divided into a control group and a drug concentration group (1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group).

(2) Treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: after the cells are attached to the wall, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment is given. The method is the same as the steps 1 and 2.

(3) Data processing: and (3) placing the 96-well plate in an IncuCyte living cell imaging system, collecting cell image dynamics at regular time, and calculating the cell fusion rate under different treatment conditions.

2. The effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell migration was used to quantify the potential for cell mass spreading.

The method comprises the following specific steps:

(1) plate preparation: according to 4X 10⁴At cell density of one/ml, HepG-2 cells were plated in 96-well plates and cultured.

(2) Scratching and administration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: after the cells were confluent, the medium was discarded, scratched, washed with buffer, and treated with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene at a concentration of 0.4. mu.M (2 IC) for 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene₅₀)。

(3) Data processing: in the IncuCyte live cell imaging system, photographs were taken at different time points and the mobility of the cells was analyzed at each time point.

3. Effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 apoptosis.

3.1Hoechst 33258 staining to detect apoptosis

The method comprises the following specific steps:

(1) plate preparation: according to 7.5X 10⁴At cell density of one/ml, HepG-2 cells were plated in six-well plates and cultured.

(2) Treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (1.56 μ M, 3.125 μ M) treatment is given.

(3) Fixing: after HepG-2 cells are treated by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48 hours, 4 percent of paraformaldehyde is added, and the mixture is fixed at normal temperature in a dark place.

(4) Dyeing: the fixative solution is discarded, and Hoechst33258 staining solution is added, and incubation is carried out at 37 ℃ in the dark.

(5) And (3) observation by a fluorescence microscope: the cells were washed with buffer, observed under a fluorescent microscope for morphology, and photographed.

3.2Annexin V/PI double staining to detect apoptosis and to distinguish early and late apoptosis, necrosis and normal cells.

The method comprises the following specific steps:

(1) plate preparation: according to 7.5X 10⁴HepG-2 cells were seeded at a cell density of one cell/ml in a six-well plate and cultured.

(2) Treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (1.56. mu.M, 3.125. mu.M, 6.25. mu.M) treatment is given.

(3) Collecting cells: and (3) treating the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48 hours, and collecting cells.

(4) Grouping: the control group (DMSO group) and the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group were added with 1 × Annexin V binding Solution, respectively. The control component is a negative control group, an Annexin V-FITC staining group, a PI staining group, an Annexin V-FITC and PI double staining group; the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene component is Annexin V-FITC and PI double staining group.

(5) Dyeing: and (4) adding Annexin V-FITC conjugate into each group in the step (4), uniformly mixing, adding PI Solution, uniformly mixing, and culturing at room temperature in a dark place.

(6) Flow detection: adding 1 × Annexin V Binding Solution, and performing machine detection within 1 h.

4. Effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell cycle

4.1PI Monostain in combination with flow cytometer for cell cycle detection

The method comprises the following specific steps:

(1) plate preparation: according to 7.5X 10⁴HepG-2 cells were seeded at a cell density of one cell/ml in a six-well plate and cultured.

(2) Treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: when the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (3.125 μ M) treatment is given.

(3) Collecting cells: cells are collected after 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts for 36 hours.

(4) Ethanol fixation: adding 70% ethanol, and fixing at 4 deg.C.

(5) Propidium iodide staining: after ethanol fixation, propidium iodide staining solution was added to each tube of cell sample, and the tube was incubated at 37 ℃ in the dark.

(6) Flow detection: cell cycle assays were performed using a flow cytometer over 1 h.

5. The change condition of related proteins in vivo after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HepG-2 cells.

5.1 Western Blot assay (Western Blot analysis)

The method comprises the following specific steps:

(1) extraction of total cellular protein:

plate preparation: according to 7.5X 10⁴HepG-2 cells were seeded at a cell density of one cell/ml in a six-well plate and cultured.

Treatment with 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene: after the cells grow to 60% -70%, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (1.56. mu.M, 3.125. mu.M, 6.25. mu.M) treatment is given.

Collecting cells: and (3) after the intervention of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h, collecting cells.

Cell lysis: adding lysis mixture (volume ratio of RIPA: PMSF: phosphatase inhibitor is 100:1:1), lysing for 30min, then performing ultrasonic treatment, and centrifuging.

(2) BCA assay protein concentration

Diluting a standard product: 0.5mg/ml protein standard was diluted.

Dilution of the protein to be tested: diluting by 40 times

Loading a 96-well plate: and adding a protein standard diluent and a protein diluent to be detected into the 96-well plate, and then adding a BCA working solution.

And (3) incubation: incubate at 37 ℃ in the dark.

Measuring absorbance by an enzyme-linked immunosorbent assay (ELIAS): the absorbance of each well at A562nm was measured, and a standard curve was drawn with the concentration as the abscissa and the absorbance (OD) as the ordinate.

(3) Denaturation of proteins

5 XLoading buffer was added to the protein samples and denatured at 100 ℃.

(4) Glue preparation

And (3) leak detection: and adding deionized water, and detecting whether glue leaks or not.

Preparing glue: respectively preparing separation gel and concentrated gel, adding the separation gel into the plate, adding the concentrated gel after solidification, inserting a 9-hole comb, and standing for gel polymerization of the concentrated gel.

(5) Electrophoretic preparation and sampling

The installation device comprises: adding 1 Xelectrophoresis liquid into the electrophoresis tank, pulling out the comb to remove air bubbles, and designing a loading pore channel.

Protein sample and Marker loading: 15ul-20ul of protein denaturation sample is loaded on each pore channel, and Marker with the same volume as protein is added.

Electrophoresis: and (3) keeping the voltage at 80V, adjusting the voltage to 120V after the bromophenol blue runs through the concentrated gel, and stopping electrophoresis when the bromophenol blue runs to the bottom of the concentrated gel and does not run out.

(6) Film transfer: removing concentrated gel without protein with a wane, spreading a layer of sponge on each of black and white color of the transfer film clamp, adding 4 layers of filter paper, soaking, spreading, placing the gel on the black transfer film clamp, activating PVDF film in methanol, transferring to the gel, closing the transfer film clamp, and transferring to the film.

(7) Sealing milk: the PVDF membrane was transferred to a milk blocking solution and blocked at room temperature.

(8) Primary antibody incubation: primary antibody is added according to the size of the membrane, and the membrane is incubated in an environment at 4 ℃.

(9) And (3) secondary antibody incubation: secondary antibody was added according to the size of the membrane and incubated at room temperature.

(10) And (3) developing: mixing ECl hypersensitive luminous liquid A and liquid B in equal volume, covering the mixture on the surface of a membrane, and performing machine and development.

(11) And (4) analyzing results: images were collected and analyzed using an Image lab gel analysis system.

5.2 Small interfering RNA (siRNA) cell transfection

Small interfering RNA (siRNA) cell transfection experiments are carried out, and the small interfering RNA specifically degrades target mRNA by combining with the target gene or the mRNA of the target gene, thereby silencing the target gene.

The method comprises the following specific steps:

(1) plate preparation: according to 7.5X 10⁴Cell density per ml, HepG-2 cells were seeded in a six-well plate and cultured.

(2) Transfection: and adding a transfection reagent after the cell density reaches 30% -50%, and performing transfection. The Transfection reagent is siRNA control, p53 siRNA #1, p53 siRNA #2, p53 siRNA #3, jetPRIME Buffer, jetPRIME Transfection reagent.

(3) After 16h of transfection, transfected cells were collected, re-counted and plated in 96-well plates, and after 10h of culture, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (1.56. mu.M, 3.125. mu.M) treatment was given.

(4) MTT assay and analytical data: and after 48h, MTT detection is carried out to calculate the survival rate, and images are collected in a fixed time mode in an IncuCyte zooming live cell imaging system and used for analyzing the survival condition of the cells.

The invention has the beneficial effects that:

(1) the invention discovers that the action mechanism of the compound 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (Mulanphenanthrene) for treating or preventing liver cancer is a new action mechanism based on an MDM2-p53 protein-protein interaction signal channel and a p53 target spot, and the invention provides a scientific theoretical basis for clinical application of the compound.

(2) Cell scratch experiments prove that the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can inhibit the migration of HepG-2 cells; the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can induce apoptosis through Hoechst33258 fluorescent staining, annexin V-FITC/PI double staining and PI single staining apoptosis detection; 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can induce G0/G1 phase block of HepG-2 cells as proved by a PI single staining cell cycle method.

(3)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene plays an anti-tumor role by activating p53 protein, and the pathway of the anti-tumor role is the p53-MDM2 pathway and the p53 action target point.

Drawings

FIG. 1 is a schematic diagram of the p53 target and MDM2-p53 protein and protein action pathways.

FIG. 2 BCA Standard Curve for protein concentration determination.

FIG. 31, results of experiment on HepG-2 cells inhibited by 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene; a is the result of the inhibition of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells; b, the inhibition effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene with different concentrations on HepG-2 cells; c, the inhibition effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells with different action times.

FIG. 41 shows that after 48h of action of 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, the cell morphology of the control group and the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group changes.

FIG. 5 shows the change of cell fusion rate at various time points after adding 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene to HepG-2 cells.

FIG. 61, 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene inhibits migration of HepG-2 cells; a, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HepG-2 cells for 24 hours, and the HepG-2 cells are observed under the microscope during migration; b, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HepG-2 cells for 24 hours, and the average mobility of the HepG-2 cells is improved. Statistical results are expressed as mean ± standard deviation, n is 3, P <0.05 compared to control.

FIG. 7 is a fluorescent microscope to observe the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on nuclei.

FIG. 81, 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene induces apoptosis in HepG-2 cells; a, annexin V-FITC/PI double staining to detect apoptosis; b, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HepG-2 cells for 48 hours, and the average apoptosis rate of the HepG-2 cells is increased. Statistical results are expressed as mean ± standard deviation, n is 3, P <0.01 compared to control.

FIG. 91, 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene block the HepG-2 cell cycle at G0/G1; A. PI single staining and flow cytometry detection of cell cycle; c, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HepG-2 cells for 48 hours, and the proportion of each cell cycle is accounted. Statistical results are expressed as mean ± standard deviation, n is 3, P <0.01 compared to control.

FIG. 101, protein expression of 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene after acting on HepG-2 cells.

FIG. 11 p53 mediates the inhibition of proliferation of HepG-2 cells by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene; transfection p53 siRNA, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene action HepG-2 cells for 48h, fusion rate curve of HepG-2 cells.

FIG. 12 survival of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene-treated HepG-2 cells after transfection with p53 siRNA for 48h, HepG-2 cells.

Detailed Description

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Examples

The sources of the drugs and reagents used in the examples of the invention are shown in Table 1, and the experimental instruments are shown in Table 2.

TABLE 1 drugs and reagents

TABLE 2 Experimental instruments

Preparation of reagent and test article

1.1 preparation of the test drugs:

1.00mg of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene powder was weighed out precisely and mixed with DMSO to prepare a 50mM solution.

1.2 preparation of MTT solution:

adding 50ml PBS into 250mg MTT powder in batches, fully dissolving to prepare 5mg/ml MTT solution, filtering with 0.22 mu m microporous filter membrane, subpackaging in 1.5ml brown EP tube, avoiding light and aseptic operation, sticking sealing membrane, storing in refrigerator at-20 deg.C for a long time, avoiding repeated freeze thawing, and storing in refrigerator at 4 deg.C for two weeks.

1.3 preparation of cell frozen stock solution:

20% fetal bovine serum, 70% DMEM high-sugar medium and 10% DMSO, and mixing well to prepare cell freezing medium.

1.4 preparation of complete Medium:

a complete culture medium is prepared by adding 10% (v/v) fetal calf serum and 1% (v/v) diabesin (penicillin and streptomycin) into a DMEM high-sugar culture medium.

1.5 preparation of propidium iodide staining solution:

(1) preparation of dye liquor for 1 sample:

(2) preparing propidium iodide staining solution according to the number of samples

1.6 Tris-glycine electrophoresis buffer (1 ×)

(1) Weighing the following reagents into a 1000mL beaker

Tris-base 3.03g

Glycine 14.4g

SDS 1.00g

(2) 1L of UP water was added and the dissolution was accelerated on a magnetic stirrer.

1.7 Tris-glycine electrotransfer buffer (1 ×)

(1) Weighing the following reagents into a 1000mL beaker

Tris-base 3.03g

Glycine 14.4g

(2) Placing in 1000ml beaker, adding 800ml UP water to dissolve, placing on magnetic stirrer to accelerate dissolution

(3) 200ml of methanol is measured again, and the volume is determined to be 1L. (note that methanol is volatile).

1.8 Membrane washing buffer TBST (1X)

(1) Weighing the following reagents into a 1000mL beaker

Tris-base 2.42g

NaCl 8.0g

(2) Placing in 1000mL beaker, adding 1L UP water, and stirring on magnetic stirrer

(3) 1mL of Tween 20 was added slowly and stirring was continued until homogeneous.

1.9 milk sealing liquid (5% skimmed milk)

(1) The following reagents were weighed into a 100mL beaker

Skimmed milk powder 2g

(2) 40ml of membrane washing buffer TBST was added and the mixture was thoroughly dissolved on a magnetic stirrer.

1.10 preparation of protein standards

(1) 0.8ml of the protein standard preparation solution was added to a tube of the protein standard (20mg BSA), and the mixture was dissolved sufficiently to prepare a 25mg/ml protein standard solution.

(2) The 25mg/ml protein standard solution was diluted to 0.5mg/ml with PBS. The diluted 0.5mg/ml protein standard solution can be stored for a long time at the temperature of minus 20 ℃.

1.11BCA working solution preparation

An appropriate amount of BCA working solution was prepared by adding 50 volumes of BCA reagent a to 1 volume of BCA reagent B (v: 50:1) according to the number of samples, and thoroughly mixed. The BCA working solution is stable within 24 hours at room temperature.

Second, basic culture of cells

Culturing human liver cancer cell HepG-2 with DMEM high-sugar complete culture medium, and culturing at 37 deg.C with 5% CO₂The cell culture box is used for culturing, and the experiment is carried out when the cells grow logarithmically.

2.1 cell Resuscitation

Taking out a tube of HepG-2 cells from a-80 ℃ refrigerator to rapidly obtain the HepG-2 cellsThawing, transferring into 2ml preheated DMEM high sugar medium, centrifuging at 800rmp/min for 5min, removing supernatant, adding new culture medium to resuspend cells, inoculating into culture flask, adding 5% CO at 37 deg.C₂Culturing in an incubator, and changing the culture solution after 24 h.

2.2 cell passages

When the cell density in the culture flask reaches 80% -90%, the culture medium is discarded, and the cells are washed 2 times with PBS. Adding pancreatin to digest cells (about 2min for timing, the pancreatin action time should not be too long). Observing cell rounding under microscope, tapping the bottle body slightly to make it fall off, immediately adding new complete culture medium to stop digestion, transferring it to centrifuge tube, centrifuging, discarding culture medium, adding new culture medium, uniformly distributing a bottle of HepG-2 cells into three new culture bottles at a ratio of 1:3, placing in 5% CO at 37 deg.C₂And continuing culturing in the incubator.

2.3 cell cryopreservation

When the cell density in the culture bottle is 80% -90% and the growth state is good, centrifuging according to the method of cell passage in the step 2.2, discarding the supernatant, adding a cell cryopreservation solution, blowing and uniformly mixing, sucking 1ml of cell suspension to a cryopreservation tube, marking the cryopreservation tube (cell name and cryopreservation date), putting the cryopreservation tube into a program gradient cryopreservation box, storing the cryopreservation tube in a-80 ℃ refrigerator for a short time, and storing the cryopreservation tube in liquid nitrogen for a long time.

2.4 cell count

10 μ l of the cell suspension was gently pipetted into a cell counting plate and counted under a microscope. The number of cells in each of the four grid lines was counted under the microscope according to the formula: (sum of cell numbers in four Large lattices/4). times.10⁴Cell density was calculated as number of cells/ml.

The method for detecting the effect of the tri-1, 5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cells based on the p53 target spot comprises the following steps:

the following steps are partially sequenced;

1. effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell proliferation

1.1 assay of MTT for in vitro antitumor Activity to test cell number，IC₅₀The value is obtained.

The detection principle is that exogenous MTT can be reduced into water-insoluble blue-purple crystalline Formazan (Formazan) by succinate dehydrogenase in mitochondria of living cells and deposited in the cells, while dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value is measured by an enzyme-labeling instrument at 490nm wavelength, and the MTT crystal formation amount is in direct proportion to the number of cells in a certain cell number range. The number of living cells was judged from the measured absorbance value (OD value), and the larger the OD value, the stronger the cell activity (in this experiment, the lower the drug toxicity).

The method comprises the following specific steps:

(1) plate preparation: HepG-2 cells in logarithmic growth phase are inoculated into a 96-well plate at an inoculation density of 4 multiplied by 10⁴one/mL, left overnight in the incubator.

(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: when the cell adherence is 50% -60%, the culture medium is discarded, and 100 μ l of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene with different concentration gradients diluted by DMEM complete culture medium is added. The concentrations of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group and the positive control group were set to 1.56. mu.M, 3.125. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M, and a blank control DMSO control group was designed to be 0.2% (v: v). Each group of 5 multiple wells. Placing at 37 deg.C and 5% CO₂The cultivation was continued in the incubator for 48 h.

(3) MTT detection: adding 10 μ l of MTT stock solution into each well in dark, shaking, mixing, standing at 37 deg.C and 5% CO₂After further 2 hours of incubation in the incubator, the old medium was discarded, 150. mu.l of DMSO was added to dissolve formazan crystals, and the resulting solution was shaken with a microplate reader for 5 minutes, followed by detection of OD at a wavelength of 490 nm.

(4) Calculating the survival rate of the cells:

(5) compound IC₅₀The determination of (1): curve fitting and IC calculation by Graphpad 8.0.2 software₅₀The value is obtained.

Results:1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene has strong proliferation inhibiting effect on HepG-2, and IC thereof₅₀The value is 0.2 +/-0.12 mu M, which is superior to that of positive drug cisplatin (IC)₅₀2.28 ± 0.36) and paclitaxel (IC)₅₀＝0.21±0.076)。

1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene intervenes in HepG-2 cells for 48h, and morphological observation under a microscope shows that as the concentration of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene increases, the number of cell death increases (figure 3A), the cell morphology changes, the cell begins to extend out of an antenna, and obvious shrinkage of a cell membrane can be observed (figure 4), which is similar to the result of apoptosis of the cell and the survival rate of the cell is reduced (figure 3B); 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene intervenes in HepG-2 cells for 12h, 24h and 48h respectively, and the survival rate curve of the HepG-2 cells gradually moves downwards (FIG. 3C).

The results show that the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can obviously inhibit the cell proliferation of HepG-2 and is in a concentration and time dependent relationship.

1.2IncuCyte live cell dynamic imaging system to detect cell fusion rate.

IncuCyte ZOOM, a long-time dynamic living cell observation system, records the real-time state of cells by a non-invasive method, records and tracks the cells in a real-time label-free manner, and automatically analyzes the functional indexes of the cells. The working principle of the method is that the whole growth process of cells is recorded through real-time microscopic shooting, images at different time points are automatically analyzed, and real-time detection indexes such as the number of the cells, the cell density, the apoptosis/death number, the fluorescence intensity and density, the migration and invasion speed of the cells and the like are quantified, so that the data of the whole growth process of the cells are obtained. Directly provides data such as images, change data, change curves and the like of the cell growth change process for experiments.

The method comprises the following specific steps:

(1) plate preparation: taking HepG-2 cells with good logarithmic phase growth state according to 4 multiplied by 10⁴And (3) paving the cells per ml in a 96-well plate, putting the cells into an incubator in an IncuCyte ZOOM zooming live cell imaging system, setting parameters and starting to take pictures, wherein the time interval of image acquisition is set to be 2h, and images are acquired by a 10X lens.

(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: after the cells adhere to the wall, the cells are taken out from the incubator and treated by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene. The method is the same as the steps 1 and 2.

(3) Data processing: cell areas in each image are automatically identified through analysis software arranged in the system, 4 pictures with different visual fields are collected in each hole, the cell fusion rate under different processing conditions is calculated, a curve graph is drawn, and the cell survival condition is indirectly reflected.

As a result: by acquiring images of HepG-2 cells treated by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (0-100 mu M) with different concentrations in real time and analyzing the cell fusion rate of the acquired images after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is treated for 48 hours, the result shows that the cell fusion rate curve gradually moves downwards along with the increase of the concentration of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, which indirectly reflects that the drug can generate concentration-dependent proliferation inhibition on hepatoma cells (figure 5).

2. Effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell migration.

The cell scratching test (round Healing) is an in vitro test method for researching cell migration, which is simple to operate and economical. The principle is that when the cells grow to be fused into a single layer, a blank area is artificially made on the fused single-layer cells, namely the scratch, and the cells at the edge of the scratch gradually enter the blank area to heal the scratch, so that the process of in vivo cell migration is simulated to a certain extent, and the method is the simplest method in an in vitro experiment for researching the cell migration.

The method comprises the following specific steps:

(1) plate preparation: taking HepG-2 cells with good logarithmic phase growth state according to 4 multiplied by 10⁴Cell density per ml, plated in 96-well plates, and put in 5% CO at 37 ℃₂In the incubator, the culture was performed overnight.

(2) Scratching and 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: after the cells were confluent, the medium was discarded, 100. mu.l PBS was added to each well and then scratched using a scratcher, and 1,5, 6-tris was administered after washing 2 times with PBS until no clumped cells were scraped under the microscopeMethoxy-2, 7-dihydroxyphenanthrene treatment, 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene concentration 0.4. mu.M (2 IC)₅₀) The control group was DMSO of the corresponding volume, and each group was provided with 5 duplicate wells.

(3) Data processing: the cells were placed in an IncuCyte live cell imaging system, observed and photographed at different time points, and the mobility of the cells at each time point was analyzed using IncuCyte ZOOM software.

As a result: after 24h scratching, HepG-2 cells migrated toward the inside of the scratched area in photographs taken by an Incucyte live cell dynamic imaging system, and the migration distance and the migration rate were automatically analyzed according to the system, which showed that the migration distance of the cells in the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group was significantly reduced compared to the control group (FIG. 6A). 0.4. mu.M (2 IC) in comparison with control DMSO₅₀) The cell mobility of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is reduced within 24 hours (^*P<0.05) (fig. 6B).

3. Effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 apoptosis.

After HepG-2 cells are treated by adding 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene, the cell morphology is changed, the cell volume is reduced, the connection disappears, the cells are separated from surrounding cells, the cells are in a shrinking state one by one, and the result is similar to the result of the apoptosis state of the cells, so that I firstly adopt Hoechst33258 to combine with a fluorescence microscope to carry out morphological observation on cell nuclei, and adopt Annexin V/PI double staining to combine with a flow cytometer to detect whether the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene induces the HepG-2 cells to die.

3.1Hoechst 33258 staining to detect apoptosis

Hoechst33258 is a non-intercalating human fluorescent dye that binds to DNA in the minor groove of an enriched region of DNA poly AT sequences in living cells. When the cell nucleus of the apoptotic cell is excited by ultraviolet light of a fluorescence microscope, the cell nucleus can be seen to be densely dyed compact granular blocky fluorescence, and the living cell is evenly dispersed fluorescence. Therefore, the method can be used for analyzing the morphological change caused by apoptosis of Hoechst33258 fluorescence staining cells.

The method comprises the following specific steps:

(1) plate preparation: taking HepG-2 cells with good growth state according to the standard of 7.5 multiplied by 10⁴Fineness of one/mlThe cells were plated in six-well plates at cell density and placed in an incubator overnight.

(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: after the cells grow to 60% -70%, washing twice with PBS, washing off floating dead cells, and adding 2ml of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene diluted by culture medium into each hole. The concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is set to be 1.56 mu M and 3.125 mu M, and the control group is DMSO with the same volume.

(3) Fixing: after HepG-2 cells are treated by 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48 hours, the culture medium is discarded, 1ml of 4% paraformaldehyde is added into each hole, and the mixture is fixed for 15min at normal temperature in a dark place.

(4) Dyeing: the fixative was discarded and washed 3 times with cold PBS for 3min each. Add 500. mu.l/well Hoechst33258 staining solution, incubate 15min at 37 ℃ in the dark.

(5) And (3) observation by a fluorescence microscope: the cells were washed 2 times with cold PBS for 3min each, and after adding 1ml of PBS, the morphology of the cells was observed under a fluorescence microscope and photographed.

The results show that the HepG-2 cells of the control group present weak and uniform blue fluorescence, while the nuclear chromatin of the HepG-2 cells of the drug-added group is condensed, the condensed and compact granular strong fluorescence and obvious irregular nuclear fragmentation phenomena can be seen in the cells, and the apoptosis phenomenon is obvious along with the increase of the drug concentration (figure 7).

3.2Annexin V/PI double staining to detect apoptosis

The principle of Annexin V detection of apoptosis is that the distribution of normal cell membrane phospholipids is asymmetric, the inner surface of the membrane contains negatively charged phospholipids (such as Phosphatidylserine (PS)), and the outer surface of the membrane contains mostly neutral phospholipids. In the early stages of apoptosis, PS within the cell membrane flips from the inside to the cell membrane surface. Annexin V is a calcium-dependent phospholipid binding protein that binds specifically to PS with high affinity. PI is a nucleic acid dye that cannot penetrate the intact cell membrane, whereas the cell membrane of early apoptotic cells is intact and dye-repellent to PI. But in cells in the middle and late stages of apoptosis and dead cells, PI can permeate the cell membrane to stain the nucleus. Therefore, the cells in the early apoptosis stage can be distinguished from other cells by using annexin V and PI simultaneously.

The method comprises the following specific steps:

(1) plate preparation: taking HepG-2 cells with good log phase state according to 7.5 × 10⁴Cell density per ml, seeded in six-well plates, and placed in an incubator overnight.

(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: after the cells grow to 60% -70%, washing twice with PBS, washing off floating dead cells, and adding 2ml of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene diluted by culture medium into each hole. 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is set at the concentration of 1.56 mu M, 3.125 mu M and 6.25 mu M, a control group is DMSO with the maximum concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene equal to the volume, each three holes are set as one group, and 3 groups of multiple holes are set for each concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene.

(3) Collecting cells: treating 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene for 48h, collecting cells, transferring supernatant into a 15ml centrifuge tube, adding precooled PBS (phosphate buffer solution) for washing for 2 times, recovering a cleaning solution into the corresponding 15ml centrifuge tube, digesting with pancreatin, adding a culture medium to stop digestion, and recovering into the corresponding 15ml centrifuge tube. 1000rmp, centrifugation at 4 ℃ for 5min, discarding the supernatant, adding 1ml of cold PBS, and repeated centrifugation 1 time.

(4) Grouping: after discarding the supernatant, 1 × Annexin V binging Solution was added. 400. mu.l of 1 × Annexin V binding Solution was added to the DMSO group, and 100. mu.l of 1 × Annexin V binding Solution was added to the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group. 100. mu.l of each cell suspension was added to a new flow tube. Wherein the DMSO component is a negative control group, an Annexin V-FITC staining group, a PI staining group, an Annexin V-FITC and PI double staining group; the drug group was designated Annexin V-FITC and PI double staining group.

(5) Dyeing: adding 5 mul Annexin V-FITC conjugate into the cell suspension according to the grouping, mixing uniformly, adding 5 mul PI Solution, mixing uniformly, and culturing at room temperature in the dark for 15 min.

(6) Flow detection: adding 400 μ l of 1 × Annexin V Binding Solution, blowing, beating, mixing, and detecting on a machine within 1 h.

The results show that: after 48h of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment, the number of early and late apoptotic cells of HepG-2 cells increased in a dose-dependent manner. After 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene was administered, the proportion of early apoptotic cells in HepG-2 cells increased from 1.33% to 26.68% and the proportion of late apoptotic cells increased from 4.05% to 17.39% in the control group. From this, it was found that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene can induce HepG-2 cell apoptosis and significantly inhibit cell proliferation thereof (fig. 8).

4. Effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on HepG-2 cell cycle.

Since cell cycle arrest is also one of the mechanisms for inhibiting tumor cell proliferation, I studied the effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on the cell cycle after HepG-2 cells by using PI single stain in combination with flow cytometry.

4.1PI Monostain in combination with flow cytometer for cell cycle detection

The method comprises the following specific steps:

(1) plate preparation: taking HepG-2 cells with good log phase state according to 7.5 × 10⁴Cell density per ml, seeded in six-well plates, and placed in an incubator overnight.

(2)1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: after the cells grow to 60% -70%, washing twice with PBS, washing off floating dead cells, and adding 2ml of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene diluted by culture medium into each hole. The concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is set to be 3.125. mu.M. The control group is DMSO with the maximum concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene equal to the volume, one group is arranged in each three-hole, and 3 groups of multiple holes are arranged in each concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene.

(3) Collecting cells: 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is acted for 36h, cells are collected, supernatant is discarded, the cells are washed for 2 times by precooled PBS, 0.25% of pancreatin without EDTA is added for digestion, a culture medium is added for stopping digestion, the cells are centrifuged for 5min at 1000rmp and 4 ℃, the supernatant is discarded, 1ml of PBS is added, the centrifugation is repeated for 1 time, and the PBS is discarded.

(4) Ethanol fixation: 1ml of 70% ethanol was added and fixed in a refrigerator at 4 ℃ overnight.

(5) Propidium iodide staining: preparing an propidium iodide staining solution according to the number of samples to be detected, centrifuging the fixed samples at the temperature of 1000rmp for 5min, removing supernatant, adding 500 mu l of the propidium iodide staining solution into each tube of cell samples, slowly and fully resuspending cell precipitates, carrying out warm bath at the temperature of 37 ℃ in a dark place for 30min, then carrying out ice bath and light-proof storage, and completing detection within 24 h.

(6) Flow detection: the excitation wavelength is 488nm, and red fluorescence is detected.

The results show that the ratio of G0/G1 increases after 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene (3.125 mu M) treatment for 36h (period G0/G1)^*P is less than 0.05), the proportion of S phase is reduced (^**P < 0.01), the ratio G2/M is reduced (^*P < 0.05), indicating that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene blocks the HepG-2 cell cycle at G0/G1. (FIG. 9).

5. The change condition of related proteins in vivo after the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on HepG-2 cells.

To further study the mode of action of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene to cause apoptosis, we examined the expression of some key proteins in the apoptotic pathway and confirmed the pathway of action by transfection of siRNA.

5.1 Western Blot assay (Western Blot analysis)

The method comprises the following specific steps:

(1) extraction of total cellular protein:

plate preparation: taking HepG-2 cells with good log phase state according to 7.5 × 10⁴Cell density per ml, seeded in six-well plates, and placed in an incubator overnight.

1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene treatment: after the cells grow to 60% -70%, washing twice with PBS, washing off floating dead cells, and adding 2ml of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene diluted by culture medium into each hole. The 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene group is set at a concentration of 1.56. mu.M, 3.125. mu.M, 6.25. mu.M, and the control group is DMSO at a maximum concentration of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene in an equal volume.

Collecting cells: after 48h of drug intervention, collecting supernatant fluid and cleaning fluid, digesting with pancreatin, adding culture medium to stop digestion, transferring to a 50ml centrifuge tube, centrifuging at 4 ℃ and 1000rpm for 5min, discarding supernatant fluid, adding cold PBS, blowing, beating and mixing uniformly, centrifuging once, adding 1ml PBS, blowing uniformly, transferring to a 1.5ml EP tube, and centrifuging at 4 ℃ and 1500rpm for 5 min.

Cell lysis: discarding the supernatant, adding lysis mixture at a volume ratio of RIPA to PMSF to phosphatase inhibitor of 100:1:1, lysing on ice for 30min, performing ultrasound with 50% energy of an ultrasonicator to completely lyse the protein, and centrifuging at 4 deg.C for 30min at 12 XG.

(2) BCA assay protein concentration

Diluting a standard product: the prepared 0.5mg/ml protein standard was diluted in the following concentration gradient.

TABLE 3 dilution of protein standards

Dilution of the protein to be tested: the dilution was 40 fold, i.e., 78. mu.l deionized water was added to 2. mu.l protein sample.

Loading a 96-well plate: and respectively adding 20 mul of protein standard diluent and protein diluent to be detected into each hole, setting three multiple holes, adding 200 mul of BCA working solution, and slowly and fully mixing uniformly to avoid bubbles.

And (3) incubation: incubate at 37 ℃ for 30min in the dark.

Measuring absorbance by an enzyme-linked immunosorbent assay (ELIAS): and (3) measuring the absorbance of each hole under A562nm by using a multifunctional microplate reader, drawing a standard curve by taking the concentration as an abscissa and the absorbance (OD) as an ordinate, and calculating the protein concentration of the sample according to the standard curve, wherein the protein concentration curve is shown in figure 2.

(3) Denaturation of proteins

According to the measured concentration, the loading amount of the protein sample is leveled by UP water, 5 Xloading buffer solution is added, 1/4 with the loading amount of the protein sample is added, and after uniform mixing, the mixture is denatured for 5min at the constant temperature of 100 ℃ in a denaturator.

(4) Glue preparation

And (3) leak detection: and (3) installing an electrophoresis tank, adding deionized water, detecting leakage, pouring out water, and sucking dry by using filter paper.

Preparing glue: preparing the separation gel and the concentrated gel according to the table 4 and the table 5 respectively, wherein each plate needs about 6.5ml of the separation gel and 1.5ml of the concentrated gel, adding the concentrated gel after the separation gel is solidified, slowly inserting a 9-hole comb, and standing for gel polymerization.

TABLE 4 preparation of 15ml SDS-PAGE gels (12%) volume of fractions (ml)

(5) Electrophoretic preparation and sampling

Adding 1 Xelectrophoresis solution 500ml into electrophoresis tank, pulling comb to remove air bubble, and designing loading pore channel.

Protein sample and Marker loading: 15ul of protein denaturation sample and Marker are loaded on each hole of the 9-hole comb, and the blank hole is filled with 1 × loading with the same volume.

Electrophoresis: and (3) keeping the voltage at 80V, adjusting the voltage to 120V after the bromophenol blue runs through the concentrated gel, and stopping electrophoresis when the bromophenol blue runs to the bottom of the concentrated gel and does not run out.

(6) Film transfer: putting the gel plate after electrophoresis into a white empty tray filled with 1 multiplied membrane transferring liquid (after being prepared, putting the gel plate into a refrigerator for refrigeration at 4 ℃), removing redundant gel by a wane according to the molecular weight marked by a Marker, sequentially laying a layer of sponge, 4 layers of filter paper, gel, a PVDF membrane (the PVDF membrane is put into methanol for activation for 20s), 4 layers of filter paper and sponge, closing the membrane transferring clamp to avoid bubbles, putting the membrane transferring clamp into a membrane transferring groove, pouring the membrane transferring liquid, and constantly transferring the membrane for 90min in an ice-water bath.

(7) Sealing milk: after the transfer, the PVDF membrane was transferred to a milk blocking solution with tweezers and blocked overnight on a shaker at room temperature and 4 ℃.

(8) Primary antibody incubation: and (3) putting the PVDF membrane into the membrane washing solution, and washing the membrane for 10min each time for 3 times. The antibodies were diluted according to the antibody instructions, filled into plastic film, sealed and incubated overnight in a shaker at 4 ℃.

(9) And (3) secondary antibody incubation: and taking the membrane out of the primary antibody, putting the membrane into membrane washing liquid, and washing the membrane on a shaking table for 3 times, 10min each time. Diluting the antibody according to the instruction, filling the diluted antibody into a plastic packaging film, sealing the plastic packaging film, and incubating the plastic packaging film for 1.5h at room temperature in a shaking table.

(10) And (3) developing: and taking the membrane out of the secondary antibody, putting the membrane into membrane washing liquid, and washing the membrane for 10min each time on a shaking table at room temperature for 3 times. Mixing ECl hypersensitive luminous liquid A and liquid B in equal volume, covering the mixture on the surface of a membrane, and performing machine and development.

(11) And (4) analyzing results: images were collected and analyzed using an Image lab gel analysis system.

The results show that: the p53 protein is up-regulated, the MDM2 protein is down-regulated, and the Bax/Bcl-2 ratio is increased. The p53 protein is up-regulated, the down-regulation of the MDM2 protein shows that 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene possibly acts on a p53-MDM2 pathway, and the increase of the Bax/Bcl-2 ratio also shows that the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene participates in the regulation and control of apoptosis after acting on HepG-2 cells (figure 10).

5.2 Small interfering RNA (siRNA) cell transfection

Small interfering RNA (siRNA), also called short interfering RNA or silencing RNA (silencing RNA), is a double-stranded RNA of 20 to 25 nucleotides in length. It prevents translation by interfering with the post-transcriptional degradation of mRNA of a particular gene expressing a nucleotide sequence complementary thereto.

The method comprises the following specific steps:

(1) plate preparation: taking HepG-2 cells in logarithmic growth phase according to 7.5X 10⁴Cell density per ml, seeded in six-well plates, placed in an incubator, cultured overnight, seeded with medium without double antibody.

(2) Transfection: after the cell density reached 30% -50%, the medium was discarded and medium containing transfection reagent was added, taking care to use medium without double antibody. Transfection reagents were prepared according to the manufacturer's instructions.

(3) After 16h of transfection, transfected cells were collected, re-counted and plated in 96-well plates, and after 10h of culture drug treatment (1.56. mu.M, 3.125. mu.M) was given. And putting the 96-well plate into an IncuCyte zooming live cell imaging system to acquire images at fixed time.

(4) MTT assay and analytical data: and after 48h, MTT detection is carried out to calculate the survival rate, and images are collected in a fixed time mode in an IncuCyte zooming live cell imaging system and used for analyzing the survival condition of the cells.

As a result: (1) the silencing p53 gene can reverse the proliferation inhibition of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on hepatoma cells.

In FIG. 11, the change in cell fusion rate after 48h of drug addition was dynamically observed using IncuCyte. According to images collected at different moments, fusion rate analysis is carried out, and results show that in HepG-2 cells, compared with cells of a Control group of Control siRNA and p53 siRNA, a cell fusion rate curve of the p53 siRNA group is higher than that of the Control group of Control siRNA, and the fact that the expression of p53 is reduced to promote the proliferation of cells is proved. After 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene is added, the cell fusion rate curve of the p53 siRNA + Drug group is higher than that of the Control siRNA + Drug group, and the result shows that the expression level of p53 can influence the proliferation effect of the 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on cells, so that the compound 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene acts on a p53 pathway.

(2) Downregulation of the expression of p53 protein, effect of 1,5, 6-trimethoxy-2, 7-dihydroxyphenanthrene on survival of HepG-2 cells.

In FIG. 12, after transfection of p53 siRNA in HepG-2 cells, the cells were treated according to the Control group, p53 siRNA group, Control siRNA + Drug group and p53 siRNA + Drug group, respectively, and the MTT cell survival rate was examined after 48 hours. The results show that: in the Control group, the survival rate of HepG-2 cells after the Control siRNA + Drug (3.125. mu.M) group was 33.53%, while the survival rate of HepG-2 cells after the p53 siRNA + Drug treatment was 39.26% (si p53#3) after the p53 siRNA transfection, the survival rate of HepG-2 cells after the Control siRNA + Drug (1.56. mu.M) group was 47.13%, while the survival rate of HepG-2 cells after the p53 siRNA + Drug treatment was 54.97% (si p53#3) after the p53 siRNA transfection (a statistical significance of the difference) (1.125. mu.M)^****P<0.0001)。

The data of the experiment were statistically analyzed using Graphpad 8.0.2 software,data are all mean. + -. standard deviationAnd (4) showing. The graphs were prepared by GraphPad 8.0.2 and Adobe Photoshop CS6 software.^*P<0.05 was considered statistically significant.