CN113476456B - Pharmaceutical composition rich in forsythiaside and application thereof - Google Patents
Pharmaceutical composition rich in forsythiaside and application thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
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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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a pharmaceutical composition rich in forsythiaside and application thereof, belonging to the technical field of biological medicine, wherein the pharmaceutical composition comprises forsythiaside and oxaliplatin, and the forsythiaside alone has proliferation inhibition effect on colon cancer cell strain HCT116 and colon cancer oxaliplatin-resistant cell strain HCT 116/L-OHP; the forsythiaside combined with oxaliplatin effect cells find that the forsythiaside can enhance the proliferation inhibition effect of oxaliplatin on HCT116/L-OHP cells, and the forsythiaside is shown to have the effect of anti-tumor drug resistance.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a pharmaceutical composition rich in forsythiaside and application thereof.
Background
The current approach to chemotherapy is one of the major clinical treatments for colorectal cancer. However, as the amount of the chemotherapeutic agent used by the patient increases, the tumor cell inhibitory effect of the chemotherapeutic agent decreases, resulting in drug resistance, which can lead to failure of chemotherapy. Therefore, the development of a novel safe and efficient drug resistance reversal agent for improving the curative effect of chemotherapy is urgently needed.
Phillygenin (Phillygenin) is the aglycone of phillyrin, the main active ingredient with high content in forsythia suspensa leaves, and is the lignan monomer compound extracted and separated from forsythia suspensa leaves by Shizuka Kitagawa (1984) and the like at first. The structure of the compound is polymerized by phenylpropanoids, namely C6-C3 derivatives, namely, two, three and four, and belongs to diepoxy type bis-tetrahydrofuran lignans, and the compound is white amorphous powder. Forsythiaside is mainly present in the wood of plants, and is often in a free state, or may be combined with sugar to form a glycoside. Forsythiasis hormone is insoluble in water and soluble in organic solvents such as benzene, petroleum ether, chloroform and ethanol.
The forsythiaside has wide pharmacological activity, and mainly has the pharmacological actions of resisting inflammation, protecting liver, resisting oxidation, reducing blood pressure, reducing blood fat, resisting cancer and the like. Studies show that the forsythiaside has certain inhibition effect on the growth of cervical cancer cells, liver cancer cells, melanoma cells, gastric cancer cells and other cells. However, the research on the reversing of tumor cell resistance by the forsythiaside is blank at present.
Disclosure of Invention
The invention aims to overcome the defects of the existing intestinal cancer treatment medicines and the application limitation of oxaliplatin, and provides a medicine composition rich in forsythiaside and application thereof.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a pharmaceutical composition rich in forsythiaside, which comprises forsythiaside (PG) and oxaliplatin (L-OHP).
Further, the chemical structural formula of the forsythiaside is as follows:
further, the concentration of the forsythiaside in the pharmaceutical composition is 30-60 mu g/mL, and the concentration of the oxaliplatin is 10-160 mu g/mL.
Further, the concentration of the forsythiaside in the pharmaceutical composition is 50 mug/mL, and the concentration of the oxaliplatin is 10-160 mug/mL.
Further, the concentration of the forsythiaside in the pharmaceutical composition is 30-60 mug/mL, and the concentration of the oxaliplatin is 20 mug/mL.
Further, the concentration of the forsythiaside in the pharmaceutical composition is 30 or 60 μ g/mL, and the concentration of the oxaliplatin is 20 μ g/mL. Further, the pharmaceutical composition has an inhibitory effect on the proliferation of HCT 116/L-OHP.
The research of the invention finds that the forsythiaside has proliferation inhibition effect on colon cancer cell strain HCT116 and colon cancer oxaliplatin-resistant cell strain HCT116/L-OHP independently; the forsythiaside combined with oxaliplatin effect cells find that the forsythiaside can enhance the proliferation inhibition effect of oxaliplatin on HCT116/L-OHP cells, and the forsythiaside is shown to have the effect of anti-tumor drug resistance.
The invention also provides application of the pharmaceutical composition in preparing medicines for preventing and treating intestinal cancer.
Further, the intestinal cancer is colorectal cancer.
Further, the pharmaceutical composition is used for preparing an anti-colorectal cancer cell proliferation inhibition drug.
The invention also provides application of the pharmaceutical composition in preparation of anti-tuberculosis/rectal cancer oxaliplatin resistant drugs.
The invention discloses the following technical effects:
the invention adopts MTT method to detect the proliferation inhibition effect of the forsythiaside with different concentrations on colon cancer cell strain HCT116 and colon cancer oxaliplatin-resistant cell strain HCT116/L-OHP alone; and different concentrations of forsythiaside combined with oxaliplatin on HCT116/L-OHP cells. The results show that the forsythiaside with different concentrations independently has inhibition effects on the proliferation of colon cancer cell strain HCT116 and colon cancer oxaliplatin-resistant cell strain HCT116/L-OHP, and the forsythiaside can increase the inhibition effect of the proliferation of oxaliplatin on HCT116/L-OHP cells, which shows that the forsythiaside has anti-tumor drug resistance.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 shows the proliferation inhibitory effect of forsythiaside on colon cancer cell line HCT 116;
FIG. 2 shows the effect of forsythiaside on the proliferation inhibition of oxaliplatin-resistant cell line HCT116/L-OHP in colon cancer;
FIG. 3 shows the proliferation inhibitory effect of oxaliplatin on colon cancer cell line HCT 116;
FIG. 4 shows the effect of oxaliplatin on inhibiting the proliferation of oxaliplatin-resistant cell line HCT116/L-OHP in colon cancer;
FIG. 5 shows the effect of forsythiaside (50. mu.g/mL) in combination with oxaliplatin (10-160. mu.g/mL) on the inhibition of proliferation of oxaliplatin-resistant cell line HCT116/L-OHP in colon cancer;
FIG. 6 shows the effect of forsythiaside (30. mu.g/mL, 60. mu.g/mL) in combination with oxaliplatin (20. mu.g/mL) on the inhibition of proliferation of oxaliplatin-resistant cell line HCT116/L-OHP against colon cancer;
FIG. 7 is a graph showing the effect of forsythiasin on colony formation of HCT116/L-OHP cells;
FIG. 8 is a graph showing the results of analysis of the effect of forsythiasin on colony formation of HCT116/L-OHP cells;
FIG. 9 shows the results of the effect of forsythiasin on apoptosis of HCT116/L-OHP cells;
FIG. 10 shows the effect of forsythiaside on the level of JAK2 protein in HCT116/L-OHP cells;
FIG. 11 shows the effect of forsythiaside on the intracellular phosphorylated JAK2(P-JAK2) protein content of HCT 116/L-OHP;
FIG. 12 shows the effect of forsythiaside in combination with oxaliplatin on HCT116/L-OHP cell morphology.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
In the invention, the forsythiaside and oxaliplatin are purchased from commercial sources.
In the following examples, the chemical structure of forsythin is:
example 1 Forsythiacin inhibits proliferation of Colon cancer cell line HCT116 and colon cancer oxaliplatin-resistant cell line HCT116/L-OHP
And (3) cell culture: human colon cancer cell line HCT116 and colon cancer oxaliplatin-resistant cell line HCT 116/L-OHP. The culture medium comprises 90% RPMI-1640 and 10% calf serum, and 1% double antibody at 37 deg.C under CO 2 5 percent of volume fraction and saturated humidity. After the cells are attached to the wall, the solution is changed for 1 time every 1-2 days, a culture medium containing 5 mu g/mL oxaliplatin medicament is added when HCT116/L-OHP cells are cultured, and after the cells grow to 80% -90%, the cells are subjected to conventional digestion and passage by 0.25% trypsin.
Cell passage: discarding the original culture solution in the culture bottle which is full of cells, adding 3-4mL PBS to wash twice, adding 1mL trypsin to digest for 1-2min, observing under a microscope, adding 2mL culture medium to stop digestion after the cells are all rounded, and blowing and mixing the cells uniformly. Sucking the cell suspension into a centrifuge tube, centrifuging for 5min at 1000r/min, removing supernatant, adding culture medium, mixing, packaging to dilute cells, and culturing in an incubator.
Preparing a sample concentration gradient: weighing forsythiaside, dissolving with DMSO (dimethyl sulfoxide), preparing into stock solution with concentration of 100mg/mL, and diluting with culture medium to obtain working solution with gradient concentration of forsythiaside. The content of DMSO is controlled below 0.2%.
Adjusting the cell suspension concentration to 5X 10 4 cells/mL, after pipetting, 100. mu.L per well, and plating to density the cells to 5000 cells/well (the marginal wells were filled with sterile PBS solution). 5% CO 2 Incubate at 37 ℃ for 24 hours until the cells adhere to the wall (96-well flat bottom plate), discard the old medium by aspiration, and add the freshly prepared new medium containing different concentrations of forsythin.
5％CO 2 Incubated at 37 ℃ for 48 hours and observed under an inverted microscope.
mu.L of MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide salt, namely thiazole blue, MTT for short, 5mg/mL) solution is added into each hole, and the culture is continued for 4 hours.
The culture medium was then carefully aspirated off the wells. Add 150. mu.L of dimethyl sulfoxide into each well, and shake for 10min at low speed on a shaking bed to dissolve the crystals sufficiently. In enzyme-linked immunoassay instrument OD 570 The absorbance value A of each well was measured at nm.
Each set was set with 5 replicate wells and plotted against absorbance value a.
Cell viability (%) ═ a Experimental group -A Blank group )/(A Control group -A Blank group ) X 100%, cell proliferation inhibition (%) 1-cell viability, tumor cell proliferation inhibition by each concentration of drug was calculated, and half inhibition IC was calculated using SPSS 23 software 50 。
Results of the experiment
The proliferation inhibiting effect of phillygenin on colon cancer cell strain HCT116 is shown in FIG. 1<0.05，**p<0.01), the forsythiaside concentrations are in sequence: 0.25, 50, 100, 200, (mu g/mL), the activity of HCT116 cells gradually decreased with the increase of the forsythiaside concentration, which indicates that the forsythiaside has proliferation inhibition effect on HCT116 cells and is concentration-dependent. The IC of the forsythiaside on the colon cancer cell line HCT116 is calculated by SPSS 23 software 50 Comprises the following steps: 120.51 μ g/mL.
The proliferation inhibition effect of forsythiaside on anti-oxaliplatin resistant cell line HCT116/L-OHP in colon cancer is shown in FIG. 2 (. about.p)<0.05，**p<0.01), the forsythiaside concentrations are in sequence: 0.25 portions of,50. The activity of HCT116/L-OHP cells is gradually reduced along with the increase of the forsythiaside concentration at 100 and 200 (mu g/mL), which shows that the forsythiaside has proliferation inhibition effect on HCT116/L-OHP cells and is concentration-dependent. The IC of the forsythiaside for the anti-oxaliplatin resistant cell strain HCT116/L-OHP of the colon cancer is calculated by SPSS 23 software 50 Comprises the following steps: 126.73 μ g/mL.
And (4) conclusion: the forsythiaside has inhibition effect on proliferation of colon cancer cell strain HCT116 and colon cancer oxaliplatin-resistant cell strain HCT116/L-OHP, and the inhibition effect is increased along with the increase of the concentration of the forsythiaside.
Example 2 detection of drug resistance of oxaliplatin to oxaliplatin-resistant cell line HCT116/L-OHP in colon cancer
Cell culture: human colon cancer cell line HCT116 and colon cancer oxaliplatin-resistant cell line HCT 116/L-OHP. The culture medium is 90% RPMI-1640 and 10% calf serum, and 1% double antibody is added at 37 deg.C under CO condition 2 5 percent of volume fraction and saturated humidity. After the cells are attached to the wall, the solution is changed for 1 time every 1-2 days, a culture medium containing 5 mu g/mL oxaliplatin medicament is added when HCT116/L-OHP cells are cultured, and after the cells grow to 80% -90%, the cells are subjected to conventional digestion and passage by 0.25% trypsin.
Adjusting the cell suspension concentration to 5X 10 4 cells/mL, drench, add 100. mu.L per well, plate to adjust cell density to 5000 cells/well, (margin wells filled with sterile PBS solution). 5% CO 2 Incubate at 37 ℃ for 24 hours until the cells adhere to the walls (96-well flat bottom plate), discard the old medium by aspiration, and add new medium containing oxaliplatin at different concentrations. 5% CO 2 Incubated at 37 ℃ for 48 hours and observed under an inverted microscope. mu.L of MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide salt, namely thiazole blue, MTT for short, 5mg/mL) solution is added into each hole, and the culture is continued for 4 hours.
The culture medium in the wells was then carefully aspirated. Add 150. mu.L of dimethyl sulfoxide into each well, and shake for 10min at low speed on a shaking bed to dissolve the crystals sufficiently. In enzyme-linked immunoassay instrument OD 570 The absorbance value A of each well was measured at nm.
Cell viability (%) ═ a Experimental group -A Blank group )/(A Control group -A Blank group ) X 100%, cell proliferation inhibition (%) 1-cell viability, tumor cell proliferation inhibition by each concentration of drug was calculated, and half inhibition IC was calculated using SPSS 23 software 50 。
Results of the experiment
Oxaliplatin with different concentrations is respectively acted on a colon cancer cell HCT116 and a colon cancer oxaliplatin-resistant cell HCT116/L-OHP for 48 hours, and the cell viability is detected by an MTT method. As shown in fig. 3 and 4 (. about.p)<0.05，**p<0.01), the inhibition effect of oxaliplatin on HCT116 and HCT116/L-OHP is concentration-dependent, and the IC of oxaliplatin on HCT116 and HCT116/L-OHP cells is calculated by SPSS 23 software 50 The values were 9.7 and 78.6. mu.g/mL, respectively, and the resistance index was 8.1.
Example 3 Forsythiacin in combination with oxaliplatin inhibitory Effect on proliferation of oxaliplatin-resistant cell line HCT116/L-OHP resistant to colon cancer
Cell culture: human colon cancer cell line HCT116 and colon cancer oxaliplatin-resistant cell line HCT 116/L-OHP. The culture medium is 90% RPMI-1640+ 10% calf serum, and 1% double antibody is added at 37 deg.C under CO 2 5 percent of volume fraction and saturated humidity. After the cells are attached to the wall, the solution is changed for 1 time every 1-2 days, the culture medium containing oxaliplatin medicaments (5 mu g/mL) is added when HCT116/L-OHP cells are changed, and after the cells grow to 80-90%, the cells are subjected to conventional digestion and passage by 0.25% trypsin.
Preparing a sample concentration gradient: weighing forsythiaside, dissolving with DMSO to prepare stock solution with concentration of 100mg/mL, and diluting with culture medium to obtain working solution containing forsythiaside with different concentrations (30 μ g/mL, 50 μ g/mL, 60 μ g/mL). The content of DMSO is controlled below 0.2%.
Adjusting the cell suspension concentration to 5X 10 4 cell/mL, blow and mix, each well add 100 u L, plate to cell density to 5000 cells/well, (margin hole with sterile PBS solution filling). 5% CO 2 Incubate at 37 ℃ for 24 hours until the cells adhere to the walls (96-well flat bottom plate), discard the old medium by aspiration, and add the freshly prepared new medium containing forsythiaside and the corresponding oxaliplatin drug. 5％CO 2 Incubated at 37 ℃ for 48 hours and observed under an inverted microscope.
mu.L of MTT solution (5mg/mL, i.e., 0.5% MTT) was added to each well and incubation was continued for 4 h.
The culture was terminated and the culture medium in the wells was carefully aspirated. Add 150. mu.L of dimethyl sulfoxide into each well, and place on a shaking bed to shake at low speed for 10min to fully dissolve crystals. In an enzyme-linked immunosorbent assay (ELISA) detector OD 570 The absorbance value A of each well was measured at nm.
Each set was set with 5 replicate wells and plotted against absorbance values.
Cell viability (%) ═ a Experimental group -A Blank group )/(A Control group -A Blank group ) X 100%, cell proliferation inhibition (%) 1-cell viability, proliferation inhibition of tumor cells was calculated for each concentration of drug, and half inhibition IC was calculated using SPSS 23 software 50 。
Results of the experiment
The results of the proliferation inhibition effect of forsythiaside (50. mu.g/mL) in combination with oxaliplatin (10-160. mu.g/mL) on oxaliplatin-resistant cell line HCT116/L-OHP in colon cancer are shown in FIG. 5 (. mu.p)<0.05，**p<0.01), wherein the abscissa refers to the concentration of oxaliplatin, forsythiaside (50 μ g/mL) in combination with oxaliplatin (10-160 μ g/mL) increases the proliferation-inhibiting effect of oxaliplatin on the oxaliplatin-resistant cell line HCT116/L-OHP of colon cancer. The IC of forsythiaside (50 mug/mL) combined with oxaliplatin (10-160 mug/mL) for the anti-oxaliplatin resistant cell strain HCT116/L-OHP for the colon cancer is calculated by SPSS 23 software 50 Comprises the following steps: 26.38 μ g/mL. Calculation of fold reversal-oxaliplatin alone IC 50 Combined action IC 50 ＝78.59/26.38＝3。
The results of the proliferation inhibition effect of forsythiaside combined with oxaliplatin on the anti-oxaliplatin drug-resistant cell strain HCT116/L-OHP in colorectal cancer are shown in FIG. 6 (p <0.05, p <0.01), the combination of forsythiaside (30. mu.g/mL, 60. mu.g/mL) and oxaliplatin (20. mu.g/mL) can reduce the activity of HCT116/L-OHP cells, and the combined effect of forsythiaside (30. mu.g/mL, 60. mu.g/mL) and oxaliplatin (20. mu.g/mL) is lower than that of HCT116/L-OHP cells when oxaliplatin alone (20. mu.g/mL). The forsythiaside is shown to increase the proliferation inhibition effect of oxaliplatin on the anti-oxaliplatin resistant cell strain HCT116/L-OHP of colon cancer.
And (4) conclusion: the forsythiaside has the effect of resisting colon cancer and oxaliplatin resistance strains, can increase the proliferation inhibition effect of oxaliplatin on colon cancer and oxaliplatin resistance cell strains HCT116/L-OHP by combining with oxaliplatin, and has the reversal multiple of 3.
Example 4 colony formation assay of cells to examine the Effect of Forsythiacin on colony formation of drug-resistant cell line HCT116/L-OHP
Experimental procedure
Cell culture: human colon cancer oxaliplatin-resistant cell line HCT116/L-OHP is resistant. The culture medium is 90% RPMI-1640+ 10% calf serum, and 1% double antibody is added at 37 deg.C under CO 2 5 percent of volume fraction and saturated humidity. After the cells are attached to the wall, the liquid is changed for 1 time every 1-2 days, the oxaliplatin medicament is added when the liquid is changed, and after the cells grow to 80% -90%, the cells are subjected to conventional digestion and passage by using 0.25% trypsin.
Preparing a sample concentration gradient: weighing forsythiaside, dissolving with DMSO to prepare stock solution with concentration of 100mg/mL, and diluting with culture medium to obtain gradient working solution containing forsythiaside with concentration of 0, 50, 100 μ g/mL.
Adjusting the concentration of the cell suspension, blowing, mixing, adding into 6-well plate, adding 2000 cells and 5% CO per well 2 And incubating for 24 hours at 37 ℃ until the cells adhere to the wall, removing the old culture medium by suction, and respectively adding 3mL of the culture medium containing the phillygenin medicament prepared in situ into each hole.
5％CO 2 Culturing at 37 deg.C for 10 days, timely replacing the fresh culture solution containing medicine according to pH change of the culture solution, and observing under an inverted microscope.
When colonies were visible to the naked eye in the dish, the culture was terminated, the culture solution was discarded, carefully washed with PBS solution 2 times, and then dried in the air. Methanol was fixed for 15 minutes, and air-dried after discarding methanol. Dyeing with Giemsa dye liquor for 10 minutes, slowly washing off the dye liquor with running water, and air drying.
Photographic records, Image J software counts the number of cell clonings, calculates the relative clonality and analyzes the results (defined as a cluster of at least 50 cells).
Relative clone formation (%) (number of experimental group colony/number of control group colony) × 100%.
Results and analysis of the experiments
The effect of forsythiaside on HCT116/L-OHP cell colony formation is shown in FIG. 7, and the analysis result of the effect of forsythiaside on HCT116/L-OHP cell colony formation is shown in FIG. 8, and it can be seen from FIGS. 7 and 8 that: the clone number of HCT116/L-OHP cell colonies decreases with the increase of the concentration of the forsythiaside, when the concentration of the forsythiaside is 50 mu g/mL, the relative clone formation rate of HCT116/L-OHP cells is 26.5%, and when the concentration of the forsythiaside is 100 mu g/mL, the relative clone formation rate of HCT116/L-OHP cells is only 0.3%, which indicates that the forsythiaside has strong inhibitory effect on the colony formation of the HCT116/L-OHP cells.
Example 4 flow cytometry to examine the Effect of Forsythiacin on apoptosis of HCT116/L-OHP cells
Experimental procedure
Preparing a sample concentration gradient: weighing forsythiaside, dissolving with DMSO to prepare stock solution with concentration of 100mg/mL, and diluting with culture medium to obtain working solution containing forsythiaside with concentration gradient of 0, 25, 50, 100 μ g/mL.
Adjusting the concentration of the cell suspension, blowing, beating and mixing uniformly, and adding into a 6-pore plate. 5% CO 2 Culturing at 37 deg.C until cell grows to 60% -70%, removing old culture medium, and adding 3mL of prepared culture medium containing phillygenin. 5% CO 2 Incubated at 37 ℃ for 48h and observed under an inverted microscope.
Collecting cells: discarding the old culture solution, washing twice with 1-2mL PBS, adding 1mL trypsin for digestion for 1min, observing under microscope, adding 2mL culture medium to stop digestion after cell rounding, and blowing and mixing the cells uniformly. Sucking the cell suspension into a centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, adding a serum-free culture medium, and uniformly mixing and resuspending.
Staining the samples according to the instructions of the apoptosis detection kit: cells were washed 2 times with pre-chilled PBS and centrifuged for 5min at 4 ℃. Collecting 1-5X 10 5 A cell. PBS was aspirated off and 100. mu.L of 1 × Binding Buffer was added to resuspend the cells. AddingAdd 5. mu.L Annexin V-FITC (Annexin V-fluorescein isothiocyanate) and 10. mu.L PI (propidium iodide) staining solution and mix gently. And (4) keeping out of the light and reacting at room temperature for 10-15 min. Add 400. mu.L of 1 XBinding Buffer, mix well and place on ice, the sample is detected by flow cytometry or fluorescence microscope within 1 hour.
Results and analysis of the experiments
The effect of forsythiasin on apoptosis of HCT116/L-OHP cells is shown in FIG. 9. Experimental results show that the forsythiaside can promote the apoptosis of HCT116/L-OHP cells, and when the concentration of the forsythiaside is 25 mu g/mL, the apoptosis rate of the HCT116/L-OHP cells is 25.52 percent, and the effect is most obvious.
Example 5 Effect of Forsythia on HCT116/L-OHP intracellular protein tyrosine kinase JAK2 and phosphorylated JAK2 was examined by Elisa experiment
Experimental procedure
Preparing a sample concentration gradient: weighing forsythiaside, dissolving with DMSO to prepare stock solution with concentration of 100mg/mL, and diluting with culture medium to obtain medicinal concentration containing forsythiaside with concentration gradient of 0, 50, 100 μ g/mL.
Adjusting the concentration of the cell suspension, blowing, beating and mixing uniformly, and adding into a 6-hole plate. 5% CO 2 Culturing at 37 deg.C until cell grows to 60% -70%, removing old culture medium, and adding 3mL of prepared culture medium containing phillygenin. 5% CO 2 Incubated at 37 ℃ for 48h and observed under an inverted microscope.
And (3) extracting total cell protein: the cells were gently scraped with a spatula, and then the cells and supernatant were transferred to a 1.5ml centrifuge tube with a pipette, placed in a 4-degree centrifuge (pre-cooled by opening the centrifuge in advance), centrifuged for 15min, and the supernatant was discarded. Prepare lysate (the lysate is RIPA lysate, which can release the protein sufficiently), add 10 μ L PMSF (phenylmethylsulfonyl fluoride, 100mM) per 1mL lysate, shake well and place on ice. (PMSF can be mixed with the lysate until no crystals are present by shaking). The prepared lysate is added to the obtained cell pellet, typically 5X 10 6 100 μ L of lysis solution was added to each cell, and then resuspended by pipetting to fully lyse the cells. Cracking on ice for 30min, shaking on oscillator for 10min, and separating at 12000 rpmAnd (5) taking the supernatant after 15min, and detecting the protein concentration by using a BCA method.
Detection was performed according to the procedures of the Elisa kit instructions.
Results and analysis of the experiments
The effect of forsythiaside on the levels of JAK2 and phosphorylated JAK2 proteins in HCT116/L-OHP cells is shown in FIGS. 10-11. The experimental results show that: with the increase of the concentration of the forsythiaside, the contents of JAK2 and phosphorylated JAK2 protein in HCT116/L-OHP cells are increased, and the fact that the forsythiaside can promote the expression of JAK 2-related protein and promote the phosphorylation of JAK2 is shown. Suggesting that forsythiaside reverses resistance in resistant cells may be associated with JAK 2-associated signaling pathways.
Example 6 Effect of Forsythiacin in combination with oxaliplatin on HCT116/L-OHP cell morphology
After forsythiaside (50. mu.g/mL) and oxaliplatin (20. mu.g/mL) were allowed to act on HCT116/L-OH cells separately and in combination, respectively, for 48 hours, the change in cell growth morphology was observed using an inverted microscope. As can be seen from FIG. 12, the cell morphology of the control group is not significantly changed by the forsythiaside or oxaliplatin, but the cell morphology of the control group combined with the forsythiaside or oxaliplatin is significantly changed, the cell gap is gradually increased, the cell density is gradually reduced, the cell volume is retracted, and the cell shape is fusiform.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (2)
1. The application of a pharmaceutical composition in preparation of anti-colorectal cancer and oxaliplatin resistant drugs is characterized in that the pharmaceutical composition consists of forsythiaside and oxaliplatin.
2. The use according to claim 1, wherein the pharmaceutical composition has an inhibitory effect on the proliferation of the colon cancer resistant oxaliplatin cell line HCT 116/L-OHP.
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