CN116549504A - Pharmaceutical composition for treating melanoma - Google Patents
Pharmaceutical composition for treating melanoma Download PDFInfo
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- CN116549504A CN116549504A CN202210112490.9A CN202210112490A CN116549504A CN 116549504 A CN116549504 A CN 116549504A CN 202210112490 A CN202210112490 A CN 202210112490A CN 116549504 A CN116549504 A CN 116549504A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/481—Astragalus (milkvetch)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/42—Cucurbitaceae (Cucumber family)
- A61K36/428—Trichosanthes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a pharmaceutical composition for inhibiting proliferation of melanoma cells, which is prepared by combining astragalus (less than 4 mg/ml) and radix trichosanthis (less than 1.28 mg/ml), so that the effect of inhibiting proliferation of A375 cells without obvious hepatotoxicity is achieved, and a new treatment scheme and theoretical basis are provided for clinical drug treatment of melanoma.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine compatibility, in particular to traditional Chinese medicine compatibility for treating melanoma.
Background
Malignant melanoma (Malignant Melanoma, MM) is an invasive malignant tumor caused by mutation of a sphingocyte which produces melanin, abnormal tyrosine metabolism, increased melanin production and accumulation in a body, and has the characteristics of high malignancy, high development speed and strong metastasis. Malignant melanoma is statistically the third in men's benign cancers and the fifth in women's benign cancers in the united states. In recent years, the incidence of cancer as a whole is decreasing, but the incidence of malignant melanoma is increasing, increasing by about 3% each year. And among all melanoma patients, 47% of patients under 65 years old and 33% of patients under 50 years old, the ill population has a tendency to be younger. Surgical treatment is the main treatment method for early melanoma, but has high postoperative recurrence rate due to strong metastatic property. Patients who are partially refused to operate or cannot operate in the late stage mostly adopt a radiotherapy and chemotherapy method, the curative effect is poor, the damage to the organism is larger, the tolerance of the patients is low, the curative effect is reduced along with the increase of the times of radiotherapy and chemotherapy, and the prognosis is poor. Thus, the search for new effective therapies is a major problem in the current clinical therapies for malignant melanoma. In addition, the effective treatment effect of the traditional Chinese medicine on malignant melanoma is gradually discovered, and the traditional Chinese medicine has a wide application prospect.
Astragalus mongholicus and radix trichosanthis are used as important qi-tonifying drugs and heat-clearing and toxicity-removing drugs widely used clinically, and are first found in Shennong's herbal channels of ancient book of traditional Chinese medicine. At present, a great deal of evidence shows that astragalus and radix trichosanthis have the anti-tumor, anti-inflammatory and antiviral effects, are low in price, have few side effects, are not easy to generate drug resistance, are also used for treating various tumors, and have the effect of inhibiting tumor growth and metastasis. The compatibility is the main method for clinically applying traditional Chinese medicines, and when certain traditional Chinese medicines are matched according to a specific proportion, the effect is better. Although astragalus root and trichosanthes root are not common pairs of traditional Chinese medicines, the application of the compatibility of the two is largely recorded in the literature, and most commonly used in the treatment of diabetes.
In the previous study of the team, radix astragali, radix trichosanthis and compatibility thereof have been found to inhibit proliferation and migration of malignant melanoma B16 cells in mice, and the compatibility effect of the two medicines is superior to that of a single traditional Chinese medicine (Zhang Qiuyan, etc., 2020). However, in the subsequent experiments, we further find that the compatibility of the two medicines has certain hepatotoxicity and cannot be applied to clinic. Therefore, how to reduce the toxicity of the compatibility of the two drugs is an urgent problem to be solved.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a traditional Chinese medicine formula for treating melanoma without obvious hepatotoxicity. Specifically, the invention provides the following technical scheme:
in a first aspect of the present invention, there is provided a pharmaceutical composition having no liver toxicity, the active ingredients of the pharmaceutical composition are astragalus and trichosanthin, wherein the final concentration of the drug of astragalus is lower than 4mg/ml, and the final concentration of the drug of trichosanthin is lower than 1.28mg/ml.
In a preferred embodiment, the astragalus root has a final concentration of 1.1-3.6mg/ml and radix trichosanthis has a final concentration of 0.137-0.863mg/ml.
In a most preferred embodiment, the astragalus has a final concentration of 1.1mg/ml and radix trichosanthis has a final concentration of 0.863mg/ml.
In a second aspect, the invention provides application of the pharmaceutical composition in preparation of medicines for promoting melanoma cell apoptosis.
In a third aspect of the invention, there is provided the use of the above pharmaceutical composition for the preparation of a medicament for inhibiting migration of melanoma cells.
In a fourth aspect, the invention provides an application of the pharmaceutical composition in preparing a medicament for improving the Bax/Bcl-2 expression ratio in melanoma cells.
In a fifth aspect, the invention provides an application of the pharmaceutical composition in preparing a medicament for reducing the activity of LDH and SOD in A375 cells and increasing the MDA content in A375 cells.
In a sixth aspect, the invention provides the use of the above pharmaceutical composition in the preparation of a medicament for inhibiting Akt activity and phosphorylation in melanoma cells.
Compared with the prior art, the invention has the following remarkable technical effects：
1. According to the invention, through a large number of compatibility grouping experiments, the influence of astragalus and radix trichosanthis on melanoma and proliferation of normal human liver cells under different compatibility conditions is analyzed in detail, so that the optimal compatibility proportion of the astragalus and the radix trichosanthis is confirmed;
2. the invention verifies the treatment effect of each compatibility formula on melanoma from multiple aspects through in-vivo experiments of cells and animals, expounds the action mechanism of the compatibility formula, provides detailed theoretical basis and dosage scheme reference for the traditional Chinese medicine treatment of clinical melanoma, and has positive significance for the clinical treatment of the melanoma and the medicine popularization of astragalus and radix trichosanthis.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 shows the effect of decoction of Astragalus membranaceus and Trichosanthis radix on cell proliferation after 48h of action on A375 cells, with P <0.05 compared with control group;
FIG. 2 shows the effect of decoction of Astragalus membranaceus and Trichosanthis radix on cell proliferation after 72h of action on A375 cells, with P <0.05 compared with control group;
FIG. 3 shows the effect of radix astragali and radix Trichosanthis on cell proliferation after 48h of A375 cells, compared to control group, with P < 0.05; the ratio of the compatibility group to the corresponding astragalus single medicine group is #P < 0.05; the ratio of the compatibility group to the corresponding single medicine group of the radix trichosanthis is delta P less than 0.05;
FIG. 4 shows the effect of the combination of astragalus and trichosanthin on cell proliferation after 48h, P <0.05 compared to control; the ratio of the compatibility group to the corresponding astragalus single medicine group is #P < 0.05; the ratio of the compatibility group to the corresponding single medicine group of the radix trichosanthis is delta P less than 0.05;
FIG. 5 shows the effect of the combination of astragalus and trichosanthin on cell proliferation after 48 h;
FIG. 6 shows the effect of astragalus root, trichosanthes root and their combination on cell proliferation cycle of A375 cells for 48h, with P <0.05 compared to control; the ratio of the compatibility group to the corresponding astragalus single medicine group is #P < 0.05; the ratio of the compatibility group to the corresponding single medicine group of the radix trichosanthis is delta P less than 0.05;
FIG. 7 shows the effect of radix astragali, radix Trichosanthis and combinations thereof on melanoma apoptosis, compared to control group, with P < 0.05; the ratio of the compatibility group to the corresponding astragalus single medicine group is #P < 0.05; the ratio of the compatibility group to the corresponding single medicine group of the radix trichosanthis is delta P less than 0.05;
FIG. 8 shows the effect of astragalus root, trichosanthes root and their combination on cell migration by A375 cells for 48h, with P <0.05 compared to control; the ratio of the compatibility group to the corresponding astragalus single medicine group is #P < 0.05; the ratio of the compatibility group to the corresponding single medicine group of the radix trichosanthis is delta P less than 0.05;
fig. 9 shows the effect of astragalus root, trichosanthes root and their combination on melanoma LDH, SOD, MDA, P <0.05 compared to control; the ratio of the compatibility group to the corresponding astragalus single medicine group is #P < 0.05; the ratio of the compatibility group to the corresponding single medicine group of the radix trichosanthis is delta P less than 0.05;
FIG. 10 shows the effect of radix astragali, radix Trichosanthis and combinations thereof on melanoma Akt protein, compared to control group, with P < 0.05; the ratio of the compatibility group to the corresponding astragalus single medicine group is #P < 0.05; the ratio of the compatibility group to the corresponding single medicine group of the radix trichosanthis is delta P less than 0.05.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
EXAMPLE 1 Effect of radix astragali, radix Trichosanthis and its compatibility on proliferation of A375 cells and human normal hepatocytes (L-02 cells)
Astragalus mongholicus and radix Trichosanthis are purchased from Beijing Tongren Tang pharmaceutical Co. Weighing 100g of astragalus and radix trichosanthis respectively, soaking in deionized water for about 20min, decocting according to a conventional traditional Chinese medicine decoction method, pouring the decoction into a 150mL beaker when the decoction is decocted to about 100mL, and concentrating to 50mL; transferring to an ultra-clean workbench, transferring the concentrated liquid medicine to an aseptic centrifuge tube, centrifuging at 1000r/min for 20min, collecting supernatant, and hermetically preserving at-20deg.C to obtain liquid medicine which is radix astragali decoction and radix Trichosanthis decoction with concentration of 2g/mL, and is drug storage concentration. Mixing 1mL of supernatant with 2mL of culture medium, injecting into a culture flask, placing into 37 ℃,5％CO 2 and (3) carrying out stationary culture in an incubator, and carrying out bacteria detection. After 24 hours, the culture medium is observed, the sterility of the liquid medicine is determined, and the liquid medicine is hermetically preserved at-20 ℃.
MTT assay
Taking human malignant melanoma A375 cells and human normal hepatocytes L-02 cells in logarithmic growth phase at 3×10 respectively 3 Individual/well and 8×10 3 The concentration of each/well is inoculated in a 96-well cell culture plate and placed at 37 ℃ and 5% CO 2 Culturing in incubator for 24 hr, changing liquid, and treating with medicine. The experiment sets a control group (serum-free culture medium), a astragalus water decoction group (the concentration is respectively 2, 4, 8, 10 and 20 mg/mL), and a radix trichosanthis water decoction group (the concentration is respectively 0.16, 0.32, 0.64, 1.28 and 2.56 mg/mL), and each group is provided with 5 parallel holes. After the medicine treatment, respectively and continuously culturing for 48 hours and 72 hours, sucking out the liquid medicine in each hole, washing for 1 time by using PBS, then adding 10 mu L of MTT solution with the mass concentration of 5mg/mL and 90 mu L of serum-free culture medium into each hole under the light-proof condition, uniformly mixing, wrapping and standing by using tinfoil, continuously culturing for 4 hours, then adding 150 mu L of DMSO into each hole, shaking for 10 minutes by using a shaking table, measuring absorbance (OD value) at 490nm on an enzyme-labeled instrument, and calculating the cell growth inhibition rate by a formula.
Inhibition (%) = (1-average absorbance value of experimental group/average absorbance value of control group) ×100%.
The concentrations (IC) corresponding to the cell growth inhibition ratios of the drugs at 0%, 25% and 50% were determined 0 、IC 25 、IC 50 ) Drug concentrations were screened for subsequent experiments. The experiment was then followed by a control group (serum-free medium); astragalus decoction group: astragalus water decoction IC 0 (1.1mg/ml)、IC 25 (3.6mg/ml)、IC 50 (14 mg/ml); radix trichosanthis decoction group: trichosanthin decoction IC 0 (0.137mg/ml)、IC 25 (0.863mg/ml)、IC 50 (2.113 mg/ml); the compatibility of astragalus and radix trichosanthis is: astragalus water decoction IC 0 +radix trichosanthis decoction IC 0 (1.1+0.137 mg/ml), astragalus root decoction IC 0 +radix trichosanthis decoction IC 25 (1.1+0.863 mg/ml), astragalus root decoction IC 0 +radix trichosanthis decoction IC 50 (1.1+2.113mg/ml)、Astragalus water decoction IC 25 +radix trichosanthis decoction IC 0 (3.6+0.137 mg/ml), astragalus root decoction IC 25 +radix trichosanthis decoction IC 25 (3.6+0.863 mg/ml), astragalus root decoction IC 25 +radix trichosanthis decoction IC 50 (3.6+2.113 mg/ml), and further detecting the effect of radix astragali, radix Trichosanthis and its compatibility on proliferation of A375 cells and L-02 cells.
MTT assay results
In terms of the inhibition of melanoma cells, after 48h of drug treatment, the MTT results show that astragalus water decoction and radix trichosanthis water decoction with different concentrations have different inhibition effects on proliferation of A375 cells. As shown in Table 1 and FIG. 1, the inhibition rates of astragalus decoction with different concentrations on proliferation of A375 cells are 26.51%, 33.85%, 37.29%, 49.33% and 57.27%, respectively, and each administration group has statistical significance (P < 0.05) compared with the control group; the inhibition rate of the trichosanthin decoction with different concentrations on the proliferation of A375 cells is 8.27%, 17.79%, 23.40%, 32.95% and 46.89%, respectively, and each administration group has statistical significance (P < 0.05) compared with the control group.
TABLE 1 influence of decoction of Astragalus membranaceus and Trichosanthis radix on cell proliferation after 48h action on A375 cellsn＝3)
Note that: p <0.05 compared to the control group
After the medicine is treated for 72 hours, MTT results show that the astragalus decoction and the radix trichosanthis decoction with different concentrations have different inhibition effects on proliferation of A375 cells. As shown in Table 2 and FIG. 2, the inhibition rates of astragalus water decoction with different concentrations on A375 cells are respectively 20.23%, 28.31%, 39.14%, 49.23% and 68.17%, and compared with a control group, each administration group has statistical significance (P < 0.05); the inhibition rates of the trichosanthin decoction with different concentrations on A375 cells are 11.48%, 21.22%, 29.39%, 43.52% and 61.11%, respectively, and each administration group has statistical significance (P < 0.05) compared with the control group.
TABLE 2 influence of decoction of Astragalus membranaceus and Trichosanthis radix on cell proliferation after 72h action on A375 cellsn＝3)
Note that: p <0.05 compared to the control group
Based on the above results, calculating corresponding IC by statistical software 0 、IC 25 IC (integrated circuit) 50 . The results are shown in Table 3, and these concentrations were individually selected for further investigation.
TABLE 3 inhibitory concentration IC of Astragalus membranaceus decoction and radix Trichosanthis decoction on A375 cells at different time of action 0 、IC 25 IC (integrated circuit) 50
As shown in table 4 and fig. 3, after 48 hours of drug treatment, the astragalus group (IC 0 、IC 25 、IC 50 ) Radix Trichosanthis group (IC) 0 、IC 25 、IC 50 ) Both astragalus root and trichosanthes root can inhibit proliferation of A375 cells, and the difference has statistical significance (P is less than 0.05). Yellow IC in compatibility group 0 +Tian IC 25 Yellow IC 0 +Tian IC 50 Yellow IC 25 +Tian IC 25 Yellow IC 25 +Tian IC 50 The inhibition rate of the group to the proliferation of A375 cells is higher than that of the corresponding astragalus group; yellow IC 25 +Tian IC 0 Yellow IC 25 +Tian IC 25 Yellow IC 25 +Tian IC 50 The inhibition rate of the group on the proliferation of A375 cells is higher than that of the corresponding radix trichosanthis group. Which is a kind ofIn yellow IC 25 +Tian IC 25 Yellow IC 25 +Tian IC 50 The inhibition effect of the group on A375 cell proliferation is different from that of corresponding astragalus root and radix trichosanthis single medicine. Yellow IC in this experiment 0 +Tian IC 50 Hehuang IC 25 +Tian IC 50 Has better proliferation inhibition capability on A375 cells.
TABLE 4 influence of the compatibility of Astragalus mongholicus and radix Trichosanthis on cell proliferation after 48h with A375 cellsn＝3)
Note that: p <0.05 compared to the control group;
the ratio of the compatibility group to the corresponding astragalus single medicine group is #P < 0.05;
the ratio of the compatibility group to the corresponding single medicine group of the radix trichosanthis is delta P less than 0.05
However, in MTT experiments on human normal hepatocytes, it was found that the concentrations of inhibition by A375 were IC 0 、IC 25 、IC 50 After the astragalus and radix trichosanthis decoction is used for treating normal liver cells L-02 cells for 48 hours, different compatibility of the astragalus and radix trichosanthis also has different effects on proliferation of human normal liver cells. As shown in Table 5 and FIG. 4, in each of the administration groups, IC in the astragalus group 0 、IC 25 Yellow IC in compatibility group 0 +Tian IC 0 Yellow IC 0 +Tian IC 25 Yellow IC 25 +Tian IC 0 Yellow IC 25 +Tian IC 25 There was no statistical difference in proliferation of human normal hepatocytes (P > 0.05). While other groups have some inhibition of proliferation of human normal hepatocytes, the inhibition of proliferation of L-02 cells is much lower than that of A375, such as yellow IC 50 The inhibition rate for A375 cells was 50%, while the inhibition rate for L-02 cells was only 10.97 (as shown in Table 5), indicating that its toxicity to normal cells was far lower than its toxicity to tumor cells. Yellow IC 25 +Tian IC 50 Compatibility of group, to A375 cellsThe theoretical inhibition rate is 75 percent, which is the formula with the highest inhibition rate in a plurality of administration groups, the inhibition rate of the compatibility on L-02 cells is 28.23 percent, and the single use of the trichosanthin IC 50 When the dosage is taken, the inhibition rate of normal cells is 46.92 percent, and after different dosages of astragalus root are added for compatibility (yellow IC 0 +Tian IC 50 Hehuang IC 25 +Tian IC 50 ) The inhibition rate of the astragalus to L-02 cells is reduced to 29.64 percent and 28.23 percent, which shows that the astragalus has the effect of reducing the toxicity of the radix trichosanthis to a certain extent in the compatibility formula.
TABLE 5 influence of the compatibility of Astragalus mongholicus and radix Trichosanthis on cell proliferation after 48hn＝6)
Note that: p <0.05 compared to the control group;
the ratio of the compatibility group to the corresponding astragalus single medicine group is #P < 0.05;
the ratio of the compatibility group to the corresponding single medicine group of the radix trichosanthis is delta P less than 0.05
FIG. 5 shows the effect of astragalus root and trichosanthes root on L-02 cell morphology after 48h of administration of each administration group, as can be seen, the control group has a higher cell number, regular cell morphology, clear cell membrane, and a lower number of dead cells, and the IC in the astragalus group is compared with the control group 0 、IC 25 Yellow IC in compatibility group 0 +Tian IC 0 Yellow IC 0 +Tian IC 25 Yellow IC 25 +Tian IC 0 Yellow IC 25 +Tian IC 25 The number of cells in the group is not reduced, and the cell morphology is regular; yellow IC 50 Tian IC 0 Hetian IC 25 The group, compared with the control group, had a reduced cell number but was not apparent, whereas it was Tianic 50 Group, yellow IC 0 +Tian IC 50 Hehuang IC 25 +Tian IC 50 The number of cells was significantly reduced, and the morphology of the cells was altered, and round cells were increased, consistent with the results in Table 5.
In summary, the MTT results described above indicate that yellow IC 25 Yellow IC 0 +Tian IC 0 Yellow IC 0 +Tian IC 25 Yellow IC 25 +Tian IC 0 Four groups can also obviously inhibit the proliferation of melanoma cells under the condition of no toxicity to human normal liver cells.
EXAMPLE 2 Effect of radix astragali, radix Trichosanthis and its compatibility on melanoma cell cycle and apoptosis
To further verify the effect of each dosing group on melanoma, we used 1×10 a375 cells 4 The density of each hole is inoculated into a 6-hole plate, and an astragalus water decoction group, a radix trichosanthis water decoction group and a radix astragali and radix trichosanthis compatibility group are experimentally set, and each group is provided with 3 compound holes. After 48h of drug treatment, the cell cycle and apoptosis changes were detected by flow cytometry. Detection of apoptosis cells were treated with an Annexin-FITC apoptosis kit. In addition, we stained nuclei with Hoechst33342 and photographed with confocal microscopy (400×) to observe apoptotic morphology.
C57BL/6 mice of 6-8 weeks of age were selected for in vivo experiments and randomly divided into 6 groups of 6, with abdominal dehairing. Normal Control Group (NCG) was subcutaneously injected with 0.1mL of PBS solution in the right flank, and the remaining mice were injected with 5X 10 5 PBS solution of individual B16-F10 cells, labeled d0. After 2 days (d 2), black spots of more than 2mm appear at the injection site, and the melanoma model is successfully established. At d3, NCG and model control (MSG) were lavaged with purified water, the other dosing groups were given a dilution with purified water of 5:3 the combination of astragalus root and radix trichosanthis decoction (low, medium and high dose groups are respectively 3.12g/kg,6.24g/kg and 12.48 g/kg), the Positive Control Group (PCG) is given with the compound cyclophosphamide tablet solution (19.5 mg/kg) 1 time a day, and the stomach is continuously irrigated for 14d. Mice were monitored every two days for body weight, tumor length, and transverse diameter, and MSG group tumor photographs were taken every 4 days. After the end of the experiment, the mice were anesthetized and blood, tumor and liver were removed. By H&E staining detects histopathological features.
The flow cytometry results are shown in Table 6 and FIG. 6, and after different concentrations of radix astragali decoction and radix Trichosanthis decoction and their compatibility A375 cells are used for 48 hr, the control group, radix astragali decoction group, radix Trichosanthis decoction group, and radix astragali decoction groupsG of radix Trichosanthis compatibility group 1 Stage, S stage and G 2 The percentage of cells in the phase is different from the total number of cells. With control group G 1 Percentage of cells in phase (64.4%) compared to yellow IC 25 Group G 1 No significant change in cell percentage (64.5%) at stage, yellow IC 50 Group G 1 The percentage of cells (71.0%) was significantly increased, which was statistically significant. Yellow IC 0 Group, radix trichosanthis group and compatibility group G of radix astragali and radix trichosanthis 1 The percentage of cells in the phase (61.4%, 62.0%, 60.2%, 38.5%, 57.2%, 53.8%, 45.4%, 57.3%, 61.7%, 52.7%) was reduced compared to the control group, which was statistically significant. With control group G 2 Percentage of cells in phase (8.9%) compared to each group G 2 The percentage of cells in the phase (15.3%, 11.7%, 13.0%, 17.2%, 20.9%, 29.5%, 14.9%, 17.8%, 20.9%, 16.4%, 11.4%, 12.5%, respectively) was increased to different extents, and was statistically significant. Yellow IC compared to the control group S phase cell percentage (25.2%) 0 Yellow IC 50 Tian IC 0 Tian IC 25 The percentage of S phase cells in the group (23.0%, 16.2%, 21.2%, 18.8%, respectively) was reduced, and IC was found to be high 50 Yellow IC 0 +Tian IC 25 Yellow IC 0 +Tian IC 50 The percentage of group S cells (32.2%, 27.9%, 31.8%, respectively) was increased, p<0.05. The percentages of the S-phase cells of the remaining groups were not significantly different from the control. Compared with the single astragalus root group, the compatibility group G 1 The percentage of cells in the phase is reduced; compared with single radix astragali group, yellow IC 0 +Tian IC 25 Yellow IC 0 +Tian IC 50 Yellow IC 25 +Tian IC 0 Group G 2 The percentage of cells in the stage is increased, which is statistically significant. Compared with single radix trichosanthis medicine group, the compatibility group removes yellow IC 25 +Tian IC 0 Group G 2 The cell number is not obviously different in the period, and other groups are lower than that of the radix trichosanthis group, so that the method has statistical significance.
TABLE 6 influence of radix astragali, radix Trichosanthis and compatibility A375 cells for 48h on cell proliferation cyclen＝3)
Note that: * Represents p <0.05 compared to the control group
# represents p <0.05 compared with the corresponding Astragalus mongholicus group
Delta represents p <0.05 compared with the corresponding single radix Trichosanthis group
The Annexin-FITC/PI double-staining flow pattern results are shown in Table 7 and FIG. 7, the percentage of normal cells in the administration group is obviously reduced compared with the percentage of normal cells in the control group, the percentage of apoptotic cells is obviously increased, and the difference is statistically significant. Yellow-removing IC compared with control group 0 Tian IC 0 Yellow IC 0 +Tian IC 0 The number of early apoptotic cells in each of the remaining dosing groups increased; yellow removing IC 0 The number of late apoptotic cells was increased in each of the remaining dosing groups. Compared with a single medicine group, the early apoptotic cell number of the compatibility group is obviously increased, and the total number of apoptotic cells is obviously increased.
Hoechst staining results showed that most of the nuclear membranes in the compatible group were significantly collapsed and the intracardiac chromatin condensed (fig. 7B). WB results show that astragalus root, radix trichosanthis and compatibility thereof can reduce Bcl-2 protein expression quantity and improve Bax protein expression level, and the Bax/Bcl-2 ratio is increased in a dose-dependent manner, so that the compatibility group effect is better. In addition, the compatibility group can also increase the expression of p53 protein. Further in vivo experimental results show that p53 and Bax protein expression levels in melanoma model mice administered with the combination are also increased (fig. 7F).
TABLE 7 influence of astragalus root, trichosanthes root and compatibility A375 cells for 48h on apoptosisn＝3)
Note that: * Represents p <0.05 compared to the control group
# represents p <0.05 compared with the corresponding Astragalus mongholicus group
Delta represents p <0.05 compared with the corresponding single radix Trichosanthis group
The results show that astragalus root, radix trichosanthis and compatibility thereof can promote apoptosis of A375 cells by regulating the expression levels of Bax, bcl-2 and p53 proteins. In mice, the optimal compatibility ratio of astragalus and radix trichosanthis can also influence the growth of tumors by promoting the expression of p53 protein or inhibiting the expression of Bcl-2. Therefore, the radix astragali and the radix trichosanthis can achieve the effect of treating the melanoma by inhibiting cell proliferation, regulating cell cycle and inducing apoptosis, and the compatibility of the two medicines has synergistic anti-tumor effect.
EXAMPLE 3 Effect of radix astragali, radix Trichosanthis and its compatibility on melanoma cell migration
Malignant melanoma is highly metastatic. Epithelial Mesenchymal Transition (EMT) is a key step in cell invasion and metastasis, with E-cadherin and N-cadherin being two key factors of EMT. Research proves that up-regulating E-cadherin protein expression and reducing N-cadherin protein expression can effectively inhibit cell migration and invasion.
To verify the effect of astragalus root, trichosanthes root and their compatibility on melanoma cell migration, we used A375 cells at 3X 10 3 Density of wells/well was inoculated into 96-well plates, cultured for 24 hours, and then a 10 μl gun head was used to scratch a cross at the center of each well, washed 2 times with PBS to remove cell debris and photographed for recording. Then, the astragalus water decoction group, the radix trichosanthis water decoction group and the astragalus and radix trichosanthis compatibility group are experimentally set, and each group is provided with 3 compound holes. And (5) culturing for 48 hours and 72 hours, observing the healing degree of scratches under a microscope, photographing and recording, and analyzing the migration condition of cells by using Image J software.
After astragalus root, radix trichosanthis and compatibility A375 cells are treated for 48 hours, the knot is observed under an inverted optical microscopeAs shown in fig. 8, the control group showed a large number of cells, and the scratch tended to heal. Yellow-removing IC compared with control group 0 Tian IC 0 Yellow IC 0 +Tian IC 0 The cell number of the other administration groups is reduced, the scratch healing capacity is poor, and the non-healing areas of the scratches are higher than those of the control group, so that the method has statistical significance. Compared with single medicine group, the compatibility group yellow IC 0 +Tian IC 25 Yellow IC 0 +Tian IC 50 Yellow IC 25 +Tian IC 50 The group scratches healed to a lower degree and the scratch distance was greater. After astragalus root, radix trichosanthis and the compatibility A375 cells are treated for 72 hours, the cell scratches of the control group heal obviously, and the IC is eliminated 0 In the group, the unhealed areas of the other administration groups are obviously higher than those of the control group. Compared with a single medicine group, the compatibility group has lower scratch healing degree and larger scratch distance.
The results of the mechanism study show that in the drug group, the E-cadherin protein expression level is increased, the snail and N-cadherin expression is reduced, and the E-cadherin/N-cadherin ratio is increased. In addition, further in vivo experimental results show that the yellow IC 25 +Tian IC 50 E-cadherin protein expression levels were increased and N-cadherin protein expression was decreased in tumor tissues of mice model for melanoma administered in the matched set (FIG. 8E).
From this, it is proved that astragalus root, radix trichosanthis and compatibility thereof can inhibit A375 cell migration by affecting E-cadherin, N-cadherin and snail protein expression, and the optimal compatibility ratio of the two drugs can also regulate the expression levels of E-cadherin and N-cadherin in tumor tissue of melanoma mice. The compatibility of the astragalus and the radix trichosanthis can obviously inhibit the migration of A375 cells and show an anti-tumor synergistic effect.
EXAMPLE 4 Effect of radix astragali, radix Trichosanthis and its compatibility on melanoma LDH, SOD, MDA
Glycolysis is required for the growth of tumor cells, and excessive glycolysis promotes cell proliferation and metastasis. LDH is a key enzyme in glycolysis and can promote malignancy of tumors by activating EMT in cancer cells. Antitumor drugs can reduce glycolysis by decreasing LDH activity, thereby inhibiting proliferation and migration of tumor cells. SOD is an important antioxidant biomarker in organisms, while MDA is often used as an indicator of oxidative damage to cell membranes. Studies have shown that antioxidants are effective against apoptosis and promote migration of melanoma cells. Therefore, we further explore the anti-tumor effect of astragalus, radix trichosanthis and the compatibility thereof by detecting the influence of the astragalus, the radix trichosanthis and the compatibility thereof on melanoma LDH, SOD, MDA.
At 1X 10 4 Individual cells/well a375 cells were seeded into 6-well plates. The experiment sets astragalus water decoction group, radix trichosanthis water decoction group and astragalus and radix trichosanthis compatibility group, and each group is provided with 3 compound holes. Cell supernatants after 48h of drug treatment were collected and serum from mice administered for 14 days. Then, an LDH kit, an SOD kit and an MDA kit are respectively used for detecting LDH activity, SOD activity and MDA content.
After astragalus root, radix trichosanthis and the compatible application thereof are used for 48 hours on human malignant melanoma A375 cells, as shown in figure 9, compared with a control group, the medicine group can reduce the activity of LDH and SOD of A375 cells and increase the MDA content. Compared with the corresponding single medicine group, the compatibility group has lower activity of cell SOD, higher MDA content and statistically significant difference.
In addition, further in vivo experimental results show that compared with the control mice, the LDH activity of the model mice is obviously increased, and the LDH activity of the other administration groups is reduced (figure 9B).
In summary, we find that astragalus and radix trichosanthis can reduce the activity of LDH and SOD of A375 cells in vitro experiments, increase MDA value, reduce the removal of ROS from organism, inhibit antioxidant defense system, increase damage to A375 cells caused by oxidative stress, and possibly regulate a series of activities such as cell proliferation, apoptosis and migration through the way. In addition, in vivo experimental results show that the LDH in the serum of the mice in the model control group is higher than that of the mice in the normal control group, and the abnormal rise of the LDH of the mice in each administration group is reduced after the administration. This shows that the compatibility of astragalus root and trichosanthes root can effectively inhibit the metastasis of melanoma.
EXAMPLE 5 Effect of radix astragali, radix Trichosanthis and its compatibility on melanoma Akt-associated Signal pathway protein expression
Akt is an important oncogene closely related to cell proliferation, migration and metabolism, and has obvious difference in expression of malignant melanoma and common nevus pigmentosus, and increases along with exacerbation of malignant melanoma, reduces activity of Akt, inhibits phosphorylation of Akt, and can promote death of malignant melanoma cells. Activated Akt plays an important role in both apoptosis and migration. Studies have shown that it can not only regulate apoptosis by affecting Bax and Bcl-2 expression, but also inhibit E-cadherein expression to promote EMT development during melanoma development. In addition, activated Akt also affects ROS production, thereby affecting melanoma cell survival.
We determined Akt protein concentration in a375 cells and mouse tissue proteins by WB. As shown in FIG. 10, after the radix astragali, the radix trichosanthis and the compatibility A375 cells are processed for 48 hours, the expression level of the cell Akt and the p-Akt protein can be reduced. After the medicine acts for 72 hours, compared with the corresponding single medicine group, the compatible group has better effect of reducing the expression of Akt and p-Akt proteins. In addition, in vivo test results showed that expression of Akt protein was also reduced in tumor tissue of mice model melanoma by co-administration (fig. 10D).
The results prove that the astragalus root, the radix trichosanthis and the compatibility thereof can reduce the expression of melanoma Akt and p-Akt proteins, thereby inhibiting the activity and phosphorylation of Akt. This may be the mechanism by which these two traditional Chinese medicines regulate malignant melanoma cell proliferation, cell cycle, apoptosis and migration. And the compatibility has synergistic effect.
In conclusion, the invention proves that astragalus (less than 4 mg/ml) and radix trichosanthis (less than 1.28 mg/ml) can both play a role in inhibiting proliferation of A375 cells, and simultaneously, L-02 cells are not influenced or slightly influenced. The method shows that the astragalus and the radix trichosanthis are non-cytotoxic medicines at a certain dosage, and the method has positive significance for popularization of the application of the two medicines. More importantly, the optimal compatibility ratio of the astragalus and the radix trichosanthis can inhibit the growth of the melanoma of the mice in vivo, and has no liver toxicity.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (9)
1. The medicinal composition without hepatotoxicity is characterized in that the active ingredients of the medicinal composition are astragalus and radix trichosanthis, wherein the final concentration of the astragalus is lower than 4mg/ml, and the final concentration of the radix trichosanthis is lower than 1.28mg/ml.
2. The pharmaceutical composition of claim 1, wherein the astragalus has a final concentration of 1.1-3.6mg/ml and the radix trichosanthis has a final concentration of 0.137-0.863mg/ml.
3. The pharmaceutical composition of claim 1, wherein the astragalus has a final concentration of 1.1mg/ml and the radix trichosanthis has a final concentration of 0.863mg/ml.
4. Use of a pharmaceutical composition according to any one of claims 1-3 for the preparation of a medicament for promoting apoptosis of melanoma cells.
5. Use of a pharmaceutical composition according to any one of claims 1-3 for the preparation of a medicament for inhibiting migration of melanoma cells.
6. Use of a pharmaceutical composition according to any one of claims 1-3 for the preparation of a medicament for increasing the ratio of Bax/Bcl-2 expression levels in melanoma cells.
7. Use of a pharmaceutical composition according to any one of claims 1-3 for the preparation of a medicament for reducing the viability of LDH and SOD in a375 cells and for increasing the MDA content in a375 cells.
8. Use of a pharmaceutical composition according to any one of claims 1-3 for the preparation of a medicament for inhibiting Akt activity and phosphorylation in melanoma cells.
9. The pharmaceutical composition capable of remarkably inhibiting the proliferation of melanoma cells is characterized in that active ingredients of the pharmaceutical composition are astragalus membranaceus and radix trichosanthis, wherein the final concentration of the astragalus membranaceus is 3.6mg/ml, and the final concentration of the radix trichosanthis is 2.113mg/ml.
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| 张秋艳等: "黄芪和天花粉对上鼠恶性黑色素瘤B16细胞增殖和迁移及E-cadherin、 N-cadherin蛋白表达的影响", 中国中医药信息杂志, vol. 27, no. 5, 31 May 2020 (2020-05-31), pages 28 - 34 * |
| 谢惠民: "临床药物新用联用大全", 31 August 1999, 人民卫生出版社, pages: 880 * |
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