CN114642698B - Application of total saponins of herba Solani Nigri in preparing drug-resistant leukemia drug - Google Patents
Application of total saponins of herba Solani Nigri in preparing drug-resistant leukemia drug Download PDFInfo
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Classifications
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- 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)
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- A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
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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
- A61P35/02—Antineoplastic agents specific for leukemia
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- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/50—Methods involving additional extraction steps
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Abstract
The invention discloses application of total saponins of black nightshade in preparation of drug-resistant leukemia drugs, wherein the drug-resistant leukemia is multi-drug-resistant leukemia, and the use concentration of the total saponins of black nightshade is more than 10 mug/mL. The inventor finds that SN has better anti-tumor activity in vivo by utilizing a leukemia drug-resistant cell strain nude mice transplantation tumor model, and finds out the expression condition of the SN on related proteins such as intracellular cycle, apoptosis, autophagy and drug resistance by measuring the inhibition effect of the SN on K562/ADR and K562 cell proliferation in vitro, and verifies the influence of the SN on autophagy or related pathways on overcoming drug resistance and cell death.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of total saponins of black nightshade in preparation of a drug-resistant leukemia drug.
Background
Tumor multidrug resistance (multidrug resistance, MDR) is one of the main causes of failure and recurrence of tumor chemotherapy, and is characterized by resistance to various other chemotherapeutics with different structures and mechanisms of action while malignant cells are resistant to one chemotherapeutic. Therefore, how to overcome the multidrug resistance of tumors is an urgent issue in cancer treatment.
Autophagy (autophagy), also known as type II apoptosis, is a mechanism of self-protection of body cells under stress conditions, a phenomenon commonly occurring during the treatment of cancer. Autophagy is a double sword for multi-drug resistant tumors according to the composition of tumor cells, the treatment mode and the change of microenvironment, and the double sword can participate in the generation of multi-drug resistance of the tumor and protect the tumor cells from being influenced by chemotherapeutics, but can also induce excessive autophagy of the cells to finally cause autophagic death of the tumor cells. Thus, modulation of autophagy to overcome multidrug resistance of tumors is a new strategy to treat various types of malignancies. However, in the related art, no technology has been disclosed for overcoming the multidrug resistance of tumors by using autophagy.
Solanum nigrum L as a traditional Chinese medicine has various biological effects, and the extract thereof has remarkable anti-tumor effect. However, the solanine has stronger cytotoxicity and has higher requirements on the dosage and the using method, thereby greatly limiting the application of the solanine in the aspect of resisting tumors.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides application of total saponins of black nightshade in preparing drug-resistant leukemia drugs. The inventor discovers that total steroid Saponin (SN) extracted and enriched from black nightshade has the function of inhibiting the proliferation of drug-resistant leukemia cells, so that the total steroid Saponin (SN) can be used as a novel drug-resistant leukemia drug to effectively solve the problem of leukemia drug resistance.
In a first aspect, the invention provides an application of total saponins of black nightshade in preparing drug for treating drug-resistant leukemia.
In the invention, the inventor proves that the SN reduces the activity of K562 and K562/ADR cells in a concentration and time dependent mode through various experiments, which proves that the SN can effectively kill general leukemia cells and drug-resistant leukemia cells and has a certain anti-leukemia drug-resistant effect.
According to a first aspect of the invention, in some embodiments of the invention, the drug-resistant leukemia comprises multidrug-resistant leukemia such as doxorubicin-resistant leukemia.
In some preferred embodiments of the invention, the total saponins of Solanum nigrum are used at a concentration of greater than 10 μg/mL.
In the present invention, 10 μg/mL of Solanum nigrum extract has significant cytotoxicity to K562/ADR cells. In addition, researches on the black nightshade extract for overcoming tumor resistance are carried out at present, and particularly, researches on the black nightshade extract for directly overcoming tumor resistance are very few. Therefore, intensive research on steroid saponins in black nightshade is necessary to find novel anti-leukemia drugs with drug resistance overcoming effects.
In some more preferred embodiments of the invention, the total saponins of Solanum nigrum are used at a concentration of greater than 20 μg/mL.
In the invention, the inventor finds that 20 mug/mL of SN can effectively inhibit the expression of BCRP, P-gp and other drug-resistant proteins in K562/ADR, the inhibition effect is even stronger than that of positive control doxorubicin, and the result shows that the SN can inhibit the drug resistance of K562/ADR cells by inhibiting the expression of BCRP, P-gp and other drug-resistant proteins.
According to a first aspect of the present invention, in some embodiments of the present invention, the preparation method of total saponins of black nightshade comprises:
(1) Reflux-extracting black nightshade olive with 60-70% alcohol for 2-4 times to obtain alcohol extractive solution;
(2) Removing ethanol in the ethanol extract, adding water, re-suspending, eluting with D101 macroporous resin column, collecting eluate, concentrating, and drying.
In some preferred embodiments of the invention, the eluent is 50% -70% ethanol eluent.
In some preferred embodiments of the invention, the eluent is 50% ethanol and 70% ethanol.
In some preferred embodiments of the present invention, the preparation method of the total saponins of black nightshade specifically comprises: and (3) taking dried black nightshade fruits, and heating to about 70 ℃ with 65% ethanol for reflux (3 times) extraction to obtain black nightshade ethanol extract. Removing ethanol in the black nightshade ethanol extract by adopting a decompression concentration mode, and adding water for resuspension to obtain black nightshade water suspension. Sequentially performing gradient elution on the black nightshade water suspension by using a D101 macroporous resin column and using water, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol, collecting the eluent of 50% ethanol and 70% ethanol, and mixing to obtain the black nightshade total saponin extract.
According to a first aspect of the invention, in some embodiments of the invention, the total saponins of black nightshade overcome leukemia resistance by activating autophagy.
In some preferred embodiments of the invention, the SN induces autophagy in cells and overcomes K562/ADR cell resistance via the (PI 3K)/Akt/mTOR/p 70S6K/4EBP1 signaling pathway.
In some preferred embodiments of the invention, the total saponin-activated autophagy of black nightshade is expressed as:
promote the conversion of LC3-I to LC 3-II;
increasing expression of Beclin-1; and
the expression of p62 was reduced.
In a second aspect, the invention provides an application of total saponins of black nightshade in preparing a cell proliferation inhibition medicament.
According to a second aspect of the invention, in some embodiments of the invention, the cells are K562/ADR cells.
In some preferred embodiments of the invention, the total saponins of Solanum nigrum inhibit cell proliferation by blocking the K562/ADR cell cycle.
In a third aspect, the invention provides an application of total saponins of black nightshade in preparing medicines for promoting apoptosis.
According to a third aspect of the invention, in some embodiments of the invention, the cells are K562/ADR cells.
In some preferred embodiments of the invention, the total saponins of Solanum nigrum promote apoptosis by increasing PARP-1, clear-caspase 3 expression, decreasing pro-caspase 3, pro-caspase 8 and pro-caspase9 expression.
In a fourth aspect, the invention provides an application of total saponins of black nightshade in preparing medicaments for promoting autophagy.
According to a fourth aspect of the invention, in some embodiments of the invention, the cells are K562/ADR cells.
In some preferred embodiments of the invention, the total saponins of Solanum nigrum promote the conversion of LC3-I to LC3-II by inhibiting the expression of p-Akt, p-mTOR, p-p70S6K and p-4EBP1, thereby promoting autophagy in the cell.
The beneficial effects of the invention are as follows:
the invention utilizes aqueous ethanol to extract black nightshade fruits, the extracted extractum is subjected to macroporous resin gradient elution, and the obtained eluent is decompressed and concentrated, so that the black nightshade steroid Saponin (SN) is obtained. The inventor finds that SN has better anti-tumor activity in vivo by utilizing a leukemia drug-resistant cell strain nude mice transplantation tumor model, and finds out the expression condition of the SN on related proteins such as intracellular cycle, apoptosis, autophagy and drug resistance by measuring the inhibition effect of the SN on K562/ADR and K562 cell proliferation in vitro, and verifies the influence of the SN on autophagy or related pathways on overcoming drug resistance and cell death.
Drawings
FIG. 1 is a high performance liquid chromatogram of the composition analysis of total saponins of Solanum nigrum.
FIG. 2 is a graph showing the inhibition of growth of a graft by SN and the time of drug delivery in an embodiment of the present invention.
Fig. 3 shows the effect of SN on tumor volume and tumor weight.
FIG. 4 shows the cytotoxic effects of SN on K562/ADR and K562 cells.
FIG. 5 shows the expression levels of BCRP and P-gp proteins in K562/ADR and K562 cells.
FIG. 6 shows the effect of different concentrations of SN on the expression of BCRP and P-gp proteins in K562/ADR cells.
FIG. 7 shows the effect of SN on G0/G1 phase associated cyclin expression of K562/ADR and K562.
FIG. 8 shows the effect of SN on expression of apoptosis-related proteins in both K562/ADR and K562 cells.
FIG. 9 is the effect of Z-VAD-FMK pretreatment on expression of apoptosis-related proteins in K562/ADR cells.
FIG. 10 is the effect of Z-VAD-FMK pretreatment on drug resistance associated protein expression in K562/ADR cells.
FIG. 11 shows the effect of SN on autophagy-related protein expression in both K562/ADR and K562 cells.
FIG. 12 is the effect of pretreatment of 3MA and CQ on apoptosis-related protein expression in K562/ADR cells.
FIG. 13 is the effect of pretreatment of 3MA and CQ on drug resistance associated protein expression in K562/ADR cells.
FIG. 14 is the effect of Z-VAD-FMK pretreatment on LC3-II expression in K562/ADR cells.
FIG. 15 shows the effect of SN on p-Akt, p-mTOR, p-p70S6K, and p-4EBP1 protein expression in both K562/ADR and K562 cells.
FIG. 16 shows the effect of SN on BCRP, P-gp, P-mTOR and LC3-II protein expression in K562/ADR nude mouse transplants.
FIG. 17 shows the effect of MHY1485 pretreatment on p-mTOR, p-p70S6K and p-4EBP1 protein expression.
FIG. 18 is the effect of MHY1485 pretreatment on expression of apoptosis-related proteins in K562/ADR cells.
FIG. 19 shows the effect of MHY1485 pretreatment on LC3-II and p62 protein expression in K562/ADR cells.
FIG. 20 shows the effect of MHY1485 pretreatment on drug resistance associated protein expression in K562/ADR cells.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to the following specific embodiments. It should be understood that the detailed description is presented herein for purposes of illustration only and is not intended to limit the invention.
The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.
Extraction, enrichment and content determination of total saponins of black nightshade
And (3) taking dried black nightshade fruits, and heating the dried black nightshade fruits to 70 ℃ by using 65% ethanol for reflux extraction to obtain black nightshade alcohol extract. Removing ethanol in the black nightshade ethanol extract by adopting a decompression concentration mode, and adding water for resuspension to obtain black nightshade water suspension. Allowing the herba Solani Nigri aqueous suspension to pass through D101 macroporous resin column, sequentially performing gradient elution with water, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol, collecting eluate of 50% ethanol and 70% ethanol, and mixing to obtain herba Solani Nigri total saponin extract. The total Saponin (SN) content of the black nightshade in the extract was determined by uv spectrophotometry at 430nm wavelength. The method uses the saponin monomer separated in the early stage of the laboratory as a reference substance, adopts a high performance liquid chromatography to analyze the components of the total saponin of the black nightshade, and has the following chromatographic conditions: detector ELSD (drift tube temperature: 95 ℃, carrier gas: compressed air, pressure: 0.1 MPa), chromatographic column: COSMIL 5C 18-MS-II (4.6 ID. Times.250 mm,5 μm), column temperature: room temperature, mobile phase: acetonitrile (a) and 0.8% trifluoroacetic acid-water (B), elution gradient conditions: 0-15 min:22% -24% (A), 15-30 min:24% -27% (A), 30-40 min:27% -29% (A), 40-45 min:29% -38% (A), 45-60 min:38% -38% (A), 60-70 min:38% -100% (A), flow rate: 1.0mL/min.
The results are shown in FIG. 1.
We identified a total of 4 major steroid saponin monomer chromatographic peaks, chemical names shown in table 1.
TABLE 1
Antitumor effect of total saponins of Solanum nigrum
In this example, the laboratory mice were female nude mice for 3-4 weeks purchased from the medical animal laboratory center in Guangdong province.
(1) Modeling an experimental animal model:
taking female nude mice of 3-4 weeks, inoculating equivalent human white blood by subcutaneous injection into left upper limbAdriamycin resistant strain K562/ADR, when cells become tumor and grow to about 100mm 3 And finishing the molding.
(2) Research on anti-tumor effect of total saponins of black nightshade:
the experimental mice after the modeling are divided into five groups randomly, and the following methods are adopted for treatment respectively:
model group: injecting the same amount of physiological saline into the abdominal cavity;
positive drug group: 2mg/kg of doxorubicin was injected intraperitoneally;
low dose group of total saponins of black nightshade: the total saponins of black nightshade extracted in the above example are infused with 62.5mg/kg by the mode of gastric administration.
Dose group of total saponins of black nightshade: the total saponins of the black nightshade, which are extracted from the above examples, are infused with 125mg/kg by the mode of gastric administration.
High dose group of total saponins of black nightshade: the total saponins of the black nightshade extracted in the above example are infused with 250mg/kg by the mode of gastric administration.
Wherein the positive drug group is administered once every 3 days for 6 times.
The experimental groups of total saponins of Solanum nigrum (low dose group, medium dose group and high dose group) were dosed once every 2 days for 7 times.
The experiment lasted for 24 days and the experimental flow chart and tumor volume changes are shown in figure 2.
Tumor volumes were weighed every other day and measured (formula:). Closely paying attention to vital signs of nude mice, dislocation is performed after the experimental period is finished, and the experimental mice are killed. Dissecting out the tumor and weighing the tumor.
The results are shown in FIG. 3.
From the results, compared with the model group, the tumor of the mice treated by SN administration has smaller obvious volume, especially the SN high-dose group, and even compared with the positive drug group, the inhibition effect on the tumor is most obvious, which indicates that SN can obviously inhibit the growth of K562/ADR transplanted tumor in vivo. From the tumor weight data obtained, it can be seen that SN-administered treatment significantly reduced tumor quality compared to positive drug treatment, especially for the high dose group, with significant differences compared to the positive drug group (p < 0.01).
Cytotoxicity of Solanum nigrum Total saponins
The effect of SN on cell proliferation and cell viability was examined using CCK-8, the specific experimental procedure was as follows:
taking K562 and K562/ADR cells at 5×10 respectively 3 The density of cells/wells was seeded in 96-well plates and placed in an incubator for overnight incubation. The total saponins of Solanum nigrum extracted in the above examples were added to wells at different concentrations (0, 5, 10, 20 and 40. Mu.g/mL) on separate days, incubated with cells for 24, 48 and 72 hours respectively, and then incubated with 10. Mu.L of CCK-8 solution per well for another 4 hours. Absorbance (OD value) was then measured at 450nm using a microplate reader.
The results are shown in FIG. 4.
The SN can be found to reduce the activity of K562 and K562/ADR cells in a concentration and time dependent manner, which indicates that the SN can effectively kill general leukemia cells and drug-resistant leukemia cells and has a certain anti-leukemia drug-resistant effect.
Research on action mechanism of SN anti-tumor drug resistance
Inoculating K562 and K562/ADR cells in logarithmic growth phase (inoculating concentration of 4×10) 5 cells/well) were placed in 6-well plates and incubated overnight in an incubator. Cells in wells were collected on alternate days for subsequent experiments.
The expression of BCRP and P-gp proteins in cells was detected separately using Western blotting. Wherein, the primary antibodies P-gp and BCRP are purchased from Wuhan Sanying (Wuhan, china) and the antibodies are used at a ratio of 1:2000 was diluted. The secondary antibody was rabbit antibody, purchased from Cell Signaling Technology (Danvers, USA) and used at 1:2000 was diluted.
The results are shown in FIG. 5.
It can be found that the expression of BCRP, P-gp, etc. resistance proteins in K562/ADR cells is significantly higher than that in K562 cells (P < 0.01), suggesting that SN is effective in inhibiting K562/ADR cells probably due to its inhibition of expression of the relevant resistance proteins.
To further verify this hypothesis, the inventors individually inoculated K562/ADR cells in the logarithmic growth phase (at an inoculation concentration of 4X 10) 5 cells/well) were placed in 6-well plates and incubated overnight in an incubator. 2mL of the total saponins of Solanum nigrum extracted in the above examples at different concentrations (5, 10 and 20. Mu.g/mL) was added to each well every other day, and positive control doxorubicin (ADR, 50. Mu.M) was set and incubated with the cells for 24 hours, after which the expression of BCRP and P-gp proteins in the cells was detected by using the western blot method. Wherein, the primary antibody P-gp and BCRP are purchased from Wuhan Sanying (Wuhan, china), and the antibody is used at a ratio of 1:2000 was diluted. The secondary antibody was rabbit antibody, purchased from Cell Signaling Technology (Danvers, USA) and used at 1:2000 was diluted. Blank controls (no SN and ADR added) were set.
The results are shown in FIG. 6.
It can be found that SN can effectively inhibit the expression of BCRP, P-gp and other drug-resistant proteins in K562/ADR, especially at a concentration of 20 μg/mL, the inhibition effect is even stronger than that of positive control doxorubicin (P < 0.01), and by this result, it can be shown that SN realizes the effect of inhibiting drug resistance of K562/ADR cells by inhibiting the expression of BCRP, P-gp and other drug-resistant proteins.
To further explore the mechanism of action of SN anti-tumor drug resistance, the inventors individually inoculated K562/ADR cells in the logarithmic growth phase (inoculation concentration 4X 10) 5 cells/well) were placed in 6-well plates and incubated overnight in an incubator. 2mL of the total saponins of Solanum nigrum extracted in the above example with a certain concentration (the concentration acting on K562/ADR cells is 10. Mu.g/mL, the concentration acting on K562 cells is 8. Mu.g/mL) was added to each well every other day, and after incubation with the cells for 0, 12, 18 and 24 hours, the expression of the G0/G1 phase-related cyclin (cyclin E2 and p 21) was detected by using a western blot method. Wherein the primary antibodies were derived from cell cycle regulatory antibody sampling kit II, the secondary antibodies were rabbit antibodies, all purchased from Cell Signaling Technology (Danvers, USA), and the antibodies were used at 1: the ratio of 1000.
The results are shown in FIG. 7.
It was found that the ratio of G0/G1 cells (p 21 expression level/cyclin E2 expression level) was significantly increased compared to SN pre-treated cells, after treatment with SN for 12, 18 and 24 hours, indicating that SN significantly increased the expression of p21 protein in K562/ADR cells and K562 cells, whereas cyclin E2 protein was significantly decreased by SN, indicating that SN inhibited cell proliferation by blocking K562/ADR and K562 cell cycles (< 0.05, < 0.01).
To further explore the mechanism of action of SN anti-tumor drug resistance, the inventors have taken K562 and K562/ADR cells in logarithmic growth phase (seeding concentration 4X 10) 5 cells/well) were placed in 6-well plates and incubated overnight in an incubator. 2mL of the total saponins of Solanum nigrum extracted in the above examples at different concentrations (5, 10 and 20. Mu.g/mL) was added to each well every other day, and positive control doxorubicin (ADR, 50. Mu.M) was set and incubated with the cells for 24 hours, and then apoptosis-related proteins (PARP-1, pro-caspase 3, clear-caspase 3, pro-caspase 8 and pro-caspase 9) were detected by the western blot method (GAPDH as an internal reference). Wherein primary anti-PARP-1 and GAPDH are purchased from Wuhan Sanying (Wuhan, china), primary anti-pro-caspase 3, clear-caspase 3, pro-caspase 8 and pro-caspase9, and secondary anti-rabbit and mouse antibodies, purchased from Cell Signaling Technology (Danvers, USA), removal of antibody GAPDH to 1:2000, the remaining antibodies were all diluted at a ratio of 1: the ratio of 1000.
The results are shown in FIG. 8.
SN can be found to significantly increase apoptosis rates in K562/ADR and K562 cells, and can be found to significantly increase PARP-1, clear-caspase 3 protein expression, and decrease pro-caspase 3, pro-caspase 8, and pro-caspase9 protein expression (< 0.05, p <0.01, p <0.001, p < 0.0001). In contrast, 50. Mu.M doxorubicin did not induce apoptosis in K562/ADR cells. The above results suggest that SN induces apoptosis in leukemia cells.
Meanwhile, in order to prove the conclusion, the inventor firstly uses pan-caspase inhibitor Z-VAD-FMK (5 mu M) to pretreat K562/ADR for 4 hours, then adds SN (16 mu g/mL) according to the test method to incubate for 24 hours, and uses a western blot method to detect the expression condition of apoptosis-related proteins. Wherein primary anti-PARP-1 and GAPDH are purchased from Wuhan Sanying (Wuhan, china), primary anti-pro-caspase 3, clear-caspase 3, pro-caspase 8 and pro-caspase9, and secondary anti-rabbit and mouse antibodies, purchased from Cell Signaling Technology (Danvers, USA), removal of antibody GAPDH to 1:2000, the remaining antibodies were all diluted at a ratio of 1: the ratio of 1000. pan-caspase inhibitor Z-VAD-FMK is available from Selleck Chemicals (Houston, TX, USA)
The results are shown in FIG. 9.
It was found that the expression of pro-caspase 3 protein was significantly restored in K562/ADR cells after addition of Z-VAD-FMK (5. Mu.M) compared to SN treated groups, decreasing the expression of clear-PARP-1, clear-caspase 3 protein (ns was not significant, # P <0.01, # P <0.05, # P <0.01, # P < 0.001), indicating that the apoptotic effect of SN on K562/ADR cells was indeed produced by inducing caspase-dependent apoptosis in K562/ADR cells.
And detecting BCRP and P-gp protein expression of the K562/ADR cells in the Z-VAD-FMK pretreatment group by using a western blot method. Wherein, the primary antibody P-gp and BCRP are purchased from Wuhan Sanying (Wuhan, china), and the antibody is used at a ratio of 1:2000 was diluted. The secondary antibody was rabbit antibody, purchased from Cell Signaling Technology (Danvers, USA) and used at 1:2000 was diluted. The pan-caspase inhibitor Z-VAD-FMK was purchased from Selleck Chemicals (Houston, TX, USA).
As a result, it was found that Z-VAD-FMK pretreatment did not have any effect on BCRP and P-gp protein expression in K562/ADR cells (fig. 10, ns was not significant, #p <0.01, # P <0.05, # P < 0.01), suggesting that caspase-dependent apoptosis may not be the main cause of SN to overcome multi-drug resistance in K562/ADR cells.
Based on the above results, the inventors examined autophagy-related proteins (LC 3, p62/SQSTM 1) and Beclin-1) expression by the western blot method (according to the above test method). Wherein, the primary antibodies LC3, p62 and Beclin-1 are purchased from Wuhan Sanying (Wuhan, china), the secondary antibody is rabbit antibody, purchased from Cell Signaling Technology (Danvers, USA), the antibodies are used as 1:2000 was diluted.
As a result, SN was found to significantly increase LC3-I to LC3-II conversion, and Beclin-1 protein expression, while decreasing p62 protein expression (fig. 11, p <0.05, p <0.01, p < 0.001). This result indicated that SN induced autophagy in K562/ADR and K562 cells.
To demonstrate the above conclusion, the inventors used the autophagy inhibitor 3MA (2 mM) or CQ (8. Mu.M) to pretreat K562/ADR cells for 4 hours, then added SN (16. Mu.g/mL) for co-incubation for 24 hours according to the above test method, and examined autophagy-related protein expression by western blot. Wherein, the primary antibody P-gp and BCRP are purchased from Wuhan Sanying (Wuhan, china), and the antibody is used at a ratio of 1:2000 was diluted. The secondary antibody was rabbit antibody, purchased from Cell Signaling Technology (Danvers, USA) and used at 1:2000 was diluted. 3MA was purchased from Sigma Chemical (St.Louis, MO, USA), CQ was purchased from Albumin (Shanghai, china).
As a result, it was found that 3MA and CQ pretreatment significantly restored the expression of pro-caspase 3 protein, while simultaneously decreasing the expression of PARP-1, clear-caspase 3 protein, and also the expression of drug-resistance related proteins (BCRP, P-gp) was restored (fig. 12 to 13, ns were not significant, #p <0.01, #p <0.05, #p < 0.01). Pretreatment in combination with Z-VAD-FMK had no effect on LC3-I conversion to LC3-II in K562/ADR cells (fig. 14, ns was not significant, ×p < 0.01), suggesting SN-induced autophagy is a key factor in overcoming drug resistance and thus promoting K562/ADR cell death.
To verify the above conclusion, the inventors examined the expression of the PI3K/Akt/mTOR signaling pathway-related proteins (p-Akt, akt, p-mTOR, mTOR, p-p70S6K, p70S6K, 4EBP1 and p-4EBP 1) of K562/ADR and K562 cells by western blot as described above. Wherein the primary antibodies p-Akt, akt, p-mTOR, mTOR, p-p70S6K, p70S6K, 4EBP1 and p-4EBP1 and the secondary antibody were rabbit antibodies were purchased from Cell Signaling Technology (Danvers, USA), divided by 4EBP1, p-4EBP1 and rabbit antibodies to 1:2000, the remaining antibodies were all diluted at a ratio of 1: the ratio of 1000.
As a result, SN was found to significantly inhibit expression of p-Akt, p-mTOR, p-p70S6K, and p-4EBP1 proteins in K562/ADR and K562 cells (fig. 15, p <0.05, p <0.01, p <0.001, p < 0.0001). And SN significantly inhibited p-mTOR protein expression in K562/ADR nude mouse transplants compared to model and doxorubicin groups, increasing LC3-I to LC3-II conversion, consistent with the results above (fig. 16, p <0.01, p < 0.001). Thus, it can be shown that SN is capable of inhibiting the PI3K/Akt/mTOR signaling pathway of leukemia cells.
The inventor firstly uses mTOR agonist MHY1485 (10 mu M) to pretreat K562/ADR cells for 4 hours, then adds SN (16 mu g/mL) to incubate for 24 hours according to the test method, and uses a western blot method to detect the expression of PI3K/Akt/mTOR signal path related proteins, apoptosis related proteins, autophagy related proteins and drug resistance related proteins. Wherein, the primary antibody P-gp and BCRP are purchased from Wuhan Sanying (Wuhan, china), and the antibody is used at a ratio of 1:2000 was diluted. The secondary antibody was rabbit antibody, purchased from Cell Signaling Technology (Danvers, USA) and used at 1:2000 was diluted. mTOR agonist MHY1485 is purchased from MedChemExpress (Shanghai, china).
As a result, p-mTOR, p-p70S6K, and p-4EBP1 proteins were found to be significantly increased following dry prognosis of MHY1485 (fig. 17, ns was not significant, #p <0.01, #p <0.05, #p < 0.01). In addition, MHY1485 also significantly restored (increased) pro-caspase 3 protein expression, decreased PARP-1, clear-caspase 3 protein expression (fig. 18, ns was not significant, #p <0.01, ×p <0.05, ×p < 0.01). More importantly, activation of p-mTOR protein expression by MHY1485 intervention significantly inhibited LC3-I conversion to LC3-II, as well as degradation of p62 protein (fig. 19, ns was not significant, #p <0.01, ##p <0.00001, #p <0.01, #p < 0.0001). Furthermore, the down-regulation of BCRP and P-gp protein expression by SN was significantly inhibited following activation of PI3K/Akt/mTOR signaling pathway (fig. 20, ns was not significant, #p <0.01, #p <0.001, #p <0.05, #p < 0.01).
In summary, SN regulates K562/ADR autophagy via PI3K/Akt/mTOR signaling pathway to overcome tumor cell resistance, ultimately inducing autophagic cell death.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (1)
1. The application of the total saponins of black nightshade in preparing the external PI3K/Akt/mTOR signal path inhibitor with non-therapeutic purpose;
the in vitro PI3K/Akt/mTOR signaling pathway inhibitor is used for inhibiting the expression of p-Akt, p-mTOR, p-p70S6K and p-4EBP1 and promoting the conversion of LC3-I to LC 3-II;
the using concentration of the total saponins of the black nightshade is more than 20 mug/mL;
the total saponin of the black nightshade is prepared by the following preparation method:
(1) Reflux-extracting black nightshade fruits by heating with 60-70% ethanol for 2-4 times to obtain an ethanol extract;
(2) Removing ethanol in the ethanol extract, adding water, re-suspending, eluting with D101 macroporous resin column, collecting eluate, concentrating, and drying to obtain herba Solani Nigri total saponins;
the eluent is 50% -70% ethanol eluent.
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Non-Patent Citations (3)
| Title |
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| Sankar Jagadeeshan等.Solanum nigrum Unripe fruit fraction attenuates Adriamycin resistance by down-regulating multi-drug resistance protein (Mdr)-1 through Jak-STAT pathway.《BMC Complementary and Alternative Medicine》.2017,(第17期),第1-9页，尤其是第3页左栏第1段，第4页左栏第2段和右栏第1段以及第8页右栏第2段. * |
| Yi Wang等.S-20, a steroidal saponin from the berries of black nightshade, exerts anti-multidrug resistance activity in K562/ADR cells through autophagic cell death and ERK activation.《Food Funct.》.2022,(第13期),第2200-2215页，尤其是第2201页左栏第2段和第2203页左栏最后一段. * |
| 朱聪等.龙葵碱对白血病K562/ADR细胞多药耐药性的逆转作用及其分子机制.《肿瘤》.2017,第37卷第1276-1281页. * |
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