CN111419865B - Application of double-split pregnane steroid compound in preparation of anti-erythroleukemia drugs - Google Patents
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Abstract
The invention discloses an application of a bis-pregnane steroid compound in preparation of an anti-erythroleukemia drug, belonging to the technical field of biology and medicine and pharmacology. The double-split pregnane type steroid compound is C21 steroid saponin BW18. The MTT detection shows that different concentrations of BW18 can inhibit cell viability of HEL, and the concentration of BW18 is dependent. Flow cytometry and Western blot detection show that BW18 mainly induces HEL cells to generate G2 phase cycle arrest by up-regulating P27 and down-regulating expression of c-Myc, cyclin E1, CDK1 and CDK2 cycle-related proteins, and simultaneously regulates expression of erythroid and megakaryocytic differentiation-related genes, promotes cells to differentiate towards erythroid and megakaryocytic systems, thereby inhibiting erythroleukemia. BW18 can be used as a potential micromolecule for treating erythroleukemia, and provides a research basis for the development of medicaments for treating erythroleukemia.
Description
Technical Field
The invention relates to the technical field of biology and medicine, in particular to an application of a double-schizopregnane steroid compound in preparation of an anti-erythroleukemia drug.
Background
Acute Erythroid Leukemia (AEL) is a subtype of Acute myeloid leukemia. Although the incidence rate of acute erythroleukemia is low, the prognosis and survival rate are very poor, and the average survival period is only 4-14 months. The current treatment methods for acute erythroleukemia mainly include conventional chemotherapy and hematopoietic stem cell transplantation. Hematopoietic stem cell transplantation therapy is difficult to be widely used clinically due to various factors such as the limitation of matching types and expensive medical expenses. At present, the acute erythroleukemia is mainly treated by conventional chemotherapy, and the clinical standard chemotherapy scheme is that anthracycline drugs are combined with cytarabine. However, due to the lack of high selectivity, conventional chemotherapeutic drugs can also cause severe damage to normal cells, resulting in toxic side effects. At the same time, due to the heterogeneity of the disease, there are certain limitations to this treatment regimen, even the appearance of drug resistance. Therefore, the research and development of high-efficiency and low-toxicity treatment medicines and the optimization of treatment schemes are particularly important for improving the long-term survival rate of AEL patients. The diversity and complexity of the structure of natural small molecular compounds endow the compounds with diversity of biological activity, so that the compounds become valuable resources for drug development. Therefore, the small molecular compound capable of resisting erythroleukemia is urgently needed to be found from natural small molecular compounds, so that a foundation is laid for the development of high-efficiency and low-toxicity erythroleukemia treatment medicines.
Disclosure of Invention
In view of the above, the invention aims to provide an application of a bis-pregnane steroid compound in preparation of an anti-erythroleukemia drug, and the bis-pregnane steroid compound C21 steroid saponin BW18 can be used as a potential small molecule for treating erythroleukemia, and provides a research basis for development of a drug for treating erythroleukemia.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides an application of a bis-pregnane steroid compound in preparation of an anti-erythroleukemia drug, wherein the bis-pregnane steroid compound is C21 steroid saponin BW18, and the chemical formula of the BW18 is shown as formula I:
preferably, the invention provides an application of the C21 steroid saponin BW18 in preparing a medicament for inhibiting HEL cell proliferation, wherein the BW18 inhibits HEL cell proliferation by inducing HEL cells to generate G2 phase cycle block.
More preferably, the BW18 induces G2 cycle arrest in HEL cells by up-regulating P27, down-regulating c-Myc, cyclin B1, cyclin D1, cyclin E1, CDK1 and CDK2 cycle-related protein expression.
Preferably, the invention also provides the application of the C21 steroid saponin BW18 in preparing a medicament for promoting differentiation of HEL cells to erythroid and megakaryocytic systems, wherein the BW18 promotes differentiation of HEL cells to erythroid systems by promoting expression of an erythroid surface marker of the HEL cells; the BW18 promotes the differentiation of the HEL cells to the megakaryocyte system by promoting the expression of surface markers of the megakaryocyte system.
More preferably, the BW18 promotes differentiation of HEL cells into erythroid cells by up-regulating the mRNA and protein levels of the erythroid-associated genes GATA1, GFI1B and EKLF in HEL cells; the BW18 promotes the differentiation of HEL cells to the megakaryoid by up-regulating the mRNA and protein levels of megakaryoid differentiation related genes GATA1, GFI1B, GP and RUNX1 in HEL cells.
Preferably, the percentage content of the BW18 in the medicament is 0.1-99.9%.
The BW18 is a double-split pregnane type steroid compound separated from cynanchum atratum roots, belongs to a natural small molecular compound, and has small side effect. Moreover, the MTT detection shows that different concentrations of BW18 can inhibit the cell viability of HEL, and the concentration is dependent. Flow cytometry and Western blot detection show that the double-schizopregnane steroid compound C21 steroid saponin BW18 induces HEL cells to generate G2 phase cycle block mainly by up-regulating P27 and down-regulating expression of C-Myc, cyclin E1, CDK1 and CDK2 cycle related proteins, and simultaneously regulates expression of erythroid and megakaryoid differentiation related genes to promote differentiation of the cells to erythroid and megakaryoid, thereby inhibiting generation of erythroleukemia. The results show that the double-schizopregnane steroid compound C21 steroid saponin BW18 can be used as a potential micromolecule for treating erythroleukemia, and provides a research basis for development of medicaments for treating erythroleukemia.
Drawings
FIG. 1 is a graph showing the inhibitory effect of different concentrations of BW18 on HEL cells, wherein the abscissa is the concentration of BW18 (. Mu.M); the ordinate represents the inhibition ratio (%); DMSO was used as a control group.
Fig. 2 shows the effect of different concentrations of BW18 on HEL cell morphology, with DMSO as control.
FIG. 3 is a graph of the growth of HEL cells at different concentrations of BW18 over time with the abscissa representing the time of action (h); the ordinate is the absorbance at 490 nm; DMSO was used as a control group.
FIG. 4 shows the effect of different concentrations of BW18 on HEL cell cycle distribution, wherein A is the effect of different concentrations of BW18 on HEL cell cycle after 48h and 72 h; b is the proportion statistics of HEL cells in different growth stages of G1, S and G2.
Figure 5 is a graph of the effect of BW18 on the expression level of HEL cell cycle associated protein, wherein the HEL cell cycle associated protein is plotted on the abscissa; the ordinate represents the relative expression level of the protein; DMSO was used as a control group.
FIG. 6 shows the effect of BW18 on HEL cell differentiation surface marker antigens, wherein A is the proportion of CD41+ cells; b is the proportion of CD61+ cells; c is the proportion of CD71+ cells; d is the proportion of CD235a + cells; the abscissa is BW18 concentration (μ M); the ordinate is positive cell rate (%); DMSO was used as a control group.
FIG. 7 shows the effect of BW18 on the expression level of genes and proteins involved in the differentiation of HEL cells into erythroid and megakaryoid, wherein A is the effect of BW18 on the erythroid differentiation of HEL cells and the mRNA level expression of megakaryoid gene; b is the effect of BW18 on the erythroid differentiation of HEL cells and the protein level expression of megakaryocyte differentiation genes.
Detailed Description
The invention provides an application of a bis-pregnane steroid compound in preparation of an anti-erythroleukemia drug, wherein the bis-pregnane steroid compound is C21 steroid saponin BW18, and the chemical formula of the BW18 is shown as formula I:
in the invention, the double-split pregnane steroid compound C21 steroid saponin BW18 is a double-split pregnane steroid compound separated from the root of cynanchum atratum. The source of the Cynanchum atratum root is not particularly limited in the present invention. In the invention, the C21 steroid saponin BW18 plays a role in resisting erythroleukemia mainly by inducing G2 phase cycle arrest, inhibiting HEL cell proliferation and promoting megakaryoid and erythroid differentiation.
The invention also provides application of the C21 steroid saponin BW18 in preparing a medicament for inhibiting HEL cell proliferation. The BW18 induces the G2 phase of HEL cells to block by regulating HEL cycle-related protein; mainly through up-regulating the expression of P27, down-regulating the expression of c-Myc, cyclin B1, cyclin D1, cyclin E1, CDK1 and CDK2, inducing the HEL cell to generate G2 phase cycle block, thereby inhibiting the proliferation of the HEL cell.
The invention also provides application of the C21 steroid saponin BW18 in preparing a medicament for promoting differentiation of HEL cells to erythroid and megakaryoid. In the invention, the BW18 promotes the differentiation of the HEL cells to the erythroid by promoting the expression of the surface marker of the erythroid of the HEL cells. In the present invention, the BW18 promotes the differentiation of HEL cells into the megakaryoid by promoting the expression of a surface marker of the megakaryoid.
In the invention, the BW18 promotes differentiation of HEL cells to erythroid by up-regulating the mRNA and protein levels of erythroid-related genes GATA1, GFI1B and EKLF in the HEL cells; the BW18 promotes the differentiation of HEL cells to the megakaryoid by up-regulating the mRNA and protein levels of megakaryoid differentiation related genes GATA1, GFI1B, GP and RUNX1 in HEL cells.
In the invention, the percentage content of the C21 steroid saponin BW18 in the medicament is preferably 0.1-99.9%, more preferably 10-90%.
In a specific embodiment of the invention, the HEL is a erythroleukemia cell line. The source of the HEL cells is not particularly limited in the present invention, and a commercially available in vitro cultured cell line that is conventional in the art may be used. The HEL described in this embodiment is a cell line that is a boon schooling in canada.
The present invention will be described in detail with reference to the following embodiments and drawings for better understanding of the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
In the following examples, unless otherwise specified, all methods are conventional.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Each experiment in the following examples was independently repeated three times, all data were expressed as mean ± SD, student's' st-test was performed with GraphPad Prism6, P <0.05 was considered statistically significant, P <0.05, P <0.01.
Example 1
The MTT method is used for determining the effect of different concentrations of BW18 on the proliferation inhibition of HEL cells.
Subjecting the red leukemia cell HEL to 8 × 10 3 The density of each well was plated in 96-well plates and treated immediately with different concentrations of 20. Mu.M, 10. Mu.M, 5. Mu.M and 2.5. Mu.M BW18 solutions. In addition, a control group was prepared, and DMSO was added in an amount of 0.1% to the control group. After 72h of culture, 20 mu L of MTT solution with the concentration of 5mg/mL is added into each well, the mixture is incubated for 4h at 37 ℃, the supernatant is centrifuged and discarded, 160 mu L of DMSO is added into each well for dissolution, and the blue-purple crystals are left to standAfter the formazan is completely dissolved, an enzyme-labeling instrument measures absorbance values OD of 490nm positions of different treatment groups and a control group, and corresponding inhibition rates are calculated to obtain a graph 1.
The calculation method of the inhibition rate comprises the following steps: inhibition rate = (control OD) 490nm Treatment group OD 490nm ) Control group OD 490nm 。
Half inhibition rate (IC) 50 ) The calculating method of (2): determination of IC corresponding to compound by using forecast function 50 。
As shown in the results of FIG. 1, HEL cells treated with different concentrations of BW18 inhibited HEL cell viability and had concentration-dependent, IC 50 The concentration was 12.45. + -. 0.82. Mu.M.
Example 2
HEL cells were arranged in a 6X 10 format 3 Density of individual/well was plated in 96-well plates and treated immediately with different concentrations of BW18 of 20. Mu.M, 10. Mu.M and 5. Mu.M, while setting a control group to which 0.1% DMSO was added. After 72h of incubation, the morphological changes of the groups of cells were visualized by taking a picture with an inverted microscope, yielding FIG. 2.
The results in figure 2 show that as BW18 concentration increased, the number of HEL cells decreased significantly, the cell volume became larger, BW18 at concentrations of 10 μ Μ and 20 μ Μ significantly inhibited cell growth, indicating that BW18 has a concentration-dependent effect on inhibition of HEL cells.
Example 3
HEL cells were arranged in a 6X 10 format 3 Density of individual/well was plated in 96-well plates and treated immediately with different concentrations of BW18 of 20. Mu.M, 10. Mu.M and 5. Mu.M, while setting a control group in which DMSO was added at 0.1% by weight. The OD values at 490nm at 0h, 12h, 24h, 36h, 48h, 72h and 96h of the treated group and the control group were measured by MTT method, respectively, and growth curves were plotted to obtain FIG. 3.
As can be seen from fig. 3, different concentrations of BW18 all inhibited proliferation of HEL cells compared to the control group, and as the concentration of BW18 increased, the growth rate of the cells gradually decreased, indicating that BW18 was concentration-and time-dependent on proliferation of HEL cells.
Example 4
HEL cells were packed at 1X 10 5 Planting in one/holeAfter culturing for 48h or 72h in 6-well plates with 20. Mu.M high BW18, 10. Mu.M medium BW18 and 5. Mu.M low BW18, respectively, the cells were harvested. Setting up a control group simultaneously, adding 0.1% DMSO to the control group. The collected cells were centrifuged at 1000rpm for 5min, washed once with pre-chilled PBS, and the cells were resuspended in 70% pre-chilled ethanol and fixed overnight at-20 ℃. After overnight, the cells were centrifuged at 1000rpm for 5min, the supernatant was discarded, washed once with PBS, centrifuged at 1000rpm for 5min, and the cell pellet was collected. The cell pellet was resuspended in 500. Mu.L PBS, 5. Mu.L RNaseA, 25. Mu.L PI and 0.25. Mu.L TritonX were added, gently mixed, incubated 30min at 37 ℃ in the dark, centrifuged at 1000rpm for 5min, the dye was discarded, resuspended in 200. Mu.L PBS, cell cycle distribution was measured by flow cytometry, and the results are shown in FIG. 4.
As can be seen from fig. 4, after 48h of BW18 treatment of HEL cells at different concentrations of high, medium and low, the proportion of G2 phase cells increased from 5.33% ± 3.04% in the low concentration treatment group to 9.94% ± 2.42%, the proportion of G2 phase cells increased to 19.29% ± 2.42% (10 μ M) in the medium concentration treatment group, and the proportion of G2 phase cells increased to 12.11% ± 2.45% in the high concentration treatment group, compared to the control group. After 72h of treatment of HEL cells with BW18 at different concentrations, the proportion of G2 phase cells increased from 8.00% ± 2.5% to 13.89% ± 2.59% in the low concentration treatment group, to 38.99% ± 2.75% in the medium concentration treatment group, and to 22.27% ± 2.48% in the high concentration treatment group. It was shown that different concentrations of BW18 solutions significantly increased the proportion of HEL cells in the G2 phase, and the BW18 blocking effect gradually increased from 5. Mu.M to 10. Mu.M, but at 20. Mu.M, the proportion of cells in the G2 phase was lower than that in the 10. Mu.M treatment group.
Example 5
HEL cells were packed at 1X 10 5 Cells were harvested after culturing in 6-well plates with 20 μ M high BW18, 10 μ M medium BW18, and 5 μ M low BW18 for 48h or 72h, respectively. Setting up a control group simultaneously, adding 0.1% DMSO to the control group. The collected cells were centrifuged at 1000rpm for 5min, washed once with pre-cooled PBS, and the cells were resuspended in 100. Mu.L PBS. Adding 2.5 μ L of CD41-FITC, CD61-APC, CD71-FITC or CD235a-APC antibody into each tube, flicking, mixing, incubating at 4 deg.C in dark for 30min, centrifuging at 1000rpm for 5min, discarding dye, and incubating with 100 μ L of the mixtureL PBS was resuspended. The levels of the megakaryocyte surface marker molecules CD41 and CD61, the erythrocyte surface glycoprotein CD235a and the transferrin receptor CD71 were measured by flow cytometry, and the results are shown in FIG. 6.
As shown in fig. 6, different concentrations of BW18 can significantly increase the proportion of CD41+, CD61+, CD71+, and CD235a + cells, indicating that BW18 can induce differentiation of the HEL erythroid and megakaryoid, and has certain concentration and time dependence.
Example 6 detection of mRNA expression levels of cell differentiation-associated genes after BW18 Effect by real-time fluorescent quantitative PCR
(1) Extraction of Total RNA
HEL cells were packed at 1X 10 5 Cells were harvested after each well in 6-well plates and treated with 20. Mu.M high BW18, 10. Mu.M medium BW18, and 5. Mu.M low BW18 for 24 h. Total cellular RNA was extracted according to TRIzol kit instructions. Taking a proper amount of dissolved total RNA, carrying out electrophoresis detection on the RNA quality, and determining the RNA concentration by using a Nano Drop2000 ultramicro spectrophotometer. The total RNA is stored at-80 ℃ for later use.
(2) cDNA was synthesized by reverse transcription, and cDNA was synthesized using TaKaRa reverse transcription kit.
(3) According to the instruction of the qRT-PCR kit, a reaction system is prepared for qRT-PCR, and the detected genes are differentiation related genes GATA1, GFI1B, EKLF, GP6 and RUNX1.
Example 7 detection of protein expression levels of cell differentiation-associated genes after BW18 Effect Using Westernblot
(1) Collecting and lysing cells: HEL cells were packed at 4X 10 5 Each well was cultured in a 10 cm-diameter dish, and after 24 hours of exposure with 20. Mu.M of high BW18 concentration, 10. Mu.M of medium BW18 concentration, and 5. Mu.M of low BW18 concentration, cells were collected. The cells were washed once with PBS and lysed on ice for 30min using RIPA lysate containing PMSF. Vortex every 5min, centrifuge at 12000rpm for 15min at 4 deg.C, and collect the supernatant.
(2) BCA protein quantification: protein quantification is carried out according to the BCA protein quantification kit instruction, after protein concentration is calculated, 5 × loading buffer is added according to the volume of a collected protein sample, and the protein is denatured in a metal bath at 100 ℃ for 5min and stored at-80 ℃ according to the volume of 5:1.
(3) SDS-PAGE electrophoresis: preparing 8-12% of separation gel and 5% of concentrated gel according to the molecular weight of the protein, adding 50 mu g of protein sample into each hole by using a micro-syringe after the gel is prepared, carrying out electrophoresis at 80V until the protein sample enters the separation gel, adjusting the voltage to 100V, and carrying out electrophoresis until bromophenol blue reaches the bottom of a gel plate.
(4) Film transfer: the PVDF membrane needs to be soaked and activated by methanol before use, glue, the PVDF membrane and filter paper are sequentially discharged by utilizing a Sanming's method, the PVDF membrane, the filter paper are placed into a membrane rotating groove, a wet membrane rotating solution is added, and membrane rotating conditions are set as follows: constant current 220mA, time 120min.
(5) And (3) sealing: after the membrane transfer is finished, immediately placing the protein membrane into a Westernblot membrane washing box which is added with TBS solution in advance, and rinsing for 1-2min to wash off the membrane transfer solution on the membrane. The membrane-washing solution was poured off, 3% BSA blocking solution was added, and blocking was carried out at room temperature for 60min.
(6) Primary antibody incubation: according to the instructions for the primary antibody, the primary antibody was diluted with 3% BSA blocking solution at an appropriate ratio, the blocking solution was aspirated off with a dropper, the diluted primary antibody was immediately added, and the mixture was incubated overnight at 4 ℃ with slow shaking on a side-shaking table.
(7) And (3) secondary antibody incubation: the primary antibody was recovered and washed with TBS solution on a side-shaking table for 5min, 3 times in total. The secondary antibody was diluted with 5% skim milk powder at a ratio of 1. The washing solution was aspirated off with a dropper, diluted secondary antibody was immediately added, slowly shaken on a side-shaking table at room temperature, and incubated for 2h in the dark.
(8) Protein detection: the secondary antibody was recovered, TBS solution was added, and the mixture was slowly shaken on a side shaking table and washed away from light for 5min for 3 times. The expression of the cycle-associated proteins c-Myc, CDK1, CDK2, cyclinB1, cyclinD1, cyclinE1 and P27 and the differentiation-associated proteins GATA1, GFI1B and RUNX1 were detected using an Odyssey imager.
The results of the mRNA and protein expression levels of the cell differentiation-associated genes obtained in examples 6 to 7 are shown in FIGS. 7 and 5, respectively.
As can be seen from fig. 5, BW18 can significantly down-regulate protein expression of c-Myc, CDK1, CDK2, cyclin B1, cyclin D1 and cyclin E1, and up-regulate protein level of P27, indicating that BW18 may induce HEL cell cycle arrest by up-regulating P27, down-regulating expression of cycle-related proteins such as c-Myc, cyclin E1, CDK1 and CDK 2.
From the results of FIG. 7, it was found that, when BW18 acts on HEL cells, the transcription factors essential for erythrocyte differentiation and megakaryocyte differentiation, GATA binding Factor 1 (GATA 1) and Growth Factor Independent 1B (GFI 1B), erythroid specific transcription Factor (EKLF), megakaryocyte marker glycoprotin VI (GP 6), and the transcription Factor of megakaryocyte differentiation, runt-related transcription Factor 1 (RUNX 1), showed a significant increase in the average dose-dependent mRNA level. The result of Western blot is consistent with the result of real-time fluorescence quantitative PCR, and the BW18 can obviously improve the protein levels of GATA1, GFI1B and RUNX1.
According to the embodiment, the double-schizopregnane steroid compound C21 steroid saponin BW18 can inhibit cell activity of HEL and has concentration dependence; mainly through up-regulating P27, down-regulating the expression of c-Myc, cyclin E1, CDK1 and CDK2 cycle-related proteins to induce the HEL cell to generate G2 phase cycle block, and simultaneously regulating the expression of erythroid and megakaryoid differentiation-related genes to promote the cell to differentiate to erythroid and megakaryoid, thereby inhibiting the generation of erythroleukemia. The active ingredient BW18 can be used as a potential small molecule for treating erythroleukemia, and provides a research basis for the development of medicaments for treating erythroleukemia.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (2)
1. The application of the double-split pregnane steroid compound as a single active ingredient in the preparation of the anti-erythroleukemia medicine is characterized in that the double-split pregnane steroid compound is C21 steroid saponin BW18, and the chemical formula of the BW18 is shown as a formula I:
2. The use according to claim 1, wherein the BW18 is present in an amount of 0.1% to 99.9% by weight of the medicament.
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