CN110742901B - Metabolism check point FBP1 agonist and application thereof in anti-tumor aspect - Google Patents
Metabolism check point FBP1 agonist and application thereof in anti-tumor aspect Download PDFInfo
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Abstract
The invention discloses a metabolism check point FBP1 agonist and application thereof in the aspect of tumor resistance. FBP1 is one of the key enzymes of gluconeogenesis, which plays an inhibitory role in the sugar degradation process by catalyzing the hydrolysis of fructose-1, 6-diphosphate. FBP1 is known to be low-expressed in various tumor cells, and is associated with the growth and proliferation of tumor cells, and the growth and proliferation of tumor cells can be inhibited by activating FBP 1. The invention provides an FBP1 activator of a metabolism check point, the activator is a natural plant polysaccharide, and a CCK-8 test proves that the FBP activator can obviously inhibit the proliferation of human lung adenocarcinoma, liver cancer and breast cancer cells and presents obvious dose dependence; western blotting experiments prove that the natural plant polysaccharide can effectively activate the expression of FBP1 protein in the tumor cells, which is probably an action mechanism for resisting human lung adenocarcinoma, liver cancer and breast cancer.
Description
Technical Field
The invention belongs to the field of medicines, relates to development and application of a metabolism check point medicine, and particularly relates to an FBP1 agonist of a metabolism check point and application of the FBP1 agonist in the aspect of tumor resistance.
Background
Glycolysis and oxidative phosphorylation are the major pathways for energy metabolism by cells. For normal cells, glucose is completely oxidized when oxygen is sufficient to produce 32 molecules of Adenosine Triphosphate (ATP); 2 molecules of ATP are produced only by glycolysis when oxygen supply is insufficient. Thus, normal cells use glycolysis, a low efficiency energy-producing pathway, only in the absence of oxygen. Tumor cells, even when they are sufficiently oxygenated, are still supplied with energy by glycolysis, which is a difference in sugar metabolism between tumor cells and normal cells, and is called the "Warburg (Warburg) effect", and since then, tumors are also considered to be a "metabolic disease". Based on this, the goal of tumor treatment by using drugs to regulate and control the carbohydrate metabolism of tumor cells has become a research hotspot in the fields of tumor cell biology and metabolism (carbohydrate metabolism regulation and tumor treatment of tumor cells, journal of biomedical engineering, 2019).
Since tumors are also a "metabolic disease," regulation of various metabolic functions of tumor cells has become an important research direction for tumor therapy. Recently, the research on the metabolism of tumor cells is very active, and the activation, differentiation, migration and the like of the tumor cells have key metabolic pathways and corresponding check points, so that the 'metabolic check points' refer to some important enzymes or receptors in the metabolic pathways, and the activity of the 'metabolic check points' directly influences the functions of the tumor cells and can regulate the proliferation, migration and invasion of the tumor cells. Then, by regulating the "metabolic checkpoint" of tumor cells to change their metabolic state, reduce the proliferation, migration and invasion activities of tumor cells, or reduce the tolerance of tumor cells to chemotherapeutic drugs, or reduce their immune escape capacity to immune cells, it is likely to be the next new key breakthrough point for improving the efficacy of cancer.
Fructose-1,6-bisphosphatase 1 (FBP 1) is one of the key enzymes of gluconeogenesis, exerting an inhibitory effect during sugar degradation by catalyzing the hydrolysis of fructose-1, 6-bisphosphate (up-regulation of FBP1 expression in the inhibition of gastric cancer cell growth, journal of gastroenterology and hepatology, 2018). FBP1 is low-expressed in various tumor cells, and is related to the growth and proliferation of the tumor cells, and the growth and proliferation of the tumor cells can be inhibited by activating the FBP 1. Zhangieyi et al have studied the effects and mechanisms of up-regulating FBP1 expression on gastric cancer cell proliferation and apoptosis, and found that FBP1 has low expression in gastric cancer cells, and up-regulating expression can inhibit cell proliferation and induce apoptosis, and the action mechanism may be related to up-regulating clear caspase-3 protein and down-regulating Cyclin D1, c-Myc protein expression (up-regulating FBP1 expression has inhibition on gastric cancer cell growth, gastroenterology and hepatology journal, 2018). The Hongzhendong et al discusses the expression of FBP1 in human Renal Clear Cell Carcinoma (RCCC) and tissues beside the RCC, and the role and clinical significance in the occurrence, development and prognosis of renal cancer, and as a result, the FBP1 and the protein thereof are found to be low expressed in the human renal cancer tissue, are related to the occurrence and development of renal cancer, and can become one of candidate markers for renal cancer prognosis (the expression change and significance of FBP1 in the renal clear cell carcinoma tissue, chongqing medicine, 2018). Liu Jia et al have studied the expression level of FBP1 gene in colorectal adenocarcinoma and the relation with clinical pathological characteristics, and the result shows that the low expression of FBP1 gene and protein may play an important role in the occurrence and development of colorectal adenocarcinoma, and is a potential therapeutic target (immunohistochemistry method and qPCR technology for detecting the expression and clinical significance of FBP1 in colorectal adenocarcinoma, chuanbei medical college report, 2017). It has been found that FBP1 expression is low in lung cancer tissues and cells, and that restoration of FBP1 expression not only inhibits glucose uptake and lactate production, but also inhibits lung cancer cell proliferation and invasion under hypoxic conditions in vitro, and inhibits lung cancer growth in vivo (Down-regulation of FBP1 by ZEB1-mediated expression to growth and invasion in lung cancer cells [ J ]. Molecular and Cellular Biochemistry, 2016).
Unfortunately, the current metabolic checkpoint FBP1 activators are reported very rarely and are clearly under-studied.
The traditional Chinese medicine LygodiiHerba is dry aerial part of Lygodium japonicum (Thunb.) Sw, lygodium microphyllum L.microphyllum R.Br. or Lygodium amansii L.flexosum Sw. Of the family of Hypsizygaceae, has the functions of clearing heat and detoxicating, and inducing diuresis for treating stranguria, and contains various chemical components such as polysaccharide, flavone, terpenoid and the like.
At present, no report of the activity of the cissampelos polysaccharose for metabolic checkpoint FBP1 activators and antitumor activity exists.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a metabolic checkpoint FBP1 agonist and application thereof in resisting tumors, particularly lung adenocarcinoma.
The above purpose of the invention is realized by the following technical scheme:
a FBP1 agonist of a metabolism check point is a cissampelos mutica polysaccharide and is prepared by the following method:
pulverizing and sieving the lygodium japonicum, taking powder, adding deionized water, carrying out hot reflux extraction, filtering an extracting solution, carrying out reduced pressure concentration, centrifuging, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol, uniformly stirring, standing for alcohol precipitation, collecting precipitate, redissolving with deionized water, centrifuging, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol, uniformly stirring, standing for alcohol precipitation, collecting precipitate, washing, redissolving with deionized water, deproteinizing by a Sevag method, and freeze-drying.
The medicinal application of the sargentgloryvine stem polysaccharide in preparing antitumor medicaments.
Further, the tumor is lung adenocarcinoma, liver cancer or breast cancer.
A pharmaceutical preparation for treating lung adenocarcinoma comprises a polysaccharide of radix Stephaniae Sinicae as active ingredient, and pharmaceutically acceptable adjuvants to make into pharmaceutically acceptable dosage forms; the golden sargentgloryvine stem polysaccharide is prepared by the following method:
pulverizing and sieving the lygodium japonicum, taking powder, adding deionized water, carrying out hot reflux extraction, filtering an extracting solution, carrying out reduced pressure concentration, centrifuging, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol, uniformly stirring, standing for alcohol precipitation, collecting precipitate, redissolving with deionized water, centrifuging, collecting supernatant, adding 3 times of volume of absolute ethyl alcohol, uniformly stirring, standing for alcohol precipitation, collecting precipitate, washing, redissolving with deionized water, deproteinizing by a Sevag method, and freeze-drying.
Further, the auxiliary material is solid, liquid or semisolid auxiliary material.
Further, the dosage forms comprise tablets, capsules and injections.
Has the beneficial effects that:
the invention provides an FBP1 activator of a metabolism check point, the activator is a natural plant polysaccharide, and CCK-8 tests prove that the activator can obviously inhibit the proliferation of human lung adenocarcinoma, liver cancer and breast cancer cells and presents obvious dose dependence; western blotting experiments prove that the sargentgloryvine stem polysaccharide can effectively activate the expression of FBP1 protein in the tumor cells, which is probably an action mechanism of the sargentgloryvine stem polysaccharide for resisting human lung adenocarcinoma, liver cancer and breast cancer.
Drawings
FIG. 1 shows the proliferation inhibition rate of Cissampelos segetum polysaccharide on human lung adenocarcinoma A549 cells at different concentrations;
FIG. 2 shows the effect of different concentrations of Cissampelos pareira polysaccharide on the expression of FBP1 protein in human lung adenocarcinoma A549 cells;
FIG. 3 shows the proliferation inhibition rate of the polysaccharide of Cissampelos pareira for HepG2 cells of human liver cancer at different concentrations;
FIG. 4 shows the effect of sargentgloryvine stem polysaccharide of different concentrations on the expression of FBP1 protein in human liver cancer HepG2 cells;
FIG. 5 shows the proliferation inhibition rate of Cissampelos pareira polysaccharide on human breast cancer MDA-MB-231 cells at different concentrations;
FIG. 6 shows the effect of different concentrations of Cissampelos Angustifolia on the expression of FBP1 protein in human breast cancer MDA-MB-231 cells.
Detailed Description
The following detailed description will be given with reference to the accompanying drawings and examples, but the scope of the invention is not limited thereto.
Example 1: preparation of golden sargentgloryvine stem polysaccharide
Crushing cissampelos mutica purchased from a traditional Chinese medicine market, sieving with a 40-mesh sieve, taking 2kg of powder, adding 15L of deionized water, carrying out hot reflux extraction for 3 times, carrying out extraction for 2 hours for the 1 st time, carrying out extraction for 1.5 hours for the 2 nd time and carrying out extraction for 1 hour for the 3 rd time, combining three extracting solutions, filtering, concentrating under reduced pressure to 5L, centrifuging at 4500rpm for 10min, collecting supernatant, adding 15L of absolute ethanol, stirring uniformly, carrying out standing alcohol precipitation for 12h, collecting precipitate, washing sequentially by absolute ethanol and 75% ethanol (volume concentration), redissolving by deionized water, deproteinizing by a Sevag method, carrying out freeze drying, obtaining 75g of polysaccharide, and determining the purity of the polysaccharide to be not less than 95% by a phenol-sulfuric acid method. The sargentgloryvine stem polysaccharide prepared in this example was used for antitumor activity test.
Example 2: preparation of golden sargentgloryvine stem polysaccharide
Crushing lygodium japonicum purchased from a traditional Chinese medicine market, sieving with a 40-mesh sieve, taking 2kg of powder, adding 10L of deionized water, carrying out hot reflux extraction for 3 times, extracting for 2h each time, combining three extracting solutions, filtering, concentrating under reduced pressure to 5L, centrifuging at 4500rpm for 10min, collecting supernatant, adding 15L of absolute ethyl alcohol, stirring uniformly, standing for alcohol precipitation for 12h, collecting precipitate, redissolving with 5L of deionized water, centrifuging at 4500rpm for 10min, collecting supernatant, adding 15L of absolute ethyl alcohol, stirring uniformly, standing for alcohol precipitation for 12h, collecting precipitate, washing sequentially with absolute ethyl alcohol and 75% ethyl alcohol (volume concentration), redissolving with deionized water, deproteinizing by a Sevag method, freezing and drying to obtain 83g of polysaccharide, wherein the polysaccharide purity is not lower than 95% when measured by a phenol-sulfuric acid method.
Example 3: preparation of polysaccharide of golden sargentgloryvine stem
Crushing lygodium japonicum purchased from a traditional Chinese medicine market, sieving with a 40-mesh sieve, taking 2kg of powder, adding 20L of deionized water, carrying out hot reflux extraction for 3 times, extracting 1h each time, combining three extracting solutions, filtering, concentrating under reduced pressure to 5L, centrifuging at 4500rpm for 10min, collecting supernatant, adding 15L of absolute ethyl alcohol, stirring uniformly, standing for alcohol precipitation for 12h, collecting precipitate, redissolving with 5L of deionized water, centrifuging at 4500rpm for 10min, collecting supernatant, adding 15L of absolute ethyl alcohol, stirring uniformly, standing for alcohol precipitation for 12h, collecting precipitate, washing sequentially with absolute ethyl alcohol and 75% ethyl alcohol (volume concentration), redissolving with deionized water, deproteinizing by a Sevag method, freezing and drying to obtain 68g of polysaccharide, wherein the polysaccharide purity is not lower than 95% when measured by a phenol-sulfuric acid method.
Example 4: inhibitory activity of cissampelos sampsonii hance polysaccharide on lung adenocarcinoma
1. Test materials and methods
1. Cell recovery and culture
Human lung adenocarcinomaA549 cells (frozen in the laboratory) were thawed and cultured in RPMI 1640 medium (HyClone; thermo Fisher Scientific) containing 10% fetal bovine serum (HyClone; thermo Fisher Scientific) and 100U/ml penicillin, 100. Mu.g/ml streptomycin at 5% CO 2 Culturing and subculturing in an incubator at 37 ℃, and taking logarithmic phase cells for subsequent experiments.
2. CCK-8 method for measuring cell proliferation activity
The procedure was followed with CCK-8 kit (Dojindo, japan). Digesting logarithmic growth phase A549 cells with pancreatin to prepare a cell suspension, inoculating 2000 cells per well into a 96-well plate, adding 100. Mu.l of a culture medium per well, and subjecting to 37 ℃ and 5% CO 2 Adherence was performed overnight in an incubator. Setting a sargentgloryvine stem polysaccharide group with drug concentration of 5 and 20 mug/mL, a negative control group (with drug concentration of 0 mug/mL) and a blank control group, wherein each group is provided with 3 multiple holes. In the polysaccharide group of the golden sargentgloryvine stem, 10 mul of the corresponding concentration drug is added into each hole, and 10 mul of the culture medium is added into each hole of the blank control group. And putting the mixture into an incubator to be cultured for 48h, taking the mixture out, adding 10 mu l of CCK-8 reagent into each hole, slightly knocking the culture plate, uniformly mixing the mixture, and incubating the mixture in the incubator for 2h. And measuring the light absorption value (A value) of 450nm by using a microplate reader, and calculating the proliferation inhibition rate of the sargentgloryvine stem polysaccharide with different concentrations on A549 cells. Proliferation inhibition =1- (chrysotile polysaccharide group a value-blank control group a value)/(negative control group a value-blank control group a value) × 100%.
3. Western blotting method for measuring FBP1 expression level in cell
Culturing human lung adenocarcinoma A549 cells with culture solutions containing 0, 5 and 20 mu g/mL of sargentgloryvine stem polysaccharide for 48h, collecting and washing each group of cells, adding lysate containing protease inhibitors for cracking, incubating on ice for 30min, centrifuging and collecting supernatant, and determining the protein concentration of a sample by using a BCA method. Equivalent amounts of protein were taken, separated on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, blocked in 1 XTBST-containing 0.1% Tween for 1h at room temperature, washed 3 times with PBS, incubated overnight at 4 ℃, washed 3 times with PBS, washed with HRP-labeled secondary antibody, washed with PBS, subjected to band analysis using a chemiluminescence imager, and the relative expression amount of FBP1 with respect to internal control β -actin was calculated and 1.00 in terms of the relative expression amount of FBP1 in 0. Mu.g/mL of the Chrysosplenium polysaccharide group cells.
4. Statistical analysis
Data analysis was performed using SPSS 19.0 software (IBM, NY). Data are expressed as mean ± sd, and differences between two sets of normally distributed continuous variables were analyzed using independent samples or paired t-test, with p < 0.05 representing significant differences.
2. Test results
1. CCK-8 method detection result
The results are shown in table 1 and fig. 1, the polysaccharide of cissampelos sampsonii hance can effectively inhibit the proliferation of human lung adenocarcinoma a549 cells, and shows obvious dose dependence, which indicates that the polysaccharide of cissampelos sampsonii hance has the activity of resisting the proliferation of human lung adenocarcinoma cells.
TABLE 1 proliferation inhibition rate of acutangular polysaccharide on human lung adenocarcinoma A549 cells at different concentrations
| 5 mu g/mL of cissampelos hancei polysaccharide | 20 mu g/mL of sargentgloryvine stem polysaccharide | |
| Proliferation inhibition ratio (%) | 33.7±3.2 | 55.4±3.8 |
2. Detection result of Western blotting method
Western blotting results are shown in FIG. 2, the expression of FBP1 protein in human lung adenocarcinoma A549 cells can be effectively activated by the cissampelos polysaccharose with different concentrations, and the cissampelos polysaccharose is an effective FBP1 agonist, which is probably a mechanism for the cissampelos polysaccharose to play a role in resisting the proliferation of human lung adenocarcinoma cells.
Example 5: inhibitory activity of sargentgloryvine stem polysaccharide on liver cancer
1. Test materials and methods
1. Cell recovery and culture
Human hepatoma HepG2 cells (frozen in the laboratory) were thawed and cultured in RPMI 1640 medium (Hyclone; thermo Fisher Scientific) containing 10% fetal bovine serum (Hyclone; thermo Fisher Scientific) and 100U/ml penicillin, 100. Mu.g/ml streptomycin at 5% CO 2 Culturing and subculturing in an incubator at 37 ℃, and taking logarithmic phase cells for subsequent experiments.
2. CCK-8 method for measuring cell proliferation activity
The procedure was followed in accordance with the CCK-8 kit (Dojindo, japan). Digesting HepG2 cells in logarithmic growth phase with pancreatin to prepare a cell suspension, inoculating 2000 cells per well into a 96-well plate, adding 100. Mu.l of a culture medium per well, and subjecting to 37 ℃ and 5% CO 2 Adherence was performed overnight in the incubator. Setting a sargentgloryvine stem polysaccharide group with drug concentration of 5 and 20 mug/mL, a negative control group (with drug concentration of 0 mug/mL) and a blank control group, wherein each group is provided with 3 multiple holes. The polysaccharide group of the golden sargentgloryvine stem is added with 10 mul of corresponding concentration drug in each hole, and the blank control group is added with 10 mul of culture medium in each hole. And putting the mixture into an incubator to be cultured for 48h, taking the mixture out, adding 10 mu l of CCK-8 reagent into each hole, slightly knocking the culture plate, uniformly mixing the mixture, and incubating the mixture in the incubator for 2h. And measuring the value of the light absorption (A value) at 450nm by using a microplate reader, and calculating the proliferation inhibition rate of the aucklandia japonica polysaccharide with different concentrations on HepG2 cells. Proliferation inhibition =1- (chrysotile polysaccharide group a value-blank control group a value)/(negative control group a value-blank control group a value) × 100%.
3. Western blotting method for measuring FBP1 expression level in cell
Culturing human liver cancer HepG2 cells with culture solution containing 0, 5 and 20 mu g/mL of sargentgloryvine stem polysaccharide for 48h, collecting and washing each group of cells, adding lysis solution containing protease inhibitor for lysis, incubating on ice for 30min, centrifuging and collecting supernatant, and determining the protein concentration of a sample by BCA method. Equivalent amounts of protein were separated on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, blocked in skimmed milk prepared with 1 XTSST containing 0.1% Tween for 1h at room temperature, washed 3 times with PBS, incubated overnight at 4 ℃, washed 3 times with PBS, washed with HRP-labeled secondary antibody, washed with PBS, subjected to banding analysis using a chemiluminescence imager, and the relative expression amount of FBP1 to endogenous reference β -actin was calculated and found to be 1.00 in terms of the relative expression amount of FBP1 in 0. Mu.g/mL of the Cissampelos polysaccharide group cells.
4. Statistical analysis
Data analysis was performed using SPSS 19.0 software (IBM, NY). Data are expressed as mean ± standard deviation, and differences between two groups of normally distributed continuous variables were analyzed by independent sample or paired t-test, with p < 0.05 representing significant differences.
2. Test results
1. CCK-8 method detection result
The results are shown in table 2 and fig. 3, the sargentgloryvine stem polysaccharide can effectively inhibit the proliferation of human liver cancer HepG2 cells, and presents obvious dose dependence, which indicates that the sargentgloryvine stem polysaccharide has the activity of resisting the proliferation of human liver cancer cells.
TABLE 2 proliferation inhibition rate of Cissampelos segetum polysaccharide with different concentrations on human liver cancer HepG2 cells
| 5 mu g/mL of cissampelos hancei polysaccharide | 20 mu g/mL of sargentgloryvine stem polysaccharide | |
| Proliferation inhibition ratio (%) | 36.1±3.5 | 60.2±4.1 |
2. Detection result of Western blotting method
Western blotting results are shown in FIG. 4, the expression of FBP1 protein in human hepatoma HepG2 cells can be effectively activated by the sargentgloryvine stem polysaccharide with different concentrations, and the sargentgloryvine stem polysaccharide is an effective FBP1 agonist, which is probably a mechanism for the sargentgloryvine stem polysaccharide to play a role in resisting the proliferation of human hepatoma cells.
Example 6: inhibitory activity of lygodium japonicum polysaccharide on breast cancer
1. Test materials and methods
1. Cell recovery and culture
Human breast cancer MDA-MB-231 cells (frozen in the laboratory) were cultured in RPMI 1640 medium (HyClone; thermo Fisher Scientific) containing 10% fetal bovine serum (HyClone; thermo Fisher Scientific) and 100U/ml penicillin, 100. Mu.g/ml streptomycin at 5% CO 2 Culturing and passage in an incubator at 37 ℃, and taking logarithmic phase cells for subsequent experiments.
2. CCK-8 method for measuring cell proliferation activity
The procedure was followed in accordance with the CCK-8 kit (Dojindo, japan). Digesting MDA-MB-231 cells in logarithmic growth phase with pancreatin to prepare a cell suspension, inoculating 2000 cells per well into a 96-well plate, adding 100. Mu.l of a medium per well, and subjecting to 37 ℃ and 5% of CO 2 Adherence was performed overnight in the incubator. Setting a sargentgloryvine stem polysaccharide group with drug concentration of 5 and 20 mug/mL, a negative control group (with drug concentration of 0 mug/mL) and a blank control group, wherein each group is provided with 3 multiple holes. The polysaccharide group of the golden sargentgloryvine stem is added with 10 mul of corresponding concentration drug in each hole, and the blank control group is added with 10 mul of culture medium in each hole. And putting the mixture into an incubator to be cultured for 48h, taking the mixture out, adding 10 mu l of CCK-8 reagent into each hole, slightly knocking the culture plate, uniformly mixing the mixture, and incubating the mixture in the incubator for 2h. Measuring the 450nm light absorption value (A value) by using an enzyme labeling instrument, and calculating the proliferation inhibition rate of the aucklandia japonica thunb polysaccharide with different concentrations on MDA-MB-231 cells. Proliferation inhibition =1- (chrysotile polysaccharide group a value-blank control group a value)/(negative control group a value-blank control group a value) × 100%.
3. Western blotting method for measuring FBP1 expression level in cell
Culturing human breast cancer MDA-MB-231 cells with culture solutions containing 0, 5 and 20 mu g/mL of sargentgloryvine stem polysaccharide for 48h, collecting and washing each group of cells, adding a lysis solution containing a protease inhibitor for lysis, incubating on ice for 30min, centrifuging and collecting a supernatant, and determining the protein concentration of a sample by a BCA method. Equivalent amounts of protein were separated on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, blocked in skimmed milk prepared with 1 XTSST containing 0.1% Tween for 1h at room temperature, washed 3 times with PBS, incubated overnight at 4 ℃, washed 3 times with PBS, washed with HRP-labeled secondary antibody, washed with PBS, subjected to banding analysis using a chemiluminescence imager, and the relative expression amount of FBP1 to endogenous reference β -actin was calculated and found to be 1.00 in terms of the relative expression amount of FBP1 in 0. Mu.g/mL of the Cissampelos polysaccharide group cells.
4. Statistical analysis
Data analysis was performed using SPSS 19.0 software (IBM, NY). Data are expressed as mean ± sd, and differences between two sets of normally distributed continuous variables were analyzed using independent samples or paired t-test, with p < 0.05 representing significant differences.
2. Test results
1. CCK-8 method detection result
The results are shown in table 3 and fig. 5, the sargentgloryvine stem polysaccharide can effectively inhibit the proliferation of human breast cancer MDA-MB-231 cells, and shows obvious dose dependence, which indicates that the sargentgloryvine stem polysaccharide has the activity of resisting the proliferation of human breast cancer cells.
TABLE 3 proliferation inhibition rate of Cissampelos segetum polysaccharide in different concentrations on human breast cancer MDA-MB-231 cells
| 5 mu g/mL of sargentgloryvine stem polysaccharide | 20 mu g/mL of sargentgloryvine stem polysaccharide | |
| Proliferation inhibition ratio (%) | 31.5±3.0 | 53.9±3.6 |
2. Detection result of Western blotting method
Western blotting results are shown in FIG. 6, the expression of FBP1 protein in human breast cancer MDA-MB-231 cells can be effectively activated by the sargentgloryvine stem polysaccharide with different concentrations, and the sargentgloryvine stem polysaccharide is an effective FBP1 agonist, which is probably a mechanism for the sargentgloryvine stem polysaccharide to play a role in resisting the proliferation of human breast cancer cells.
FBP1 is one of the key enzymes of gluconeogenesis, which plays an inhibitory role in the sugar degradation process by catalyzing the hydrolysis of fructose-1, 6-diphosphate. It is known that FBP1 is underexpressed in various tumor cells and is associated with the growth and proliferation of tumor cells, and that the growth and proliferation of tumor cells can be inhibited by activating FBP 1. The invention provides an FBP1 activator of a metabolism check point, the activator is a natural plant polysaccharide, and a CCK-8 test proves that the FBP activator can obviously inhibit the proliferation of human lung adenocarcinoma, liver cancer and breast cancer cells and presents obvious dose dependence; western blotting test proves that the natural plant polysaccharide can effectively activate the expression of FBP1 protein in the tumor cells, which is probably an action mechanism for resisting human lung adenocarcinoma, liver cancer and breast cancer.
Example 7: pharmaceutical preparation of golden sargentgloryvine stem polysaccharide
A pharmaceutical preparation of polysaccharide of climbing fern is prepared by using the polysaccharide of climbing fern prepared in any one of embodiments 1 to 3 as an active ingredient, and preparing the active ingredient into a pharmaceutically acceptable dosage form by using pharmaceutically acceptable auxiliary materials; the auxiliary material is solid, liquid or semisolid, and the preparation form comprises tablets, capsules and injection.
The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.
Claims (1)
1. Use of a metabolic checkpoint FBP1 agonist for the preparation of a medicament against lung adenocarcinoma; wherein, the FBP1 agonist of the metabolic checkpoint is a lygodium japonicum polysaccharide, and is prepared by the following method: crushing the lygodium japonicum, sieving with a 40-mesh sieve, taking 2kg of powder, adding 15L of deionized water, carrying out hot reflux extraction for 3 times, carrying out extraction for 2h for the 1 st time, carrying out extraction for 1.5h for the 2 nd time, carrying out extraction for 1h for the 3 rd time, combining three extracting solutions, filtering, concentrating under reduced pressure to 5L, centrifuging at 4500rpm for 10min, collecting supernatant, adding 15L of absolute ethyl alcohol, stirring uniformly, adding 15L of absolute ethyl alcohol, standing for alcoghohol precipitation for 12h, collecting precipitate, washing sequentially with absolute ethyl alcohol and 75% of ethyl alcohol, redissolving with deionized water, deproteinizing by a Sevag method, and freeze-drying.
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| 响应面分析法优化海金沙草多糖的提取工艺;武芸等;《食品科学》;20101231;第31卷(第18期);第109页左栏部分；第108页左栏部分第1段 * |
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