CN115252634A - Application of ouabain G in preparation of Nrf2 inhibitor and medicine for treating diseases related to abnormal activation of Nrf2 - Google Patents
Application of ouabain G in preparation of Nrf2 inhibitor and medicine for treating diseases related to abnormal activation of Nrf2 Download PDFInfo
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Abstract
The invention discloses application of ouabain G in preparation of Nrf2 inhibitors and medicines for diseases related to Nrf2 abnormal activation, and relates to the field of medicines. According to the invention, the inhibition effect of ouabain G on Nrf2 and the influence of ouabain G on cancer cells are evaluated through a series of experiments, and the result shows that ouabain G can obviously reduce the activity and expression of Nrf2 in-vivo and in-vitro models, and can effectively inhibit the proliferation and growth capacity of endometrial cancer cells. The toxic hairy flower glycoside G is proved to be capable of being used for preparing Nrf2 inhibitors and anticancer drugs, and the toxic hairy flower glycoside G is used clinically for many years as other drugs, safe, reliable and good in application prospect.
Description
Technical Field
The invention belongs to the field of medicines, relates to a new application of ouabain G, and particularly relates to an application of ouabain G in preparation of Nrf2 inhibitors and medicines for diseases related to Nrf2 abnormal activation.
Background
According to the '2015 Chinese malignant tumor prevalence analysis' published in 2019, the incidence rate of malignant tumors is on a rapid increase trend nowadays, the death of malignant tumors accounts for 23.91% of all the causes of death of residents, and the death of malignant tumors is in a continuously rising state in recent decades. In addition, the onset of malignant tumors increases with age in the age distribution, and rapidly increases from 40 years of age. Therefore, the search for effective antitumor drugs and treatment methods becomes an important topic that cannot be avoided in current life medicine.
Nuclear transcription factor E2-related factor 2 (Nuclear factor 2-related factor 2, nrf 2) is a transcription factor with a highly conserved basic leucine zipper structure, and is a central regulatory factor of antioxidant response in the body. Many studies have shown that Nrf2 is abnormally activated in tumor cells, thereby promoting tumorigenesis, progression, and being closely related to chemotherapeutic resistance of tumors. Therefore, the Nrf2 activity can be effectively inhibited, and the expression of the Nrf2 can be reduced, so that the Nrf2 activity can be used as a new direction for treating tumors. Many compounds that inhibit Nrf2 activity, such as flavonoids, brucellol (brusatol), ochratoxin a (OTA), glucocorticoids, etc., have been found through studies, but many of these compounds have deficiencies that limit their use as Nrf2 inhibitors in the treatment of cancer, such as: the flavonoid inhibits Nrf2 activity unstably, and the effect of inhibiting Nrf2 by OTA is unclear and high in toxicity.
Therefore, there is a need to find a novel Nrf2 inhibitor with definite and reliable effect and high safety for preparing an antitumor product with good therapeutic effect and safety.
Disclosure of Invention
Aiming at the problem that the number of the existing Nrf2 inhibitors with good effect and high safety is small, the invention provides a new application of ouabain G, namely the application of ouabain G in preparing Nrf2 inhibitors and anticancer drugs. Ouabain G is a cardiac glycoside drug that has been used clinically for many years. The use concentration, incompatibility, adverse reaction and other related information of the Chinese medicinal composition in human bodies are relatively complete. However, through examination of relevant documents at home and abroad, no research report about that ouabain G can inhibit the activity of Nrf2 and can be further prepared into Nrf2 inhibitors and anticancer drugs for cancer treatment is found.
The first purpose of the invention is to provide application of ouabain G in preparation of an Nrf2 inhibitor, and application of the Nrf2 inhibitor in preparation of a radiotherapy or chemotherapy sensitizer.
The invention also aims to provide application of ouabain G in preparing medicines for treating diseases related to abnormal activation of Nrf 2.
Preferably, the disease is endometrial cancer.
Preferably, the ouabain G is used alone.
Preferably, the ouabain G is used in the form of a pharmaceutical composition.
Preferably, the medicine is a medicinal preparation prepared from ouabain G and medicinal auxiliary materials.
The ouabain G is used for inhibiting the activity and expression of Nrf 2.
The ouabain G is used for inhibiting the proliferation and invasion capacity of cancer cells.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the inhibition effect of ouabain G on Nrf2 and the influence of ouabain G on cancer cells are evaluated through a series of experiments, and the result shows that ouabain G can obviously reduce the activity and expression of Nrf2 and can effectively inhibit the proliferation and growth of endometrial cancer cells. The toxic hairy flower glycoside G is proved to be capable of being used for preparing Nrf2 inhibitors and anticancer drugs, and the toxic hairy flower glycoside G is used clinically for many years as other drugs, safe, reliable and good in application prospect.
Drawings
FIG. 1 is a chemical structural diagram of Ouabain G (Ouabain) used in the embodiment of the present invention.
FIG. 2 shows that ouabain G inhibits Nrf2 activity in luciferase reporter gene experiments.
FIG. 3 shows the expression of Nrf2 and its downstream gene proteins 48 hours after various concentrations of ouabain G treated endometrial cancer cells.
FIG. 4 shows that Nrf2 and its downstream gene protein are expressed after G-treatment of endometrial cancer cells for different periods of time by ouabain.
FIG. 5 is a graph of the effect of ouabain G on endometrial cancer cell proliferation; wherein the content of the first and second substances,
a represents the effect of ouabain G on Ishikawa cancer cell proliferation;
b shows the effect of ouabain G on the proliferation of AN3CA cancer cells.
FIG. 6 shows the colony formation assay of ouabain G on Ishikawa and AN3CA cells.
FIG. 7 is a statistic of the number of clones of ouabain G acting on Ishikawa and AN3CA cells; wherein the content of the first and second substances,
a represents the statistics of the number of clones of ouabain G acting on Ishikawa cells;
b shows the statistics of the number of clones that ouabain G acted on AN3CA cells.
FIG. 8 shows the experimental process of tumor formation and administration in nude mice.
FIG. 9 is a graph of tumor growth.
FIG. 10 is a diagram of isolated tumor mass after administration of ouabain G or normal saline.
FIG. 11 shows the isolated tumor body weight statistics after ouabain G or normal saline administration.
Figure 12 immunohistochemistry analysis nude mice xenografted tumorigenic tissue protein expression after each group of treatments.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.
The research of the invention discovers that Ouabain G (Ouabain) is combined with Nrf2 through ubiquitin ligase E3, so that Nrf2 is ubiquitinated and degraded, therefore, the invention provides a new application of Ouabain G, namely the application of Ouabain G in preparing Nrf2 inhibitors and medicines for treating diseases related to Nrf2 abnormal activation; the Nrf2 inhibitor can be used for preparing radiotherapy and chemotherapy sensitizers; preferably, the disease is endometrial cancer.
The ouabain G can be used independently when used for preparing anti-cancer drugs; can also be used in the form of pharmaceutical compositions; or mixing with medicinal adjuvants, and making into medicinal preparation, such as tablet, capsule, granule, pill, suppository, membrane, colloid, unguent, powder, injection, mixture, oral liquid, syrup, medicated liquor, sol, and emulsion.
The medicinal preparation contains effective dose of ouabain G, and the rest is pharmaceutically acceptable, nontoxic and inert carriers and excipients for human and animals. The pharmaceutically acceptable carrier or excipient is one or more selected from solid, semi-solid and liquid diluents, and nontoxic and inert pharmaceutically acceptable carriers and excipients for fillers.
The medicine can inhibit the activity and expression of Nrf 2; can also inhibit the proliferation and growth ability of cancer cells.
According to the invention, the inhibition effect of ouabain G on Nrf2 is evaluated through a series of experiments, and the result shows that ouabain G can effectively inhibit the proliferation and growth of cancer cells in-vivo and in-vitro models, can reduce the activity and expression of Nrf2, and verifies the inhibition effect of ouabain G on Nrf2 and the treatment effect on cancer.
The following are detailed descriptions of the experimental materials, experimental procedures and experimental results of the present invention. In the examples of the present invention, reagents and consumables are commercially available products unless otherwise specified.
(I) test materials
1. Cell line
In this example, human type I endometrial cancer cell lines Ishikawa and AN3CA were stored in the Zymaphos laboratory of the assisted reproductive department in the clinical center of gynecological department, a first human hospital affiliated with the Shanghai university medical college, and commercially available cells were also used.
2. Laboratory animal
The experimental animal used in the invention is a SPF-grade 6-week-old female BALC/c-nu nude mouse. The experimental animal center of the first people hospital affiliated with Shanghai transportation university medical college is ordered to Shanghai public health clinic center and is uniformly raised in the SPF-level area of the experimental animal center. The animal experiment passes the ethical approval of the hospital, and has an ethical (IACUC) number of 2021AW011.
3. Ouabain G reagent
Ouabain G (Ouabain) used in the present invention was purchased from Sigma-Aldrich official website, and its chemical structure is shown in fig. 1.
Inhibition of Nrf2 activity by ouabain G
1. Experimental methods
In the experiment, tert-butyl hydroquinone (tBHQ) is used as an Nrf2 inducer, and the influence of ouabain G on the activity of luciferase induced by the tBHQ, namely the influence of ouabain G on the activity of Nrf2, is detected. The specific experimental process is as follows:
2. Results of the experiment
The results are shown in fig. 2, and show by comparison that: the ouabain G with different concentrations can effectively inhibit the activity of Nrf 2-dependent luciferase, and the inhibition capability is increased along with the increase of the concentration of the ouabain G. The toxic hairy flower glycoside G can effectively inhibit the activity of Nrf2, and the inhibition effect is stronger along with the increase of the concentration of the toxic hairy flower glycoside G.
(III) the curculigoside G can reduce the protein level of Nrf2 and downstream target genes thereof
1. Experimental methods
(1) 6X 10 inoculation in 6-well plates6Endometrial cancer cells (Ishikawa and AN3CA cells) in logarithmic growth phase are added with 2ml of culture solution for conventional culture; after 24 hours, adding ouabain G with different concentrations, and continuing culturing; after 48 hours, the culture medium is discarded, and 1ml of PBS solution is added into each hole for cleaning for 2 times; adding 100 mul of lysate containing protease and phosphatase inhibitor into each hole, scraping adherent cells, and gently transferring the cells and the lysate into a precooled microcentrifuge tube; after shaking for 30 minutes at 4 ℃, centrifuging for 10 minutes at 12000rpm at 4 ℃, sucking the supernatant to a newly precooled microcentrifuge tube and recording the volume; adding a proper amount of loading buffer into each pipe according to the volume, and then boiling in a boiling water bath for 10 minutes to fully denature; subpackaging, and storing at-80 deg.C for a long period; the recovered protein was subjected to gel electrophoresis detection and data was analyzed.
(2) 6X 10 inoculation in 6-well plates6Endometrial cancer cells (Ishikawa and AN3CA cells) in logarithmic growth phase are added with 2ml of culture solution for conventional culture; after 24 hours, 100nM ouabain G was added at each time point, and the culture was continued until the duration of the action of ouabain G was 12, 24, 48 hours, respectively. Collecting protein and performing gel electrophoresis operation as before.
2. Results of the experiment
(1) The result is shown in figure 3, the Nrf2 protein expression is reduced after ouabain G action, and the inhibition effect is dose-dependent. The protein expression quantity of Nrf2 is obviously reduced by ouabain G at 1 mu M, and the lower the protein expression quantity of Nrf2 is along with the increase of concentration; meanwhile, the protein expression amounts of downstream genes AKR1C1 and GCLM regulated by Nrf2 nuclear transcription factor are also obviously reduced, and HO-1 in Ishikawa cells has no obvious change. In AN3CA cells, HO-1 is in a gradient down-regulation trend. The ouabain G is shown to have obvious inhibition effect on Nrf 2.
(2) The results are shown in fig. 4, and in both cells, proteins of Nrf2 and its downstream target gene were gradually decreased with the prolonged action time of ouabain G.
(IV) Effect of ouabain G on endometrial cancer cell proliferation
1. Experimental methods
2000 endometrial cancer cells (Ishikawa and AN3CA cells) in logarithmic growth phase are inoculated in a 96-well plate, and 200 mul of culture solution is added for conventional culture; adding ouabain G (5 repeats per concentration) of 10nM, 100nM, 1000nM and 10000nM the next day, and culturing; after 48 hours, the medium was discarded, 100. Mu.l of a 4 ℃ precooled 10% TCA solution was added to each well, which was then fixed in a 4 ℃ freezer for 1 hour, taken out and the fixative discarded, washed 5 times with deionized water and dried at room temperature; adding 0.4% SRB dye solution 100 μ l per well, dyeing for 30 min, discarding the dye solution, washing with 1% acetic acid for 5 times, and air drying at room temperature; adding 100 mu L of 10mM Tris-base alkali liquor into each hole, and placing on a horizontal shaker for 20 minutes until the dye is completely dissolved; absorbance at 540nm was measured with a microplate reader and the values were recorded.
2. Results of the experiment
As shown in FIG. 5, ouabain G significantly inhibited cell proliferation compared to the control group 48 hours after treating Ishikawa and AN3CA cells with ouabain G at different concentrations. After statistical analysis, the Ishikawa cell proliferation inhibiting effect is obvious (P is less than 0.01) when the acting concentration of ouabain G is 10 nM. When the compound acts on AN3CA cells, the drug concentration of 100nM can obviously inhibit proliferation (P < 0.01). In addition, the ouabain G proliferation inhibitory effect is a dose-dependent effect.
(V) Effect of ouabain G on cloning ability of endometrial cancer cells
1. Experimental methods
After endometrial cancer cells in logarithmic growth phase are digested by pancreatin, a complete culture medium (a basic culture medium +10% fetal calf serum) is resuspended into a cell suspension, and counting is carried out; inoculating 800 cells/well in a 6-well plate culture plate; after 24 hours, various concentrations of ouabain G were added to the wells, 3 replicate wells per concentration, and solvent (DMSO) control; continuously culturing for 14 days or until the number of cells in most single clones is more than 50, changing the culture medium every 3 days in the middle and observing the cell state; after cloning, washing with PBS for 1 time, adding 1ml of 4% paraformaldehyde into each hole for fixing for 30-60 minutes, and washing with PBS for 1 time; adding 1ml of crystal violet staining solution into each hole, and staining cells for 10-20 minutes; washing the cells for several times with PBS, air-drying, and scanning with a scanner; directly counting clones by naked eyes, and finally calculating the clone formation rate; clone formation rate = (number of clones/number of seeded cells) × 100%.
2. Results of the experiment
As shown in fig. 6 and 7, the cell clone formation rate was significantly lower in the group to which different concentrations of ouabain G were added compared to the control group (solvent group), and the cell clone formation rate was lower as the ouabain G concentration increased. Further proves that ouabain G can effectively reduce the proliferation capacity of cancer cells.
(VI) in the nude mouse xenogeneic subcutaneous tumor transplantation model, ouabain G slows down the proliferation of endometrial cancer cells
1. Experimental methods
Referring to fig. 8, ishikawa cells were injected subcutaneously into axilla of female nude mice to construct a xenograft subcutaneous tumor model. After the tumor body is formed, dividing the nude mice into 4 groups, and injecting ouabain G into the abdominal cavity every other day for administration, wherein the concentration of ouabain G in the 3 groups is 1mg/kg, 2mg/kg and 4mg/kg respectively. The control group was injected with an equal volume of physiological saline, and the nude mice were weighed and the tumor volume was measured. Nude mice were sacrificed after 9 doses, tumors were stripped and weighed.
2. Results of the experiment
The length and length of tumor body were measured every 1 day from the day of the first administration, and the volume of tumor body was calculated. The tumor growth curve is shown in fig. 9, the tumor volume growth of each group of subcutaneous Ishikawa cell tumors has no obvious difference, the intraperitoneal injection of ouabain G can effectively inhibit the tumor volume growth, and the higher the ouabain G concentration is, the slower the tumor proliferation is in the range of the effect concentration of ouabain G in the experiment. When the acting concentration of ouabain G is 1mg/kg, the growth of endometrial cancer cells can be obviously inhibited. Compared with the normal saline control group, the tumor volume growth trend of the group injected with ouabain G in the abdominal cavity is reduced, and the average tumor volume of the group with ouabain G4 mg/kg concentration measured at 26 days is obviously smaller than that of the normal saline control group.
After the nude mice were sacrificed and the tumor bodies were taken out, the sizes of the tumor bodies of the groups are shown in fig. 10. Tumor bodies were further weighed, and the results are shown in FIG. 11. The above results demonstrate that ouabain G can still slow down the proliferation rate of endometrial cancer cells in vivo.
Effect of (hepta) ouabain G on Nrf2 expression in nude mouse xenograft tumorigenic tissues
1. Experimental methods
And (5) performing immunohistochemical staining analysis on tumor bodies taken from the xenografted tumor tissues of the nude mice in each group in the experiment (six).
2. Results of the experiment
The results are shown in FIG. 12 (brown-yellow is the antigen-antibody binding site). The expression of Nrf2 in the group injected with ouabain G is obviously lower than that of the group injected with normal saline, and the expression of Nrf2 is also reduced along with the increase of the injection concentration of ouabain G. In addition, the expression of Proliferating Cell Nuclear Antigen (PCNA) in the group of injected ouabain was down-regulated compared to the group of injected normal saline, and ouabain G was further demonstrated to be able to inhibit endometrial cancer proliferation in an in vivo model.
In conclusion, ouabain G can effectively inhibit the activity and expression of Nrf2, can effectively inhibit the proliferation capacity of cancer cells in vivo and in vitro, and can be used for preparing Nrf2 inhibitors and anti-cancer drugs.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Claims (9)
1. Application of ouabain G in preparation of Nrf2 inhibitor.
2. The use of claim 1, wherein the Nrf2 inhibitor is used in the preparation of a radio-or chemo-sensitizer.
3. Application of ouabain G in preparation of medicines for treating diseases related to Nrf2 abnormal activation.
4. The use of claim 3, wherein the disease is endometrial cancer.
5. The use according to claim 3, wherein ouabain G is used alone.
6. The use according to claim 3, wherein said ouabain G is used in the form of a pharmaceutical composition.
7. The use of claim 3, wherein the medicament is a pharmaceutical preparation prepared from ouabain G and pharmaceutical excipients.
8. The use according to any one of claims 3 to 7, wherein ouabain G is used to inhibit activity and expression of Nrf2 in cancer cells.
9. The use according to any one of claims 3 to 7, wherein ouabain G is used to inhibit the proliferative capacity of cancer cells.
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