Application of combination of triterpenoid saponin extract and oxaliplatin in preparation of medicine for treating liver cancer

Abstract

The invention discloses application of a triterpenoid saponin extract in preparation of a medicine for treating liver cancer, and belongs to the technical field of biomedicine.The Chinese pulsatilla saponin D (SB365) shown in the formula (I) is a novel triterpenoid saponin extract extracted and identified from Chinese pulsatilla roots, is small in toxic and side effect, can induce liver cancer cell apoptosis and has a good proliferation inhibition effect on liver cancer cells; on the other hand, the SB365 can inhibit an HR pathway by inhibiting an AKT / ATM / DDR signal transduction axis and combining RAD51 protein in a targeted manner, so that the sensitivity of oxaliplatin to hepatocellular carcinoma is improved.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, specifically to the application of the combination of triterpenoid saponin extracts and oxaliplatin in the preparation of drugs for treating liver cancer. Background Art

[0002] Pulsatilla chinensis is a traditional Chinese herbal medicine that has been widely used in clinical practice of traditional Chinese medicine. It has the effects of "cooling blood", detoxifying, anti-infective, anti-viral, and enhancing immunity. In ancient China, it was used to treat diseases such as intestinal amoeba bacterial infection, malaria, and cancer.

[0003] Pulsatoside D (SB365), a triterpenoid saponin compound extracted from the traditional Chinese herbal medicine Pulsatilla chinensis, has been widely studied and proven to have significant anti-tumor activity. However, although it shows good anti-tumor effects in in vitro experiments and animal models, there is still a lack of systematic research on its treatment of liver cancer, especially its potential in clinical applications has not been fully developed.

[0004] In the existing technology, the treatment methods for liver cancer mainly include surgical resection, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, etc. However, these treatment methods all have many limitations. For example, surgical resection is only applicable to patients with early-stage liver cancer, and the postoperative recurrence rate is relatively high; although chemotherapy and radiotherapy can inhibit tumor growth to a certain extent, their toxic and side effects are significant, and the damage to normal tissue cells is large, which limits their clinical applications; although targeted therapy and immunotherapy provide new directions for the treatment of liver cancer, their curative effects are still limited, and the treatment costs are relatively high, making it difficult to meet the clinical needs of the majority of patients. Summary of the Invention

[0005] To solve one of the technical problems existing in the prior art, this application provides the application of the combination of triterpenoid saponin extracts and oxaliplatin in the preparation of drugs for treating liver cancer.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In the first aspect of this application, there is provided the application of triterpenoid saponin extracts in the preparation of drugs for treating or adjuvantly treating liver cancer, wherein the triterpenoid saponin extracts are compounds represented by formula (I), their pharmaceutically acceptable salts, their stereoisomers, or their prodrug molecules:

[0008]

[0009] More specifically, in the above technical solution, the functions of the triterpenoid saponin extracts include: inhibiting the contents of AKT signaling pathway-related proteins and DDR proteins in Huh7 and SMMC-7721 liver cancer cells.

[0010] More specifically, in the above technical solution, the functions of the triterpenoid saponin extract further include: aggravating DNA damage in liver cancer cells.

[0011] More specifically, in the above technical solution, the AKT signaling pathway-related proteins include: AKT, p-AKTS473, and p-mTOR; the DDR-related proteins include: p-ATM, ATM, DNA-PKcs, and RAD51.

[0012] More specifically, in the above technical solution, the dosage forms of the drug include: injections, tablets, capsules, kits, or patches. Those skilled in the art can prepare the above drug compositions into various dosage forms according to specific circumstances, and the preparation method is well-known in the art, so it will not be elaborated in this application.

[0013] In a second aspect, the present application proposes an application of a triterpenoid saponin extract in the preparation of a synergist for inducing apoptosis in liver cancer cells, and the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

[0014]

[0015] In a third aspect, the present application proposes an application of a triterpenoid saponin extract in the preparation of a synergist for inhibiting the proliferation of liver cancer cells, and the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

[0016]

[0017] In a fourth aspect, the present application proposes an application of a triterpenoid saponin extract in the preparation of a liver cancer chemosensitizer for sensitizing oxaliplatin, and the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

[0018]

[0019] In a fifth aspect, the present application proposes an application of a combination of a triterpenoid saponin extract and oxaliplatin in the preparation of a drug for treating or adjuvant treating liver cancer, and the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

[0020]

[0021] The "pharmaceutically acceptable salts" are conventional non-toxic salts formed by the reaction of the compound of general formula (I) with inorganic acids or organic acids. For example, the conventional non-toxic salts can be prepared by reacting the compound of general formula (I) with inorganic acids or organic acids. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid, etc., and the organic acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid, etc.; or the sodium salts, potassium salts, calcium salts, aluminum salts or ammonium salts formed by reacting the compound of general formula (I) with inorganic bases after forming esters with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid; or the methylamine salts, ethylamine salts or ethanolamine salts formed by reacting the compound of general formula (I) with organic bases; or the corresponding inorganic acid salts formed by reacting the compound of general formula (I) with lysine, arginine, ornithine after forming esters and then reacting with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid or the corresponding organic acid salts formed by reacting with formic acid, acetic acid, picric acid, methanesulfonic acid and ethanesulfonic acid.

[0022] Stereoisomers refer to different stereoisomers obtained by changing the configurations of multiple chiral centers in the SB365 molecule. Stereoisomers may have different pharmacokinetic properties (such as absorption, distribution, metabolism and excretion) in vivo, but as long as their core structures are similar to SB365, they can still retain their anti-tumor activity. Therefore, stereoisomers are also regarded as having the same pharmacological effects as SB365.

[0023] Prodrug molecules refer to compounds that can release SB365 after metabolism or chemical reactions in vivo. The design of prodrugs is usually to improve the stability, targeting or reduce the toxic and side effects of drugs. After the prodrugs are converted into the active ingredient SB365 in vivo, they can still exert their anti-tumor effects.

[0024] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0025] 1. The inventors found that SB365 significantly inhibits the contents of proteins related to the AKT signaling pathway and key proteins of the DDR signaling pathway in Huh7 and SMMC-7721 liver cancer cells, and increases the contents of the DNA damage marker γ-H2AX and the apoptosis marker Cleaved-PARP. And SB365 can target and bind to the RAD51 protein to inhibit the HR pathway.

[0026] 2. SB365 significantly inhibits the cell viability of Huh7 and SMMC-7721 cells, with a higher degree of inhibition on the cell viability of SMMC-7721 cells. The IC 50 is only 1.108 μM at 48 h.

[0027] 3. SB365 can inhibit the AKT / ATM / DDR pathway, exacerbate DNA damage, and sensitize oxaliplatin, significantly inhibiting the proliferation of liver cancer cells and increasing the apoptosis of liver cancer cells. Description of the Drawings

[0028] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following described drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0029] Figure 1 It is a schematic diagram of SB365 inhibiting the AKT and DDR pathways and targeting and binding to the RAD51 protein in the HR pathway;

[0030] Figure 2 It is a schematic diagram of SB365 increasing the sensitivity of liver cancer cells to oxaliplatin;

[0031] Figure 3 It is a schematic diagram of animal experiments proving that SB365 improves the efficacy of oxaliplatin against liver cancer. Detailed Embodiments

[0032] In order to more clearly understand the technical content of the present invention, the following specific examples are given in detail in conjunction with the drawings. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. All kinds of common chemical reagents used in the examples are commercially available products.

[0033] SB365 is a triterpenoid saponin extract from the traditional Chinese herbal medicine Pulsatilla chinensis. Its chemical formula is C 47 H 76 O 17 . A 10 mM concentrated SB365 solution was purchased from MCE Company (purity: 98.60%) and stored at -80 °C. When used in experiments, a part of the mother liquor was taken and diluted with DMSO to a 1 mM working concentration.

[0034]

[0035] Experimental Materials and Methods

[0036] Cell lines: The cell lines used in the experiment were human liver cancer Huh7 cells and SMMC-7721 cells, both of which were adherent growth cells. They were provided by the Biochemical Laboratory of the Basic Medical College of Jinan University and stored at ultra-low temperature.

[0037] Table 1 Antibodies required for the experiment and their suppliers:

[0038]

[0039]

[0040] Methods: Western blotting was used to detect the protein content in Huh7 cells and SMMC-7721 cells after treatment with SB365 alone or in combination with oxaliplatin. MTT assay was used to detect the effects of SB365 alone or in combination with oxaliplatin on the survival rate of liver cancer cells. Cell colony formation assay was used to detect the effects of SB365 alone or in combination with oxaliplatin on the proliferation ability of Huh-7 cells. Cell thermal migration assay was used to identify the action target of SB365. Annexin V-FITC / PI staining and flow cytometry were used to detect the apoptosis rate of Huh-7 cells after treatment with SB365 in combination with oxaliplatin. Animal experiments were conducted to detect the effects of SB365 in combination with oxaliplatin on the growth volume and weight of xenograft tumors of Huh7 liver cancer cells in nude mice.

[0041] Western blotting assay:

[0042] (1) Protein extraction. Cells in the logarithmic growth phase were seeded in 6-well plates, grouped, and after overnight adherent culture, drug irradiation treatment was carried out. After 24 - 48 h, the supernatant culture medium was aspirated, and the excess culture medium was washed away with PBS. The cells were collected and lysed with RIPA strong lysis buffer containing 1% protease inhibitor and 1% protein phosphatase inhibitor, and lysed on ice for 30 min. Then the lysed cells were transferred to a centrifuge tube and centrifuged at a pre-cooled high-speed centrifuge (4°C, 12,000 rpm) for 15 min to obtain the protein supernatant. The protein concentration of the protein supernatant was measured using a BCA micro protein concentration assay kit. Then, 1 / 4 volume of 5×SDS-PAGE protein loading buffer was added, and after mixing, it was heated at 100°C in a metal bath for 5 min to denature the protein. The obtained protein samples were stored at -80°C.

[0043] (2) Gel electrophoresis and membrane transfer. Assemble the 4-20% precast protein gel with the electrophoresis device, add 1× electrophoresis buffer to both the inner and outer tanks, then load the Rainbow Protein Maker and protein samples together for electrophoresis. Electrophoresis (120V) for about 30 min until the indicator band is close to the bottom of the precast gel. Stop electrophoresis, take out the gel, cut the gel containing the target protein according to the Maker bands, place it flat on a 0.45 or 0.22 μm PVDF membrane (select a membrane with different thicknesses according to the molecular weight of the target protein, and the membrane is pre-soaked and activated with methanol). After confirming that the membrane and gel are completely adhered, insert them into the membrane transfer clip, place it in the membrane transfer tank, fill it with 1× transfer buffer, and transfer the membrane at a constant current of 250 mA for 90 min. The periphery of the membrane transfer tank needs to be filled with ice for cooling.

[0044] (3) Blocking and antibody incubation. After membrane transfer, soak the membrane in 5% blocking milk and incubate it on a shaker at room temperature for 1 h. Then wash off the excess milk with 1× TBST buffer, soak the membrane in the corresponding primary antibody dilution and incubate it overnight (4℃). After the primary antibody incubation, wash the membrane 3 times with 1× TBST buffer. Then put the membrane into the corresponding secondary antibody dilution and incubate it on a shaker at room temperature for 1.5 h. After that, wash the membrane 3 times with 1× TBST buffer again.

[0045] (4) Protein band luminescence and development. After washing the membrane, apply the hypersensitive ECL chemiluminescence solution on the membrane, expose it through a fully automatic chemiluminescence image analysis system, collect the image and analyze the result bands.

[0046] MTT assay:

[0047] (1) Experimental grouping: Design experimental groups, control groups and zero calibration groups. In the control group, add cells and the volume of DMSO in the maximum drug concentration group. In the zero calibration group, only add the culture medium.

[0048] (2) Seed the cells in the logarithmic growth phase at 3000 cells per well evenly in a 96-well plate. Set 5 replicates for each group, keep the liquid volume in each well consistent, fill the outer ring of the well plate with 200 μL of 1× PBS to reduce the evaporation of the culture medium, and place it in the incubator overnight for cell attachment. Then treat the cells according to the experimental requirements (drug treatment), and then culture them in the incubator for 48 h.

[0049] (3) After culturing for 48 h, add 20 μL of MTT solution to each well and continue to place it in the incubator at 37℃ for 4 h. Discard the supernatant, and add 150 μL of DMSO solution to each well. After the formazan purple crystals are fully dissolved, use a multi-functional microplate reader to detect the absorbance at 570 nm. Subtract the absorbance value of the zero calibration group from the absorbance value of each well to get the effective value. Calculate the average absorbance value of each group, calculate the survival rate of the cells in each group, and calculate the IC using GraphPad Prism 9.1 50And perform statistical analysis, with the mean ± standard deviation (Mean ± SD) representing the values of each group.

[0050] Cell colony formation assay:

[0051] (1) Set up a control group and an experimental group. Take cells in the logarithmic growth phase and seed them evenly into a six-well plate at a density of approximately 500 - 1000 cells per well. Shake the plate gently to spread the cells evenly, and then place it in an incubator overnight to allow the cells to adhere to the plate.

[0052] (2) After the cells adhere, treat the cells according to the experimental requirements (drug treatment), keeping the volume of the culture medium in each group consistent. Continue to culture for 7 - 14 days until the clone spots in the control group reach a size of 0.3 - 1.0 mm.

[0053] (3) Remove the culture medium, wash the wells 2 - 3 times with PBS. Add an appropriate amount of paraformaldehyde solution to each well, fix at room temperature for 30 min, then remove the fixative and wash with PBS. Add an appropriate amount of 0.1% crystal violet solution to each well and stain for 30 min. Remove the staining solution, then gently rinse the plate with running water, air dry, and take pictures or scan to save the images. Add 30% glacial acetic acid to dissolve the crystal violet completely. Then transfer the solution from each well to three replicate wells of a 96-well plate, and read the absorbance OD value at a wavelength of 450 nm on a microplate reader to represent the clone value. Use GraphPad Prism 9.1.1 to plot the graph, and the mean ± standard deviation (Mean ± SD) represents the values of each group.

[0054] Annexin V-FITC / PI staining and flow cytometry assay:

[0055] (1) Set up a control group and an experimental group. Take cells in the logarithmic growth phase and seed them evenly into a six-well plate at a density of approximately 200,000 cells per well. Place it in an incubator overnight to allow the cells to adhere to the plate. After the cells adhere, treat the cells according to the experimental requirements (drug treatment).

[0056] (2) After 24 h or 48 h, take out the six-well plate and collect the culture medium in the wells into a centrifuge tube. Wash each well 2 times with 1 mL of 1×PBS, and collect the aspirated PBS into the centrifuge tube as well. Add an appropriate amount of trypsin to the wells to digest the cells. Transfer the cells to the centrifuge tube and centrifuge at 1000 rpm for 5 min. Note that the operation should be gentle to avoid physical damage to the cells.

[0057] (3) Discard the supernatant, add 1 mL of PBS to resuspend the cells, repeat centrifugation and discard the supernatant again, retaining the cell pellet. Use the Annexin V / PI double-staining cell apoptosis kit, and sequentially add Binding Buffer, Annexin V, and PI staining solution at a volume ratio of 100:1:1 to suspend and mix the cells evenly. Keep the process in the dark and incubate at room temperature for 10 min. Then, use an intelligent analytical flow cytometer for on-machine detection, analyze the apoptosis of the cells, and draw a graph.

[0058] Cell thermal migration experiment:

[0059] (1) Take tumor cells in the logarithmic growth phase, divide them into an experimental group and a control group. Digest the cells and centrifuge, discard the supernatant, and then use the culture medium to resuspend the cell pellet. The number of cells in both groups is kept the same. Add an appropriate concentration of SB365 drug to the cell suspension of the experimental group for treatment, and add an equal amount of DMSO to the cell suspension of the control group. Incubate in an incubator for 2 h.

[0060] (2) Centrifuge the two groups of cells, discard the supernatant, resuspend the cells with 1 mL of PBS, divide the two groups of cells evenly into 9 parts, and heat them gradiently in a thermal cycler (the gradient heating temperature range can be designed by yourself, such as setting 9 temperature ranges between 40 °C and 80 °C). Heat each range for 3 min, incubate at room temperature for 3 min, and then quickly freeze.

[0061] (3) Freeze-thaw the cells 3 times repeatedly between liquid nitrogen and room temperature, 3 min each time, to lyse the cells. Then centrifuge the lysate at 12,000 rpm at 4 °C for 10 min, take the supernatant, add 1 / 4 volume of 5×SDS-PAGE protein loading buffer, denature the protein in a metal bath at 100 °C for 5 min, and then perform Western blotting experiments to detect the changes in the protein contents of AKT, RAD51, β-actin, etc. with temperature. Finally, use ImageJ software to calculate the gray value of the protein band, and use GraphPad Prism software to draw the CETSA curve and perform thermal stability analysis.

[0062] Animal experiment:

[0063] (1) Establish a nude mouse xenograft tumor model by subcutaneous injection of tumor cells. Use BALB / c-nu nude mice, and subcutaneously inoculate 400×10 4 Huh7 cells into each mouse; when the tumor grows to about 100 mm 3 , perform grouping.

[0064] (3) Animal experiments: Divided into a control group, an SB365 (5 mg / kg) group, an oxaliplatin (5 mg / kg) group, and an SB365 combined with oxaliplatin group. The two drugs were intraperitoneally injected once every other day, with a dosage of 5 mg / kg each time, for a total of 9 drug administrations. During the experiment, the tumor growth was observed. The tumor volume was measured every 2 days, and the longest diameter a and the shortest diameter b of the tumor were measured. The tumor volume V was calculated according to the formula a*b 2 / 2, and the tumor growth curve was plotted. On the 19th day, the mice were euthanized (anesthetized and decapitated by dislocation of the posterior cervical spine) according to the experimental animal ethics, the tumor tissues were dissected and weighed; some mouse blood samples were collected for the detection of liver and kidney function indexes.

[0065] Data processing and statistical analysis:

[0066] The experimental data were statistically analyzed and processed using software SPSS 21.0 and GraphPad Prism 9.1. A representative experiment from three independent experiments was presented, and the data were expressed as mean ± standard deviation (Mean ± SD). The differences between two groups of samples were tested by Student's t-test, and the differences between more than two groups of samples were compared by One Way ANOVA. A P value < 0.05 was considered statistically significantly different; image processing was performed using software Adobe Illustrator 2021; schematic diagrams were drawn using FigDraw software.

[0067] Example 1

[0068] To clarify the effect of SB365 on liver cancer, the inventors detected the protein content in Huh7 and SMMC-7721 liver cancer cells after treatment with different concentrations of SB365 for 24 h by Western blotting experiment. As Figure 1 shown in A, SB365 significantly inhibited the contents of AKT signaling pathway-related proteins (AKT, p-AKT S473, p-mTOR) and DDR key proteins (p-ATM, ATM, DNA-PKcs, RAD51, etc.) in Huh7 and SMMC-7721 liver cancer cells, and increased the contents of DNA damage marker γ-H2AX and apoptosis marker Cleaved-PARP. This indicates that SB365 can inhibit the AKT and DDR pathways in liver cancer cells, aggravate DNA damage and induce apoptosis.

[0069] Next, the MTT experiment was used to detect the effect of SB365 on cell viability. As Figure 1 shown in B, the 48 h IC of SB365 on Huh7 and SMMC-7721 cells 50They were 2.529 μM and 1.108 μM respectively. Further, the inhibitory effect of SB365 on the proliferation of Huh7 cells was detected by colony formation assay. As Figure 1 shown in D, low-concentration SB365 could significantly inhibit the proliferation of Huh7 cells and had a good anti-hepatocellular carcinoma effect.

[0070] To identify the target of SB365, the inventors performed Cellular Thermal Shift Assay (CETSA). The principle of CETSA is that when a drug molecule binds to a target protein, it usually becomes stable. As the temperature increases, the protein bound to the drug molecule shows less protein thermal degradation compared to the protein not bound to the drug molecule, that is, the content of undegraded protein is higher and the thermal degradation curve of this protein shifts to the right. Through the CETSA experiment, it was found that compared with the control group, there was no obvious shift in the temperature degradation curve of AKT protein in Huh7 hepatocellular carcinoma cells in the SB365 treatment group, while the temperature degradation curve of RAD51 protein shifted significantly to the right, as Figure 1 shown in C. This indicates that SB365 binds to RAD51 protein, a key molecule of the target HR, rather than AKT protein. In addition to inhibiting the AKT / mTOR pathway, SB365 can also target and bind to RAD51 protein to inhibit the HR pathway.

[0071] Example 2

[0072] Oxaliplatin is a chemotherapeutic drug that exerts its anti-tumor effect mainly by damaging DNA. Therefore, to verify whether SB365 can sensitize oxaliplatin by inhibiting the AKT / ATM / DDR pathway and aggravating the DNA damage of hepatocellular carcinoma cells caused by oxaliplatin, the inventors found through MTT assay that SB365 (1 μM) combined with oxaliplatin could more significantly reduce the survival rate of Huh7 hepatocellular carcinoma cells compared with oxaliplatin alone, and the IC 50 of oxaliplatin for Huh7 cells decreased from 20.95 μM to 3.217 μM. As Figure 2 shown in B, there was an obvious sensitizing effect.

[0073] To more intuitively show the effect of SB365 combined with oxaliplatin on hepatocellular carcinoma cells, the inventors treated Huh7 hepatocellular carcinoma cells with SB365 (1 μM) and oxaliplatin (15 μM) respectively, and observed the cell morphology under a 100× low-power microscope after 24 h. As Figure 2As shown in C, the proliferation of liver cancer cells treated with SB365 or oxaliplatin alone was inhibited, the cell density decreased, and a small amount of apoptosis occurred. However, in the group treated with the combination of SB365 and oxaliplatin, obvious massive cell floating and death were observed, indicating that the combination of SB365 and oxaliplatin has a good synergistic lethal effect. Annexin V-FITC / PI staining and flow cytometry experiments further confirmed that the combination of SB365 (1 μM) and oxaliplatin (10 μM) significantly increased the apoptosis rate of Huh7 cells, as Figure 2 shown in D. The colony formation assay showed that the combination of SB365 (0.3 μM) and low-dose oxaliplatin (1 μM) could synergistically inhibit the colony proliferation of Huh7 cells, as Figure 2 shown in E.

[0074] The Western blot experiment further revealed the mechanism of action of the combination of SB365 and oxaliplatin. As Figure 2 shown in A, SB365 inhibited the protein levels of AKT, p-AKT S473, mTOR, ATM, DNA-PKcs, and RAD51, and increased the levels of the DNA damage marker γ-H2AX and the apoptosis marker Cleaved-PARP caused by oxaliplatin, showing a good sensitizing effect. This indicates that SB365 sensitizes oxaliplatin by inhibiting the AKT / ATM / DDR pathway and aggravating DNA damage.

[0075] Example 3

[0076] To further verify the efficacy of SB365 in sensitizing oxaliplatin, the inventors conducted a nude mouse xenograft tumor experiment. After nude mice were inoculated with tumors, the nude mice were treated with the combination of SB365 (5 mg / kg / time, intraperitoneal injection) and oxaliplatin (5 mg / kg / time, intraperitoneal injection). The results showed that the combination treatment group significantly inhibited the tumor growth volume and tumor weight of subcutaneous xenograft tumors in nude mice. The tumor volume and tumor weight of the combination treatment group were significantly lower than those of the other three groups, as Figure 3 shown in B-D. This indicates that the combination of SB365 and oxaliplatin has a good synergistic anti-hepatocellular carcinoma effect, and SB365 is a good chemosensitizer for oxaliplatin. The body weights of the mice in each group did not significantly decrease during the experiment, as Figure 3 shown in E. Blood samples were taken from the mice in each group to detect the levels of AST (aspartate aminotransferase), ALT (alanine aminotransferase), and SCr (serum creatinine) to evaluate liver and kidney functions. As Figure 3 shown in F-G, the detection results showed that the indicators of each group did not increase significantly and were all within the normal range, suggesting that the combination of SB365 and oxaliplatin has low toxicity and has little effect on the liver and kidney functions of mice.

[0077] In this application, the inventors first found through Western blotting experiments that SB365 can significantly inhibit the AKT, p-AKT S473, and p-mTOR proteins in Huh 7 and SMMC-7721 liver cancer cells, as well as key proteins in the DDR pathway such as ATM, DNA-PKcs, RAD51, etc., inhibit the damage repair of double-stranded DNA, and exacerbate DNA damage. MTT experiments found that SB365 significantly inhibits the cell viability of liver cancer cell lines, among which the inhibitory effect on the cell viability of SMMC-7721 cells is the highest, and the IC 50 is only 1.108 μM. The colony formation experiment further proved that SB365 can significantly inhibit the proliferation of Huh-7 cells. In addition, the inventors used the CETSA experiment to study the action target of SB365 and found that the action target of SB365 is the RAD51 protein. All these indicate that SB365 has the potential to become a new anti-liver cancer drug.

[0078] Oxaliplatin belongs to the commonly used DNA damage therapy for liver cancer. Therefore, the inventors also found through MTT experiments that the combination of SB365 and oxaliplatin can significantly reduce the survival rate of Huh7 cells compared with oxaliplatin alone, making the IC of oxaliplatin on Huh7 cells 50It decreased from 20.95μM to 3.217μM. The cell morphology changes of Huh7 cells were observed under a low-power microscope after 24 hours of treatment with SB365, oxaliplatin and the combination of the two. It was found that liver cancer cells treated with SB365 or oxaliplatin alone showed proliferation inhibition, decreased cell density and a small amount of cell apoptosis, while liver cancer cells in the SB365 combined with oxaliplatin treatment group showed obvious large-scale cell floating and death. Annexin V-FITC / PI staining and flow cytometry experiments also further confirmed that SB365 combined with oxaliplatin would significantly increase the apoptosis rate of Huh7 cells. The clone formation experiment showed that SB365 combined with low-concentration oxaliplatin could synergistically inhibit the clonal proliferation of Huh7 cells. Western blot experiments further revealed the mechanism of action of SB365 combined with oxaliplatin. SB365 inhibited the protein content of AKT, p-AKT (Ser473), mTOR, ATM, DNA-PKcs, and RAD51, and increased the content of γ-H2AX, a DNA damage marker caused by oxaliplatin, and Cleaved-PARP, a cell apoptosis marker. This shows that SB365 sensitizes oxaliplatin by inhibiting the AKT / ATM / DDR pathway and aggravating DNA damage. The results of the Huh7 cell nude mouse transplant tumor experiment showed that the SB365 combined with oxaliplatin treatment group could significantly inhibit the tumor growth volume and tumor weight of subcutaneous transplant tumors in nude mice, and both were significantly lower than those in the other three groups. The body weight of mice in each group did not decrease significantly during the experiment, and the levels of AST, ALT, and SCr in the blood of mice in each group did not increase significantly, all within the normal range. This shows that SB365 combined with oxaliplatin has low toxicity, has little effect on the liver and kidney function of mice, and SB365 is a good oxaliplatin chemotherapy sensitizer.

[0079] In summary, the inventors found that SB365 not only has a good anti-tumor effect on hepatocellular carcinoma, but also can improve the efficacy of oxaliplatin on hepatocellular carcinoma by inhibiting the AKT pathway and inhibiting the DNA damage response, without significantly increasing toxic side effects. This study clarifies the anti-tumor effect and molecular mechanism of the triterpenoid saponin extract SB365 extracted from Pulsatilla chinensis in hepatocellular carcinoma cells, as well as the mechanism of enhancing the efficacy of oxaliplatin on hepatocellular carcinoma cells, providing experimental basis for the future application of SB365 in the clinical treatment of liver cancer.

[0080] The above-described embodiments only express several embodiments of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

1. Use of a triterpenoid saponin extract in the preparation of a drug for treating or assisting the treatment of liver cancer, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

2. The use according to claim 1, characterized in that: The functions of the triterpenoid saponin extract include: inhibiting the content of AKT signaling pathway related proteins and DDR proteins in Huh7 and SMMC-7721 liver cancer cells.

3. The use according to claim 2, characterized in that: The triterpenoid saponin extract also has the function of aggravating DNA damage in liver cancer cells.

4. The use according to claim 2, characterized in that: The AKT signaling pathway related proteins include: AKT, p-AKT S473 and p-mTOR; the DDR related proteins include: p-ATM, ATM, DNA-PKcs and RAD51.

5. The use according to any one of claims 1 to 4, characterized in that: The dosage forms of the drug include: injection, tablet, capsule, kit or patch.

6. Use of a triterpenoid saponin extract in the preparation of a synergist for inducing apoptosis of liver cancer cells, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

7. Use of a triterpenoid saponin extract in the preparation of a synergist for inhibiting the proliferation of liver cancer cells, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

8. Use of a triterpenoid saponin extract in the preparation of an anti-liver cancer sensitizer for sensitizing oxaliplatin, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

9. Use of a triterpenoid saponin extract in combination with oxaliplatin in the preparation of a drug for treating or assisting the treatment of liver cancer, wherein the triterpenoid saponin extract is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

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