Pharmaceutical composition and use thereof

Abstract

A pharmaceutical composition and the use thereof. The pharmaceutical composition comprises curcumin and a B7-H3 gene inhibitor. On the basis of the B7-H3 gene inhibitor, curcumin is added to reduce the affect of the tumor microenvironment of solid tumors such as non-small cell lung cancer; and curcumin and the B7-H3 gene inhibitor have a potential synergistic effect on anti-tumor activity.

Description

A pharmaceutical combination and use thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a pharmaceutical combination and use thereof. BACKGROUND

[0002] In today's rapid development of medical technology, cancer is still one of the major diseases threatening human life and health around the world. Among them, non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer. In China, the number of new cases and deaths of lung cancer accounts for the first place. Therefore, the prevention and treatment of lung cancer is serious and urgent. Although some clinical progress has been made in conventional treatment, due to its unique physiological conditions and frequent tumor escape, the overall survival rate of NSCLC is still not optimistic. Metastatic spread is one of the reasons for the failure of treatment of most NSCLC patients. Lung cancer cells often tend to metastasize to certain major organs and tissues, such as bone, brain, lung and liver. Although most NSCLC patients receive multiple treatments including surgery, radiotherapy, chemotherapy, targeted therapy, etc., NSCLC still has low cure rate, high recurrence rate and high mortality. Although new strategies for treating NSCLC are being tried, the real challenge of conquering NSCLC still faces severe challenges.

[0003] At present, targeted immunotherapy for cancer has made significant and rapid development in recent years. In the treatment of NSCLC, immunotherapy represented by chimeric antigen receptor T cell immunotherapy (CAR-T) has shown certain application prospects and has redefined the traditional NSCLC treatment regimen. CAR-T constructs tumor target recognition and activation messenger to T cells, so that T cells acquire the function of specifically recognizing and killing tumor cells. However, due to the MHC restriction of T cells and the restriction of the immune suppressive tumor microenvironment (TME) in the patient's body on the exertion of anti-tumor immune activity, not all patients can benefit from such immunotherapy. At the same time, due to the complex microenvironment of tumor, this immunotherapy will still be limited by the occurrence of tumor immune escape.

[0004] B7-H3 is a newly discovered immune checkpoint molecule. B7-H3 is involved in the shaping and development of tumor microenvironment, regulates the function of T cells and NK cells, and leads to the occurrence of tumor cell immune escape. Overexpression in various tumor types such as lung cancer, breast cancer and colon cancer, and less expression in normal cells make B7-H3 an ideal target for cancer immunotherapy. PD-L1 and B7-H3 can stimulate the aerobic glycolysis of tumor cells by activating the PI3K-AKT-mTOR signaling pathway. In HeLa cells, the glycolytic enzyme ENO1 was found to interact with B7-H3. Down-regulation of B7-H3 in HeLa cells reduces ATP and lactic acid levels, and also reduces c-Myc and lactate dehydrogenase A levels. In addition, B7-H3 also promotes the Warburg effect by increasing glucose uptake and lactic acid production. At the same time, B7-H3 can also enhance tumor cell glycolysis by up-regulating HIF-1α and its downstream targets Glut1 and PFKFB3.

[0005] Currently, there are numerous studies on the anti-tumor properties of natural products, which are derived from their proven effective pharmacological effects and fewer side effects. Among them, traditional Chinese medicine has a history of more than 1000 years, and has various pharmacological effects, such as liver and kidney protection, anti-inflammatory, anti-cancer, antioxidant and antibacterial activities. Although some studies have reported the anti-tumor mechanisms and pathways of effective components of traditional Chinese medicine, i.e. traditional Chinese medicine monomers, their direct molecular targets and specific mechanisms are still unclear. Traditional Chinese medicine, especially Chinese herbal medicine, has been increasingly used in China, some other Asian countries and European countries. It has been proven to enhance the therapeutic effects of chemotherapy, radiotherapy, targeted therapy and immunotherapy, while also reducing the damage to patients caused by the above therapies. Chinese herbal medicine can treat cancer by inhibiting tumor progression and improving the immune system of the organism. More and more studies have shown that many Chinese herbal medicines have a beneficial impact on immune regulation. There is also much evidence that traditional Chinese medicine monomers, as well as extracts and preparations, provide new insights into the clinical application of traditional Chinese medicine from the perspective of immune regulation, and the key mechanism is to regulate the immune system of cancer patients.

[0006] Given the crucial role of immune cells in cancer development, as well as the cancer treatment effects of traditional Chinese medicine through immune regulation, exploring the rational application of immunotherapy and traditional Chinese medicine monomers has far-reaching significance and prospects. Therefore, elucidating the mechanisms of metabolic changes in cells during targeted immunotherapy and their interactions with related signaling pathways, and thus more rationally applying immunotherapy combined with traditional Chinese medicine to the treatment of solid tumors such as NSCLC, is our main research content.

[0007] SUMMARY

[0008] The present application provides a pharmaceutical combination and its use.

[0009] In a first aspect, the present application provides a pharmaceutical combination comprising curcumin and a B7-H3 gene inhibitor.

[0010] Curcumin is a diketone compound extracted from the rhizomes of Zingiberaceae and Araceae plants. It is a rare pigment with a diketone structure in the plant kingdom. From a chemical point of view, it is a natural phenol, which is typically yellow. It is easily soluble in acetic acid, ketone, base and chloroform, and insoluble in water at acidic and neutral pH. Due to its hydrophobicity, it can diffuse through the cell membrane to the endoplasmic reticulum, mitochondria and nucleus, where it exerts its effects. Curcumin is often used as an element of dietary supplements and a component of cosmetics. A large number of documents have shown that curcumin has inhibitory effect on various cancers. At the same time, curcumin has been proven to have an impact on the tumor microenvironment, but due to its low bioavailability, single curcumin cannot play a good inhibitory role in the development of tumors. Curcumin has also been reported to affect different signaling pathways and molecular targets involved in the development of various cancers. The present application combines curcumin with a B7-H3 gene inhibitor, which has a potential synergistic effect on anti-tumor activity.

[0011] Preferably, the B7-H3 gene inhibitor comprises a nucleic acid molecule.

[0012] Preferably, the targeting sequence of the B7-H3 gene inhibitor comprises the sequence set forth in SEQ ID NO. 1.

[0013] SEQ ID NO. 1: CAACGAGCAGGGCTTGTTT.

[0014] Preferably, the form of the pharmaceutical combination comprises any one of the following forms:

[0015] (1) the B7-H3 gene inhibitor and curcumin are prepared into independent preparations, respectively; or

[0016] (2) the B7-H3 gene inhibitor and curcumin are formulated into a compound preparation.

[0017] Preferably, the pharmaceutical combination further comprises an excipient.

[0018] Preferably, the excipient comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

[0019] In a second aspect, the present application provides use of the pharmaceutical combination according to the first aspect in the preparation of a medicament for treating non-small cell lung cancer.

[0020] In a third aspect, the present application provides use of the pharmaceutical combination according to the first aspect in the preparation of an inhibitor of human non-small cell lung cancer cells.

[0021] According to the research results of the present application, the pharmaceutical combination described in the present application can inhibit the activity of human non-small cell lung cancer cells, that is, a simple test preparation is prepared for inhibiting the activity of human non-small cell lung cancer cells, and the inhibitor claimed in the present application is not used to eliminate the cause or lesion, that is, the use in the preparation of the inhibitor is for non-therapeutic purposes.

[0022] In a fourth aspect, the present application provides use of the pharmaceutical combination according to the first aspect in the preparation of an inhibitor of GPX4 gene or GSDMD gene.

[0023] According to the research results of the present application, the pharmaceutical combination described in the present application can inhibit the expression of GPX4 protein and GSDMD protein, so the pharmaceutical combination can be used as a potential inhibitor of GPX4 gene and GSDMD gene, a simple test preparation is prepared for inhibiting the expression of GPX4 gene and GSDMD gene, and the inhibitor claimed is not used to eliminate the cause or lesion, that is, the use in the preparation of the inhibitor is for non-therapeutic purposes.

[0024] In a fifth aspect, the present application provides use of the pharmaceutical combination according to the first aspect in the preparation of an inhibitor of CASPASE-1 gene.

[0025] According to the research results of the present application, the pharmaceutical combination described in the present application can inhibit the expression of CASPASE-1 protein, so the pharmaceutical combination can be used as a potential inhibitor of CASPASE-1 gene, a simple test preparation is prepared for inhibiting the expression of CASPASE-1 gene, and the inhibitor claimed is not used to eliminate the cause or lesion, that is, the use in the preparation of the inhibitor of CASPASE-1 gene is for non-therapeutic purposes.

[0026] In a sixth aspect, the present application provides use of curcumin in the preparation of an inhibitor of B7-H3 gene, GPX4 gene, GSDMD gene, CASPASE-1 gene or human non-small cell lung cancer cells.

[0027] According to the research results of the present application, curcumin can inhibit the expression of B7-H3 protein, GPX4 protein, GSDMD protein and CASPASE-1 protein, and can inhibit the activity of human non-small cell lung cancer cells, so curcumin can be used as a potential B7-H3 gene inhibitor, GPX4 gene inhibitor, GSDMD gene inhibitor, CASPASE-1 gene inhibitor, and human non-small cell lung cancer cell inhibitor, that is, a simple test preparation is prepared for inhibiting the expression of B7-H3 gene, GPX4 gene, GSDMD gene and CASPASE-1 gene, and inhibiting the activity of human non-small cell lung cancer cells. The claimed inhibitor is not used to eliminate the cause or lesion, that is, the application in preparing the inhibitor for non-therapeutic purposes.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] (1) The B7-H3 gene silencing, overexpression and normal expression cell models are constructed by using cell transfection technology, the influence of B7-H3 expression level on cell migration ability is evaluated by cell scratch test, it is found that B7-H3 gene silencing low expression has antitumor activity effect, and can effectively inhibit the migration ability of NSCLC cells, and B7-H3 gene overexpression has pro-tumor activity effect.

[0030] (2) It is found by CCK-8 test that curcumin can inhibit the growth and proliferation ability of NSCLC cells; cell colony formation test shows that curcumin can significantly inhibit the single cell colony formation ability of NSCLC cells.

[0031] (3) It is found that curcumin and B7-H3 gene inhibitor have potential antitumor activity synergistic effect. BRIEF DESCRIPTION OF DRAWINGS

[0032] Fig. 1 is a result graph of the inhibitory effect of different concentrations of curcumin on A549 cells.

[0033] Fig. 2 is a result graph of the inhibitory effect of different concentrations of curcumin on H1703 cells.

[0034] Fig. 3 is a result graph of the inhibitory effect of different concentrations of curcumin on HCC827 cells.

[0035] Fig. 4 is a result graph of the inhibitory effect of different concentrations of curcumin on PC9 cells.

[0036] Fig. 5 is a result graph of the cell scratch test of A549 cells after treatment with 10 μM curcumin and B7-H3 silencing or overexpression.

[0037] Fig. 6 is a statistical result graph of the cell scratch test of A549 cells after treatment with 10 μM curcumin and B7-H3 silencing or overexpression.

[0038] Figure 7 is a graph of the results of a cell scratch experiment of H1703 cells after treatment with 10 μΜ curcumin and silencing or overexpression of B7-H3.

[0039] Figure 8 is a graph of the statistical results of a cell scratch experiment of H1703 cells after treatment with 10 μΜ curcumin and silencing or overexpression of B7-H3.

[0040] Figure 9 is a graph of the results of a cell scratch experiment of HCC827 cells after treatment with 10 μΜ curcumin and silencing or overexpression of B7-H3.

[0041] Figure 10 is a graph of the statistical results of a cell scratch experiment of HCC827 cells after treatment with 10 μΜ curcumin and silencing or overexpression of B7-H3.

[0042] Figure 11 is a graph of the results of a cell scratch experiment of PC9 cells after treatment with 10 μΜ curcumin and silencing or overexpression of B7-H3.

[0043] Figure 12 is a graph of the statistical results of a cell scratch experiment of PC9 cells after treatment with 10 μΜ curcumin and silencing or overexpression of B7-H3.

[0044] Figure 13 is a graph of the results of the inhibition of clone formation of non-small cell lung cancer cells by different concentrations of curcumin.

[0045] Figure 14 is a graph of the statistical results of the clone inhibition of A549 cells by different concentrations of curcumin.

[0046] Figure 15 is a graph of the statistical results of the clone inhibition of PC9 cells by different concentrations of curcumin.

[0047] Figure 16 is a graph of the statistical results of the clone inhibition of HCC827 cells by different concentrations of curcumin.

[0048] Figure 17 is a graph of the statistical results of the clone inhibition of H1703 cells by different concentrations of curcumin.

[0049] Figure 18 is a graph of the effects of silencing or overexpression of B7-H3 on the expression of P110, AKT, B7-H3, GSDMD, and GAPDH proteins.

[0050] Figure 19 is a graph of the effects of silencing or overexpression of B7-H3 on the expression of B7-H3, AKT, CASPASE-1, GPX4, and GAPDH proteins.

[0051] Figure 20 is a graph of the results of a Trans-well experiment of A549 cells after treatment with 10 μΜ curcumin and silencing or overexpression of B7-H3. DETAILED DESCRIPTION

[0052] The technical solutions of the present application are further illustrated by specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0053] The following content involves the following abbreviations, which are annotated as follows:

[0054] ① CON: the experimental control group without any treatment.

[0055] ② CUR: the administration group with curcumin administration of 10 μM.

[0056] ③ SI: the treatment group after 50 nM SiRNA inhibits B7-H3 protein expression.

[0057] ④ SI+CUR: the treatment group after 50 nM SiRNA inhibits B7-H3 protein expression and curcumin administration of 10 μM is added.

[0058] ⑤ OE: the B7-H3 overexpression treatment group.

[0059] ⑥ OE+CUR: the treatment group after B7-H3 overexpression treatment and curcumin administration of 10 μM is added.

[0060] Example 1

[0061] CCK-8 cytotoxicity detection test

[0062] 1.1 Experimental principle

[0063] CCK-8 (CELL COUNTING KIT 8) is a commonly used experimental method for studying cell proliferation and cytotoxicity detection test. The most important chemical in the CCK-8 kit is WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt). WST-8 can be oxidized by NADH in the respiratory chain of living cells to form water-soluble yellow formazan, and the amount of formazan is proportional to the number of living cells and shows a linear relationship within a certain range. The more living cells, the darker the color. The absorbance at 450 nm can directly reflect the number of living cells, and then calculate the effective inhibition rate of the drug. +

[0064] 1.2 Experimental steps

[0065] I. Cell plating: obtain cell suspension by cell subculture method, count and dilute; inoculate 200 μL of cell suspension into a 96-well plate at 3×10 3 cells / well, set up 3 replicate wells, and add the same amount of PBS to the outermost circle; mix the wells gently in a "∞" trajectory, then place them in an incubator for about 24 h to adhere.​

[0066] II. Experimental treatment: According to the experimental purpose, curcumin administration treatment was carried out, and the control group was replaced with an equal amount of culture medium; and was placed back into the incubator for continuous culture for about 24 h.

[0067] III. CCK-8 detection: 10 μL of CCK-8 detection reagent was added to each well in the dark, and was placed into the incubator for continuous incubation for 2 h; the absorbance at 450 nm was measured by the enzyme label instrument, and the data was derived and analyzed.

[0068] 1.3 Data analysis

[0069] Inhibition rate = [(control well - experimental well) / (control well - blank well)] x 100%

[0070] Blank well: no cells, only medium, CCK-8, for correcting system error; control well: no treatment cells, medium, CCK-8; transfection experimental well: transfection treatment cells, medium, CCK-8; drug administration experimental well: drug administration treatment cells, medium, CCK-8.

[0071] The results are shown in Figures 1-4, and A549 cells, H1703 cells, HCC827 cells, and PC9 cells are all human non-small cell lung cancer cells, and curcumin has an inhibitory effect on the activity of several cells.

[0072] Example 2

[0073] Cell scratch test

[0074] 2.1 Experimental principle

[0075] The cell scratch test is an in vitro test method for studying cancer cell migration and invasion. When a dense monolayer of cells is formed, a clear cell scratch is artificially made on the cell layer. In subsequent culture, the cells will continuously migrate to fill the scratch, and the scratch morphology is observed and photographed at regular time intervals. By comparing the scratch healing at different time points, the effects of different drug concentrations on the cell migration ability of different cells can be compared.

[0076] 2.2 Experimental steps

[0077] I. Cell plating: Invert the 6-well plate, and draw 5-6 parallel lines in each well with a MARK pen; obtain cell suspension by cell subculture method, count and dilute; inoculate 2 mL of cell suspension into the 6-well plate at 4 x 10 5 cells / well.

[0078] II. Experimental treatment: According to the experimental purpose, transfection or drug administration treatment was carried out. The control group was replaced with an equal amount of culture medium; and was placed back into the incubator for continuous culture for about 24 h.

[0079] III. Scratch wound: Sterilize ruler stand on 6-well plate, use 10 μL gun head against the ruler, gun tip perpendicular to the bottom of the well, scratch across the dense cell layer once, form clear cell scratch; discard the medium, add 1 mL PBS to wash the cells per well until the scratch is clear and no cell residues remain; discard the PBS, add 2 mL low serum medium per well, use the CCD microscopic image acquisition system to collect the scratch edge image and record as 0 h; add 10 μM curcumin to the treatment group, replace the same amount of medium to the control group; return to the incubator for continuous culture, collect the scratch edge image at 12 h and 24 h.

[0080] 2.3 Data processing

[0081] Adjust the image parameters with Photoshop, add a ruler, and mark the scratch edge for comparison.

[0082] The results are shown in Figures 5-12. Both the B7-H3 gene inhibitor and curcumin can inhibit the migration ability of cells, and the combination of the B7-H3 gene inhibitor and curcumin can more significantly inhibit the migration ability, and the two have a potential synergistic effect.

[0083] Example 3

[0084] 3. Plate clone formation test

[0085] 3.1 Experimental principle

[0086] Single adherent cells can survive but are not necessarily proliferative. The cell population formed by the proliferation of single cells in vitro is a cell clone, and the proliferation ability of tumor cells shows a certain population dependence. After the dispersed single cells adhere, they are continuously cultured for a period of time under the treatment of different concentrations of curcumin, and the clone formation is stained and photographed for recording, so that the clone formation inhibition ability of different concentrations of drugs can be compared.

[0087] 3.2 Experimental steps

[0088] I. Cell plating: Obtain cell suspension by cell passage method, count and dilute; inoculate 2 mL of cell suspension per 1000 cells per well in a 12-well plate to ensure uniform dispersion of cells without clumping; place in an incubator for about 24 h for adhesion.

[0089] II. Experimental treatment: According to the set concentration gradient (0 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM), perform curcumin administration treatment in the corresponding wells, and replace the same amount of medium in the control group; return to the incubator for continuous culture for 8 days, and replace the drug-containing medium every 2 days to maintain the drug concentration.

[0090] III. Clone staining: discard the culture medium, add 1 mL of PBS to each well, and wash twice on a shaker for 3 min each time; add 500 μL of 4% paraformaldehyde to each well, and fix for 15 min; discard the paraformaldehyde, add 1 mL of PBS to each well, and wash twice on a shaker for 3 min each time; discard the PBS, invert twice on a blotting paper, and place in a 37°C oven to dry for 5 min; add 500 μL of crystal violet staining solution to each well, and stand at room temperature for 40 min in the dark; discard the staining solution, add 1 mL of PBS to each well, and wash twice on a shaker for 3 min each time; discard the PBS, invert twice on a blotting paper, and take a photo after standing at room temperature overnight.

[0091] The results are shown in Figures 13-17. As can be seen from the results, curcumin can inhibit the formation of non-small cell lung cancer cell clones.

[0092] Example 4

[0093] Western blotting test

[0094] 4.1 Experimental principle

[0095] Western blotting is a qualitative and semi-quantitative test method for protein expression, which is widely used in gene expression research at the protein level, antibody activity detection, etc. The protein sample to be tested is separated by polyacrylamide gel electrophoresis, then transferred to a polyvinylidene fluoride (PVDF) membrane, then combined with the target protein using specific primary antibodies, and then combined with specific secondary antibodies with detection groups to perform chemiluminescence detection. By detecting the differences in the expression of B7-H3 and related proteins in different treated cells, the potential mechanism of B7-H3 and curcumin at the protein level is explored.

[0096] 4.2 Experimental steps

[0097] I. Glue preparation: 1.5 mm glass plates are washed in advance and placed in an oven to dry, ensuring that they are clean, dry, and free of stains. The glue preparation frame clamps the glass plates to ensure that the glue does not leak. Prepare 8% separation glue according to the formula, pour it in, and seal it with distilled water to remove air bubbles. Let it stand for 30 min to allow the separation glue to solidify. Prepare 5% concentrated glue according to the formula, insert the 10-hole or 15-hole comb, and let it stand for 30 min to allow the concentrated glue to solidify.

[0098] II. Collecting cell proteins: Place the hole plate on ice, wash the cells 3 times with 1 mL of pre-cooled PBS per well; add 1:50 of the proteinase inhibitor cocktail (50x) and phosphatase inhibitor (50x) to the lysis buffer; add 60 μL of lysis buffer to each well, and scrape the cells on the bottom of the hole plate with a cell scraper for about 40 s; collect the protein extract in an ep tube, and centrifuge at 4°C and 12000 rpm for 30 min.

[0099] III. BCA quantification: Prepare standard protein gradient (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 mg / mL); prepare BCA working solution: 200 μL BCA working solution per well, A and B are prepared at a ratio of 50:1; dilute protein samples: dilute protein extract with pure water or PBS by 5 times; add PBS to the periphery of the 96-well plate, add 20 μL protein standard or sample to the other wells, then add 200 μL BCA working solution, cover with tin foil paper to avoid light, and place in a 37°C incubator for 30 min; measure the OD value of the 96-well plate at 562 nm with a microplate reader, prepare a standard curve, and calculate the target protein concentration;

[0100] IV. Sample preparation: According to the protein concentration to be obtained, calculate the original concentration, and prepare a sample solution containing Loading buffer at 10 μg / μL; place in a 90°C water bath to denature the protein for standby use.

[0101] V. Gel electrophoresis: Place the SDS PAGE gel into the electrophoresis tank and add the electrophoresis liquid; electrophorese, concentrate the gel: 80 V, 30 min; separate the gel: 100 V, 45 min.

[0102] VI. Membrane transfer: After methanol activates the PVDF membrane for 30 s, pry open the glass plate, cut off the concentrated gel, and place it into the membrane transfer liquid for equilibration; fix according to the order of sponge, two layers of filter paper, separation gel, membrane, two layers of filter paper, and sponge; place the electrophoresis tank in an ice bath for membrane transfer at 100 V for 90 min.

[0103] VII. Immunoreaction (including blocking): immerse the PVDF membrane obtained in the previous step in 5% skim milk / TBST, and incubate at room temperature for 1 h; wash the membrane with TBST for 3 times, add specific primary antibody, and incubate at 4°C overnight; wash the membrane with TBST for 3 times, each for 5 min; add specific secondary antibody, incubate at room temperature for 1 h, and wash the membrane with TBST for 3 times, each for 5 min.

[0104] VIII. Exposure: mix the developing solution and drop it onto the membrane, expose it to the gel imaging system, and export the data for storage.

[0105] The results are shown in FIGS. 18-19. GAPDH was used as a control. As shown in the figures, curcumin had an inhibitory effect on the expression of B7-H3 gene, GSDMD, CASPASE-1 gene, and GPX4 gene. The B7-H3 gene inhibitor had an inhibitory effect on the expression of B7-H3 gene, GSDMD, CASPASE-1 gene, and GPX4 gene. Curcumin and the B7-H3 gene inhibitor had a potential synergistic effect on the above effects.

[0106] Example 5

[0107] Trans-well experiment

[0108] 5.1 Principle of the experiment

[0109] After different treatments, tumor cells were inoculated in the upper chamber of the culture medium without serum, and fetal bovine serum (FBS) or certain specific chemokines were added to the lower chamber. In order to obtain more nutrients, tumor cells would migrate to the lower chamber with high nutritional components. The amount of cells entering the lower chamber was counted to reflect the migration ability of tumor cells.

[0110] 5.2 Experimental steps

[0111] I. Cell plating: obtain cell suspension by cell subculture method, count and dilute; inoculate 1 mL cell suspension in a 24-well plate at 1 × 10 5 cells / well.

[0112] II. Experimental treatment: according to the purpose of the experiment, transfection or drug treatment was performed. The control group was replaced with an equal amount of culture medium; and then returned to the incubator for continuous culture for about 24 h.

[0113] III. Trans-well counting and transfer: take one chamber to a 24-well plate, absorb 100 μL of FBS-free medium to the upper chamber of the chamber for hydration; obtain cell suspension in the conventional way (terminate digestion and resuspend with serum-free culture medium); count cells: 10 μL of cell and trypan blue suspension to a counting plate; inoculate 100 μL of cell suspension to the upper chamber of each chamber at 1.5 × 10 4 cells / well; absorb 100 μL of suspension to the upper chamber, and 600 μL of 30% FBS medium to the lower chamber, mark well, and gently place the plate in the medium for 16 h.

[0114] IV. Trans-well photography: after 16 h, remove the chamber upside down on the absorbent paper to remove the culture medium, gently wipe the inside of the chamber with a cotton swab, absorb 600 μL of 4% paraformaldehyde into an empty 24-well plate, gently place the chamber, fix for 30 min; place the fixed chamber upside down on the absorbent paper to remove the fixing solution, rinse the chamber in a 6 cm dish containing PBS for 1 time; absorb a certain amount of 0.1% crystal violet for 10 min, rinse with PBS for 3 times, and wipe clean with a cotton swab; place the dried chamber on a glass slide, find 5 fields of cells under a 10-fold microscope, and take a photo.

[0115] The results showed that curcumin and B7-H3 gene inhibitors could inhibit the migration ability of A549 cells, and the combination of curcumin and B7-H3 gene inhibitors could more significantly inhibit the migration ability of cells.

[0116] The applicant declares that the present application is illustrated by the above examples of a pharmaceutical combination and its use, but the present application is not limited to the above examples, i.e. it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

[0117] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments. Within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0118] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, various possible combinations are not described again in the present application.

Claims

1. A pharmaceutical combination comprising curcumin and a B7-H3 gene inhibitor.

2. The pharmaceutical combination of claim 1, wherein, The B7-H3 gene inhibitor comprises a nucleic acid molecule.

3. The pharmaceutical combination of claim 1 or 2, wherein, The targeted sequence of the B7-H3 gene inhibitor comprises a sequence as set forth in SEQ ID NO.

1.

4. The pharmaceutical combination according to any one of claims 1-3, wherein, The form of the pharmaceutical combination comprises any one of the following forms: (1) the B7-H3 gene inhibitor and the curcumin are prepared into independent formulations, respectively; or (2) the B7-H3 gene inhibitor and the curcumin are formulated into a compound preparation.

5. The pharmaceutical combination according to any one of claims 1-4, wherein, The pharmaceutical combination further comprises an excipient.

6. The pharmaceutical combination according to claim 5, wherein, The excipient comprises any one of or a combination of at least two of a pharmaceutically acceptable carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizing agent, an osmotic pressure adjusting agent, a surfactant, a coating material, a coloring agent, a pH adjusting agent, an antioxidant, a bacteriostatic agent, or a buffer. 7.Use of the pharmaceutical combination according to any one of claims 1-6 in the preparation of a medicament for treating non-small cell lung cancer. 8.Use of the pharmaceutical combination according to any one of claims 1-6 in the preparation of a human non-small cell lung cancer cell inhibitor. 9.Use of the pharmaceutical combination according to any one of claims 1-6 in the preparation of a GPX4 gene inhibitor or a GSDMD gene inhibitor. 10.Use of the pharmaceutical combination according to any one of claims 1-6 in the preparation of a CASPASE-1 gene inhibitor. 11.Use of curcumin in the preparation of a B7-H3 gene inhibitor, a GPX4 gene inhibitor, a GSDMD gene inhibitor, a CASPASE-1 gene inhibitor, or a human non-small cell lung cancer cell inhibitor.

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