Pharmaceutical composition for treating pancreatic cancer

Abstract

The invention relates to a pharmaceutical composition for treating pancreatic cancer, and belongs to the technical field of biological medicines. The mass ratio of the resveratrol to the glucosamine is (2.28 mg-45.6 mg): (179.17 mg-895.85 mg), and the pharmaceutical composition group is applied to preparation of products for treating pancreatic cancer or preventing pancreatic cancer metastasis. Compared with a single medication group, the combined application of the resveratrol and the glucosamine has the advantages that the metastasis and invasion capability of pancreatic cancer cells can be more obviously inhibited, and the influence on normal cells is smaller. Researches show that the resveratrol realizes an anti-tumor effect by inhibiting the expression of a ligand in a TGF-beta (Transforming Growth Factor-beta) pathway; and the glucosamine inhibits N-glycosylation modification and further inhibits the stability of a receptor in a TGF-beta pathway so as to realize an anti-tumor effect. And a synergistic interaction effect is achieved through multiple targets on the same path. Therefore, the method has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a pharmaceutical composition for treating pancreatic cancer. Background Art

[0002] With the changes in living environment and eating habits, digestive tract malignancies have gradually become the most common tumor among many cancers. Currently, pancreatic cancer is mainly treated with surgery and chemotherapy, but the drugs are highly toxic and have a low overall response rate. The selection of targeted drugs is extremely limited, and the effect of improving patient survival is not significant.

[0003] Traditional Chinese medicine plays an important role in the treatment of advanced gastrointestinal malignancies. Studies have shown that traditional Chinese medicine can effectively reduce toxic side effects, effectively intervene in treatment, and delay cancer metastasis. In recent years, the anti-cancer effect of resveratrol, an extract from Polygonum cuspidatum, has attracted widespread attention. Resveratrol has anti-cancer effects both in vivo and in vitro. In pancreatic cancer, the anti-cancer effect of resveratrol is mainly through downregulating signaling pathways such as AKT, MAPK, and TGF-β, thereby inhibiting the EMT process and effectively inhibiting cell proliferation, migration and invasion, blocking the cell cycle, and causing cell apoptosis. Preliminary clinical trials have shown that resveratrol is well tolerated, but its bioavailability is low. What is more serious is that the results of resveratrol in the clinical treatment of prostate cancer, colon cancer, and breast cancer metastasis are not ideal. In order to solve the above clinical problems, it is urgent to find a comprehensive understanding of the anti-tumor mechanism of resveratrol and a scheme for reducing toxicity and enhancing efficacy of resveratrol.

[0004] Glucosamine is a natural product and is currently widely used as a daily health product. It is a necessary substance for the synthesis of proteoglycans in the matrix of human articular cartilage. Glucosamine is an important component of glycoproteins and proteoglycans. It can regulate multiple signaling pathways and play a certain pharmacological role in various diseases such as cancer, cardiovascular disease, neurodegenerative diseases, skin diseases and bacterial infections. The effect of glucosamine on pancreatic cancer has not been reported. In tumor models such as colon cancer and breast cancer, glucosamine has shown anti-tumor effects, mainly manifested in inhibiting cancer cell proliferation and inducing apoptosis, inducing autophagic death of cancer cells, reversing tumor resistance, anti-tumor angiogenesis and inhibiting the expression of matrix metalloproteinases. Glucosamine, as an effective and non-toxic potential anticancer drug, has a wide range of application value. However, the research on the anti-tumor mechanism of glucosamine is not clear.

[0005] The main feature of Chinese medicine in anti-tumor is multi-point micro-effect, so how to reasonably integrate and coordinate to achieve the goal of cascade amplification of drug efficacy is the focus of current research. There are many plants in natural products, such as knotweed, grape skin and grape seeds, which contain high levels of resveratrol, which have anti-tumor and anti-inflammatory effects. Turtle shell mainly contains polysaccharides and calcium, and glucosamine is the main monosaccharide component of turtle shell polysaccharide. Animal medicine and food varieties such as deer antler, antler, antler glue, sea cucumber, chicken gizzard also contain glucosamine ingredients, and they show anti-tumor effects. Summary of the invention

[0006] The invention provides a pharmaceutical composition for treating pancreatic cancer, in particular a composition of resveratrol and glucosamine for treating pancreatic cancer.

[0007] The technical solution adopted by the present invention is that the mass ratio of resveratrol to glucosamine is resveratrol:glucosamine=(2.28mg~45.6mg):(179.17mg~895.85mg).

[0008] The invention relates to the use of the drug combination group in preparing a product for treating pancreatic cancer or preventing pancreatic cancer metastasis.

[0009] The product of the present invention is medicine, health care product, food or cosmetics.

[0010] The concentration ratio of resveratrol to glucosamine in the present invention is (10 μM~200 μM): (1 mM~5 mM).

[0011] When the present invention is used in combination, the drug concentration ratio of resveratrol to glucosamine is 100 μM:2.5 mM.

[0012] The invention comprises active ingredients and auxiliary materials.

[0013] The active ingredients of the present invention are resveratrol and glucosamine, and the auxiliary materials include one or more of fillers, adhesives, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, fragrances, preservatives, dispersants, film formers, plasticizers, pore formers, opacifiers, retardants, and solvents.

[0014] The invention discloses an application of the drug combination group in preparing a drug for inhibiting signals of a TGF-β signaling pathway.

[0015] The invention discloses an application of the drug combination group in preparing a drug for inhibiting signals of a MAPK signal pathway.

[0016] The product of the present invention includes a combination of natural plants or animals (including but not limited to deer antlers, grapes and other plants or animals containing resveratrol or glucosamine) containing two effective ingredients, resveratrol and glucosamine, which is used to prepare drugs and health products, foods or cosmetics for treating or preventing pancreatic cancer metastasis.

[0017] The advantages of the present invention are: the experiment proves that the pharmaceutical composition of resveratrol and glucosamine can more significantly inhibit TGF-β pathway signal transduction, migration and infiltration of pancreatic cancer cells, and cell survival rate compared with their single medication groups, while having little effect on normal cells. The present invention finds that the resveratrol and glucosamine composition not only produces a synergistic effect, but also shows a synergistic effect, that is, the effect of the effect is greater than the sum of the two individual drug effects, and glucosamine can effectively improve the anti-cancer function of resveratrol. Under the same dosage of resveratrol, the drug effect of the combined medication group is much higher than the effect of the two drugs alone, and at a lower drug concentration, it shows a strong anti-cancer activity, especially reducing the migration and invasion of pancreatic cancer cells. Further research from the target to the mechanism of the action pathway shows a good synergistic anti-tumor effect, indicating that the pharmaceutical composition is safe and effective, has a good practical application value, and provides a research direction for the development of new drugs and the new use of old drugs.

[0018] Through mechanism studies, it was found that the synergistic effect of the combination of resveratrol and glucosamine is achieved by targeting different targets on the same pathway, thereby achieving a low-dose, long-term, and effective inhibitory effect and increasing anti-tumor efficacy. In pancreatic cancer, resveratrol inhibits the activation of the TGF-β pathway and the migration of cancer by inhibiting the expression of TGF-β ligands; glucosamine inhibits the stability of the TGF-β kinase receptor by inhibiting N-glycosylation modification, thereby inhibiting the TGF-β pathway and anti-tumor development. Omics studies have found that the combined use of the two increases the anti-pancreatic cancer pathway, and that glucosamine further increases the effect of resveratrol in inhibiting the signals of the TGF-β and MAPK signaling pathways and promoting cell apoptosis.

[0019] At the same drug dose, the effect of the drug combination is significantly better than that of resveratrol or glucosamine alone, and the combined treatment is about 1.5-2 times the effect of resveratrol alone. About 15% of pancreatic cancer patients have defects in the TGF-β pathway. Experiments have found that resveratrol combined with glucosamine also has a significant inhibitory effect on the migration and infiltration ability of these tumor cells, which is significantly higher than the effect of using the drug alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention and in combination with the accompanying drawings. To facilitate the display of various drugs in the drawings, the drugs in the drawings are abbreviated as glucosamine GAN and resveratrol RES; Figure 1 This is the inhibitory effect of resveratrol and glucosamine alone and in combination on the proliferation of PANC-1 pancreatic cancer cells. Note: administration / blank***P<0.001; Figure 2 This is the effect of resveratrol and glucosamine alone and in combination on the invasion ability of PANC-1 pancreatic cancer cells, Note: A: control group; B: glucosamine 2.5 mM; C and D: resveratrol 50 μM and 100 μM; E and F: glucosamine combined with resveratrol 50 μM and 100 μM; G: statistical analysis of migration data of each group; H: number of reduced migrating cells. Administration / blank *P<0.05, **P<0.01, ***P<0.001; Figure 3 The effect of resveratrol and glucosamine alone and in combination on the migration ability of PANC-1 pancreatic cancer cells, A: control group; B: glucosamine 2.5 mM; C and D: resveratrol 50 μM and 100 μM; E and F: glucosamine combined with resveratrol 50 μM and 100 μM; G: statistical analysis of invasion data of each group; H: reduced number of invasive cells, drug / blank***P<0.001; Figure 4-1 This is the effect of resveratrol and glucosamine alone and in combination on the healing ability of PANC-1 pancreatic cancer cells. Note: A: The healing status of PANC-1 pancreatic cancer cells 12 and 24 hours after resveratrol and glucosamine were administered alone and in combination; B: 12-hour data statistical analysis chart; C: 24-hour data statistical analysis; administration / blank *P<0.05, ***P<0.001; Figure 4-2 This is the effect of resveratrol and glucosamine alone and in combination on the healing ability of Mia Paca2 pancreatic cancer cells. Note: A: The healing of MIA pancreatic cancer cells after resveratrol and glucosamine alone and in combination for 12 and 24 hours; B: 12-hour data statistical analysis chart; C: 24-hour data statistical analysis chart. **P<0.01, ***P<0.001 for drug administration compared with blank; Figure 5The effect of resveratrol and glucosamine alone and in combination on the cloning ability of PANC-1 pancreatic cancer cells, Note: A: control group; B: glucosamine 2.5 mM; C and D: resveratrol 50 μM and 100 μM; E and F: glucosamine combined with resveratrol 50 μM and 100 μM; G: statistical analysis of cloning data of each group; H: number of weakened cloned cells; administration compared with blank ***P<0.001; Figure 6-1 The figure shows the regulatory effects of resveratrol and glucosamine alone and in combination on PANC-1 pancreatic cancer cell proteins (A) and genes (B). Note: The numbers are the folds of gene or protein expression regulated compared with the control group. The positive and negative values ​​are positive and negative regulation, respectively. The larger the value, the greater the regulation fold. Figure 6-2 This is a diagram showing the regulatory effects of resveratrol and glucosamine alone and in combination on the protein (A) and gene (B) pathways of PANC-1 pancreatic cancer cells. Note: The darker the color of the dots in the figure, the more significant the regulation of the pathway, and the further to the right the dots are, the more genes or proteins are regulated in the pathway; Figure 7 This is the effect of resveratrol and glucosamine alone and in combination on TGF-β pathway-related proteins. Note: A: smad2 and p-smad2 protein levels in PANC-1 cells of each group; B: corresponding statistical analysis chart; drug administration / blank ***P<0.001; Figure 8 This is the effect of resveratrol and glucosamine alone and in combination on the proteins related to the migration and apoptosis pathway of PANC-1 pancreatic cancer cells. Note: A: p-p38, Bax and Bcl2 protein levels in PANC-1 cells of each group; B: p-p38 expression analysis; C: BAX / Bcl2 expression analysis. Drug administration / blank ***P<0.001; Fig. 9 This is the effect of grape seed and deer antler capsule on the migration and cloning ability of PANC-1 pancreatic cancer cells. Note: C and D are the statistics of cloning and invasion data respectively; drug administration / blank *P<0.05, **P<0.01, ***P<0.001; Fig.10 This is the effect of grape skin and antler film on the migration and cloning ability of PANC-1 pancreatic cancer cells. Note: C and D are the statistics of cloning and invasion data respectively; drug administration / blank* P <0.05,*** P <0.001. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the experimental examples are not intended to limit the present invention.

[0022] Materials involved in the present invention: PANC-1 pancreatic cancer cells were purchased from Wuhan Punosai Life Science Technology Co., Ltd., pancreatic enzymes were purchased from Sigma, batch number 0000306113, DMEM culture medium was purchased from Gibco, batch number 6124122, CCK8 was purchased from Dalian Meilun, batch number MA0218-Feb-J, PBS was purchased from Dalian Meilun, batch number MA0015, double antibody was purchased from Solebao, batch number 240001001, fetal bovine serum (FBS) was purchased from CLARK, batch number FB2015, dimethyl sulfoxide Dimethyl sulfoxide (DMSO) was purchased from Sigma, batch number SHBL3599, resveratrol was purchased from Shandong Sikejie Biotechnology Co., Ltd., batch number DADTU, glucosamine hydrochloride was purchased from Shandong Sikejie Biotechnology Co., Ltd., batch number HUKVM, cell invasion chamber (Transwell chamber) was purchased from Zhenxuan, batch number 28823800AD, 4% paraformaldehyde universal tissue fixative was purchased from Biosharp, and 1% ammonium oxalate crystal violet staining solution was purchased from Yuanye Biotechnology, batch number O28HR13713A.

[0023] Experimental Example 1: Effects of resveratrol and glucosamine alone and in combination on the proliferation of PANC-1 pancreatic cancer cells Experimental methods: A total of 6 groups were set up, namely control group, 2.5 mM glucosamine hydrochloride, 100 μmol / L resveratrol, 200 μmol / L resveratrol, 2.5 mM glucosamine hydrochloride + 100 μmol / L resveratrol, and 2.5 mM glucosamine hydrochloride + 200 μmol / L resveratrol. 3 Cells / well) were inoculated in a 96-well plate for 12 hours. Each group was given drugs at their respective concentrations. After 3 consecutive days of drug administration, cell viability was measured using a cell counting kit-8 (CCK-8) to evaluate proliferation ability.

[0024] Experimental results: The experimental results showed that glucosamine had little ability to inhibit the proliferation of PANC-1 cells, while different concentrations of resveratrol could inhibit the proliferation of PANC-1 cells. The ability to inhibit proliferation increased with increasing concentrations. The combined administration of resveratrol and glucosamine greatly enhanced the ability of resveratrol to inhibit proliferation, increasing by 1.25 times ( Figure 1), indicating that after combined administration, the same dosage concentration has an enhanced effect on inhibiting the proliferation of PANC-1 pancreatic cancer cells, showing a synergistic effect, that is, the drug dosage is reduced under the same efficacy, while the toxicity of the drug is reduced.

[0025] Experimental Example 2: Effects of resveratrol and glucosamine alone and in combination on the migration ability of PANC-1 pancreatic cancer cells Experimental methods: The experimental groups were control group, resveratrol 50 and 100 μM, glucosamine 2.5 mM alone, and glucosamine 2.5 mM combined with 50 and 100 μM. Cells in logarithmic growth phase were prepared into single cell suspension and inoculated into six-well plates (cell density: 2×10 5 After incubation for 12 h, 2 mL of the corresponding concentrations of the five groups of different drugs and the blank control group culture medium were added, and the cells were cultured for 48 h to prepare the cell suspension. The tumor cells of each treatment group were inoculated in the transwell plate chamber with 200 μL serum-free culture medium (cell density: 2.5×10 4 500 μL of complete medium containing 20% ​​FBS was added to the lower chamber. After 24 h, the cells in the lower chamber were fixed with 4% polymethanol for 30 min and then stained with 0.1% crystal violet stain for 30 min. The pictures were taken and analyzed by ImageJ software.

[0026] Experimental results: The results showed that the combined administration of resveratrol and glucosamine significantly enhanced the inhibitory effect on PANC-1 cell migration, and the inhibitory effect was much stronger than that of glucosamine and resveratrol alone, which was twice the sum of the two individual effects. Figure 2 ), showing an unexpected synergistic effect.

[0027] Experimental Example 3: Effects of resveratrol and glucosamine alone and in combination on the invasive ability of PANC-1 pancreatic cancer cells Experimental methods: The experimental grouping was the same as in Experimental Example 2. The prefabricated transwell plate with matrix gel was placed in the incubator for 2 h to prepare the cell suspension. The tumor cells of each treatment group were inoculated in the upper chamber with 200 μL serum-free culture medium (cell density: 5×10 4 100 μL of complete medium containing 20% ​​FBS was added to the lower chamber. After 24 h, the cells in the lower chamber were fixed with 4% polymethanol for 30 min and then stained with 0.1% crystal violet stain for 30 min. The pictures were taken and analyzed by ImageJ software.

[0028] Experimental results: The results of the analysis showed that (glucosamine 2.5 mM, resveratrol 100 μM, and resveratrol combined with glucosamine all had an inhibitory effect on PANC-1 cell invasion.) The inhibitory effect of resveratrol combined with glucosamine on PANC-1 cell invasion was significantly enhanced, and the intensity of the inhibitory effect was much greater than that of glucosamine alone and resveratrol alone, which was 1.1 times the sum of the intensity of the two individual administrations ( Figure 3 ), showing an unexpected synergistic effect.

[0029] Experimental Example 4: Effects of resveratrol and glucosamine alone and in combination on the healing ability of PANC-1 and Mia Paca2 pancreatic cancer cells Experimental methods: The experimental grouping was the same as that of Experiment 2. Cells in the logarithmic growth phase were used to prepare a single cell suspension. The cells were inoculated into a six-well plate with 5-6 straight lines drawn on the bottom (cell density: 5×10 5 10 μL pipette tip was used to scratch the cells perpendicular to the pre-drawn line after they were full, and the scratched cells were removed. 2 mL of RPMI-1640 complete medium containing 2% serum of the drug and control group was added for culture. The scratch positions were recorded at 0 h, 6 h, 12 h, 24 h, and 48 h, and analyzed by ImageJ software.

[0030] Experimental results: The analysis results showed that at 6 h and 12 h, only the 50 μM resveratrol combined with glucosamine and the 100 μM resveratrol combined with glucosamine administration groups significantly inhibited the healing ability of PANC-1 pancreatic cancer cells, while glucosamine alone and resveratrol 50 μM and 100 μM alone had no inhibitory effect. At 24 h, the combined administration of resveratrol and glucosamine significantly enhanced the ability to inhibit the healing of PANC-1 cells and Mia Paca2 pancreatic cancer cells, and the intensity of the inhibitory effect on the healing ability was much greater than that of glucosamine alone and resveratrol alone, which was 1.04 and 1.03 times the sum of the intensity of the two individual administrations ( Figure 4-1 , 4-2 ), showing an unexpected synergistic effect.

[0031] Experimental Example 5: Effects of single administration and combined administration of resveratrol and glucosamine on the cloning ability of PANC-1 pancreatic cancer cells Experimental methods: The experimental grouping was the same as that of Experiment 2. After the cells were inoculated in a six-well plate for 12 h, 2 mL of the culture medium of the above five groups of different drugs and the blank control group was added. The cells were cultured for 48 h to prepare the cell suspension, and then inoculated into a new six-well plate (cell density: 5×10 2The cells were fixed with 1 mL of 4% paraformaldehyde at room temperature for 30 min and then stained with 0.1% crystal violet solution and photographed to count the number of cloned cells.

[0032] Experimental results: The experimental results showed that the control group had strong cell cloning ability and a large number of cell clusters. The combined administration of resveratrol and glucosamine significantly enhanced the ability to inhibit PANC-1 cell cloning. The intensity of the inhibitory effect was much greater than that of glucosamine and resveratrol alone, which was 1.18 times the sum of the two individual administration intensities ( Figure 5 ), indicating that combined administration significantly enhanced the ability of PANC-1 pancreatic cancer cell cloning.

[0033] Example 5: Bi-omics analysis of the regulation of PANC-1 pancreatic cancer cells by resveratrol and glucosamine alone or in combination Experimental methods: Transcriptomics experimental method: 2.75×10 6 PANC-1 pancreatic cancer cells were inoculated into culture dishes at a density of 10 cells per 100 μM. A control group and three treatment groups (glucosamine 2.5 mM, resveratrol 100 μM, and glucosamine 2.5 mM combined with resveratrol 100 μM) were set up for 3 consecutive days. Total RNA was extracted using Trizol reagent and sequenced using the HiSeq 4000 platform (IllμMina). The DESeq2 algorithm was used to screen differentially expressed genes and enrich pathways.

[0034] Proteomics experimental method: The cell grouping and processing method was the same as that described for the transcriptome. Three days after administration, the cells were rinsed with 4°C precooled PBS, digested with trypsin, and centrifuged to remove the supernatant. The cell pellet was used as a proteomics sample and detected using a Thermoorbitrap astral mass spectrometer to obtain the raw data (.raw) of mass spectrometry. The DIA-NN software (Direct DIA) was used to interpret the raw data and search the species library. The T-test was used to screen differential proteins and enrich pathways.

[0035] Experimental results: Proteomics results screened the differential proteins of glucosamine and resveratrol in regulating PANC-1 pancreatic cancer cells by fold change (FC)>2 and P value<0.05. The results showed that in addition to the proteins regulated by the separate administration of glucosamine and resveratrol, the combined administration of the two newly added regulatory proteins SCAND1, PTPDC1, LSM11, LRR1, LHFPL6, ECM1, CNTNAP4, and USF3. The regulatory effect of the combined administration on THSD7A, IMMP1L, SIMC1, DKK1, ZRSR2P1, ST6GALNAC2, and SLC30A4 proteins was significantly higher than the sum of the efficacy of the two alone, and the intensity of action was enhanced by more than 1.2 times ( Figure 6-1 A). This indicates that the combined use of the two drugs produces a new regulatory protein effect beyond the sum of the two drugs.

[0036] Proteomic differential protein pathway enrichment analysis showed that the combined use of the two drugs, in addition to the pathways regulated by the two drugs alone, newly added regulatory pathways such as ferroptosis pathway, apoptosis pathway, LC-mediated transmembrane transport, programmed cell death, NOTCH signaling pathway, Wnt signaling pathway, proteoglycans in cancer, epithelial-mesenchymal transition in cancer cells, AMPK signaling pathway, and glucocorticoid receptor pathway. The regulatory effect of the combined administration on the NRF2 pathway and integrin cell surface interaction signaling pathway was significantly higher than the sum of the efficacy of the two drugs alone, and the intensity of action was enhanced by more than 2 times ( Figure 6-2 A), these pathways can promote the degradation of BCL-2 under pro-apoptotic stimulation through the TGF-β signaling pathway to trigger cell apoptosis, and play an important role in inhibiting the occurrence and development of pancreatic cancer, indicating that the combined use of the two drugs produces a new regulatory pathway effect beyond the sum of the two.

[0037] Transcriptomics results screened the differentially expressed genes in PANC-1 pancreatic cancer cells regulated by glucosamine alone, resveratrol alone, and combined administration of glucosamine and resveratrol through FC>2 and P value<0.05. The results showed that the combined administration had a significantly higher regulatory effect on AC104695.2, C11orf96, AC092645.1, AC005586.2, COCH, ANKFN1, AC010198.1, RF00019, FLNC, AC016134.1, AP003108.1, CACNA1C, DNAH6, AC005330.1, AL590666.2, CD274, FLT1, C3orf70, AL355512.1, CCRL2, DYSF, GRM2, and ADGRF4 than the sum of the effects of the two alone, and the intensity of action was enhanced by more than 15 times ( Figure 6-1B), indicating that the combined effect of the two drugs produced new regulatory gene effects beyond the sum of the two drugs.

[0038] Transcriptomics differential gene pathway enrichment analysis showed that the combined use of the two drugs not only had the pathways regulated by the two drugs separately, but also added a new regulatory pathway for the regulation of the terpene skeleton biosynthesis pathway, which is closely related to the induction of cancer cell apoptosis. At the same time, the regulatory effect of the combined administration on the TGF-β signaling pathway, axon guidance, oxidative phosphorylation, retrograde endocannabinoid signaling, prion protein and other signaling pathways was more than 5 times that of the efficacy of glucosamine and resveratrol alone, showing an absolute advantage of combined administration, indicating that the combined use of the two drugs produced a new regulatory pathway effect beyond the sum of the two drugs ( Figure 6-2 B) These pathways can trigger apoptosis by promoting the degradation of BCL-2 in response to pro-apoptotic stimuli through the TGF-β signaling pathway.

[0039] Experimental Example 7: Effects of resveratrol and glucosamine alone and in combination on the TGF-β pathway The TGF-β signaling pathway significantly regulated by the combined drug administration group in the multi-omics study is a transmembrane signal transduction pathway, and its family members can activate downstream Smad2 and overexpress phosphorylated Smad2 (p-Smad2), thereby promoting tumor growth and metastasis. This experimental example describes the effects of resveratrol and glucosamine alone and in combination on the TGF-β pathway by detecting the gene expression of TGF-β1 and 2 and the protein expression of Smad2 and p-Smad2.

[0040] Experimental methods: PANC-1 pancreatic cancer cells were divided into control group 1, control group 2, glucosamine 2.5 mM, resveratrol 100 μM single administration group and glucosamine 2.5 mM and resveratrol 100 μM combined administration group. After three consecutive days of administration, 100 pM TGF-β1 was added to the other groups except control group 1, and total protein was extracted for 30 min. Smad2 and p-Smad2 were detected by WB method. β-actin was used as the internal reference protein, and the protein expression was analyzed according to the gray value of the band.

[0041] Experimental results: WB experiments showed that the expression of Smad2 was not affected by the outside world, which was in line with the laws of nature. Cells not stimulated by TGF-β1 did not express p-Smad2, while cells stimulated by TGF-β1 expressed a large amount of p-Smad2. Glucosamine alone did not affect the expression of p-Samd2 (cells stimulated by TGF-β1). Although resveratrol alone reduced the expression of p-Smad2 (cells stimulated by TGF-β1), the combined administration of the two reduced the expression of p-Smad2 by 1.8 times that of either alone ( Figure 7 ), achieving an unexpected regulatory effect.

[0042] Experimental Example 8: Effects of resveratrol and glucosamine alone or in combination on MAPK and apoptosis pathways The newly added significantly regulated MAPK and apoptosis pathways in the combined drug administration group in the multi-omics study play an important role in the occurrence and development of cancer. The phosphorylation of key proteins in the MAPK pathway (p-p38 protein) plays an important role in cell apoptosis, affecting the ratio of Bcl family proteins in this cell apoptosis protein. This ratio forms a "molecular switch" for cell apoptosis. The ratio of apoptosis-promoting and apoptosis-inhibiting proteins in cancer cells (Bax / Bcl-2) directly determines whether cancer cells survive. The larger the ratio, the more it promotes cancer cell apoptosis. This experimental example uses the apoptosis kit and WB to detect the Bax / Bcl-2 protein expression ratio to illustrate the effects of resveratrol and glucosamine alone and in combination on the apoptosis pathway.

[0043] Experimental methods: Cell apoptosis and its effect on Bax / Bcl-2 ratio: PANC-1 pancreatic cancer cells were divided into a control group (glucosamine 2.5 mM, resveratrol 100 μM alone) and a glucosamine 2.5 mM and resveratrol 100 μM combined administration group. After three consecutive days of administration, p-p38, BAX and Bcl-2 were detected by WB. β-actin was used as the internal reference protein, and the Bax / Bcl-2 protein expression ratio was analyzed according to the gray value of the band.

[0044] Experimental results: Compared with the control group, the p-P38 expression level of the combined drug was significantly lower than the sum of the two drugs alone, and the effect strength was enhanced by 1.3 times ( Figure 8 B), while the Bax / Bacl2 ratio increased in the single and combined administration of resveratrol and glucosamine, and the Bax / Bacl2 ratio of the combined administration was significantly higher than the sum of the two single administrations, and the effect intensity was enhanced by 1.2 times, indicating that the combined administration group had a more significant regulation of Bax / Bacl2. This shows that the combined use of the two drugs has achieved an unexpected effect on the Bax / Bacl2 ratio ( Figure 8 C).

[0045] Experimental Example 9: Preparation of Grape Seed and Deer Antler Capsules and Their Effects on the Migration and Cloning Ability of PANC-1 Pancreatic Cancer Cells Extracts of plant and animal medicines (traditional Chinese medicines or items with medicinal and edible properties) containing glucosamine and resveratrol are made into health products for preventing cancer or tumor metastasis after radiotherapy, chemotherapy and surgery.

[0046] Experimental methods: Take 200g of grape seeds and 200g of deer antler, add 4 times the amount of water and boil three times, filter, concentrate and dry under reduced pressure to get dry powder, add starch to mix and granulate, put into capsules, take 2 capsules twice a day, for preventing and treating cancer or tumor recurrence after radiotherapy and chemotherapy.

[0047] Cell invasion and cloning experiments provide a theoretical basis for the development of grape seed and deer antler capsule health products. A total of 4 groups were set up, namely the control group, high-dose, medium-dose, and low-dose groups of grape seed and deer antler capsule. PANC-1 pancreatic cancer cells in each treatment group were inoculated in the upper chamber with 200 μL serum-free culture medium (cell density: 5×10 4 100 μL of complete medium containing 20% ​​FBS was added to the lower chamber. After 24 h, the cells in the lower chamber were fixed with 4% polymethanol for 30 min and then stained with 0.1% crystal violet stain for 30 min. The pictures were taken and analyzed by ImageJ software.

[0048] After 12 h of inoculation in a six-well plate, 2 mL of the culture medium of the above-mentioned grape seed and deer antler capsules with different concentrations and the control group was added, and the cells were cultured for 48 h to prepare the cell suspension, which was then counted and inoculated into a new six-well plate (cell density: 5×10 2 The cells were fixed with 1 mL of 4% paraformaldehyde at room temperature for 30 min and then stained with 0.1% crystal violet to investigate the effect of grape seed and deer antler capsule on the PANC-1 pancreatic cancer cell clones.

[0049] Experimental results: The analysis results showed that the control group had strong cell cloning ability and more cell clusters, and the grape seed and deer antler capsule dose-dependently reduced the colony formation of PANC-1 pancreatic cancer cells ( Fig. 9 A, C), inhibited cell invasion ability ( Fig. 9 B, D), indicating that the health product can significantly inhibit the cloning ability of PANC-1 pancreatic cancer cells.

[0050] Experimental Example 10: Preparation of health products for cancer prevention or tumor metastasis prevention after radiotherapy, chemotherapy and surgery made of grape skin and antler gum extract and its effect on the migration and cloning ability of PANC-1 pancreatic cancer cells Experimental methods: Take 200g of grape peel and 200g of deer antler glue, add 4 times the amount of water and boil three times, filter, concentrate and dry under reduced pressure to get dry powder, add starch to mix and granulate, press into candy tablets, take 2 tablets twice a day, for preventing and treating cancer or tumor recurrence after radiotherapy and chemotherapy.

[0051] CCK-8 was used to detect cell viability and cell cloning experiments to provide a theoretical basis for the development of health products of grape skin and deer antler film. A total of 4 groups were set up, namely the control group, high-dose, medium-dose and low-dose groups of grape skin and deer antler film. PANC-1 pancreatic cancer cells in each treatment group were inoculated in the upper chamber with 200 μL serum-free culture medium (cell density: 5×10 4 100 μL of complete medium containing 20% ​​FBS was added to the lower chamber. After 24 h, the cells in the lower chamber were fixed with 4% polymethanol for 30 min and then stained with 0.1% crystal violet stain for 30 min. The pictures were taken and analyzed by ImageJ software.

[0052] After 12 h of inoculation in a six-well plate, 2 mL of the above-mentioned culture medium of different concentrations of grape skin and deer antler films and the control group were added, and the cells were cultured for 48 h to prepare the cell suspension, which was then counted and inoculated into a new six-well plate (cell density: 5 × 10 2 The cells were fixed with 1 mL 4% paraformaldehyde at room temperature for 30 min and then stained with 0.1% crystal violet to investigate the effect of grape skin and antler film on the PANC-1 pancreatic cancer cell clones.

[0053] Experimental results: The cells in the control group had strong cloning ability and a large number of cell clusters. Grape skin and antler film dose-dependently reduced the colony formation of PANC-1 pancreatic cancer cells ( Fig.10 A, C), inhibited cell invasion ability ( Fig.10 B, D), indicating that the health product can significantly inhibit the cloning ability of PANC-1 pancreatic cancer cells.

[0054] Experimental Example 11 The mass ratio of resveratrol and glucosamine is resveratrol:glucosamine = 2.28 mg:179.17 mg.

[0055] Experimental Example 12 The mass ratio of resveratrol to glucosamine is resveratrol: glucosamine = 45.6 mg: 895.85 mg.

[0056] Experimental Example 13 The mass ratio of resveratrol and glucosamine is resveratrol:glucosamine = 23.94 mg:537.51 mg.

[0057] Experimental Example 14: Preparation method of a composition of grape dregs extract and sea cucumber extract Take the grape skin residue and extract it with water (or alcohol) three times, each time for 1 hour. Concentrate the extract and dry it. Add starch or sucrose to the sea cucumber and make it into granular tea or pressed candy tablets.

Claims

1. A pharmaceutical composition for treating pancreatic cancer, characterized in that: The mass ratio of resveratrol and glucosamine is resveratrol:glucosamine = (2.28mg~45.6mg): (179.17mg~895.85mg).

2. Use of the drug combination set as claimed in claim 1 in preparing a product for treating pancreatic cancer or preventing pancreatic cancer metastasis.

3. The use according to claim 2, characterized in that: The product is a medicine, a health product, a food or a cosmetic.

4. The use according to claim 2, characterized in that: The concentration ratio of resveratrol to glucosamine is (10 μM~200 μM): (1 mM~5 mM).

5. The use according to claim 4, characterized in that: When used in combination, the drug concentration ratio of resveratrol and glucosamine is 100μM:2.5mM.

6. The pharmaceutical composition according to claim 1, characterized in that: Including active ingredients and excipients.

7. The pharmaceutical composition according to claim 6, characterized in that: The active ingredients are resveratrol and glucosamine, and the auxiliary materials include one or more of fillers, adhesives, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, fragrances, preservatives, dispersants, film formers, plasticizers, pore formers, opacifiers, retardants, and solvents.

8. Use of the drug combination set as claimed in claim 1 in preparing a drug for inhibiting signals of the TGF-β signaling pathway.

9. Use of the drug combination set as claimed in claim 1 in preparing a drug for inhibiting signals of a MAPK signaling pathway.

10. The product according to claim 2 comprises simultaneously or separately: a combination of natural plants or animals containing two effective ingredients, resveratrol and glucosamine, for preparing medicines and health products, foods or cosmetics for treating or preventing pancreatic cancer metastasis.

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