Medicine for resisting liver metastasis of intestinal cancer and application thereof

Abstract

The invention discloses a medicine for resisting intestinal cancer liver metastasis and application thereof, relates to an anti-cancer medicine and application thereof, and is applied to the field of medicine preparation. The invention aims to provide a low-toxicity second-line therapeutic drug after the first-line therapeutic drug for treating liver metastasis of intestinal cancer is resistant. According to the medicine, sunflower stem marrow polysaccharide is combined with intra-tumor rhodopseudomonas palustris, the material basis for inhibiting tumor proliferation is mainly the metabolite 5-aminolevulinic acid of the rhodopseudomonas palustris, and the material basis for inhibiting intestinal cancer from metastasis to liver is mainly the sunflower stem marrow polysaccharide metabolite acetic acid and long-chain fatty acid. The main mechanism for resisting intestinal cancer liver metastasis is cell apoptosis caused by protein such as ROS damaged mitochondrial membrane and the like generated by activation of 5-ALA; the main mechanism of resisting intestinal cancer liver metastasis is that long-chain fatty acid acts on DNA topoisomerase I to influence cell DNA replication. The method is applied to the pharmaceutical field.

Description

Technical Field

[0001] The present invention relates to a medicine for resisting liver metastasis of intestinal cancer and application thereof. Background Art

[0002] In recent years, the incidence of colorectal cancer has shown a rapid upward trend. With the improvement of living standards and changes in living habits, the incidence of colorectal cancer has become younger. This situation is not optimistic and must be paid attention to. We must pay more attention to prevention in our daily lives. In addition, although traditional colorectal cancer treatments can improve the five-year survival rate of patients, they cannot guarantee the quality of life of patients. Chemotherapy has serious toxic side effects on patients; radiotherapy causes strong side effects in patients; targeted therapy requires specific gene mutations and has drug resistance problems; immunotherapy has a narrow spectrum of application and can cause some complications; and gene therapy has problems such as low introduction rate and high cost. Therefore, finding a safe, effective and relatively low-cost drug is an urgent problem that needs to be solved. Summary of the Invention

[0003] The purpose of the present invention is to provide a low-toxic second-line therapeutic drug for treating colorectal cancer liver metastasis after the first-line therapeutic drug becomes resistant.

[0004] The present invention provides a drug for preventing liver metastasis of colorectal cancer, which comprises drug A and drug B. The drug A is sunflower stem pith polysaccharide, and the drug B is a resuspension of Rhodopseudomonas palustris.

[0005] This study uses sunflower stem pith as raw material, extracting crude polysaccharides from it using a water-extraction and alcohol-precipitation method and optimizing the process. Subsequently, the photosynthetic bacterium Rhodopseudomonas palustris was used to determine its ability to produce 5-ALA and its ability to target tumors, thereby confirming its biosafety. Furthermore, in vitro studies were conducted on CT26.WT cells treated with bacterial culture supernatant / 5-ALA combined with crude polysaccharides from sunflower stem pith to analyze their antiproliferative and synergistic effects. Furthermore, the in vivo therapeutic activity of Rhodopseudomonas palustris combined with crude polysaccharides from sunflower stem pith against colorectal cancer liver metastasis was evaluated using a mouse model of colorectal cancer liver metastasis.

[0006] The advantages of the present invention over the prior art are:

[0007] The drug of this invention combines sunflower stem pith polysaccharide with intratumoral Rhodopseudomonas palustris, a bacterium with tumor tissue targeting capabilities. The primary mechanism for inhibiting tumor proliferation is 5-ALA, a metabolite of Rhodopseudomonas palustris. The primary mechanism for inhibiting colorectal cancer metastasis to the liver is acetic acid and long-chain fatty acids, metabolites of sunflower stem pith polysaccharide. The mechanism of action against colorectal cancer liver metastasis is primarily due to ROS generated by 5-ALA activation, which damages mitochondrial membrane proteins and causes cell apoptosis. The mechanism of action against colorectal cancer liver metastasis is primarily due to long-chain fatty acids affecting cellular DNA replication through their action on DNA topoisomerase I. Because the combination is highly effective and low toxic, it has the potential to become a second-line drug for treating colorectal cancer liver metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The standard curves are polysaccharide (A) and protein (B);

[0009] Figure 2 Preparation and identification of intratumoral Rhodopseudomonas palustris; A is the solid culture medium for preliminary screening and the solid culture medium after strain purification; B is the mass spectrometry of the strain;

[0010] Figure 3 This is the LC / MS spectra of 5-ALA standard in positive ion mode;

[0011] Figure 4 is the positive ion mode LC / MS spectrum of the sample;

[0012] Figure 5 5-ALA standard curve (A) and 5-ALA concentration in bacterial culture supernatant (B) (n=3);

[0013] Figure 6 Effects of sunflower stem pith crude polysaccharide combined with Rhodopseudomonas palustris on body weight (A), survival (B), liver weight (C), and metastasis inhibition rate (D) in mice with colorectal cancer liver metastasis model (n=6, * P<0.05, ** P<0.01, *** P<0.001, **** P < 0.0001; Blank: blank, Saline: normal saline, HSPP: sunflower stem pith crude polysaccharide solution, RP: Rhodopseudomonas palustris, CAP: capecitabine);

[0014] Figure 7 Images of the liver after treatment with saline, sunflower stem pith polysaccharide (HSPP), intratumoral Rhodopseudomonas palustris (RP), sunflower stem pith polysaccharide combined with intratumoral Rhodopseudomonas palustris (HSPP+RP), sunflower stem pith polysaccharide combined with intratumoral Rhodopseudomonas palustris plus X-ray irradiation (HSPP+RP-X-ray), and capecitabine (CAP);

[0015] Figure 8 H&E-stained sections of the heart, liver, spleen, lung, and kidney of each group (magnification: 200x; Blank: blank, Saline: normal saline, HSPP: sunflower stem pith crude polysaccharide solution, RP: Rhodopseudomonas palustris, CAP: capecitabine);

[0016] Figure 9 H&E-stained sections of liver metastases in each group (scale: 1000 μm, circled area: metastatic tumor area; Blank: blank, Saline: normal saline, HSPP: sunflower stem pith crude polysaccharide solution, RP: Rhodopseudomonas palustris, CAP: capecitabine);

[0017] Figure 10 PCA score graphs are (A) serum, (B) liver (Blank: blank control, HSPP: sunflower stem pith polysaccharide, RP: tumor endophyte);

[0018] Figure 11 PLS-DA score graph and cross-validation and permutation test results (A) serum, (B) liver (Blank: blank control, HSPP: sunflower stem pith crude polysaccharide solution, RP: Rhodopseudomonas palustris);

[0019] Figure 12 OPLS-DA score diagram of each drug-treated group and the blank control group (HSPP: sunflower stem pith polysaccharide, RP: tumor endophyte, Serum: serum sample, Liver: liver homogenate sample);

[0020] Figure 13 The OPLS-DA permutation test results of each drug-treated group and the blank control group are shown (HSPP: sunflower stem pith polysaccharide, RP: tumor endophyte, Serum: serum sample, Liver: liver homogenate sample);

[0021] Figure 14 Volcano plot of differential metabolic screening of each drug-treated group relative to the blank control group (HSPP: sunflower stem pith polysaccharide, RP: tumor endophyte, Serum: serum sample, Liver: liver homogenate sample);

[0022] Figure 15 Cluster heatmap of each drug group relative to the blank control group (A) serum, (B) liver (H: sunflower stem pith polysaccharide, R: tumor endophyte, HR: combined use);

[0023] Figure 16 A new compound M in serum samples 212 Chemical structure of

[0024] Figure 17 A new compound M in serum samples 212 Positive ion mode LC / MS spectra; (A: total ion current; B: primary mass spectrum; C: secondary mass spectrum, m / z 377.2642);

[0025] Figure 18 is the growth inhibition rate of drugs on CT26.WT cells (48h / 72h); HSPP: sunflower stem pith crude polysaccharide solution, RPS: bacterial culture supernatant freeze-dried powder solution, 5-FU: 5-fluorouracil;

[0026] Figure 19 Effects of lyophilized powder solution of bacterial culture supernatant (RPS) and sunflower stem pith polysaccharide solution (HSPP) alone or in combination on the proliferation of CT26.WT cells (48h);

[0027] Figure 20 The effects of RPS and HSPP alone or in combination on the colony formation of CT26.WT cells (n=3, *P<0.05, **P<0.01 vs. Saline, # P<0.05 vs. 5-FU; Saline: normal saline, HSPP: (2 mg / mL) sunflower stem pith polysaccharide, RPS: (200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS-PDT: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder-(18 J / cm 2 ) Photodynamic therapy, 5-FU: (20 μM) 5-fluorouracil);

[0028] Figure 21 Figure 2 shows the cell apoptosis analysis by flow cytometry (A) and the apoptosis rate statistics (B) (n=3, **P<0.01 vs. Saline, ## P<0.01 vs. 5-FU; Saline: normal saline, HSPP: (2 mg / mL) sunflower stem pith polysaccharide, RPS: (200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS-PDT: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder -(18 J / cm 2 ) Photodynamic therapy, 5-FU: (20 μM) 5-fluorouracil);

[0029] Figure 22 Cell migration experiment (A: cell migration microscopy imaging ×100; B: cell migration rate) (n=3, **P<0.01 vs. Saline, ## P<0.01 vs. 5-FU; Saline: normal saline, HSPP: (2 mg / mL) sunflower stem pith polysaccharide, RPS: (200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS-PDT: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder -(18 J / cm 2 ) Photodynamic therapy, 5-FU: (20 μM) 5-fluorouracil);

[0030] Figure 23 High-content imaging of intracellular ROS production (scale bar: 1000 μm, Saline: physiological saline, HSPP: (2 mg / mL) sunflower stem pith polysaccharide, RPS: (200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant lyophilized powder, HSPP+RPS: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant lyophilized powder, HSPP+RPS-PDT: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant lyophilized powder-(18 J / cm 2 ) Photodynamic therapy, 5-FU: (20 μM) 5-fluorouracil);

[0031] Figure 24 Flow cytometry analysis of intracellular reactive oxygen species (A), superimposed histogram (B) and statistical analysis of mean fluorescence intensity (C) (n=3, **P<0.01 vs. HSPP+RPS, ## P<0.01 vs. 5-FU; Saline: normal saline, HSPP: (2 mg / mL) sunflower stem pith polysaccharide, RPS: (200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS-PDT: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder -(18 J / cm 2 ) Photodynamic therapy, 5-FU: (20 μM) 5-fluorouracil);

[0032] Figure 25 Flow cytometric analysis of mitochondrial membrane potential (A) Overlay histogram (B) (Saline: normal saline, HSPP: (2 mg / mL) sunflower stem pith polysaccharide, RPS: (200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS-PDT: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder-(18 J / cm 2 ) Photodynamic therapy, 5-FU: (20 μM) 5-fluorouracil).

[0033] Figure 26 Cell cycle was analyzed by flow cytometry (Saline: normal saline, HSPP: (2 mg / mL) sunflower stem pith polysaccharide, RPS: (200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS: (2 mg / mL) HSPP+(200 μg / mL) intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder, HSPP+RPS-PDT: 2 mg / mL HSPP+200 μg / mL intratumoral Rhodopseudomonas palustris culture supernatant freeze-dried powder-(18 J / cm 2 ) Photodynamic therapy, 5-FU: (20 μM) 5-fluorouracil). DETAILED DESCRIPTION

[0034] Specific embodiment 1: In this embodiment, a drug for preventing liver metastasis of colorectal cancer is divided into drug A and drug B. The drug A is sunflower stem pith polysaccharide, and drug B is a resuspension of Rhodopseudomonas palustris.

[0035] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that the method for extracting sunflower pith polysaccharide is as follows: 1. Extract dried sunflower pith powder with hot water to obtain an extract; after cooling, filter and collect the filtrate, concentrate under reduced pressure, let it stand, centrifuge, dry the resulting precipitate, and extract it three times with 95% ethanol by volume, then three times with 80% ethanol by volume, and dry it to obtain a crude sunflower pith polysaccharide sample; 2. Dissolve the crude sunflower pith polysaccharide sample in distilled water to obtain a crude sunflower pith polysaccharide solution, add Sevag reagent, shake, let it stand to allow the solution to separate, remove protein, and centrifuge. The resulting precipitate is dried and set aside to obtain sunflower pith polysaccharide. The other steps are the same as those in specific embodiment 1.

[0036] Specific embodiment 3: This embodiment differs from specific embodiment 1 or 2 in that the amount of Sevag reagent added is 1 / 4 of the volume of the sunflower stem pith crude polysaccharide solution. The other steps are the same as those of specific embodiment 1 or 2.

[0037] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the Sevag reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:1. The other steps are the same as those of specific embodiments 1 to 3.

[0038] Specific embodiment 5: The difference between this embodiment and specific embodiments 1 to 4 is that the preparation method of the resuspension of Rhodopseudomonas palustris is as follows: when the volume of the subcutaneous tumor of the tumor-bearing mouse model reaches about 400mm 3 During the experiment, a Rhodopseudomonas palustris bacterial solution was injected into the tail vein. After incubation, the tumor was crushed and homogenized. The homogenized solution was diluted with sterile PBS and then spread on TSA solid medium for anaerobic cultivation under light. A single red colony was selected for further purification and culture, and then resuspended in physiological saline to obtain a Rhodopseudomonas palustris suspension. The steps are the same as those in Specific Embodiments 1 to 4.

[0039] Specific embodiment 6: This embodiment uses the drug for treating colorectal cancer liver metastasis as a photodynamic drug.

[0040] The following examples are used to verify the beneficial effects of the present invention:

[0041] Example 1: Extraction of sunflower stem pith polysaccharides

[0042] The extraction method of sunflower stem pith polysaccharide is as follows: first, taking dried sunflower stem pith powder and extracting it with hot water, wherein the mass ratio of material to liquid is 1:40, the number of extractions is 2, the extraction temperature is 90°C, and the extraction time is 3.0h to obtain an extract; then waiting for the extract to cool, filtering and collecting the filtrate, concentrating under reduced pressure, adding anhydrous ethanol to make the overall ethanol content of the solution 80%, standing for 24h, centrifuging at 4000r / min for 10min, drying the obtained precipitate, and refluxing and extracting it with 95% by volume ethanol; third, The method comprises the following steps: first, dissolving the crude polysaccharide powder of sunflower pith in distilled water, adding Sevag reagent (1 / 4 of the solution volume), shaking for 10 minutes, standing for the solution to separate into layers, removing the milky white denatured protein and organic solvent in the interlayer, repeating the process several times until the milky white denatured protein almost ceases to precipitate, centrifuging at 4000 rpm for 10 minutes, and drying the resulting precipitate to obtain sunflower pith polysaccharide. The sunflower pith polysaccharide is prepared into a solution with a concentration of 0.1 mg / mL, measuring the absorbance at 562 nm, substituting the absorbance into the standard curve, and calculating the protein content of the crude polysaccharide solution of sunflower pith after protein removal.

[0043] The polysaccharide concentration standard curve was drawn with the polysaccharide concentration (mg / mL) as the horizontal axis and the absorbance as the vertical axis, as shown in the following figure: Figure 1 A. The experimental data were processed to obtain the regression equation: y = 60.72x + 0.02061, where R 2 =0.9996. Based on this standard curve, the concentration of polysaccharides in the solution was measured, and then the polysaccharide content in the sunflower stem pith crude polysaccharide sample was calculated. It was found that the polysaccharide content of the sunflower stem pith crude polysaccharide prepared according to the extraction conditions of 1:40 solid-liquid ratio, 90°C extraction temperature, 3.0h extraction per time, and repeated extraction twice can reach 838.15±110.06mg / g (n=3). A protein concentration standard curve was drawn with protein concentration (mg / mL) as the horizontal axis and absorbance as the vertical axis, as shown in Figure 3. Figure 1 B. The regression equation is: y = 1.041x + 0.2063, where R 2 =0.9973. Based on this standard curve, the protein concentration in the solution can be measured and the protein content of the crude polysaccharide from sunflower stem pith can be calculated. The final protein content of the crude polysaccharide sample was 108.6±9.71 mg / g. After protein removal using the Sevag method, the protein content in the crude polysaccharide sample was reduced to 17.43±1.56 mg / g (n=3). This demonstrates that the Sevag method can effectively remove free protein from crude polysaccharide from sunflower stem pith.

[0044] Example 2: Establishment of subcutaneous tumor-bearing mouse model and preparation and identification of tumor-endophytic bacteria

[0045] 1. Strain recovery and activation: Rhodopseudomonas palustris CGMCC1.167 was purchased from Guangdong Provincial Microbial Culture Collection Center. Using aseptic operation, the strain was taken out of the sealed freeze-dried tube, and the surface of the tube was wiped with alcohol cotton for disinfection. The top of the freeze-dried tube was placed on the outer flame of an alcohol lamp for rotational heating. Then, 2-3 drops of sterile distilled water were added to rupture the tube wall. 0.5 mL of the prepared TSA liquid culture medium was aspirated with a sterile pipette to fully resuspend the freeze-dried powder of the bacteria until it was completely dissolved.

[0046] Transfer the dissolved bacterial suspension to a 10mL sterile centrifuge tube, fill with culture medium, seal, and incubate in a 30°C incubator under 45W tungsten light for 3 days. After incubation, inoculate 1mL of the bacterial suspension at a 1% ratio into 100mL of sterile TSA liquid medium and culture according to the above conditions. Maintain good bacterial growth and set aside.

[0047] 2. Establishment of Subcutaneous Tumor-Bearing Mouse Model and Preparation of Tumor-Intratumoral Bacteria

[0048] Male BALB / c mice aged 4-6 weeks and weighing 18-22 g were selected and mouse colon cancer CT26.WT cells in the logarithmic growth phase were resuspended in physiological saline to adjust the cell concentration to about 5×10 6 / mL, and store in an ice water bath at 4°C until use. Urethane was dissolved in physiological saline to prepare a 15% (w / v) urethane solution. After filtering through a 0.22μm filter membrane, mice were anesthetized by intraperitoneal injection at a dose of 10mL / kg. 0.2mL of cell suspension was subcutaneously injected to establish a subcutaneous tumor-bearing mouse model. When the volume of the subcutaneous tumor in the mouse reached about 400mm 3 The concentration of Rhodopseudomonas palustris bacteria was adjusted to about 5×10 9 CFU / mL, 0.2 mL / mouse was injected into the tail vein. After 2 days, the mice were euthanized, and the subcutaneous tumors were removed under a sterile environment, cut into pieces and placed in 5 mL of sterile PBS solution for ice-water bath homogenization. After homogenization, the solution was diluted 10 times with sterile PBS.

[0049] Take 100 μL of the diluted homogenate and spread it on 1.3% TSA solid culture medium, then cover it with a layer of 2.0% TSA solid culture medium containing 0.1% sodium deoxycholate to ensure the growth advantage of the target tumor endogenous bacteria. Place the culture dish in a 30°C incubator under light and anaerobic cultivation for about 7 days. Characteristic red colonies can be observed. Pick a single red colony for further purification and culture, and store it in a 4°C refrigerator for subsequent identification and drug administration.

[0050] 3. Identification of Tumor Endophytic Bacteria

[0051] Tumor-endophytic bacteria were identified using morphological methods and microbial mass spectrometry. For mass spectrometry, samples were loaded for testing after pre-treatment. A small number of colonies were first picked with a sterile 10μL pipette tip and spread in a clockwise direction onto a metal target plate, forming a thin film on the target. After drying, the sample was covered with 1μL of formic acid, then dried again and covered with 1μL of matrix solution. After drying, the sample was tested on a microbial mass spectrometer.

[0052] The results of the preparation of intratumoral Rhodopseudomonas palustris were as follows Figure 2 As shown in (A), red and pink characteristic colonies of Rhodopseudomonas palustris grew on TSA solid medium coated with diluted mouse subcutaneous tumor homogenate. A single red colony was picked and purified to obtain intratumoral Rhodopseudomonas palustris (RP).

[0053] The purified intratumoral Rhodopseudomonas palustris was identified by microbial mass spectrometry, and the identification spectrum was as follows Figure 2As shown in (B), the identification results showed that the strain was Rhodopseudomonas palustris, a member of the genus Rhodopseudomonas. This indicated that the tumor-endophytic Rhodopseudomonas palustris was successfully prepared. The strain was expanded and preserved for in vivo bioactivity experiments.

[0054] 4. Qualitative analysis of 5-ALA production capacity of Rhodopseudomonas palustris

[0055] The ability of intratumoral Rhodopseudomonas palustris to produce 5-ALA was qualitatively analyzed using high performance liquid chromatography-mass spectrometry (HPLC-MS) in positive ion detection mode. Figure 3 and Figure 4 The total ion current, primary and secondary mass spectra of 5-ALA standard and intratumoral Rhodopseudomonas palustris culture supernatant in positive ion mode, respectively.

[0056] like Figure 3 As shown, A: total ion current; B: primary mass spectrum; C: secondary mass spectrum. Upper: m / z 132.07; lower: m / z 114.05. In positive ion mode, 5-ALA standard (M) peaks at a retention time of 0.60 min. Its primary mass spectrum mainly observes two ions, [M+H] + (m / z 132.07) and [M+H-H2O] + (m / z 114.05). Scanning with m / z 132.07 as the secondary precursor ion revealed that the m / z 132.07 ion further fragmented into [M+H-H2O] + (m / z 114.05) ion, [M+H-H2O-CO] + (m / z 86.06) ion, [M+H-H2O-CO-H2O] + (m / z 68.05) ion and [M+H-H2O-CO-CH2NH3] + (m / z55.02) and other fragment ion peaks; scanning with m / z114.05 ion as the secondary parent ion found that the fragmentation of m / z 114.05 ion was similar, and continued to fragment into [M+H-H2O-CO] + (m / z 86.06) ion, [M+H-H2O-CO-H2O] + (m / z 68.05) ion and [M+H-H2O-CO-CH2NH3] +(m / z 55.02) and other fragment ion peaks. This indicates that under certain conditions, the parent ion m / z 132.07 can first remove a H2O to obtain a product ion m / z 114.05, which may then continue to fragment to produce a neutral fragment C=O, obtaining m / z 86.06. The ion peaks m / z 68.05 and m / z 55.02 may be obtained by removing a H2O or cleavage at the β-position of the amino group at m / z 86.06, respectively.

[0057] Correspondingly, the sample results are as follows Figure 4 As shown, A: total ion current; B: primary mass spectrum; C: secondary mass spectrum. Upper: m / z 132.07; Lower: m / z 114.06. In positive ion mode, the peak with a retention time of 0.92 min is the characteristic peak of 5-ALA in the supernatant of the culture fluid of intratumoral Rhodopseudomonas palustris. Its primary mass spectrum also observed [M+H] + (m / z 132.07) and [M+H-H2O] + (m / z 114.06), and then scanned with m / z 132.07 and m / z 114.06 ions as secondary parent ions. The m / z 132.07 ion continued to fragment into [M+H-H2O] + (m / z 114.06) ion, [M+H-H2O-CO] + (m / z 86.06) ion, [M+H-H2O-CO-H2O] + (m / z 68.05) ion and [M+H-H2O-CO-CH2NH3] + (m / z 55.02) and other fragment ion peaks; m / z 114.06 ion fragmented into [M+H-H2O-CO] + (m / z 86.06) ion, [M+H-H2O-CO-H2O] + (m / z 68.05) ion and [M+H-H2O-CO-CH2NH3] + (m / z 55.02) and other fragment ion peaks. Comparison showed that the ion peak characteristics were the same as those of the 5-ALA standard, indicating that intratumoral Rhodopseudomonas palustris has the ability to produce 5-ALA.

[0058] 5. Quantitative analysis of 5-ALA production capacity of Rhodopseudomonas palustris

[0059] Drawing of 5-ALA standard curve

[0060] Preparation of 5-ALA standard solution: Accurately weigh 10 mg of 5-aminolevulinic acid hydrochloride standard into a 10 mL volumetric flask. Add water to the mark and shake well to obtain a 1.0 g / L 5-ALA reference solution. Dilute the solution with water to 1, 2, 4, 6, 8, and 10 mg / L, respectively, and shake well for later use.

[0061] Ehrlich's Reaction: Ehrlich reagent reacts with compounds containing amino groups (-NH2) to form a colorimetric product. Add 300 μL of each prepared 5-ALA standard of varying concentrations to a 1.5 mL EP tube. Add 400 μL of sodium acetate solution and 35 μL of acetylacetone to each tube, mix thoroughly, and incubate in a boiling water bath for 15 minutes. After cooling, add an equal volume of Ehrlich reagent and react for 10 minutes.

[0062] The absorbance of the solution after the reaction was measured at 554 nm using a spectrophotometer, and a protein content standard curve was drawn with 5-ALA concentration (mg / L) as the horizontal axis and absorbance as the vertical axis, as shown in the following example: Figure 5 (A) The experimental data were processed to obtain the regression equation: y = 0.09561x + 0.134, where R 2 =0.9988. Based on this standard curve, the 5-ALA content in the sample can be measured.

[0063] Determination of 5-ALA content in bacterial culture supernatant

[0064] The activated cultured tumor endobacteria suspension was inoculated into a conical flask containing 100 mL of sterile TSA liquid culture medium at a ratio of 2%, and then cultured in a constant temperature incubator at 30°C under 45W tungsten light. Samples were taken at 0, 24, 48, 72, 96, and 120 hours, centrifuged at 8000 rpm for 10 minutes, and 300 μL of the supernatant was taken. The 5-ALA content in the bacterial culture supernatant was determined according to the same method based on the above standard curve. Figure 5 As shown in (B), the 5-ALA concentrations in the supernatant of intratumoral Rhodopseudomonas palustris at 24, 48, 72, 96, and 120 h of incubation were (0.3±0.13) mg / L, (0.82±0.12) mg / L, (1.57±0.22) mg / L, (2.08±0.32) mg / L, and (1.93±0.25) mg / L, respectively (n=3). The 5-ALA content decreased slightly after 96 h, possibly due to the susceptibility of 5-ALA to degradation under prolonged light exposure.

[0065] Determination of 5-ALA content in freeze-dried powder of bacterial culture supernatant

[0066] After 72 hours of incubation, the supernatant of the bacterial culture was placed in a 10 cm disposable plastic Petri dish. The dish was left uncovered and completely wrapped with plastic wrap to secure it. A number of holes were punched in the plastic wrap using a syringe needle to allow for the passage of water vapor during freeze-drying. The dish was then placed in a -40°C freezer for 24 hours until the supernatant was completely frozen. The frozen supernatant was then freeze-dried in a vacuum freeze dryer to a powder for later use. Three batches of the prepared freeze-dried powder were reconstituted with deionized water to prepare a freeze-dried powder solution (RPS) of intratumoral Rhodopseudomonas palustris. The absorbance at 554 nm was measured and substituted into the 5-ALA standard curve equation: y = 0.09561x + 0.134. The 5-ALA content in 1.0 g of RPS was calculated to be approximately 0.04 ± 0.01 mg / mL, indicating that the culture supernatant after 72 hours of incubation contained 4 ± 1% 5-ALA (n = 3). In the above example, crude polysaccharides from sunflower stem pith were extracted using a water extraction and alcohol precipitation method. According to the extraction conditions of a solid-liquid ratio of 1:40, an extraction temperature of 90°C, an extraction time of 3.0 h, and two extractions, the polysaccharide content in the crude polysaccharide from sunflower stem pith could reach 838.15±110.06 mg / g. After deproteinization by the Sevag method, the residual protein content in the crude polysaccharide sample was 17.43±1.56 mg / g (n=3). (2) A CT26.WT subcutaneous tumor-bearing mouse model was established. After tail vein injection of the strain, the tumor was isolated and homogenized. Red characteristic colonies were screened in TSA medium containing 0.1% sodium deoxycholate. After purification and microbial mass spectrometry identification, intratumoral Rhodopseudomonas palustris (RP) was successfully prepared and preserved for in vivo experiments. (3) The ability of intratumoral Rhodopseudomonas palustris to produce 5-ALA was verified by HPLC-MS. The 5-ALA content in the supernatant after 72 h of culture was determined by spectrophotometry to be 4±1% (n=3).

[0067] Example 3: In vivo activity test of Rhodopseudomonas palustris combined with sunflower polysaccharide in inhibiting liver metastasis of colorectal cancer

[0068] Mouse colon cancer CT26.WT cells in the logarithmic growth phase were washed with sterile PBS and digested with 1 mL of trypsin (0.25%, containing EDTA). Digestion was stopped when the cells were observed to be in a single, compacted state under a microscope. 5 mL of fresh culture medium was added to terminate the digestion. The cells were pipetted repeatedly and evenly dispersed, then transferred to a centrifuge tube and centrifuged at 1200 rpm for 5 min. The supernatant was discarded and an appropriate amount of pre-cooled physiological saline was added to resuspend the cells to adjust the cell concentration to approximately 1 × 10 6 / mL, and store in an ice water bath at 4℃ until use.

[0069] The spleen implantation method was used to establish a mouse model of colorectal cancer liver metastasis. The mice used in the experiment were male BALB / c mice aged 4-6 weeks and weighing 18-22 g.

[0070] 1. Grouped medication

[0071] The mice with the established model were randomly divided into six groups, with six mice in each group, including a negative control group (Saline), a sunflower stem pith crude polysaccharide group (HSPP), a Rhodopseudomonas palustris group (RP), a Rhodopseudomonas palustris combined with sunflower stem pith crude polysaccharide group (HSPP+RP), a Rhodopseudomonas palustris combined with sunflower stem pith crude polysaccharide plus X-ray irradiation group (HSPP+RP-X-ray), and a capecitabine group (CAP). In addition, a group of mice without the established model served as a blank control group (Blank).

[0072] Drug administration began on the second day after model establishment (dose day 1). CAP and HSPP were administered by gavage, while the other drug treatments were administered by tail vein or tail vein administration plus irradiation. The dosage and frequency of administration for each group were as follows: negative control group: Saline, 0.2 mL / animal by tail vein injection every 3 days from day 1 to day 9, for a total of 3 doses; sunflower stem pith crude polysaccharide group: HSPP, 250 mg / kg, 0.2 mL / animal by gavage every day from day 1 to day 7, for a total of 7 doses; Rhodopseudomonas palustris group: RP, 5×10 9 CFU / mL, 0.2 mL / animal by tail vein injection every 3 days from 1st to 9th day, for a total of 3 doses; Rhodopseudomonas palustris combined with sunflower stem pith polysaccharide group: HSPP, 250 mg / kg, 0.2 mL / animal by oral gavage every day from 1st to 7th day, for a total of 7 doses; RP, 5×10 9 CFU / mL, 0.2 mL / animal by tail vein injection every 3 days from day 1 to day 9, for a total of 3 doses; Rhodopseudomonas palustris combined with sunflower stem pith crude polysaccharide plus X-ray irradiation group: HSPP, 250 mg / kg, 0.2 mL / animal by oral gavage every day from day 1 to day 7, for a total of 7 doses; RP, 5×10 9 CFU / mL, 0.2 mL / animal was injected into the tail vein every 3 days from day 1 to day 9, for a total of 3 doses. X-ray irradiation was also administered continuously from day 1 to day 9 at 20 mGy / animal for a total of 9 doses, for a total of 180 mGy / animal (the total radiation dose was less than 1% of the clinical chemotherapy radiation dose, so the effect of a single radiation dose on tumors was negligible). The positive control group received CAP at 60 mg / kg, 0.2 mL / animal was continuously gavaged from day 1 to day 7, for a total of 7 doses. The blank group received no treatment and was maintained normally.

[0073] 2. Analysis of weight changes

[0074] The mice were divided into groups and weighed on the day of modeling, and weighed once on the second day after modeling (first day of administration). Thereafter, the mice in the Saline group, HSPP group, RP group, HSPP+RP group, HSPP+RP-X-ray group, and CAP group were weighed once every 3 days until day 17. The weight changes of mice in each group are shown in Figure 2. Figure 6 As shown in Figure A, mice in all groups experienced weight loss during the first three days due to unavoidable trauma during surgery, including bleeding. After three days, weight recovery generally began. The Saline, HSPP, and RP groups experienced faster weight gain. The other drug-treated groups experienced slower weight gain, but still higher than the CAP group. This suggests that the toxicity and side effects of the combined drug may be less pronounced than those of the active drug, CAP. Starting on day 8 after modeling, the average weight of mice in each group began to increase to varying degrees, indicating the development of hemorrhagic ascites and increasingly pronounced abdominal distension, leading to gradual weight gain. At this time, weight gain was slower in the HSPP + RP-X-ray group, indicating a superior inhibitory effect on hemorrhagic ascites and tumor growth (n = 6, **P < 0.01 vs. HSPP + RP-X-ray).

[0075] 3. Survival Outcomes

[0076] To illustrate the therapeutic effect of the combination of the two drugs on liver metastasis of colorectal cancer (n=6, *P<0.05, **P<0.01, ****P<0.0001). The survival time of mice in each group was recorded and the Kaplan-Meier method in Prism 8.0 software was used to analyze the survival of the model mice. The results are shown in the figure. Figure 6 As shown in B: The average survival times of mice in the Saline group, HSPP group, RP group, HSPP+RP group, HSPP+RP-X-ray group and CAP group were (17.8±2.1) d, (24.2±3.5) d, (27.3±4.4) d, (32.7±2.5) d, (36.0±4.3) d and (28.2±2.5) d, respectively. Combined with the analysis of the weight change trend and survival period of each group of mice, it was observed that the single treatment groups (HSPP group and RP group) were superior to the positive control CAP group in maintaining weight stability, and their survival period was similar to that of the CAP group, suggesting that they had in vivo anti-tumor activity comparable to CAP and lower toxicity; it is worth noting that the average survival period of mice in the combined treatment groups (HSPP+RP group and HSPP+RP-X-ray group) was significantly prolonged compared with the CAP group, and the weight change trend was more stable, indicating that the combination of the two drugs had a better therapeutic effect on colorectal cancer liver metastasis and relatively low drug toxicity (n=6, **P<0.01, ****P<0.0001).

[0077] 4. Synergistic effect determination results

[0078] The average liver weight and metastasis inhibition rate of each group of mice are as follows Figure 6 As shown in Figures C and 6D, the liver weights of the Blank, Saline, HSPP, RP, HSPP+RP, HSPP+RP-X-ray, and CAP groups were (1.40±0.23) g, (7.49±0.89) g, (4.18±0.35) g, (4.98±0.68) g, (2.92±0.28) g, (2.60±0.46) g, and (4.29±0.43) g, respectively. Liver weight analysis showed that compared with the Saline group, the mean liver weights of mice in all treatment groups decreased to varying degrees (n=6, P<0.01 vs. Saline), suggesting that both single and combined treatments have a certain inhibitory effect on the growth of colorectal cancer liver metastases. Notably, among the single-drug groups, the HSPP group demonstrated a significant anti-metastatic effect in vivo, with the smallest mean liver weight, even lower than the positive control CAP group (n=6, P<0.05 vs. CAP), suggesting that HSPP may have a strong inhibitory activity against colorectal cancer liver metastasis in vivo. Furthermore, the combined drug groups demonstrated an even more significant metastasis-inhibiting effect, with mean liver weights in the HSPP+RP and HSPP+RP-X-ray groups significantly lower than those in the CAP group (n=6, P<0.05 vs. CAP). Correspondingly, metastasis inhibition analysis showed that the metastasis inhibition rates in the HSPP, RP, HSPP + RP, HSPP + RP-X-ray, and CAP groups were 54.30±5.81%, 41.16±11.12%, 75.05±4.67%, 80.25±7.52%, and 52.49±6.99% groups, respectively. The HSPP + RP-X-ray group significantly outperformed the CAP group (n=6, P<0.01 vs. CAP). This trend is consistent with the results of prolonged mean survival and reduced mean liver weight, suggesting that the combined use of HSPP and RP may synergistically more effectively inhibit the progression of liver metastases. Inhibition rate (%) = (liver weight of negative control group - liver weight of drug-treated group) / (liver weight of negative control group - liver weight of blank group) × 100%.

[0079] The King's formula is used to evaluate whether the combined drug has a synergistic effect in in vivo experiments. The formula is as follows:

[0080]

[0081] Ea, Eb, and Ea+b represent the metastasis inhibition rates of the HSPP group, RP group, and combined administration group on mice with liver metastasis models of colorectal cancer, respectively. The drug interaction effect was evaluated by the synergy index q value. When q > 1.15, it indicated a synergistic effect when used in combination; when 0.85 < q < 1.15, it indicated an additive effect when used in combination; when q < 0.85, it indicated an antagonistic effect when used in combination.

[0082] The interaction patterns of different treatment regimens were calculated and analyzed by the King's formula. The results showed that when HSPP and the RP group were used in combination, they had an additive anti-tumor effect (q = 1.094, 0.85 < q < 1.15); while when the HSPP + RP - X-ray group was used in combination, they had a synergistic anti-tumor effect (q = 1.167, q > 1.15). This synergistic effect was manifested in the treatment results as follows: compared with the positive control CAP group, the average survival time of the HSPP + RP - X-ray group was significantly prolonged (n = 6, P < 0.01, vs. CAP), the change in mouse body weight was more stable (n = 6, P < 0.01, vs. Saline), the average liver weight was lower and the number of metastatic foci was less (n = 6, P < 0.05, vs. CAP).

[0083] 5. Observation of liver specimens

[0084] Taking the Saline group as the negative control and the CAP group as the positive control, the distribution of liver metastatic nodules in each group was as Figure 7 shown.

[0085] Compared with the mice in the blank group, the liver of the Saline group showed typical metastatic pathological features, with grayish-white tumor nodules of different sizes densely distributed on the surface. Due to the infiltration of metastatic foci, the liver volume was larger than that of the blank group and the texture of the organ was hardened.

[0086] It is worth noting that both the HSPP group and the RP group inhibited the number of liver metastatic tumor nodules to varying degrees, but the forms they showed were different. The metastatic foci in the HSPP group were often smaller and floating on the liver surface; the metastatic foci in the RP group were larger and deeper inside the liver, indicating that their mechanisms of action in vivo may be different. The number of metastatic foci in the HSPP + RP group was less than that in the single-drug group, and the HSPP + RP - X-ray group further improved the treatment effect. Its metastatic foci were significantly fewer than those in the single-drug administration group and the positive control group, greatly improving the pathological condition and quality of life of mice with liver metastasis of colorectal cancer, proving that the combination of HSPP and RP has good in vivo anti-liver metastasis activity against colorectal cancer.

[0087] 6. Results of drug toxicity analysis

[0088] The hearts, livers, spleens, lungs, and kidneys of mice in each group were stained with H&E to investigate the safety of HSPP and RP used alone or in combination. The experimental results were as Figure 8As shown. No obvious lesions were found in the hearts and lungs of mice in each group. The liver of the Saline group was severely damaged due to the invasion of CT26.WT cells, and the spleen lymphocytes of the CAP group increased, indicating that the mice in the CAP group had congestive splenomegaly. In addition, the renal glomeruli in the CAP group showed internal congestion, and some renal glomeruli only retained their outlines, indicating that the CAP group may cause kidney damage. Comparing the pathological results of various tissues and organs in the Blank group, it can be seen that the tissue sections of HSPP combined with RP or RP-X-ray did not show severe damage. It is speculated that the two drugs will not cause organic lesions in various organs of mice when used alone or in combination.

[0089] 7. HE staining of liver pathological sections

[0090] H&E staining and panoramic scanning of tissue sections were used to observe the morphological changes of liver metastases in the Blank, Saline, HSPP, RP, HSPP+RP, HSPP+RP-X-ray, and CAP groups. Figure 9 .

[0091] Liver tissue sections in the Saline group showed a large number of metastatic CT26.WT cells, and the resulting metastatic lesions were the largest, indicating very severe liver metastasis from colorectal cancer. In the HSPP group, the number of CT26.WT cell aggregates was reduced compared to the Saline group, and the overall area of ​​the metastatic lesions was smaller. In the RP group, the metastatic lesions were larger in area, but the number of metastatic lesions was smaller. The number of metastatic CT26.WT cells and the overall area of ​​metastatic lesions in the HSPP combined with RP or RP-X-ray groups were smaller than in the single-drug group. Furthermore, the HSPP + RP-X-ray group was able to further reduce the number and area of ​​metastatic lesions compared to the positive control CAP group. Therefore, the combined drug therapy has a certain inhibitory effect on the metastasis of CT26.WT cells, and the results of pathological sections are consistent with the liver metastasis inhibition rate and the observation of liver tumor nodules.

[0092] 8. Study on the toxicity of intratumoral Rhodopseudomonas palustris

[0093] The mice in the RP group maintained good survival throughout the entire dosing cycle, and no mouse deaths were observed during the dosing cycle. Whole blood collected from the mice after the last dose was analyzed using a veterinary fully automatic blood cell analyzer. The analysis results are shown in Table 1. Compared with the blank control group, the number of immune-related cells such as white blood cells (WBC), neutrophils (NE), and lymphocytes (LY) in the RP group showed a moderate increase. This phenomenon is completely consistent with the characteristics of live bacteria administration stimulating the body's nonspecific immune response. However, all blood cell indicators are still within the normal reference range, indicating that long-term, multi-dose administration has little effect on the survival of the mice and their blood routine indicators. It is inferred that RP administration via the tail vein route has reliable in vivo safety.

[0094] The above results indicate that HSPP combined with RP or RP-X-ray significantly inhibits colorectal cancer liver metastasis through synergistic enhancement, and the toxic side effects are lower than the positive control.

[0095] Table 1

[0096]

[0097] Example 4 Metabolomics study on the inhibition of colorectal cancer liver metastasis by intratumoral Rhodopseudomonas palustris combined with sunflower stem pith polysaccharide

[0098] In this study, 3-nitrophenylhydrazine (3-NPH) was derivatized with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). HPLC-MS was used to detect differential metabolites in mouse serum and liver. Spectral and peak area screening was combined with principal component analysis and orthogonal partial least squares-discriminant analysis to identify differentially regulated metabolites after administration, clarifying the material basis for the anti-liver metastasis effect of HSPP combined with RP. MetaboAnalyst 6.0 (https: / / www.metaboanalyst.ca / ) was used to analyze the metabolites of the initially screened mouse serum and liver homogenate samples. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were first performed to detect intergroup distribution differences and visualize the overall distribution trends of the samples. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was then used to separate the samples and predict the clusters. This allowed for better comparison of the degree of discrete clustering between the treated and blank groups to assess intergroup variability. The significantly upregulated differential metabolites in mouse serum and liver were screened using a concentration-fold change (FC) > 10.0 relative to the blank control group, P < 0.05, and a variable importance projection (VIP) > 1. Volcano plots and cluster heat maps were constructed to visually display the changes in metabolites.

[0099] 1. Principal Component Analysis (PCA)

[0100] After modeling, 100 μL of serum from each group of mice was added to 1 mL of 70% acetonitrile (ACN) aqueous solution and homogenized in an ice-water bath. After vortex mixing at 1000 rpm for 3 minutes, the mixture was centrifuged at 10,000 rpm for 15 minutes. 100 μL of the supernatant was placed in a reaction tube, 50 μL of 3-NPH (200 mM) and 25 μL of EDC (120 mM) were added, and the mixture was reacted in a 60°C water bath for 30 minutes. The mixture was cooled to room temperature, filtered through a 0.22 μm filter, and refrigerated until further use. Mouse serum derivatization samples were obtained. 50 mg of liver samples from each group of mice were added to 500 μL of 70% acetonitrile (ACN) aqueous solution and homogenized in an ice-water bath. The samples were then derivatized as described above and refrigerated until further use. This yielded liver homogenate derivatization samples.

[0101] Non-targeted metabolomics analysis was performed on the serum-derivative samples and liver homogenate-derivative samples of the blank control group (Blank), the single-drug group (HSPP, RP), and the combined-drug group (HSPP+RP). First, the data were dimensionality reduced by unsupervised principal component analysis (PCA) to obtain the corresponding score map ( Figure 10 ), which directly reflects the original distribution state of each group of samples at the metabolic level and shows the differences between different groups of samples. Figure 10 As shown, the blank control group and each drug-treated group exhibited clear separation on the PC1-PC2 plane in mouse serum and liver samples. The scatter between the HSPP and RP groups was also significant, indicating significant differences in metabolites between the two groups, suggesting that the two monotherapy regimens may act through different metabolic pathways. Acetic acid was the most influential principal component in serum samples, with significantly higher levels in the HSPP and HSPP+RP groups compared to the Blank group (P<0.01). 5-ALA was the most influential principal component in liver samples, with significantly higher levels in the RP and HSPP+RP groups compared to the Blank group (P<0.01).

[0102] 2. Partial Least Squares-Discriminant Analysis (PLS-DA)

[0103] Compared with PCA, PLS-DA is a supervised discriminant method, such as Figure 11 As shown in the figure, the horizontal and vertical axes in PLS-DA represent the scores of the first and second principal components, respectively. Clear separation between the drug-treated groups and the blank control group is evident in both serum and liver samples. The separation between the HSPP and RP groups also indicates significant differences in the levels of different metabolites after drug administration. 5-ALA has the highest variable projected importance (VIP) value in both serum and liver samples, indicating that it is the primary component influencing classification.

[0104] In order to prevent the analysis model from overfitting, the analysis adopted a cross-validation method and performed a 100-reaction permutation test on the model. Figure 11 , when the number of components is 5, the serum sample R 2 =0.955; liver sample R 2 =0.969, both greater than 0.9. The result of the model permutation test is P<0.01, which proves that the quality of the PLS-DA model is good and there is no overfitting phenomenon. The next step of analysis can be carried out.

[0105] 3. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA)

[0106] In order to further verify the differences between the samples of each drug group and the blank control group, the single drug group (HSPP, RP) and the combined drug group (HSPP+RP) were subjected to supervised OPLS-DA analysis with the blank control group (Blank). Figure 12 As shown, the horizontal axis represents the predicted principal component score of the first principal component, representing the inter-group differences of the samples. The greater the inter-group differences, the longer the horizontal distance of the samples. The vertical axis represents the scores of the orthogonal principal components, representing the intra-group differences of the samples. The smaller the intra-group differences, the shorter the vertical distance. It can be observed that each drug-treated group showed significant differences from the blank control group, and the drug-treated group samples contained metabolites with significantly increased levels. This demonstrates that the HSPP group, the RP group, and the HSPP + RP group all have significantly different metabolites compared to the blank control group. The VIP values ​​of the differential metabolites between each drug-treated group and the blank control group were obtained and used as an important indicator for screening significantly differential metabolites.

[0107] In order to prevent the analysis model from overfitting, the analysis adopted a cross-validation method and performed a 100-reaction permutation test on the model. Figure 13 As shown, where R 2 Y and Q 2 The values ​​of are all greater than 0.85, and the P value of the permutation test is 2 Y and Q 2 The results were all less than 0.05, which showed that the quality of the OPLS-DA model was good and there was no overfitting phenomenon, so the next step of analysis could be carried out.

[0108] 4. Screening of significantly different metabolites

[0109] (I) Results of drawing metabolic difference volcano map and cluster heat map

[0110] In order to further accurately identify the differential metabolites between the drug-treated group and the blank control group, multivariate statistical analysis was applied to perform a t-test on the data to obtain the P value. The VIP values ​​obtained by OPLS-DA were classified and screened under the conditions of P < 0.05 and VIP > 1 to obtain the differential metabolites between the groups. In order to more intuitively show the trend of differential metabolites, a metabolic difference volcano diagram was drawn based on the peak area of ​​the substances screened in the LC-MS data. Figure 14 As shown, the horizontal axis Log2 (FC) is the fold change of each substance in this group compared with the blank control group, and the vertical axis -Log10 (P) is reflected by the P value of the t test. Red represents significantly upregulated metabolites.

[0111] Similarly, draw a cluster heat map, such as Figure 15 As shown, the color change of each component in the heat map is determined by the value of Log2(FC), and dark red represents significantly upregulated metabolites.

[0112] (II) Analysis results of significant metabolites

[0113] Using the volcano plot and cluster heat map as reference, the significantly upregulated differential metabolites were screened based on the criteria of fold change (FC) greater than 10.0, VIP>1, and P<0.05 in the metabolite concentration of the drug group relative to the blank control group. As shown in Tables 2 and 3, it can be concluded that in serum samples, adrenic acid (Adrenic Acid) and docosahexaenoic acid (DHA) in the HSPP group were significantly upregulated; the 5-ALA and DHA contents in the RP group and the combination group were significantly upregulated; in liver samples, the stearic acid (Stearic Acid), linolenic acid (Linolenic Acid), oleic acid (Oleic acid), myristic acid (Myristic acid), arachidonic acid (ARA), and DHA contents in the HSPP group were all significantly upregulated; the 5-ALA, DHA, and ARA contents in the RP group and the combination group were all significantly upregulated.

[0114] Table 2 List of significantly upregulated metabolites with concentrations increased more than 10-fold (serum)

[0115]

[0116] Table 3 List of significantly upregulated metabolites with concentrations increased more than 10-fold (liver)

[0117]

[0118] (III) Identification results of new compounds in differentially metabolized products

[0119] The screening and identification of new compounds in the differentially metabolized products were performed using samples without derivatization and then subjected to HPLC-MS detection. After collecting sample data from different batches of mouse blood, the negative control group was used as a blank control and the system software Compound Discoverer was used for screening. The screening conditions were set as "mass error <5ppm, peak area ≥10 7 , matching degree ≥85”, and 6 unique compounds were screened out from the blood samples of mice in the combined drug group, among which compound M 212 It was identified as a new compound (retention time = 15.308 min, peak area = 11048739). Figure 16 , shown in 17, compound M 212 The molecular weight is C 23 H 36 O4, m / z 377.2642 is its [M+H] + The molecular ion peak of , the deviation between its theoretical value and the measured value is 4.7ppm. 212 After breaking the O-CH2 bond and the C2-C3 bond, an ion peak of m / z 60.0813 (C2H3O2+H + ), M 212 After breaking the CO bond in the ester, an ion peak of m / z 74.064 (C3H5O2+H + ). It can be seen from the secondary mass spectrum that C 13 -C 14 Bond rupture means removal of C7H 13 The O group (113.3177) was then hydrogenated to generate a group with m / z 264.9525; C 12 -C 13 The bond breaks and the oxygen atom in propylene oxide is removed to generate a group with m / z 232.9270; the group with m / z 232.9270 is further dehydrated to generate an ion peak with m / z 214.9167; M 212 After breaking the CO bond in the ester, an ion peak of m / z 74.064 (C3H5O2+H + ), the ion peak is added with water and depleted with hydrogen (C3H5O2+H + +H2O-H) is the peak at m / z 90.9767.

[0120] The compound information (CCCCCC1OC1C\C=C / C\C=C / C\C=C / CCC(=O)OC1COC1) was substituted into the Pubchem online database for search, but no chemical structure matching the compound was found. The new compound M was predicted based on the online reverse fishing target database (https: / / prediction.charite.de / ) 212 The targets and therapeutic diseases of IL-67 are shown in Table 4 below.

[0121] Table 4 Predicted in vivo targets

[0122]

[0123]

[0124] M 212 Among the first 41 targets, 26 are related to tumors, especially the target DNA topoisomerase I, which is closely related to the treatment of colon cancer and metastatic colon cancer. 212 It is one of the material bases of anti-tumor activity.

[0125] This example demonstrates that the material basis for the anti-colonial liver metastasis activity of HSPP combined with RP includes DHA, ARA, and 5-ALA. DHA can inhibit tumors by enhancing immune cell function and inhibiting invasion and migration pathways, while ARA can modulate inflammation and the immune microenvironment through its metabolites, and can promote or inhibit metastasis under specific conditions. 5-ALA, as a photosensitizer, exerts anti-tumor effects by absorbing light energy and entering an excited state.

[0126] Example 5: In vitro activity test of Rhodopseudomonas palustris combined with sunflower polysaccharide in inhibiting liver metastasis of colorectal cancer

[0127] 1. Cytotoxicity assay

[0128] The MTT colorimetric method was used to determine the inhibitory effect of sunflower stem pith polysaccharide (HSPP) and Rhodopseudomonas palustris culture supernatant freeze-dried powder (RPS) on the proliferation of CT26.WT cells in a short period of time.

[0129] Select cells in good growth condition, wash them when they have grown to more than 80% of the culture flask area, add 1 mL of 0.25% trypsin to digest the cells, add fresh cell culture medium to resuspend the cells and centrifuge at 1200 rpm for 5 minutes, add appropriate amount of fresh culture medium to resuspend the cells and adjust the cell concentration to 5×10 4 Cells / mL. 200 μL of cell suspension was inoculated into each well of a 96-well plate. 200 μL of sterile PBS was added to the outermost wells of the plate to prevent cell evaporation. The 96-well plate was placed in a CO2 cell incubator (37°C, 5% CO2). Cell growth was observed after 24 hours. Dosing was initiated when the cell density per well reached 70% or higher.

[0130] Sunflower stem pith polysaccharide (HSPP), Rhodopseudomonas palustris culture supernatant freeze-dried powder (RPS), and 5-FU were gradiently diluted and added to a 96-well plate to obtain the following concentrations: RPS (50μg / mL, 100μg / mL, 200μg / mL, 400μg / mL, 600μg / mL), HSPP (1mg / mL, 2mg / mL, 4mg / mL, 8mg / mL, 10mg / mL), 5-FU (5μM, 10μM, 20μM, 30μM, 40μM), and a blank control group (Saline). Each treatment group was set up with 3 replicates. After administration, the 96-well plate was placed in a CO2 cell culture incubator (37°C, 5% CO2) and cultured for 48h and 72h, respectively.

[0131] In a separate 96-well plate, cells were seeded using the same method. Combination therapy was then administered. Based on preliminary studies and the results of cell proliferation inhibition with either drug alone, the HSPP concentration was fixed at 2 mg / mL, and the RPS concentrations were varied to 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 600 μg / mL. A blank control group (Saline) was established, and three replicates were set for each treatment group. After drug administration, the 96-well plate was placed in a CO2 cell culture incubator (37°C, 5% CO2) and incubated for 48 hours.

[0132] Remove the 96-well plate, aspirate the culture medium from each well with a pipette, add 200 μL of fresh culture medium and 20 μL of MTT solution (5 mg / mL) to each well, and continue incubating in a CO2 cell culture incubator for 4 hours. Remove the 96-well plate again, aspirate the supernatant from each well, add 150 μL of dimethyl sulfoxide to each well, and shake on a horizontal shaker for 5-10 minutes.

[0133] The growth inhibitory effect of different concentrations of monomers on CT26.WT cells at 48 h and 72 h was determined, and the half-maximal inhibitory concentration (IC50) of each drug was calculated using Prism 8 software. 50 ), the results are shown in Table 5. At the same time, the cell activity curve was drawn, as shown in Figure 18 As shown in the figure, the inhibitory effect of HSPP on CT26.WT cells increased in a dose-dependent manner at 48 and 72 hours, but the inhibitory effect was not significant, requiring high doses or prolonged treatment to achieve an inhibitory effect of more than 50% on CT26.WT cells. RPS had a stronger inhibitory effect on CT26.WT cells, achieving an inhibition rate of more than 50% within 48 hours and at lower dosage concentrations.

[0134] Table 5 Cytotoxicity of drugs

[0135]

[0136] 2. Results of drug synergistic inhibition of cell proliferation and calculation of the combination index (CI)

[0137] Based on the anti-proliferative effects of different concentrations of the drug on CT26.WT cells when acting alone, 2 mg / mL HSPP was combined with RPS to investigate the synergistic anti-proliferative effect of Rhodopseudomonas palustris combined with sunflower stem pith polysaccharide on CT26.WT cells.

[0138] First, different concentrations of RPS and a fixed concentration of 2 mg / mL HSPP were used to treat CT26.WT cells for 48 h. The effects on cell proliferation were detected by MTT assay, and the half-maximal inhibitory concentration (IC50) of RPS after the combination was calculated using Prism 8 software. 50 ), and the combination index (CI) was calculated using Compu Syn 1.0 software.

[0139] As shown in Table 6, Figure 19 As shown in the figure, compared with the anti-proliferative ability of drugs alone on CT26.WT cells, combined use of drugs can further enhance the inhibitory effect. At this time, the IC 50 The value was 105.1 μg / mL, and the anti-tumor activity was increased to about 1.5 times that of the single use.

[0140] The CI values ​​of RPS and HSPP (2 mg / mL) co-administered at different ratios are shown in Table 6. Figure 19 As shown in Figure B, when the RPS concentration exceeds 200 μg / mL, the CI is less than 1, indicating that the combination has a synergistic anti-tumor effect. Furthermore, as the RPS ratio increases, the combination index decreases, and the synergistic effect increases. This suggests that appropriately increasing the RPS ratio during combined use can enhance the anti-tumor effect.

[0141] Table 6 Combination index of combined drugs (48h)

[0142]

[0143] 3. Cell colony formation assay

[0144] The plate colony formation assay was used to determine the ability of sunflower stem pith polysaccharide (HSPP) and Rhodopseudomonas palustris culture supernatant freeze-dried powder solution (RPS) to inhibit the proliferation of CT26.WT cells for a long time.

[0145] The effects of RPS and HSPP alone or in combination on CT26.WT cell proliferation over a longer period of time were investigated by colony formation experiments. The fewer colonies formed, the stronger the long-term inhibitory effect of the drug on CT26.WT cells. Figure 20As shown, after 10 days of treatment, the Saline group, HSPP group, RPS group, HSPP+RPS group, and HSPP+RPS-PDT group [PDT: after drug administration, a low-intensity 647 nm red light excitation light source that does not damage cells was used for 3 min (100 mW, total light dose 18 J / cm 2 The average colony numbers of the HSPP and 5-FU groups were 91.33±7.09, 76.00±4.58, 38.33±5.13, 20.00±5.57, 12.66±4.04, and 28.66±3.05, respectively. A large number of colonies were formed in the negative control wells, while the number of colonies decreased in the single or combined drug groups. The effect of the HSPP+RPS-PDT group was significantly greater than that of the single drug group and the positive control group (n=3, *P<0.05, **P<0.01 vs. Saline, # P < 0.05 vs. 5-FU), which proves that the combined administration of HSPP and RPS also has a good activity in inhibiting the proliferation of CT26.WT cells over a longer period of time.

[0146] 4. Cell apoptosis experiment

[0147] Annexin V-FITC and PI dyes were used for double staining and analyzed on a flow cytometer. The third quadrant belongs to the range of normal viable cells (Annexin V-FITC negative, PI negative); the fourth quadrant belongs to the range of early apoptotic cells, indicating that the cell membrane begins to be damaged (Annexin V-FITC positive, PI negative); the first quadrant belongs to the range of late apoptotic or completely dead cells (Annexin V-FITC positive, PI positive); and the fourth quadrant belongs to the range of cells with only the nucleus left due to cell membrane rupture caused by external damage (Annexin V-FITC negative, PI positive). Figure 21 As shown, 24 hours after administration, the apoptosis rates of cells in the Saline group, HSPP group, RPS group, HSPP+RPS group, HSPP+RPS-PDT group, and 5-FU group were 2.96±1.38%, 37.26±3.12%, 42.09±2.73%, 44.69±2.47%, 63.29±2.89%, and 54.70±1.56%, respectively. Among them, apoptosis in the HSPP group was mainly manifested in early apoptosis; apoptosis in the RPS group was mainly manifested in late apoptosis; the increase in apoptosis rate in the HSPP+RPS group was not significant compared with RPS alone, which indicates that HSPP may not be a cytotoxic drug, and its anti-intestinal cancer liver metastasis activity has little to do with promoting apoptosis. The apoptosis rate in the HSPP+RPS-PDT group was significantly increased and exceeded that of the positive control group 5-FU (n=3, ##P<0.01 vs. 5-FU), which may be related to the activation of 5-ALA, the main metabolite contained in RPS.

[0148] 5. Transwell migration assay

[0149] Image J software was used to count the number of migrated cells and calculate the migration rate: Migration rate (%) = (number of migrated cells in the blank group - number of migrated cells in the drug group) / number of migrated cells in the blank group × 100%.

[0150] The experimental results are as follows Figure 22 As shown in the results, 24 hours after administration, the cell migration rates of the HSPP group, RPS group, HSPP+RPS group, HSPP+RPS-PDT group, and 5-FU group were 37.21±6.45%, 40.28±6.42%, 26.61±4.61%, 26.42±7.28%, and 54.14±7.91%, respectively. The migration rates of all the treatment groups were lower than those of the 5-FU group. The HSPP group was able to achieve a significantly lower migration rate than the 5-FU group at a low cytotoxic dose (n=3, ## However, when the two were combined and illuminated, the migration rate of CT26.WT cells did not decrease significantly compared to the combined treatment, suggesting that 5-ALA, a major metabolite of RP, may not be significantly involved in the inhibition of migration. Therefore, it is speculated that HSPP, when administered in combination, primarily inhibits colon cancer cell metastasis. This is consistent with the results of in vivo activity experiments showing smaller liver metastases and lighter liver weights in mice treated with HSPP.

[0151] The in vitro experimental results of this example show that HSPP and RPS inhibit CT26.WT proliferation and induce apoptosis through synergistic action, wherein 5-ALA photodynamic activation can enhance this effect, but the anti-migratory mechanism is independent of the 5-ALA pathway.

[0152] Example 6: Exploring the mechanism of HSPP combined with RP in inhibiting liver metastasis of colorectal cancer based on metabolomics data

[0153] 1. High-content imaging analysis

[0154] A high-content imaging analysis system was used to detect the production of reactive oxygen species in CT26.WT cells 24 hours after administration to each drug group. The DCFH-DA probe itself has no fluorescence and can freely cross the cell membrane. After entering the cell, it can be hydrolyzed by the esterase in the cell to produce DCFH. DCFH cannot pass through the cell membrane, allowing the probe to be loaded into the cell. The reactive oxygen species in the cell can oxidize the non-fluorescent DCFH to produce DCF with green fluorescence. The fluorescence of DCF can be detected to know the level of reactive oxygen species in the cell. The high-content imaging results are as follows: Figure 23As shown, no significant fluorescence was observed in the Saline and HSPP groups, with high-content imaging analysis detecting mean fluorescence intensities within the field of view of 385±37 and 448±45, respectively. A certain degree of fluorescence was observed in both the PRS and HSPP+RPS groups, with high-content imaging analysis detecting mean fluorescence intensities within the field of view of 1110±116 and 1154±177, respectively. The most pronounced fluorescence signal was observed in the HSPP+RPS-PDT group, with a mean fluorescence intensity within the field of view of 7953±302, significantly higher than that in the 5-FU group (1673±282) and the other treatment groups (n=3, P<0.01).

[0155] High-content imaging analysis revealed significant production of reactive oxygen species (ROS) in CT26.WT cells following administration of HSPP + RPS-PDT. It is hypothesized that the mechanism by which HSPP combined with RP inhibits colorectal cancer liver metastasis may involve activation of RP's primary metabolite, 5-ALA, leading to the production of ROS that kill tumor cells. This is consistent with the metabolomics findings showing significant upregulation of 5-ALA levels in both the RP and combination groups, and also explains why the HSPP combined with RP-X-ray showed greater anti-colorectal cancer liver metastasis activity than the RP combined group in in vivo activity experiments.

[0156] 2. Reactive oxygen species (ROS) content analysis results

[0157] Flow cytometry was used to analyze the ROS content produced in CT26.WT cells 24 hours after administration of each drug group. The analysis results are as follows: Figure 24 As shown, the trends in the reactive oxygen species levels in each group were consistent with the results of high-content imaging. The mean fluorescence intensities of cells in the Saline, HSPP, RPS, HSPP+RPS, HSPP+RPS-PDT, and 5-FU groups were 2127.8±164.2, 3787.3±470, 11627.3±1799.3, 13623±1269.8, 76364.6±2831, and 15825.6±1598.8, respectively. The reactive oxygen species levels in the HSPP+RPS-PDT group were significantly higher than those in the HSPP+RPS and 5-FU groups (n=3, **P<0.01 vs. HSPP+RPS, ## P < 0.01 vs. 5-FU). The results once again illustrate that one of the mechanisms of HSPP combined with RP in the treatment of colorectal cancer liver metastasis is that the main metabolic component of RP, 5-ALA, is activated by X-ray or infrared light to produce reactive oxygen species, thereby killing tumor cells.

[0158] 3. Mitochondrial membrane potential test results

[0159] The mitochondrial membrane potential of cells was detected by Rhodamine 123 fluorescent probe to explore the inhibitory effect of each drug group on mitochondria. When the mitochondrial membrane potential is normal, it can accumulate in the mitochondria to produce yellow-green fluorescence. When the mitochondrial membrane potential is lost or decreased, Rhodamine 123 will be released by the cells. The fluorescence intensity is detected by the 488 / 525nm channel of the flow cytometer to reflect the mitochondrial membrane potential. The test results are as follows: Figure 25 As shown, the abscissa represents fluorescence intensity, and the ordinate represents the number of cells at the corresponding fluorescence intensity. Overall, the data are divided into two peaks: the peak with lower fluorescence intensity corresponds to abnormal cells with impaired mitochondrial membrane potential, while the peak with higher fluorescence intensity corresponds to cells with normal mitochondrial membrane potential. Twenty-four hours after administration, the percentages of cells with impaired membrane potential were 34.1%, 37.39%, 42.74%, 44.14%, 71.78%, and 45.69% in the saline, HSPP, RPS, HSPP+RPS, HSPP+RPS-PDT, and 5-FU groups, respectively. The percentage of cells with abnormal membrane potential in the HSPP+RPS-PDT group was significantly higher than in the HSPP+RPS and 5-FU groups (P < 0.01). This suggests that activation of 5-ALA, a major metabolite of RP, can produce free radicals that damage mitochondria, leading to a decrease in mitochondrial membrane potential.

[0160] Reactive oxygen species (ROS) and mitochondrial membrane potential detection showed that the ROS level in the HSPP+RPS-PDT group was significantly higher than that in the other groups (P<0.01), and the proportion of cells with abnormal mitochondrial membrane potential was 71.78%, indicating that 5-ALA induced cell apoptosis by damaging mitochondria through ROS after activation, explaining the phenomenon of enhanced anti-tumor activity under light activation.

[0161] 3. Cell cycle detection

[0162] The results of cell cycle detection showed that ( Figure 26), the proportions of G1, S and G2 phases of CT26.WT cells in each group 24 hours after administration were: Saline group (44.67%, 14.58% and 40.30%), HSPP group (76.03%, 4.72% and 19.16%), RPS group (69.48%, 8.99% and 21.34%), HSPP+RPS group (77.18%, 6.72% and 15.92%), HSPP+RPS-PDT group (78.57%, 6.37% and 14.96%), and 5-FU group (75.90%, 21.67% and 2.26%). HSPP significantly arrested CT26.WT cells in the G1 phase (76.03%) and reduced the proportions of the S phase (4.72%) and G2 phase (19.16%) (P<0.01 vs. Saline), while there was no significant difference in the cycle distribution between the HSPP+RPS group and the HSPP+RPS-PDT group (P>0.05), indicating that the target of 5-ALA is weakly related to DNA replication.

[0163] 4. Molecular docking

[0164] To investigate the interactions between DHA, ARA, and 5-ALA and their respective targets, molecular docking was performed using AutoDockVina software. The docking results showed that the three compounds could enter the binding domains of different target proteins, primarily through hydrogen bonding and hydrophobic interactions. Lower docking binding energy values ​​indicate stronger binding between the compound and the protein target.

[0165] The results showed that DHA and ARA bound to nuclear factor κB subunit p105 with ΔG of -4.9 kcal / mol and -3.9 kcal / mol, respectively; bound to DNA topoisomerase I with ΔG of -6.1 kcal / mol and -5.7 kcal / mol, respectively; 5-ALA bound to nuclear factor κB subunit p105 and DNA-(apurinic or apyrimidinic site) lyase with ΔG of -4.8 kcal / mol and -3.8 kcal / mol, respectively, revealing its molecular mechanism of inhibiting tumor replication and metastasis.

[0166] In summary, it can be seen that the core mechanism of HSPP combined with RP in inhibiting colorectal cancer proliferation is that ROS generated by 5-ALA activation damages mitochondria and induces apoptosis; the anti-liver metastasis mechanism is that HSPP and / or its metabolite fatty acids target the nuclear factor κB subunit p105 protein, inhibiting the migration and invasion of colorectal cancer.

Claims

1. A drug for treating colorectal cancer liver metastasis, characterized in that The medicine is divided into medicine A and medicine B, medicine A is sunflower stem pith polysaccharide, and medicine B is a heavy suspension of Rhodopseudomonas palustris.

2. The drug for treating colorectal cancer liver metastasis according to claim 1, characterized in that: The method for extracting sunflower stem pith polysaccharide comprises the following steps:

1. extracting dried sunflower stem pith powder through hot water to obtain an extract; filtering and collecting the filtrate after cooling, concentrating under reduced pressure, standing, centrifuging, drying the obtained precipitate, extracting it three times with 95% by volume ethanol through reflux, and then extracting it three times with 80% by volume ethanol through reflux, and drying to obtain a crude sunflower stem pith polysaccharide sample; 2. dissolving the crude sunflower stem pith polysaccharide sample with distilled water to obtain a crude sunflower stem pith polysaccharide solution, adding Sevag reagent, shaking, standing to wait for the solution to separate, removing protein and centrifuging, drying the obtained precipitate for later use, and obtaining sunflower stem pith polysaccharide.

3. The drug for treating colorectal cancer liver metastasis according to claim 2, characterized in that: The amount of Sevag reagent added is 1 / 4 of the volume of the crude polysaccharide solution of sunflower stem pith.

4. A drug for treating colorectal cancer liver metastasis according to claim 2 or 3, characterized in that: Sevag reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:

1.

5. The drug for treating colorectal cancer liver metastasis according to claim 1, characterized in that: The preparation method of the resuspension of Rhodopseudomonas palustris is as follows: when the volume of the subcutaneous tumor of the tumor-bearing mouse model reaches about 400 mm 3 When the tumor was grown, the tail vein was injected with a liquid of Rhodopseudomonas palustris. After culture, the tumor was crushed and homogenized. The homogenized liquid was diluted with sterile PBS and then spread on TSA solid culture medium for light anaerobism culture. A single red colony was picked for further purification and culture, and then resuspended with physiological saline to obtain a resuspension of Rhodopseudomonas palustris.

6. Use of the drug according to claim 1 as a photodynamic drug for treating liver metastasis of colorectal cancer.

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