Inonotus obliquus acidic polysaccharide IOP-2 with thyroid cancer resisting activity and application of inonotus obliquus acidic polysaccharide IOP-2

Abstract

The invention discloses inonotus obliquus acidic polysaccharide IOP-2 with thyroid cancer resisting activity and application of the inonotus obliquus acidic polysaccharide IOP-2, and belongs to the field of biological medicine. According to the invention, a method for extracting inonotus obliquus polysaccharide by boiling water and precipitating the inonotus obliquus polysaccharide by ethanol is adopted, a DEAE cellulose column purification technology is combined, and the principle that macromolecular polysaccharide is insoluble in an organic solvent is utilized, so that the separation of the inonotus obliquus polysaccharide is successfully realized, and the inonotus obliquus acidic polysaccharide IOP-2 with thyroid cancer resisting activity is obtained; the monosaccharide of the inonotus obliquus acidic polysaccharide IOP-2 is prepared from the following components in percentage by mole: 0.71 percent of fucose, 3.33 percent of rhamnose, 2.18 percent of arabinose, 2.92 percent of galactose, 80.59 percent of glucose, 3.46 percent of xylan and 6.81 percent of mannose. In-vivo and in-vitro experiments prove that the traditional Chinese medicine composition has a remarkable thyroid cancer resisting effect, and a novel treatment method is provided for clinical treatment of thyroid cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an acidic polysaccharide IOP-2 from Inonotus obliquus with anti-thyroid cancer activity and its applications. Background Technology

[0002] Inonotus obliquus, also known as Chaga mushroom, is a unique fungus with a long history of traditional Chinese medicine. It has attracted significant attention in modern scientific research due to its high levels of anti-tumor and antioxidant activity, as well as its various bioactive components (polysaccharides, triterpenes, polyphenols, etc.). Existing research reveals its potential in immunomodulation, anti-inflammation, anti-tumor activity (at the cellular and animal levels), and hypoglycemia. Many studies have investigated tumor cells using fungal monomers or extracts, finding that the active components of the fungus can inhibit tumor cell proliferation and differentiation and promote cancer cell apoptosis, demonstrating certain value in clinical treatment. However, different active components of the same fungus exhibit significantly different therapeutic effects on different types of tumors due to their chemical structure, mechanism of action, and the heterogeneity of tumor cells. For example, triterpenoids in Ganoderma lucidum (such as ganoderic acid) can combat liver and breast cancer by inducing apoptosis and inhibiting angiogenesis, while polysaccharides (such as β-glucan) enhance immune surveillance against lung or colon cancer by activating immune cells (such as macrophages and NK cells). Similarly, cordycepin in Cordyceps sinensis selectively inhibits the proliferation of leukemia cells by interfering with RNA synthesis, but its adenosine components may be more effective against solid tumors (such as gliomas) because they can penetrate the blood-brain barrier and regulate the tumor microenvironment. Furthermore, protein-bound polysaccharides (PSK) in Trametes versicolor have been shown to enhance the sensitivity of chemotherapeutic drugs to gastric cancer, while another component, Trametes versicolor glycopeptide (PSP), inhibits cervical cancer progression by regulating the Th1 / Th2 immune balance. This difference is also reflected in pharmacokinetic properties: lipid-soluble components (such as sterols in fungi) are more likely to accumulate in adipose tissues such as the breast or prostate, while water-soluble components (such as polysaccharides) are at higher concentrations in organs with rich blood flow, such as the liver and kidneys. Therefore, the specificity of the antitumor effects of fungi depends not only on the molecular targets of the components (such as topoisomerase inhibition and epigenetic regulation) but also on the molecular subtype of the tumor (such as the sensitivity of HER2-positive breast cancer to fungal terpenes). This complexity suggests that the antitumor effects of fungal active ingredients require more high-quality, large-scale experimental data to confirm in order to achieve precision treatment.

[0003] Thyroid cancer is a malignant tumor originating from thyroid tissue (mainly follicular epithelial cells or parafollicular C cells). The thyroid gland, located in the front of the neck on either side of the trachea, is shaped like a butterfly and is an important endocrine organ responsible for secreting thyroid hormones, regulating key physiological processes such as metabolism, growth, and development. In recent years, the incidence of thyroid cancer has shown a significant upward trend, with a relatively young age of onset, making it one of the more common malignant tumors. Current research has found that various monomeric components of traditional Chinese medicine have shown potential in laboratory studies against thyroid cancer, such as flavonoids (baicalin, wogonin, and quercetin), saponins (ginsenosides and notoginseng saponins), polyphenols (curcumin and resveratrol), and alkaloids (berberine and tripterygium wilfordii). These small-molecule active ingredients mainly work through mechanisms such as inhibiting proliferation, inducing apoptosis, inhibiting metastasis, and angiogenesis. Currently, these components are mainly being explored as potential adjuvant therapies or candidate molecules for new drug development. However, the focus of basic research is on finding more effective and safer drugs, and overcoming the challenges of existing treatments (such as radioactive iodine resistance). The antitumor effects of Inonotus obliquus (especially its active ingredient, Inonotus obliquus polysaccharide) on thyroid cancer have not yet yielded experimental results, and no relevant reports have been published, warranting further investigation. Summary of the Invention

[0004] The purpose of this invention is to provide an acidic polysaccharide IOP-2 from Inonotus obliquus with anti-thyroid cancer activity and its applications, thereby addressing the problems existing in the prior art. This invention isolates and obtains an acidic polysaccharide IOP-2 from Inonotus obliquus with anti-non-small cell lung cancer activity. In vitro and in vivo experiments have verified its significant anti-thyroid cancer effect, providing a new treatment method for the clinical treatment of thyroid cancer.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an Acidic Polysaccharide IOP-2 from Inonotus obliquus with anti-thyroid cancer activity. The monosaccharide composition of the Acidic Polysaccharide IOP-2 from Inonotus obliquus, by molar percentage, includes fucose 0.71%, rhamnose 3.33%, arabinose 2.18%, galactose 2.92%, glucose 80.59%, xylan 3.46%, and mannose 6.81%.

[0007] The preparation method includes the following steps:

[0008] After ultra-fine pulverization of the fruiting bodies of Inonotus obliquus, the mixture was soaked in ethanol, boiled with distilled water, and the extract was concentrated after filtration. The supernatant was collected after ultra-high speed centrifugation, precipitated with ethanol solution, centrifuged again, and the precipitate was collected. The precipitate was subjected to DEAE cellulose column chromatography, eluted successively with double-distilled water, 0.1 mol / L and 0.3 mol / L NaCl solutions, and the 0.3 M NaCl solution eluent was collected to obtain the Inonotus obliquus polysaccharide IOP-2.

[0009] When the supernatant is precipitated with an ethanol solution, the volume concentration of ethanol in the system is 80%.

[0010] Furthermore, the distilled water is added according to a material-to-liquid ratio of fruiting body powder: distilled water = 1g: 50mL.

[0011] Furthermore, the conditions for ultra-high speed centrifugation are 9000 rpm for 10 min.

[0012] Furthermore, the conditions for the second centrifugation are 8000 rpm for 15 min.

[0013] The present invention also provides the use of the aforementioned Inonotus obliquus acidic polysaccharide IOP-2 in the preparation of a medicament for treating thyroid cancer.

[0014] The present invention also provides a drug for treating thyroid cancer, the active ingredient of which includes the aforementioned Inonotus obliquus acidic polysaccharide IOP-2.

[0015] The present invention discloses the following technical effects:

[0016] This invention employs boiling water extraction and ethanol precipitation of Inonotus obliquus polysaccharides, combined with DEAE cellulose column purification technology. Utilizing the principle that macromolecular polysaccharides are insoluble in organic solvents, the invention successfully separated Inonotus obliquus polysaccharides and obtained an acidic polysaccharide, IOP-2, from Inonotus obliquus with anti-thyroid cancer activity.

[0017] This invention, through cell experiments, confirmed that the acidic polysaccharide IOP-2 from Inonotus obliquus has an inhibitory effect on thyroid cancer cells. Further analysis revealed that IOP-2 inhibits the occurrence and development of thyroid cancer by regulating the Bax / Bcl-2 / Caspase-3 mitochondrial apoptosis pathway. This invention provides a novel treatment method for the clinical treatment of thyroid cancer. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Here is the molecular configuration diagram of IOP-2;

[0020] Figure 2 The results show the molecular weight and uniformity of IOP-2.

[0021] Figure 3 The results of monosaccharide composition analysis of IOP-2;

[0022] Figure 4 The image shows the results of FT-IR detection of functional groups in IOP-2;

[0023] Figure 5 The effect of crude polysaccharide IOP from Inonotus obliquus on the inhibitory activity of four types of tumor cells;

[0024] Figure 6 The effects of IOP-W, IOP-1, and IOP-2 (1000 μg / mL, 48 h) on the morphology of thyroid cancer C643 cells were investigated.

[0025] Figure 7 The effects of IOP-W, IOP-1, and IOP-2 on the inhibitory activity of thyroid cancer C643 cells on proliferation;

[0026] Figure 8 The effect of IOP-2 (1000 μg / mL, 48 h) on the nuclear morphology of C643 thyroid cancer cells;

[0027] Figure 9 The effect of IOP-2 (1000 μg / mL, 48 h) on apoptosis in thyroid cancer C643 cells;

[0028] Figure 10 The effect of IOP-2 on tumor growth in C643 thyroid cancer-bearing mice;

[0029] Figure 11 The effect of IOP-2 on the survival status of C643 thyroid cancer-bearing mice; where A represents average body weight; B represents food intake; and C represents water intake.

[0030] Figure 12 To detect the blood supply around the tumor in tumor-bearing mice after IOP-2 administration using a laser speckle system;

[0031] Figure 13 To investigate the effect of IOP-2 on the expression of mitochondrial apoptosis-related proteins in tumor tissues of thyroid cancer-bearing mice. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] The Inonotus obliquus was collected from the Changbai Mountain area of ​​Jilin Province and identified as Inonotus obliquus by Professor Liu Yang of Jilin Agricultural University.

[0038] Other biological materials included thyroid cancer cell line C643 (CL-0648, Pronosei Biotechnology Co., Ltd.), liver cancer cell line HepG2 (CL-0627, Pronosei Biotechnology Co., Ltd.), lung cancer cell line NCI-H460 (CL-0299, Pronosei Biotechnology Co., Ltd.), pancreatic cancer cell line MIAPaCa-2 (CL-0627, Pronosei Biotechnology Co., Ltd.), fetal bovine serum (164210, Pronosei Biotechnology Co., Ltd.), and matching culture medium 1640 (PM150110, Pronosei Biotechnology Co., Ltd., China). DAPI fluorescent staining solution (KGA1808-50, Jiangsu Kaiji Biotechnology Co., Ltd., China) and Annexin V-FITC / PI double staining apoptosis detection kit (KGA1102-100, Jiangsu Kaiji Biotechnology Co., Ltd., China) were also used. Immunofluorescence and protein antibodies were also provided (ABclonal Biotechnology Co., Ltd., Wuhan, China).

[0039] Example 1: Extraction, separation and molecular weight determination of Inonotus obliquus polysaccharide IOP-2

[0040] 1. Extraction and separation of Inonotus obliquus polysaccharide IOP-2

[0041] The fruiting bodies of *Inonotus obliquus* were ultra-finely pulverized, and the powder was soaked in ethanol for 3 days. Then, distilled water was added at a ratio of fruiting body powder to distilled water of 1 g: 50 mL, and the mixture was heated to boiling point for extraction. This extraction was repeated 5 times, 2 hours each time. The extracts were combined, filtered twice through a filter cloth, concentrated to 1 / 5 of the original volume, and centrifuged at 9000 rpm for 10 min. The supernatant was retained for later use. Four times the volume of 95% ethanol was added to the supernatant to precipitate the ethanol, bringing the final concentration to 80%. The mixture was stirred thoroughly with a glass rod and allowed to stand overnight at 4°C for ethanol precipitation. The next day, it was centrifuged at 9000 rpm for 15 min, and the precipitate was collected. The precipitate was dissolved in a small amount of water, freeze-dried, and used to obtain the crude polysaccharide IOP.

[0042] 500 mg of crude polysaccharide was fully dissolved and subjected to DEAE cellulose DE-52 ion exchange column chromatography. Double-distilled water, 0.1 mol / L, and 0.3 mol / L NaCl solutions were used as eluents successively, maintaining a stable flow rate. An automated sampler was used to collect the eluents, yielding three fractions: IOP-W, IOP-1, and IOP-2. The sugar content in each tube of the automated sampler was determined using the phenol-sulfuric acid method, and fractions with the same composition were combined. After lyophilization, molecular sieve chromatography was performed on a G200 dextran gel column to finally obtain lyophilized powders of the three fractions. IOP-W and IOP-1 have been disclosed in Chinese patents with authorization publication numbers CN118615316B and CN119350519B, respectively.

[0043] 2. Identification of the molecular weight, monosaccharide composition, and structure of IOP-2

[0044] (1) Molecular configuration of IOP-2

[0045] After purification by G200 dextran gel column chromatography, molecular conformation analysis revealed that the slope of IOP-2 was 0.03 ± 0.00, indicating that the molecular conformation of the Inonotus obliquus acidic polysaccharide IOP-2 tends to be spherical. Figure 1 .

[0046] (2) Polysaccharide molecular weight detection

[0047] The molecular weight of polysaccharides was determined by gel permeation chromatography (GPC). A Wyatt Sec-Mals Dawn Heleos-II laser detector and a differential refractive index detector were used for sample detection. The chromatographic column was a Shodex OHpak SB-806, with 0.1 M sodium sulfate and 0.2% sodium azide as the mobile phase, and the flow rate was set to 0.5 mL / min. The concentration of the polysaccharide sample was 2 mg / mL, and the total injection volume was 500 μL. Dextran 40000 was used as a reference standard for normalization, and the run time was set to 35 min. Finally, the absolute molecular weights of the four polysaccharides in this study were calculated using the principle of light scattering.

[0048] The results are as follows Figure 2 The molecular weight of Inonotus obliquus polysaccharide IOP-2 is 55.1822 kDa.

[0049] (3) Determination of monosaccharide composition

[0050] The monosaccharide composition of polysaccharide samples was determined by high performance liquid chromatography. The monosaccharide standards included mannose, rhamnose, glucuronic acid, glucose, arabinose, galacturonic acid, galactose, xylose, and fucose.

[0051] Before the experiment, the solution was prepared. The detailed steps are as follows:

[0052] Preparation of Standards: Take a clean 15mL centrifuge tube, add 8mL of sterile water, and then add 100mg each of fucose, arabinose, glucose, fructose, galactose, ribose, xylose, mannose, galacturonic acid, glucuronic acid, and mannuronic acid in sequence. After dissolving, bring the volume to 10mL to prepare a 10mg / mL mixed standard stock solution. Dilute the above mixed standard stock solution 100 times to prepare a 100μg / mL working solution. Take the above solution and dilute it according to the following gradient, then fill it into 1.5mL EP tubes.

[0053] Sample pretreatment: Weigh 5 mg of polysaccharide sample, add the prepared 2M TFA solution, and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Wash with methanol, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water, transfer to a clean chromatographic vial, and await instrumental analysis.

[0054] The chromatographic detection system used was a Thermo ICS5000+ ion chromatography system (Thermo Fisher Scientific, USA), employing Dionex... TM CarboPac TM A PA20 liquid chromatography column was used, with an injection volume of 20 μL. Mobile phase A (H2O) and mobile phase B (100 mM NaOH) were used. The column temperature was set at 30 °C, and the monosaccharide components were analyzed and detected using an electrochemical detector.

[0055] Monosaccharide composition analysis revealed that IOP-2 comprises fucose, rhamnose, arabinose, galactose, glucose, xylan, and mannose, accounting for 0.71%, 3.33%, 2.18%, 2.92%, 80.59%, 3.46%, and 6.81% of the total molar percentage of IOP-2, respectively. Figure 3 ).

[0056] (4) Structural identification of IOP-2

[0057] The main functional groups present in IOP-2 were detected by infrared spectroscopy to understand its structural characteristics. FT-IR analysis revealed a peak at 3416.76 cm⁻¹. -1 The presence of a distinct strong absorption peak indicates the existence of intermolecular and internal hydrogen bonds, a typical characteristic of hydroxyl stretching vibrations. 3134.2 cm⁻¹ -1 The absorption peak at 2918.25 cm⁻¹ further confirms the hydrogen bonding effect of the hydroxyl groups on the sugar chain. -1 The absorption peak at 1617.02 cm⁻¹ corresponds to the stretching vibration of the CH bond. -1 The absorption peak at 1263.62 cm⁻¹ indicates the presence of C=O stretching vibration, suggesting that IOP-2 may contain acid-linked dextran. -1 The absorption peak at 1067.41 cm⁻¹ is due to the angular vibration absorption of the methyl group (CH) attached to the carboxyl group. -1 The absorption peak corresponds to the stretching vibration of the hydroxyl group in the pyranose ring, indicating that the monosaccharide exists in the form of pyranose. 619.52 cm⁻¹ -1 The absorption peak at this point indicates that the glycosidic bond in the sugar chain may be in the β-configuration, which is one of the typical characteristics of polysaccharide structure. Figure 4 ).

[0058] Example 2: Antitumor activity of Inonotus obliquus polysaccharide IOP-2

[0059] 1. Inhibitory activity of crude polysaccharide from Inonotus obliquus against different tumor cell types

[0060] Four tumor cell lines were selected: HepG2 liver cancer cells, NCI-H460 lung cancer cells, MIAPaCa-2 pancreatic cancer cells, and C643 thyroid cancer cells. The effect of crude polysaccharide IOP from Inonotus obliquus on the proliferation ability of tumor cells was detected by the CCK-8 assay.

[0061] Cell lines and cell culture: HepG2 liver cancer cells, NCI-H460 lung cancer cells, MIAPaCa-2 pancreatic cancer cells, and C643 thyroid cancer cells were cultured at 37°C under constant temperature and humidity and 5% carbon dioxide in RPMI-1640 (containing 100 μg / mL streptomycin and 100 units / mL penicillin G) and 10% fetal bovine serum, respectively. Cells were passaged 1:3 and cultured continuously after reaching a confluence of 90%.

[0062] Cell proliferation assay: HepG2 liver cancer cells, NCI-H460 lung cancer cells, MIAPaCa-2 pancreatic cancer cells, and C643 thyroid cancer cells (all 2×10⁻⁶ cells) were detected. 3 Cells were cultured in 96-well plates (cells / well) for 24 hours. The original culture medium was then replaced with a drug-treated medium containing different concentrations of crude polysaccharide IOP (0, 500, and 1000 μg / mL). Negative, positive, and blank controls were included: negative control cells were cultured normally without any drugs; positive control cells were treated with doxorubicin hydrochloride (DOX) instead of Inonotus obliquus polysaccharide (DOX) at a dose of 5 μM; blank control cells were not seeded and no drugs were added. After 24 hours of seeding, C643 cells were co-incubated with 5 μM DOX and different concentrations of IOP (0, 500, and 1000 μg / mL). Cell viability was assessed using a CCK-8 assay kit after 48 hours of co-incubation. The CCK-8 reagent was added to the culture medium at a 1:10 ratio, and the cells were incubated at 37°C for 30 minutes. The absorbance (A) at 450 nm was calculated using the following formula:

[0063] Proliferation inhibition rate (%) = ([A) 阴性对照 -A 实验组 ] / [A 阴性对照 -A 空白对照 ])×100%.

[0064] Each of the above groups has 5 replicate wells, and the experiment is repeated at least 3 times in parallel.

[0065] The results are as follows Figure 5IOP exhibited inhibitory activity against the proliferation of four tumor cell lines, but showed the strongest inhibitory effect (48h) against thyroid cancer cells C643, reaching 85.42±4.91%. The inhibition rates against liver cancer cells HepG2, lung cancer cells NCI-H460, and pancreatic cancer cells MIAPaCa-2 were 39.82±3.46%, 42.31±4.61%, and 59.24±6.87%, respectively. This indicates that IOP's inhibitory effect on thyroid cancer cells C643 is significantly superior to that against other tumor cell lines. Further in-depth research will be conducted using thyroid cancer cells C643.

[0066] 2. Inhibitory activity of different Inonotus obliquus polysaccharides against tumor cells

[0067] Cell line and cell culture: Thyroid cancer cells C643 were cultured at 37°C under constant temperature and humidity in RPMI-1640 (containing 100 μg / mL streptomycin and 100 units / mL penicillin G) and 10% fetal bovine serum, with a carbon dioxide concentration of 5%. Cells were passaged 1:3 and cultured continuously once the confluence reached 90%.

[0068] Cell proliferation assay: C643 cells (2×10⁻⁶) 3 Cells were cultured in 96-well plates (cells / well) for 24 hours. The original culture medium was then replaced with a medium containing different concentrations of IOP-W, IOP-1, and IOP-2 (0, 500, and 1000 μg / mL). Negative controls, positive controls, and blank controls were included: negative control cells were cultured normally without any drugs; positive control cells were treated with doxorubicin hydrochloride (DOX) instead of Inonotus obliquus polysaccharide, with a DOX dose of 5 μM; blank control cells were not seeded and no drugs were added. After 24 hours of seeding, C643 cells were co-incubated with 5 μM DOX and different concentrations of IOP-W, IOP-1, and IOP-2 (0, 500, and 1000 μg / mL). Cell viability was assessed using a CCK-8 assay kit after 24 and 48 hours of co-incubation. The CCK-8 reagent was added to the culture medium at a 1:10 ratio, and the cells were incubated at 37°C for 30 minutes. The absorbance (A) at 450 nm was calculated using the following formula:

[0069] Proliferation inhibition rate (%) = ([A) 阴性对照 -A 实验组 ] / [A 阴性对照 -A 空白对照 ])×100%.

[0070] Each of the above groups has 5 replicate wells, and the experiment is repeated at least 3 times in parallel.

[0071] Microscopic observation after 48 hours of co-incubation with IOP-2 showed that the C643 cells co-cultured with IOP-2 exhibited abnormal morphology, including shrinkage and fragmentation, and a significantly reduced cell density. Figure 6The effects of each isolated and purified IOP component on the inhibitory activity of thyroid cancer C643 were further examined using the CCK-8 assay (48 h). The results are as follows: Figure 7 The results showed that IOP-2 exhibited the best activity, with a proliferation inhibition rate of 87.89±2.76% at a concentration of 1000 μg / mL, which was significantly higher than that of the two polysaccharide components IOP-W and IOP-1. Furthermore, the antitumor activity of all three components was concentration-dependent.

[0072] 3. IOP-2 significantly induced apoptosis in C643 cells.

[0073] To verify the effect of IOP-2 on apoptosis of thyroid cancer C643 cells, DAPI fluorescence staining was performed using IOP-2 at concentrations of 500 and 1000 μg / mL, respectively.

[0074] DAPI fluorescence staining experiment: C643 cells were stained with 1×10⁻⁶ DAPI fluorescence staining assay. 4 Cells were seeded at a density of 100 cells / well in culture dishes containing culture medium and incubated for 24 hours. Subsequently, the original medium was replaced with drug-treated medium, and the cells were co-incubated with IOP-2 for 24 and 48 hours. Afterward, the cells were washed with PBS, fixed with methanol, and washed again. 80 μL of LDAPI was added to each well, stained in the dark for 5 minutes, and then washed once with PBS. Finally, images were taken using a Nikon inverted fluorescence microscope (100× and 200× magnification), with 5 images retained for each drug concentration.

[0075] The results showed that after IOP-2 administration, the morphology of C643 cell nuclei began to change, with chromatin aggregation, nuclear pyknosis, and the appearance of bright blue apoptotic bodies. The number of apoptotic bodies in the experimental group was significantly higher than that in the control group, and increased with increasing drug concentration (see [link to study]). Figure 8 ).

[0076] Inducing apoptosis is an important pathway to inhibit tumor cell proliferation. Flow cytometry analysis of the apoptosis rate:

[0077] C643(2×10 5 Cells (1 cell / well) were cultured in RPMI-1640 medium for 24 h, then replaced with drug-treated medium. After co-incubating with IOP-1 (1000 μg / mL) for 48 h, the cells were washed twice with PBS, and reagents were added according to instructions. The mixture was thoroughly mixed and incubated at room temperature in the dark for 5–15 min. The experiment was repeated at least three times.

[0078] The results are as follows Figure 9When C643 cells were treated with 1000 μg / mL IOP-2, the number of viable cells decreased significantly, the apoptosis rate changed significantly (P<0.05), and the total apoptosis rate increased significantly, reaching a maximum of 18.92±2.19%. Nuclear fluorescence staining and flow cytometry results indicated that IOP-2 could significantly induce apoptosis in C643 cells.

[0079] Example 3: Effects of Inonotus obliquus polysaccharide IOP-2 on thyroid cancer-bearing mice

[0080] 1. Tumor-bearing mouse modeling and drug administration

[0081] Male C57BL / 6 mice aged 4–6 weeks were randomly divided into groups of 5. Each group received an inoculation of C643 cells (10-10) every 7 days via the right axilla. 7 Mice were vaccinated for 3 consecutive weeks. Starting in the 4th week, they were treated with IOP-2 (400 mg / kg polysaccharide concentration) once daily by gavage for 28 consecutive days. The control group received saline by gavage. Food intake, water consumption, and body weight were measured weekly. Tumor size was monitored every 2 weeks using small animal MRI. Tumor growth was tracked using a small animal MRI system. Body weight was measured 72 hours after the last administration, and the mice were then sacrificed. After confirmation of death, the mice were dissected, and the tumor tissue was completely removed.

[0082] During the modeling period, the survival status, body weight, food intake, and tumor size of tumor-bearing mice were monitored. Results are as follows: Figure 10 After 4 weeks of treatment, mice in the control group were administered physiological saline by gavage, and the tumors in these mice significantly increased in size, from 33.645 mm. 2 Reduced to 67.834mm 2 In contrast, the tumor volume in mice treated with IOP-2 was significantly reduced, from 46.011 mm. 2 Reduced to 29.539mm 2 The above results indicate that IOP-2 can inhibit the proliferation of C643 tumor cells in tumor-bearing mice. Furthermore, observation of the mice's survival status revealed that the average body weight and food and water intake of the control group decreased as the tumor progressed, while all indicators of the treated group increased. These results suggest that IOP-2 not only inhibits tumor growth in tumor-bearing mice but also effectively improves their survival status, demonstrating significant therapeutic effects. Figure 11 As shown.

[0083] 2. Effects of IOP-2 on blood flow around tumor tissue in tumor-bearing mice

[0084] The blood supply around tumor tissue in tumor-bearing mice was detected using a laser speckle blood flow imaging system. The results showed that before treatment, the blood supply around the tumor tissue was abundant, and the laser intensity values ​​were relatively high (215.00±37.97 in the control group and 221.05±24.10 in the treatment group). After treatment, as the tumor shrank, the blood flow around the tumor tissue decreased, with values ​​of 231.45±21.75 in the control group and 194.72±12.98 in the treatment group (P<0.05). This indicates that the blood flow around the tumor tissue in the IOP-2 group was reduced, which may be related to the reduction in tumor volume. (See attached figures). Figure 12 See Table 1.

[0085] Table 1. Effects of IOP-2 on blood supply around tumor tissue in thyroid cancer-bearing mice.

[0086] control group IOP-2 Group 0 weeks 215.00±37.97 221.05±24.10 4 weeks 231.45±21.75 194.72±12.98*

[0087] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0088] 3. IOP-2 affects the expression of apoptosis-related genes in tumor tissues of C643 thyroid cancer-bearing mice.

[0089] Based on the interaction between IOP-2 and C643 cells described above, combined with animal experiments, it was found that IOP-2 can inhibit the proliferation and apoptosis of C643 cells and suppress tumor growth, which may be achieved by regulating multiple genes and proteins. Therefore, apoptosis gene Q-PCR was performed on tumor tissues extracted from tumor-bearing mice to detect the expression levels of apoptosis-related genes Bcl-2, Bax, and Caspase-3 in the tumor, elucidating its anti-tumor mechanism. The results are shown in Table 2. Compared with the control, the expression levels of apoptosis-related genes Bcl-2, Bax, and Caspase-3 in the tumor were significantly altered. Specifically, the expression of the pro-apoptotic gene Bax was significantly increased, while Bcl-2 was significantly decreased, and the expression of Caspase-3 was also significantly increased.

[0090] Table 2. Effects of IOP-2 on the expression of apoptosis-related genes in tumor tissues of thyroid cancer-bearing mice.

[0091] Control IOP-2 Bcl-2 1 0.72±0.13* Bax 1 3.15±1.02** Caspase-3 1 3.98±2.14**

[0092] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0093] 4. IOP-2 affects the expression of mitochondrial apoptosis-related proteins in tumor tissues of C643 thyroid cancer-bearing mice.

[0094] Based on the Q-PCR results, the proteins Bax, Bcl-2, and Caspase-3, which are closely related to mitochondrial apoptosis, were first detected.

[0095] Western blotting: Cell samples were collected from each culture group, and protein samples were extracted and analyzed using Western blotting. After electrophoresis, protein samples were transferred to polyvinylidene fluoride (PVDF) membranes for 1.5 hours. The membranes were then blocked with skim milk for 2 hours, incubated overnight with primary antibody, washed four times with PBST, and co-incubated with secondary antibody for 2 hours. Finally, ECL was used for color development, and protein expression was visualized and quantitatively analyzed using a Tanon-5200 imaging system (Tanon, China).

[0096] The results showed a significant increase in the pro-apoptotic protein Bax, while Bcl-2 significantly decreased, and Caspase-3 expression also significantly increased, indicating an increased trend of mitochondrial apoptosis. Western Blot results are as follows: Figure 13 See Table 3. The above results indicate that IOP-2-induced apoptosis in C643 cells may be related to the regulation of the expression of proteins involved in mitochondrial apoptosis, such as Bax, Bcl-2, and Caspase-3.

[0097] Table 3. Analysis of mitochondrial apoptosis-related protein expression in tumor tissues of thyroid cancer-bearing mice using IOP-2.

[0098] Control IOP-2 Bcl-2 1 0.32±0.25* Bax 1 4.26±1.78** Caspase-3 1 5.81±1.92***

[0099] Note: Mean ± standard deviation, n≥3; *P<0.05; **P<0.01; ***P<0.001 compared with the control group.

[0100] The above examples demonstrate that the IOP-2 component of Inonotus obliquus acidic polysaccharide has significant inhibitory activity against C643 thyroid cancer cells, and the antitumor activity of IOP-2 was confirmed in vivo in tumor-bearing mice. Preliminary mechanistic studies have explored that IOP-2 inhibits the development and progression of thyroid cancer by regulating the Bax / Bcl-2 / Caspase-3 mitochondrial apoptosis pathway. In conclusion, IOP-2 provides a novel treatment option for the clinical treatment of thyroid cancer.

[0101] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An acidic polysaccharide IOP-2 from Inonotus obliquus with anti-thyroid cancer activity, characterized in that, The monosaccharide composition of the Inonotus obliquus acidic polysaccharide IOP-2, in molar percentage, includes fucose 0.71%, rhamnose 3.33%, arabinose 2.18%, galactose 2.92%, glucose 80.59%, xylan 3.46%, and mannose 6.81%. The preparation method includes the following steps: After ultra-fine pulverization of the fruiting bodies of Inonotus obliquus, the mixture was soaked in ethanol, boiled with distilled water, and the extract was concentrated after filtration. The supernatant was collected after ultra-high speed centrifugation, precipitated with ethanol solution, centrifuged again, and the precipitate was collected. The precipitate was subjected to DEAE cellulose column chromatography, eluted successively with double-distilled water, 0.1 mol / L and 0.3 mol / L NaCl solutions, and the 0.3 M NaCl solution eluent was collected to obtain the Inonotus obliquus polysaccharide IOP-2. When the supernatant is precipitated with an ethanol solution, the volume concentration of ethanol in the system is 80%.

2. The Birch Inonotus acidic polysaccharide IOP-2 according to claim 1, characterized in that, Add the distilled water according to the ratio of fruiting body powder to distilled water = 1g: 50mL.

3. The Birch Inonotus acidic polysaccharide IOP-2 according to claim 1, characterized in that, The conditions for ultra-high speed centrifugation were 9000 rpm for 10 min.

4. The Birch Inonotus acidic polysaccharide IOP-2 according to claim 1, characterized in that, The conditions for the second centrifugation were 8000 rpm for 15 min.

5. The use of the Inonotus obliquus acidic polysaccharide IOP-2 as described in any one of claims 1-4 in the preparation of a medicament for treating thyroid cancer.

6. A drug for treating thyroid cancer, characterized in that, The active ingredient includes the Inonotus obliquus acidic polysaccharide IOP-2 as described in any one of claims 1-4.

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