Application of dictamnine in preparing medicines for resisting osteosarcoma and osteolysis diseases caused by osteosarcoma

Abstract

The invention provides application of dictamnine in preparing medicines for resisting osteosarcoma and osteolysis diseases caused by osteosarcoma, belongs to the technical field of medicines, and studies the regulation effect of dictamnine on osteosarcoma and osteolysis diseases caused by osteosarcoma, dictamnine can inhibit proliferation, migration and invasion of K7M2 (mouse osteosarcoma cells), and can be used for preparing medicines for treating osteosarcoma and osteolysis diseases caused by osteosarcoma. TRAP staining shows that dictamnine inhibits formation and bone resorption of osteoclasts induced by RANKL in a dose-dependent manner, and meanwhile, the dictamnine can intervene in maturation and differentiation of osteoclasts induced by bone marrow macrophages (BMMs) in vitro. The establishment of a mouse tibial plateau osteosarcoma-bearing model shows that dictamnine can protect osteolysis caused by osteosarcoma. Through HE dyeing and histopathology result analysis, compared with a tumor-bearing mouse group, the dictamnine treatment group obviously reduces the number of osteoclasts in bone tissues. The medicine disclosed by the invention is a preparation consisting of dictamnine serving as an active component and a medicinal carrier.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of dithianine in preparing drugs for resisting osteosarcoma and osteolysis caused by osteosarcoma. Background Art

[0002] Osteosarcoma (OS) refers to the appearance of osteoid or immature bone produced by malignant mesenchymal cells in the skeleton. It is the most common primary bone solid malignancy. The annual incidence of osteosarcoma in the general population is 2 to 3 million, which is higher during adolescence. The annual incidence reaches a peak of 8 to 11 million at the age of 15-19, accounting for 15% of all solid cancers in this age group. The frequency of males affected is 1.4 times that of females. Osteosarcoma can occur in any bone, most commonly in the epiphysis of long bones, such as the distal femur, proximal tibia and proximal humerus, of which 50% originate around the knee. Local pain, swelling and limited joint movement are typical signs and symptoms of osteosarcoma. At the same time, osteosarcoma has a high rate of tumor cell proliferation, neovascularization and tumor-induced bone lysis. For patients with osteolytic tumors, pathological fractures may be the first sign of osteosarcoma.

[0003] Bones are important organs for body movement, organ protection, hematopoiesis, and maintenance of mineral homeostasis. Bone tissue is constantly remodeled during the life cycle of an organism to maintain its corresponding structure and function. The homeostasis of bone remodeling is maintained by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. When osteoclasts are overactivated and osteoblasts are underactivated, the balance of bone remodeling will be broken, causing bone metabolic diseases such as osteolysis.

[0004] Osteoclasts are the main effector cells that cause bone loss. Osteoclasts originate from the hematopoietic stem cell line and are formed by the fusion of macrophage precursors. The formation of osteoclasts requires the participation of multiple cytokines: the survival and proliferation of osteoclast precursors depend on the stimulation of macrophage colony-stimulating factor (M-CSF), and the binding of receptor activator of nuclear factor-κB ligand (RANKL) to its receptor RANK promotes the formation of osteoclasts. The interaction between the two can activate downstream cell signaling cascades such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK), further activating nuclear factor of T cell c1 (NFATc1) and proto-oncogene proteins to regulate the expression of osteoclast-related genes including tartrate-resistant acid phosphatase (TRAP), cathepsin K (Ctsk), vacuolar membrane H+-ATPase V0 domain d2 subtype (Atp6v0d2), matrix metalloproteinase-9 (Mmp9) and dendritic cell-specific transmembrane protein (Dcstamp), which plays a key role in the maturation of osteoclasts.

[0005] Current treatments for osteosarcoma include surgical resection, doxorubicin, cisplatin, ifosfamide, and large amounts of methotrexate combined with leukophagic rescue. Although the continuous advancement of imaging technology, biomedical engineering technology, and the positive effects of preoperative chemotherapy have led to a major shift in osteosarcoma resection from amputation to limb-saving surgery, resection for osteosarcoma is still very challenging due to the huge risk of local recurrence and frequent complications after reconstruction. The drugs used in systemic comprehensive treatment have high chemotherapy toxicity and emetic properties, and many other drugs need to be introduced for supportive treatment, such as the introduction of serotonin antagonists and dexamethasone to treat highly emetogenic chemotherapy, and the introduction of opioids to control tumor pain and chemotherapy-induced granulocytopenia. In short, it is of great clinical significance to find drugs that can prevent and treat osteosarcoma and osteolytic diseases caused by osteosarcoma. Chinese herbal medicine is a great treasure house of traditional Chinese medicine and even world medicine. The discovery of Chinese medicine monomers provides an effective way and broad ideas for creating new drugs, preventing and treating tumors, and preventing osteolytic diseases. In recent years, in order to find a treatment method with reliable efficacy, few toxic side effects and low cost, more and more researchers have turned their attention to active ingredients derived from natural medicines.

[0006] Dioscorea alkaloids are a natural furanquinoline alkaloid isolated from the root bark of Dictamni and are the most abundant component in the root bark of Dictamni. Dioscorea alkaloids have multiple biological activities, including inhibiting inflammatory responses, slowing viral replication, and inhibiting bacterial and fungal infections and killing cancer cells. Studies have reported the inhibitory effects of dioscorea alkaloids on pancreatic cancer, colon cancer, and small cell lung cancer cells, but there is still a blank in the treatment of osteosarcoma. In addition, dioscorea alkaloids are similar in structure to psoralens and Zanthoxylum bungeanum toxins, and a large number of literatures have reported the bone protective effects of the latter two monomers. However, as a structural analogue of these two, dioscorea alkaloids are very scarce in the study of the skeletal system. Therefore, it is necessary to design the application of dioscorea alkaloids in the preparation of anti-osteosarcoma and osteolytic diseases caused by osteosarcoma, and dioscorea alkaloids can be developed as drugs for the preparation of treatments for osteosarcoma and its osteolytic damage. Summary of the invention

[0007] The purpose of the present invention is to provide the use of distilled water in the preparation of drugs for treating osteosarcoma and osteosarcoma-induced osteolysis, to study the regulatory effect of distilled water on osteosarcoma cell lines and osteoclasts, and to design the use of distilled water in anti-tumor and bone protection drugs.

[0008] By studying the regulatory effect of distilled water on osteosarcoma and osteosarcoma-induced osteolysis, it was found that distilled water can inhibit the proliferation, migration and invasion of K7M2. TRAP staining showed that distilled water inhibited RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner. From the mechanism of action, distilled water acts on osteoclasts by inhibiting the ERK signaling pathway activated by RANKL, and down-regulating the expression of osteoclast maturation and differentiation marker genes (Fos, NFATc1, Ctsk, Mmp9, Acp5, Dcstamp). Then, by establishing a mouse tibial plateau osteosarcoma tumor-bearing model, it was shown that distilled water can protect against osteosarcoma-induced osteolysis.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] Application of dioscorea alkaloids in the preparation of anti-osteosarcoma drugs.

[0011] Application of dioscorea alkaloids in the preparation of drugs for preventing and treating bone loss diseases caused by osteoclasts.

[0012] Application of dioscorea alkaloids in the preparation of drugs for treating osteosarcoma and osteolysis caused by osteosarcoma.

[0013] Furthermore, the medicine is a preparation consisting of distilled water alkaline as an active ingredient and a pharmaceutical carrier.

[0014] Furthermore, the drug is in the form of oral dosage form, injection, suppository or external dosage form.

[0015] Furthermore, the pharmaceutical carrier is lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, water, methylparaben, propylparaben, talc, magnesium stearate or mineral oil.

[0016] The regulatory effect of distilled water on osteosarcoma and osteosarcoma-induced osteolysis was studied. It was found that distilled water could inhibit the proliferation, migration and invasion of K7M2. TRAP staining showed that distilled water inhibited RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner. From the mechanism of action, distilled water acts on osteoclasts by inhibiting the ERK signaling pathway activated by RANKL, and down-regulated the expression of osteoclast maturation and differentiation marker genes (Fos, NFATc1, Ctsk, Mmp9, Acp5, Dcstamp). The establishment of a mouse tibial plateau osteosarcoma tumor-bearing model showed that distilled water could protect against osteosarcoma-induced osteolysis.

[0017] The present invention studies the regulatory effect of distilled water on osteosarcoma and osteolysis caused by osteosarcoma. Distilled water can inhibit the proliferation, migration and invasion of K7M2 (mouse osteosarcoma cells). Through TRAP staining, it is found that distilled water inhibits RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner, and can interfere with the maturation and differentiation of osteoclasts induced by bone marrow macrophages (BMMs) in vitro. By establishing a tibial plateau osteosarcoma tumor-bearing model of mice, it is shown that distilled water can protect osteolysis caused by osteosarcoma. From the analysis of HE staining and histopathological results, compared with the tumor-bearing mouse group, the distilled water treatment group significantly reduced the number of osteoclasts in bone tissue. The drug of the present invention is a preparation composed of distilled water as an active ingredient and a pharmaceutical carrier.

[0018] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0019] The present invention found that distilled water inhibited the proliferation, migration, emigration, osteoclast formation and bone resorption of osteosarcoma cells in a dose-dependent manner; it was found that distilled water could inhibit osteosarcoma by intervening in the PI3K / AKT signaling pathway in osteosarcoma cells, and at the same time intervene in the ERK signaling pathway in osteoclasts to inhibit osteoclasts. In order to further prove the effect of distilled water on osteosarcoma cells and osteoclasts, a mouse osteosarcoma model was established. The results showed that the distilled water group could prevent and treat osteosarcoma and its induced bone dissolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The present invention is a graph that inhibits the proliferation of K7M2 cells in a dose-dependent manner;

[0021] Figure 2 The present invention inhibits the movement and secretion of osteosarcoma K7M2 cells by downregulating the phosphorylation of PI3K and AKT;

[0022] Figure 3 The present invention is a functional diagram for inhibiting the generation of osteoclasts and bone resorption;

[0023] Figure 4 It is a graph showing that the present invention inhibits the phosphorylation of ERK in the MAPK pathway and the expression of downstream osteoclast-related genes and proteins;

[0024] Figure 5 The present invention inhibits the growth of osteosarcoma and the secretion of TGF-β in vivo without liver and kidney toxicity;

[0025] Figure 6 The present invention reduces osteosarcoma-induced bone lytic damage by inhibiting osteoclasts. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0027] 1. Materials and Methods

[0028] Reagents

[0029] Dictyostelium was purchased from Chengdu Desit Biotechnology Co., Ltd. (Sichuan, China). RAW 264.7 was purchased from American Type Culture Collection (Maryland, USA). Mouse MCSF was purchased from R&D Systems (Minneapolis, USA). α-MEM was purchased from Thermo Fisher, and fetal bovine serum (FBS) was purchased from TRACE (Sydney, Australia). Recombinant GST-rRANKL was purified in our laboratory. Recombinant human M-CSF required for induction of human peripheral blood mononuclear cells was purchased from Chemicon (California, USA), and fetal bovine serum, penicillin-streptomycin, and L-glutamine were purchased from Invitrogen (California, USA). IκBα and NFATc1 antibodies were purchased from Santa Cruz Biotechnology Com. (California, USA). V-ATPase d2 antibody was synthesized and purified in our laboratory.

[0030] 1) Isolation, culture and differentiation of bone marrow macrophages in vitro

[0031] Bone marrow macrophages (BMMs) were isolated from the bone marrow of C57BL / 6J mice. The experimental methods were approved by the Animal Ethics Committee of Guangxi Medical University. BMMs were cultured in α-MEM complete medium containing 10% fetal bovine serum, 5 μg / mL penicillin, 50 U / mL streptomycin, and 10 ng / mL L-CSF at 37°C with 5% CO. 2 Incubate in incubator.

[0032] After the cells were confluent, BMMs (6×10 3 ) were inoculated in 96-well plates and divided into negative control group, positive control group and drug-treated group. The negative control group did not add RANKL, the positive control group only added RANKL, and the drug-treated group added RANKL and different concentrations of dioscorea. The medium was changed once every 2 days. After 7 days, the cells were fixed with 4% paraformaldehyde for 20 minutes and TRAP staining solution was added for staining for 1 hour. Under an optical microscope, cells with ≥3 nuclei in TRAP-positive multinucleated cells were defined as osteoclasts and counted.

[0033] 2) CCK8 assay to determine the effect of damipine on osteoclast precursors and K7M2 cell proliferation

[0034] BMMs(6×10 3 ) were inoculated in a 96-well plate and cultured overnight. The culture medium (containing different concentrations of dihydrochloride) was replaced and incubated for 48 hours. Then CCK8 reagent was added to each well and incubated for 2 hours, and finally detected by ELISA.

[0035] 3) EdU staining experiment

[0036] Phosphate buffered saline (PBS) was purchased from Solarbio (Beijing, China). Triton X-100 and EdU staining kits were purchased from Beyotime (Shanghai, China). K7M2 cells were plated at 3 × 10 3 Cells were seeded in 96-well plates at a density of 10 cells and treated with dihydrochloride for 24 hours after adhesion. EdU working solution was added to the cells and incubated for 4 hours according to the instructions of the kit. The cells were then fixed with 4% PA, washed with 3% PBS, and permeabilized with 0.3% Triton X-100 for 15 minutes. Click reaction solution was added and incubated in the dark for 30 minutes, followed by staining with 1x Hoechst33342 for 10 minutes in the dark. Images were acquired using a Bio-Tek imaging system.

[0037] 4) Cloning experiment

[0038] K7M2 cells were seeded in 24-well plates at a density of 250 cells per well. After compliance, they were treated with dithianine for 24 hours, and then switched to complete medium and incubated in an incubator. After two weeks, the cells were fixed with 4% PA and stained with crystal violet solution (Beyotime, Shanghai, China), and then photographed.

[0039] 5) AnnexinV-FITC detection and cell cycle analysis based on flow cytometry

[0040] Annexin V-FITC apoptosis detection kit and cell cycle kit were purchased from Beyotime Biotechnology (Shanghai, China).

[0041] The cells were digested with trypsin-EDTA solution and washed with PBS, then resuspended in annexin V-FITC conjugate at a concentration of 4×105 cells / mL. 5 μL of annexin V-FITC and 10 μL of propidium iodide staining solution were added to the total volume of 485 μL of cell suspension. After incubation for 1 hour, the cells were analyzed using a flow cytometer (Beckman Coulter, USA).

[0042] After digestion, the cells were fixed with 75% cold ethanol overnight at 4°C. K7M2 cells were then resuspended in propidium iodide staining medium and incubated at 37°C in the dark for 30 min before being detected by flow cytometry.

[0043] 6) Wound healing assay

[0044] K7M2 cells were resuspended to a density of 1×105 cells / mL, and 100 μL of cell suspension was added to each chamber. After attachment, the medium was replaced with serum-free DMEM, and the cells were treated with different doses of dihydrochloride. Cell images were captured at 0 h, 24 h, and 48 h using an inverted optical microscope, and wound healing was evaluated using ImageJ software.

[0045] 7) Transwell assay

[0046] K7M2 cells were resuspended in serum-free DMEM at a density of 2×105 cells / mL. A total of 100 μL of cell suspension was added to each Transwell chamber, while 650 μL of complete DMEM was added to each well of a 24-well plate. After 48 h of distilled water treatment, the chambers were fixed with 4% PA, stained with crystal violet, and examined under a microscope to assess invasion.

[0047] Matrigel was purchased from R&D Systems (Minneapolis, MN, USA). For invasion assays, complete medium was mixed with Matrigel at a ratio of 7:1. A total of 100 μL of the Matrigel mixture was added to each Transwell chamber, and after solidification, subsequent steps were performed according to the migration assay protocol.

[0048] 8) Reverse transcription-polymerase chain reaction

[0049] BMMs (1×10 6 ) were inoculated in 6-well plates and cultured overnight. The culture medium (containing different concentrations of dihydrochloride 5 and 10 μM) was replaced and incubated for 7 days. The cells were lysed with Trizol and total RNA was extracted. The RNA was reverse transcribed into cDNA using reverse transcriptase containing oligo-dT primers. Then, primers of different genes were added to amplify the cDNA by polymerase chain reaction. β-actin was used as an internal reference.

[0050] The following are the primer sequences for different genes:

[0051]

[0052] 9) Bone resorption detection

[0053] BMMs were divided into 1×10 5 / well concentration in a 96-well plate seeded with 0.75mm thick bovine bone plates for overnight incubation. Add dihydrochloride and incubate for 2 weeks, fix with 4% paraformaldehyde, and add TRAP staining solution for staining for 15 minutes. Under an optical microscope, cells with ≥3 nuclei in TRAP-positive multinucleated cells were defined as osteoclasts and counted. After washing and drying, bone resorption pits were detected with a Philips XL30 scanning electron microscope, and the percentage of bone resorption area was calculated using Scion Image software.

[0054] 10) Western blotting

[0055] BMMs were seeded in 6-well plates overnight. For short-acting proteins (ERK), distilled water was added for pre-incubation for 1 hour, and RANKL was added for stimulation at 0, 10, 20, 30, 60, and 120 minutes; for long-acting proteins (c-FOS, NFATc1, CTSK, and MMP9), distilled water and RANKL were added for stimulation at 0, 1, 3, and 5 days. Cell lysate was added after the detection time point to collect the protein.

[0056] Then, SDS-PAGE gel was prepared, protein samples were loaded, and the membrane was transferred. After blocking with 5% skim milk powder at room temperature for 1 hour, the membrane was washed with TBST, and then incubated with relevant antibodies, and finally developed by ECL luminescence.

[0057] 11) Construction of osteosarcoma tumor-bearing mouse model

[0058] The C57BL / 6J mouse experiment was approved by the Ethics Committee of Guangxi Medical University. Twenty-four 4-week-old male mice were randomly divided into 4 groups (i.e., sham operation group, control group, cisplatin treatment group, and distilled alcohol treatment group), with 6 mice in each group. Except for the sham operation group, each mouse was injected with 10% chloral hydrate intraperitoneally and then injected with K7M2 osteosarcoma cell suspension on the tibial plateau. The drugs were administered every other day. Intraperitoneal injections were performed according to body weight and grouping. The sham operation group and the control group were injected with 1% DMSO, the cisplatin group was injected with cisplatin (10 mg / kg), and the distilled alcohol group was injected with distilled alcohol (10 mg / kg). After 2 weeks of administration, the mice were killed and their tibiae were removed. The tibiae were fixed with 4% paraformaldehyde for 24 hours and then examined by micro-CT scanning.

[0059] 12) Micro-CT analysis

[0060] The mouse tibia was washed three times with 1×PBS and placed in a test tube for scanning. The Bruker 1176 micro-CT was used for scanning, with scanning conditions of 50KV-550μA, 0.5mm aluminum filter, and a spatial resolution of 9μM. The data was reconstructed into CT images using NRecron; the CTAn analysis software calibrated the sample's Hall value and density. The samples rebuilt using CTAn software (Bruker) were analyzed. The following is the method of parameter analysis: The study area of ​​trabeculae and cortical bone was delineated based on the bottom of the growth plate. The study area of ​​trabeculae is the area from 0.5 mm to 1.5 mm below the bottom of the growth plate. The representative study area of ​​trabeculae was delineated from the peripheral cortical bone by drawing a curve. The study area of ​​trabeculae was binarized by a constant threshold, and then quantitatively analyzed by CTAn software.

[0061] 13) Bone tissue morphology analysis

[0062] Tibiae were fixed with 4% paraformaldehyde for 24 hours and rinsed with 1×PBS 3 times. 14% EDTA was added for decalcification for 7 days. Samples were embedded in paraffin and stained with HE or TRAP. Quantitative analysis was performed using BIOQUANTOSTEO software to obtain data such as osteoclasts / BS and N.Oc / BS

[0063] 14) Statistical processing

[0064] All experimental data were repeated 3 times. The results of each parameter were processed and analyzed by Graphpad Prism software, and the results were expressed as mean ± standard deviation (x ± SEM); hypothesis testing was set at P < 0.05 for statistical significance.

[0065] 2. Experimental Results

[0066] 1) Dioscorea alata affects the proliferation and apoptosis of K7M2 cells.

[0067] To evaluate the anti-osteosarcoma effect of distilled water, K7M2 cells were treated with distilled water at concentrations of 0, 5, 10, 20, 40, and 80 μM for 24 and 48 h. CCK-8 assay showed that distilled water inhibited the proliferation and viability of K7M2 cells, and the inhibitory effect was proportional to the treatment dose. The half-maximal inhibitory concentration (IC50) of distilled water in K7M2 cells was 27.64±2.59 μM at 24 h and 27.82±4.15 μM at 48 h.

[0068] 5-Ethynyl-2'-deoxyuracil (EdU) can be used to label newly synthesized deoxyribonucleic acid (DNA). 22 After 24 h of distilled water treatment, DNA synthesis in K7M2 cells was inhibited, and newly synthesized DNA decreased in a dose-dependent manner. Colony formation assays were performed to evaluate the long-term antiproliferative effects of distilled water on K7M2 cells, revealing that distilled water significantly inhibited colony formation ability in a dose-dependent manner. These findings suggest that distilled water exerts an antiproliferative effect on K7M2 cells in a dose-dependent manner.

[0069] After treatment with 40 μM DTM for 24 h, the apoptotic cell population increased from 4.05 ± 1.01% to 13.30 ± 1.55%, indicating that the antiproliferative effect of DTM may be mediated by apoptosis. Cell cycle analysis showed that the G0 / G1 phase cell population was significantly reduced, the S phase cell population was significantly increased, and the proportion of G2 / M phase cells remained unchanged. S phase arrest may represent the key mechanism by which DTM inhibits DNA synthesis in K7M2 cells.

[0070] 2) Dioscorea alkaloids inhibit the migration, invasion and secretion of TGF-β in K7M2 cells through the PI3K / AKT signaling pathway.

[0071] Using a wound healing assay, the migration ability of K7M2 cells was evaluated, revealing that diffusin significantly inhibited migration and delayed the healing area in a dose-dependent manner. Transwell assays confirmed that diffusin reduced the migration and invasion of K7M2 cells in a dose-dependent manner.

[0072] To elucidate the mechanism by which distilled alcohol affects K7M2 cells, RNA transcriptome sequencing was performed on K7M2 cells with and without distilled alcohol intervention, and then KEGG enrichment analysis of DEGs was performed, indicating that distilled alcohol may affect K7M2 cells through the PI3K / AKT signaling pathway. To verify the KEGG results, we used Western blotting to examine the changes in PI3K / AKT pathway proteins after treatment with different concentrations of distilled alcohol for 24 h, and the results showed that the levels of phosphorylated PI3K and AKT were downregulated in a concentration-dependent manner. Sequencing data and PCR validation further showed that distilled alcohol inhibited the secretion of TGF-β by K7M2 cells. Taken together, these findings indicate that distilled alcohol can inhibit the phosphorylation of PI3K and AKT, thereby hindering the movement and secretion of K7M2 cells during osteosarcoma progression.

[0073] 3) Dioscorea alkali inhibits the formation of osteoclasts and their bone resorption function.

[0074] To verify whether distilled water directly affects osteoclast formation, we first confirmed that the inhibitory effect of distilled water on osteoclasts was not due to a decrease in the activity or number of BMMs. The results of the CCK8 assay showed that distilled water had no significant toxicity to BMMs at concentrations between 0 and 50 μM from 48 to 96 h. Subsequently, we co-cultured BMMs with increasing concentrations of distilled water (0, 10, 20, 30, and 40 μM), and after 5 days of RANKL induction, we observed a significant dose-dependent decrease in the number and area of ​​TRAP-positive mature osteoclasts (nuclei ≥ 3) under distilled water intervention.

[0075] To evaluate the effect of distilled water on osteoclast bone resorption function, we plated osteoclasts on bovine bone slices under 0, 20, and 40 μM distilled water conditions. After 14 days, TRAP staining and electron microscopy were performed. With consistent TRAP-positive nuclei, we found that the osteoclast area on the bone slices and the area of ​​resorption pits observed by electron microscopy decreased in a concentration-dependent manner after distilled water intervention.

[0076] 4) Dioscorea glabra downregulates the expression of osteoclast-related genes and downstream proteins by inhibiting the phosphorylation of ERK.

[0077] We incubated BMMs with 20 and 40 μM diosmin and RANKL for 5 days, and then performed RT-PCR analysis to evaluate the expression of osteoclast-specific genes. The results showed that diosmin significantly reduced the expression levels of genes such as Acp5, Fos, Nfatc1, Ctsk, Mmp9, Dcstamp, and Atp6v0d2 ( Figure 5 A). Meanwhile, Western blot analysis showed that EMCL treatment led to a significant decrease in the protein expression of NFATc1, MMP9, c-FOS, and CTSK on days 3 and 5. Collectively, these findings further confirmed the inhibitory effect of damipine on RANKL-induced osteoclastogenesis.

[0078] After treatment with 40 μM dithiocarbazine and RANKL stimulation at different time points, ERK phosphorylation was significantly inhibited after 5 minutes of RANKL stimulation. These results strongly suggest that the inhibitory effect of dithiocarbazine on osteoclasts may be achieved by inhibiting ERK phosphorylation within the MAPK pathway.

[0079] 5) Dioscorea alkaloids inhibit the growth of osteosarcoma in vivo without showing liver or kidney toxicity.

[0080] We used the tibial plateau tumor model in C57BL / 6J mice to confirm the anti-osteosarcoma effect of dithianine in vivo. All mice survived until the end of the experiment. To facilitate the observation of the effects of osteosarcoma on the bone and because of the difficulty in isolating tumors implanted in the tibial plateau, pale, hard, round masses located at the junction of the tibia and femur were considered osteosarcoma tumors. Tumor volume was calculated using the formula volume = 0.5 × length × width × width.

[0081] Compared with the model group, both distilled alcohol and cisplatin treatment resulted in a significant reduction in tumor volume and hindlimb weight, with the cisplatin group showing a more significant reduction, indicating that distilled alcohol can inhibit osteosarcoma in vivo; however, at the same 10 mg / kg dose, its anticancer effect was weaker than that of cisplatin. Serum ELISA results showed that distilled alcohol intervention also downregulated TGF-β levels in tumor-bearing mice. These results are consistent with the results of in vitro experiments.

[0082] Histological examination of liver and kidney tissues by HE staining showed no significant differences in liver and kidney morphology between diffusin-treated mice and mice in the sham or model groups, indicating that diffusin can inhibit osteosarcoma without liver or kidney toxicity. In contrast, treatment with the same dose of cisplatin resulted in significant weight loss and the presence of a large number of balloon-like hepatocytes, suggesting that cisplatin has greater side effects than diffusin at the same dose when exerting its anticancer effect.

[0083] 6) Dictyosteline reduces osteosarcoma-induced bone lytic damage in vivo by inhibiting osteoclast activity.

[0084] To investigate the effect of distilled water on osteoclasts, we performed TRAP staining on bone tissue sections from the femorotibial junction and quantified the fraction of osteoclast area in the bone marrow cavity relative to the trabecular surface area. We also counted osteoclasts in the tumor site outside the bone marrow cavity. Our results showed that the number of osteoclasts around the trabecular bone and within the tumor in the tumor-bearing group was significantly increased compared with the Sham group, while distilled water treatment resulted in a significant decrease in osteoclasts, indicating that distilled water inhibits the production of osteoclasts in the osteosarcoma microenvironment.

[0085] High-resolution Micro-CT scanning was used to reconstruct the structural features of osteolytic lesions to further elucidate the protective effect of distilled water on osteosarcoma-induced bone resorption. Osteosarcoma caused significant damage to both cortical and trabecular bones, and the distilled water treated group showed more intact trabecular and cortical bone structure. We quantified various morphometric parameters associated with osteolysis, including trabecular bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), and BV / TV of cancellous bone. The results showed that tumor-bearing mice had severe bone loss in the tibial plateau, with significantly reduced or completely destroyed trabeculae and significantly enlarged trabecular spaces. Compared with the model group and cisplatin-treated groups, distilled water intervention showed a strong rescue effect on osteosarcoma-related bone loss.

[0086] Discussion

[0087] In contrast to the rapid advances observed in the treatment of other solid tumors, the prognosis of osteosarcoma patients has not improved significantly over the past three decades. This stagnation can be attributed to the presence of a high level of cellular heterogeneity in osteosarcoma cells, as well as the complex molecular and genetic mechanisms associated with disease onset and progression. As osteosarcoma is better understood, oncologists have become increasingly aware of the challenges of singularly targeting osteosarcoma cells to improve patient outcomes. As a result, recent research and therapeutic efforts have shifted their focus to the interactions between cells in the tumor microenvironment. This includes studying intercellular communication involving osteoclasts in the tumor microenvironment to predict the prognosis of osteosarcoma patients and identifying biomaterials or therapeutics with anti-osteosarcoma and anti-resorptive properties.

[0088] The PI3K / AKT signaling pathway is often overactivated in osteosarcoma, promoting tumor proliferation, metastasis, invasion, cell cycle progression, apoptosis inhibition, angiogenesis, and chemoresistance. In this study, we determined that dioscorea alata could inhibit the progression of osteosarcoma by inhibiting the PI3K / AKT pathway.

[0089] Disruption of the physiological homeostasis of bone remodeling is one of the main characteristics of osteosarcoma. Bone remodeling is a continuous and dynamic process that is highly coupled to osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Under physiological conditions, bone remodeling helps maintain the homeostasis between anabolic and catabolic activities, ensures normal calcium levels in the body, and plays a vital role in maintaining the structural integrity and immune support of bones. In the bone microenvironment of osteosarcoma, osteosarcoma cells secrete a large amount of RANKL, which promotes the differentiation and functional activation of osteoclasts. In our in vitro and in vivo experiments, we demonstrated that the osteosarcoma environment promotes the formation and function of osteoclasts, while damipine inhibits osteoclast maturation and its bone resorption activity, thereby preventing osteosarcoma-induced bone destruction.

[0090] TGF-β is one of the most abundant growth factors in bone and plays a complex role in both osteosarcoma and osteoclasts, being a key component of the vicious cycle between the two. Dioscorea serrata reduced the level of TGF-β in the serum of tumor-bearing mice in vitro and downregulated the expression of TGF-β in K7M2 cells. This may also represent one of the potential mechanisms of the antitumor and bone-protective effects of dioscorea serrata.

[0091] Cisplatin is a platinum-based genotoxic agent that inhibits DNA replication by causing single-strand or double-strand damage to DNA, thereby inhibiting tumor cell proliferation. Therefore, cisplatin treatment is often associated with side effects such as hepatotoxicity, ototoxicity, nephrotoxicity, neurotoxicity, gastrointestinal toxicity, and bone loss. In this study, dioscorea demonstrated that dioscorea was able to inhibit osteosarcoma without hepatotoxicity or nephrotoxicity, while also showing enhanced bone protection compared to cisplatin, which may be due to its independent inhibitory effect on osteoclasts.

[0092] In summary, as a natural bioactive small molecule, damipine can simultaneously inhibit the proliferation and progression of osteosarcoma and block the formation and function of osteoclasts. This provides a new possibility for breaking the vicious cycle between osteosarcoma and osteoclasts and provides a promising candidate for drug development for the treatment of osteosarcoma.

[0093] Figure 1 (A) Molecular structure of distilled water. (B, C) The CCK-8 method was used to determine the viability of K7M2 cells after treatment with different concentrations of distilled water for 24 and 48 hours, proving that distilled water can inhibit the activity of osteosarcoma cells K7M2. (D, E) The half inhibitory concentration (IC50) was calculated based on the CCK-8 results of distilled water treatment, which was 27.64±2.59μM at 24 hours and 27.82±4.15μM at 48 hours. (F, G) Representative images of EdU-labeled K7M2 cells (scale bar = 200μm) and quantitative analysis of EdU-positive cells, proving that distilled water dose-dependently inhibits the DNA replication of K7M2 cells. (H, I) Representative images of colonies stained with 0.5% crystal violet solution (scale bar = 100μm) and quantitative analysis of cell clone formation experiments, proving that distilled water can inhibit the survival and cloning ability of K7M2 cells;

[0094] Figure 2Inhibits the motility and secretion of osteosarcoma K7M2 cells by downregulating the phosphorylation of PI3K and AKT. (AC) Representative images of K7M2 cell wound healing assay (scale bar = 200 μm) and quantitative analysis of the migration ability of K7M2 cells after 24 and 48 hours of distilled water treatment, demonstrating that distilled water inhibits the motility and migration ability of K7M2 cells. (DF) Representative images of crystal violet-stained cells in Transwell assay (scale bar = 2 mm) and quantitative analysis of the migration and invasion rates of K7M2 cells after distilled water treatment, indicating that distilled water can inhibit the migration and invasion of K7M2 cells. (G) Transcriptome sequencing of K7M2 cells treated with or without distilled water was performed, and KEGG pathway enrichment analysis of differentially expressed genes was performed, showing that K7M2 may affect K7M2 cells through the PI3K / AKT signaling pathway. (HJ) Western blotting images showed the expression levels of p-PI3K, PI3K, p-AKT, AKT and β-actin proteins in K7M2 cells, and the quantitative analysis of p-PI3K / PI3K and p-AKT / AKT bands verified that distilled water concentration-dependently downregulated the expression levels of phosphorylated PI3K and phosphorylated AKT. (K, L) The expression level of TGF-β1 gene in K7M2 cells was analyzed based on transcriptome sequencing results, and the relative expression level of TGF-β1 gene after treatment with different concentrations of distilled water was detected by PCR experiments. The results showed that distilled water could inhibit the expression of TGF-β1;

[0095] Figure 3 In the figure, (A, B) CCK8 assay of the effect of DTM on the cell viability of BMMs after 48 hours and 96 hours, demonstrating that distilled water has no killing effect on BMMs. (C) Representative TRAP staining images of RANKL-induced osteoclasts with or without distilled water treatment, where cells with ≥3 nuclei are considered mature osteoclasts (scale bar = 2 mm, scale bar in enlarged images = 200 μm). (D-E) Quantitative analysis of the number and area of ​​mature osteoclasts after distilled water treatment, demonstrating that distilled water reduces the number and area of ​​osteoclasts induced by BMMs without reducing the number of BMMs. (F) Representative electron microscopy images of resorption pits in bone resorption experiments (scale bar = 400 μm, scale bar in enlarged images = 50 μm) and TRAP staining images of bovine bone sections treated with different concentrations of distilled water (scale bar = 200 μm, scale bar in enlarged images = 20 μm). (G) Statistical analysis of the number of cell nuclei, mature osteoclast area, and bone resorption area (cell nuclei ≥ 3 were considered mature OCs) on bovine bone slices, demonstrating that damipine can inhibit osteoclast maturation and bone resorption function;

[0096] Figure 4Inhibits the phosphorylation of ERK in the MAPK pathway and the expression of downstream osteoclast-related genes and proteins. (A) The relative expression levels of various genes related to osteoclasts after treatment with different concentrations of distilled water, proving that distilled water inhibits osteoclastogenesis possibly due to the downregulation of these genes. (B, C) Representative Western blot images showing the effects of distilled water on ERK protein in the MAPK pathway at different time points of RANKL stimulation and quantitative analysis of the relative grayscale ratio of phosphorylated bands to total protein bands, proving that the inhibitory effect of distilled water on osteoclasts is due to the inhibition of ERK protein phosphorylation at an early stage. (D, E) Representative Western blotting images and corresponding quantitative analysis of osteoclast-related proteins after 0, 1, 3, and 5 days of RANKL stimulation with and without distilled water intervention, proving that the inhibitory effect of distilled water on osteoclastogenesis may be due to the downregulation of the expression of proteins such as c-FOS, NFATc1, CTSK, and MMP9;

[0097] Figure 5 In vivo, it inhibited the growth of osteosarcoma and the secretion of TGF-β without liver and kidney toxicity. (AC) At the end of distilled water treatment, the tumor of mouse osteosarcoma was measured and photographed, and the tumor volume and tumor-bearing hind limbs were quantitatively analyzed, proving that distilled water can inhibit the development of osteosarcoma in vivo. (D, E) Representative HE staining images of tibial tissue sections after distilled water treatment and quantitative analysis of K7M2 cell infiltration area in HE staining sections, proving that distilled water reduced the proliferation and invasion of osteosarcoma cells. (F) Quantitative analysis of serum TGF-β levels by ELISA proved that distilled water treatment downregulated the expression level of TGF-β in osteosarcoma-bearing mice. (G) Quantitative analysis of mouse body weight, compared with the effect of cisplatin on the mouse body, distilled water treatment did not cause weight loss in mice. (H, I) Representative HE staining images of liver and kidney and quantitative analysis of the number of abnormal hepatocytes in liver sections, compared with the hepatocyte necrosis caused by injection of cisplatin, distilled water treatment did not show obvious liver and kidney toxicity.

[0098] Figure 6 In the study, dioscorea alleviated osteosarcoma-induced osteolytic damage by inhibiting osteoclasts. (AC) Representative images of TRAP staining on bone tissue sections and quantitative analysis of osteoclast surface area / bone surface (OC.S / BS) and the number of osteoclasts in tumors, demonstrating that dioscorea reduces osteoclasts in osteosarcoma-bearing mice. (D, E) Representative images of a three-dimensional reconstructed model of tibial trabeculae in osteosarcoma-bearing mice and quantitative analysis of trabecular bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th), demonstrating that dioscorea has a salvage effect on osteosarcoma-induced osteolytic damage.

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of dioscorea alkaloids in the preparation of anti-osteosarcoma drugs.

2. The application of dioscorea alkali in the preparation of drugs for preventing and treating bone loss diseases caused by osteoclasts.

3. The application of dioscorea alkaloids in the preparation of drugs for treating osteosarcoma and osteolysis caused by osteosarcoma.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine is a preparation consisting of distilled water alkaloids as an active ingredient and a pharmaceutical carrier.

5. The use according to claim 4, characterized in that: The medicine is in oral dosage form, injection, suppository or topical dosage form.

6. The use according to claim 4, characterized in that: Pharmaceutical carriers are lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, water, methylparaben, propylparaben, talc, magnesium stearate or mineral oil.

Images