Silicon ion preparation for treating chronic pancreatitis, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a silicon ion preparation for treating chronic pancreatitis and a preparation method and application thereof. The silicon ion preparation has excellent effects of promoting regeneration of pancreatic acinar cells and inhibiting inflammation and fibrosis of pancreatic tissue. The silicon mineral preparation prepared according to the concentration can be used for treating chronic pancreatitis and preventing fibrosis, and has great application prospects. The silicon ion preparation provided in the embodiment can treat chronic pancreatitis and prevent fibrosis. The active material can significantly treat the activity of pancreatitis cells, so as to form healthy pancreatic tissue. The silicon ion preparation comprises that the concentration of silicon ions in the silicon ion preparation is 5 ppm-800 ppm, and the silicon ion preparation is prepared by releasing silicon ions from a silicon mineral preparation.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to a silicon ion preparation for the treatment of chronic pancreatitis, its preparation method and application. Background Technology

[0002] Pancreatitis is a fibrotic inflammatory syndrome caused by multiple factors. Its main pathological features include atrophy, destruction of pancreatic acini, and interstitial fibrosis. Clinical manifestations include recurrent upper abdominal pain, insufficient secretion from both the inner and outer pancreas, and pancreatic duct stones. Ultimately, it leads to a reduced quality of life and shortened life expectancy, and chronic pancreatitis increases the risk of pancreatic cancer.

[0003] Currently, the diagnostic and treatment agents used for chronic pancreatitis include stem cell therapy or small molecule chemical drugs. However, these therapies, which involve stem cell therapy or injection of small molecule chemical drugs, are mainly symptomatic treatments to relieve symptoms, and their clinical application is limited due to toxicological factors. Summary of the Invention

[0004] In view of this, embodiments of this application provide a silicon ion preparation for treating chronic pancreatitis, its preparation method, and its application, in order to solve the problems in the prior art.

[0005] The first aspect of this application provides a silicon ion preparation for treating chronic pancreatitis, comprising: the concentration of silicon ions in the silicon ion preparation is 5ppm-800ppm, and the silicon ion preparation is prepared by releasing silicon ions from a silicon mineral preparation.

[0006] In conjunction with the first aspect, in a first possible implementation of the first aspect, the silicate preparation comprises silicate-based bioceramics.

[0007] In conjunction with the first possible implementation of the first aspect, in the second possible implementation of the first aspect, silicate-based bioceramics include one or more of calcium silicate, zinc silicate, magnesium feldspar, and 45S5 bioactive glass.

[0008] In conjunction with the first aspect, in a third possible implementation of the first aspect, the concentration of silicon ions in the silicon ion preparation is 60 ppm.

[0009] The second aspect of this application provides a method for preparing a silicon ion preparation. The method is used to prepare the silicon ion preparation described in the first aspect. The method includes: obtaining one or more silicon mineral preparations; thoroughly mixing the silicon mineral preparations with PBS or culture medium according to a preset concentration of silicon ions in the silicon ion preparation to obtain a mixture; and obtaining the supernatant of the mixture to obtain a silicon ion preparation that meets the preset concentration requirements.

[0010] A third aspect of this application provides the use of a silicon ion preparation as described in the first aspect in the preparation of a preparation for treating chronic pancreatitis.

[0011] In conjunction with the third aspect, in the first possible implementation of the third aspect, the chronic pancreatitis includes one or more of chronic calcifying pancreatitis, chronic obstructive pancreatitis, and chronic inflammatory pancreatitis.

[0012] The beneficial effects of the embodiments in this application compared with the prior art are:

[0013] This application provides a silicon ion preparation for treating chronic pancreatitis, its preparation method, and its application. The silicon ion preparation comprises a silicon ion concentration of 5 ppm to 800 ppm, and is prepared by releasing silicon ions from a silicon mineral preparation. In this embodiment, the silicon ion preparation with a silicon ion concentration in the range of 5 ppm to 800 ppm exhibits excellent effects in promoting pancreatic acinar cell regeneration and inhibiting inflammation and fibrosis of pancreatic tissue. The silicon mineral preparation prepared according to this concentration can be used to treat chronic pancreatitis and prevent fibrosis, showing great application potential. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a schematic diagram illustrating the effect of calcium silicate (CS) ion solutions of different concentrations on pancreatic acinar cells, provided in an embodiment of this application.

[0016] Figure 2 This is a schematic diagram illustrating the effect of calcium silicate (CS) ion solutions of different concentrations on macrophages, provided in an embodiment of this application.

[0017] Figure 3 This is a schematic diagram illustrating the effect of calcium silicate (CS) ion solutions of different concentrations on pancreatic stellate cells, provided in an embodiment of this application.

[0018] Figure 4 This is an example of an embodiment of the present application showing the immunofluorescence staining results of α-SMA and Col1α1 on damaged pancreatic acinar cells after co-culturing with a 1 / 64 concentration of CS ion solution;

[0019] Figure 5This is a diagram showing the results of reducing the expression of macrophage phenotype M1-related cytokines using CS ion solution according to an embodiment of this application;

[0020] Figure 6 This is a graph showing the therapeutic effect of the CS ion solution provided in one embodiment of this application on mice with chronic pancreatitis.

[0021] Figure 7 This is a graph showing the effect of CS ion solution provided in one embodiment of this application on apoptosis and regeneration of pancreatic acinar cells;

[0022] Figure 8 This is a graph showing the effect of CS ion solution provided in an embodiment of this application on the inhibition of macrophage M1 transformation. Detailed Implementation

[0023] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of this application. However, those skilled in the art will understand that this application may also be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods have been omitted so as not to obscure the description of this application with unnecessary detail.

[0024] The technical solutions provided in this application will be explained in detail below with reference to specific embodiments.

[0025] This application provides a silicon ion preparation for treating chronic pancreatitis, wherein the concentration of the silicon ion preparation is 5ppm-800ppm, and the silicon ion preparation is prepared by releasing silicon ions from a silicon mineral preparation.

[0026] In some embodiments, the silicate mineral preparation comprises silicate bioceramics, including one or more of calcium silicate, zinc silicate, magnesium feldspar, and 45S5 bioactive glass.

[0027] In this embodiment, silicate bioceramics possess excellent anti-inflammatory properties, and these materials have been widely used in the treatment of diabetic wounds, osteoarthritis, and other diseases. Silicon ions are the key ions responsible for this effect. Since silicate bioceramics are the primary biomaterials that release silicon ions, this embodiment uses these materials as the main raw material for preparing silicon ion preparations.

[0028] In other embodiments, the concentration of silicon ions in the silicon ion preparation for treating chronic pancreatitis is 60 ppm. Experiments have shown that when the concentration of silicon ions in the silicon ion preparation is 60 ppm, its effect on treating chronic pancreatitis is better.

[0029] This application also provides a method for preparing a silicon ion preparation, which includes the following steps S100 to S300.

[0030] S100, to obtain one or more silicon mineral preparations.

[0031] The silica mineral preparation includes one or more of calcium silicate, zinc silicate, magnesium feldspar, and 45S5 bioactive glass. During preparation, one of them can be selected according to actual needs, such as using calcium silicate as raw material, or multiple of them can be selected, such as using calcium silicate and zinc silicate as raw materials.

[0032] S200: Based on the preset concentration of silicon ions in the silicon ion preparation, the silicon mineral preparation is thoroughly mixed with PBS or culture medium to obtain a mixed solution.

[0033] First, based on the required concentration of silicon ions in the desired silicon ion formulation, the preset concentration is 5ppm-800ppm. For example, if the required concentration of silicon ions in the desired silicon ion formulation is 200ppm, then 200ppm is the preset concentration of silicon ions in the silicon ion formulation.

[0034] Then, based on the preset concentration of silicon ions, select the corresponding PBS or culture medium, and thoroughly mix the silicon mineral preparation with it to obtain a mixture.

[0035] S300: Obtain the supernatant of the mixture to obtain a silicon ion preparation that meets the preset concentration requirements.

[0036] In some embodiments, obtaining the supernatant of the mixture can be achieved by immersing the mixture in water for a certain period of time, after which the supernatant separates from the substrate. Alternatively, the mixture can be centrifuged to obtain the supernatant.

[0037] It should be understood that the sequence number of each step in the above embodiments does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0038] The silicon ion preparation provided in this application is used to prepare a treatment for chronic pancreatitis, which includes one or more of chronic calcific pancreatitis, chronic obstructive pancreatitis, and chronic inflammatory pancreatitis.

[0039] The following describes the preparation of the silicon ion preparation provided in the embodiments of this application using calcium silicate (CS) as an example, and elaborates on the application of the silicon ion preparation in the preparation of a preparation for treating chronic pancreatitis.

[0040] This application embodiment is based on the above-described method for preparing silicon ion preparations, using calcium silicate to prepare calcium silicate ion solutions, i.e., silicon ion preparations. Specifically, a series of gradient dilution ion extracts of calcium silicate were prepared using the above method. Exemplarily, in this embodiment, calcium silicate powder was added to serum-free DMEM (Dulbecco's Modified Eagle Medium) (DMEM; Gibco) or PBS (Gibco) at a solid / liquid ratio of 200 mg / mL at 37°C for 24 hours. Then, the mixture was centrifuged at 4000 rpm for 5 minutes. The supernatant was collected and sterilized through a filter membrane (Millipore; 0.22 μm) to obtain the CS ion solution. Cell experiments were conducted using acinar DMEM medium (containing 2.5% fetal bovine serum, 1% penicillin-streptomycin mixture, and 0.25 mg / mL trypsin inhibitor) or DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). Different dilutions of calcium silicate ion solutions of 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, and 1 / 256 were prepared.

[0041] In this embodiment, the effects of different concentrations of CS ion solution on the cell viability of pancreatic acinar cells (PACs), macrophages (264.7), and pancreatic stellate cells (PSCs) were experimentally verified.

[0042] Specifically, in one embodiment of this application, mouse pancreatic acinar cells (PACs) are prepared from mice with severe chronic pancreatitis using a collagenase digestion method. This involves washing the pancreas twice in Hanke's balanced salt solution (HBSS), then rapidly dissecting the mouse pancreas and cutting it into 1-3 mm segments. 3Small pieces were prepared. Then, 10 mL of collagenase IA solution (HBSS 1x, containing 10 mM HEPES, 200 U / mL collagenase IA, and 0.25 mg / mL trypsin inhibitor) was added to digest the pancreatic slices. When the pancreatic tissue was completely separated, the enzyme reaction was stopped with cold buffer wash solution (HBSS 1x containing 5% fetal bovine serum (FBS) and 10 mM HEPES). The cell mixture was filtered through a 100 μm filter and seeded into 6-well plates with DMEM medium containing 2.5% FBS, 1% penicillin-streptomycin mixture, and 0.25 mg / mL trypsin inhibitor. Primary mouse pancreatic stellate cells (PSCs) were obtained from mouse pancreas. Briefly, mouse pancreatic tissue was extracted under aseptic procedures, washed with fetal bovine serum (Gibco), cut into 0.5–1 mm³ pieces, and cultured in 6-well plates. The medium was changed the next day. After primary PSCs emerged, tissue blocks were placed in new 6-well plates for further culture, followed by the culture of primary PSCs and passage. This study used PSCs passaged 2-5 times. RAW 264.7 cells were obtained from the American Cell Collection Center (ATCC, VA, USA). PSCs and Raw264.7 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humid environment with 5% CO2, with the culture medium changed every other day. Each generation of Raw264.7 cells could be used for further experiments. PACs (3*10) were then... 5 / hole), PSCs (1*10) 4 / hole) and Raw 264.7 (1*10 4 (Each well) was seeded in a 96-well plate, and the medium contained different concentrations (1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, and 1 / 256) of diluted CS ion solution. At the preset time point, the medium was removed with fresh medium containing CCK8 (Cell Counting Kit-8) assay solution (10:1), and the plates were incubated at 37°C for another 2 hours. The absorbance of the medium was measured at 450 nm using a microplate reader (SpectraMax, Molecular Devices).

[0043] In this embodiment, CS ion solution was used to perform immunofluorescence staining of α-SMA and Col1α1 on damaged pancreatic acinar cells after co-culture.

[0044] Specifically, in one embodiment of the present invention, cell slides were fixed in 4% paraformaldehyde for 30 min, washed three times with PBS, infiltrated with 1% Triton X-100 for 30 min, blocked with 5% bovine serum albumin at 37°C for 2 h, and then incubated overnight in a dark, humidified room at 4°C with anti-Col1α1 antibody (Cst, #72026, 1:100) and anti-α-smooth muscle actin antibody (Cst, #19245, 1:100). The cell sections were then incubated with secondary antibodies (Alexa Fluor488 goat anti-rabbit IgG or Cy3 goat anti-rabbit antibody (ServiceBio, China)) at room temperature in the dark for 2 hours. Cell nuclei were stained with DAPI, and cell sections were mounted with glycerol. Finally, the cell slides were sealed with glycerol. Cells were observed using a fluorescence microscope (Olympus, Japan).

[0045] This embodiment uses experiments to verify the effect of CS ion solution on inhibiting the NF-κB signaling pathway in macrophages.

[0046] Specifically, in one embodiment of this application, Raw 264.6 (1*10) 5 M1 polarized macrophages were seeded at 100 ng / mL LPS (Sigma, USA) for 1 hour in 6-well plates. The cell culture medium was then changed to CS ionized solution (1 / 8 CS and 1 / 16 CS) or control DMEM. After 24 hours, cells were harvested, and the expression of IL-1β, IL-6, TNF-α, and iNOS was detected by qRT-PCR. Total RNA was extracted from cells or mouse pancreatic tissue using Trizol reagent (Novazan, China), and its concentration was measured using a nano-titration 1000 reader (Thermo Science). 1 μg of ribonucleic acid was then reverse transcribed into cDNA using a reverse transcription kit (Novazan, China). Highly purified primers were purchased from (Shanghai Sangon Biotech, China) and processed at Light... Triple qRT-PCR amplifications were performed on a 480II system (Roche, Sandhofer, Germany).

[0047] In this embodiment, the therapeutic effect of CS ion solution on mice with chronic pancreatitis was verified through experiments.

[0048] Specifically, in one embodiment of this application, the specific steps include: selecting mild and severe pancreatitis mice (23-29g, 14-18 weeks old) from the University of Texas MD Anderson Cancer Center, and randomly dividing them into the following four groups after weight matching: (1) mild chronic pancreatitis control group (2) mild chronic pancreatitis treatment group (3) severe chronic pancreatitis control group (4) severe chronic pancreatitis treatment group. Each group of mice was given tamoxifen (3mg / 40g body weight) for 5 days to induce chronic pancreatitis gene expression, and after a 7-day rest, they were injected intraperitoneally with tamoxifen (50ug / kg, 4h / time, once every 2 days, for 4 consecutive weeks). After the last injection of tamoxifen, CS ions (8μL / g, once every 2 days, for 4 weeks) were given. Then the mice were sacrificed, and two days after the last injection of tamoxifen, histological and immunohistochemical staining was performed. The specific steps were: immediately fixing the tissue in 4% paraformaldehyde and embedding it in paraffin. Five-micrometer sections were taken and stained with hematoxylin and eosin (H&E) for histopathological evaluation. For acinar atrophy and fibrosis in chronic pancreatitis, the degree of pancreatic damage was evaluated and analyzed using optical microscopy according to previously published criteria. Immunohistochemical staining with Sirius red (ab150681, Abcam) and Ki67 (ab15580, Abcam) was performed on paraffin-embedded pancreatic sections. Macrophages were immunohistochemically stained with F4 / 80 antibody. Staining quantification was performed using ImageJ analysis software.

[0049] This embodiment uses experiments to verify the effects of CS ion solution on pancreatic acinar cell apoptosis and regeneration.

[0050] Specifically, in one embodiment of this application, pancreatic tissue is harvested post-surgery, fixed in 4% paraformaldehyde at room temperature, and then embedded in paraffin. Pancreatic samples are sectioned to 5 μm and stained using a TUNEL apoptosis detection kit (Novazan, Jiangsu, China) according to the manufacturer's protocol. Cells are counterstained with DAPI (blue) and TUNEL-positive nuclei (green). Apoptosis of PACs is observed using a fluorescence microscope (Olympus, Japan). Three regions are randomly selected from each section for total nucleus count and TUNEL+ nucleus count, and the apoptosis index (TUNEL+ nuclei / total nuclei × 100%) is calculated.

[0051] The following explanation, based on the experimental results described above, further elaborates on the application of CS ion solution in the preparation of a treatment for chronic pancreatitis in this embodiment.

[0052] It should be noted that the following examples further illustrate this application in detail. Similarly, it should be understood that the following examples are only for further explanation of this application and should not be construed as limiting the scope of protection of this application. Any non-essential improvements and adjustments made by those skilled in the art based on the above content of this application fall within the scope of protection of this application. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0053] Example 1:

[0054] Effects of different concentrations of CS ion solution on the cell viability of pancreatic acinar cells (PACs), macrophages (264.7), and pancreatic stellate cells (PSCs).

[0055] (1) Cell isolation and culture: Mouse pancreatic acinar cells (PACs) were freshly prepared from mice with severe chronic pancreatitis using collagenase digestion. Briefly, after washing twice in Hanke's balanced salt solution (HBSS), the mouse pancreas was rapidly dissected and cut into 1–3 mm³ pieces. Then, 10 mL of collagenase IA solution (HBSS 1x, containing 10 mM HEPES, 200 U / mL collagenase IA, and 0.25 mg / mL trypsin inhibitor) was added to digest the pancreatic sections. When the pancreatic tissue was completely isolated, the enzymatic reaction was stopped with cold-buffered washing solution (HBSS 1x containing 5% fetal bovine serum (FBS) and 10 mM HEPES). The cell mixture was filtered through a 100 μm filter and seeded into 6-well plates with DMEM medium containing 2.5% FBS, 1% penicillin-streptomycin mixture, and 0.25 mg / mL trypsin inhibitor. Primary mouse pancreatic stellate cells (PSCs) were obtained from the mouse pancreas. In summary, mouse pancreatic tissue was extracted under aseptic conditions, washed with fetal bovine serum (Gibco), and then cut into 0.5–1 mm³ pieces. The tissue was cultured in 6-well plates. The culture medium was changed the next day. After primary PSCs emerged, the tissue pieces were placed in new 6-well plates for further culture, and then the primary PSCs were cultured and passed through. PSCs passaged 2–5 times were used in this study. RAW264.7 cells were obtained from the American Cell Collection Center (ATCC, VA, USA). PSCs and Raw264.7 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humid environment with 5% CO₂, with the culture medium changed every other day. Each generation of Raw264.7 cells was used for further experiments.

[0056] (2) Preparation of dilution solutions: Calcium silicate (CS) ion solutions were prepared using calcium silicate (CS). Specifically, a series of gradient dilutions of calcium silicate ion extracts were prepared. In short, calcium silicate powder was added to serum-free DMEM medium (DMEM; Gibco) or PBS (Gibco) at 37°C for 24 hours at a solid / liquid ratio of 200 mg / mL. The mixture was then centrifuged at 4000 rpm for 5 minutes. The supernatant was collected and sterilized through a filter membrane (Millipore; 0.22 μm) to obtain CS ion solutions. Calcium silicate ion solutions of different dilutions (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, and 1 / 256) were prepared for cell experiments using acinar DMEM medium (containing 2.5% fetal bovine serum, 1% penicillin-streptomycin mixture, and 0.25 mg / mL trypsin inhibitor) or DMEM supplemented with 10% FBS and 1% PS.

[0057] (3) Cell viability: PACs (3*10 5 / hole), PSCs (1*10) 4 / hole) and Raw 264.7 (1*10 4 (Each well) was seeded in a 96-well plate, and the medium contained different concentrations (1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, and 1 / 256) of diluted CS ion solution. At the preset time point, the medium was removed with fresh medium containing CCK8 (Cell Counting Kit-8) assay solution (10:1), and the plates were incubated at 37°C for another 2 hours. The absorbance of the medium was measured at 450 nm using a microplate reader (SpectraMax, Molecular Devices).

[0058] The results showed that diluting CS ion solutions with DMEM at 1 / 8, 1 / 16, 1 / 32, and 1 / 64 increased PAC cell viability within 24 hours. (See [link to relevant documentation]). Figure 1 As shown in the figure. Similarly, CS ion solutions at dilutions of 1 / 4, 1 / 8, 1 / 16, 1 / 32, and 1 / 64 CS significantly stimulated the survival rate of Raw264.7 cells at 24 and 48 hours, see [reference]. Figure 2 As shown in the figure. This suggests that CS can promote the cell viability of PACs and RAW264.7 over a wide concentration range. Furthermore, compared to the control group, CS ion solution did not promote the cell viability of PSCs; see [reference needed]. Figure 3 As shown in the figure. Furthermore, in the initial concentration of CS ion solution, the cell viability of all three cell types was inhibited, mainly due to their higher pH values.

[0059] Example 2:

[0060] In this embodiment, CS ion solution was used to perform immunofluorescence staining of α-SMA and Col1α1 on damaged pancreatic acinar cells after co-culture. The experiment showed that CS ion solution reduced the production of extracellular matrix (ECM) by downregulating PSC activation through intercellular communication.

[0061] Specifically:

[0062] (1) Seed PSCs cells in 6-well plates at a rate of 1.5*106 cells / well, and then add culture medium, 1 / 64 CS ion solution, damaged PACs cells, and 1 / 64 CS ion solution + damaged PACs cells in sequence.

[0063] (2) Immunofluorescence staining of α-SMA and Col1α1: Cell culture plates were fixed in 4% paraformaldehyde for 30 min, washed three times with PBS, infiltrated with 1% Triton X-100 for 30 min, blocked with 5% bovine serum albumin at 37°C for 2 h, and then incubated overnight in a dark, humidified room at 4°C with anti-Col1α1 antibody (Cst, #72026, 1:100) and anti-α-smooth muscle actin antibody (Cst, #19245, 1:100). Cell sections were then incubated with secondary antibodies (Alexa Fluor488 goat anti-rabbit IgG or Cy3 goat anti-rabbit antibody (ServiceBio, China)) at room temperature in the dark for 2 h. Cell nuclei were stained with DAPI, and cell sections were mounted with glycerol. Finally, cell slides were mounted with glycerol. Cells were observed using a fluorescence microscope (Olympus, Japan).

[0064] The results showed that 1 / 64CS ion solution significantly reduced the concentrations of α-SMA and Col1α1. However, when PSCs were co-cultured with damaged PACs, the expression of α-SMA and Col1α2 increased compared to the control group. Conversely, 1 / 64CS ion solution significantly inhibited the excessive secretion of α-SMA and Col1α1 mediated by damaged PACs. (See [link to relevant documentation]). Figure 4 As shown in the figure. The above results indicate that CS ion solution can directly inhibit the activation of PSCs, thereby inhibiting pancreatic fibrosis.

[0065] Example 3:

[0066] In this embodiment, the effect of CS ion solution on inhibiting the NF-κB signaling pathway in macrophages was verified by experiments. The experiments showed that CS ion solution can directly or indirectly affect macrophage polarization through the NF-κB signaling pathway and intercellular communication.

[0067] Specifically:

[0068] (1) Effect of CS ion solution on inhibition of NF-κB signaling pathway in macrophages (Raw 264.7): Raw264.6 (1*10 5 M1 polarized macrophages were seeded into 6-well plates with 100 ng / mL LPS (Sigma, USA) for 1 hour. The cell culture medium was then changed to CS ionized solution (1 / 8 CS and 1 / 16 CS) or control DMEM. After 24 hours, cells were harvested, and the expression of IL-1β, IL-6, TNF-α, and iNOS was detected by qRT-PCR.

[0069] (2) Quantitative Real-Time PCR: Total RNA was extracted from cells or mouse pancreatic tissue using Trizol reagent (Novazan, China), and its concentration was measured using a Nanotitling 1000 reader (Thermo Science). Then, 1 μg of ribonucleic acid was reverse transcribed into cDNA using a reverse transcription kit (Novazan, China). Highly purified primers were purchased from (Shanghai Sangon Biotech, China) and processed using Light... Triple qRT-PCR amplifications were performed on a 480II system (Roche, Sandhofer, Germany).

[0070] from Figure 3 It was found that 1 / 8CS ion solution significantly reduced the mRNA levels of IL-1β, TNF-α, and iNOS in Raw264.7. (See [link to relevant documentation]). Figure 5 As shown in the figure, this demonstrates that CS ion solution can regulate M1 polarization of macrophages by inhibiting the NF-κB signaling pathway.

[0071] Example 4:

[0072] In this embodiment, the therapeutic effect of CS ion solution on mice with chronic pancreatitis was verified by experiments. The experiments showed that CS ion solution reduced acinar atrophy and fibrosis in mice with different degrees of chronic pancreatitis (CP).

[0073] Specifically:

[0074] (1) Establishment, treatment, and performance evaluation of animal models: Mild and severe pancreatitis mice (23-29g, 14-18 weeks old) were obtained from the University of Texas MD Anderson Cancer Center and randomly divided into four groups after weight matching: mild chronic pancreatitis control group, mild chronic pancreatitis treatment group, severe chronic pancreatitis control group, and severe chronic pancreatitis treatment group. Each group of mice was given tamoxifen (3mg / 40g body weight) for 5 days to induce chronic pancreatitis gene expression. After a 7-day rest period, they were intraperitoneally injected with tamoxifen (50ug / kg, 4h / time, every 2 days for 4 weeks). Following the last tamoxifen injection, mice were given CS ions (8μL / g, every 2 days for 4 weeks). The mice were then sacrificed, and analysis was performed 2 days after the last tamoxifen injection. The experimental procedures and the ethical handling of the mice were approved by the Animal Ethics Committee of Changhai Hospital.

[0075] (2) Histological and Immunohistochemical Staining: Tissues were immediately fixed in 4% paraformaldehyde and embedded in paraffin. Sections were taken at 5 μm for histopathological evaluation using hematoxylin and eosin (H&E) staining. For acinar atrophy and fibrosis in chronic pancreatitis, the extent of pancreatic damage was evaluated using optical microscopy according to previously published criteria. Immunohistochemical staining with Sirius Red (ab150681, Abcam) and Ki67 (ab15580, Abcam) was performed on paraffin sections of the pancreas. Macrophages were immunohistochemically stained with F4 / 80 antibody. Staining quantification was performed using ImageJ analysis software.

[0076] from Figure 6 As can be seen, when mice developed mild and severe chronic pancreatitis, the weight of these mice in the control group gradually decreased. Conversely, CS ion solution significantly prevented weight loss in mice with severe chronic pancreatitis. This is because LSL / Cre mice had more severe CP than Cre mice, which made the weight loss trend more pronounced. See [link to relevant documentation]. Figure 6 As shown in Figure A. In the optical photographs, we can clearly observe that the CS ion solution effectively prevented pancreatic atrophy, both in mice with mild and severe chronic pancreatitis. See Figure A. Figure 6 As shown in B. Quantitative statistical results also showed that, compared with the CS group, the PBS group had a significantly lower pancreatic weight / body weight percentage (0.376 ± 0.03% in Cre mice and 0.51% ± 0.03% in LSL / Cre mice). More importantly, the pancreatic weight / body weight percentage in the CS group was close to the normal value (1%), indicating that CS ionized solution can alleviate pancreatic damage during CP, see [reference]. Figure 6As shown in Figure C. Histopathological changes in the pancreas of mice in each group were examined by H&E staining. The results showed that, compared with the CS treatment group, the control group had a large number of inflammatory cells infiltrating the pancreas, leading to fibrotic lesions and atrophy of the acinar tissue. (See Figure C for details.) Figure 6 As shown in D. Furthermore, compared to the PBS group, the CS ion solution significantly reduced Sirius red positive staining in pancreatic tissue, indicating that CS can effectively inhibit excessive collagen deposition to avoid pancreatic fibrosis. See [reference needed]. Figure 6 As shown in E.

[0077] Example 5:

[0078] In this embodiment, the effects of CS ion solution on pancreatic acinar cell apoptosis and regeneration were verified by experiments. The experimental results showed that CS ion solution reduced pancreatic acinar cell (PAC) apoptosis and promoted regeneration.

[0079] Specifically:

[0080] (1) TUNEL staining analysis: TUNEL assay was used to detect apoptosis of mouse pancreatic acinar cells (PACs). Pancreatic tissue was harvested after surgery, fixed in 4% paraformaldehyde at room temperature, and then embedded in paraffin. Pancreatic samples were sectioned to 5 μm and stained with a TUNEL apoptosis detection kit (Novazan, Jiangsu, China) according to the manufacturer's protocol. Cells were counterstained with DAPI (blue) and TUNEL-positive nuclei (green). TUNEL staining was used to identify apoptotic PACs, and Ki67 staining was used to label regenerating PACs. Apoptosis of PACs was observed using a fluorescence microscope (Olympus, Japan). Three regions were randomly selected from each section for total nucleus count and TUNEL+ nucleus count, and the apoptosis index (TUNEL+ nucleus count / total nucleus count × 100%) was calculated.

[0081] (2) Immunohistochemical staining with Ki67 (ab15580, Abcam) was performed on paraffin sections of the pancreas. The staining was quantified using ImageJ analysis software.

[0082] from Figure 7 As can be seen, CS ion solution significantly reduced TUNEL-positive cells in mice with mild and severe chronic pancreatitis. (See [reference]) Figure 7 As shown in A, and with the addition of Ki67-positive cells, see [reference]. Figure 7 B and Figure 7 As shown in C.

[0083] Example 6:

[0084] This embodiment also verified that the inhibition of macrophage infiltration and M1 polarization by CS ion solution is closely related to the progression of pancreatic fibrosis.

[0085] Specifically:

[0086] (1) The tissue was fixed in 4% paraformaldehyde and embedded in paraffin. 5 μm sections were taken, and macrophages were immunohistochemically stained with F4 / 80 antibody. The staining was quantified using ImageJ analysis software.

[0087] from Figure 8 It can be seen that CS ion solution significantly reduced F4 / 80 positive cells in Cre and LSL / Cre mice. (See [reference needed]) Figure 8 As shown in Figure A. Quantitative analysis showed that, compared with the PBS group, Cre mice and Cre / LSL mice with positive staining regions of F4 / 80 had a reduction of CS by more than 9.8 times. See Figure A. Figure 8 As shown in B.

[0088] In summary, the embodiments of this application provide a silicon ion preparation for treating chronic pancreatitis, its preparation method, and its application. This silicon ion preparation exhibits excellent effects in promoting the regeneration of pancreatic acinar cells and inhibiting inflammation and fibrosis in pancreatic tissue. Silicon mineral preparations prepared at this concentration can be used to treat chronic pancreatitis and prevent fibrosis, showing great application potential. Furthermore, the silicon ion preparation provided in this embodiment can treat chronic pancreatitis and prevent fibrosis; such active materials can significantly treat the activity of pancreatic cells, aiming to form healthy pancreatic tissue.

[0089] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this application.

Claims

1. The application of a silicon ion preparation in the preparation of a treatment for chronic pancreatitis, wherein the concentration of silicon ions in the silicon ion preparation is 5 ppm-800 ppm, the silicon ion preparation is prepared by releasing silicon ions from calcium silicate, and the preparation method of the silicon ion preparation is as follows: Calcium silicate powder was added to serum-free DMEM or PBS at 37°C for 24 hours at a solid / liquid ratio of 200 mg / mL. The mixture was then centrifuged at 4000 rpm for 5 minutes, and the supernatant was collected and sterilized through a filter membrane to obtain a calcium silicate ion solution.

2. The application according to claim 1, characterized in that, The chronic pancreatitis includes one or more of the following: chronic calcific pancreatitis, chronic obstructive pancreatitis, and chronic inflammatory pancreatitis.

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