Application of Xuebijing injection in preparation of medicine for improving survival of pancreatic beta cells in inflammatory microenvironment

Abstract

The invention relates to the technical field of crossing of traditional Chinese medicine and modern transplantation medicine, in particular to application of Xuebijing injection in preparation of a medicine for improving pancreatic beta cell survival in an inflammatory microenvironment. Aiming at a core pathological link of inducing beta cell apoptosis by inflammatory factor storm (IL-1beta / TNF-alpha / IFN-gamma) in pancreatic islet transplantation, the survival rate and the function of beta cells in an inflammatory microenvironment are remarkably improved by inhibiting expression of apoptosis key proteins such as Cleved Caspase-3 / PARP, and an innovative treatment strategy is provided for relieving pancreatic islet inflammatory injury after clinical pancreatic islet transplantation.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of traditional Chinese medicine and modern transplantation medicine, and in particular to the application of Xuebijing injection in the preparation of drugs that improve the survival of pancreatic β cells in an inflammatory microenvironment. Background Technology

[0002] Islet transplantation has become a promising treatment for patients with type 1 diabetes (T1D), potentially enabling insulin independence and improving glycemic control. Despite these encouraging results, early post-transplantation islet damage due to inflammatory responses can impair graft function and long-term transplant success rates. Early innate immune responses reduce the overall efficacy of islet transplantation. Although advances in immunosuppressive therapy and anti-inflammatory strategies have been made, islet damage caused by inflammation cannot be completely avoided. Furthermore, most current drugs are monoclonal antibodies, which are expensive, require stringent storage conditions, and carry the risk of immunosuppression. In particular, intrahepatic islet transplantation via the portal vein releases pro-inflammatory factors such as IL-1β, TNF-α, and IFN-γ locally, activating apoptosis-related pathways and inducing islet cell apoptosis.

[0003] Xuebijing Injection is a compound traditional Chinese medicine injection extracted from safflower, red peony root, chuanxiong rhizome, danshen root, and angelica root. It is a clinically approved standardized Chinese herbal extract with clear multi-target anti-inflammatory and organ-protective effects. It has been included in the National Diagnosis and Treatment Protocol for Novel Coronavirus Infection, significantly reducing the mortality rate of critically ill patients and improving multi-organ function. However, existing research focuses on indications such as sepsis and COVID-19 infection, and its mechanism of action and application in the field of islet transplantation have not yet been systematically explored.

[0004] Based on the clarified pharmacodynamic material basis of Xuebijing, this invention is the first to apply it to the field of islet transplantation. By inhibiting β-cell apoptosis in the inflammatory microenvironment, it breaks through the efficacy limitations of existing single-target drugs and provides a "traditional Chinese and Western medicine integration" solution for the prevention and treatment of post-transplant islet inflammatory damage that is both highly effective and safe. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides the application of Xuebijing injection in the preparation of drugs that improve pancreatic β-cell survival in an inflammatory microenvironment. This invention targets the core pathological step of β-cell apoptosis induced by cytokine storm (IL-1β / TNF-α / IFN-γ) during islet transplantation. By inhibiting the expression of key apoptosis proteins such as Cleaved Caspase-3 / PARP, it significantly improves the survival rate and function of β-cells in an inflammatory microenvironment, providing an innovative treatment strategy for alleviating islet inflammation damage after clinical islet transplantation.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention also provides the application of Xuebijing injection in the preparation of drugs that improve pancreatic β-cell survival in an inflammatory microenvironment.

[0008] Preferably, the dosage of the Xuebijing injection is 50 μL / mL.

[0009] Preferably, the inflammatory microenvironment is induced by inflammatory factors.

[0010] Preferably, the inflammatory factors include IL-1β, TNF-α, and IFN-γ.

[0011] Preferably, the amount of IL-1β used is 10 ng / mL, the amount of TNF-α used is 25 ng / mL, and the amount of IFN-γ used is 100 ng / mL.

[0012] Preferably, the induction time is 24 hours.

[0013] The beneficial effects of this invention are:

[0014] This invention targets the core pathological link of cytokine storm (IL-1β / TNF-α / IFN-γ)-induced β-cell apoptosis in islet transplantation. By inhibiting the expression of key apoptosis proteins such as Cleaved Caspase-3 / PARP, it significantly improves the survival rate and function of β cells in the inflammatory microenvironment, providing an innovative treatment strategy to alleviate islet inflammation damage after clinical islet transplantation. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 The results of CCK8 in in vitro NIT cell lines treated with Xuebijing under the stimulation of inflammatory factors, as well as the function of NIT cells and the transcription level of inflammation-related genes;

[0017] Figure 2 The expression of apoptosis-related proteins in in vitro NIT cell lines after treatment with Xuebijing under the stimulation of inflammatory factors, and the quantitative statistical graph;

[0018] Figure 3 The apoptosis of mouse islet cell clusters after treatment with Xuebijing under in vitro inflammatory cytokine stimulation, and the quantitative statistical graphs;

[0019] Figure 4 Changes in the transcriptional levels of β-cell-related functional genes and inflammation-related genes in mouse pancreatic islets after treatment with Xuebijing under inflammatory cytokine stimulation.

[0020] Terminology Explanation:

[0021] CCK-8: Cell Counting Kit-8 is a cell proliferation and activity assay kit based on water-soluble tetrazolium salt. It indirectly reflects the number and status of live cells by measuring cell metabolic activity.

[0022] TUNEL: Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling; TUNEL staining is a classic molecular biology technique used to detect apoptosis. Its core principle is to visualize the location and number of apoptotic cells under a microscope by labeling broken DNA strands. Detailed Implementation

[0023] This invention provides the application of Xuebijing injection (hereinafter referred to as XBJ) in the preparation of drugs to improve pancreatic β-cell survival in an inflammatory microenvironment. In this invention, the Xuebijing injection was purchased from China Resources Tianjin Pharmaceutical Co., Ltd. In this invention, the preferred dosage of the Xuebijing injection is 50 μL / mL.

[0024] In this invention, the inflammatory microenvironment is preferably induced by inflammatory factors. In this invention, the inflammatory factors preferably include IL-1β, TNF-α, and IFN-γ. In this invention, the amount of IL-1β used is preferably 10 ng / mL, the amount of TNF-α used is preferably 25 ng / mL, and the amount of IFN-γ used is preferably 100 ng / mL. In this invention, the induction time is preferably 24 h.

[0025] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1

[0027] In vitro NIT cell apoptosis inhibition experiment

[0028] Materials and Methods

[0029] 1. Cell Culture: NIT-1 cells (purchased from the American Type Culture Collection (ATCC)) were cultured in DMEM / F-12 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) in a 5% CO2 incubator at a constant temperature of 37°C. After passage, NIT-1 cells in the exponential growth phase were harvested and the cell density was adjusted to 8 × 10⁶ cells / year. 5 Inflammatory factor damage was induced after inoculating the cells at a density of 1 / mL into 6-well plates and incubating them at 37°C in a 5% CO2 incubator for 24 h.

[0030] 2. Grouping and Treatment: ① Blank control group (no treatment); ② Inflammation model group (20 ng / mL TNF-α + 10 ng / mL IL-1β + 100 ng / mL IFN-γ); ③ XBJ treatment group (25 ng / mL TNF-α + 10 ng / mL IL-1β + 100 ng / mL IFN-γ model + 50 μL / mL Xuebijing injection). Adherent cells in the logarithmic growth phase were digested, resuspended, and counted. They were then seeded at an appropriate density in cell culture plates and cultured at 37°C, 5% CO2, until approximately 70% confluence or the cells entered the stationary phase. Subsequently, the required concentrations of inflammatory factors (TNF-α, IL-1β, IFN-γ) working solutions were aseptically prepared. The old culture medium in the original culture plates was discarded and replaced with fresh DMEM / F-12 complete medium containing 10% FBS and specific concentrations of inflammatory factors. The XBJ treatment group also received the corresponding concentration of Xuebijing injection. Return the cells to the incubator and continue culturing for 24 hours. After treatment, discard the culture medium containing the factor, gently wash the cells 1-2 times with pre-cooled PBS, and then collect the cells using TRIzol for subsequent RNA extraction.

[0031] 3. Detection of inflammation and functional gene transcription expression levels: After discarding the culture medium and washing the cells, add an appropriate amount of TRIzol reagent to the cells in the culture plate, mix well by pipetting, and transfer the lysate to an RNase-free centrifuge tube; then add chloroform at a ratio of 0.2 mL chloroform per 1 mL TRIzol, vortex vigorously to mix, and let stand at room temperature for 5 minutes; then centrifuge at 4℃, 12,000 g for 15 minutes, and carefully aspirate the colorless aqueous phase to a new tube; add an equal volume of pre-chilled isopropanol, mix well, and let stand at room temperature for 10 minutes to precipitate RNA; centrifuge again at 4℃, 12,000 g for 10 minutes, and a small amount of white RNA precipitate will be visible at the bottom of the tube; discard the supernatant, add 1 mL of pre-chilled 75% ethanol to gently wash the precipitate; finally, centrifuge at 4℃, 7,500 g for 5 minutes, discard the ethanol, dry the precipitate at room temperature for 5-10 minutes, dissolve the RNA precipitate with an appropriate amount of RNase-free water, determine the concentration and purity, and store at -80℃. Subsequently, reverse transcription and real-time quantitative PCR (qRT-PCR) were used to analyze the mRNA expression levels of inflammatory factors such as Mafa, TNF-α, and Ccl-2, with β-actin as an internal reference gene.

[0032] 4. Detection of Apoptosis-Related Protein Expression Levels: NIT cells were lysed using RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor), and the supernatant was collected by centrifugation. Protein concentration was quantified using the BCA method. After denaturation at 95°C, proteins were transferred to a PVDF membrane via 10% SDS-PAGE gel electrophoresis and wet transfer. The membrane was blocked with 5% skim milk for 1 h, followed by incubation with primary antibody overnight at 4°C and HRP-labeled secondary antibody for 1 h at room temperature. ECL chemiluminescence imaging was performed, and the grayscale ratio of the target protein to the internal control β-actin was quantified using ImageJ software. Results are expressed as relative expression levels. This method was used to verify the regulatory effect of the drug on apoptosis proteins, supporting the core mechanism of "reducing β-cell apoptosis" in the claims.

[0033] 5. CCK8 assay for cell viability: NIT cells were cultured at 5 × 10⁻⁶ cells / mL. 4 The cells were seeded at a density of 1 cell per well in a 96-well plate and cultured for 24 hours. Then, the cells were cultured for another 24 hours according to the grouping treatment mentioned above. 10% volume of CCK-8 solution was added to each well and the cells were incubated at 37°C for 1.5 hours in the dark. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader.

[0034] 6. Data Analysis: Statistical analysis was performed using Graphpadprism 10 software. A p-value < 0.05 was considered statistically significant.

[0035] result:

[0036] To simulate the inflammatory microenvironment encountered by transplanted islets, this embodiment employed an in vitro TNF-α+IL-1β+IFN-γ stimulation model. Under cytokine stimulation, Xuebijing effectively reduced NIT cell damage caused by cytokine stimulation, as measured by CCK8. Figure 1 (A). Furthermore, Xuebijing can upregulate the expression of the Mafa gene, which is related to β-cell function, and downregulate the expression of the pro-inflammatory cytokine genes TNF-α and Ccl-2. Figure 1 (BD).

[0037] The apoptosis markers cleaved PARP and cleaved caspase-3 were detected by Western blotting. Compared with the control group, the expression of apoptosis markers was significantly increased in NIT cells treated with cytokines, but the expression rate was significantly decreased in the Xuebijing treatment group. Figure 2 ).

[0038] Example 2

[0039] In vitro mouse pancreatic islet cell apoptosis inhibition experiment

[0040] 1. Islet Isolation and Purification: Pancreatic Perfusion and Isolation: 8-10 week old C57BL / 6 mice were euthanized by cervical spondylosis and placed supine on the operating table of a surgical microscope. The abdominal skin was disinfected with 75% alcohol. A V-shaped incision was made in the abdomen to fully expose the abdominal cavity. Under the microscope, the duodenum was located, and the junction of the duodenum and common bile duct was found and clamped with hemostatic forceps. The porta hepatis was fully exposed, and the common bile duct was freed. A 25G syringe needle was connected to a 5mL syringe, 5mL of collagenase P solution was drawn up, and the tip of the needle was bent at a 15° angle and inserted into the common bile duct. 4mL of collagenase P solution was injected to fully perfuse the pancreas. The pancreas was bluntly dissected from the duodenum, stomach, and spleen. The entire pancreas was cut off and placed in a 50ml sterile centrifuge tube and placed on ice. Pancreas digestion: The centrifuge tube containing the mouse pancreas was placed in a 37℃ water bath for 12 minutes. Then, the centrifuge tube was quickly removed, wiped with 75% alcohol, and transferred to a clean bench. 20mL of pre-cooled 1640 solution containing 10% fetal bovine serum was added to stop digestion. The centrifuge tube was quickly manually shaken 40 times. 30mL of 1640 solution containing 10% fetal bovine serum at 4℃ was added, and the mixture was centrifuged at 4℃, 1000r / min for 1 minute. Discard the supernatant, add 10 ml of pre-cooled RPMI-1640 solution containing 10% fetal bovine serum, and resuspend using a sterile Pasteur pipette. Filter the suspension through a 40-mesh sieve. Combine the suspensions of 3-4 pancreas in a centrifuge tube, centrifuge at 1000 rpm for 1 min at 4°C, and discard the supernatant. Islet purification: Resuspend the above precipitate in 15 mL of Histopaque-1077 solution, and resuspend using a sterile Pasteur pipette. Slowly add 10 mL of RPMI-1640 culture medium solution along the wall of the centrifuge tube. Once the solution in the centrifuge tube separates into layers, centrifuge at 2400 rpm for 20 min at 25°C. Slowly remove the centrifuge tube; the islets will be visible suspended in the lower Histopaque-1077 solution layer. Using a power pipette, transfer the Histopaque-1077 solution containing islets from the lower layer to another 50 mL sterile centrifuge tube. Add 35-40 mL of pre-chilled 1640 solution containing 10% fetal bovine serum (FBS), and centrifuge at 1000 rpm for 1 min at 4 °C. Islets will precipitate after centrifugation. Wash once more with pre-chilled 1640 solution containing 10% FBS. Finally, transfer the islets to a cell culture dish and further purify them manually under an optical microscope. Culture in 1640 solution containing 10% FBS until ready for use.

[0041] 2. Grouping and Treatment: ① Blank control group (no treatment); ② Inflammation model group (20 ng / mL TNF-α + 10 ng / mL IL-1β + 100 ng / mL IFN-γ); ③ XBJ treatment group (25 ng / mL TNF-α + 10 ng / mL IL-1β + 100 ng / mL IFN-γ model + 50 μL / mL Xuebijing injection). Primary mouse islet cells were isolated and extracted and seeded at an appropriate density in non-adhesive cell culture plates, and cultured for 12 h at 37℃ and 5% CO2. Subsequently, the required concentrations of inflammatory factors (TNF-α, IL-1β, IFN-γ) working solutions were aseptically prepared, and the old culture medium in the original culture plates was discarded and replaced with fresh 1640 complete medium containing 10% FBS and specific concentrations of inflammatory factors. The XBJ treatment group simultaneously received the corresponding concentration of Xuebijing injection. Return the cells to the incubator and continue culturing for 24 hours. After treatment, discard the culture medium containing the factor, gently wash the cells 1-2 times with pre-cooled PBS, and fix the pancreatic islet cells in 4% paraformaldehyde.

[0042] 3. TUNEL staining to detect pancreatic islet cell apoptosis: Islet cell clusters fixed with 4% paraformaldehyde were washed with PBS and permeabilized with 1% Triton X-100, then equilibrated with Equilibration Buffer. Incubation buffer containing rTdT enzyme and fluorescently labeled -dUTP was then added, and the mixture was incubated at 37°C in a humidified chamber for 60 minutes to specifically incorporate the fluorescent label into the DNA fragments of apoptotic cells. After the reaction, the incubation was terminated with 2X SSC and the cells were thoroughly washed with PBS. The cells were then incubated with DAPI staining solution for 15 minutes, washed with PBS, and the autofluorescence was quenched. Images were taken using a Nikon A1+ confocal microscope, and the proportion of apoptotic cells within the islets in different groups was recorded.

[0043] 6. Data Analysis: Statistical analysis was performed using Graphpadprism 10 software. A p-value < 0.05 was considered statistically significant.

[0044] result:

[0045] Since β cells constitute the absolute majority of pancreatic islet cell clusters and are the main targets of inflammatory damage, the effect of Xuebijing injection on islet cell apoptosis induced by inflammatory factors was assessed using TUNEL staining. TUNEL staining showed that the number of TUNEL-positive transplanted islet cells in the XBJ treatment group was significantly lower than that in the control group. Figure 3 (A, B). This finding corroborates our functional experimental results, and the reduction in apoptosis directly explains the mechanism of its functional improvement, further supporting the conclusion that Xuebijing has a protective effect on β cells.

[0046] Example 3

[0047] In vitro mouse pancreatic β-cell insulin secretion function protection experiment

[0048] 1. Islet Isolation and Purification: Pancreatic Perfusion and Isolation: 8-10 week old C57BL / 6 mice were euthanized by cervical spondylosis and placed supine on the operating table of a surgical microscope. The abdominal skin was disinfected with 75% alcohol. A V-shaped incision was made in the abdomen to fully expose the abdominal cavity. Under the microscope, the duodenum was located, and the junction of the duodenum and common bile duct was found and clamped with hemostatic forceps. The porta hepatis was fully exposed, and the common bile duct was freed. A 25G syringe needle was connected to a 5mL syringe, 5mL of collagenase P solution was drawn up, and the tip of the needle was bent at a 15° angle and inserted into the common bile duct. 4mL of collagenase P solution was injected to fully perfuse the pancreas. The pancreas was bluntly dissected from the duodenum, stomach, and spleen. The entire pancreas was cut off and placed in a 50ml sterile centrifuge tube and placed on ice. Pancreas digestion: The centrifuge tube containing the mouse pancreas was placed in a 37℃ water bath for 12 minutes. Then, the centrifuge tube was quickly removed, wiped with 75% alcohol, and transferred to a clean bench. 20mL of pre-cooled 1640 solution containing 10% fetal bovine serum was added to stop digestion. The centrifuge tube was quickly manually shaken 40 times. 30mL of 1640 solution containing 10% fetal bovine serum at 4℃ was added, and the mixture was centrifuged at 4℃, 1000r / min for 1 minute. Discard the supernatant, add 10 ml of pre-cooled RPMI-1640 solution containing 10% fetal bovine serum, and resuspend using a sterile Pasteur pipette. Filter the suspension through a 40-mesh sieve. Combine the suspensions of 3-4 pancreas in a centrifuge tube, centrifuge at 1000 rpm for 1 min at 4°C, and discard the supernatant. Islet purification: Resuspend the above precipitate in 15 mL of Histopaque-1077 solution, and resuspend using a sterile Pasteur pipette. Slowly add 10 mL of RPMI-1640 culture medium solution along the wall of the centrifuge tube. Once the solution in the centrifuge tube separates into layers, centrifuge at 2400 rpm for 20 min at 25°C. Slowly remove the centrifuge tube; the islets will be visible suspended in the lower Histopaque-1077 solution layer. Using a power pipette, transfer the Histopaque-1077 solution containing islets from the lower layer to another 50 mL sterile centrifuge tube. Add 35-40 mL of pre-chilled 1640 solution containing 10% fetal bovine serum (FBS), and centrifuge at 1000 rpm for 1 min at 4 °C. Islets will precipitate after centrifugation. Wash once more with pre-chilled 1640 solution containing 10% FBS. Finally, transfer the islets to a cell culture dish and further purify them manually under an optical microscope. Culture in 1640 solution containing 10% FBS until ready for use.

[0049] 2. Grouping and Treatment: ① Blank control group (no treatment); ② Inflammation model group (20 ng / mL TNF-α + 10 ng / mL IL-1β + 100 ng / mL IFN-γ); ③ XBJ treatment group (25 ng / mL TNF-α + 10 ng / mL IL-1β + 100 ng / mL IFN-γ model + 50 μL / mL Xuebijing injection). Primary mouse islet cells were isolated and extracted and seeded at an appropriate density in non-adhesive cell culture plates, and cultured for 12 h at 37℃ and 5% CO2. Subsequently, the required concentrations of inflammatory factors (TNF-α, IL-1β, IFN-γ) working solutions were aseptically prepared, and the old culture medium in the original culture plates was discarded and replaced with fresh 1640 complete medium containing 10% FBS and specific concentrations of inflammatory factors. The XBJ treatment group simultaneously received the corresponding concentration of Xuebijing injection. The cells were returned to the incubator and cultured for another 24 hours. After the treatment, the culture medium containing the factor was discarded, and the pancreatic islets were gently washed 1-2 times with pre-cooled PBS. Then, the cells were collected using TRIzol for subsequent RNA extraction and transcriptome sequencing.

[0050] 3. Transcriptome Sequencing: Total RNA was extracted using TRIzol reagent, and RNA quality was assessed using NanoDrop ND-1000. RNA integrity (RIN value > 7.0) was analyzed using an Agilent 2100 Bioanalyzer, and further validated by denaturing agarose gel electrophoresis. cDNA libraries were constructed using qualified, high-quality RNA samples, and then 2×150bp paired-end sequencing was performed on an Illumina Novaseq 6000 platform. Bioinformatics analysis was performed using the OmicStudio online tool. Differentially expressed genes (DEGs) were identified using DESeq2 software, with a screening threshold set at a false discovery rate (FDR) < 0.05 and log2|fold change| ≥ 2. All figures were generated based on R4.1.3 on the OmicStudio platform. 4. Data Analysis: Statistical analysis was performed using Graphpadprism 10 software. P < 0.05 was considered statistically significant.

[0051] result:

[0052] To simulate the inflammatory microenvironment encountered by transplanted islets, this embodiment employed an in vitro TNF-α+IL-1β+IFN-γ stimulation model. Under cytokine stimulation, Xuebijing upregulated the expression of multiple genes related to pancreatic β-cell function and downregulated the expression of pro-inflammatory cytokine genes such as CXCL-10 and IL-6. Figure 4 )

[0053] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of Xuebijing Injection in the preparation of drugs that improve pancreatic β-cell survival in an inflammatory microenvironment.

2. The application according to claim 1, characterized in that, The dosage of Xuebijing injection is 50 μL / mL.

3. The application according to claim 1, characterized in that, The inflammatory microenvironment is induced by inflammatory factors.

4. The application according to claim 3, characterized in that, The inflammatory factors include IL-1β, TNF-α, and IFN-γ.

5. The application according to claim 4, characterized in that, The dosage of IL-1β is 10 ng / mL, the dosage of TNF-α is 25 ng / mL, and the dosage of IFN-γ is 100 ng / mL.

6. The application according to claim 3, characterized in that, The induction time is 24 hours.

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