Application of Xuebijing Injection in the Preparation of Drugs for Inhibiting Immediate Blood-Mediated Inflammatory Response Following Portal Vein Islet Transplantation

Abstract

This invention relates to the interdisciplinary field of transplant medicine and pharmacology, and particularly to the application of Xuebijing injection in the preparation of drugs that inhibit the immediate blood-mediated inflammatory response after portal vein islet transplantation. This invention innovatively proposes its application in the prevention and treatment of the immediate blood-mediated inflammatory response in islet transplantation. By synergistically inhibiting complement activation, coagulation cascade, and inflammatory cell infiltration through multiple targets, it overcomes the efficacy bottleneck of traditional single anticoagulation or anti-inflammatory strategies, providing a patented 'integrated traditional Chinese and Western medicine' solution with both highly efficient regulation and safety advantages for overcoming critical early post-transplant inflammatory damage.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of transplantation medicine and pharmacology, and in particular to the application of Xuebijing injection in the preparation of drugs that inhibit immediate blood-mediated inflammatory responses after portal vein islet transplantation. Background Technology

[0002] Xuebijing Injection is a compound preparation composed of extracts from five traditional Chinese medicines: safflower, red peony root, chuanxiong rhizome, salvia miltiorrhiza root, and angelica sinensis root. Developed based on the theory of "treating both bacteria, toxins, and inflammation," it obtained a Class II New Drug Certificate in 2004 and was approved for the treatment of sepsis and multiple organ dysfunction syndrome (MODS). Its core pharmacological effects include: multi-target anti-inflammatory effects: inhibiting the release of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6, downregulating the expression of adhesion molecules such as ICAM-1, and blocking leukocyte infiltration; regulation of the NF-κB pathway: reducing systemic inflammatory response syndrome by inhibiting IκBα phosphorylation and p65 nuclear translocation; organ protection: demonstrating its ability to reduce damage to multiple organs such as the heart, kidneys, and liver in models of sepsis and severe COVID-19.

[0003] In the field of islet transplantation, the pathological mechanism of immediate blood-mediated inflammation involves a cascade amplification of coagulation, inflammation, and complement, requiring multi-target intervention. Immediate blood-mediated inflammation leads to over 50% islet graft loss shortly after transplantation, thereby impairing graft function and long-term transplant success rates. Early innate immune responses reduce the overall surgical outcome. Despite advances in immunosuppressive therapy and anti-inflammatory strategies, immediate blood-mediated inflammation remains a major challenge in improving islet graft survival.

[0004] When islet grafts come into contact with the bloodstream, they immediately trigger an inflammatory response characterized by the release of pro-inflammatory cytokines and the activation of immune cells such as neutrophils and macrophages. This innate immune activation has a direct cytotoxic effect on islet cells and also promotes the initiation of an adaptive immune response. Simultaneously, the interaction between inflammation and coagulation creates a cascade amplification effect, where each pathway enhances the others, leading to rapid and significant islet loss. This synergistic amplification effect is key to the destructive nature of the immediate blood-mediated inflammatory response and underscores the urgent need for novel interventions capable of interrupting this cycle and improving the efficacy of islet transplantation.

[0005] Xuebijing injection has demonstrated potent anti-inflammatory and antithrombotic effects in diseases such as sepsis and acute lung injury. In clinical applications, Xuebijing has shown significant efficacy in treating various acute inflammatory injuries, especially in cases of severe infections (such as sepsis), but its application in islet transplantation has not yet been investigated. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides the application of Xuebijing injection in the preparation of drugs that inhibit blood-mediated inflammatory responses immediately following portal vein islet transplantation. This invention innovatively proposes its application in the prevention and treatment of blood-mediated inflammatory responses immediately following islet transplantation. By synergistically inhibiting complement activation, coagulation cascade, and inflammatory cell infiltration through multiple targets, it overcomes the efficacy bottleneck of traditional single anticoagulation or anti-inflammatory strategies, providing a patented 'integrated traditional Chinese and Western medicine' solution with both high-efficiency regulation and safety advantages for overcoming critical early post-transplant inflammatory damage.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of Xuebijing injection in the preparation of a drug that inhibits the immediate blood-mediated inflammatory response after portal vein islet transplantation.

[0009] Preferably, the Xuebijing injection inhibits thrombus deposition around the islets after portal vein islet transplantation.

[0010] Preferably, the Xuebijing injection inhibits leukocyte infiltration around the islets after portal vein islet transplantation.

[0011] Preferably, the Xuebijing injection inhibits the mRNA expression level of inflammation-related factors at the transplantation site after portal vein islet transplantation.

[0012] Preferably, the inflammation-related factors include TNF-α, HMGB1, IL-6, COX-2, and MIP-1β.

[0013] Preferably, the dosage of the Xuebijing injection is 16µL / g.

[0014] The beneficial effects of this invention are:

[0015] This invention targets the core pathological mechanism of "coagulation-inflammation cascade amplification" in the immediate blood-mediated inflammatory response (tissue factor-mediated platelet aggregation, leukocyte recruitment around pancreatic islet grafts, and upregulation of inflammatory gene transcription levels at the transplantation site). Through the synergistic effect of multiple components of Xuebijing, it reduces post-transplant thrombosis and inflammatory response.

[0016] This invention innovatively proposes to apply it to the prevention and treatment of immediate blood-mediated inflammatory responses in islet transplantation. By synergistically inhibiting complement activation, coagulation cascade reactions, and inflammatory cell infiltration through multiple targets, it breaks through the efficacy bottleneck of traditional single anticoagulation or anti-inflammatory strategies. It provides a patented 'integrated traditional Chinese and Western medicine' solution with both high-efficiency regulation and safety advantages for overcoming critical inflammatory damage in the early post-transplantation period. Attached Figure Description

[0017] 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.

[0018] Figure 1 HE staining results of liver sections after islet transplantation via portal vein, as well as thrombotic infiltration around the islets and scoring statistics.

[0019] Figure 2 The image shows the results of CD45 immunohistochemical staining of liver sections after pancreatic islet transplantation via the portal vein, as well as a statistical graph of the number of CD45-positive infiltrating cells.

[0020] Figure 3 This is the result of detecting the transcriptional level of inflammation-related genes at the islet transplantation site after islet transplantation via the portal vein. Detailed Implementation

[0021] This invention provides the use of Xuebijing injection in the preparation of a medicament for inhibiting the immediate blood-mediated inflammatory response following portal vein islet transplantation. In this invention, the Xuebijing injection preferably inhibits periislet thrombus deposition after portal vein islet transplantation. In this invention, the Xuebijing injection preferably inhibits periislet leukocyte infiltration after portal vein islet transplantation. In this invention, the Xuebijing injection preferably inhibits the mRNA expression levels of inflammation-related factors at the transplantation site after portal vein islet transplantation. In this invention, the inflammation-related factors preferably include TNF-α, HMGB1, IL-6, COX-2, and MIP-1β. In this invention, the preferred dosage of the Xuebijing injection is 16 µL / g.

[0022] In this invention, the Xuebijing injection was purchased from China Resources Tianjin Pharmaceutical Co., Ltd.

[0023] 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. Example 1

[0024] Xuebijing injection inhibits peri-islet thrombus encapsulation after portal vein islet transplantation.

[0025] 1. Rat Pancreatic Islet Isolation and Purification: Pancreatic Perfusion and Isolation: Male SD rats (250g-280g) were euthanized with carbon dioxide 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 10mL syringe was filled with 10mL of collagenase P solution, and the tip of the needle was bent at a 45° angle and inserted into the common bile duct. 10mL 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 50 ml sterile centrifuge tube and placed on ice. Pancreatic digestion: The centrifuge tube containing the rat pancreas was placed in a 37°C water bath for 13 min. Then, the centrifuge tube was quickly removed, wiped with 75% alcohol, and transferred to a clean bench. 20 mL of pre-cooled 1640 solution containing 10% fetal bovine serum was added to stop digestion. The centrifuge tube was quickly manually shaken 40 times. 30 mL of 1640 solution containing 10% fetal bovine serum at 4°C was added, and the mixture was centrifuged at 1000 r / min for 1 min at 4°C. 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, centrifuge at 1000 r / min 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 r / min for 20 min at 25°C. Slowly remove the centrifuge tube; the islets will be visible suspended in the lower Histopaque-1077 solution. 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 RPMI-1640 solution containing 10% fetal bovine serum (FBS). Centrifuge at 1000 r / min for 1 min at 4°C. Islets will precipitate after centrifugation. Wash once with pre-chilled RPMI-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 RPMI-1640 solution containing 10% FBS until ready for use.

[0026] 2. Recipient grouping and treatment: ① Blank control group (no treatment): The same volume of physiological saline was injected into the tail vein at the same time point; ② Xuebijing injection intervention group: 16 μL / g of Xuebijing injection was injected into the tail vein 1 hour before transplantation. Mice were euthanized 6 hours after transplantation, and the livers were removed, fixed, and cryopreserved for later use.

[0027] 3. Transportal islet transplantation: C57BL / 6J mice were induced to develop a diabetic model by intraperitoneal injection of streptozotocin (200 mg / kg). A blood glucose level >20 mmol / L for 2 consecutive days confirmed the successful establishment of the diabetic model. After the mice were fully anesthetized, the laparotomy was performed along the midline to fully expose the portal vein. The separated rat islets were aspirated into a 0.45 mm diameter transportal infusion needle, suspended in physiological saline containing 1% FBS, and the portal vein was punctured. The islets were slowly infused. After the needle was removed, hemostasis was achieved by applying pressure with a coagulation sponge, and the abdomen was closed with 6-0 sutures.

[0028] 4. Liver Fixation and Sectioning: Immediately after explantation, the liver tissue was fixed in 10 times its volume of 4% paraformaldehyde for 48 hours. It was then dehydrated sequentially with 70%, 80%, 95%, and 100% ethanol, followed by xylene clearing. The tissue was placed in an embedding cassette with the portal vein transverse section facing down, and paraffin wax was injected at 65°C. The cassette was then rapidly cooled and shaped on an ice table. The embedded liver tissue was sectioned using a microtome to a thickness of 5 μm per section. The sections were then mounted on detachable glass slides, dried overnight in a 37°C oven, and stored in a sealed container at 4°C.

[0029] 5. HE staining: Paraffin sections were dewaxed with xylene I / II (10 min each), hydrated with graded ethanol (100%→95%→80%→70%, 5 min each), and rinsed with PBS for 5 min; Hematoxylin staining: Immersed in hematoxylin staining solution for 5 min, rinsed with running water to regain blue color; Differentiation and blue color: Differentiated with 1% hydrochloric acid ethanol for 10 s; Eosin staining: Stained with eosin staining solution for 2 min, dehydrated with graded ethanol (80%→95%→100%); Clearing and mounting: Cleared with xylene (I / II, 5 min each), and mounted with neutral resin.

[0030] result:

[0031] The status of peripancreatic thrombus deposition was assessed using a semi-quantitative scoring scheme. Figure 1 In this study, it was observed that XBJ (Xuebijing Injection) treatment reduced peripancreatic thrombus deposition from an average score of 1.49 to 1.23. Figure 1 (B, C). Notably, in the XBJ treatment group, 46.19% of pancreatic islet thrombi had a score of 0, while in the control group it was only 34.95%. Example 2

[0032] Xuebijing injection inhibits leukocyte infiltration around islets after portal vein islet transplantation.

[0033] 1. Rat Pancreatic Islet Isolation and Purification: Pancreatic Perfusion and Isolation: Male SD rats (250g-280g) were euthanized with carbon dioxide 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 10mL syringe was filled with 10mL of collagenase P solution, and the tip of the needle was bent at a 45° angle and inserted into the common bile duct. 10mL 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 50 ml sterile centrifuge tube and placed on ice. Pancreatic digestion: The centrifuge tube containing the rat pancreas was placed in a 37°C water bath for 13 min. Then, the centrifuge tube was quickly removed, wiped with 75% alcohol, and transferred to a clean bench. 20 mL of pre-cooled 1640 solution containing 10% fetal bovine serum was added to stop digestion. The centrifuge tube was quickly manually shaken 40 times. 30 mL of 1640 solution containing 10% fetal bovine serum at 4°C was added, and the mixture was centrifuged at 1000 r / min for 1 min at 4°C. 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, centrifuge at 1000 r / min 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 r / min for 20 min at 25°C. Slowly remove the centrifuge tube; the islets will be visible suspended in the lower Histopaque-1077 solution. 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 RPMI-1640 solution containing 10% fetal bovine serum (FBS). Centrifuge at 1000 r / min for 1 min at 4°C. Islets will precipitate after centrifugation. Wash once with pre-chilled RPMI-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 RPMI-1640 solution containing 10% FBS until ready for use.

[0034] 2. Recipient grouping: ① Negative control group: The same volume of physiological saline was injected into the tail vein at the same time point; ② Xuebijing injection intervention group: 16 μL / g of Xuebijing injection was injected into the tail vein 1 hour before transplantation. Mice were euthanized 6 hours after transplantation, and the livers were removed, fixed, and cryopreserved for later use.

[0035] 3. Transportal islet transplantation: C57BL / 6J mice were induced to develop a diabetic model by intraperitoneal injection of streptozotocin (200 mg / kg). A blood glucose level >20 mmol / L for 2 consecutive days confirmed the successful establishment of the diabetic model. After the mice were fully anesthetized, the laparotomy was performed along the midline to fully expose the portal vein. The separated rat islets were aspirated into a 0.45 mm diameter transportal infusion needle, suspended in physiological saline containing 1% FBS, and the portal vein was punctured. The islets were slowly infused. After the needle was removed, hemostasis was achieved by applying pressure with a coagulation sponge, and the abdomen was closed with 6-0 sutures.

[0036] 4. Liver Fixation and Sectioning: Immediately after explantation, the liver tissue was fixed in 10 times its volume of 4% paraformaldehyde at 4°C for 48 h. It was then dehydrated sequentially with 70%, 80%, 95%, and 100% ethanol, followed by xylene clearing. The tissue was placed in an embedding cassette with the portal vein transverse section facing down, and paraffin wax was injected at 65°C. The cassette was then rapidly cooled and shaped on an ice table. The embedded liver tissue was sectioned using a microtome to a thickness of 5 μm per section. The sections were then mounted on detachable glass slides, dried overnight in a 37°C oven, and stored in a sealed container at 4°C.

[0037] 5. Paraffin sections were dewaxed with xylene I / II (10 min each), hydrated with a gradient of ethanol (100%→95%→80%→70%, 5 min each), and rinsed with PBS for 5 min. After dewaxing and hydration, the sections were immersed in sodium citrate buffer (pH 6.0), microwaved for repair, and allowed to cool naturally. Endogenous enzyme blocking: incubated with 3% H2O2 at room temperature for 10 min, and rinsed with PBS. Primary antibody incubation: anti-CD45 antibody (1:200) was added and incubated overnight at 4°C. Secondary antibody reaction: HRP-labeled secondary antibody (1:500) was incubated at room temperature for 1 h. DAB working solution was added, and the staining time was controlled under a microscope, with the reaction stopped by running water. Counterstaining and mounting: hematoxylin counterstaining for 1 min, hydrochloric acid ethanol differentiation, and neutral resin mounting.

[0038] result:

[0039] This invention involves staining adjacent sections of insulin-positive sections with CD45 (a common leukocyte antigen and inflammatory marker) to assess the infiltration of immune cells around transplanted islets. Figure 2 The results showed that, compared with the control group, the XBJ treatment group had a significant reduction in leukocyte infiltration around the islets of Langerhans. Example 3

[0040] Xuebijing injection inhibits the transcription level of inflammation-related genes at the transplantation site after portal vein islet transplantation.

[0041] 1. Rat Pancreatic Islet Isolation and Purification: Pancreatic Perfusion and Isolation: Male SD rats (250g-280g) were euthanized with carbon dioxide 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 10mL syringe was filled with 10mL of collagenase P solution, and the tip of the needle was bent at a 45° angle and inserted into the common bile duct. 10mL 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 50 ml sterile centrifuge tube and placed on ice. Pancreatic digestion: The centrifuge tube containing the rat pancreas was placed in a 37°C water bath for 13 min. Then, the centrifuge tube was quickly removed, wiped with 75% alcohol, and transferred to a clean bench. 20 mL of pre-cooled 1640 solution containing 10% fetal bovine serum was added to stop digestion. The centrifuge tube was quickly manually shaken 40 times. 30 mL of 1640 solution containing 10% fetal bovine serum at 4°C was added, and the mixture was centrifuged at 1000 r / min for 1 min at 4°C. Discard the supernatant, add 10 mL of pre-chilled RPMI-1640 solution containing 10% fetal bovine serum, and resuspend using a sterile Pasteur pipette. Filter the suspension through a 40-mesh sieve, 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; islets will be visible suspended in the lower Histopaque-1077 solution. Transfer the lower Histopaque-1077 solution containing islets to another 50 mL sterile centrifuge tube using a power pipette, add 35-40 mL of pre-chilled RPMI-1640 solution containing 10% fetal bovine serum, and centrifuge at 1000 rpm for 1 min at 4°C. After centrifugation, islets of pancreas precipitate can be observed. The islets are then washed once with pre-cooled RPMI-1640 solution containing 10% fetal bovine serum. Finally, the islets are transferred to cell culture dishes and further manually selected and purified under an optical microscope. They are then cultured in RPMI-1640 solution containing 10% fetal bovine serum for later use.

[0042] 2. Recipient grouping: ① Negative control group: The same volume of physiological saline was injected into the tail vein at the same time point; ② Xuebijing injection intervention group: 16 μL / g of Xuebijing injection was injected into the tail vein 1 hour before transplantation. Mice were euthanized 6 hours after transplantation, and their livers were removed and fixed for later use.

[0043] 3. Transportal islet transplantation: C57BL / 6J mice were induced to develop a diabetic model by intraperitoneal injection of streptozotocin (200 mg / kg). A blood glucose level >20 mmol / L for 2 consecutive days confirmed the successful establishment of the diabetic model. After the mice were fully anesthetized, the laparotomy was performed along the midline to fully expose the portal vein. The separated rat islets were aspirated into a 0.45 mm diameter transportal infusion needle, suspended in physiological saline containing 1% FBS, and the portal vein was punctured. The islets were slowly infused. After the needle was removed, hemostasis was achieved by applying pressure with a coagulation sponge, and the abdomen was closed with 6-0 sutures.

[0044] 4. Detection of inflammatory gene transcriptional expression levels: Total RNA was extracted from the liver using TRIzol reagent, and the mRNA expression levels of inflammatory factors such as TNF-α, HMGB1, IL-6 and COX-2 were analyzed by reverse transcription and real-time quantitative PCR (qRT-PCR), with β-actin as an internal reference gene.

[0045] Results: The inflammatory response at the transplant site was assessed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that, compared with the control group, the mRNA expression of several key inflammatory markers was significantly reduced in the XBJ (Xuebijing injection) treatment group. Figure 3 ).

[0046] 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. The use of Xuebijing injection in the preparation of a medicament for inhibiting the blood-mediated inflammatory response immediately after portal vein islet transplantation. The Xuebijing injection inhibits thrombus deposition around islets after portal vein islet transplantation. The Xuebijing injection inhibits leukocyte infiltration around islets after portal vein islet transplantation. The Xuebijing injection inhibits the mRNA expression level of inflammation-related factors at the transplantation site after portal vein islet transplantation. The inflammation-related factors include TNF-α, HMGB1, IL-6, COX-2, and MIP-1β.

2. Use according to claim 1, characterized in that, The amount of the Xuebijing injection used is 16 µL / g.

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