Application of Xuebijing injection in preparation of medicine for inhibiting instant blood-mediated inflammatory response after portal vein pancreas islet transplantation

Abstract

The invention relates to the technical field of transplantation medicine and pharmacology crossing, in particular to application of Xuebijing injection in preparation of a medicine for inhibiting instant blood-mediated inflammatory response after portal vein pancreas islet transplantation. According to the application, the application is innovatively applied to prevention and treatment of instant blood-mediated inflammatory response in pancreas islet transplantation, complement activation, blood coagulation cascade and inflammatory cell infiltration are synergistically inhibited through multiple targets, and the curative effect bottleneck of a traditional single anti-coagulation or anti-inflammatory strategy is broken through; the invention provides a Chinese and western medicine combined patent solution which has the advantages of efficient regulation and safety for overcoming early-stage key inflammatory injury after transplantation.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medical transplantation and pharmacology, and particularly relates to application of Xuebijing injection in preparation of a medicine for inhibiting blood-mediated inflammatory reaction immediately after islet transplantation in portal vein. BACKGROUND

[0002] Xuebijing injection is a compound preparation composed of safflower, red peony root, chuanxiong rhizome, salvia miltiorrhiza and angelica sinensis, and is developed based on the theory of treating bacteria, toxin and inflammation simultaneously. In 2004, Xuebijing injection was awarded a national new drug certificate of type II, and was approved for treating sepsis and multiple organ dysfunction syndrome (MODS). The core pharmacological effects of Xuebijing injection include: multi-target anti-inflammatory: inhibiting the release of pro-inflammatory factors such as TNF-alpha, IL-1beta and IL-6, down-regulating the expression of adhesion molecules such as ICAM-1, and blocking leukocyte infiltration; regulating the NF-kappa B pathway: reducing systemic inflammatory response syndrome by inhibiting IKBalpha phosphorylation and p65 nuclear translocation; organ protection: in models of sepsis and severe COVID-19, it is proved that Xuebijing injection can reduce the damage of multiple organs such as heart, kidney and liver.

[0003] In the field of islet transplantation, the pathological mechanism of immediate blood-mediated inflammatory reaction involves the cascade amplification of blood coagulation, inflammation and complement, and requires multi-target intervention. Immediate blood-mediated inflammatory reaction leads to more than 50% of islet grafts lost in a short time after transplantation, thereby damaging the function of the graft and the long-term success rate of transplantation. Early innate immune response reduces the effectiveness of the entire operation. Despite the progress in immunosuppressive therapy and anti-inflammatory strategies, immediate blood-mediated inflammatory reaction remains a major challenge to improve the survival rate of islet grafts.

[0004] When islet grafts come into contact with blood, an inflammatory reaction is immediately triggered, which is 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 adaptive immune response. At the same time, the interaction between inflammation and coagulation forms a cascade amplification effect, in which each pathway enhances the other, leading to rapid and massive loss of islets. This synergistic amplification effect is the key to the destructive nature of immediate blood-mediated inflammatory reaction, and highlights the urgent need for new interventions that can interrupt this cycle and improve the effectiveness of islet transplantation.

[0005] In diseases such as sepsis and acute lung injury, Xuebijing injection has shown strong anti-inflammatory and antithrombotic effects. In clinical applications, Xuebijing has shown significant efficacy in treating various acute inflammatory injuries, especially in the case of severe infections such as sepsis, but it has not been studied in the field of islet transplantation. SUMMARY

[0006] In order to solve the above problems, the application provides the use of Xuebijing injection in the preparation of a medicine for inhibiting blood-mediated inflammatory reaction immediately after portal vein islet transplantation.

[0007] In order to achieve the above object, the application provides the following technical scheme. The application provides the use of Xuebijing injection in the preparation of a medicine for inhibiting blood-mediated inflammatory reaction immediately after portal vein islet transplantation.

[0008] Preferably, the Xuebijing injection inhibits thrombus deposition around islets after portal vein islet transplantation.

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

[0010] Preferably, the Xuebijing injection inhibits the mRNA expression level of an inflammation-related factor at a transplantation site after portal vein islet transplantation.

[0011] Preferably, the inflammation-related factor includes TNF-alpha, HMGB1, IL-6, COX-2 and MIP-1beta.

[0012] Preferably, the use amount of the Xuebijing injection is 16 muL / g.

[0013] The application has the following beneficial effects. The application is directed to the core pathological mechanism (tissue factor-mediated platelet aggregation, leukocyte recruitment around islet grafts and up-regulation of transcription level of inflammatory genes at a transplantation site) of blood coagulation-inflammation cascade amplification in blood-mediated inflammatory reaction, and reduces thrombosis and inflammatory reaction after transplantation through the synergistic effect of multiple components of Xuebijing.

[0014] The application is innovatively applied to the prevention and treatment of blood-mediated inflammatory reaction in islet transplantation, and inhibits complement activation, blood coagulation cascade reaction and inflammatory cell infiltration through multiple targets, breaks through the efficacy bottleneck of traditional single anticoagulation or anti-inflammatory strategy, and provides a 'combination of traditional Chinese and Western medicine' patent solution with high-efficiency regulation and safety advantage for overcoming key inflammatory damage in the early stage after transplantation. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical scheme in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced.

[0016] Figure 1 HE staining results of liver sections after portal vein transplantation of islets, and thrombus infiltration around islets and scoring statistics.

[0017] Figure 2 CD45 immunohistochemical staining results of liver sections after portal vein transplantation of islets, and CD45 positive cell infiltration number statistics chart.

[0018] Figure 3 Detection results of inflammation-related gene transcription levels at islet transplantation site after portal vein transplantation of islets. DETAILED DESCRIPTION

[0019] The application provides application of Xuebijing injection in preparation of a medicine for inhibiting blood-mediated inflammatory reaction immediately after portal vein transplantation of islets. In the application, the Xuebijing injection preferably inhibits thrombus deposition around islets after portal vein transplantation of islets. In the application, the Xuebijing injection preferably inhibits leukocyte infiltration around islets after portal vein transplantation of islets. In the application, the Xuebijing injection preferably inhibits mRNA expression levels of inflammation-related factors at a transplantation site after portal vein transplantation of islets. In the application, the inflammation-related factors preferably include TNF-α, HMGB1, IL-6, COX-2 and MIP-1β. In the application, the use amount of the Xuebijing injection is preferably 16 μL / g.

[0020] In the application, the Xuebijing injection is purchased from Tianjin Huatong Pharmaceutical Co., Ltd.

[0021] In order to further illustrate the application, the application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the application. Example 1

[0022] Xuebijing injection inhibits thrombus wrapping around islets after portal vein transplantation of islets: 1. Rat pancreatic islet isolation, purification: perfusion and isolation of pancreas: 250g-280g male SD rats were euthanized with carbon dioxide, placed on the operating microscope operating table on their backs, and the abdominal skin was disinfected with 75% alcohol. A "V" shaped incision was made with surgical scissors to open the full-thickness of the abdomen, and the abdominal cavity was fully exposed. The duodenum was located under the microscope, and the junction of the duodenum and common bile duct was found and clamped with a hemostat. The hepatic portal was fully exposed and the common bile duct was freed. A 10 mL syringe was used to draw 10 mL of collagenase P solution, and the needle tip was bent at a 45° angle for 2 cm. The needle was inserted into the common bile duct, and 10 mL of collagenase P solution was injected to fully perfuse the pancreas. The pancreas was bluntly separated at the duodenum, stomach, and spleen, and the entire pancreas was cut off and placed in a 50 ml sterile centrifuge tube for ice bath; pancreas digestion: the centrifuge tube containing the rat pancreas was placed in a 37°C water bath for 13 minutes, then quickly removed and transferred to a clean bench after wiping with 75% alcohol. Add 20 mL of pre-cooled 1640 solution containing 10% fetal bovine serum to terminate digestion, quickly shake the centrifuge tube by hand 40 times, add 30 mL of 4°C 1640 solution containing 10% fetal bovine serum, and centrifuge at 1000 r / min for 1 min. Discard the supernatant, add 10 ml of pre-cooled 1640 solution containing 10% fetal bovine serum, and blow it into a suspension with a sterile Pasteur pipette. Filter the suspension with a 40-mesh sieve, and discard the supernatant after centrifugation at 4°C, 1000 r / min for 1 min; islet purification: resuspend the above precipitate with 15 mL of Histopaque-1077 solution, and blow the suspension evenly with a sterile Pasteur pipette. Slowly add 10 mL of RPMI-1640 medium solution along the wall of the centrifuge tube. When the solution in the centrifuge tube appears to be layered, centrifuge at 25°C, 2400 r / min for 20 min. Slowly remove the centrifuge tube, and you can see the islets suspended in the lower Histopaque-1077 solution. Use an electric pipette to transfer the lower Histopaque-1077 solution containing the islets to another 50 mL sterile centrifuge tube, add 35-40 mL of pre-cooled 1640 solution containing 10% fetal bovine serum, and centrifuge at 4°C, 1000 r / min for 1 min. After centrifugation, the islets can be seen to precipitate, and they are washed once with pre-cooled 1640 solution containing 10% fetal bovine serum. Finally, the islets are transferred to a cell culture dish and further purified under a light microscope; use 1640 solution containing 10% fetal bovine serum for culture.

[0023] 2. Recipient group processing: ① blank control group (no treatment): same time point tail vein injection of the same volume of normal saline; ② Xuebijing injection intervention group: 1 hour before transplantation, Xuebijing injection 16 μL / g was injected into the tail vein. 6 hours after transplantation, the mice were euthanized, and the mouse liver was removed, fixed and stored for use.

[0024] 3. Portal vein islet transplantation: C57BL / 6J mice were intraperitoneally injected with streptozotocin (200 mg / kg) to induce a diabetic model, and diabetes was confirmed to be successfully established when the blood glucose was >20 mmol / L for 2 consecutive days. After the mice were fully anesthetized, the portal vein was fully exposed along the midline of the abdomen. The separated rat islets were taken into a 0.45 mm inner diameter portal infusion needle, suspended in physiological saline containing 1% FBS, and slowly infused into the portal vein. After the needle was removed, a blood clot sponge was used to compress the bleeding, and the abdomen was closed with a 6-0 suture.

[0025] 4. Liver fixation and sectioning: Immediately after the liver was removed, it was immersed in 10 times the volume of 4% paraformaldehyde for fixation. After 48 hours of fixation, the liver was dehydrated in 70%, 80%, 95%, and 100% ethanol, and then cleared with xylene. The tissue was placed in an embedding box with the portal vein cross-section facing down, and 65°C paraffin was injected and quickly cooled on an ice platform. The embedded liver tissue was sectioned using a microtome, with a thickness of 5 μm per slice. The sections were attached to the surface of a non-removable glass slide and dried in a 37°C oven overnight, and then stored at 4°C.

[0026] 5. HE staining: The paraffin sections were deparaffinized with xylene I / II (10 minutes each), hydrated with gradient ethanol (100%→95%→80%→70%, 5 min each), and rinsed with PBS for 5 minutes. Hematoxylin staining: immerse in hematoxylin staining solution for 5 min, and rinse with running water to return to blue. Differentiate the blue: differentiate with 1% hydrochloric acid ethanol for 10 s. Eosin staining: stain with eosin staining solution for 2 min, and dehydrate with gradient ethanol (80%→95%→100%). Clear and mount: clear with xylene (5 min each), and mount with neutral balsam.

[0027] Results: The status of thrombus deposition around the islets was evaluated using a semi-quantitative scoring scheme Figure 1 A), and the present application observed that XBJ (Xiaobijing injection) treatment reduced the thrombus deposition around the islets from an average score of 1.49 to 1.23 Figure 1 B, C). Notably, 46.19% of the islet thrombus deposition in the XBJ treatment group was 0, while only 34.95% in the control group. Example 2

[0028] Xiaobijing injection inhibits leukocyte infiltration around islets after portal vein islet transplantation: 1. Rat pancreatic islet isolation, purification: perfusion and isolation of pancreas: 250g-280g male SD rats were euthanized with carbon dioxide, placed on the operating microscope operating table on their backs, and the abdominal skin was sterilized with 75% alcohol. A "V" shaped incision was made with surgical scissors to open the full-thickness of the abdomen, and the abdominal cavity was fully exposed. The duodenum was located under the microscope, and the junction of the duodenum and common bile duct was clamped with a hemostat. The hepatic portal was fully exposed, and the common bile duct was freed. A 10 mL syringe was used to draw 10 mL of collagenase P solution, and the needle tip was bent at a 45° angle. The needle was inserted into the common bile duct, and 10 mL of collagenase P solution was injected to fully perfuse the pancreas. The pancreas was bluntly separated at the duodenum, stomach, and spleen, and the entire pancreas was cut off and placed in a 50 mL sterile centrifuge tube for ice bath. Pancreatic digestion: The centrifuge tube containing the rat pancreas was placed in a 37°C water bath for 13 minutes, then quickly removed and transferred to a sterile bench after wiping with 75% alcohol. Add 20 mL of pre-cooled 1640 solution containing 10% fetal bovine serum to terminate digestion, quickly shake the centrifuge tube by hand 40 times, add 30 mL of 4°C 1640 solution containing 10% fetal bovine serum, and centrifuge at 1000 r / min for 1 min. Discard the supernatant, add 10 mL of pre-cooled 1640 solution containing 10% fetal bovine serum, and blow the suspension with a sterile Pasteur pipette. Filter the suspension with a 40-mesh sieve, and centrifuge at 4°C, 1000 r / min for 1 min. Discard the supernatant; islet purification: add 15 mL of Histopaque-1077 solution to resuspend the above-mentioned precipitate, and blow the suspension evenly with a sterile Pasteur pipette. Slowly add 10 mL of RPMI-1640 medium solution along the wall of the centrifuge tube. When the solution in the centrifuge tube appears to be layered, centrifuge at 25°C, 2400 r / min for 20 min. Slowly remove the centrifuge tube, and the islets can be seen suspended in the lower Histopaque-1077 solution. Use an electric pipette to transfer the lower Histopaque-1077 solution containing the islets to another 50 mL sterile centrifuge tube, add 35-40 mL of pre-cooled 1640 solution containing 10% fetal bovine serum, and centrifuge at 4°C, 1000 r / min for 1 min. After centrifugation, the islets can be seen precipitated, and they are washed once with pre-cooled 1640 solution containing 10% fetal bovine serum. Finally, the islets are transferred to a cell culture dish for further manual selection and purification under a light microscope; use 1640 solution containing 10% fetal bovine serum for culture.

[0029] 2. Recipient group processing: ① Negative control group: same volume of normal 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. The mice were euthanized 6 hours after transplantation, and the mouse liver was removed, fixed and stored for use.

[0030] 3. Portal vein islet transplantation: C57BL / 6J mice were intraperitoneally injected with streptozotocin (200 mg / kg) to induce diabetes model, and diabetes model was confirmed to be successfully established when blood glucose was > 20 mmol / L for 2 consecutive days; after the mice were fully anesthetized, the portal vein was fully exposed along the abdominal midline; the separated rat islets were sucked into a 0.45 mm inner diameter portal infusion needle, suspended in physiological saline containing 1% FBS, punctured the portal vein, and slowly infused the islets; after the needle was pulled out, a blood sponge was used to compress and stop bleeding, and the abdomen was closed with a 6-0 suture.

[0031] 4. Liver fixation and sectioning: Immediately after the liver was removed, it was immersed in 10 times the volume of 4% paraformaldehyde for fixation, and fixed at 4°C for 48 h, and then dehydrated in 70%, 80%, 95% and 100% ethanol, and transparentized with xylene. The tissue was placed in an embedding box, the portal vein was cut transversely, and the surface was downward, 65°C paraffin was injected, and the ice platform was rapidly cooled and shaped. The embedded liver tissue was sectioned using a microtome, with a thickness of 5 μm per slice, and the sections were attached to the surface of a non-removable glass slide, dried in a 37°C oven overnight, and stored at 4°C.

[0032] 5. Paraffin sections were deparaffinated with xylene I / II (10 minutes each), hydrated with gradient ethanol (100%→95%→80%→70%, 5 min each), and rinsed with PBS for 5 minutes; after deparaffination and hydration, the sections were immersed in sodium citrate buffer (pH 6.0), microwave repaired, and naturally cooled; endogenous enzyme blocking: 3% H2O2 was incubated at room temperature for 10 min, and PBS was washed; primary antibody incubation: anti-CD45 antibody (1:200) was added dropwise, and incubated at 4°C overnight; secondary antibody reaction: HRP-labeled secondary antibody (1:500) was incubated at room temperature for 1 h; DAB working solution was added dropwise, and the color development time was controlled under a microscope, and then terminated with running water; re-staining and mounting: stained with hematoxylin for 1 min, differentiated with hydrochloric acid ethanol, and mounted with neutral resin.

[0033] Results: The present application uses CD45 (a common leukocyte antigen and an inflammation marker) to stain the adjacent section of the insulin positive section to evaluate the immune cell infiltration around the transplanted islets Figure 2 ). The results show that compared with the control group, the leukocyte infiltration around the islets in the XBJ treatment group is significantly reduced. Example 3

[0034] Xuebijing injection inhibits the transcription level of inflammation-related genes at the transplantation site after portal vein islet transplantation: 1. Rat pancreatic islet isolation, purification: perfusion and separation of pancreas: 250g-280g male SD rats were euthanized with carbon dioxide, placed on the operating microscope operating table on their backs, and the abdominal skin was disinfected with 75% alcohol. A "V" shaped incision was made with surgical scissors to open the full-thickness of the abdomen, and the abdominal cavity was fully exposed. The duodenum was located under the microscope, and the junction of the duodenum and common bile duct was clamped with a hemostat. The hepatic portal was fully exposed and the common bile duct was freed. A 10 mL syringe was used to draw 10 mL of collagenase P solution, and the needle tip was bent at a 45° angle for 2 cm. The needle was inserted into the common bile duct, and 10 mL of collagenase P solution was injected to fully perfuse the pancreas. The pancreas was bluntly separated at the duodenum, stomach, and spleen, and the entire pancreas was cut off and placed in a 50 ml sterile centrifuge tube for ice bath; pancreatic digestion: the centrifuge tube containing the rat pancreas was placed in a 37°C water bath for 13 minutes, then quickly removed and transferred to a clean bench after wiping with 75% alcohol. Add 20 mL of pre-cooled 1640 solution containing 10% fetal bovine serum to terminate digestion, quickly shake the centrifuge tube by hand 40 times, add 30 mL of 4°C 1640 solution containing 10% fetal bovine serum, and centrifuge at 1000 r / min for 1 min. Discard the supernatant, add 10 ml of pre-cooled 1640 solution containing 10% fetal bovine serum, and blow the suspension with a sterile Pasteur pipette. Filter the suspension with a 40-mesh sieve, discard the supernatant after centrifugation at 4°C, 1000 r / min for 1 min; islet purification: resuspend the above precipitate with 15 mL of Histopaque-1077 solution, blow the suspension evenly with a sterile Pasteur pipette, slowly add 10 mL of RPMI-1640 medium solution along the wall of the centrifuge tube, and wait for the solution in the centrifuge tube to separate into layers. Centrifuge at 25°C, 2400 r / min for 20 min. Slowly remove the centrifuge tube, and the islets can be seen suspended in the lower Histopaque-1077 solution. Use an electric pipette to transfer the lower Histopaque-1077 solution containing the islets to another 50 mL sterile centrifuge tube, add 35-40 mL of pre-cooled 1640 solution containing 10% fetal bovine serum, and centrifuge at 4°C, 1000 r / min for 1 min. After centrifugation, the islets can be seen precipitated, and they are washed once with pre-cooled 1640 solution containing 10% fetal bovine serum. Finally, the islets are transferred to a cell culture dish and further purified under a light microscope; use 1640 solution containing 10% fetal bovine serum for culture.

[0035] 2. Recipient group processing: ① Negative control group: same time point tail vein injection of the same volume of normal saline; ② Xuebijing injection intervention group: 1 hour before transplantation, 16 μL / g of Xuebijing injection was injected into the tail vein. 6 hours after transplantation, the mice were euthanized, and the mouse liver was removed and fixed for use.

[0036] 3. Portal vein islet transplantation: C57BL / 6J mice were intraperitoneally injected with streptozotocin (200 mg / kg) to induce diabetes model, and diabetes model was confirmed to be successfully established when blood glucose was >20 mmol / L for 2 consecutive days; after the mice were fully anesthetized, the portal vein was fully exposed along the abdominal midline; the separated rat islets were sucked into a 0.45 mm inner diameter portal infusion needle, suspended in physiological saline containing 1% FBS, punctured the portal vein, and slowly infused the islets; after the needle was pulled out, a blood sponge was used to compress the bleeding, and the abdomen was closed with a 6-0 suture.

[0037] 4. Inflammation gene transcription expression level detection: total RNA was extracted from liver using TRIzol reagent, and the mRNA expression levels of TNF-α, HMGB1, IL-6 and COX-2 and other inflammatory factors were analyzed by reverse transcription and real-time fluorescence quantitative PCR (qRT-PCR), with β-actin as an internal reference gene.

[0038] Results: The inflammatory response at the transplantation site was evaluated by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that the mRNA expression of several key inflammatory markers was significantly reduced in the XBJ (Xuebijing injection) treatment group compared to the control group Figure 3 ).

[0039] Although the above embodiments make a detailed description of the present application, it is only a part of the embodiments of the present application, not all embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. Use of Xuebijing injection in the preparation of a medicament for inhibiting blood-mediated inflammatory response immediately after portal vein islet transplantation.

2. Use according to claim 1, characterized in that, The Xuebijing injection inhibits thrombus deposition around islets after portal vein islet transplantation.

3. Use according to claim 1, characterized in that, The Xuebijing injection inhibits leukocyte infiltration around islets after portal vein islet transplantation.

4. Use according to claim 1, characterized in that, The Xuebijing injection inhibits the mRNA expression level of inflammation-related factors at the transplantation site after portal vein islet transplantation.

5. Use according to claim 4, characterized in that, The inflammation-related factors include TNF-α, HMGB1, IL-6, COX-2 and MIP-1β.

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

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