Application of bilobalide in preparation of organ protection liquid

Abstract

The invention discloses an application of bilobalide in preparation of an organ protection solution. According to the application, a mouse liver ischemia reperfusion injury model is adopted; the bilobalide can obviously relieve a plurality of indexes such as liver cell injury caused by liver ischemia reperfusion, pathological injury of a liver tissue structure, liver cell apoptosis, liver inflammatory cell infiltration (neutrophil and macrophage), inflammatory factor expression (secretion of inflammatory cytokines (TNF-alpha and IL-1beta) and the like, and has an obvious protective effect on the liver ischemia reperfusion injury. Research is carried out by combining rat orthotopic liver transplantation with a low-temperature mechanical perfusion model, and experimental research finds that after the liver perfusion preserving fluid containing bilobalide is used for perfusing an in-vitro liver by using a low-temperature mechanical perfusion instrument, the liver function after transplantation can be remarkably protected, and liver injury is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of organ transplantation, and particularly relates to the application of bilobalide in the preparation of organ protective solution. Background Art

[0002] Hepatic ischemia-reperfusion injury (HIRI) is a critical and complex challenge during liver transplantation, significantly impacting the postoperative recovery and long-term prognosis of liver transplant patients. HIRI can lead to severe liver tissue damage and dysfunction, and even threaten the patient's life. The pathogenesis of HIRI involves complex pathophysiological processes, including cellular oxidative stress, inflammatory response, and apoptosis.

[0003] Numerous studies have demonstrated that hypothermic mechanical perfusion can reduce the severity of liver ischemia-reperfusion injury, significantly prolong the preservation time of donor livers, and improve transplant success rates. Furthermore, hypothermic mechanical perfusion can be used not only as a method for preserving donor livers but also as a therapeutic approach, by adding specific drugs to the donor liver perfusion solution to further repair and improve the quality of the donor liver. Currently, the commonly used donor liver perfusion solutions include HTK solution and UW solution, which have been effective in prolonging the preservation of donor livers.

[0004] Patent application CN116789888A discloses an anti-free radical polymer capable of alleviating organ ischemia-reperfusion injury, as well as its preparation method and uses. This invention targets mitochondria, binds free radicals, and significantly reduces liver ischemia-reperfusion injury. Adding this polymer to organ preservation fluid can also reduce liver cold ischemia damage.

[0005] The invention application with publication number CN117581858A discloses a cold preservation solution and its use in reducing ischemia-reperfusion injury to cells, tissues, or organs. By adding phosphorylcholine, quinoline-4-carboxylic acid, and sodium tauroursodeoxycholate to the preservation solution, it was found that the cold preservation of cells, tissues, and / or organs using the preservation solution with these substances increased cell survival rate, effectively reduced cell apoptosis caused by cold preservation and rewarming, reduced mitochondrial ROS, maintained cell membrane integrity, effectively reduced mitochondrial damage caused by cold preservation and rewarming, reduced ischemia-reperfusion injury, and promoted liver regeneration. This has significant significance for the advancement of organ transplant preservation technology.

[0006] In order to further improve the success rate and effect of liver transplantation, reduce postoperative complications, and expand the scope of use of donor livers, improving the donor liver perfusion preservation fluid has become a hot topic and difficulty in the current field of liver disease research.

[0007] Bilobalide (BB) is one of the main components of Ginkgo biloba extract. It is a sesquiterpenoid compound, CAS: 33570-04-6, molecular formula: C 15 H 18 O8, the structural formula is shown in Formula Ⅰ.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to provide a perfusion preservation fluid that can effectively improve ischemia-reperfusion injury of transplanted liver donors, apply it to the cold preservation process of liver transplantation, improve the quality of donor livers, and reduce the abandonment rate.

[0010] The present invention has found that bilobalide has a protective effect on ischemia-reperfusion injury of the liver and can be added to the perfusion preservation solution of the donor liver to improve the protective effect.

[0011] Ischemia-reperfusion during liver transplantation can cause early damage to the transplanted liver, leading to liver dysfunction. Research in this study demonstrates that bilobalide can significantly improve liver ischemia-reperfusion injury. The primary mechanism is that bilobalide effectively inhibits tissue damage, apoptosis, and inflammation caused by HIRI. This study further improves ischemia-reperfusion-induced liver cell damage by adding a series of ingredients, including bilobalide, to HTK solution.

[0012] The present invention first provides the use of bilobalide in the preparation of an organ protection solution. Preferably, the organ protection solution is used to protect the liver from ischemia-reperfusion injury. When used, the organ protection solution is used to perfuse and protect an isolated organ.

[0013] The present invention further provides an organ protective solution comprising bilobalide and an isotonic liquid for providing the organ protective solution with an isotonic effect. Preferably, the isotonic liquid is HTK solution, i.e., a histidine-tryptophan-ketoglutarate solution. The concentration of bilobalide is 500 mg / L.

[0014] The organ protection solution, based on a volume of 1000 ml, comprises 500 mg of bilobalide, 1 g of sodium penicillin, 20,000 U of heparin, 10 mg of N-acetylcysteine, and 5 ml of dimethyl sulfoxide, and the volume is made up to 1000 ml with HTK solution.

[0015] The present invention also provides an ex vivo organ preservation method, wherein the ex vivo organ is perfused with the organ preservation solution prior to transplantation. The perfusion temperature is 2°C to 6°C, preferably 4°C, and the perfusion time is 1 hour. Preferably, the organ is a liver.

[0016] The inventors of this application used a mouse liver ischemia-reperfusion injury model and found through experimental studies that bilobalide can significantly reduce multiple indicators such as liver cell damage, pathological damage to liver tissue structure, hepatocyte apoptosis, liver inflammatory cell infiltration (neutrophils, macrophages), and inflammatory factor expression (secretion of inflammatory cytokines (TNF-α and IL-1β)) caused by liver ischemia-reperfusion, and has a significant protective effect on liver ischemia-reperfusion injury. Using a rat orthotopic liver transplantation combined with a low-temperature mechanical perfusion model, the inventors found that the liver perfusion preservation solution containing bilobalide can significantly protect post-transplant liver function and reduce liver damage after perfusion of the isolated liver using a low-temperature mechanical perfusion apparatus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 To detect the serum ALT and AST levels of mice in the sham group, HIRI group, HIRI+BB group, and HIRI+NAC group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (the same below).

[0018] Figure 2 HE staining of liver tissue from mice in each group at 40x and 200x magnification. Scale bars: 200 μm and 50 μm. The degree of liver injury was assessed using the Suzuki score. Black arrows indicate inflammatory cell infiltration. Green arrows indicate necrotic hepatocytes, and yellow arrows indicate hepatic sinusoidal congestion.

[0019] Figure 3 Figure 3. Tunel staining of liver tissue from mice in each group at 40x and 200x magnification. Scale bars: 200 μm and 50 μm. Apoptotic cells were observed at 200x magnification and statistically analyzed.

[0020] Figure 4 The expression levels of cleaved-caspase 3 in liver tissues of mice in each group were analyzed at 400x magnification. Scale bar: 50 μm.

[0021] Figure 5 is the expression levels of IL-1β and Tnf-α in liver tissues of mice in each group.

[0022] Figure 6 Neutrophil infiltration levels in liver tissues of mice in each group at 400x magnification, with statistical analysis performed. Scale bar: 50 μm.

[0023] Figure 7 Figure 4 shows the macrophage infiltration levels in the liver tissues of mice in each group at 400x magnification, with statistical analysis performed. Scale bar: 50 μm.

[0024] Figure 8To detect the serum ALT and AST levels of rats in HTK group and HTK+ group. DETAILED DESCRIPTION

[0025] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the following detailed description of the technical solution of the invention is provided. However, this description should not be construed as limiting the scope of the invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0026] Bilobalide BB was purchased from MedChemExpress Co., Ltd., CAS No. 33570-04-6, Catalog No. HY-N0076. N-Acetylcysteine ​​(NAC) was purchased from MedChemExpress Co., Ltd., CAS No. 616-91-1, Catalog No. HY-B0215. HTK solution was purchased from Puchuan Medical Technology (Shanghai) Co., Ltd.

[0027] Example 1: Testing the Effect of Bilobalide on Alleviating Hepatic Warm Ischemia-Reperfusion Injury

[0028] 1. Animals

[0029] Forty healthy male C57BL / 6 mice (7–8 weeks old) weighing 20 ± 2 g were purchased from the Animal Experiment Center of Zhejiang Academy of Medical Sciences (Hangzhou, China). All animals were housed in a standard environment with free access to food and water and a 12-h / 12-h light / dark cycle.

[0030] 2. Experimental Grouping and Model Establishment

[0031] 2.1 Experimental Grouping

[0032] Forty C57BL / 6 mice (20 ± 2 g) were randomly divided into the following four groups:

[0033] (1) Sham group: Mice were intraperitoneally injected with 300 μl of normal saline 6 hours and 1 hour before laparotomy, and only underwent laparotomy and closure, but no HIRI was performed;

[0034] (2) Hepatic ischemia-reperfusion injury group (HIRI group): mice were intraperitoneally injected with 300 μl of normal saline 6 h and 1 h before ischemia;

[0035] (3) Hepatic ischemia-reperfusion injury + bilobalide group (HIRI+BB group): mice were intraperitoneally injected with 30 mg / kg BB 6 h and 1 h before ischemia;

[0036] (4) Hepatic ischemia-reperfusion injury + N-acetylcysteine ​​(NAC) group (HIRI+NAC group): Mice were intraperitoneally injected with 200 mg / kg NAC 6 hours and 1 hour before ischemia.

[0037] 2.2 Establishment of 70% liver warm ischemia-reperfusion injury model in mice

[0038] Mice were fasted for 12 hours before surgery and had free access to water. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. After satisfactory anesthesia, the mice were fixed to the operating table with tape. The mice were placed in a supine position, the abdomen was prepared, and the skin and muscle layers were cut layer by layer along the linea alba to expose the liver hilum. The hepatic artery and portal vein of the left and middle lobes of the liver were found. The hepatic pedicles of the left and middle lobes of the liver were clamped with a non-invasive vascular clamp, causing approximately 70% ischemia in the liver. Macroscopic observation showed that the blocked liver lobe turned grayish white compared to the right lobe of the liver where blood flow was not blocked, indicating that blood flow was successfully blocked. The mice were placed on a 37°C warming blanket to maintain body temperature. After 90 minutes of ischemia, the vascular clamp was removed and the abdomen was closed. The reperfusion time was 6 hours.

[0039] 3. Sample collection and testing items

[0040] 3.1 Liver function tests

[0041] Six hours after reperfusion, the mice's eyes were enucleated and blood samples were collected from each group. After standing at room temperature for 30 minutes, all blood samples were centrifuged at 3000 rpm for 15 minutes, and serum was collected. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were measured using an automated blood biochemical analyzer (Shenzhen Raydu Life Science).

[0042] 3.2 Hematoxylin and eosin (HE) staining of liver tissue Liver specimens were taken, fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, and sliced. The slices were then dewaxed, stained with hematoxylin and eosin, dehydrated and sealed. The slices were scanned and imaged using a digital slide scanner (Olympus, Tokyo, Japan). Pathologists analyzed the histological samples and used the Suzuki score to assess the degree of liver necrosis. The Suzuki score criteria are shown in Table 1.

[0043] Table 1

[0044] Numerical evaluation Sinusoidal congestion Cell vacuolization Cell necrosis 0 none none none 1 Very few Very few Single cell necrosis 2 Mild Mild -30% cell necrosis 3 Moderate Moderate -60% cell necrosis 4 severe severe >60% cell necrosis

[0045] 3.3 Tunel method

[0046] Paraffin sections of liver tissue were stained with TUNEL to detect hepatocyte apoptosis according to the manufacturer's instructions (ServiceBio, China). Sections were scanned and imaged using a digital slide scanner (Olympus, Tokyo, Japan). Images were analyzed using ImageJ software. A distinct yellow staining of the nuclei indicated that TUNEL-positive cells were undergoing apoptosis. The percentage of TUNEL-positive hepatocytes relative to the total number of hepatocytes was determined.

[0047] 3.4 Immunofluorescence staining

[0048] Liver samples were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. Immunofluorescence staining was performed with anti-Ly6g and anti-F4 / 80 antibodies at 4°C overnight. After incubation with a fluorophore-conjugated secondary antibody, nuclei were stained with DAPI. Finally, sections were scanned and imaged using a digital slide scanner (Olympus, Tokyo, Japan) to visualize the fluorescence staining.

[0049] 3.5 Detection of inflammatory factors in liver tissue

[0050] The liver tissue was added to PBS (10 times the weight of the tissue) and mechanically homogenized. After centrifugation, the supernatant was collected and TNF-α and IL-1β were detected using ELISA kits to evaluate the expression levels of inflammatory factors in the liver tissue.

[0051] 4. Experimental Results

[0052] 4.1 Bilobalide can alleviate liver ischemia-reperfusion injury

[0053] Six hours after reperfusion, we tested the serum liver function biochemical indicators (ALT, AST) of mice in each group. In order to determine the optimal dosage concentration for mice, we conducted a gradient administration experiment. The evaluation of the BB dosage gradient (10, 20 and 30 mg / kg) showed that 30 mg / kg had the best protective effect ( Figure 1 Therefore, all subsequent experiments used a concentration of 30 mg / kg. Compared with the sham-operated group, serum ALT and AST levels were significantly increased in the HIRI, HIRI+BB, and HIRI+NAC groups. The HIRI+BB and HIRI+NAC groups showed significantly lower serum ALT and AST levels than the HIRI group, with the HIRI+BB group showing a more significant reduction in ALT and AST than the HIRI+NAC group.

[0054] HE pathological results showed that the liver damage in the HIRI group was more severe than that in the HIRI+BB group, with a larger area of ​​necrosis. Suzuki score further confirmed this observation ( Figure 2 These results confirmed the liver protective effect of BB against HIRI.

[0055] 4.2 Bilobalide can alleviate cell apoptosis induced by liver ischemia-reperfusion injury

[0056] Apoptosis is a fundamental phenomenon in HIRI. The in situ terminal deoxynucleotidyl transferase (TUNEL) assay was used to examine the effect of BB on apoptosis in HIRI-induced liver tissue sections. Compared with the sham-operated group, the number of TUNEL-positive cells in the HIRI group increased significantly ( Figure 3 The number of TUNEL-positive cells decreased after BB treatment (p<0.05). In addition, the expression of apoptosis protein cleaved-caspase 3 in the liver tissue of rats in the HIRI group increased, and the expression was significantly reduced after BB treatment ( Figure 4 Taken together, these results indicate that BB reduces HIRI-induced apoptosis.

[0057] 4.3 Bilobalide can reduce inflammation during work-related ischemia-reperfusion injury

[0058] During reperfusion, a series of inflammatory syndrome reactions are activated, producing many inflammatory cytokines, further aggravating the damage to liver tissue and hindering its function. Compared with the sham operation group, the expression levels of TNF-α and IL-1β in liver tissue of the HIRI group were significantly increased ( Figure 5 ). The expression levels of Tnf-α and IL-1β in the HIRI+BB group were significantly lower than those in the HIRI group. In addition, Ly-6g staining showed that the neutrophil infiltration in the HIRI+BB group was significantly inhibited compared with the HIRI group ( Figure 6 F4 / 80 staining showed that the macrophage infiltration in the HIRI+BB group was significantly reduced compared with the HIRI group ( Figure 7 ). Therefore, based on our data, we concluded that BB alleviated the inflammatory response during HIRI.

[0059] In summary, in the partial liver ischemia-reperfusion injury model, the bilobalide of the present invention can protect the liver and reduce ischemia-reperfusion injury, alleviate cell apoptosis caused by HIRI, and reduce the inflammatory response of the liver during HIRI. Therefore, bilobalide can be used to alleviate liver ischemia-reperfusion injury.

[0060] Example 2: Testing the Effect of a Novel Ex Vivo Liver Perfusion Preservation Solution on Alleviating Cold Ischemia-Reperfusion Injury in Transplanted Liver

[0061] 1. Animals

[0062] Forty healthy male Sprague-Dawley rats weighing 250–300 g were purchased from the Animal Experiment Center of Zhejiang Academy of Medical Sciences (Hangzhou, China). All animals were housed in a standard environment with free access to food and water and a 12-h / 12-h light / dark cycle.

[0063] 2. Experimental Grouping and Model Establishment

[0064] 2.1 Experimental Grouping

[0065] Twenty SD rats (250 g to 300 g) were randomly divided into the following two groups:

[0066] (1) Control group (HTK group): The donor liver was perfused with HTK solution at low temperature for 1 hour before transplantation;

[0067] (2) Experimental group (perfusion fluid group): The donor liver was perfused mechanically in a novel perfusion preservation fluid for 1 hour at low temperature before transplantation.

[0068] The composition of the perfusion preservation solution is shown in Table 2.

[0069] Table 2

[0070] Element concentration bilobalide 500mg Penicillin sodium 1g heparin 20000U N-acetylcysteine 10mg Dimethyl sulfoxide (DMSO) 5ml HTK fluid Fill up to 1000ml

[0071] 2.2 Establishment of rat liver transplantation combined with hypothermia-mechanical perfusion injury model

[0072] 2.2.1 Obtaining donor liver: Anesthetize the rat by injecting 0.8 ml / 100 g body weight of anesthetic into the abdominal cavity.

[0073] The donor rat is fixed, disinfected, opened, and the liver is freed. A V-shaped incision is made with microscissors after separating the common bile duct, and a bile duct stent is inserted. The bile duct stent is fixed with needle and thread, and the common bile duct is severed. The right renal vein and the right suprarenal vein are ligated (both ligatures should be as close to the inferior vena cava as possible). The donor is systemically heparinized. The aorta is punctured, and the liver is slowly irrigated with 10ml of HTK solution. After proper irriga- tion , the entire liver will turn white. Continue to cut the connective tissue at the back of the liver to fully separate the donor liver. After removing the donor liver, place it in a low-temperature mechanical perfusion apparatus filled with organ preservation solution at 0-4°C for storage.

[0074] 2.2.2 Donor liver preparation and hypothermic perfusion: Insert an intravenous cannula into the portal vein and secure with a 5-0 silk slipknot. Connect the cannula to the perfusion apparatus and begin perfusion for 1 hour. Parameters are set as follows: constant flow: 1 ml / min; organ reservoir temperature: 4°C; perfusion time: 1 hour. After perfusion, cannulate the portal vein and infrahepatic inferior vena cava. Place traction lines on both sides of the infrahepatic inferior vena cava openings for future use.

[0075] 2.2.3 Recipient hepatectomy

[0076] The recipient rat is anesthetized, the abdomen is opened, the liver is dissected, and the upper end of the common bile duct is ligated. The proper hepatic artery is sutured with microsurgical forceps. The right adrenal vein is ligated. A traction strip is placed behind the superior and inferior vena cava. Using curved microsurgical forceps, the inferior vena cava is lifted infrahepatically. The inferior vena cava segment between the right renal vein and the right adrenal vein is clamped, thus occluding the inferior vena cava infrahepatically. The portal vein is then occluded, and the anhepatic phase begins. Hepatic blood is flushed into the systemic circulation via the portal vein (inject 2 ml of saline). After the liver is isolated, it is removed. The recipient rat's abdominal cavity is inspected for bleeding points and hemostasis is performed appropriately.

[0077] 2.2.4 Donor liver implantation

[0078] Gauze was placed over the peritoneal cavity of the rat after the liver was removed. Perfusion of the donor liver was stopped, the indwelling needle was removed, and the donor liver was placed on the gauze. The donor liver was then sutured to the suprahepatic superior vena cava of the recipient rat using the traction sutures on the donor liver.

[0079] After suturing and fixing both sides of the superior and inferior vena cava, perform continuous suture of the superior and inferior vena cava. First, use one ligature to suture the posterior end of the vein on the opposite side and tie a knot to fix it. Then, suture the anterior end continuously, leaving a small opening when the suturing is completed. Use a syringe to inject an appropriate amount of normal saline into the superior and inferior vena cava through the small opening to eliminate bubbles in the vein. Then continue suturing to complete the front end suture. After tying a knot and fixing it, cut off the excess sutures on both sides to complete the closure of the superior and inferior vena cava. Both the portal vein and the inferior vena cava are cannulated and fixed with 5-0 silk thread. After cutting a V-shaped opening in the recipient bile duct with microscissors, the donor bile duct stent is inserted and fixed with 5-0 silk thread. Close the abdominal cavity and complete the operation.

[0080] 3. Sample collection and testing items

[0081] 3.1 Liver function tests

[0082] Six hours after reperfusion, the mouse eyeballs were enucleated, and blood samples were collected from each group of rats. After standing at room temperature for 30 minutes, all blood samples were centrifuged at 3000 rpm for 15 minutes, and serum was collected. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were measured using an automated blood biochemical analyzer (Shenzhen Raydu Life Science).

[0083] 3.2 Organ preservation effect

[0084] like Figure 8 The prepared organ preservation perfusate served as the experimental group (new perfusate group), and serum ALT and AST levels were significantly lower than those in the control group (HTK group) treated with the simple HTK solution. These results indicate that the new organ preservation perfusate is significantly more effective in protecting liver transplant recipients from cold ischemia-reperfusion injury than HTK solution after perfusion at 4°C for one hour.

Claims

1. Application of bilobalide in the preparation of organ protective solution.

2. The use according to claim 1, It is characterized in that The organ protection solution is used for protecting the liver from ischemia-reperfusion injury.

3. The use according to claim 1, It is characterized in that When in use, the organ protection solution is used to perform perfusion protection on the isolated organ.

4. An organ protection solution, It is characterized in that The organ protection solution comprises bilobalide and an isotonic liquid for providing the organ protection solution with an isotonic effect.

5. The organ protection solution according to claim 4, It is characterized in that The isotonic liquid is HTK solution.

6. The organ protection solution according to claim 4, It is characterized in that The concentration of bilobalide is 500 mg / L.

7. The organ protection solution according to claim 4, It is characterized in that Based on a volume of 1000 ml, the components include 500 mg of bilobalide, 1 g of penicillin sodium, 20000 U of heparin, 10 mg of N-acetylcysteine, 5 ml of dimethyl sulfoxide, and HTK solution is added to make up to 1000 ml.

8. A method for preserving an organ in vitro, It is characterized in that The organ after ex vivo transplantation is perfused in the organ protection solution according to any one of claims 4 to 7.

9. The method for preserving an organ in vitro according to claim 8, It is characterized in that The temperature during infusion is 2℃~6℃; the infusion time is 1h.

10. The method for preserving an organ in vitro according to claim 8, It is characterized in that The organ is the liver.

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