Preserving fluid for relieving early injury of fatty degeneration transplanted liver

Abstract

The invention discloses a preserving fluid for relieving ischemia-reperfusion injury of transplanted fatty liver, and belongs to the technical field of biomedicine. The invention provides an application of a fibroblast growth factor 21 in preparation of a medicine for preventing and treating liver injury caused by ischemia-reperfusion or a liver transplanted organ preserving fluid. Hepatic cell injury caused by ischemia-reperfusion can be effectively improved through exogenous supplement of the fibroblast growth factor 21; particularly, aiming at the fatty degeneration transplanted liver, the traditional Chinese medicine composition not only can inhibit color tissue damage, cell apoptosis and inflammation caused by ischemia-reperfusion, but also can improve lipid accumulation in the liver, reverse the fatty degeneration of the liver and improve the fatty degeneration donor liver transplantation curative effect.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a preservation solution for alleviating early damage of fatty degenerated transplanted liver. Background Art

[0002] Liver transplantation (LT) is the most effective radical treatment for patients with end-stage liver disease. Early injury to the transplanted liver is common after LT, which can lead to early allograft dysfunction and is closely related to poor prognosis of the transplanted liver and recipient. Ischemia-reperfusion (I / R) injury is the main cause of early injury to the transplanted liver and the main determinant of liver shortage.

[0003] In recent years, in order to overcome the contradiction between organ demand and supply, major transplant centers have increased the use of fatty livers year by year. However, fatty livers are more sensitive to I / R injury and prone to graft dysfunction, which seriously affects the efficacy of this type of liver transplantation. In the context of fatty degeneration, the occurrence of I / R injury is more like a "second blow", and the damaged liver is prone to some more aggressive forms of the disease, such as non-alcoholic steatohepatitis and hepatocellular carcinoma.

[0004] However, there are currently no approved drugs for the prevention and treatment of I / R-induced liver injury in clinical practice.

[0005] Static cold storage (SCS) is currently the mainstream method for preserving liver donors in vitro in clinical practice. It uses low temperature to reduce cell metabolic rate and oxygen consumption, thereby reducing damage to the donor liver during storage. However, even in a low-temperature environment, cells still perform anaerobic respiration at a lower rate, leading to intracellular ATP depletion and lactic acid accumulation, and ultimately causing cell damage and loss of graft function. At the same time, the lack of shear stress stimulation caused by dynamic blood flow may reduce the production of endogenous nitric oxide and aggravate endothelial cell damage during reperfusion. Currently, SCS is only suitable for short-term organ preservation, and the effect is poor.

[0006] The results of many studies have shown that perfusion fluid combined with nitric oxide and various vasodilators can significantly improve liver vascular microcirculation, enhance liver function, and reduce the risk of post-transplantation complications. A study from Massachusetts General Hospital also showed that the "delipidation cocktail" strategy combined with perfusion fluid can achieve liver cell delipidation in a short period of time and significantly improve the function of fatty liver transplants. It can be seen that in-depth analysis of the molecular mechanism of the lipid metabolism regulatory network of liver cells, identification of related key regulatory factors, and the development of new intervention strategies based on cold preservation technology, while actively promoting the development of multi-center, leading clinical research, will be an important research direction for improving the quality of fatty liver donors and reducing their abandonment rate in the future.

[0007] Fibroblast growth factor 21 (FGF21) is a secretory factor mainly produced by hepatocytes. It plays an important role in maintaining the body's energy metabolism homeostasis and has the function of regulating tissue repair and regeneration. To date, more and more evidence has shown that FGF21 has shown good therapeutic effects in various disease models, such as atherosclerosis, microvascular injury and stroke, and the potential mechanism of its pharmacological action has not yet been elucidated. At present, the therapeutic effects of FGF21 are mainly anti-inflammatory, anti-oxidative, maintaining energy metabolism homeostasis and promoting tissue repair. The effect and mechanism of FGF21 on liver I / R injury are still unclear and need further study. Summary of the invention

[0008] The purpose of the present invention is to provide a drug that can effectively improve ischemia-reperfusion injury of transplanted liver, especially fatty liver donor, and apply it to the cold storage process of liver transplantation to improve the quality of fatty liver donor and reduce the abandonment rate.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The present invention provides the use of fibroblast growth factor 21 in preparing a medicine for preventing and / or treating liver damage caused by ischemia-reperfusion.

[0011] The fibroblast growth factor 21 can be a recombinant protein, which is a protein produced in host cells using recombinant DNA or RNA genetic engineering technology and is a modified form of natural protein.

[0012] Furthermore, the liver injury is ischemia-reperfusion injury of the transplanted liver. Ischemia-reperfusion during liver transplantation can cause early injury to the transplanted liver and lead to dysfunction of the transplanted liver.

[0013] The present invention shows that exogenous supplementation of fibroblast growth factor 21 protein can significantly improve liver ischemia-reperfusion injury.

[0014] Furthermore, the liver injury is ischemia-reperfusion injury of fatty liver. Fatty degeneration will aggravate ischemia-reperfusion injury. The present invention shows that exogenous supplementation of fibroblast growth factor 21 protein can effectively inhibit tissue damage, cell apoptosis and inflammation caused by I / R, and can also improve lipid accumulation in the liver and reduce triglyceride levels, reversing fatty degeneration of the liver.

[0015] Furthermore, the transplanted liver is a fatty liver transplanted liver.

[0016] Furthermore, the manifestations of liver damage include: sinus congestion, edema, cell vacuolation, increased number of hepatocyte necrosis areas and apoptotic cells, and immune cell infiltration. Exogenous supplementation of fibroblast growth factor 21 protein can significantly alleviate the above symptoms.

[0017] The present invention also provides the use of fibroblast growth factor 21 in the preparation of liver transplant organ preservation solution. The present invention improves liver cell damage caused by ischemia-reperfusion by adding fibroblast growth factor during the preservation process of transplanted liver.

[0018] Furthermore, the preservation solution is a preservation solution for static cold preservation. The preservation solution is composed of a conventional organ preservation solution such as UW solution or histidine-tryptophan-ketoglutarate solution (HTK solution) and fibroblast growth factor 21. Taking rats as an example, 1 mg of fibroblast growth factor 21 is added to the preservation solution per kilogram of body weight.

[0019] Furthermore, the preservation solution is a preservation solution for static cold preservation of fatty liver donors. Studies have shown that static cold preservation of fatty liver donors in an organ preservation solution containing fibroblast growth factor 21 can not only significantly improve the inherent immune damage of transplanted liver, but also improve lipid accumulation in the liver.

[0020] The present invention also provides a preservation solution for alleviating early damage of fatty degeneration transplanted liver, comprising fibroblast growth factor 21 recombinant protein and isotonic liquid.

[0021] Furthermore, the isotonic liquid is a histidine-tryptophan-ketoglutarate liquid.

[0022] The present invention has the following beneficial effects:

[0023] The present invention discloses for the first time that fibroblast growth factor 21 is an effective intervention target for early liver transplant injury. Exogenous supplementation of fibroblast growth factor 21 can effectively improve liver cell injury caused by ischemia-reperfusion; especially for fatty liver transplants, it can not only inhibit tissue damage, cell apoptosis and inflammation caused by ischemia-reperfusion, but also improve lipid accumulation in the liver, reverse fatty degeneration of the liver, and improve the success rate of fatty liver transplantation. Therefore, the application of fibroblast growth factor 21 in the preparation of drugs or liver transplant organ preservation solutions for preventing and treating liver injury caused by ischemia-reperfusion has important application value in the field of liver transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1Schematic diagram of the construction of the mouse I / R injury model and drug treatment in Example 1. Mice in the fibroblast growth factor 21 recombinant protein (rFGF21) pretreatment group were intravenously injected with rFGF21 (0.5 mg / kg) 1.5 h before surgery, n=6 mice / group.

[0025] Figure 2 Representative HE staining of liver sections of mice in each group 6 hours after reperfusion in the mouse I / R injury model in Example 1 (A) and quantitative evaluation using Suzuki score (B), ***p<0.001.

[0026] Figure 3 Representative TUNEL staining of liver sections of mice in each group 6 hours after reperfusion in the mouse I / R injury model in Example 1 (A) and comparison of the number of TUNEL+ cells per high-power field in each group (B), ***p<0.001.

[0027] Figure 4 Comparison of serum ALT (A) and AST (B) levels of mice in each experimental group 6 hours after reperfusion in the mouse I / R injury model in Example 1, ***p<0.001;

[0028] Figure 5 The Western blot in Example 1 was used to detect the expression levels of BCL-2 and Cleaved caspase-3 in the liver of each group.

[0029] Figure 6 Schematic diagram of the construction of the mouse fatty liver I / R injury model and drug treatment in Example 2. Mice in the rFGF21 pretreatment group were intraperitoneally injected with rFGF21 (0.5 mg / kg, 2 weeks); n=5 mice / group.

[0030] Figure 7 Comparison of serum ALT (A) and AST (B) levels in mice of each experimental group 6 hours after reperfusion in the mouse fatty liver I / R injury model in Example 2, **p<0.01, ***p<0.001.

[0031] Figure 8 Representative HE staining, TUNEL staining and MPO-IHC staining of liver sections of mice in each group 6 hours after reperfusion in the mouse fatty liver I / R injury model in Example 2 (A) and quantitative evaluation of liver I / R injury using Suzuki score, number of TUNEL+ cells and number of MPO+ cells per high-power field, respectively (B), ***p<0.001, ****p<0.0001.

[0032] Fig. 9Oil red O staining of liver sections of mice in each group 6 hours after reperfusion in the mouse fatty liver I / R injury model in Example 2 (A), comparison of NAFLD activity scores of each group (B) and comparison of serum TG content (C), **p<0.01, ***p<0.001.

[0033] Fig.10 This is a schematic diagram of the construction and intervention treatment of the rat steatotic donor liver orthotopic liver transplantation model in Example 3. During the liver transplantation, the donor liver was cold-stored in vitro for 4 hours and then implanted; n=5 rats / group.

[0034] Fig.11 Comparison of serum ALT (A) and AST (B) levels of rats in each experimental group 24 hours after liver transplantation in Example 3, *p<0.05, ***p<0.001.

[0035] Fig.12 Representative HE staining, TUNEL staining, MPO-IHC staining and Oil Red O staining of liver sections of rats in each group 24 hours after orthotopic liver transplantation in Example 3.

[0036] Fig.13 The Suzuki score, the number of TUNEL+ and MPO+ cells per high-power field, and the NAFLD activity score were used to quantitatively evaluate liver I / R injury in Example 3, **p<0.01, ****p<0.0001. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0039] The experimental methods involved in the following embodiments are as follows:

[0040] 1. Hematoxylin-eosin staining (HE): First, soak the liver tissue sections in the prepared xylene I and xylene II for 5 minutes each, then soak in 100% ethanol for 1 minute, 95% ethanol for 1 minute, 80% ethanol for 1 minute, and 75% ethanol for 1 minute; stain with hematoxylin for 5 minutes, rinse lightly with tap water, and the tissue can be seen to change from red to blue; differentiate in 1% hydrochloric acid alcohol for 3 seconds and return to blue in tap water for 10 minutes; stain with eosin for 1 minute, then rinse with tap water; dehydrate in alcohol gradient, 75% ethanol for 30 seconds; 80% ethanol for 30 seconds; 95% ethanol for 30 seconds; 100% ethanol for 30 seconds; transparent: place in xylene I and xylene II for 2 minutes each; seal: seal with neutral resin in a fume hood; injury scoring standard refers to Suzuki's score.

[0041] 2. Terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling (TUNEL) apoptosis detection: For adherent cells, the cells were fixed with 4% paraformaldehyde for about 30 minutes after washing with PBS once, and then 0.3% Triton X-100 was added after washing with PBS once, and incubated at room temperature for 5 minutes; for paraffin sections, the dewaxing process was as described below for immunohistochemical staining, and then 20 μg / mL DNase-free proteinase K was added, placed in a 37°C incubator for 20 minutes, and then washed three times with PBS; reaction: a circle of appropriate tissue size was drawn on the section with an immunohistochemical pen, and then about 50 μL of TUNEL detection solution was added to the sample, and incubated in a 37°C incubator in the dark for 60 minutes; after completion, PBS was washed three times; sealing: the sections were sealed with anti-fluorescence quencher sealing solution; observation: the sections were placed under a fluorescence microscope for observation and photography (excitation wavelength 450 nm, detection wavelength 565 nm).

[0042] 3. Immunohistochemistry (IHC): After slicing, mounting and baking, soak the tissue sections in xylene I and II for 10 min each, and then soak them in alcohol concentration gradient (100% ethanol-100% ethanol-95% ethanol-80% ethanol-70% ethanol) for 5 min each; immerse the sections completely in PBS solution, wash three times for 5 min each time; place the tissue sections in 0.01 mol / L citric acid repair solution (pH 6.0), preheat the water bath to 95°C, fix for 40 minutes, and wait for it to cool naturally; immerse the slice completely in PBS solution, wash three times for 5 minutes each time; use a suitable immunohistochemistry pen to draw appropriate circles around the slice tissue; add 3% hydrogen peroxide to the tissue slice and completely cover the tissue, and place it in a dark room for reaction for 10 minutes; immerse the slice completely in PBS solution, wash three times for 5 minutes each time; add the primary antibody diluted with PBS solution (the blank control only adds PBS solution) to the slice tissue and incubate at 4°C overnight; immerse the slice completely in PBS solution, wash three times for 5 minutes each time; add an appropriate amount of prepared secondary antibody to the slice tissue and incubate at 37°C for 1 hour; immerse the slice completely in PBS solution The slices were washed three times for 5 minutes each time; DAB color developing solution was prepared, and after being added to the slices, the slices were carefully observed under a microscope for color development. After color development was completed, the slices were placed in tap water to stop the color development process; after being passed through tap water for 30 seconds, the slices were stained with hematoxylin for 5 minutes, and then passed through tap water again until the color of the tissue on the slices changed from red to blue; after differentiation with 1% hydrochloric acid alcohol for 3 seconds, the slices were returned to blue under tap water for 10 minutes; the slices were placed in an alcohol concentration gradient (70% ethanol-80% ethanol-90% ethanol-100% ethanol-100% ethanol) for dehydration, and soaked for 1 minute respectively; the slices were placed in xylene I and xylene II for 2 minutes each; after natural drying in a well-ventilated place, the slices were sealed with resin and stored away from light; the immunohistochemical staining results were scored by two other researchers.

[0043] 4. Oil Red O staining of liver tissue: Fresh liver tissue was embedded with OCT embedding agent; the embedded specimen was placed on the support table and sliced ​​with a freezing microtome (generally -22°C), with a slice thickness of 5 μm, and stored in a -20°C refrigerator; Preparation of Oil Red O staining working solution: Prepare Oil Red O staining working solution according to the ratio of Oil Red O solution and ddH2O of 3:2, mix well, let it stand for a while, and then filter it with a 0.22 μm filter to ensure that it is used within 2 hours; add an appropriate amount of staining washing solution to cover the frozen section for 20 seconds, remove the staining washing solution, cover the Oil Red O staining working solution on the section, and stain for 15 minutes; after staining, wash with ddH2O, and then observe and take pictures under a microscope; finally, evaluate and analyze according to the NAFLD Activity Score (NAS).

[0044] 5. Liver function biochemical test: Alanine transaminase (ALT) and aspartate aminotransferase (AST) in liver function biochemical test are all tested by Mindray Medical's BS-220 fully automatic biochemical analyzer.

[0045] 6. Statistical methods: SPSS26.0 and GraphPad Prism 9 software were used for statistical analysis. Normal distribution quantitative data were expressed as mean ± standard deviation (x ± s), and independent sample t test was used for inter-group comparison; skewed distribution quantitative data were expressed as median (interquartile range) (M (IQR)), and Mann-Whitney test was used for inter-group comparison. Chi-square test was used for comparison of categorical variable data. Kaplan-Meier method was used to calculate survival rate and draw related survival curves, and Log-rank test was used for survival rate differences. All p values ​​were two-tailed, and p < 0.05 was considered statistically significant.

[0046] The FGF21 recombinant protein (rFGF21) used in the following examples is recombinant human fibroblast growth factor 21 purchased from Suzhou Jinan Protein Technology Co., Ltd., produced by a mammalian expression system, encoding the target gene Ala29-Ser210 (protein NCBI number Q9JJN1), with a 6His tag at the C-terminus; HTK solution was purchased from Puchuan Medical Technology (Shanghai) Co., Ltd.

[0047] Example 1: Exogenous supplementation of rFGF21 protein improves liver I / R-mediated tissue pathological damage

[0048] Considering that the absence of FGF21 can lead to worsening of transplant liver injury, this example constructed a mouse liver I / R injury model to further study whether supplementation of FGF21 can improve liver I / R injury.

[0049] 1. Experimental methods

[0050] The method for constructing the mouse liver I / R injury model pretreated with rFGF21 was as follows: Figure 1 As shown, normal C57BL / 6 mice weighing about 20 g were selected to construct a liver I / R injury model and divided into three experimental groups: a sham operation group, an I / R+0.9% saline group, and an rFGF21 pretreatment group; the rFGF21 pretreatment group was intravenously injected at a dose of 0.5 mg / kg 1.5 h before surgery, and the corresponding solvent of rFGF21 was saline (0.9% NaCl).

[0051] Establishment of mouse ischemia-reperfusion (I / R) injury model: C57BL / 6 mice weighing about 20 g were selected and fasted 12 h before surgery; 0.2 mL of 4% chloral hydrate was injected intraperitoneally to anesthetize the mice before surgery; the mice were skinned and completely disinfected with 75% alcohol, and then a midline incision was made to open the abdominal cavity of the mice, and a peritoneal dilator was placed to expose the liver. The intestinal tract was moved to one side with a cotton swab to expose the first liver portal, and the hepatic pedicles of the left and middle lobes of the liver were freed; the portal vein and hepatic artery of the left and middle lobes of the liver were clamped with non-invasive microvascular clamps. The artery was clamped to make about 70% of the liver ischemic. After a few seconds of clamping, the left and middle lobes of the liver became lighter in color, indicating successful ischemia. The abdominal cavity was temporarily closed and the surgical area was covered with wet gauze, and the mouse was placed on an electric blanket to maintain body temperature. After the mouse liver was continuously ischemic for 90 minutes, the abdominal cavity was opened, the microvascular clamp used for clamping was quickly loosened, and then the abdominal muscles and skin were sutured layer by layer to close the abdominal cavity, and disinfection was performed to complete the operation. After 6 hours of blood flow recovery in the ischemic liver, the mouse was killed after blood was drawn from the eyeball, and the ischemic liver of the mouse was completely removed for subsequent experiments.

[0052] Liver tissue sections were prepared for HE staining and TUNEL staining; serum ALT and AST levels were measured; Western blot was used to detect the expression levels of apoptosis-related proteins in the liver.

[0053] 2. Experimental results

[0054] like Figure 2 As shown, compared with the control group mice, liver I / R injury was significantly alleviated after rFGF21 pretreatment, as manifested by reduced sinus congestion, edema, and cell vacuolation or necrosis.

[0055] like Figure 3 As shown, TUNEL staining also indicated a decrease in the number of apoptotic cells.

[0056] like Figure 4 As shown, 6 hours after reperfusion, the ALT and AST levels of the rFGF21 pretreatment group decreased by 74.7% and 67.0% respectively compared with those of the control group.

[0057] like Figure 5 As shown, Western blot showed that after I / R injury, the expression of anti-apoptotic protein BCL-2 was upregulated and the expression of pro-apoptotic protein C-Caspase3 was downregulated in the liver of the rFGF21 pretreatment group compared with the control group.

[0058] The above results fully demonstrate that exogenous supplementation of rFGF21 protein can significantly improve liver I / R injury. Example 2: Exogenous supplementation of rFGF21 protein significantly reduces fatty liver I / R injury and improves fatty degeneration

[0059] In this example, a mouse fatty liver I / R injury model was constructed to evaluate the effect of exogenous rFGF21 supplementation on fatty liver I / R injury.

[0060] 1. Experimental methods

[0061] Fatty liver model in mice and rats: 4-week-old C57BL / 6 mice were selected and raised in an SPF animal room; they were deprived of food but not water for 3 hours; on the first day, the normal diet: high-fat diet was 5:1; on the second day, the normal diet: high-fat diet was 2:1; on the third day, the normal diet: high-fat diet was 1:1; on the fourth day, the diet was completely changed to high-fat diet; after 8 weeks of high-fat diet feeding, 3 mice were randomly selected, and the fatty liver model was successfully established by liver HE staining and Oil Red O staining. SD rats weighing about 120g were selected and raised in an SPF animal room; they were deprived of food but not water for 3 hours; on the first day, the normal diet: high-fat diet was 5:1; on the second day, the normal diet: high-fat diet was 2:1; on the third day, the normal diet: high-fat diet was 1:1; on the fourth day, the diet was completely changed to high-fat diet; after 8 weeks of high-fat diet feeding, 3 rats were randomly selected, and the fatty liver model was successfully established by liver HE staining and Oil Red O staining.

[0062] Construction of mouse fatty liver I / R injury model and administration of FGF21 recombinant protein Figure 6 As shown, after fatty liver mice were established after 8 weeks of high-fat diet feeding, a liver I / R injury model was constructed and divided into three experimental groups: a sham operation group, an I / R+0.9% saline group, and an rFGF21 pretreatment group; mice were intraperitoneally injected with rFGF21 at a dose of 0.5 mg / kg daily for 2 consecutive weeks before surgery, and the corresponding solvent of rFGF21 was saline (0.9% NaCl).

[0063] Then, liver tissue sections were prepared for HE staining, TUNEL staining and MPO-IHC staining; liver frozen sections were prepared for Oil Red O staining; and serum ALT, AST and TG levels were measured.

[0064] 2. Experimental results

[0065] like Figure 7 As shown in the figure, after I / R injury, the serum ALT and AST levels of mice in the rFGF21 pretreatment group were significantly lower than those in the control group.

[0066] like Figure 8 As shown, HE staining and TUNEL staining results showed that fewer hepatocyte necrosis areas and apoptotic cells were observed in the liver sections of the rFGF21 pretreatment group; MPO-IHC staining results showed that the infiltration degree of neutrophils in the I / R-injured liver of the rFGF21 pretreatment group was significantly lower than that of the control group, indicating that the innate immune damage in fatty liver was improved.

[0067] like Fig. 9 As shown, Oil Red O staining revealed that the livers of the rFGF21 pretreatment group showed more dispersed lipid microvesicles and smaller lipid vacuoles compared with the control group, and the NAFLD activity score was also significantly reduced ( Fig. 9 A and B). Combined with the comparative analysis of serum TG, it was also confirmed that the degree of liver steatosis in the rFGF21 pretreatment group was milder ( Fig. 9 C).

[0068] These results indicate that exogenous supplementation of rFGF21 can effectively inhibit tissue damage, cell apoptosis and inflammation caused by I / R, while also improving lipid accumulation in the liver, reducing triglyceride levels, and reversing liver fatty degeneration.

[0069] Example 3: FGF21 recombinant protein storage solution effectively reduces fatty liver transplant injury

[0070] In this example, a rat fatty liver transplantation model was constructed to evaluate the effect of FGF21 recombinant protein preservation solution on the repair and function improvement of marginal liver donors during liver transplantation.

[0071] 1. Experimental methods

[0072] Construction of orthotopic liver transplantation model in rats with fatty liver and intervention treatment Fig.10 As shown, after rats with fatty liver were established after 8 weeks of high-fat diet feeding, an orthotopic liver transplantation model was constructed and divided into three experimental groups: sham operation group, HTK solution group and HTK solution + rFGF21 group; the HTK solution + rFGF21 group was 100mL HTK solution with 1mg / kg rFGF21 added (calculated according to the weight of the donor rat). During the liver transplantation process, the donor liver was implanted after 4 hours of cold storage in vitro. The method is as follows:

[0073] Establishment of rat orthotopic liver transplantation (OLT) model:

[0074] (1) Preparation of donors and recipients: SD rats weighing about 450 g and fed a high-fat diet for 8 weeks were selected as donors for rat OLT, and SD rats weighing slightly larger than the donors and fed a normal diet were selected as recipients. They were fasted from 12 hours before surgery and were prohibited from drinking water from 4 hours before surgery.

[0075] (2) Anesthesia: The donor and recipient rats were anesthetized with isoflurane using a small animal inhalation anesthesia machine;

[0076] (3) Donor surgery: After the donor rat is fully anesthetized, the rat is fixed and disinfected, and then the abdominal cavity is entered along the midline of the abdomen, and an abdominal dilator is placed to expose the liver; the ligaments between the liver and the surrounding tissues and organs are severed, and the left phrenic vein is ligated and cut; the thoracic segment of the abdominal aorta is clamped with a vascular clamp, and the indwelling needle is punctured above the celiac trunk and fixed with a vascular clamp. 10 mL of 4°C UW solution is slowly perfused, and the inferior vena cava is cut as the outflow tract; after the perfusion is completed, the left and right renal veins are ligated and severed respectively, the infrahepatic inferior vena cava, portal vein, and superior and inferior vena cava are freed and severed, and the common bile duct is severed and ligated; the separated donor liver is placed in 100 mL of 4°C HTK solution or rFGF21 repair solution and stored cold for 4 h for further processing;

[0077] (4) Liver repair: The “double-cuff method” reported by Kamada was used to trim the ex vivo liver. The portal vein cuff and the infrahepatic inferior vena cava cuff were effectively fixed. The vascular wall was covered on the cuff by eversion and fixed. 10 mL of 4°C pre-cooled HTK solution was slowly perfused through the portal vein. The trimmed donor liver was placed in a 4°C refrigerator for later use.

[0078] (5) Recipient subhepatic liver: After the recipient rat is anesthetized, the abdominal cavity is entered through the midline of the abdomen, and the common bile duct and common hepatic artery are separated and ligated. The inferior vena cava below the liver is blocked with a non-damaging vascular clamp above the right renal vein, and the portal vein is blocked above the pyloric vein to enter the anhepatic phase; the main trunk of the portal vein is punctured and the liver is flushed with normal temperature saline until the liver turns white, and then the superior and inferior vena cava above the liver are blocked with a non-damaging vascular clamp, and then the entire liver is separated; the anhepatic phase should be controlled within 30 minutes;

[0079] (6) Donor liver implantation: Use 10-0 non-damaging sutures to continuously anastomose the superior and inferior vena cava of the liver connecting the donor and recipient; use the "cuff method" to complete the reconstruction of the portal vein and inferior vena cava; open the portal vein, and the anhepatic period ends. Use the biliary stent method to reconstruct the common bile duct. After checking that there is no active bleeding in the recipient's abdominal cavity, the abdomen can be closed; the recipient is placed in an incubator for postoperative recovery;

[0080] (7) Sampling: 24 h after the blood flow of the transplanted liver was restored, the rats were killed and blood was drawn from the inferior vena cava. The rat transplanted liver was completely removed for subsequent experiments.

[0081] Among them, FGF21 recombinant protein preservation solution: 100 mL of HTK solution was added at a dose of 1 mg / kg as the FGF21 recombinant protein repair solution for each rat fatty degeneration donor liver.

[0082] 2. Experimental results

[0083] like Fig.11 As shown in the figure, in the early stage after liver transplantation, the serum ALT and AST levels of rats in the FGF21 recombinant protein preservation solution group were significantly lower than those in the HTK solution group.

[0084] like Fig.12 and Fig.13 As shown, HE staining and TUNEL staining results showed that fewer hepatocyte necrosis areas and apoptotic cells were observed in the transplanted liver sections of the FGF21 recombinant protein preservation solution group. MPO-IHC staining results showed that the degree of neutrophil infiltration in the transplanted liver of the FGF21 recombinant protein preservation solution group was significantly lower than that of the control group, suggesting that the inherent immune damage in the fatty degeneration donor liver was improved. Oil red O staining found that the transplanted liver of the FGF21 recombinant protein preservation solution group showed more dispersed lipid microvesicles and smaller lipid vacuoles than that of the HTK solution group, but there was no statistically significant difference in the NAFLD activity score.

[0085] These results indicate that the FGF21 recombinant protein preservation solution group can effectively improve tissue damage, cell apoptosis and inflammation caused by the liver transplantation process, and also has certain potential for improving lipid accumulation in the liver.

Claims

1. Use of fibroblast growth factor 21 in the preparation of a drug for preventing and / or treating ischemia-reperfusion-induced liver injury.

2. The use according to claim 1, characterized in that The liver injury is ischemia-reperfusion injury of fatty liver.

3. The use according to claim 1, characterized in that The liver injury is ischemia-reperfusion injury of transplanted liver.

4. The use according to claim 3, characterized in that The transplanted liver is a fatty liver.

5. The use according to any one of claims 1 to 4, characterized in that: The manifestations of liver damage include: sinus congestion, edema, cell vacuolation, increased areas of hepatocyte necrosis and apoptotic cell numbers, and immune cell infiltration.

6. Use of fibroblast growth factor 21 in the preparation of liver transplantation organ preservation solution, characterized in that: Fibroblast growth factor 21 improves hepatocyte injury induced by ischemia-reperfusion.

7. The use according to claim 6, characterized in that The storage solution is a storage solution for static cold storage.

8. The use according to claim 6, characterized in that The preservation solution is a preservation solution used for static cold preservation of fatty degenerated donor livers.

9. A preservation solution for reducing early damage to fatty liver transplantation, characterized in that: Includes fibroblast growth factor 21 recombinant protein and isotonic fluid.

10. The preservation solution according to claim 9, characterized in that The isotonic liquid is histidine-tryptophan-ketoglutarate liquid.

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