Organ storage liquid and application thereof

Abstract

The invention discloses an organ storage solution and application thereof. The organ storage solution comprises Pan23 polypeptide and an organ preservation solution. The organ storage liquid provided by the invention can be used for reducing in-vitro injury of organs or tissues, reducing stress response of endoplasmic reticulum in cells, reducing formation of mitochondria-endoplasmic reticulum coupling bodies (MAMs), reducing formation of lipid droplets in the cells and cold injury of the cells, playing a role in protecting the mitochondria, improving generation of antioxidant enzymes in the cells and playing a role in protecting the cells.

Description

[0001] This application claims the priority of Chinese Patent Application No. 2022112689202 filed on October 17, 2022. This application cites the entire text of the above Chinese Patent Application. Technical Field

[0002] The present invention relates to the technical field of cell biology, and in particular to an organ storage solution and application thereof. Background Art

[0003] Organ transplantation is the only effective treatment for various end-stage organ diseases, but the severe shortage of donors has become one of the key issues restricting the development of organ transplantation. The short storage time and poor preservation effect of ex vivo organs are one of the main reasons for the shortage of donor organs. A large number of potential donors are abandoned due to the lack of more effective preservation measures. At the same time, the preservation effect of the donor liver directly affects the quality of the donor liver, and the quality of the donor liver is directly related to the success of the recipient's transplant surgery.

[0004] UW (University of Wisconsin solution) preservation solution is the "gold standard" for organ preservation solution in transplantation. It has been used clinically for nearly 20 years and can effectively preserve the liver, pancreas, and kidneys for 2 days. UW organ preservation solution is an intracellular fluid type organ preservation solution, containing high K + 、Na + 、H2PO4 - OH - To neutralize the H produced in cells under anaerobic metabolism + . The electrolyte components in the intracellular fluid type preservation fluid are similar to the intracellular electrolyte concentration, which can reduce the exchange of intracellular and extracellular electrolytes, thereby keeping the cells in a quiescent state. UW preservation fluid contains non-permeable substances including lactobionic acid, kapok sugar, and hydroxyethyl starch, which can inhibit cell edema during low-temperature preservation. It contains glutathione and adenosine, which helps to improve the ability of the graft to resist ischemia and reperfusion injury and maintain cell energy metabolism. However, some organs (10-15% of livers and 20-30% of kidneys) still have graft dysfunction after UW preservation fluid irrigation. In addition to the factors of the donor organ itself, the preservation effect of UW preservation fluid itself also needs to be improved. In theory, UW preservation fluid can preserve the donor liver for 20-24 hours, but there is cold preservation damage during the static cold storage (SCS) process based on the organ preservation fluid. Excessive cold storage time, that is, prolonged cold ischemia time, can cause biliary complications, graft loss of function, and even death of the recipient. The ideal cold storage time for donor livers should not exceed 8 hours. In clinical practice, the storage time for donor livers generally does not exceed 12 to 15 hours.

[0005] Celsior organ preservation solution was successfully developed by Pasteur-Merieux in 1994. It is an extracellular fluid type organ preservation solution. Celsior solution is high in sodium and low in potassium, similar to extracellular fluid, and has low viscosity, which makes it easy to diffuse quickly and evenly into the interstitial space, allowing the preserved organs to cool evenly in a short time, thereby improving the perfusion effect. Celsior solution contains glutamate, which can be converted with α-ketoglutaric acid, which is an intermediate product of the tricarboxylic acid cycle. It can synthesize ATP through the tricarboxylic acid cycle, respiratory chain and oxidative phosphorylation. Therefore, it helps to synthesize ATP during ischemia-reperfusion and alleviates the lack of energy in cells.

[0006] At present, hypothermic cold ischemia preservation is still the most important technology for preserving organs. The low temperature of 6-8℃ and the chemical composition of the solution are the basis of cold ischemia preservation. Although hypothermic preservation can slow down the speed of biochemical reactions, it cannot stop cell metabolism or prevent cell death. The harmful processes caused by ischemia and hypothermia continue, such as cell edema, interstitial edema and cell acidosis. This requires the improvement of the composition of the solution to avoid or reduce such damage. Studies have shown that 4℃ preservation is harmful to cells because it destroys the Na-K-ATP pump and mitochondrial adenosine convertase, destroys the stability of the intracellular environment, and induces cell stress response. At the same time, cell edema or interstitial edema can reduce the rate of gas exchange. Researchers have added other chemicals such as dexamethasone, glutathione, allopurinol, etc. to the preservation solution to reduce oxygen free radicals, reduce cell stress damage, and reduce reperfusion injury. Therefore, it is of positive and far-reaching significance to improve the existing organ preservation solution or to research and develop new organ preservation solutions. Summary of the invention

[0007] In order to solve the defects of the prior art that cold ischemia of tissue or organ preservation fluid causes intracellular stress response leading to cell damage and low tissue or organ preservation quality, the present application provides an organ storage fluid and its application, wherein the organ storage fluid contains a novel ATP channel inhibitor Pan23 polypeptide. When the tissue or organ is preserved with the organ storage fluid, the opening of the Panx1 membrane channel on the cell membrane is inhibited, thereby inhibiting the release of ATP and reducing the stress response of cells in the tissue or organ, which can ultimately reduce cell damage, prolong the organ preservation time, and improve the preservation quality.

[0008] In order to solve the above problems, one of the solutions of the present invention provides an organ storage solution, which comprises Pan23 polypeptide and organ preservation solution.

[0009] In a specific embodiment, the amino acid sequence of the Pan23 polypeptide is shown in SEQ ID NO:1.

[0010] In some embodiments, the amino acids in the amino acid sequence are in L-form.

[0011] In some embodiments, the content of Pan23 polypeptide is ≥30 μM, such as 50 μM, 100 μM or 200 μM, preferably 50 μM.

[0012] In some embodiments, the organ preservation solution is an extracellular fluid-mimicking organ preservation solution or an intracellular fluid-mimicking organ preservation solution.

[0013] In a preferred embodiment, the extracellular fluid-mimicking organ preservation solution is Celsior preservation solution, and the intracellular fluid-mimicking organ preservation solution is University Wisconsin (UW) preservation solution.

[0014] In order to solve the above problems, the second solution of the present invention provides a method for preserving tissues or organs, which comprises the following steps: placing the free tissues or organs after lavage in the organ storage solution as described in one of the solutions of the present invention for preservation; the temperature of the organ storage solution is 1 to 10°C, preferably 4°C.

[0015] In specific embodiments, the organ is the liver.

[0016] In order to solve the above problems, the third solution of the present invention provides a method for preparing the organ storage solution described in the first solution of the present invention, and the preparation method comprises the following steps: adding Pan23 polypeptide to an organ preservation solution such as UW preservation solution or Celsior preservation solution; the final concentration of the Pan23 polypeptide is ≥30 μM, for example, 50 μM, 100 μM or 200 μM, preferably 50 μM.

[0017] In order to solve the above problems, the fourth solution of the present invention provides the use of Pan23 polypeptide or the organ storage solution as described in one of the solutions of the present invention in preserving tissues or organs.

[0018] In specific embodiments, the organ is the liver.

[0019] In a preferred embodiment of one of the schemes of the present invention, when the organ preservation fluid is an extracellular fluid-mimicking organ preservation fluid, the organ storage fluid is at least prepared from Celsior preservation fluid and Pan23; when the organ preservation fluid is an intracellular fluid-mimicking organ preservation fluid, the organ storage fluid is at least prepared from UW preservation fluid and Pan23.

[0020] In a preferred embodiment of one of the schemes of the present invention, the final concentration of Pan23 is ≥30 μM, such as 50 μM, 100 μM or 200 μM.

[0021] In a specific embodiment of one of the schemes of the present invention, the UW preservation solution comprises 50 g / L hydroxyethyl starch, 100 mmol / L lactobionic acid, 25 mmol / L potassium dihydrogen phosphate, 5 mmol / L magnesium sulfate, 30 mmol / L raffinose, 5 mmol / L adenosine, 3 mmol / L glutathione, 150 mmol / L potassium hydroxide, and an osmotic pressure of 3163 mOsm / L; the Celsior preservation solution comprises 80 mmol / L lactobionic acid, 1-3 mmol / L potassium dihydrogen phosphate (KH2PO4·2H2O), 13 mmol / L magnesium sulfate (MgSO4·7H2O), 100 mmol / L sodium glutamate, 5 mmol / L adenosine, 3 mmol / L glutathione, and 150 mmol / L potassium hydroxide, and an osmotic pressure of 3163 mOsm / L. 10 H 13 N5O4) 10mmol / L, mannitol 60mmol / L, glutathione 3mmol / L, potassium hydroxide (KOH) 15mmol / L, glutamate 20mmol / L and histidine 30mmol / L.

[0022] In a preferred embodiment of the fourth scheme of the present invention, the tissue or organ is a tissue or organ that has been subjected to cold ischemia for 8 to 24 hours; preferably 8 to 12 hours, and more preferably 8 to 10 hours.

[0023] In a preferred embodiment of the fourth embodiment of the present invention, the preservation is selected from one or more of the following:

[0024] (1) Reduce the levels of AST and ALT in perfusate or serum;

[0025] (2) Upregulation of intracellular peroxidase gene expression and peroxisomes;

[0026] (3) Reduce the formation of autophagosomes;

[0027] (4) Inhibit the opening of cell membrane channel protein Panx1, reduce intracellular ATP release, maintain the stability of the intracellular environment, and reduce the stress response of tissues and organs;

[0028] (5) Improve the 24-hour survival rate after transplantation;

[0029] (6) Reduce the damage of bile acid to cells;

[0030] (7) Reduce hepatocyte bridging necrosis, inflammatory cell infiltration, and abnormal morphology of hepatic cords and lobules;

[0031] (8) Less mitochondrial damage, MAMs, endoplasmic reticulum stress, and cellular autophagy response.

[0032] In order to solve the above problems, the fifth solution of the present invention further provides the use of Pan23 polypeptide or the organ preservation solution as described in one of the solutions of the present invention in promoting the regenerative function of the organ after transplantation.

[0033] In the present invention, the organ storage solution refers to the modified organ preservation solution. The organ storage solution and organ preservation solution are only used to distinguish whether it is modified on the basis of the organ preservation preparation in the prior art, and have no other practical meaning. The organ preservation solution in the present invention refers to the preparation for preserving organs in the prior art.

[0034] In the present invention, μM=μm / ml.

[0035] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0036] The reagents and raw materials used in the present invention are commercially available.

[0037] The positive and progressive effect of the present invention is that the organ storage solution provided by the present invention can effectively reduce cell stress response and cell damage when preserving tissues or organs. Preferably, the storage solution can reduce cell endoplasmic reticulum stress, reduce the formation of endoplasmic reticulum mitochondria coupline (MAMs), reduce intracellular lipid droplet formation, and reduce cell cold damage caused by ice crystal formation during 4°C storage. At the same time, mitochondrial damage is reduced, the level of cellular antioxidant enzymes is increased, the cell autophagy reaction is reduced, the cell protection effect is exerted, the postoperative survival rate is improved, the AST and ALT levels in serum are reduced, and the bile acid damage to liver cells is effectively reduced, and liver cell bridging necrosis, inflammatory cell infiltration, abnormal morphology of liver cords and lobules are reduced. For example, the preservation effect on the liver is far superior to that of conventional preservation solutions that do not contain Pan23 polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Serum liver transaminase test showed that the polypeptide compound Pan23 reduced ALT and AST, indicating that liver cell damage was reduced.

[0039] Figure 2 q-PCR was used to detect the mRNA of antioxidant enzymes in liver tissue, including superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and heme oxygenase-1 (HO-1). (12h) q-PCR detection showed that the polypeptide compound Pan23 increased the mRNA level of antioxidant enzymes in hepatocytes.

[0040] Figure 3Electron microscopic observation of liver tissue cell structure (12h) showed that compared with UW preservation solution and Celsior preservation solution, the addition of polypeptide compound Pan23 can reduce the formation of intracellular endoplasmic reticulum-mitochondrial coupling (MAMs) and reduce endoplasmic reticulum stress. In liver cells preserved with UW preservation solution and Celsior preservation solution, the internal cristae structure of mitochondria disappeared, mitochondria were vacuolated, and the structure was incomplete. After the addition of polypeptide compound Pan23, the mitochondrial structure was intact and the damage was reduced.

[0041] Figure 4 Electron microscopy observation of lipid droplet formation in liver cells (12h) showed that the polypeptide compound Pan23 reduced intracellular lipid droplet formation compared with UW preservation solution.

[0042] Figure 5 Electron microscopy observation of ice crystal formation in liver tissue cells (12h) showed that compared with UW preservation solution, the polypeptide compound Pan23 reduced the cell cold damage caused by ice crystal formation when stored at 4 degrees.

[0043] Figure 6 Electron microscopic observation of autophagosome formation in liver tissue cells (12h) showed that a large number of autophagosomes were formed in liver cells preserved with Celsior preservation solution; the polypeptide compound Pan23 reduced the formation of a large number of peroxisomes in cells and played a cytoprotective role.

[0044] Figure 7 This is a simplified diagram of the experimental process for rats.

[0045] Figure 8 The effect of Pan23 on the 24-hour survival rate of rats receiving liver transplantation with different cold ischemia times. The 24-hour survival rate of rats receiving liver transplantation with different cold ischemia times and storage conditions (8 pairs of experimental rats in each group).

[0046] Fig. 9 The effect of Pan23 peptide on rat liver graft function. A. AST and ALT concentrations in rat liver lavage fluid at different cold ischemia time points (lavage fluid is 50 ml); B. Serum ALT and AST concentrations in rats receiving livers with different cold ischemia time 24 hours after surgery (values ​​are shown as mean ± standard error; *P<0.05, **P<0.01, ***P<0.001, 5 experimental rats in each group).

[0047] Fig.10 The effect of Pan23 polypeptide on serum bile acid concentration in rats. Serum bile acid concentration in rats after 4-6 hours of cold ischemia and 24 hours of reperfusion of donor liver (values ​​are shown as mean ± standard error; *P<0.05, **P<0.01; 4 experimental rats in each group).

[0048] Fig.11The morphological effects of Pan23 polypeptide on cold ischemic injury of rat liver transplantation. A. HE staining of liver tissue after 8-10 hours of cold ischemia of the donor liver; B. F. Suzuki score of HE staining of liver tissue; C. The condition of transplanted livers in the two groups of rats 24 hours after 8-10 hours of cold ischemia of the donor liver (values ​​are shown as mean ± standard error; *P<0.05; HE staining scale: 1000μm; 5 experimental rats in each group).

[0049] Fig.12 The effect of Pan23 peptide on rat liver regeneration. A. Liver tissues of transplanted rats in each group with 4-6 hours and 8-10 hours of cold ischemia were obtained for Ki67 immunohistochemical staining and quantitative analysis of positive cell density 24 hours after surgery; B. Ki67 protein immunoblot analysis and quantitative analysis of protein expression level were performed on liver tissues of transplanted rats in each group 24 hours after surgery; C. Transmission electron microscopy was used to observe the hepatocytes of transplanted rats in each group 24 hours after surgery (values ​​are shown as mean ± standard error; *P<0.05, **P<0.01; immunohistochemical scale: 200μm / 20μm, electron microscope scale: 1μm / 2μm; 3 experimental rats in each group).

[0050] Fig.13 The state of the background hepatocytes of the rat donor liver under transmission electron microscopy. The organelles and ultrastructural morphology of the background hepatocytes of the rat donor liver under transmission electron microscopy (box: autophagosomes with no clear origin; scale bar: 2μm).

[0051] Fig.14 The state of liver cells in donor liver of rats under electron microscope after 4-6 hours of cold ischemia. A. Organelles and ultrastructural morphology of liver cells in donor liver of rats in control group after 4-6 hours of cold ischemia under transmission electron microscope; B. Organelles and ultrastructural morphology of liver cells in donor liver of rats in experimental group after 4-6 hours of cold ischemia under transmission electron microscope (box: autophagosomes of uncertain origin and multi-layered autophagosomes; circle: lipid droplet autophagosomes; arrow: MAMs; scale: 1μm / 2μm).

[0052] Fig.15 The status of liver cells in rat donor livers after 8-10 hours of cold ischemia under electron microscopy. A. Organelles and ultrastructural morphology in liver cells of rat donor livers in the control group after 8-10 hours of cold ischemia under transmission electron microscopy; B. Organelles and ultrastructural morphology in liver cells of rat donor livers in the experimental group after 8-10 hours of cold ischemia under transmission electron microscopy (box: autophagosomes of uncertain origin; one arrow at the bottom of the second picture: MAMs; two arrows at the top of the second picture: peroxisomes; scale: 500nm / 1μm / 2μm).

[0053] Fig.16The status of hepatocytes in rat donor livers 24 hours after transplantation under electron microscope. A. Organelles and ultrastructural morphology in hepatocytes of rat control group donor livers after 4-6 hours of cold ischemia and reperfusion for 24 hours under transmission electron microscope; B. Organelles and ultrastructural morphology in hepatocytes of rat donor livers in experimental group after 4-6 hours of cold ischemia and reperfusion for 24 hours under transmission electron microscope (circles in A2 and A4, small circles in B4: endoplasmic reticulum autophagosomes; large circles on the left side of B4: lipid droplet autophagosomes; arrows in A2, arrows above A4, arrows below B2: MAMs; arrows below A4: lipid droplets; arrows above B2: peroxisomes; scale: 500nm / 1μm / 2μm).

[0054] Fig.17 The state of liver cells in rat donor livers 24 hours after transplantation under electron microscope. A. Organelles and ultrastructural morphology in liver tissue cells of rat control group donor livers after 8-10 hours of cold ischemia and reperfusion for 24 hours under transmission electron microscope; B. Organelles and ultrastructural morphology in liver tissue cells of rat donor livers in experimental group after 8-10 hours of cold ischemia and reperfusion for 24 hours under transmission electron microscope (box: autophagosomes of uncertain origin; arrows: peroxisomes; scale: 1μm / 2μm).

[0055] Fig.18 The effect of Pan23 polypeptide on autophagic response of rat liver graft cells. A. Immunohistochemical staining of Beclin-1 in background, cold ischemia and reperfusion liver tissues of rat liver donors with 4-6 hours of cold ischemia; B. Immunohistochemical score (H-Score) of Beclin-1 in the group with 4-6 hours of cold ischemia of the liver donors; C. Immunohistochemical staining of Beclin-1 in background, cold ischemia and reperfusion liver tissues of rat liver donors with 8-10 hours of cold ischemia; D. Immunohistochemical score (H-Score) of Beclin-1 in the group with 8-10 hours of cold ischemia of the liver donors (values ​​are shown as mean ± standard error; *p<0.05, **p<0.01; scale: 20μm; 3 experimental mice in each group).

[0056] Fig.19 The effect of Pan23 polypeptide on inflammatory factors and antioxidant enzymes in rat liver. A. RT-qPCR detection of relative mRNA expression levels of Panx1 and inflammatory factors in liver; B. RT-qPCR detection of relative mRNA expression levels of antioxidant enzymes in liver (values ​​are presented as mean ± standard error; *p<0.05, **p<0.01, ***p<0.001; 5 experimental mice in each group; μM=μm / ml).

[0057] Fig. 20The effect of Pan23 polypeptide on inflammatory factors and antioxidant enzymes in rat liver. Luminex multi-protein factor was used to detect the ratio of each inflammatory factor protein in the liver tissue to the total liver tissue protein (values ​​are presented as mean ± standard error; *p<0.05; 5 experimental mice in each group; μM=μm / ml).

[0058] Fig.21 The effect of Pan23 polypeptide on rat transplanted hepatocyte apoptosis. A. Bcl-2 immunohistochemical staining and immunohistochemical score (H-Score) of rat background liver tissues after 4-6 hours and 8-10 hours of cold ischemia and 24 hours of transplantation reperfusion; B. Tunel immunohistochemical staining and quantitative analysis of positive cell density of rat background liver tissues after 4-6 hours of cold ischemia and 24 hours of transplantation reperfusion; C. Tunel immunohistochemical staining and quantitative analysis of positive cell density of rat background liver tissues after 8-10 hours of cold ischemia and 24 hours of transplantation reperfusion (values ​​are shown as mean ± standard error; *p<0.05, **p<0.01; 3 experimental mice in each group; μM=μm / ml). DETAILED DESCRIPTION

[0059] Example 1 Liver preservation test

[0060] Mice (C57BL / 6, 8-week-old male mice) were anesthetized using Ward R500 universal small animal anesthesia machine. After successful anesthesia, the mice were fixed on the operating table, the abdomen was shaved, and a midline cross incision was made. First, the xiphoid process was lifted with appendix forceps to expose the upper abdomen, and the small intestine was pushed to the left to expose the portal cavity and the inferior vena cava. The liver was freed from the surrounding tissues. The liver was irrigated once with 4°C sterile PBS perfusion solution through the portal vein, and blood was drawn from the inferior vena cava below the level of the right renal vein. Then the liver was irrigated with the following four preservation solutions respectively. After the irriga-tion, the liver was yellow. The superior and inferior vena cava, the infrahepatic inferior vena cava, the portal vein and the hepatic artery were cut off. At this time, the liver was completely free and the liver was placed in the following four 4°C preservation solutions:

[0061] (1) UW preservation solution (manufacturer: Organ discovery, catalog number: PBR-0060-123), composition: 50 g / L hydroxyethyl starch, 100 mmol / L lactobionic acid, 25 mmol / L potassium dihydrogen phosphate, 5 mmol / L magnesium sulfate, 30 mmol / L raffinose, 5 mmol / L adenosine, 3 mmol / L glutathione, 150 mmol / L potassium hydroxide; osmotic pressure: 3163 mOsm / L.

[0062] (2) UW storage solution + 50 μM Pan23 (SEQ ID NO: 1);

[0063] (3) Celsior preservation solution (Institut Georges Lopez SAS, catalog number: SL220033) contains: lactobionic acid 80 mmol / L, potassium dihydrogen phosphate (KH2PO4·2H2O) 1-3 mmol / L, magnesium sulfate (MgSO4·7H2O) 13 mmol / L, sodium glutamate 100 mmol / L, adenosine (C 10 H 13 N5O4) 10mmol / L, mannitol 60mmol / L, glutathione 3mmol / L, potassium hydroxide (KOH) 15mmol / L, glutamate 20mmol / L and histidine 30mmol / L.

[0064] (4) Celsior storage solution + 50 μM Pan23.

[0065] Perfusion fluid collection: Portal cannulation of mice, remove the liver, and place it in the containers of the above four preservation solutions. Use a syringe to extract liquid from the preservation container at 4h, 8h, 12h, 24h, and 48h, and pour it into the portal vein cannulation. Collect the preservation solution that flows out of the liver after perfusion, which is the liver tissue perfusion solution, and store it at -80 degrees for later use. Liver tissue collection: Cut a small piece of tissue at 4h, 8h, 12h, and 24h, fix the tissue with 5% glutaraldehyde solution, and store it at 4 degrees for observation; quick-freeze with liquid nitrogen and store it at -80 degrees for later use.

[0066] The experiment was divided into two groups: (1) UW preservation solution + 50μM Pan23 group and control UW preservation solution group; (2) Celsior preservation solution + 50μM Pan23 group and control Celsior preservation solution group. Roche Cobas6000 automatic biochemical analyzer was used to measure ALT and AST in the preservation solution obtained by lavage. The experimental results are shown in Figure 1Compared with UW preservation solution, the ALT levels of liver tissue perfusate at 8h (150.65±6.32 vs. 191.23±4.45, p<0.001), 12h (162.71±11.42 vs. 255.23±12.11, p<0.001), and 24h (210.45±9.27 vs. 299.17±11.53, p<0.001) after Pan23 was added to the preservation solution were all lower than the ALT levels of UW perfusate at the corresponding time points (p<0.05). The AST levels in the liver perfusate of Pan23 preserved for 8 h (103.83±5.28 vs. 189.98±7.25, p<0.001), 12 h (96.47±6.10 vs. 261.27±5.55, p<0.001), and 24 h (116.20±5.76 vs. 287.15±9.71, p<0.001) were all lower than those in the UW perfusate at the corresponding time points (p<0.05). Compared with Celsior preservation solution, the ALT levels of liver tissue perfusate at 8h (153.65±17.45 vs. 286.73±10.75, p<0.001), 12h (151.50±30.57 vs. 315.85±15.90, p<0.001) and 24h (262.11±17.34 vs. 355.10±15.90, p<0.001) of Pan23 preservation after adding Pan23 preservation solution were all lower than the ALT levels of UW perfusate at the corresponding time points (p<0.05). The AST levels in the liver tissue perfusate after Pan23 preservation at 8h (111.05±22.94 vs. 205.05±9.85, p<0.001), 12h (98.33±12.24 vs. 310.91±16.64, p<0.001) and 24h (250.75±53.60 vs. 383.08.15±23.05, p<0.001) were all lower than those in the UW perfusate at the corresponding time points (P<0.05). The results of enzymatic detection of liver tissue perfusate showed that adding Pan23 to the intracellular fluid preservation fluid UW or the extracellular fluid preservation fluid Celsior could effectively reduce the damage of ex vivo liver tissue cells.

[0067] 12h liver tissue was selected, genome was extracted, and q-PCR was used to detect the mRNA expression level of hepatocyte antioxidant enzymes. The results showed that Pan23 blocking Panx1 channel could significantly upregulate the expression of peroxidase genes in hepatocytes (P<0.05)( Figure 2 ).

[0068] Electron microscopic examination method: liver tissue specimens were collected 12 hours later, cut into 1 mm3 pieces, and quickly placed in 2.5% glutaraldehyde fixative, fixed at 4°C for more than 3 hours; washed 3 times with 0.1M phosphate buffer, and then fixed with 1% osmium acid at 4°C for 3 hours; then washed 3 times with buffer, dehydrated with ethanol step by step, and embedded with Epon 812 resin. The embedded blocks were placed on an ultrathin slicer (Leica UC6) for sectioning, the ultrathin section thickness was 70nm, and the slices were picked up with a Fanghua membrane copper mesh. After staining with uranyl acetate for 30 minutes and lead citrate for 15 minutes, the liver tissue cell structure was observed under a transmission electron microscope (Thermofisher Talos120) at 120kV, showing that in the UW preservation solution group, a large number of MAMs were generated in the cells, the endoplasmic reticulum stress response was severe, the mitochondrial structure was destroyed, the number of mitochondria decreased, and the mitochondrial damage was severe. Adding Pan23 can effectively reduce endoplasmic reticulum stress response, reduce the formation of MAMs in liver cells, and reduce liver cell mitochondrial damage ( Figure 3 ); while adding Pan23 reduced the formation of lipid droplets in liver cells ( Figure 4 ), and thus the formation of ice crystals in liver tissue cells is reduced ( Figure 5 ). Electron microscopy showed that compared with Celsion preservation solution, adding Pan23 can also effectively reduce hepatocyte mitochondrial damage, reduce endoplasmic reticulum stress response, and reduce the formation of MAMs in hepatocytes ( Figure 3 ); Adding Pan23 can effectively reduce the formation of autophagosomes in hepatocytes ( Figure 6 ).

[0069] The liver mitochondrial metabolism is relatively active and more difficult to preserve than other tissues or organs. Therefore, it can be inferred that Pan23 also has beneficial effects on other tissues or organs.

[0070] Cold ischemia after ex vivo tissues and organs will cause ATP to be released outside the cells, thereby inducing a low ATP environment inside the cells, destroying the homeostasis inside the cells, causing stress reactions inside the cells, and leading to cell damage. The mechanism of action of Pan23 is to inhibit the opening of the cell membrane channel protein Panx1, reduce the release of ATP inside the cells, maintain the stability of the intracellular environment, reduce the stress reactions of tissues and organs, and thus play a protective role in tissue cells. Therefore, Pan23 can also be compounded with other preservation solutions to obtain the same beneficial effects.

[0071] Example 2 Protective effect and mechanism of Pan23 polypeptide on rat liver donor in vitro and after transplantation

[0072] Next, a rat syngeneic liver transplantation model was established (the process diagram is shown in the figure). Figure 7 ), using 6-10 week old Lwies (RT1 1) Male rats, donors 150-180g, recipients 180-200g. The protective effect and mechanism of Pan23 peptide on rat liver transplantation before and after surgery were studied by analyzing the 24-hour survival rate of rats receiving liver donors with different cold ischemia time groups, serum / lavage fluid liver enzyme detection, liquid chromatography-mass spectrometry serum bile acid detection, liver tissue pathological sections / immunohistochemistry, liver tissue qPCR, protein luminex multifactor detection, transmission electron microscopy technology to observe the ultrastructure of hepatocytes and protein blotting.

[0073] (1) Effect of Pan23 peptide on 24-hour survival rate of rats after liver transplantation with different cold ischemia times

[0074] Animal livers have strong self-repair and regeneration capabilities. Usually, rat livers can basically recover their original volume within one week after 70% liver resection (GRANELLI-PIPERNO A. In situ hybridization for interleukin2and interleukin 2 receptor mRNA in T cells activated in the presence or absence of cyclosporin A [J]. J Exp Med, 1988, 168 (5): 1649-1658.). Combined with the surgeon's experience, it was found that rats that met the criteria for 24-hour survival experimental animals after surgery were able to continue to survive for a long time (> 30 days). Therefore, in order to avoid misjudgment caused by the reduction of phenotypic differences caused by rapid liver repair, 24 hours after surgery was selected to calculate the survival rate and collect experimental samples. According to the different cold ischemia (CI) time of the donor liver and the preservation solution (UW; UW+Pan23), the patients were divided into 7 groups (n=8 / group), namely, UW-CI(0-2)h group (Control group), UW-CI(4-6)h group, (UW+Pan23)-CI(4-6)h group, UW-CI(8-10)h group, (UW+Pan23)-CI(8-10)h group, UW-CI(12-14)h group and (UW+Pan23)-CI(12-14)h group. The concentration of Pan23 was 50μM, and 2.5% volume of Pan23 drug solvent was added to the UW solution control group. The 24-hour survival rate of each group after surgery ( Figure 8), for 0-2 hours of cold ischemia, the 24-hour survival rate of both groups of rats after surgery was 100%; for 4-6 hours of cold ischemia, there was no difference in the survival rate between the two groups. For 8-10 hours of cold ischemia, the survival rate of the Pan23 group was significantly higher than that of the solvent control group; while for 12-14 hours of cold ischemia, the survival rates of both groups were low and there was no difference. Considering that the 4-6 hours of cold ischemia is short, the low liver damage is not enough to cause a difference in postoperative survival rate; on the contrary, the liver damage in both groups for 12-14 hours of cold ischemia was more serious, and the use of Pan23 could not effectively improve the postoperative survival rate; while the liver damage in the group with 8-10 hours of cold ischemia was between excessively severe and excessively mild, and Pan23 polypeptide effectively reduced the ex vivo cold ischemic injury of rat liver and improved the 24-hour survival rate after transplantation.

[0075] (2) Effects of Pan23 peptide on rat liver allograft cell function and tissue damage

[0076] According to the results of postoperative survival rate of rats, the following study focused on the differences in the liver protection effects of UW (control group) and UW+Pan23 (50μM, experimental group) at three cold ischemia times of CI0-2h, CI4-6h and CI8-10h. The liver lavage fluid and serum 24 hours after surgery of these 6 groups of experimental mice were collected to detect AST and ALT respectively (n=5 / group). The liver enzyme test results of the liver lavage fluid suggested ( Fig. 9 A): Compared with the UW+Pan23 group, the UW group showed an upward trend in transaminases at 4-6 hours of cold ischemia, but there was no significant difference (P<0.05). At 8-10 hours of cold ischemia, the difference between the two groups increased, and the AST and ALT in the UW group were significantly higher than those in the Pan23 group (P<0.001). Compared with the lavage fluid, the difference in serum transaminases between the UW and UW+Pan23 groups 24 hours after surgery was small ( Fig. 9 B), among which ALT and AST had no significant difference between the two groups at 4-6 hours of cold ischemia, but at 8-10 hours, the UW group began to show an increasing trend, and the serum ALT in the UW group increased significantly (P<0.05).

[0077] Serum samples of rats in the control group and experimental group (n=4 / group) were further collected at 4-6 hours cold ischemia of the donor liver and 24 hours after transplantation (CI4-6h & IRI24h) and sent to the laboratory of Shanghai Renji Hospital for analysis of 15 bile acids (cholic acid CA, glycocholic acid GCA, taurocholic acid TCA, chenodeoxycholic acid CDCA, glycochenodeoxycholic acid GCDCA, taurochenodeoxycholic acid TCDCA, deoxycholic acid DCA, glycodeoxycholic acid GDCA, taurodeoxycholic acid TDCA, lithocholic acid LCA, glycolithocholic acid GLCA, taurolithocholic acid TLCA, ursodeoxycholic acid UDCA, glycoursodeoxycholic acid GUDCA, tauroursodeoxycholic acid TUDCA) by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Fig.10 The order of hydrophilicity of bile acids is: UDCA>CA>CDCA>DCA>LCA. Hydrophobic bile acids have stronger inhibitory effects than hydrophilic bile acids and are more likely to cause liver cell damage. When there is hepatic parenchymal cell lesions, liver function is impaired and liver cell death increases, resulting in decreased synthesis of total bile acid (CA). Hepatic cell damage leads to damage of 12-α-hydroxylase, increased chenodeoxycholic acid and its conjugated forms, and therefore, the ratio of total bile acid (CA) / total chenodeoxycholic acid (CDCA) is less than 1. The smaller the ratio, the more severe the liver cell damage (SU B, JACINTO E, HIBI M, et al. JNK is involved in signal integration during costimulation of T lymphocytes [J]. Cell, 1994, 77 (5): 727-736.; SU B, KARIN M. Mitogen-activated protein kinase cascades and regulation of gene expression [J]. Curr Opin Immunol, 1996, 8(3): 402-411.), in this experiment, the ratio of the control group was significantly lower than that of the experimental group (P<0.05); lithocholic acid (LCA) is a hydrophobic bile acid with strong hepatotoxicity. The results of this experiment showed that the LCA in the serum of the control group was significantly higher than that of the experimental group (P<0.01). Therefore, the results of CA / CDCA and LCA together suggest that Pan23 can effectively reduce the damage of bile acid to liver cells and has a protective effect on rat transplanted liver function.

[0078] HE pathological sections of liver tissue (before transplantation) after 8-10 hours of cold ischemia showed ( Fig.11 A), UW showed larger areas of hepatocyte bridging necrosis, inflammatory cell infiltration, and abnormal morphology of hepatic cords and lobules than UW+Pan23 group; according to F.Suzuki scoring criteria ( Fig.11 B) quantified the degree of damage of the two groups of samples, indicating that the control group had a higher score (P<0.05) and more severe cold ischemic damage to liver tissue. There was no significant difference in the appearance of the livers of the two groups of mice surviving 24 hours after surgery (after transplantation). Fig.11 C).

[0079] (3) Effect of Pan23 polypeptide on the regeneration function of hepatocytes in rat liver transplantation

[0080] The previous experimental results suggest that Pan23 can reduce cold ischemia injury of transplanted liver and protect liver function. It also has a positive effect on the recovery of liver function in transplanted mice 24 hours after surgery. It is further considered that Pan23 polypeptide may have a positive effect on liver tissue regeneration after transplantation. Rats in the control group (UW) and experimental group (Pan23 50μM) received liver transplantation after 4-6 hours and 8-10 hours of cold ischemia for 24 hours (CI4-6h&24h, CI8-10h&24h). Liver tissues of 4 groups of rats (n=3 / group) were taken for immunohistochemistry of Ki67 ( Fig.12 A) Western blot experiment ( Fig.12 B) and transmission electron microscopy observations ( Fig.12 C), found that the expression of Ki67 in liver cells of UW+Pan23 group was higher than that of UW group, and the expression of Ki67 in CI8-10h&24h groups was significantly different in immunohistochemistry (P<0.05) and western blot (P<0.01). Considering that the prolonged cold ischemia time and increased liver damage may make the difference in Pan23 promoting the regeneration and repair of the donor liver more obvious. Under the electron microscope, it can be observed that more cells in CI4-6h&24h experimental group showed binucleate nuclei, and vigorous mitosis can be observed in some cells of CI8-10h&24h experimental group ( Fig.12 C). Therefore, it is suggested that cold storage of Pan23 polypeptide grafts has a positive effect on the regeneration of liver tissue after transplantation in rats.

[0081] (4) Effects of Pan23 peptide on mitochondria and stress response of rat liver transplant cells

[0082] Previous work has shown that Pan23 can reduce cold ischemia injury in transplanted liver, improve hepatocyte function before and after transplantation, and promote regeneration and repair after liver injury. Cold ischemia and reperfusion injury usually involve intracellular oxidative stress response, mitochondrial damage and inflammatory response. To further explore the relevant mechanisms, transmission electron microscopy was used to observe the background, cold preservation and 24 hours after surgery of liver transplanted mice with cold ischemia for 4-6 hours and 8-10 hours. The purpose was to explore the effect of Pan23 added to the preservation solution on important organelles, ultrastructure and intracellular environment in liver cells at different cold ischemia times.

[0083] Rat background liver tissue:

[0084] Background liver tissue is the liver tissue of the lower half of the papillary lobe of the liver obtained uniformly after the donor surgery and before the liver perfusion and blood removal. The results of transmission electron microscopy observation of background liver tissue before cold ischemia for 4-6 hours (CI4-6h) and 8-10 hours (CI8-10h) suggest that ( Fig.13): UW (control group) and UW+Pan23 (experimental group, Pan23 50μM) at CI4-6h showed normal liver cells, and the intracellular microstructure, nucleus, endoplasmic reticulum, mitochondria, and autophagosomes were clearly visible. Some cells contained a large number of autophagosomes (boxes), which is inferred to be the body's stress response to laparotomy injury and the reason for maintaining the stability of the body environment by producing autophagosomes to remove damaged cells after liver surgery. There was no significant difference between the control group and the experimental group at CI8-10h.

[0085] Rat cold ischemic liver tissue:

[0086] Cold ischemic liver tissue is the upper part of the papillary lobe that is cryopreserved in the corresponding preservation solution after the donor liver is obtained and uniformly cut before liver transplantation. Cold ischemic liver tissue at CI4-6h and CI8-10h under transmission electron microscopy ( Fig.14 ): CI4-6h control group ( Fig.14 A) The ultrastructure of the sample cells is relatively normal ( Fig.14 A1), the mitochondria and endoplasmic reticulum structures are clear, but MAMs ( Fig.14 A2, arrows) appeared, and there were multi-layered autophagosomes in the cells ( Fig.14 A3, box) appeared; the cell structure of the experimental group samples was normal ( Fig.14 B), the microstructure is complete ( Fig.14 B1), the nucleus, endoplasmic reticulum, and mitochondria are clearly visible, with the morphology of mitochondria and endoplasmic reticulum being very good, not many autophagosomes, and less MAMs generated. However, it was observed that lipid droplets in some cells began to autophagically decompose ( Fig.14 B2, circle).

[0087] CI8-10 control group ( Fig.15 A) A large number of MAMs can be seen in liver tissue cells ( Fig.15 A1, A2, A4, arrows), hepatocytes undergo autophagy and generate a large number of autophagosomes ( Fig.14 A1, A2, A4, boxes); experimental group ( Fig.15 B) The number of MAMs produced was reduced compared with the control group ( Fig.15 B2, an arrow below), the endoplasmic reticulum-related stress response was alleviated, the autophagy of liver cells was weakened, and mitochondria with clear inner ridge structures were seen in some cells ( Fig.15 B3, B4), and peroxisomes were found in the cells ( Fig.15 B2, two arrows above).

[0088] In general, as the cold ischemia time prolonged, the mitochondrial damage, MAMs, endoplasmic reticulum stress and cellular autophagy response of liver tissue at CI8-10h were enhanced compared with CI4-6h. For the same cold ischemia time point, the mitochondrial damage, MAMs, endoplasmic reticulum stress and cellular autophagy response of the Pan23 drug-containing group were alleviated compared with the control group. Among them, peroxisomes appeared in the CI8-10h experimental group. As a new organelle, it has a strong effect in resisting oxidative stress damage.

[0089] Liver tissue of rat donor liver transplantation 24 hours later:

[0090] The liver tissue at 24 hours of reperfusion (IRI24h) was obtained under isoflurane anesthesia 24 hours after the donor liver was transplanted into the syngeneic recipient Lwies rat. Transmission electron microscopic observation of liver tissue at 24 hours after CI4-6h surgery (CI4-6h&IRI24h) and 24 hours after CI8-10 surgery (CI8-10h&IRI24h) Fig.16 ): CI4-6h&IRI24h control group ( Fig.16 The most obvious change in the cells in A) is that the endoplasmic reticulum begins to swell, vacuolate, and autophagy occurs inside ( Fig.16 The circle of A2 and the large circle on the left of A4 show that the cells are dying, accompanied by a large number of MAMs ( Fig.16 The arrow of A2, the arrow above A4) and the local formation of a large number of lipid droplets ( Fig.16 A4, an arrow below); in comparison, the cell morphology of the experimental group is relatively complete ( Fig.16 B), mitochondria and endoplasmic reticulum are abundant. It was observed that lipid droplets began to disintegrate and form larger autophagosomes ( Fig.16 B4, circle), the endoplasmic reticulum began to expand, but to a lesser extent than the control group ( Fig.16 B2, an arrow below), small autophagosomes began to appear in the endoplasmic reticulum of some cells ( Fig.16 B4, a large circle on the left), cells with a small number of MAMs and a small number of immature peroxisomes ( Fig.16 B2, an arrow above).

[0091] CI8-10h&IRI24h control group ( Fig.17 A) shows that the hepatocyte nuclear chromatin is condensed and edged severely, there is cell apoptosis, the internal structure of mitochondria is dissolved, and the cell tissue structure is severely damaged ( Fig.17 A1, A2, A3); the overall state of liver cells in the experimental group improved ( Fig.17 B), the amount of MAMs generated in the cells decreased compared with the control group, the endoplasmic reticulum was slightly swollen, the related stress response was alleviated, and the autophagy of hepatocytes was weakened ( Fig.17 B1, B2). The cell substructure is relatively intact (chromatin / mitochondria), and a large number of peroxisomes can be seen in the cells ( Fig.17 B3, B4, arrows), the damage and structural destruction of liver cells were significantly alleviated.

[0092] In general, from the perspective of intracellular ultrastructure, the degree of hepatocyte injury after 24h reperfusion was higher than that after cold ischemia alone. As the cold ischemia time prolonged, the degree of hepatocyte injury 24h after surgery increased overall. For the same cold ischemia time point, the apoptosis state, mitochondrial damage, MAMs, endoplasmic reticulum stress and autophagy reaction of the experimental group after 24h reperfusion were significantly reduced compared with those of the control group. Among them, a large number of peroxisomes were observed in the CI8-10h experimental group 24h after surgery, suggesting that Pan23 enhances the protective effect of cells against oxidative stress.

[0093] (5) Effect of Pan23 peptide on autophagic response of rat liver allograft cells

[0094] Under the electron microscope, it can be seen that under the condition of cold ischemia of liver tissue, Pan23 closes the Panx1 protein channel, which will affect cell autophagy, and can weaken the overall autophagic reaction in cells under the condition of cold ischemia, such as endoplasmic reticulum autophagy, but can promote the autophagy of lipid droplets formed by cell dysfunction. Therefore, the liver tissues of rats in the control group (UW) and experimental group (UW+Pan23) at CI4-6h and CI8-10h with background, cold ischemia, and reperfusion for 24 hours were sent to Shanghai Ruchuang Biotechnology Co., Ltd. for immunohistochemical staining of Beclin-1, an important regulatory factor of autophagy (n=3 / group). The results are shown below ( Fig.18 ): There was no significant difference in CI4-6h between background, cold ischemia and reperfusion for 24 hours ( Fig.18 A). And the results of CI8-10h ( Fig.18 B) The immunohistochemical score of the experimental group of cold ischemic liver tissue was significantly lower than that of the control group (P<0.01), which was consistent with the results of electron microscopy observation. It may be that Pan23 shuts down Panx1 to reduce liver cell damage caused by prolonged cold ischemia, thereby delaying the appearance of protective autophagy. The results of liver tissue reperfused for 24 hours showed that the experimental group was higher (P<0.05). It was considered that the control group had severe liver damage due to long-term cold ischemia itself, and the damage was further aggravated after reperfusion. Therefore, the cell function decreased, resulting in a decrease in autophagy function. Pan23 shutting down Panx1 can weaken cold ischemia and reperfusion injury, so the liver cell function damage is reduced and protective autophagy (such as lipid droplet autophagy) can be induced, so there is a trend of enhanced autophagy.

[0095] (6) Pan23 polypeptide affects the expression of inflammatory factors and antioxidant enzymes in rat cold ischemic liver. In the past, there have been many studies on the regulation of inflammatory response and ROS in the Panx1 protein channel. Inflammatory response and ROS are important mechanisms of ischemic injury and reperfusion injury. Therefore, it is considered whether Pan23 polypeptide can affect the changes of inflammatory factors and antioxidant-related enzymes in the liver. A total of 3 groups (n = 5 / group) were divided into: the liver donor UW liquid cold preservation control group, the UW+CBX (100μM) cold preservation group and the UW+Pan23 (50μM) cold preservation group. The Invitrogen ProcartaPlex immunoassay kit was used to extract RNA and total protein from rat liver tissue with cold ischemia for 8-10 hours (before transplantation), and the protein concentration was quantified. qPCR experiments and Luminex multi-protein factor quantification were further performed to detect the expression of related inflammatory factors and antioxidant enzymes. The qPCR results showed that ( Fig.19 A) The IL-1α, IL-6, IL-10 and MCP-1 in the Pan23 group and the CBX group were significantly lower than those in the control group (P<0.05, P<0.001, P<0.05, P<0.01), while the expression of Panx1 was not significantly different from that in the control group (P>0.05), suggesting that Pan23 reduces the inflammatory response of the cold ischemic liver by closing the Panx1 protein channel in the liver tissue; in addition, the expression of multiple antioxidant genes ( Fig.19 B), including SOD-1, Gpx-1, HO-1, CAT and PON1 were significantly increased compared with the control group (P<0.05), which once again confirmed that Pan23 closing Panx1 channel protein can alleviate the oxidative stress response of liver tissue under ischemic and hypoxic state by inducing the enhancement of cellular antioxidant function.

[0096] Luminex protein multifactor detection tips ( Fig. 20 )Pan23 can significantly reduce the protein expression of IL-6, MCP-1 and TNF-α in cold ischemic liver tissue, further verifying that Pan23 can reduce the inflammatory response in cold ischemic liver tissue.

[0097] (7) Effects and mechanisms of Pan23 peptide on apoptosis of rat hepatocytes after transplantation

[0098] Previous studies have found that compared with the control group, the histological results of the transplanted liver treated with Pan23 drugs during cold ischemia and 24 hours after transplantation showed a decrease in the extent of liver cell necrosis, liver enzyme and bile acid tests showed improved liver function, better organelle status in liver cells under electron microscopy, reduced oxidative stress response, reduced cell damage, more mitochondria and clearer structure. Therefore, it is considered that Pan23 peptide may play an important role in liver cell apoptosis and mitochondrial apoptosis pathways.

[0099] Effect of Pan23 polypeptide on apoptosis of transplanted hepatocytes in rats

[0100] In order to explore the effect of cold ischemia of the donor liver with Pan23 peptide in the preservation solution on hepatocyte apoptosis, four groups (n=3 / group) were designed: a control group (UW) and an experimental group (UW+Pan23, 50μM) of donor livers at CI4-6h, a control group (UW) and an experimental group (UW+Pan23, 50μM) of donor livers at CI8-10h. The background tissue (0h) and liver tissue 24 hours after transplantation (CI4-6h / CI8-10h&IRI24h) were taken from the four groups of donor livers and sent to Shanghai Ruchuang Biotechnology Co., Ltd. for immunohistochemical staining of Bcl-2, an important regulatory protein of cell apoptosis, and Tunel histochemical staining. The results are as follows ( Fig.21 ): Compared with the control group, the Bcl-2 expression levels of livers with cold ischemia for 4-6 hours and 8-10 hours were significantly increased 24 hours after transplantation ( Fig.21 A, P<0.05); Tunel immunohistochemistry results also suggest that Pan23 peptide pretreatment can reduce cell apoptosis after liver transplantation ( Fig.21 B, C). Quantitative analysis of apoptosis negative regulator protein Bcl-2 and Tunel staining also showed that the cold ischemia time was prolonged, the apoptosis of graft cells was aggravated, and the difference between the experimental group and the control group increased (CI4-6h&IRI24h:P<0.05, CI8-10h&IRI24h:P<0.01). The more obvious the anti-apoptosis protection effect after Pan23 intervention was ( Fig.21 A, B, C).

Claims

1. An organ storage solution, characterized in that: The organ storage solution comprises Pan23 polypeptide and organ preservation solution.

2. The organ storage solution according to claim 1, characterized in that: The amino acid sequence of the Pan23 polypeptide is shown in SEQ ID NO:

1.

3. The organ storage solution according to claim 2, characterized in that: The amino acids in the amino acid sequence are in L-form.

4. The organ storage solution according to any one of claims 1 to 3, characterized in that: The content of Pan23 polypeptide is ≥30 μM, such as 50 μM, 100 μM or 200 μM, preferably 50 μM.

5. The organ storage solution according to any one of claims 1 to 4, characterized in that: The organ preservation solution is an extracellular fluid-mimicking organ preservation solution or an intracellular fluid-mimicking organ preservation solution; Preferably, the extracellular fluid-mimicking organ preservation solution is Celsior preservation solution, and the intracellular fluid-mimicking organ preservation solution is UW preservation solution.

6. A method for preserving tissue or organ, characterized in that: The method comprises the following steps: placing the freed tissue or organ after lavage in the organ storage solution as claimed in any one of claims 1 to 5 for storage; The temperature of the organ storage solution is 1-10°C, preferably 4°C.

7. The method according to claim 6, characterized in that The organ is the liver.

8. The method for preparing the organ storage solution according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: adding Pan23 polypeptide into an organ preservation solution such as UW preservation solution or Celsior preservation solution; The final concentration of the Pan23 polypeptide is ≥30 μM, such as 50 μM, 100 μM or 200 μM, preferably 50 μM.

9. Use of Pan23 polypeptide or the organ storage solution according to any one of claims 1 to 5 in preserving tissues or organs.

10. The use according to claim 9, characterized in that The organ is the liver.

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