An improved method for kidney preservation solution

Abstract

The present invention discloses an improved method for kidney preservation solution, belonging to the technical field of organ preservation solutions. The method comprises adding a ganoderma lucidum extract and an epimedium extract to HC-A solution. The ganoderma lucidum extract contains ganoderma lucidum polysaccharides and total ganoderma lucidum triterpenes, while the epimedium extract contains icariin and epimedin. The improved method of the present invention, which adds the ganoderma lucidum extract and the epimedium extract to the HC-A solution, can reduce ischemic damage during cryopreservation of isolated kidneys by protecting mitochondrial structure, increasing calpain activity, and inhibiting cell apoptosis, oxidative stress, and inflammatory reactions. This method improves the preservation effect of HC-A solution for isolated kidneys, prolongs their preservation time, and thus increases the survival rate of kidney transplantation. This method provides a clinical approach to finding a safe and effective method for preserving donor kidneys.

Description

Technical Field

[0001] The invention belongs to the technical field of organ preservation fluids, and in particular relates to a method for improving kidney preservation fluids. Background Art

[0002] Kidney transplantation is currently one of the best treatments for renal failure. The quality of kidney preservation plays a crucial role in transplant success, leading to increasing attention for improving renal preservation solutions. The most successful organ preservation solution in China is HC-A (hypertonic citrate adenine solution), jointly developed by the Changzheng Hospital affiliated with the Second Military Medical University and the Shanghai Central Blood Station. It is based on the international formula of Ross solution and shares the same composition as Ross solution. HC-A incorporates three modifications: a reduction in osmotic pressure to 380 mmol / kg, the addition of 0.38 mmol / L adenine, and an adjustment of pH to 7.0. These improvements, based on advances in renal cold energy metabolism research at the time and the physiological characteristics of the Chinese kidney, led to significant success. HC-A's key features include the addition of adenine as an energy substrate, low viscosity for optimal ex vivo perfusion, simple preparation, ease of use, and low cost. However, with the deepening understanding of organ preservation and the deepening of the understanding of ischemia-reperfusion injury during organ preservation and organ transplantation, higher requirements are being placed on the development of organ preservation fluids. In the face of a severe shortage of transplant donors, the key to solving this problem is to expand the supply, improve kidney preservation effectiveness, and enhance the vitality and quality of transplanted kidneys. Therefore, it is imperative to develop kidney preservation fluids that have excellent preservation effects and are cost-effective.

[0003] Patent application number 2016800501657 discloses: a preservative for an organ or tissue comprising (A) quercetin and (B) at least one saccharide selected from the group consisting of fructose and sucrose; a preservative solution for an organ or tissue comprising the preservative; and a method for preserving an organ or tissue, the method comprising immersing the organ or tissue in a liquid mixture comprising (A) quercetin and (B) at least one saccharide selected from the group consisting of fructose and sucrose. The organ or tissue is selected from the heart, liver, kidney, pancreas, or pancreatic islets. The invention patent application with application number 2018800468554 discloses: a preservation solution for preserving cells, tissues and / or organs, comprising: (i) water for injection; (ii) at least one sugar; (iii) at least one component with pH buffering properties; (iv) optionally at least one component with calcium transport blocking properties or anticalcium activity; (v) salicylic acid in free form or salt form, or aspirin; (vi) glutamic acid in free form or salt form, or glutamine; provided that acetamide is not present, and / or if aspirin is present, glutamine is not present; if glutamine is present, aspirin is not present; the above-mentioned organs are liver, kidney, small intestine and / or pancreas. Summary of the Invention

[0004] The purpose of the present invention is to provide an improved method for kidney preservation solution. By adding Ganoderma lucidum extract and Epimedium brevicornum extract to HC-A solution, the ischemic damage of isolated kidneys during the low-temperature storage period can be reduced by protecting mitochondrial structure, increasing calpain activity, inhibiting cell apoptosis, oxidative stress response and inflammatory response.

[0005] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0006] The invention discloses an improved method for preparing kidney preservation solution. Ganoderma lucidum extract and epimedium extract are added to HC-A solution. The Ganoderma lucidum extract contains Ganoderma lucidum polysaccharide and total triterpenes, and the epimedium extract contains icariin and epimedin.

[0007] HC-A solution does not contain active substances that can resist damage from oxygen free radicals. The present invention has found that adding Ganoderma lucidum extract and Epimedium brevicornum extract to HC-A solution can improve the preservation effect of HC-A solution on isolated kidneys, prolong its preservation time, and thus increase the survival rate of kidney transplants, providing clinical research for a safe and effective method for preserving donor kidneys. This is primarily because the combined effects of Ganoderma lucidum and Epimedium brevicornum extracts: 1) protect the structural integrity of mitochondria in ischemia-reperfusion kidneys and safeguard energy metabolism in renal cortical cells during cryopreservation of isolated kidneys; 2) increase mitochondrial calpain activity in the renal cortex, preventing calcium overload; 3) upregulate Bcl-2 expression and downregulate Bax expression, thereby increasing the Bcl-2 / Bax ratio and reducing apoptosis in renal tubular epithelial cells; 4) increase the activity of the antioxidant enzyme SOD in isolated renal tissue, thereby reducing the content of malondialdehyde (MDA), a lipid peroxidation product in isolated renal tissue; and 5) downregulate the levels of the proinflammatory cytokines TNF-α, IL-1β, and HMGB1, while upregulating the anti-inflammatory cytokine IL-10. Therefore, the addition of Ganoderma lucidum and Epimedium brevicornum extracts to HC-A solution can mitigate ischemic damage during cryopreservation of isolated kidneys by protecting mitochondrial structure, increasing calpain activity, and inhibiting apoptosis, oxidative stress, and inflammation.

[0008] Preferably, the Ganoderma lucidum extract contains at least 8 wt % of Ganoderma lucidum polysaccharides and 3 wt % of Ganoderma lucidum total triterpenes.

[0009] Preferably, the epimedium extract contains icariin, epimedin A and / or epimedin B and / or epimedin C.

[0010] Preferably, the epimedium extract contains at least 55 wt % icariin and 40 wt % epimedin.

[0011] Preferably, a method for improving kidney preservation solution comprises the following steps:

[0012] S1: preparing the Ganoderma lucidum extract into a Ganoderma lucidum extract solution with a concentration of 1.0-3.5 g / mL;

[0013] S2: preparing the epimedium extract into an epimedium extract solution with a concentration of 0.2-1.8 g / mL;

[0014] S3: Add Ganoderma lucidum extract and Epimedium extract to HC-A solution, adjust the pH value to 7.3-7.8 with phosphoric acid, and disinfect to obtain the improved kidney preservation solution.

[0015] More preferably, the volume ratio of HC-A solution, Ganoderma lucidum extract and Epimedium extract is 100:1.5-3.0:1.5-3.0.

[0016] Preferably, the kidney preservation solution is a refrigerated preservation solution.

[0017] The present invention also discloses the use of a ganoderma lucidum extract combined with an epimedium extract in preventing or treating acute ischemic renal failure in humans or non-human animals. The ganoderma lucidum extract contains ganoderma lucidum polysaccharides and ganoderma lucidum total triterpenes, and the epimedium extract contains icariin and epimedin.

[0018] The invention also discloses the use of ganoderma lucidum extract and epimedium extract in preparing kidney preservation solution. The ganoderma lucidum extract contains ganoderma lucidum polysaccharide and ganoderma lucidum total triterpenes, and the epimedium extract contains icariin and epimedin.

[0019] The invention also discloses that the kidney preservation solution is used for preserving kidneys in the absence of blood supply.

[0020] The present invention also discloses the above-mentioned method for preventing isolated kidney damage after reperfusion or reducing its severity, comprising preserving the isolated kidney in the kidney preservation solution according to claim 1.

[0021] Since the present invention adopts the method of adding the ganoderma lucidum extract and the epimedium extract to the HC-A solution, the present invention has the following beneficial effects: 1) protecting the integrity of the mitochondrial structure of the ischemia-reperfusion kidney and protecting the energy metabolism of the renal cortical cells during the cryopreservation of the isolated kidney; 2) improving the activity of the mitochondrial calpain of the renal cortex and preventing calcium overload; 3) upregulating the expression of Bcl-2 and downregulating the expression of Bax, thereby upregulating the Bcl-2 / Bax ratio and reducing the apoptosis of the renal tubular epithelial cells; 4) improving the activity of the antioxidant enzyme SOD in the isolated kidney tissue and thereby reducing the content of malondialdehyde (MDA), a cellular lipid peroxidation product in the isolated kidney tissue; and 5) downregulating the content of the pro-inflammatory cytokines TNF-α, IL-1β and HMGB1 and upregulating the content of the anti-inflammatory cytokine IL-10. Therefore, the present invention provides an improved method for kidney preservation solution, in which Ganoderma lucidum extract and Epimedium brevicornum extract are added to HC-A solution, which can reduce ischemic damage of isolated kidneys during low-temperature storage by protecting mitochondrial structure, increasing calcium enzyme activity, inhibiting cell apoptosis, oxidative stress response and inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of SOD activity determination in kidney tissue;

[0023] Figure 2 is the result of determination of MDA content in kidney tissue;

[0024] Figure 3 is the TNF-α content in kidney tissue;

[0025] Figure 4 is the IL-1β content in kidney tissue;

[0026] Figure 5 is the HMGB1 content in kidney tissue;

[0027] Figure 6 is the IL-10 content in kidney tissue. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods, but this should not be understood as the scope of the above-mentioned subject matter of the present invention being limited to the following implementation examples. All technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0029] Example 1:

[0030] An improved method for kidney preservation solution

[0031] Raw materials and reagents: HC-A solution, purchased from Shanghai Changzheng Hospital; Ganoderma lucidum extract (Lingzhi Kangbao powder), containing 10 g of Ganoderma lucidum polysaccharides and 8 g of Ganoderma lucidum total triterpenes per 100 g, purchased from Fujian Xianzhilou Biotechnology Co., Ltd.; Epimedium extract, purchased from Xi'an Yueda Plant Technology Co., Ltd., containing 56 g of icariin, 6 g of epimedin A, 19 g of epimedin B, and 19 g of epimedin C per 100 g.

[0032] S1: using water for injection, preparing the Ganoderma lucidum extract into a Ganoderma lucidum extract solution with a concentration of 2.4 g / mL;

[0033] S2: using water for injection, preparing the epimedium extract into an epimedium extract solution with a concentration of 1.2 g / mL;

[0034] S3: Add 0.48 mL of Ganoderma lucidum extract and 0.4 mL of Epimedium extract to 20 mL of HC-A solution, adjust the pH to 7.4 with phosphoric acid, and sterilize under high temperature and high pressure (109°C, 45 min) to obtain the improved kidney preservation solution.

[0035] Example 2:

[0036] An improved method for kidney preservation solution

[0037] Raw materials and reagents: same as in Example 1.

[0038] S1: using water for injection, preparing the Ganoderma lucidum extract into a Ganoderma lucidum extract solution with a concentration of 2.4 g / mL;

[0039] S2: using water for injection, preparing the epimedium extract into an epimedium extract solution with a concentration of 1.2 g / mL;

[0040] S3: Add 0.48 mL of Ganoderma lucidum extract and 0.5 mL of Epimedium extract to 20 mL of HC-A solution, adjust the pH to 7.4 with phosphoric acid, and sterilize under high temperature and high pressure (109°C, 45 min) to obtain the improved kidney preservation solution.

[0041] Example 3:

[0042] An improved method for kidney preservation solution

[0043] Raw materials and reagents: same as in Example 1.

[0044] S1: using water for injection, the Ganoderma lucidum extract was prepared into a Ganoderma lucidum extract solution with a concentration of 2.4 g / mL;

[0045] S2: using water for injection, preparing the epimedium extract into an epimedium extract solution with a concentration of 1.2 g / mL;

[0046] S3: Add 0.48 mL of Ganoderma lucidum extract and 0.3 mL of Epimedium extract to 20 mL of HC-A solution, adjust the pH to 7.4 with phosphoric acid, and sterilize under high temperature and high pressure (109°C, 45 min) to obtain the improved kidney preservation solution.

[0047] Example 4:

[0048] An improved method for kidney preservation solution

[0049] Raw materials and reagents: HC-A solution, purchased from Shanghai Changzheng Hospital; Ganoderma lucidum extract (Lingzhi Kangbao powder), containing 10 g of Ganoderma lucidum polysaccharides and 8 g of Ganoderma lucidum total triterpenes per 100 g, purchased from Fujian Xianzhilou Biotechnology Co., Ltd.; Epimedium extract, purchased from Beijing Shen Aoji Pharmaceutical Biotechnology Co., Ltd., containing 58 g of icariin, 6 g of epimedin A, 11 g of epimedin B, and 25 g of epimedin C per 100 g.

[0050] S1: using water for injection, preparing the Ganoderma lucidum extract into a Ganoderma lucidum extract solution with a concentration of 2.4 g / mL;

[0051] S2: using water for injection, preparing the epimedium extract into an epimedium extract solution with a concentration of 1.2 g / mL;

[0052] S3: Add 0.48 mL of Ganoderma lucidum extract and 0.4 mL of Epimedium extract to 20 mL of HC-A solution, adjust the pH to 7.4 with phosphoric acid, and sterilize under high temperature and high pressure (109°C, 45 min) to obtain the improved kidney preservation solution.

[0053] Example 5:

[0054] An improved method for kidney preservation solution

[0055] Raw materials and reagents: HC-A solution, purchased from Shanghai Changzheng Hospital; Ganoderma lucidum extract (Lingzhi Kangbao powder), containing 10g of Ganoderma lucidum polysaccharides and 8g of Ganoderma lucidum total triterpenes per 100g, purchased from Fujian Xianzhilou Biotechnology Co., Ltd.; Epimedium extract, purchased from Xi'an Yueda Botanical Technology Co., Ltd., containing 56g of icariin, 6g of epimedin A, 19g of epimedin B, and 19g of epimedin C per 100g; stilbene glycosides, purchased from Shanghai Ruiyong Biotechnology Co., Ltd. Stilbene glycosides are the main active ingredients of Polygonum multiflorum. Their addition to HC-A solution can further alleviate calcium overload. This may be because the addition of stilbene glycosides can help improve the activity of renal cortical mitochondrial calcium enzymes in the modified kidney preservation solution, while enhancing the effects of Ganoderma lucidum extract and epimedium extract on inflammatory responses, ultimately reducing ischemic damage during the cryopreservation period of isolated kidneys. Preferably, a method for improving kidney preservation solution comprises the following steps:

[0056] S1: preparing the Ganoderma lucidum extract into a Ganoderma lucidum extract solution with a concentration of 1.0-3.5 g / mL using water for injection;

[0057] S2: using water for injection to prepare the epimedium extract into an epimedium extract solution with a concentration of 0.2-1.8 g / mL;

[0058] S3: using water for injection, preparing stilbene glycoside solution with a concentration of 2.5-10 mg / mL;

[0059] S4: Add Ganoderma lucidum extract, Epimedium extract and stilbene glycoside solution to HC-A solution, adjust the pH value to 7.3-7.8 with phosphoric acid, and disinfect to obtain the improved kidney preservation solution.

[0060] More preferably, the volume ratio of HC-A solution, Ganoderma lucidum extract and Epimedium extract is 100:1.5-3.0:1.5-3.0:1.5-3.0.

[0061] In this embodiment, a method for improving kidney preservation solution comprises the following steps:

[0062] S1: using water for injection, preparing the Ganoderma lucidum extract into a Ganoderma lucidum extract solution with a concentration of 2.4 g / mL;

[0063] S2: using water for injection, preparing the epimedium extract into an epimedium extract solution with a concentration of 1.2 g / mL;

[0064] S3: using water for injection, preparing stilbene glycoside solution with a concentration of 5 mg / mL;

[0065] S4: Add 0.48 mL of Ganoderma lucidum extract, 0.4 mL of Epimedium extract and diphenylethylene glycoside solution to 20 mL of HC-A solution, adjust the pH to 7.4 with phosphoric acid, and sterilize with high temperature and high pressure (109°C, 45 min) to obtain the improved kidney preservation solution.

[0066] Test Example 1:

[0067] 1. Determination of colloid osmotic pressure and electrolytes in modified renal preservation solution

[0068] The results of the colloid osmotic pressure and electrolyte measurement of the improved renal preservation solutions are shown in Table 1. As can be seen from Table 1, the colloid osmotic pressure of the improved renal preservation solutions of Examples 1-5 is slightly higher than that of HC-A solution, and the ion composition and ion concentration of the improved renal preservation solutions of Examples 1-5 are similar to those of HC-A solution.

[0069] Table 1 Colloid osmotic pressure and electrolytes of modified kidney preservation solution

[0070]

[0071] 2. Determining the Stability of the Modified Kidney Preservation Solution

[0072] After storage at 4°C for 7 days, the electrolytes in the improved kidney preservation solution were measured, and the results are shown in Table 1. As can be seen from Table 1, there was no significant change in the electrolytes in the improved kidney preservation solution of Examples 1-5, indicating that the components of the improved kidney preservation solution of Examples 1-5 were stable during the low-temperature storage process.

[0073] Table 2 Electrolytes in modified kidney preservation solution after storage at 4°C for 7 days

[0074]

[0075] Test Example 2:

[0076] Effects of modified kidney preservation solution on cryopreservation of isolated rat kidney

[0077] 1. Experimental Animals

[0078] Clean-grade male SD rats, weighing 200 ± 10 g, were purchased from Guangdong Medical Laboratory Animal Center.

[0079] 2. Main Reagents

[0080] Chloral hydrate: purchased from Sinopharm Chemical Reagent Co., Ltd.

[0081] Heparin sodium injection: purchased from Changzhou Qianhong Biochemical Pharmaceutical Co., Ltd., 1.6 mL of heparin sodium injection (10,000 U) was dissolved in 100 mL of normal saline (100 U / mL), shaken well, and stored at 0-4°C until use;

[0082] Mitochondrial respiratory control rate kit: purchased from Shanghai Jiemei Gene Biology Research Institute;

[0083] Coomassie brilliant blue stock solution and calcium ion determination kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0084] Bax and Bcl-2 polyclonal antibodies were purchased from Beijing Zhongshan Biotechnology Co., Ltd.

[0085] MDA kit, SOD kit, TNF-α kit, IL-1β kit, and anti-inflammatory factor IL-10 kit were purchased from R&D Company in the United States.

[0086] HMGB1 kit: purchased from IBL, Germany.

[0087] 3. Methods

[0088] 1. Establishment of a Simple Cryopreservation Model of Isolated Kidney in Experimental Animals

[0089] 1.1 Experimental Grouping: According to the different improved renal preservation solutions, the experiments were divided into HCA preservation solution group (Group K), Example 1 improved renal preservation solution group (Example 1 Group), Example 2 improved renal preservation solution group (Example 2 Group), Example 3 improved renal preservation solution group (Example 3 Group), Example 4 improved renal preservation solution group (Example 4 Group) and Example 5 improved renal preservation solution group (Example 5 Group), with 12 rats in each group; according to the different preservation times, the rats were further randomly divided into 24 h group and 72 h group, with 6 rats in each group.

[0090] 1.2 Kidney Removal

[0091] In situ perfusion model of rat kidney

[0092] Donors were allowed to eat or drink water before surgery. Thirty minutes before surgery, anesthesia was administered intraperitoneally with 10% chloral hydrate at 0.3 mL / 100 g. Surgery began 3 minutes after stabilization of anesthesia. The rats were secured on the operating table, and the abdominal skin was prepared with a razor. A midline incision was made and the skin was separated. The rectus abdominis and peritoneum were then incised from the xiphoid process of the sternum to the upper edge of the pubic bone. This incision was made in a large cross-shaped pattern, extending laterally from the middle to the left and right. The abdominal aorta was isolated and occluded below. A cannula was inserted and secured 1.5 cm below the left kidney. After the injection of 1500 U of sodium heparin, gravity lavage was performed with HCA preservation solution and aFGF compound preservation solution, respectively, at a flow rate of 12 mL / min and a pressure of 7.84-9.81 kPa (80-100 cm H2O column). The perfusion lasted for 5 minutes. At the same time, the abdominal aorta near the thoracic cavity is occluded, and the inferior vena cava is cut below the level of the renal vein to provide drainage for the perfusion fluid. Adequate renal lavage is indicated when the kidney surface turns pale and the fluid exiting the renal vein is clear. After lavage, the kidney is removed and stored at 4°C in HCA preservation solution or modified kidney preservation solution for 24 or 72 hours, respectively.

[0093] 2. Renal Histopathological Observation

[0094] 2.1 Specimen collection

[0095] The kidney was cut transversely through the middle and divided into upper and lower parts. The upper part was used for pathological observation, and the lower part was quickly stored in liquid nitrogen for index detection.

[0096] 2.2 Renal histopathological observation

[0097] After the kidneys were stored at 4°C for 24 and 72 hours, renal cortical specimens were obtained according to light microscopic pathology requirements and fixed with 10% neutral formaldehyde. Paraffin sections were prepared according to routine procedures and stained with HE for observation under a light microscopy.

[0098] Pathologists performed a blinded analysis of the examination results. Light microscopic sections were used to observe basic morphological patterns: cytoplasmic vacuolation, exposed basement membrane, cell necrosis, tubular dilation, and cell shedding. Scoring was performed based on these pathological morphologies: no abnormalities or damage affecting no more than 10% of the sample was scored as 1; mild damage affecting 10-20% of the sample was scored as 2; damage affecting 25-50% of the sample was scored as 3; damage affecting 50-75% of the sample was scored as 4; and damage affecting more than 75% of the sample was scored as 5. The sum of the scores was used to create the sample score. Three samples from each case were scored, and the average of these scores was used as the final pathological examination score, providing a comprehensive assessment of the extent of renal injury.

[0099] 2.3 The results of kidney tissue pathology scores of each group are shown in Table 3. It can be seen that the renal cortex pathology scores of each group after 24 hours of kidney preservation solution were significantly lower than those after 72 hours; the renal cortex pathology scores of Examples 1-4 groups were all lower than those of the HC-A solution group, which indicates that the addition of Ganoderma lucidum extract and Epimedium extract to HC-A solution is beneficial to kidney preservation; the renal cortex pathology score of Example 5 group was slightly higher than that of Example 1 group, which indicates that the addition of diphenyl glycosides has a beneficial effect on the preservation effect of Ganoderma lucidum extract and Epimedium extract.

[0100] Table 3 Renal tissue pathological scores in each group

[0101]

[0102] 3. Testing of Mitochondrial Respiratory Control Rate in Kidney Tissue

[0103] 3.1 Isolation of renal cell mitochondria: After the kidneys were stored at 4°C for 24 h and 72 h, the renal cortex was accurately weighed and minced, the blood was removed, and 9 times normal saline was added to make a homogenate. The homogenate was centrifuged at 4°C and 3000 r / min for 10 min. The supernatant was collected and centrifuged at 4°C and 4000 r / min for 15 min. The precipitate was mixed with 0.25 mol / L sucrose solution and centrifuged at 4°C and 4000 r / min for 10 min. The precipitate was the mitochondria.

[0104] 3.2 Methods: 1) Add the medium solution of the mitochondrial respiratory control rate quantitative detection reagent to the reaction glass tank, stir with a micromagnetic device, mix thoroughly, seal the reaction tank, and record the oxygen concentration. The initial saturated oxygen concentration is 0.24 μmol molecular oxygen / ml (25°C); 2) Inject the mitochondria to be tested, and then add the mitochondrial respiratory control rate quantitative detection reagent IV state substrate solution to start IV state respiration; 3) Add the mitochondrial respiratory control rate quantitative detection reagent III state substrate solution to start III state respiration. The detection is terminated when the oxygen concentration drops rapidly and the downward slope shows an inflection point.

[0105] 3.3 Mitochondrial Respiratory Control Rate: The respiratory control rate, also known as the respiratory regulation ratio, is the ratio of the respiratory rate in state III (ADP addition) to the respiratory rate in state IV (ADP depletion). The RCR of normal mitochondria ranges from 3 to 10. A decreased RCR indicates impaired mitochondrial ATP synthesis and respiratory impairment; an increased RCR indicates vigorous cellular activity and accelerated metabolism. It is a sensitive indicator for assessing mitochondrial structural and functional integrity, as well as the degree of oxidative phosphorylation coupling.

[0106] 3.4 Analysis of Results: The results of the mitochondrial respiratory control rate measurement during kidney cryopreservation are shown in Table 4. It can be seen that after 24 hours of cryopreservation, the mitochondrial respiratory control rates of groups 1-4 were slightly higher than those of the HC-A solution group, indicating that the kidney preservation solutions of Examples 1-5 were superior to those of HC-A solution in preserving renal cortical mitochondrial integrity. After 72 hours of cryopreservation, the mitochondrial respiratory control rates of groups 1-4 were significantly higher than those of the HC-A solution group, indicating that the kidney preservation solutions of Examples 1-5 were superior to those of HC-A solution in preserving renal cortical mitochondrial integrity. These results indicate that the addition of Ganoderma lucidum extract and Epimedium brevicornum extract to HC-A solution can protect the structural integrity of mitochondria and the degree of coupling of oxidative phosphorylation in ischemia-reperfusion kidneys, protect the energy metabolism of renal cortical cells during cryopreservation of isolated kidneys, and promote kidney preservation. Furthermore, after 24 and 72 hours of cryopreservation, the mitochondrial respiratory control rates of group 5 were slightly higher than those of Example 1, indicating that the kidney preservation solution of Example 5 was superior to that of Example 1 in preserving renal cortical mitochondrial integrity. This suggests that the addition of stilbene glycosides enhances the preservation effect of Ganoderma lucidum extract and Epimedium brevicornum extract.

[0107] Table 4 Mitochondrial respiratory control rate during kidney cryopreservation

[0108]

[0109] 4. Determination of Calcium Ion Content in Renal Cortical Mitochondria

[0110] 4.1 Isolation of renal cortical mitochondria to prepare mitochondrial homogenate: After the kidneys were stored at 4°C for 24 and 72 hours, the renal cortex was accurately weighed and minced. The blood was removed and homogenized with 9x normal saline. The homogenate was centrifuged at 3000 rpm at 4°C for 10 minutes. The supernatant was collected and centrifuged at 4000 rpm at 4°C for 15 minutes. The precipitate was mixed with 0.25 mol / L sucrose solution and centrifuged at 4000 rpm at 4°C for 10 minutes. The precipitate was the mitochondria. The renal cell mitochondria were suspended at 2-3 mg of mitochondrial protein per ml and pulverized with an ultrasonicator to prepare the mitochondrial homogenate.

[0111] 4.2 Tissue protein determination: Coomassie Brilliant Blue reagent was used. The reagent configuration is shown in Table 5. The reagent was added to each test tube, mixed, and allowed to stand for 10 minutes. The blank tube was zeroed at 595 nm and the absorbance of each tube was measured at a 1 cm optical path. The mitochondrial homogenate protein content was calculated according to the following formula:

[0112] .

[0113] Table 5 Reagent configuration table

[0114]

[0115] 4.3 Determination of calcium ions in renal cortical mitochondria: The reagents were prepared as shown in Table 6. The reagents were added to each test tube, mixed well, and allowed to stand for 10 minutes. The blank tube was zeroed at 610 nm and the absorbance of each tube was measured at a light path of 1 cm. The calcium ion content in the mitochondria was calculated according to the following formula:

[0116] .

[0117] Table 6 Reagent configuration table

[0118]

[0119] 4.4 Analysis of Results: The results of the determination of calcium ion content in renal cortical mitochondria during storage are shown in Table 7. It can be seen that after 24 hours of cryopreservation, the calcium ion content in renal cortical mitochondria of Examples 1-4 groups was slightly lower than that of the HC-A solution group; after 72 hours of cryopreservation, the calcium ion content in renal cortical mitochondria of Examples 1-4 groups was significantly lower than that of the HC-A solution group. The above results indicate that the addition of Ganoderma lucidum extract and Epimedium extract to HC-A solution is beneficial for alleviating calcium overload. This may be because Ganoderma lucidum extract and Epimedium extract can increase the activity of calpain in renal cortical mitochondria, which is beneficial for kidney preservation. In addition, after 24 hours and 72 hours of cryopreservation, the calcium ion content in renal cortical mitochondria of Example 5 group was lower than that of Example 1. This indicates that the addition of diphenylethylene glycosides can further alleviate calcium overload. This may be because the addition of diphenylethylene glycosides can help increase the activity of calpain in renal cortical mitochondria of the modified kidney preservation solution, which has a beneficial effect on the preservation effect of Ganoderma lucidum extract and Epimedium extract.

[0120] Table 7 Mitochondrial respiratory control rate during kidney cryopreservation

[0121]

[0122] 5. Determination of Bcl-2 and Bax Expression in Kidney Tissue

[0123] 5.1 Methods: 1) Kidney tissue specimens were fixed with 10% neutral formaldehyde, embedded in paraffin, and serially sectioned at 4 μm thickness. Paraffin sections were then deparaffinized and hydrated. 2) Endogenous peroxidase was removed by incubation with 3% H₂O₂ at room temperature for 10 min, followed by three washes in distilled water for 3 min each. 3) Antigen retrieval was performed by microwave retrieval with 0.01 M citrate antigen retrieval buffer for 10 min each, followed by three thaws at 98°C. After cooling, the sections were washed once with distilled water and then three times with PBS for 3 min each. 4) Primary antibody was added and the sections were incubated overnight at 4°C. The sections were then incubated at room temperature for 30 min. Wash with PBS for 3 minutes three times; 5) Add secondary antibody (universal IgG antibody-HRP multimer secondary antibody), incubate at 37°C for 25 minutes, and wash with PBS for 3 minutes three times; 6) DAB color development; 7) Lightly counterstain with hematoxylin, dehydrate, and clear as usual; 8) After drying, mount the slides and observe under a microscope; 9) Image data processing: Strictly follow the kit instructions. After DAB color development, observe under a microscope. A negative control was performed by omitting the primary antibody, counterstaining with hematoxylin, dehydrating, clearing, mounting the slides, and observing under a microscope. Positive cells were identified by brown-yellow cytoplasmic staining. Five non-overlapping fields at high magnification (×400) were selected from the positive expression area for each section. The average absorbance (OD) value was calculated using an image analysis system and used as a semi-quantitative parameter to reflect Bcl-2 and Bax expression.

[0124] 5.2 Analysis of Results: Expression of Bcl-2, Bax and Bcl-2 / Bax in Renal Tissue As shown in Table 8, after 24 h of cryopreservation, compared with the HC-A solution group, the expression of Bcl-2 in the renal tissue of the Example 1-4 groups increased, the expression of Bax decreased, and the overall ratio of Bcl-2 / Bax increased, with Bcl-2 / Bax > 1.5. After 72 h of cryopreservation, compared with the HC-A solution group, the expression of Bcl-2 in the renal tissue of the Example 1-4 groups increased, the expression of Bax decreased, and the overall ratio of Bcl-2 / Bax increased, with Bcl-2 / Bax > 1.2. These results indicate that the addition of Ganoderma lucidum extract and Epimedium brevicornum extract to HC-A solution can upregulate the expression of Bcl-2 and downregulate the expression of Bax, thereby upregulating the Bcl-2 / Bax ratio and reducing the apoptosis of renal tubular epithelial cells. In addition, after 24 h of cryopreservation, compared with the Example 1 group, the expression of Bcl-2 in the kidney tissue of the Example 5 group increased, the expression of Bax decreased, and the overall ratio of Bcl-2 / Bax increased, Bcl-2 / Bax>1.9; after 72 h of cryopreservation, compared with the Example 1 group, the expression of Bcl-2 in the kidney tissue of the Example 5 group increased, the expression of Bax decreased, and the overall ratio of Bcl-2 / Bax increased, Bcl-2 / Bax>1.5; the above results indicate that diphenylethylene glycosides can enhance the effects of Ganoderma lucidum extract and Epimedium extract.

[0125] Table 8 Expression of Bcl-2 and Bax and Bcl-2 / Bax in kidney tissue

[0126]

[0127] 6. Determination of SOD and MDA in kidney tissue

[0128] 6.1 Preparation of homogenate: After the kidneys have been stored at 4°C for 24 h and 72 h, accurately weigh the renal cortex and mince it. Remove the blood and add 9 times the amount of normal saline to make a homogenate. Centrifuge at 4°C, 3000 rpm for 10 min. The supernatant is the 10% homogenate. Take 0.1 ml of the supernatant and add 0.9 ml of normal saline to dilute it to a 1% homogenate for testing.

[0129] 6.2 Renal tissue protein determination: Follow the method in 4.2.

[0130] 6.3 Determination of SOD activity in kidney tissue: Reagents were prepared as shown in Table 9. Reagents were added to each test tube and mixed thoroughly. The tubes were then placed in a 37°C constant temperature water bath for 40 minutes. 2 mL of color developer was added to each tube, mixed thoroughly, and allowed to stand for 10 minutes. The absorbance of each tube was measured at 550 nm with distilled water at a 1 cm optical path, and the SOD activity was calculated according to the following formula:

[0131] .

[0132] Table 9 Reagent configuration table

[0133]

[0134] 6.4 Determination of MDA Content in Renal Tissue: For protein content determination, refer to the method in 4.2. Add the reagent to each test tube, mix thoroughly, and then place in a 95°C water bath for 40 minutes. Remove and cool in running water. Centrifuge at 4000 rpm for 10 minutes. Pipette the supernatant into a colorimetric tube. Adjust the light path to 532 nm at a 1 cm wavelength with distilled water and measure the absorbance of each tube. Calculate the MDA content according to the following formula:

[0135]

[0136] 6.5 Result analysis: The results of SOD activity determination in kidney tissue are as follows Figure 1 The results of MDA content determination in kidney tissue are shown in Figure 2 As shown. Figure 1 and Figure 2As can be seen, after 24 hours of cryopreservation, the SOD activity (>100 U / mg) in the renal tissue of Groups 1-4 was significantly higher than that of the HC-A solution group, while the MDA content (<5 nmol / mg) in the renal tissue of Groups 1-4 was significantly lower than that of the HC-A solution group. After 72 hours of cryopreservation, the SOD activity (>85 U / mg) in the renal tissue of Groups 1-4 was significantly higher than that of the HC-A solution group, while the MDA content (<7.5 nmol / mg) in the renal tissue of Groups 1-4 was significantly lower than that of the HC-A solution group. After 24 and 72 hours of cryopreservation, the SOD activity and MDA content in Group 5 were comparable to those in Group 1. These results demonstrate that the addition of Ganoderma lucidum extract and Epimedium brevicornum extract to HC-A solution can increase the activity of the antioxidant enzyme SOD in isolated renal tissue, thereby reducing the content of malondialdehyde (MDA), a product of cellular lipid peroxidation, in isolated renal tissue. Furthermore, stilbene glycosides have no adverse effect on the technical effect of Ganoderma lucidum extract and Epimedium brevicornum extract in improving the antioxidant properties of HC-A solution.

[0137] 7. Determination of inflammatory factor levels in renal tissue

[0138] 7.1 Preparation of homogenate: After the kidneys have been stored at 4°C for 24 and 72 hours, accurately weigh the renal cortex and mince it. Remove the blood and add 9 times normal saline to make a homogenate. Centrifuge at 4°C, 3000 rpm for 10 minutes and collect the supernatant for testing.

[0139] 7.2 Determination of Inflammatory Factor Levels: TNF-α, IL-1β, HMGB1, and IL-10 levels were determined using the ELISA method, strictly following the kit instructions. For TNF-α determination, the specific steps are as follows: 1) Bring all reagents to room temperature to prepare sample reagents, mix the working solution, and prepare the standard. 2) Clean the microplate: Place 300 μL of wash solution in the microplate for 30 seconds, then discard the wash solution and pat the microplate dry with absorbent paper for later use. 3) Sample addition: Add 50 μL of buffer and 50 μL of sample to each well, except for the standard wells. Sample addition should be continuous and complete within 15 minutes. 4) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, and incubate at room temperature for 2 h; 5) Wash: Discard the liquid and add 300 μL of wash solution to each well, wash six times, and pat dry with absorbent paper; 6) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, and incubate at room temperature for 45 min; 7) Add enzyme: Add 100 μL of horseradish peroxidase marker to each well; 8) Color development: Add 100 μL of substrate solution to each well, mix well, and incubate at room temperature in the dark for 15 min; 9) Stop and measure: Add 100 μL of stop solution to each well to stop the reaction, and measure the optical density of each well in sequence at a wavelength of 450 nm on a microplate reader within 20 min.

[0140] 7.3 Results Analysis: Contents of TNF-α, IL-1β, HMGB1, and IL-10 in renal tissues Figure 3-6 As shown, it can be seen that: after 24 h of low-temperature storage, the TNF-α, IL-1β, and HMGB1 contents in the renal tissues of the Example 1-4 groups were significantly lower than those of the HC-A solution group, and the IL-10 content in the renal tissues of the Example 1-4 groups was significantly higher than that of the HC-A solution group; after 72 h of low-temperature storage, the TNF-α, IL-1β, and HMGB1 contents in the renal tissues of the Example 1-4 groups were significantly lower than those of the HC-A solution group, and the IL-10 content in the renal tissues of the Example 1-4 groups was significantly higher than that of the HC-A solution group; after 24 h and 72 h of low-temperature storage, the TNF-α, IL-1β, and HMGB1 contents in the renal tissues of the Example 5 group were all lower than those of the Example 1 group, and the IL-10 content in the renal tissues of the Example 5 group was higher than that of the Example 1 group. The above results indicate that the addition of Ganoderma lucidum extract and Epimedium extract to HC-A solution can downregulate the levels of proinflammatory cytokines TNF-α, IL-1β and HMGB1, and upregulate the level of anti-inflammatory cytokine IL-10; the addition of stilbene glycoside can further downregulate the levels of proinflammatory cytokines TNF-α, IL-1β and HMGB1, and upregulate the level of anti-inflammatory cytokine IL-10, thereby enhancing the effects of Ganoderma lucidum extract and Epimedium extract on the inflammatory response, and ultimately reducing ischemic damage during the cryopreservation period of isolated kidneys.

[0141] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0142] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for improving kidney preservation solution, comprising the following steps: S1: preparing the Ganoderma lucidum extract into a Ganoderma lucidum extract solution with a concentration of 1.0-3.5 g / mL; S2: preparing the epimedium extract into an epimedium extract solution with a concentration of 0.2-1.8 g / mL; S3: adding the Ganoderma lucidum extract and Epimedium extract to the HC-A solution, adjusting the pH to 7.3-7.8 with phosphoric acid, and sterilizing to obtain the improved kidney preservation solution; The ganoderma lucidum extract contains ganoderma lucidum polysaccharides and total triterpenes of ganoderma lucidum; the epimedium extract contains icariin and epimedium; the epimedium includes epimedium A, epimedium B and epimedium C; the volume ratio of the HC-A solution, ganoderma lucidum extract and epimedium extract is 100:1.5-3.0:1.5-3.0; The ganoderma lucidum extract contains at least 8wt% ganoderma lucidum polysaccharides and 3wt% total triterpenes of ganoderma lucidum; the epimedium extract contains at least 55wt% icariin and 40wt% epimedin; The kidney preservation solution also contains diphenylethylene glycoside; the diphenylethylene glycoside is prepared into a diphenylethylene glycoside solution with a concentration of 2.5-10 mg / mL.

2. Use of a Ganoderma lucidum extract combined with an Epimedium brevicornum extract in the preparation of an improved kidney preservation solution obtained by the improved method of claim 1, characterized in that: The ganoderma lucidum extract contains at least 8wt% of ganoderma lucidum polysaccharide and 3wt% of ganoderma lucidum total triterpenes; the epimedium extract contains at least 55wt% of icariin and 40wt% of epimedium; the epimedium includes epimedium A, epimedium B and epimedium C.

3. The improved kidney preservation solution obtained by the improved method of claim 1 is used to preserve kidneys in the absence of blood supply.

4. A method for preventing or reducing the severity of isolated kidney injury after reperfusion, comprising preserving the isolated kidney in the improved kidney preservation solution obtained by the improved method of claim 1.

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