Construction and application of a mouse tail distal ischemic preconditioning model for renal protection

Abstract

The present invention discloses a method for constructing a mouse tail distal ischemia preconditioning renal protection model and its application, which belongs to the field of ischemia preconditioning technology, and its purpose is to solve the technical problem of poor stability and reliability of renal protection experimental results in the prior art. It is by respectively constructing a tail distal ischemia preconditioning group, a septic acute kidney injury model group and a tail control group; when constructing the tail distal ischemia preconditioning group, a tourniquet is tied on the mouse tail, and blood flow is restored by blocking blood flow and repeating the cycle; and blood and kidney tissue of mice in each group are collected for testing, serum creatinine and serum urea nitrogen indicators are measured, renal tubular pathological damage is assessed, and the expression level of renal reactive oxygen species is assessed. This method proves that tail ischemia reperfusion preconditioning can improve renal pathological damage and reduce renal tubular damage in septic acute kidney injury, inhibit renal oxidative stress in septic acute kidney injury, and reduce the inflammatory response of septic acute kidney injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of ischemic preconditioning, and relates to the construction of a renal protection model based on ischemic preconditioning in mice, and in particular to a construction method and application of a renal protection model based on distal ischemic preconditioning in the tail of mice. Background Art

[0002] Ischemic preconditioning (IPC) is a protective mechanism that enhances the tolerance of organs or tissues to subsequent, more severe ischemic injury through a brief, non-lethal ischemic stimulus. Its core principle is to stimulate endogenous adaptive responses. By activating cellular signaling pathways and reducing oxidative stress and inflammatory responses, it can protect organs from subsequent ischemia-reperfusion injury, thereby alleviating damage caused by long-term ischemia. It is widely used to protect vital organs such as the heart, brain, and liver. However, IPC acts directly on target organs, which can trigger a stress response, damage vascular structure, and lead to adverse prognosis, limiting its clinical application.

[0003] In the prior art, when ischemia-reperfusion is performed on the target organ (i.e., the kidney), a corresponding experimental device is usually required. The utility model patent with application number 202021983041.4 discloses an experimental table for animal ischemia-reperfusion experiments, which includes a flat plate, and two parallel slide rails along the front-to-back direction are fixedly installed on the top surface of the flat plate. Two first sliders are respectively installed on the left and right slide rails. A cylinder with an upper end opening is fixedly installed above the first slider. A rotating shaft is rotatably connected between the front and rear inner walls of the cylinder. A circular wheel is mounted on the rotating shaft, and a section of arc-shaped teeth is provided below the circular wheel. Worms with mutually perpendicular axes are meshed below the arc-shaped teeth. First through holes are provided on the outer sides of the side walls of the cylinder corresponding to the worms. The outer ends of the worms pass through the first through holes and are fixedly installed with coaxial turntables. The utility model patent with application number 202120170725.0 also discloses an animal ischemia-reperfusion experimental device, which includes a spring clamp and an air pump. The spring clamp is composed of a first clamping plate and a second clamping plate connected by a torsion spring. The right sides of the first clamping plate and the second clamping plate are respectively fixedly connected with a first handle and a second handle. The bottom of the inner wall of the first clamping plate and the top of the inner wall of the second clamping plate are both fixedly connected with a mounting frame, and the interior of the two mounting frames are respectively fixedly connected with a first airbag and a second airbag.

[0004] For ischemia-reperfusion, in addition to the corresponding experimental equipment, the establishment of an animal model is also crucial. The purpose of establishing an ischemia animal model is to generate renal ischemia-reperfusion injury in animals, thereby observing the growth of cells that can repair kidney damage in the animals and the effect of repairing kidney damage.

[0005] In the prior art, there is a lack of animal models for renal ischemia-reperfusion in animals, but there are other animal models. For example, the invention patent application with application number 202110816267.8 discloses a model and method for constructing an infrarenal abdominal aortic aneurysm in rats via a retroperitoneal approach. The model fully frees and ligates the branches of the perfused abdominal aorta with a mousse thread, fully exposes the surgical field of view with a mastoid spreader, first blocks the proximal end of the abdominal aorta with a microvascular clamp, then squeezes the blood in the perfused segment blood vessels to the distal end with a microhemostatic forceps, and then blocks the distal end of the abdominal aorta with a microvascular clamp. The blood vessels completely collapse, indicating that the blood vessels are well sealed. Then, 0.2 mL of 10 U of elastase is extracted with a disposable insulin syringe, and the syringe needle is slightly bent with a vascular clamp to make it slightly "L" shaped. The abdominal aorta is punctured. After successful puncture, 0.1 mL of elastase containing 5 U is slowly injected to fully fill the perfused segment. The puncture needle is fixed and retained for 20 minutes. If a small amount of elastase extravasates during this period, it is promptly replenished to ensure that the perfused segment remains fully filled. After the procedure, the intravascular medication is withdrawn, the needle is removed, and the puncture site is covered with gelatin sponge. Pressure is applied with a cotton ball. The distal vascular clamp is removed first, followed by the proximal vascular clamp. After several minutes of observation, if no active bleeding is observed, a small gauze strip soaked in 1.0 mol / L sterile calcium chloride is applied to the surface of the perfused segment. After 10 minutes, the small gauze strip is removed, the retroperitoneal space is cleaned, and if there is no bleeding, the incision is closed layer by layer. This approach can shorten the perfusion time to 20 minutes. After the perfusion is completed, local pressure can stop the bleeding. In addition, the invention patent application with application number 202411332193.0 also discloses a method for constructing an animal model of myocardial ischemia, which includes: calculating the morphological characteristics of control mesenchymal stem cells in the preprocessed image, performing principal component analysis on the morphological characteristics, and analyzing the cell activity corresponding to the principal component through the output of the activity analysis model; constructing an animal three-dimensional model of an animal with myocardial injury, and collecting the culture environment of the experimental mesenchymal stem cells; using the culture environment to select the growth environment in the animal with myocardial injury, collecting the environmental image of the growth environment, and selecting the transplantation position of the animal with myocardial injury from the environmental image; determining the three-dimensional position of the transplantation position from the animal three-dimensional model, constructing a transplantation path from the surface position of the animal three-dimensional model to the three-dimensional position, and transplanting the experimental mesenchymal stem cells to the transplantation position to obtain an in situ transplanted animal model.

[0006] Referring to the above-mentioned animal model construction method, when constructing an animal model of ischemia-reperfusion, it will be based on abdominal aortic ischemia, lower limb femoral artery ischemia, etc. modeling is implemented. Although it can observe and study the mechanism of kidney protection and screen related therapeutic drugs through two models: tourniquet-induced abdominal / lower limb ischemia and direct abdominal aortic ischemia / lower limb femoral artery ischemia; however, this method still has certain defects, mainly reflected in: 1. Trauma and injury. Both methods will cause significant trauma to the animal's abdominal / lower limb, which is not conducive to the animal's subsequent survival and recovery. 2. Nerve damage: Tourniquet-induced abdominal / lower limb ischemia will compress the nerves. Prolonged ischemia may cause nerve ischemia and hypoxia, causing nerve fiber damage and necrosis, resulting in abdominal / lower limb muscle weakness and paralysis. The surgical operation of abdominal / lower limb femoral artery ischemia may also damage peripheral nerves, leading to symptoms such as numbness, tingling, and decreased sensation in the area innervated by the compressed nerve. 3. Operation and stability: These methods require high surgical skills and postoperative care, increasing experimental difficulty and workload, and are prone to adverse consequences and increased experimental complexity. 4. Stability and reproducibility: These methods are highly invasive to animals, resulting in unstable animal conditions, large variability in results between experiments, and poor experimental stability and reproducibility. Therefore, it is necessary to develop an animal model that is less invasive and more convenient to operate, thereby improving the reliability and scientific nature of the experiments. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for constructing a mouse tail distal ischemic preconditioning renal protection model and its application in order to solve the technical problems that the models constructed in the prior art cause great trauma to animals, are difficult to operate on, and are difficult to fixate animals, resulting in poor stability and reliability of experimental results.

[0008] In order to achieve the above-mentioned purpose, the present invention specifically adopts the following technical solutions:

[0009] A method for establishing a mouse tail distal ischemic preconditioning renal protection model comprises the following steps:

[0010] Step 1: Select and group materials;

[0011] Several healthy male C56BL / 6J mice weighing 23-25 ​​g and aged 6-8 weeks were selected and randomly divided into a tail control group, a sepsis-induced acute kidney injury model group, and a tail distal ischemia preconditioning group.

[0012] Step 2, induction of anesthesia;

[0013] The mice in the distal tail ischemia preconditioning group were anesthetized by slow intraperitoneal injection of urethane at a concentration of 15-25% (the standard dosage was 1.5-2.0 mg / kg); the mice in the sepsis acute kidney injury model group and the tail control group were not anesthetized.

[0014] Step 3, constructing an animal model;

[0015] The distal tail ischemia preconditioning group was established: After anesthetizing the mice in the distal tail ischemia preconditioning group, a tourniquet was placed on the tail to block blood flow for 4-8 minutes. The tourniquet was then released to restore blood flow for 4-8 minutes (blockage time = recovery time). This "ischemia-reperfusion" cycle was repeated for 3-6 cycles. 12-20 minutes after the end of the ischemia-reperfusion cycle, 8-12 mg / kg of lipopolysaccharide (LPS) was intraperitoneally injected (80-120 μL / mouse). The time interval between anesthesia and LPS injection was defined as T.

[0016] A septic acute kidney injury model group was established: after the same duration as the distal tail ischemia preconditioning group, mice were intraperitoneally injected with lipopolysaccharide (LPS) of equal concentration and dosage (i.e., after the mice in the distal tail ischemia preconditioning group were anesthetized, lipopolysaccharide (LPS) was injected after a time T).

[0017] A tail control group was constructed: a tourniquet was placed on the mouse tail but not tied to maintain normal blood flow in the mouse tail; after the same duration as the distal tail ischemia preconditioning group, the mice were injected with an equal amount of normal saline through the abdominal cavity (i.e., after the mice in the distal tail ischemia preconditioning group were anesthetized, normal saline was injected after a time T, and the injection volume of normal saline (ul) was the same as the injection volume of lipopolysaccharide (LPS)).

[0018] When injecting lipopolysaccharide LPS, the specific injection method is as follows:

[0019] Grasp and secure the mouse with your left hand, with the abdomen facing upward and the head lower than the tail to avoid damage to internal organs. Disinfect the mouse's abdomen with an alcohol cotton ball and insert the syringe approximately 0.3-0.7 cm on either side of the mouse's linea alba. Push the needle 3-5 mm subcutaneously and then pierce the mouse's abdominal cavity at an angle of 40-48 degrees to the skin. A sense of empty space should be felt during insertion. If no liquid refluxes when the needle plug is withdrawn, slowly inject the drug solution. After the injection is complete, rotate the needle and slowly withdraw it to prevent liquid leakage.

[0020] Step 4: data collection and testing for model effect evaluation;

[0021] Twelve to 20 hours after intraperitoneal injection of lipopolysaccharide (LPS) and normal saline, blood and kidney tissues of mice were collected simultaneously.

[0022] After the collected blood is centrifuged, the supernatant is taken and the serum creatinine and serum urea nitrogen indicators are measured (the specific method for measuring serum creatinine and serum urea nitrogen indicators based on the supernatant can be directly applied to the existing technology without the need for creative work) to evaluate the renal function of mice in each group.

[0023] The collected kidney tissue is divided into three parts:

[0024] The first part is used to extract protein and RNA, detect renal tubular injury markers such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), and detect the expression levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (the specific method of using kidney tissue to detect the expression levels of renal tubular injury markers and inflammatory factors can be directly applied to existing technologies without the need for creative work):

[0025] When using kidney tissue to extract protein, the required tissue homogenate preparation conditions are as follows: place 20-30 mg of kidney tissue in a 2-5 ml grinding tube, add 450-600 ul of protein lysate, add 1-2 4 mm stainless steel grinding balls and 2-4 3 mm stainless steel grinding balls, place in a high-speed tissue grinder at 3-6°C for grinding, the grinding conditions are 60-70 Hz, 50-75 s, and grind 3-4 times.

[0026] When using kidney tissue to extract RNA, the required tissue homogenate preparation conditions are as follows: place 15-20 mg of kidney tissue in a 2-5 ml grinding tube, add 300-380 μl of lysis buffer, add 1-2 4 mm stainless steel enzyme-free grinding balls and 2-4 3 mm stainless steel enzyme-free grinding balls, and place in a high-speed tissue grinder at room temperature for grinding. The grinding conditions are 60-70 Hz, 50-75 seconds, and grinding 3-4 times.

[0027] The second part is used to prepare paraffin sections, which are then stained with hematoxylin and eosin (H&E) to score renal tubular injury and assess the pathological damage of the renal tubules (the specific methods for scoring renal tubular injury and assessing renal tubular pathological damage using renal paraffin sections can be directly applied to existing technologies without requiring any creative work);

[0028] The third part is used to prepare kidney frozen sections by OCT embedding, and evaluate the expression level of renal reactive oxygen species (ROS) by dihydroethidium (DHE) staining (among which, the specific method of evaluating the expression level of renal reactive oxygen species using kidney frozen sections can be directly applied to existing technology without the need for creative work).

[0029] Furthermore, the method further comprises step 5;

[0030] Step 5, analysis and comparison;

[0031] First, the sepsis acute kidney injury model group was compared with the tail control group to analyze the serum creatinine and serum urea nitrogen levels; if the serum creatinine in the sepsis acute kidney injury model group increased by more than 2 times, and the statistical test showed that the difference between the two groups was statistically significant, then the sepsis acute kidney injury model group was successfully constructed; otherwise, the construction failed;

[0032] The sepsis acute kidney injury model group was then compared with the tail distal ischemia preconditioning group, and the serum creatinine and serum urea nitrogen levels of the two groups were analyzed to verify the improvement effect on renal function of sepsis acute kidney injury; the RNA and protein expression levels of renal tubular injury markers and inflammatory factors were detected to see whether they were inhibited by distal ischemia preconditioning; combined with the renal pathological damage situation, renal tubular damage score, and renal reactive oxygen species expression level, it was determined whether distal ischemia preconditioning could reduce renal pathological damage and inhibit the expression level of renal ROS.

[0033] Furthermore, in step 3, a bilateral lower limb distal ischemia preconditioning group and a lower limb control group were also constructed;

[0034] A bilateral lower limb distal ischemia preconditioning group was established: after the mice in the bilateral lower limb distal ischemia preconditioning group were anesthetized, the lower limbs of the mice were tied with a tourniquet and the blood flow was blocked for 4-8 minutes, and then the tourniquet was released to restore the blood flow for the same period of time, and this "ischemia-reperfusion" process was repeated for 3-6 cycles; 12-20 minutes after the end of the ischemia-reperfusion cycle, lipopolysaccharide (LPS) at a concentration of 8-12 mg / kg was injected intraperitoneally (the dosage standard was 80-120 μl / mouse).

[0035] A lower limb control group was constructed: a tourniquet was placed on both lower limbs of the mouse but not tied to maintain normal blood flow in the mouse tail; after the same duration as that of the distal tail ischemia preconditioning group, an equal amount of normal saline was injected into the mouse through the abdominal cavity (i.e., after the mice in the distal tail ischemia preconditioning group or the distal lower limb ischemia preconditioning group were anesthetized, normal saline was injected after time T, and the injection volume of normal saline (ul) was the same as the injection volume of lipopolysaccharide (LPS)).

[0036] In step 5, the sepsis acute kidney injury model group was first compared with the tail control group and the lower limb control group; then the sepsis acute kidney injury model group was compared with the bilateral lower limb distal ischemia preconditioning group and the tail distal ischemia preconditioning group, respectively.

[0037] The model constructed according to the above construction method is used to study the protective mechanism of the kidneys by distal ischemic preconditioning of the mouse tail or to screen drugs for treating / protecting kidney diseases.

[0038] The beneficial effects of the present invention are as follows:

[0039] 1. After experiments, the model was constructed using the method of this application, with a high success rate, and it can be effectively verified that: tail ischemia-reperfusion preconditioning can improve renal pathological damage and reduce renal tubular damage in septic acute kidney injury, inhibit renal oxidative stress in septic acute kidney injury, and reduce the inflammatory response of septic acute kidney injury.

[0040] 2. Compared with the existing lower limb ischemia model, the present invention uses the mouse tail blood vessels, which are richly distributed and have a clear source of blood supply. The tail distal ischemia preconditioning group constructed using this part is relatively simple to model, causes little trauma to animals, has low difficulty in surgery and nursing, is easier for animals to adapt and recover, and is less likely to develop serious complications. It can effectively achieve distal ischemia preconditioning while reducing harm to experimental animals; by adopting a tail blood vessel model with less impact on animals, it reduces the interference of factors such as animal stress on the experimental results, can more stably and accurately simulate the distal ischemia preconditioning process, and the results between different experiments are less different, which improves the stability and repeatability of the model, and is more conducive to accurately evaluating the protective effect of distal ischemia preconditioning on the kidneys. At the same time, it also better complies with animal ethical requirements and reflects concern for the welfare of experimental animals.

[0041] 3. In the present invention, a control group, a septic acute kidney injury model group, a bilateral lower limb distal ischemia preconditioning group, and a tail distal ischemia preconditioning group are reasonably set up. During the pretreatment and model construction process, standardized and mutually controlled operating procedures are set for different groups (such as clearly stipulating the dosage of anesthetic drugs, the number of ischemia-reperfusion cycles, the time interval, and the timing and dosage of intraperitoneal injection of LPS, etc.). Through rigorous grouping and standardized processing procedures, the renal protective equivalence of tail distal ischemia preconditioning and bilateral lower limb distal ischemia preconditioning for septic acute kidney injury can be verified more scientifically and accurately.

[0042] 4. In the present invention, during the model evaluation stage, not only traditional renal function indicators such as serum creatinine and serum urea nitrogen are detected, but also tubular injury markers such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), as well as the expression levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are detected; at the same time, combined with renal pathological sections (H&E staining of paraffin sections for tubular injury scoring, DHE staining of frozen sections to evaluate the expression level of renal reactive oxygen species), a comprehensive evaluation system is established from multiple dimensions, which can more comprehensively and in-depth evaluate the protective effect of distal tail tissue ischemic preconditioning on the kidney, which is more scientific and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a schematic diagram showing the effects of the ischemic group and the control group on blood flow in the present invention;

[0044] Among them, A is the blood flow of the tail of mice in the tail ischemia group and the tail control group, B is the blood flow rate of the tail of mice in the tail ischemia group and the tail control group; C is the blood flow of the lower limbs of mice in the lower limb ischemia group and the lower limb control group, D is the blood flow rate of the lower limbs of mice in the lower limb ischemia group and the lower limb control group;

[0045] Figure 2 Schematic diagram of the effects of the lower limb control group, model group and lower limb ischemia group on renal function and renal injury in mice;

[0046] Among them, A is a schematic diagram of the construction of the lower limb ischemia group, B is a schematic diagram of the effects of creatinine and urea nitrogen, C is a schematic diagram of the staining analysis comparison, and D is a schematic diagram of the effects of renal tubular injury score and tubular injury marker levels;

[0047] Figure 3 Schematic diagram of the effects of the tail control group, model group, and tail ischemia group on renal function and renal injury in mice in the present invention;

[0048] Among them, E is a schematic diagram of the construction of the tail ischemia group, F is a schematic diagram of the effects of creatinine and urea nitrogen, G is a schematic diagram of the staining analysis comparison, and H is a schematic diagram of the effects of renal tubular injury score and tubular injury marker levels;

[0049] Figure 4 Schematic diagram of the effects of the lower limb control group, model group and lower limb ischemia group on the expression levels of inflammatory factors in the present invention;

[0050] Among them, A is a schematic diagram of the effects of oxidative stress (ROS), B is a schematic diagram of the effects of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) mRNA, C is a schematic diagram of the effects of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) proteins, and D is a schematic diagram of the protein abundance of interleukins and tumor necrosis factors;

[0051] Figure 5 This is a schematic diagram of the effects of the tail control group, model group, and tail ischemia group on the expression levels of inflammatory factors in the present invention;

[0052] Among them, E is a schematic diagram of the effects of oxidative stress (ROS), F is a schematic diagram of the effects of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) mRNA, G is a schematic diagram of the effects of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) proteins, and H is a schematic diagram of the protein abundance of interleukins and tumor necrosis factors. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0054] Therefore, based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0055] Example 1

[0056] This example provides a method for constructing a mouse tail distal ischemic preconditioning renal protection model, aiming to explore the effects of tail distal ischemia-reperfusion on renal injury. Specifically, the method includes the following steps:

[0057] Step 1: Select and group materials;

[0058] Several healthy male C56BL / 6J mice weighing 23 g and aged 6 weeks were selected and randomly divided into a tail control group, a sepsis-induced acute kidney injury model group, and a tail distal ischemia preconditioning group.

[0059] Step 2, induction of anesthesia;

[0060] Mice in the distal tail ischemia preconditioning group were anesthetized with 18% urethane (1.6 mg / kg) injected slowly intraperitoneally. Mice in the sepsis-induced acute kidney injury model group and the distal tail control group were not anesthetized.

[0061] In this step, considering that the distal tail ischemia preconditioning group required ischemia and reperfusion, to minimize pain inflicted on the mice and to reduce the potential for overreactions due to pain that could impact the experimental results, as well as ethical concerns, only the mice in the ischemia preconditioning groups (including the distal tail ischemia preconditioning group in this example and the bilateral lower limb distal ischemia preconditioning group in the following examples) were anesthetized, while the mice in the model and control groups were not anesthetized. Furthermore, preliminary studies have shown that whether or not the mice were anesthetized had no effect, or a very small effect, on the final experimental outcome, and the effect was negligible, completely negligible.

[0062] Step 3, constructing an animal model;

[0063] A distal tail ischemia preconditioning group was established: after the mice in the distal tail ischemia preconditioning group were anesthetized, a tourniquet was used to tie the tail of the mice to block the blood flow for 5 minutes, and then the tourniquet was released to restore the blood flow for 5 minutes (blocked blood flow time = blood flow recovery time). This "ischemia-reperfusion" process was repeated for a total of 4 cycles; 15 minutes after the end of the ischemia-reperfusion cycle, 9 mg / kg lipopolysaccharide (LPS) was injected intraperitoneally (the dosage standard was 90 μl / mouse).

[0064] A septic acute kidney injury model group was established: after the same duration as the distal tail ischemia preconditioning group, mice were intraperitoneally injected with lipopolysaccharide (LPS) of equal concentration and dosage (i.e., after the mice in the distal tail ischemia preconditioning group were anesthetized, lipopolysaccharide (LPS) was injected after a time T).

[0065] A tail control group was constructed: a tourniquet was placed on the mouse tail but not tied to maintain normal blood flow in the mouse tail; after the same duration as the distal tail ischemia preconditioning group, the mice were injected with an equal amount of normal saline through the abdominal cavity (i.e., after the mice in the distal tail ischemia preconditioning group were anesthetized, normal saline was injected after a time T, and the injection volume of normal saline (ul) was the same as the injection volume of lipopolysaccharide (LPS)).

[0066] When injecting lipopolysaccharide LPS, the specific injection method is as follows:

[0067] Grasp and secure the mouse with your left hand, with the abdomen facing upward and the head lower than the tail to avoid damage to internal organs. Disinfect the mouse's abdomen with an alcohol cotton ball and insert the syringe approximately 0.4 cm on either side of the mouse's linea alba. Push the needle 4 mm subcutaneously and then pierce the mouse's abdominal cavity at a 42° angle to the skin. A sense of empty space should be felt during insertion. If no liquid refluxes when the needle plug is withdrawn, slowly inject the drug solution. After the injection is complete, rotate the needle and slowly withdraw it to prevent liquid leakage.

[0068] Step 4: data collection and testing for model effect evaluation;

[0069] 14 hours after intraperitoneal injection of lipopolysaccharide (LPS) and normal saline, the blood and kidney tissues of the mice were collected simultaneously.

[0070] After the collected blood is centrifuged, the supernatant is taken and the serum creatinine and serum urea nitrogen indicators are measured (the specific method for measuring serum creatinine and serum urea nitrogen indicators based on the supernatant can be directly applied to the existing technology without the need for creative work) to evaluate the renal function of mice in each group.

[0071] The collected kidney tissue is divided into three parts:

[0072] The first part is used to extract protein and RNA, detect renal tubular injury markers such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), and detect the expression levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (the specific method of using kidney tissue to detect the expression levels of renal tubular injury markers and inflammatory factors can be directly applied to existing technologies without the need for creative work):

[0073] When using kidney tissue to extract protein, the required tissue homogenate preparation conditions are as follows: place 24 mg of kidney tissue in a 3 ml grinding tube, add 480 ul of protein lysate, put in 1 4 mm stainless steel grinding ball and 2 3 mm stainless steel grinding balls, place in a high-speed tissue grinder at 4°C for grinding, the grinding conditions are 62 Hz, 55 s, and grind 3 times.

[0074] When using kidney tissue to extract RNA, the required tissue homogenate preparation conditions are as follows: place 16 mg of kidney tissue in a 3 ml grinding tube, add 320 ul of lysis buffer, put in 1 4 mm stainless steel enzyme-free grinding ball and 2 3 mm stainless steel enzyme-free grinding balls, place in a high-speed tissue grinder at room temperature for grinding, the grinding conditions are 63 Hz, 56 s, and grind 3 times.

[0075] The second part is used to prepare paraffin sections, which are then stained with hematoxylin and eosin (H&E) to score renal tubular injury and assess the pathological damage of the renal tubules (the specific methods for scoring renal tubular injury and assessing renal tubular pathological damage using renal paraffin sections can be directly applied to existing technologies without requiring any creative work);

[0076] The third part is used to prepare kidney frozen sections by OCT embedding, and evaluate the expression level of renal reactive oxygen species (ROS) by dihydroethidium (DHE) staining (among which, the specific method of evaluating the expression level of renal reactive oxygen species using kidney frozen sections can be directly applied to existing technology without the need for creative work).

[0077] Step 5, analysis and comparison;

[0078] First, the sepsis acute kidney injury model group was compared with the tail control group to analyze the serum creatinine and serum urea nitrogen levels; if the serum creatinine in the sepsis acute kidney injury model group increased by more than 2 times, and the statistical test showed that the difference between the two groups was statistically significant, then the sepsis acute kidney injury model group was successfully constructed; otherwise, the construction failed;

[0079] The sepsis acute kidney injury model group was then compared with the tail distal ischemia preconditioning group, and the serum creatinine and serum urea nitrogen levels of the two groups were analyzed to verify the improvement effect on renal function of sepsis acute kidney injury; the RNA and protein expression levels of renal tubular injury markers and inflammatory factors were detected to see whether they were inhibited by distal ischemia preconditioning; combined with the renal pathological damage situation, renal tubular damage score, and renal reactive oxygen species expression level, it was determined whether distal ischemia preconditioning could reduce renal pathological damage and inhibit the expression level of renal ROS.

[0080] The model was constructed using the method of this embodiment. After testing and analysis, the model was successfully constructed, and the conclusion was that tail ischemia-reperfusion preconditioning can improve renal pathological damage and reduce renal tubular damage in septic acute kidney injury, inhibit renal oxidative stress in septic acute kidney injury, and reduce the inflammatory response in septic acute kidney injury.

[0081] Example 2

[0082] This example provides a method for establishing a mouse tail distal ischemic preconditioning renal protection model, aiming to investigate the effects of lower limb ischemia-reperfusion on renal injury. Specifically, the following steps are included:

[0083] Step 1: Select and group materials;

[0084] Several healthy male C56BL / 6J mice weighing 24 g and aged 7 weeks were selected and randomly divided into a lower limb control group, a sepsis acute kidney injury model group, and a bilateral lower limb distal ischemia preconditioning group.

[0085] Step 2, induction of anesthesia;

[0086] Mice in the bilateral distal ischemic preconditioning group were anesthetized with a slow intraperitoneal injection of 20% urethane (1.8 mg / kg). Mice in the sepsis-induced acute kidney injury model group and the tail control group were not anesthetized.

[0087] Step 3, constructing an animal model;

[0088] A bilateral lower limb distal ischemia preconditioning group was established: after the mice in the bilateral lower limb distal ischemia preconditioning group were anesthetized, the lower limbs of the mice were tied with a tourniquet and the blood flow was blocked for 6 minutes, and then the tourniquet was loosened to restore the blood flow for the same period of time, and this "ischemia-reperfusion" process was repeated for 5 cycles; 16 minutes after the end of the ischemia-reperfusion cycle, 10 mg / kg lipopolysaccharide (LPS) was injected intraperitoneally (the dosage standard was 100 ul / mouse).

[0089] A septic acute kidney injury model group was established: after the same duration as the distal ischemia preconditioning group, mice were intraperitoneally injected with lipopolysaccharide (LPS) of equal concentration and dosage (i.e., after the mice in the distal ischemia preconditioning group were anesthetized from the tail, lipopolysaccharide (LPS) was injected after a time T).

[0090] A lower limb control group was constructed: a tourniquet was placed on the lower limbs of the mouse but not tied to maintain normal blood flow in the mouse tail; after the same duration as that of the distal ischemia preconditioning group, an equal amount of normal saline was injected into the mouse through the abdominal cavity (i.e., after the mice in the distal tail ischemia preconditioning group or the distal ischemia preconditioning group were anesthetized, normal saline was injected after time T, and the injection volume of normal saline (ul) was the same as the injection volume of lipopolysaccharide (LPS)).

[0091] When injecting lipopolysaccharide LPS, the specific injection method is as follows:

[0092] Grasp and secure the mouse with your left hand, with the abdomen facing upward and the head lower than the tail to avoid damage to internal organs. Disinfect the mouse's abdomen with an alcohol cotton ball and insert the syringe approximately 0.5 cm on either side of the mouse's linea alba. Push the needle 4 mm subcutaneously and then pierce the mouse's abdominal cavity at a 44° angle to the skin. A sense of empty space should be felt during insertion. If no liquid refluxes when the needle plug is withdrawn, slowly inject the drug solution. After the injection is complete, rotate the needle and slowly withdraw it to prevent liquid leakage.

[0093] Step 4: data collection and testing for model effect evaluation;

[0094] 15 hours after intraperitoneal injection of lipopolysaccharide (LPS) and normal saline, the mice were killed and their blood and kidney tissues were collected simultaneously.

[0095] After the collected blood is centrifuged, the supernatant is taken and the serum creatinine and serum urea nitrogen indicators are measured (the specific method for measuring serum creatinine and serum urea nitrogen indicators based on the supernatant can be directly applied to the existing technology without the need for creative work) to evaluate the renal function of mice in each group.

[0096] The collected kidney tissue is divided into three parts:

[0097] The first part is used to extract protein and RNA, detect renal tubular injury markers such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), and detect the expression levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (the specific method of using kidney tissue to detect the expression levels of renal tubular injury markers and inflammatory factors can be directly applied to existing technologies without the need for creative work):

[0098] When using kidney tissue to extract protein, the required tissue homogenate preparation conditions are as follows: place 25 mg of kidney tissue in a 4 ml grinding tube, add 520 ul of protein lysate, put in 2 4 mm stainless steel grinding balls and 3 3 mm stainless steel grinding balls, place in a high-speed tissue grinder at 4°C for grinding, the grinding conditions are 65 Hz, 62 s, and grind 4 times.

[0099] When using kidney tissue to extract RNA, the required tissue homogenate preparation conditions are as follows: place 18 mg of kidney tissue in a 3 ml grinding tube, add 340 ul of lysis buffer, put in 2 4 mm stainless steel enzyme-free grinding balls and 3 3 mm stainless steel enzyme-free grinding balls, and place in a high-speed tissue grinder at room temperature for grinding. The grinding conditions are 65 Hz, 62 s, and grinding 4 times.

[0100] The second part is used to prepare paraffin sections, which are then stained with hematoxylin and eosin (H&E) to score renal tubular injury and assess the pathological damage of the renal tubules (the specific methods for scoring renal tubular injury and assessing renal tubular pathological damage using renal paraffin sections can be directly applied to existing technologies without requiring any creative work);

[0101] The third part is used to prepare kidney frozen sections by OCT embedding, and evaluate the expression level of renal reactive oxygen species (ROS) by dihydroethidium (DHE) staining (among which, the specific method of evaluating the expression level of renal reactive oxygen species using kidney frozen sections can be directly applied to existing technology without the need for creative work).

[0102] Step 5, analysis and comparison;

[0103] First, the sepsis acute kidney injury model group was compared with the lower limb control group to analyze the serum creatinine and serum urea nitrogen levels; if the serum creatinine in the sepsis acute kidney injury model group increased by more than 2 times, and the statistical test showed that the difference between the two groups was statistically significant, the sepsis acute kidney injury model group was successfully established; otherwise, the establishment failed;

[0104] The sepsis acute kidney injury model group was then compared with the bilateral lower limb distal ischemia preconditioning group, and the serum creatinine and serum urea nitrogen levels of the two groups were analyzed to verify the improvement effect on renal function of sepsis acute kidney injury; the RNA and protein expression levels of renal tubular injury markers and inflammatory factors were detected to see whether they were inhibited by distal ischemia preconditioning; combined with the renal pathological damage, renal tubular damage score, and renal reactive oxygen species expression level, it was determined whether bilateral lower limb distal ischemia preconditioning could reduce renal pathological damage and inhibit the expression level of renal ROS.

[0105] The model was constructed using the method of this embodiment. After testing and analysis, the model was successfully constructed, and the conclusion was that lower limb ischemia-reperfusion preconditioning can improve renal pathological damage and reduce renal tubular damage in septic acute kidney injury, inhibit renal oxidative stress in septic acute kidney injury, and reduce the inflammatory response in septic acute kidney injury.

[0106] Example 3

[0107] This example provides a method for establishing a mouse tail distal ischemic preconditioning renal protection model, with the goal of exploring and comparing the effects of tail and lower limb ischemia-reperfusion on renal injury. Specifically, the following steps are included:

[0108] Step 1: Select and group materials;

[0109] Several healthy male C56BL / 6J mice weighing 25 g and aged 8 weeks were selected and randomly divided into lower limb control group, tail control group, sepsis acute kidney injury model group, bilateral lower limb distal ischemia preconditioning group and tail distal ischemia preconditioning group.

[0110] Step 2, induction of anesthesia;

[0111] Mice in both the bilateral lower limb and tail distal ischemic preconditioning groups were anesthetized with a slow intraperitoneal injection of 24% urethane (1.8 mg / kg). Mice in the sepsis-induced acute kidney injury model group, the lower limb control group, and the tail control group were not anesthetized.

[0112] Step 3, constructing an animal model;

[0113] A distal tail ischemia preconditioning group was established: after the mice in the distal tail ischemia preconditioning group were anesthetized, a tourniquet was used to tie the mouse tail to block the blood flow for 7 minutes, and then the tourniquet was released to restore the blood flow for 7 minutes (blocked blood flow time = restored blood flow time). This "ischemia-reperfusion" process was repeated for a total of 6 cycles; 18 minutes after the end of the ischemia-reperfusion cycle, 10 mg / kg of lipopolysaccharide (LPS) was injected into the mice intraperitoneally (the dosage standard was 110 μl / mouse).

[0114] A bilateral lower limb distal ischemia preconditioning group was established: after the mice in the bilateral lower limb distal ischemia preconditioning group were anesthetized, the lower limbs of the mice were tied with tourniquets and the blood flow was blocked for 7 minutes, and then the tourniquets were released to restore the blood flow for the same period of time. This "ischemia-reperfusion" process was repeated for 6 cycles; 18 minutes after the end of the ischemia-reperfusion cycle, the mice were injected with 10 mg / kg lipopolysaccharide (LPS) (the standard dosage was 110 ul / mouse) through the abdominal cavity.

[0115] A septic acute kidney injury model group was established: after the same duration as the distal tail ischemia preconditioning group, mice were intraperitoneally injected with lipopolysaccharide (LPS) of equal concentration and dosage (i.e., after the mice in the distal tail ischemia preconditioning group were anesthetized, lipopolysaccharide (LPS) was injected after a time T).

[0116] A tail control group was constructed: a tourniquet was placed on the mouse tail but not tied to maintain normal blood flow in the mouse tail; after the same duration as the distal tail ischemia preconditioning group, the mice were injected with an equal amount of normal saline through the abdominal cavity (i.e., after the mice in the distal tail ischemia preconditioning group were anesthetized, normal saline was injected after a time T, and the injection volume of normal saline (ul) was the same as the injection volume of lipopolysaccharide (LPS)).

[0117] A lower limb control group was constructed: a tourniquet was placed on both lower limbs of the mouse but not tied to maintain normal blood flow in the mouse tail; after the same duration as that of the distal tail ischemia preconditioning group, an equal amount of normal saline was injected into the mouse through the abdominal cavity (i.e., after the mice in the distal tail ischemia preconditioning group or the distal lower limb ischemia preconditioning group were anesthetized, normal saline was injected after time T, and the injection volume of normal saline (ul) was the same as the injection volume of lipopolysaccharide (LPS)).

[0118] When injecting lipopolysaccharide LPS, the specific injection method is as follows:

[0119] Grasp and secure the mouse with your left hand, with the abdomen facing upward and the head lower than the tail to avoid damage to internal organs. Disinfect the mouse's abdomen with an alcohol cotton ball and insert the syringe approximately 0.6 cm on either side of the mouse's linea alba. Push the needle 5 mm subcutaneously and then pierce the mouse's abdominal cavity at a 46° angle to the skin. A sense of empty space should be felt during insertion. If no liquid refluxes when the needle plug is withdrawn, slowly inject the drug solution. After the injection is complete, rotate the needle and slowly withdraw it to prevent liquid leakage.

[0120] Step 4: data collection and testing for model effect evaluation;

[0121] Eighteen hours after intraperitoneal injection of lipopolysaccharide (LPS) and normal saline, the mice were killed and their blood and kidney tissues were collected simultaneously.

[0122] After the collected blood is centrifuged, the supernatant is taken and the serum creatinine and serum urea nitrogen indicators are measured (the specific method for measuring serum creatinine and serum urea nitrogen indicators based on the supernatant can be directly applied to the existing technology without the need for creative work) to evaluate the renal function of mice in each group.

[0123] The collected kidney tissue is divided into three parts:

[0124] The first part is used to extract protein and RNA, detect renal tubular injury markers such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), and detect the expression levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (the specific method of using kidney tissue to detect the expression levels of renal tubular injury markers and inflammatory factors can be directly applied to existing technologies without the need for creative work):

[0125] When using kidney tissue to extract protein, the required tissue homogenate preparation conditions are as follows: place 28 mg of kidney tissue in a 4 ml grinding tube, add 580 ul of protein lysate, put in 2 4 mm stainless steel grinding balls and 4 3 mm stainless steel grinding balls, place in a high-speed tissue grinder at 6°C for grinding, the grinding conditions are 68 Hz, 70 s, and grind 4 times.

[0126] When using kidney tissue to extract RNA, the required tissue homogenate preparation conditions are as follows: place 18 mg of kidney tissue in a 5 ml grinding tube, add 370 ul of lysis buffer, put in 2 4 mm stainless steel enzyme-free grinding balls and 4 3 mm stainless steel enzyme-free grinding balls, and place in a high-speed tissue grinder at room temperature for grinding. The grinding conditions are 68 Hz, 70 s, and grinding 4 times.

[0127] The second part is used to prepare paraffin sections, which are then stained with hematoxylin and eosin (H&E) to score renal tubular injury and assess the pathological damage of the renal tubules (the specific methods for scoring renal tubular injury and assessing renal tubular pathological damage using renal paraffin sections can be directly applied to existing technologies without requiring any creative work);

[0128] The third part is used to prepare kidney frozen sections by OCT embedding, and evaluate the expression level of renal reactive oxygen species (ROS) by dihydroethidium (DHE) staining (among which, the specific method of evaluating the expression level of renal reactive oxygen species using kidney frozen sections can be directly applied to existing technology without the need for creative work).

[0129] Step 5, analysis and comparison;

[0130] First, the sepsis acute kidney injury model group was compared with the tail control group and the lower limb control group to analyze the serum creatinine and serum urea nitrogen levels; if the serum creatinine in the sepsis acute kidney injury model group increased by more than 2 times (that is, the serum creatinine in the sepsis acute kidney injury model group was greater than 2 times the serum creatinine in the two control groups), and the statistical test showed that the difference between the two groups was statistically significant, then the sepsis acute kidney injury model group was successfully established; otherwise, the establishment failed;

[0131] The septic acute kidney injury model group was then compared with the bilateral lower limb distal ischemia preconditioning group and the tail distal ischemia preconditioning group, and the serum creatinine and serum urea nitrogen levels of the two groups were analyzed to verify the improvement effect on renal function of septic acute kidney injury; the RNA and protein expression levels of renal tubular injury markers and inflammatory factors were detected to see whether they were inhibited by distal ischemia preconditioning; combined with the renal pathological damage, renal tubular damage score, and renal reactive oxygen species expression level, it was determined whether distal ischemia preconditioning could reduce renal pathological damage and inhibit the expression level of renal ROS.

[0132] The model was constructed using the method of this embodiment. After testing and analysis, the model was successfully constructed, and the conclusion was that distal tail ischemia preconditioning and lower limb ischemia-reperfusion preconditioning can improve renal pathological damage and reduce renal tubular damage in septic acute kidney injury, inhibit renal oxidative stress in septic acute kidney injury, and reduce the inflammatory response in septic acute kidney injury; and lower limb ischemia-reperfusion preconditioning has similar protective effects on the kidneys as tail ischemia-reperfusion preconditioning.

[0133] Test example:

[0134] A method for establishing a mouse tail distal ischemic preconditioning renal protection model comprises the following steps:

[0135] Step 1: Select and group materials;

[0136] Thirty-six healthy male C56BL / 6J mice weighing 25 g and aged 8 weeks were purchased from Chengdu Jicui Pharmaceutical Co., Ltd. and randomly divided into tail control group, lower limb control group, sepsis acute kidney injury model group, bilateral lower limb distal ischemia preconditioning group and tail distal ischemia preconditioning group.

[0137] Step 2, induction of anesthesia;

[0138] Mice in the bilateral lower limb distal ischemic preconditioning group and the tail distal ischemic preconditioning group were anesthetized by a slow intraperitoneal injection of 25% urethane (1.75 mg / kg). Mice in the sepsis acute kidney injury model group, the lower limb control group, and the tail control group were not anesthetized.

[0139] Step 3, constructing an animal model;

[0140] In this experiment, two batches of animal models were constructed, each with three groups, for a total of six groups, each with six healthy mice. The two batches of animal models were: the first batch of animal models included a lower limb control group, a sepsis-induced acute kidney injury model group, and a bilateral lower limb distal ischemia preconditioning group; the second batch of animal models included a tail control group, a sepsis-induced acute kidney injury model group, and a tail distal ischemia preconditioning group. The construction methods of each group were as follows:

[0141] A distal tail ischemia preconditioning group (abbreviated as the tail ischemia group in the figure) was established: after the mice in the distal tail ischemia preconditioning group were anesthetized, a tourniquet was tied around the mouse tail to block blood flow for 5 minutes, and then the tourniquet was released to restore blood flow for 5 minutes. This "ischemia-reperfusion" process was repeated for a total of 4 cycles; 15 minutes after the end of the ischemia-reperfusion cycle, 10 mg / kg of lipopolysaccharide (LPS) was injected intraperitoneally into the mice (the dosage standard was 100 μL / mouse).

[0142] A bilateral lower limb distal ischemia preconditioning group (abbreviated as the lower limb ischemia group in the attached figure) was established: after the mice in the bilateral lower limb distal ischemia preconditioning group were anesthetized, the lower limbs of the mice were tied with a tourniquet and the blood flow was blocked for 5 minutes, and then the tourniquet was released to restore the blood flow for 5 minutes. This "ischemia-reperfusion" process was repeated for 4 cycles; 15 minutes after the end of the ischemia-reperfusion cycle, the mice were injected intraperitoneally with the same concentration and dosage of standard lipopolysaccharide LPS.

[0143] A sepsis-induced acute kidney injury model group (abbreviated as the model group in the attached figure) was established: after the same duration as the distal tail ischemia preconditioning group (i.e., 55 minutes after anesthesia), mice were intraperitoneally injected with lipopolysaccharide (LPS) of equal concentration and dosage.

[0144] A tail control group (abbreviated as the tail control group in the accompanying figure) was constructed: a tourniquet was placed on the mouse tail but not tied to maintain normal blood flow in the mouse tail; after the same duration as the distal tail ischemia preconditioning group (i.e., 55 minutes after anesthesia), the mice were injected with an equal amount of normal saline through the abdominal cavity (i.e., the injection volume of normal saline (ul) was the same as the injection volume of lipopolysaccharide (LPS) (ul)).

[0145] A lower limb control group (abbreviated as the lower limb control group in the attached figure) was constructed: a tourniquet was placed on the upper and lower limbs of the mouse but not tied to maintain normal blood flow in the mouse tail; after the same length of time as the distal tail ischemia preconditioning group (i.e., 55 minutes after anesthesia), the mice were injected with an equal amount of normal saline through the abdominal cavity.

[0146] When injecting lipopolysaccharide LPS, the specific injection method is as follows:

[0147] Grasp and secure the mouse with your left hand, with the abdomen facing upward and the head lower than the tail to avoid damage to internal organs. Disinfect the mouse's abdomen with an alcohol cotton ball and insert the syringe approximately 0.5 cm to either side of the mouse's linea alba. Push the needle 3-5 mm subcutaneously (the needle should be advanced based on a sense of empty space upon insertion). Then, insert the syringe into the mouse's abdominal cavity at an angle of approximately 45° to the skin, with a sense of empty space upon insertion. If no liquid refluxes when the needle plug is withdrawn, slowly inject the drug solution. After the injection is complete, rotate the needle and slowly withdraw it to prevent liquid leakage.

[0148] When constructing each group, pay attention to the blood flow of the corresponding mouse tails and lower limbs in the tail control group, lower limb control group, bilateral lower limb distal ischemia preconditioning group and tail distal ischemia preconditioning group at any time, and obtain the following: Figure 1 The chart shown.

[0149] Step 4: data collection and testing for model effect evaluation;

[0150] Sixteen hours after intraperitoneal injection of lipopolysaccharide (LPS) and normal saline, the mice were killed, and their blood and kidney tissues were collected simultaneously.

[0151] The collected blood was centrifuged, and the supernatant was taken to measure serum creatinine and serum urea nitrogen indicators to evaluate the renal function of mice in each group.

[0152] The collected kidney tissue is divided into three parts:

[0153] The first part is used to extract protein and RNA, detect renal tubular injury markers such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), and detect the expression levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α):

[0154] When using kidney tissue to extract protein, the required tissue homogenate preparation conditions are as follows: place about 30 mg of kidney tissue in a 2 ml grinding tube, add 500 ul of protein lysate, put in 1 4 mm stainless steel grinding ball and 2 3 mm stainless steel grinding balls, place in a high-speed tissue grinder at 4°C for grinding, the grinding conditions are 60 Hz, 60 s, and grind 4 times.

[0155] When using kidney tissue to extract RNA, the required tissue homogenate preparation conditions are as follows: place about 20 mg of kidney tissue in a 2 ml grinding tube, add 350 ul of lysis buffer, put in 1 4 mm stainless steel enzyme-free grinding ball and 2 3 mm stainless steel enzyme-free grinding balls, place in a high-speed tissue grinder at room temperature for grinding, the grinding conditions are 60 Hz, 60 s, and grind 4 times.

[0156] The second part was used to prepare paraffin sections, which were stained with hematoxylin and eosin (H&E) to score the renal tubular injury and evaluate the pathological damage of the renal tubules;

[0157] The third part was used to prepare kidney frozen sections by OCT embedding and evaluate the expression level of renal reactive oxygen species (ROS) by dihydroethidium (DHE) staining.

[0158] Step 5, analysis and comparison;

[0159] First, the sepsis acute kidney injury model group was compared with the tail control group and the lower limb control group, and the serum creatinine and serum urea nitrogen levels were analyzed. If the serum creatinine in the sepsis acute kidney injury model group increased by more than 2 times, and the statistical test showed that the difference between the two groups was statistically significant, the sepsis acute kidney injury model group was successfully established; otherwise, the establishment failed.

[0160] The septic acute kidney injury model group was then compared with the bilateral lower limb distal ischemia preconditioning group and the tail distal ischemia preconditioning group, and the serum creatinine and serum urea nitrogen levels of the three groups were analyzed and compared to verify the improvement effect on renal function of septic acute kidney injury; the RNA and protein expression levels of renal tubular injury markers and inflammatory factors were detected to see whether they were inhibited by distal ischemia preconditioning; combined with the renal pathological damage, renal tubular damage score, and renal reactive oxygen species expression level, it was determined whether distal ischemia preconditioning could reduce renal pathological damage and inhibit the expression level of renal ROS.

[0161] Test results:

[0162] 1. Blood flow velocity analysis:

[0163] After constructing each group, the blood velocity was compared between the tail control group and the tail distal ischemia preconditioning group, and between the lower limb control group and the bilateral lower limb distal ischemia preconditioning group. Figure 1 As shown in Figures A and C, there was no obvious blood flow through the tail and lower limbs of the mouse after the tourniquet was tied up. Figures B and D show that the blood flow rate in the tail and lower limbs of the untied mouse was faster, while the blood flow rate in the tail and lower limbs of the tied mouse was significantly reduced, and was about half of the blood flow rate when not tied up.

[0164] 2. Analysis of Renal Function and Renal Damage:

[0165] The blood and kidney tissues of mice in each group were analyzed for renal function and renal damage. Figure 2 、 Figure 3 The chart shown.

[0166] For the analysis of lower limb ischemia-reperfusion, Figure 2As shown; As can be seen from Figure B, the serum creatinine in the model group is more than 2 times that of the lower limb control group, so the sepsis acute kidney injury model group was successfully constructed; As can be seen from Figure C, in the sepsis acute kidney injury model group, the renal tubular epithelial cells were swollen and necrotic, vacuolar degeneration occurred, and the renal tubular lumen was dilated. Lower limb ischemia reperfusion preconditioning significantly alleviated the above-mentioned renal tubular pathological damage; As can be seen from Figure D, in the sepsis acute kidney injury model group, the mRNA expression levels of renal tubular injury indicators KIM-1 and NGAL were significantly increased, and lower limb ischemia reperfusion preconditioning significantly downregulated the expression levels of KIM-1 and NGAL mRNA. Therefore, Figure 2 It is proved that lower limb ischemia-reperfusion preconditioning can improve renal pathological damage and reduce renal tubular damage in septic acute kidney injury.

[0167] For the analysis of tail ischemia-reperfusion, e.g. Figure 3 As shown; As can be seen from Figure F, the serum creatinine in the model group is more than 2 times that of the tail control group, which once again proves that the sepsis acute kidney injury model group was successfully constructed; As can be seen from Figure G, in the sepsis acute kidney injury model group, the renal tubular epithelial cells were swollen and necrotic, vacuolar degeneration occurred, and the renal tubular lumen was dilated. The tail ischemia-reperfusion preconditioning significantly alleviated the above-mentioned renal pathological damage; As can be seen from Figure H, in the sepsis acute kidney injury model group, the mRNA expression levels of renal tubular injury indicators KIM-1 and NGAL were significantly increased, and the tail ischemia-reperfusion preconditioning significantly downregulated the expression levels of KIM-1 and NGAL mRNA. Therefore, Figure 3 It was demonstrated that tail ischemia-reperfusion preconditioning could improve renal pathological damage and reduce renal tubular damage in septic acute kidney injury.

[0168] 3. Analysis of oxidative stress and inflammation:

[0169] Oxidative stress and inflammation analysis were performed on the kidney tissues of mice in each group. Figure 4 、 Figure 5 The chart shown.

[0170] For the analysis of lower limb ischemia-reperfusion, Figure 4 As shown; As can be seen from Figure A, in the sepsis acute kidney injury model group, DHE staining of renal tissue showed that the renal oxidation level was significantly increased, and lower limb ischemia reperfusion preconditioning significantly inhibited the renal oxidative stress level. Lower limb ischemia reperfusion preconditioning can inhibit the renal oxidative stress of sepsis acute kidney injury; As can be seen from Figure BD, in the sepsis acute kidney injury model group, the expression levels of renal tissue inflammatory factors (IL-6, TNF-α) mRNA and protein were significantly increased, and lower limb ischemia reperfusion preconditioning significantly inhibited the expression level of renal inflammatory factors. Therefore, Figure 4It is proved that lower limb ischemia-reperfusion preconditioning can inhibit renal oxidative stress and reduce the inflammatory response of sepsis-induced acute kidney injury.

[0171] For the analysis of tail ischemia-reperfusion, e.g. Figure 5 As shown; Figure E shows that in the sepsis acute kidney injury model group, DHE staining of renal tissue showed that the renal oxidation level was significantly increased, and tail ischemia reperfusion preconditioning significantly inhibited the renal oxidative stress level, proving that tail ischemia reperfusion preconditioning can inhibit renal oxidative stress in sepsis acute kidney injury; Figure FG shows that in the sepsis acute kidney injury model group, the expression levels of renal tissue inflammatory factors (IL-6, TNF-α) mRNA and protein were significantly increased, and tail ischemia reperfusion preconditioning significantly inhibited the expression level of renal inflammatory factors. Therefore, Figure 5 It was demonstrated that tail ischemia-reperfusion preconditioning can inhibit renal oxidative stress and reduce the inflammatory response of sepsis-induced acute kidney injury.

[0172] In addition, Figure 4 The lower limb ischemia group and Figure 5 A comparative analysis was conducted between the lower limb ischemia-reperfusion group and the tail ischemia-reperfusion group. From the perspective of renal oxidative stress indicators, both lower limb ischemia-reperfusion preconditioning and tail ischemia-reperfusion preconditioning could inhibit renal oxidative stress in septic acute kidney injury; from the perspective of renal inflammation, both groups were significantly lower than the septic acute kidney injury model group. The above data prove that both lower limb ischemia-reperfusion preconditioning and tail ischemia-reperfusion preconditioning can inhibit renal oxidative stress and inflammatory response in septic acute kidney injury, that is, lower limb ischemia-reperfusion preconditioning and tail ischemia-reperfusion preconditioning have similar protective effects on the kidneys; however, compared with lower limb ischemia-reperfusion preconditioning, tail ischemia-reperfusion preconditioning causes less trauma to animals, is less difficult to operate and care for, is easier for animals to adapt and recover, and is less likely to develop serious complications, thereby reducing harm to experimental animals; in addition, tail ischemia-reperfusion can effectively reduce animal stress responses, reduce the interference of factors such as animal stress responses on experimental results, and can more stably and accurately simulate the remote ischemic preconditioning process. The results between different experiments are slightly different, which improves the stability and repeatability of the model and is more conducive to accurately evaluating the protective effect of remote ischemic preconditioning on the kidneys.

[0173] Analysis of test results:

[0174] In this study, we analyzed the effects of lower limb ischemia-reperfusion preconditioning and tail ischemia preconditioning on renal function (creatinine, urea nitrogen), oxidative stress, and inflammatory factors. We found that tail ischemia preconditioning significantly reduced serum creatinine and serum urea nitrogen in patients with septic acute kidney injury, improved renal function, and alleviated oxidative stress and inflammatory responses. Compared with existing lower limb ischemia preconditioning, it demonstrated comparable protective effects. However, lower limb ischemia-reperfusion preconditioning has certain drawbacks: first, it can cause significant trauma to the animal's abdomen / lower limbs, hindering subsequent survival and recovery; second, it can easily induce nerve damage, such as ischemic necrosis of nerves and paresthesia in the innervated area caused by tourniquet compression or surgical manipulation; third, the high requirements for operation and postoperative care increase the difficulty and workload of the experiment; and fourth, the severe trauma and unstable state of the animals result in poor experimental stability and reproducibility. By adopting the tail vascular model with less impact on animals, the interference of factors such as animal stress on the experimental results is reduced, and the remote ischemic preconditioning process can be simulated more stably and accurately. The results between different experiments are less different, which improves the stability and repeatability of the model and is more conducive to accurately evaluating the protective effect of remote ischemic preconditioning on the kidneys.

Claims

1. A method for constructing a mouse tail distal ischemic preconditioning renal protection model, characterized in that: The following steps are involved: Step 1: Select and group materials; Several healthy male mice were selected and randomly divided into a tail control group, a sepsis-induced acute kidney injury model group, and a tail distal ischemia preconditioning group. Step 2, induction of anesthesia; Mice in the distal tail ischemia preconditioning group were anesthetized by slowly injecting urethane intraperitoneally; mice in the sepsis acute kidney injury model group and the tail control group were not anesthetized. Step 3, constructing an animal model; The distal tail ischemia preconditioning group was established: After anesthetizing the mice in the distal tail ischemia preconditioning group, the tails were tied with a tourniquet to block blood flow for 4, 5, or 7 minutes, and then the tourniquet was released to restore blood flow for 4, 5, or 7 minutes. This "ischemia-reperfusion" process was repeated for 4 or 6 cycles. 15 or 18 minutes after the end of the ischemia-reperfusion cycle, lipopolysaccharide (LPS) was injected intraperitoneally at a dose of 90, 100, or 110 μL / mouse. The sepsis-induced acute kidney injury model group was established: after the same duration as the distal tail ischemia preconditioning group, mice were intraperitoneally injected with lipopolysaccharide (LPS) of equal concentration and dosage; A tail control group was constructed: a tourniquet was put on the mouse tail but not tied to maintain normal blood flow in the mouse tail; after the same length of time as the distal tail ischemia preconditioning group, the mice were injected with an equal amount of normal saline through the abdominal cavity.

2. The method for constructing a mouse tail distal ischemic preconditioning renal protection model according to claim 1, characterized in that: In step 2, the concentration of injected urethane is 15-25% urethane, and urethane is injected according to a dosage standard of 1.5-2.0 mg / kg.

3. The method for constructing a mouse tail distal ischemic preconditioning renal protection model according to claim 1, characterized in that: In step 3, when constructing the distal tail ischemia preconditioning group, the mouse tail was tied with a tourniquet to block blood flow for 4 minutes, and then the tourniquet was released to restore blood flow for 4 minutes. This "ischemia-reperfusion" process was repeated for 4 cycles. 15 minutes after the end of the ischemia-reperfusion cycle, lipopolysaccharide (LPS) of the same concentration and dosage was injected intraperitoneally.

4. The method for constructing a mouse tail distal ischemic preconditioning renal protection model according to claim 1, wherein: In step 3, a bilateral lower limb distal ischemia preconditioning group and a lower limb control group were also established; The distal ischemic preconditioning group was established: After the mice in the distal ischemic preconditioning group were anesthetized, their lower limbs were tied with tourniquets to block blood flow for 5 or 7 minutes, and then the tourniquets were released to restore blood flow for 5 or 7 minutes. This "ischemic-reperfusion" process was repeated for 4 or 6 cycles. 15 or 18 minutes after the end of the ischemic-reperfusion cycle, lipopolysaccharide (LPS) was injected intraperitoneally at a dose of 100 or 110 μL / mouse. A lower limb control group was constructed: a tourniquet was placed on both lower limbs of the mice but not tied to maintain normal blood flow in the tail of the mice; after the same length of time as the distal tail ischemia preconditioning group, the mice were injected with an equal amount of normal saline through the abdominal cavity.

5. The method for constructing a mouse tail distal ischemic preconditioning renal protection model according to claim 4, characterized in that: In step 3, when lipopolysaccharide (LPS) was intraperitoneally injected into the distal tail ischemia preconditioning group, the sepsis acute kidney injury model group, and the bilateral distal lower limb ischemia preconditioning group, the LPS concentration injected was 8-12 mg / kg.

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