Application of plasma-derived EVs after RIPC in preparation of medicine for preventing and / or treating kidney injury

Abstract

The invention provides application of plasma-derived EVs after RIPC in preparation of a medicine for preventing and / or treating kidney injury, and belongs to the technical field of biological medicine. The RIPC plasma-derived EVs provided by the invention can be used for preparing medicines for preventing and / or treating kidney injury caused by unilateral ureteral obstruction combined with pelvic lipopolysaccharide (LPS) injection. In-vivo and in-vitro experiments prove that compared with normal plasma-derived EVs, the plasma-derived EVs after RIPC shows a remarkable and better protection effect on U-L model kidney injury and LPS-induced HK-2 cell injury. Therefore, by taking the EVs from the plasma after RIPC as the active ingredient, the application has a wide prospect in developing a novel treatment medicine for treating the obstruction complicated with the inflammatory renal injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of plasma-derived EVs after RIPC in the preparation of drugs for the prevention and / or treatment of kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a common critical illness in clinical practice, with a high incidence rate and a strong correlation with increased patient mortality. During procedures such as percutaneous nephrolithotomy (PCNL) or diseases like sepsis, the kidneys may experience various pathological impacts, including obstruction and endotoxin exposure, leading to complex inflammation and apoptosis, ultimately resulting in renal failure. Currently, treatment for AKI is primarily supportive, lacking highly effective specific therapeutic drugs. Therefore, developing new renal protection strategies is of significant clinical importance.

[0003] Remote ischemic preconditioning (RIPC) is an endogenous protective phenomenon that induces protection for vital target organs such as the heart, brain, and kidneys by inducing transient, non-invasive ischemia-reperfusion circulation in distal organs such as limbs. Studies have shown that RIPC can reduce the incidence of acute kidney injury in patients after cardiac surgery, and its protective effect against renal ischemia-reperfusion injury (IRI) has also been confirmed in animal models. However, the renal protective mechanism of RIPC is very complex and has not yet been fully elucidated. The current mainstream view is that RIPC releases certain bioactive substances into the bloodstream from the site (e.g., limbs), and these substances reach the kidneys via the bloodstream, exerting protective effects through anti-inflammatory and anti-apoptotic mechanisms. However, the specific characteristics of these key protective substances and their delivery mechanisms in vivo have remained a bottleneck in this field of research. In recent years, extracellular vesicles (EVs) have received widespread attention as key carriers of intercellular communication. EVs are nanoscale membrane vesicles secreted by cells, carrying bioactive molecules such as proteins, nucleic acids, and lipids, which can be taken up by target cells, thereby regulating their function. Existing studies suggest that EVs may participate in the organ protection process of RIPC. For example, some literature reports that stem cell-derived EVs or EVs derived from local venous blood after pre-ischemia of specific organs have a protective effect against kidney injury. However, existing technologies have obvious limitations and unresolved issues: (1) Existing studies are mostly focused on EVs derived from specific cells (such as stem cells) or venous blood of specific organs, while there is a lack of clear and systematic verification on whether EVs derived from systemic circulating plasma (peripheral venous blood) after limb RIPC, which is simpler to operate and more universal in clinical practice, have a kidney-protective function. (2) Existing technologies are mostly focused on the classic renal ischemia-reperfusion injury (IRI) model, while the effectiveness of RIPC-derived EVs in kidney injury models that simulate the complex pathological process of obstruction combined with inflammation in surgeries such as PCNL is still unknown. (3) Existing technologies have not revealed whether there is an essential difference in the renal protective efficacy between plasma EVs after RIPC (RIPC-EVs) and normal plasma EVs (Nor-EVs). If Nor-EVs themselves have the same protective efficacy, then the RIPC process loses its key scientific value and necessity for application.

[0004] Currently, it remains unclear whether plasma-derived EVs after RIPC possess unique renal protective effects, whether their effects are significantly superior to plasma EVs under normal physiological conditions, and whether they can be effectively applied to novel renal injury models such as obstruction combined with inflammation. These are all core issues that urgently need to be addressed. Therefore, resolving these issues is crucial for the successful development of drugs based on RIPC-EVs. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the use of plasma-derived extracellular vesicles after remote ischemic preconditioning in the preparation of drugs for the prevention and / or treatment of kidney injury.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of plasma-derived extracellular vesicles after remote ischemic preconditioning in the preparation of drugs for the prevention and / or treatment of kidney injury.

[0007] Preferably, the kidney injury is kidney injury induced by unilateral ureteral obstruction combined with intrarenal pelvic lipopolysaccharide injection.

[0008] Preferably, the drug is administered via intravenous injection.

[0009] More preferably, the effective dose of the drug is 1 μg to 1000 μg of the extracellular vesicles per kilogram of body weight.

[0010] Preferably, the drug can inhibit lipopolysaccharide-induced apoptosis in human renal tubular epithelial cells.

[0011] More preferably, the effective dose of the drug is 1 × 10 5 Individual renal tubular epithelial cells were used with 0.1 μg to 50 μg of the extracellular vesicles.

[0012] The present invention provides a pharmaceutical composition for the prevention and / or treatment of kidney injury induced by unilateral ureteral obstruction combined with intraperitoneal lipopolysaccharide injection, the pharmaceutical composition comprising an effective dose of remotely ischemic preconditioning plasma-derived extracellular vesicles and a pharmaceutically acceptable carrier.

[0013] Preferably, the extracellular vesicles derived from plasma after remote ischemic pretreatment are obtained by ultra-high speed centrifugation from the plasma of individuals after remote ischemic pretreatment.

[0014] More preferably, the extracellular vesicles derived from plasma after the remote ischemic pretreatment express at least one of the marker proteins CD81, TSG101, and CD63.

[0015] More preferably, the peak particle size of the extracellular vesicles derived from plasma after remote ischemic pretreatment is 100 nm to 150 nm.

[0016] Compared with the prior art, the present invention has the following advantages: (1) This invention is the first to discover and confirm that plasma-derived extracellular vesicles (RIPC-EVs) following remote ischemic preconditioning (RIPC) can be used as active ingredients in the preparation of drugs for the prevention and / or treatment of specific types of kidney injury. This discovery clarifies RIPC-EVs from an endogenous substance into a drug with a defined therapeutic function, providing a novel approach to material selection and drug development for clinical treatment. This invention validated the efficacy of RIPC-EVs in a novel unilateral ureteral obstruction combined with intrapelvic lipopolysaccharide (UL) kidney injury model. Experimental results showed that, compared to the model group (UL), the RIPC-EVs treatment group (U-L+RIPC-EVs) significantly reduced serum creatinine (Scr) and blood urea nitrogen (BUN) levels and significantly alleviated the renal tissue pathological injury score. This demonstrates that RIPC-EVs have a clear therapeutic effect on complex kidney injuries commonly seen in clinical practice, driven by both obstruction and infection factors.

[0017] (2) The RIPC-EVs of this invention exhibit multiple protective effects. Experimental results show that they can significantly reduce neutrophil infiltration in renal tissue (reduction in the number of MPO-positive cells) and the level of the inflammatory factor IL-6 in plasma. Simultaneously, RIPC-EVs can significantly reduce the number of apoptotic cells in renal tissue (reduction in the number of TUNEL-positive cells). In cell experiments, RIPC-EVs can reduce the apoptosis rate of HK-2 cells, regulate the Bcl-2 / Bax protein ratio, and downregulate cleaved-caspase 3 expression. In the HK-2 cell model treated with LPS, it can significantly promote cell proliferation (increase in the number of EdU-positive cells). This invention also found that the protective effect of RIPC-EVs is significantly better than that of EVs derived from normal plasma (Nor-EVs). Comparative experimental data clearly show that in the UL model, there are no statistically significant differences in various indicators between the Nor-EVs treatment group (U-L+Nor-EVs) and the model group (UL), indicating that Nor-EVs are essentially ineffective. The RIPC-EVs treatment group (U-L+RIPC-EVs) showed significantly better results than the model group and the Nor-EVs group in all assessment indicators. This result demonstrates that RIPC pretreatment is a necessary condition for endowing plasma EVs with strong renal protective function, and its effect is not inherent in all plasma EVs.

[0018] (3) Experiments of this invention have confirmed that EVs extracted from the plasma of healthy individuals (including rats and humans) treated with RIPC also have a protective effect against kidney damage in another individual. This indicates that RIPC-EVs have low immunogenicity and good biocompatibility, laying a solid foundation for their development as drugs and avoiding the cumbersome and costly process of personalized preparation from autologous sources. Attached Figure Description

[0019] Figure 1 The diagrams are animal experimental models. A in the diagram is a model of a rat model of kidney injury caused by unilateral ureteral obstruction combined with intra-ureteral lipopolysaccharide injection (UL). B is a model to explore the protective effect of RIPC-EVs on kidney injury in the UL model. Figure 2 To construct the rat UL model, Figure A shows the actual operation diagram of the rat UL model, and Figure B shows that LPS can be successfully injected into the rat renal pelvis through the ureter; Figure 3 To establish an animal model of pre-ischemic limbs in rats, Figure A shows Doppler blood flow imaging of the rat hind limbs before ischemia (top image) and Doppler blood flow imaging of the hind limbs after ischemia (bottom image). Figure B shows the assessment by laser Doppler ultrasound. Figure C shows the comparison of blood flow velocity before and after ischemia in the rat hind limb animal model, compared with the control group. p<0.001; Figure 4 To illustrate the renal tissue damage and inflammatory cell infiltration in different treatment groups, Figure A shows PAS staining of renal tissue in different treatment groups, Figure B shows the expression of NGAL in renal tissue in different treatment groups detected by immunohistochemistry, Figure C shows the infiltration of MPO-positive cells in renal tissue in different treatment groups detected by immunohistochemistry, and Figure D shows the expression of TUNEL-positive cells in renal tissue in different treatment groups detected by immunohistochemistry. Sham represents the sham-operated group, UL represents the renal injury model group with unilateral ureteral obstruction combined with intra-renal lipopolysaccharide injection, and RIPC+UL represents the renal injury model group with RIPC+unilateral ureteral obstruction combined with intra-renal lipopolysaccharide injection. The field of view was the renal tubular cortex, with 9 rats in each group and a magnification of 20×. Figure 5 The UL model can induce kidney injury in rats, and lower limb RIPC can exert a nephroprotective effect. Figure A shows the comparison of serum creatinine (Scr) levels in rats of different treatment groups; B shows the comparison of blood urea nitrogen (BUN) levels in different treatment groups; C shows the comparison of plasma inflammatory factor IL-6 levels in different treatment groups; D shows the comparison of plasma inflammatory factor TNF-α levels in different treatment groups; E shows the comparison of kidney tissue injury scores in different treatment groups; F shows the comparison of NGAL expression levels (integrated optical density value, IOD value, semi-quantitative staining intensity) in kidney tissue in different treatment groups; G shows the comparison of MPO-positive cell infiltration in different treatment groups; H shows the expression of TUNEL-positive cells in kidney tissue in different treatment groups; 9 rats were in each group. p<0.05, p<0.01; Figure 6For the identification of extracellular vesicles in plasma, A in the figure shows the morphological characteristics of EVs from plasma in the control group observed by transmission electron microscopy (TEM), B shows the particle size distribution of EVs from plasma in the control group detected by nanoparticle tracking analysis (NTA), C shows the morphological characteristics of EVs from plasma in the RIPC group observed by transmission electron microscopy (TEM), D shows the particle size distribution of EVs from plasma in the RIPC group detected by nanoparticle tracking analysis (NTA), and E shows the detection of CD81, TSG101, and CD63 marker proteins in EVs by Western blotting. Figure 7 To observe the uptake of PKH26-labeled EVs by kidney tissue cells using fluorescence microscopy, 0h in the figure represents the fluorescence result of EVs injected via the tail vein without PKH26 staining, and 24h represents the fluorescence result of PKH26-labeled EVs injected into rats 24 hours later; red represents PKH26-labeled EVs; magnification 20×. Figure 8 To illustrate how RIPC-derived EVs from rat plasma improve the degree of kidney injury and local inflammation in the UL model, Figure A shows PAS staining of rat kidney tissue from different treatment groups; B shows the expression of NGAL in rat kidney tissue from different treatment groups; C shows the infiltration of MPO-positive cells in rat kidney tissue from different treatment groups; and D shows the expression of TUNEL-positive cells in rat kidney tissue from different treatment groups. Sham represents the sham-operated group; UL represents the unilateral ureteral obstruction combined with intra-renal pelvic lipopolysaccharide injection kidney injury model group; U-L+Nor-EVs represents U-L+control group rat plasma-derived EVs; and U-L+RIPC-EVs represents UL group + RIPC group rat plasma-derived EVs. Each group consisted of 6 rats; magnification was 20×. Figure 9 This figure shows the results of how plasma-derived EVs improved the degree of kidney injury in a UL model after RIPC in rats. Figure A compares serum creatinine levels in different treatment groups; B compares kidney tissue injury scores in different treatment groups; C shows the expression of the inflammatory factor IL-6 in rat plasma in different treatment groups; D shows the expression of the inflammatory factor TNF-α in rat plasma in different treatment groups; E compares kidney tissue injury scores in different treatment groups; F compares the expression level of NGAL (semi-quantitative staining intensity based on integrated optical density) in kidney tissue in different treatment groups; G shows the infiltration of MPO-positive cells in the kidney tissue of the four groups of rats; and H shows the expression of TUNEL-positive cells in the kidney tissue of rats in different treatment groups. Each group consisted of 6 rats. p<0.05, p<0.01, p<0.001, # p<0.05 compared with the Sham group; Figure 10To compare the viability counts of HK-2 cells under different concentrations of LPS intervention, p<0.001 compared with LPS 0 μg / ml; Figure 11 The figure shows the effects of LPS intervention on HK-2 cell apoptosis at different concentrations. Figure A shows the apoptosis of HK-2 cells 24 hours after LPS intervention at different concentrations, as detected by flow cytometry. Figure B shows the effect of LPS intervention at different concentrations on the apoptosis rate of HK-2 cells. p<0.001 compared with LPS 0 μg / ml; Figure 12 For the identification of extracellular vesicles derived from human plasma, Figure A shows the morphological characteristics of EVs derived from control group plasma observed by transmission electron microscopy (TEM), B shows the particle size distribution of EVs derived from control group plasma detected by nanoparticle tracking analysis (NTA), C shows the morphological characteristics of EVs derived from RIPC group plasma observed by transmission electron microscopy (TEM), D shows the particle size distribution of EVs derived from RIPC group plasma detected by nanoparticle tracking analysis (NTA), and E shows the detection of CD81, TSG101, and HSP70 marker proteins in EVs by Western blotting. Figure 13 To observe the uptake of PKH26-stained EVs by HK-2 cells using a fluorescence microscope; Figure 14 To detect the uptake of human plasma-derived EVs by HK-2 cells using flow cytometry, Figure A shows the fluorescence intensity at different time points detected by flow cytometry, and Figure B shows the uptake of EVs by HK-2 cells at different time points. Figure 15 To reduce LPS-induced apoptosis in HK-2 cells by human plasma-derived EVs in the RIPC group, Figure A shows the apoptosis of HK-2 cells in different treatment groups as detected by flow cytometry; B shows the comparison of apoptosis rates of HK-2 cells in different treatment groups; and C shows the expression levels of apoptosis-related proteins Bcl-2, Bax, and cleaved-caspas3 in HK-2 cells in different treatment groups as detected by Western blotting. Sham represents the control group, LPS represents the LPS 50 μg / ml intervention group, LPS+RIPC-EVs represents the LPS group + RIPC group human plasma-derived EVs, and LPS+Nor-EVs represents LPS + control group human plasma-derived EVs. p<0.01, p<0.001, #p<0.05, compared with the Sham group; Figure 16To reduce the inflammation level of HK-2 cells after LPS intervention in human plasma-derived EVs in the RIPC group, Figure A shows the expression of IL-6 in the supernatant of HK-2 cells in different treatment groups, and Figure B shows the expression of TNF-α in the supernatant of HK-2 cells in different treatment groups. p<0.05, p<0.01; #p<0.05, compared with the Sham group; Figure 17 To demonstrate the ability of human plasma-derived EVs in the RIPC group to promote the proliferation of HK-2 cells after LPS intervention, Figure A shows the proliferation of HK-2 cells in different treatment groups as detected by EDU, and Figure B shows the number of EDU-positive cells in different treatment groups. p<0.01, p<0.001; #p<0.05, compared with the Sham group. Detailed Implementation

[0020] This invention provides the application of plasma-derived extracellular vesicles obtained after remote ischemic preconditioning (RIPC) in the preparation of drugs for the prevention and / or treatment of kidney injury. The remote ischemic preconditioning (RIPC) described in this invention refers to one or more brief, non-invasive ischemia-reperfusion cycles performed on a limb (including but not limited to the upper or lower limb). The ischemia time can be 3-10 minutes, the reperfusion time can be 3-10 minutes, and the number of cycles can be 1-6. After the RIPC operation, blood is preferably collected within 0-60 minutes.

[0021] In this invention, the kidney injury is preferably kidney injury induced by unilateral ureteral obstruction combined with intrarenal pelvic lipopolysaccharide injection.

[0022] In this invention, the drug can be administered via various routes, including but not limited to intravenous injection and local perfusion. Preferably, the drug is administered via intravenous injection to achieve the distribution of the extracellular vesicles throughout the body and to target and accumulate in damaged kidney tissue. "Intravenous injection" refers to all intravenous injection methods, including but not limited to tail vein injection and sublingual vein injection for non-human mammals, such as rats and mice. For human patients, peripheral intravenous injection and central venous infusion can be used.

[0023] In this invention, the effective dose of the drug is preferably 1 μg to 1000 μg of the extracellular vesicles per kilogram of body weight. The effective dose referred to in this invention is the amount of extracellular vesicles (EVs) required to prevent and / or treat kidney damage. Based on extensive animal studies, the administration dose of the drug is 1 μg to 1000 μg of the extracellular vesicles per kilogram of body weight. It should be noted that this range covers all doses that have been validated as effective in animal models (such as rats). When applied to humans, standard drug development guidelines must be followed, and the starting dose for humans must be determined through scientific interspecies dose conversion. For example, the body surface area normalization method, which is recognized in the art, can be used for interspecies dose conversion to derive the equivalent dose for humans.

[0024] In this invention, the drug can inhibit lipopolysaccharide-induced apoptosis of human renal tubular epithelial cells.

[0025] In this invention, the effective dose of the drug is 1 × 10 5 Individual renal tubular epithelial cells are treated with 0.1 μg to 50 μg of the extracellular vesicles. In the cellular application of this invention, the effective dose refers to the amount of EVs used that significantly inhibits LPS-induced HK-2 cell apoptosis, reduces the release of inflammatory factors, and promotes cell proliferation.

[0026] This invention provides a pharmaceutical composition for the prevention and / or treatment of kidney injury induced by unilateral ureteral obstruction combined with intrarenal pelvic lipopolysaccharide injection. The pharmaceutical composition preferably comprises an effective dose of extracellular vesicles derived from remote ischemic preconditioning plasma and a pharmaceutically acceptable carrier. In this invention, the carrier includes, but is not limited to, PBS, physiological saline, glucose solution, etc. The pharmaceutical composition can be administered via parenteral routes such as tail vein injection or renal artery injection. Preferably, the protein concentration of EVs in the pharmaceutical composition can be adjusted as needed, for example, prepared as a suspension of 1 μg / μl for in vivo injection in animals.

[0027] In this invention, extracellular vesicles derived from plasma after remote ischemic pretreatment are obtained by ultracentrifugation. As a preferred embodiment, the steps for obtaining extracellular vesicles from plasma after remote ischemic pretreatment include plasma sample pretreatment and extracellular vesicle (EV) extraction. The plasma from the remotely ischemic pretreated individual is obtained by performing limb remote ischemic pretreatment (RIPC) on the individual, collecting blood immediately after the RIPC or within 10 minutes, and obtaining plasma by allowing it to stand and centrifuging. The individual includes experimental animals or humans. Taking rats as an example, the limb remote ischemic pretreatment (RIPC) uses a 4-cycle "5 minutes of bilateral limb ischemia / 5 minutes of reperfusion" protocol. The EVs successfully enriched by this invention are a pale yellow, translucent, adherent substance that is difficult to observe directly with the naked eye, rather than a large amount of white solid.

[0028] In this invention, the extracellular vesicles derived from plasma after remote ischemic pretreatment express at least one of the marker proteins CD81, TSG101, and CD63.

[0029] In this invention, the peak particle size of the extracellular vesicles derived from plasma after remote ischemic pretreatment is 100 nm to 150 nm. Extensive experiments have shown that not all EVs of all sizes possess equivalent renal protective activity. The 100 nm to 150 nm peak particle size range effectively enriches the most functional subpopulations of small vesicles, such as exosomes, induced by RIPC.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0032] Example 1: Application of plasma-derived EVs after RIPC in drugs protecting against kidney injury induced in a UL model. 1. Materials and Methods 1.1 Experimental Materials 1.1.1 Laboratory Animals This experiment used 8-week-old male Sprague-Dawley (SD) rats, weighing 180-225g, purchased from the Comparative Medicine Center of Yangzhou University, and classified as specific pathogen-free. They were housed in isolation at the Southeast University Animal Laboratory, with ample feed and drinking water provided. The ambient temperature was 24℃ and the humidity was 50%. The experiment was conducted one week after the rats were housed. This experiment was conducted in accordance with animal ethics guidelines. See the animal experiment diagram below. Figure 1 .

[0033] 1.2 Experimental Methods 1.2.1 Establish a stable rat model of unilateral ureteral obstruction combined with lipopolysaccharide injection induced by renal pelvis (UL kidney injury model). Rats were fasted for 8 hours and deprived of water for 4 hours before surgery. A 1% pentobarbital injection (50 mg / kg) was administered intraperitoneally. Vital signs such as heartbeat and respiration were monitored. After successful anesthesia, the rats were placed supine on a warming table, their limbs immobilized, and the abdomen prepared, disinfected with iodine, and draped with a sterile sheet. A midline abdominal incision was made one finger-width below the xiphoid process. Ophthalmic scissors were used to cut through the skin, muscle layer, and peritoneum to enter the abdominal cavity. A simple retractor made of four paperclips was used to open the abdominal cavity. A moistened cotton swab was used to move the intestines into the right abdominal cavity, and a moistened cotton ball was inserted to restrict intestinal movement and expand the field of vision in the left abdominal cavity. The left kidney was exposed, and the left ureter was carefully dissected, noting the accompanying blood vessels. The ureter was ligated with a 5-0 absorbable suture at the point where it crossed the iliac vessels. Wait 5 minutes until the proximal ureter dilates slightly. Then, use a 34G needle to insert into the dilated ureter and inject LPS suspension (10mg / ml, 20mg / kg per animal). If significant dilation of the proximal ureter and renal pelvis is observed, and there is no obvious leakage from the surrounding tissue, the surgery is considered successful. Figure 2 A) The ureter was ligated again above the puncture point using 5-0 absorbable sutures. All cotton balls and other foreign objects in the abdominal cavity were removed. 1 ml of physiological saline was injected into the abdominal muscle using a syringe. The peritoneum and muscle layer of the rat were then sutured, followed by skin suturing. The rat was disinfected with povidone-iodine, kept warm, and revived. After recovery, the rat was returned to its cage and fed a normal diet. It was euthanized after 24 hours. During the initial modeling period, crystal violet staining solution was injected into the ureter. Subsequently, the left kidney was removed, and the renal pelvis was cut open along the largest section. The renal pelvis was found to be filled with crystal violet staining solution, indicating successful fluid injection into the renal pelvis. Figure 2 B). In the sham surgery group, the incision was sutured immediately after the left ureter was separated, without any treatment of the ureter, and the remaining steps were the same; subsequently, EVs from the plasma before and after RIPC were injected via the tail vein before modeling.

[0034] 1.2.2 Construction of a rat model of bilateral hind limb ischemia preconditioning After successful anesthesia of rats, tourniquets were applied to the proximal ends of both hind limbs for 5 minutes, and then the tourniquets were removed to restore perfusion for 5 minutes. (One cycle is 10 minutes, with a 5-minute ischemic cycle and a 5-minute reperfusion cycle, for a total of 4 cycles.) During this period, laser Doppler ultrasound technology was used to control the blood flow during ischemia to be reduced to about 20% of the baseline. In the sham surgery group, the surgical procedure can be to anesthetize the animals without applying tourniquets, and then proceed with the next step after the same anesthesia time.

[0035] 1.2.3 Collect blood and tissue specimens Rats were euthanized 24 hours post-surgery, and specimens were collected. The rats were anesthetized, the incision was disinfected with povidone-iodine, the sutures were removed, and the abdominal cavity was opened layer by layer to expose it. The intestines were gently pushed open with a moistened cotton ball, which was then used to fix the intestines in place, fully exposing the inferior vena cava. Blood was drawn from an EDTA blood collection tube (purple tube) connected to a lancet, keeping the lancet stable and the tip as close to the proximal end as possible, collecting a sufficient blood sample. Typically, 5-8 ml of blood was collected from each rat. The blood sample was allowed to stand at room temperature for 10 minutes, then centrifuged (3000 rpm, 4°C) for 10 minutes. The supernatant plasma was then aspirated using a micropipette and transferred to a pre-chilled EP tube at 4°C and frozen at -20°C for use in rat biochemical assays. Care was taken to draw blood gently, avoiding pressure to prevent hemolysis. If the supernatant was pale red, it should be discarded. Because only a small amount of inferior vena cava blood can be drawn from each rat, and individual differences are significant, this experiment adopted an equal-volume mixing method to reduce error. Specifically, 20 rats were selected and randomly divided into a control group and a RIPC group, with 10 rats in each group. Equal volumes of plasma from each group were mixed to form one sample; that is, whole blood was drawn immediately after the RIPC procedure from 10 rats, and the plasma was mixed into one sample after centrifugation. The EVs extracted from this sample were designated as the RIPC-EVs sample. Nor-EVs samples were extracted using the same method. The extracted samples were stored at -80°C and used in a concentrated manner to avoid repeated freeze-thaw cycles.

[0036] After collecting blood samples, 2 ml of high-concentration chloral hydrate was rapidly injected into the peritoneal cavity of the rat. Once death was confirmed, the left kidney was quickly removed and placed on ice. The perirenal tissue was dissected, and the tissue surface was rinsed with pre-cooled PBS. The kidney tissue was then evenly divided into two parts along the largest cross-section. Part of the kidney tissue was placed in cryovials and transferred to a -80°C freezer for cryopreservation. The remaining kidney tissue was fixed and preserved in 4% paraformaldehyde solution. Immunohistochemical analysis was performed on the tissue samples, and biochemical parameters of the blood samples were measured.

[0037] 1.2.4 Extraction of extracellular vesicles (EVs) (1) Extraction: Extract rat inferior vena cava blood according to steps 1.2.3. Use EDTA blood collection tubes to collect 5-8 ml of whole blood. Let it stand at 4℃ for 10 minutes, then centrifuge at 3000 rpm (10 minutes, 4℃). Transfer the supernatant plasma to a centrifuge tube and discard the hemolyzed sample (pink supernatant). Keep the pipette tip 5 mm above the layer to avoid aspirating platelets. Then perform gradient centrifugation steps: 2000g (20 minutes, 4℃) and 10000g (30 minutes, 4℃). Use a 20 ml syringe to aspirate the supernatant after centrifugation and filter it through a 0.22 μm pore size filter membrane to remove large particles. Place the supernatant sample into an ultracentrifuge tube, level it to 0.001 g, and centrifuge at 110000g (1 hour, 4℃). Resuspend the precipitate with PBS (filtered) at 110000g (1 hour, 4℃). Aspirate the liquid from the tube and use 100 μl of PBS. The precipitate that adheres to the tube wall after PBS (filtered) resuspension is mostly pale yellow. After resuspension in PBS, it is confirmed to be EVs by testing. The extracted EVs samples are stored at -80°C to avoid repeated freeze-thaw cycles.

[0038] (2) Identification: The morphology of the extracted EVs samples was observed by transmission electron microscopy, the particle size distribution and concentration were determined by nanoparticle tracking analysis, and the expression of EVs marker proteins (CD81, TSG101, CD63) was detected by WB method.

[0039] 1.2.5 Fluorescently labeled EVs The extracted EVs were fluorescently labeled (red light) using the PKH26 kit.

[0040] 1.2.6 Measurement of EV protein concentration in rat plasma The concentration of EVs protein derived from rat plasma was measured using BCA.

[0041] 1.2.7 EVs administered via tail vein injection to rats Add filtered PBS to the EVs (PKH26 labeled for tissue uptake experiments) for quantitative resuspension to 1 μg / μl. RIPC-EVs are derived from inferior vena cava plasma collected immediately after RIPC in rats. Anesthetize the rats and wipe them back and forth with alcohol swabs until the tail vein is dilated and clearly visible. Tightly fix the rat's tail, adjust the bevel of the 1ml syringe needle to face upwards, and carefully remove air bubbles. During injection, insert the needle horizontally, tighten the tail with your left hand, and choose the distal 1 / 3 of the tail for insertion, as the skin is thin there and entry into the blood vessel is relatively easy. If the needle is in the blood vessel, a small amount of blood return can be seen at the tip of the syringe. If blood return is observed, inject slowly. After injection, press the wound with an alcohol swab. Pay attention to the infusion rate and inject slowly. The injection volume is 200 μl of EVs (200 μg) suspension per rat.

[0042] 1.2.8 In vivo tracking of EVs PKH-26-labeled EVs were injected into rats via the tail vein (200 μg EVs), and a UL model was immediately established. The rats were sacrificed 24 hours later, and frozen sections of rat kidney tissue were prepared to observe tissue uptake.

[0043] Set the cryostat temperature to -20℃, embed rat kidneys in OCT embedding medium, freeze rapidly, and then section; cut the frozen tissue into 8μm thick slices, attach them to glass slides, and immediately perform subsequent nuclear staining experiments; Add PBS to the frozen section and react for 10 minutes; blot dry with filter paper, fix with paraformaldehyde (4%) for 20 minutes; wash gently 3 times with PBS, blot dry with filter paper; add nuclear staining solution to the section, cover with a coverslip; transport in a dark box in the dark, and observe under a fluorescence microscope.

[0044] 1.3 Statistical Analysis Data were analyzed using SPSS (22.0) software and plotted using GraphPad Prism 8.0 software. Quantitative data were expressed as mean ± standard deviation. Normality was tested using the Shapiro-Wilk test, and paired t-tests were used for pairwise comparisons within each group. All statistical analyses were two-tailed tests, and p < 0.05 was considered statistically significant.

[0045] 2. Results Analysis 2.1 A rat model of remote ischemic preconditioning of both hind limbs was successfully established. A rat model of recurrent intrahepatic pulmonary embolism (RIPC) was established by tourniquet application to both hind limbs. Laser Doppler ultrasound was used to detect blood flow in the hind limbs before and after RIPC. The tourniquet tightness was adjusted to control blood flow reduction to approximately 20% of baseline during ischemia. Figure 3 A and 3B. Laser Doppler ultrasound assessment showed that, compared to the control group, the RIPC group had significantly reduced lower limb blood flow ( (p<0.001), see Figure 3 C. This study successfully established a stable rat hind limb RIPC model.

[0046] 2.2 Construction of a Kidney Injury Model (UL) and Study on the Kidney Protective Effect of Remote Ischemic Preconditioning PAS staining results of renal tissue: In the Sham group, the glomerular and tubular structures were basically normal, the morphology of the tubular epithelial cells was basically normal, there was no obvious brush border loss or defect in the lumen, no obvious dilation, and no obvious inflammatory cell infiltration in the interstitium. Compared with the Sham group, the UL group showed significant swelling of renal tubular cells, partial degeneration and necrosis, brush border loss, narrowing or dilation of the lumen, formation of casts in the lumen, and inflammatory cell infiltration in the interstitium. The renal tissue structure of the RIPC+UL group was relatively intact, the renal tubular epithelium showed mild edema, and the degeneration and necrosis were significantly reduced compared with the UL group. The brush border was relatively intact, the casts were reduced, some interstitial congestion was observed, and a small amount of inflammatory cell infiltration was present. Figure 4 A). For example Figure 4 As shown in Figure B, immunohistochemical detection of NGAL expression in kidney tissue revealed that the IOD value in the Sham group was significantly higher than that in the UL group, while the IOD value in the RIPC+UL group was significantly lower than that in the UL group. Figure 5 F). Myeloperoxidase (MPO) staining clearly showed the infiltration of neutrophils or monocytes in kidney tissue. MPO-positive cells were significantly increased in the kidney tissue of the UL group, and RIPC effectively reduced the infiltration of inflammatory cells in kidney tissue induced by UL. Figure 4 C). Terminal deoxynucleotidyl transferase dUTP nick-end marker (TUNEL) was used to detect apoptotic cells in the kidneys, and RIPC significantly reduced the number of apoptotic cells in the kidneys of the UL group. Figure 4 D).

[0047] like Figure 5 As shown: Compared with the Sham group, the UL group rats showed significantly more severe kidney damage, as evidenced by elevated levels of urea nitrogen and creatinine. Figure 5 A and Figure 5 B), Kidney tissue damage score ( Figure 5 E), expression of damage markers in kidney tissue, and significantly increased infiltration of inflammatory and apoptotic cells (E). Figure 5 G and Figure 5 H). This indicates that the UL model successfully induced unilateral kidney injury in rats. Bilateral hind limb RIPC significantly improved the degree of kidney injury in the UL model. Furthermore, plasma IL-6 and TNF-α levels in the UL group were significantly higher than those in the Sham group (H). Figure 5 C and Figure 5 (D) RIPC significantly reduced the level of IL-6 in the plasma of rats after UL modeling, suggesting that RIPC may exert a systemic anti-inflammatory effect in animal models.

[0048] 2.3 Extraction and identification of extracellular vesicles from rat plasma after remote ischemic preconditioning This invention has found that plasma and endothelial cells (EVs) extracted from the right renal vein of rats after pre-ischemia of the right kidney can protect against ischemia-reperfusion injury in allogeneic rat kidneys, while plasma without EVs has no protective effect. Based on this research, this invention isolates EVs from plasma in normal and RIPC groups and observes their effects in a ulcerative renal injury model.

[0049] Transmission electron microscopy (TEM) revealed cup-shaped, double-membrane vesicles in both samples, mostly around 120 nm in diameter. Figure 6 A and Figure 6 C). The nanoparticle tracking analyzer (NTA) detected that the vesicle size roughly followed a normal distribution, ranging from 30 to 300 nm. The peak value for the Normal group was 106.9 nm, and the peak value for the RIPC group was 127.0 nm. Figure 6 B and Figure 6 D). Western blot analysis of the pale yellow precipitate obtained by ultracentrifugation revealed positive expression of three EV surface marker proteins: CD81, TSG101, and CD63. Figure 6 E).

[0050] 2.4 In the UL model, damaged kidney tissue cells can take up extracellular vesicles. Fluorescence microscopy observation of frozen kidney sections, such as Figure 7 As shown, PKH26-labeled EVs (red) accumulated in damaged kidney tissue cells in the UL model 24 hours after tail vein injection, suggesting that EVs can be taken up by damaged kidney tissue cells, which may mediate the nephroprotective effect of RIPC.

[0051] 2.5 Extracellular vesicles derived from rat plasma after remote ischemic preconditioning have allogeneic renal protective effects. PAS staining under a light microscope revealed that the renal tissue in the U-L+RIPC-EVs group was structurally intact except for renal tubular dilation. Mild edema of the renal tubular epithelial cells was observed, with fewer necrotic portions, a small amount of brush border sloughing, and some interstitial edema. Compared with the UL group, the renal pathological changes in the U-L+RIPC-EVs group were significantly reduced, while the renal pathological changes in the U-L+Nor-EVs group were similar to those in the UL group. Figure 8 A). For example Figure 8 As shown in Figure B, NGAL expression in the renal tissue of the U-L+RIPC-EVs group was significantly lower than that in the UL group, while expression in the U-L+Nor-EVs group was similar to that in the UL group. Figure 8 C and Figure 8 As shown in Figure D, the number of MPO-positive cells and apoptotic cells in the U-L+Nor-EVs group was significantly lower than that in the UL group and the U-L+Nor-EVs group.

[0052] like Figure 9 ( Figure 9 A- Figure 9 As shown in Figure H), compared with the UL group, the U-L+RIPC-EVs group significantly reduced kidney damage, as evidenced by lower creatinine levels and a significantly lower renal tubular injury score. Further experiments showed that the expression of NGAL, MPO positivity, and the number of apoptotic cells were significantly lower in the U-L+RIPC-EVs group than in the UL group, while these indicators were not significantly different between the U-L+Nor-EVs group and the UL group.

[0053] In summary, this invention successfully isolated extracellular vesicles (EVs) from the plasma returning from the inferior vena cava of rats using ultracentrifugation, and identified the EVs based on morphology, particle size analysis, and surface markers. The EVs isolated by this invention conform to the general characteristics of extracellular vesicles, and no significant difference in particle size was found between the EVs extracted from the Normal group and the RIPC group. This invention injected EVs extracted from the plasma of rats in the Normal and RIPC groups into rats undergoing a ulnar vesicle (UL) model via the tail vein. The results showed that the EVs extracted from the RIPC group had a significant renal protective effect compared to the Normal group. Comparing the UL group and the U-L+Nor-EVs group, the RIPC-EVs injection group significantly reduced renal function (Scr), the degree of renal tissue damage (tissue damage score and NGAL expression), and the number of inflammatory cells in the renal tissue (MPO staining) and the number of apoptotic cells (Tunel staining). This invention, by using PKH26-labeled EVs, found that damaged renal tissue cells can take up EVs, further supporting the possibility that the renal protective effect of RIPC is mediated by EVs. By measuring the concentrations of IL-6 and TNF-α in rat plasma, it was found that RIPC and plasma-derived EVs after RIPC reduced IL-6 expression 24 hours after rat modeling, suggesting that RIPC may have weakened the systemic inflammatory response in rats.

[0054] Example 2: Application of plasma-derived EVs after RIPC in exerting anti-apoptotic effects on LPS-treated HK-2 cells. 1. Experimental Methods 1.1 Construction of LPS-intervention HK-2 cell damage model To ensure the accuracy and reproducibility of the experiment, cells in good condition and with a pebble-like shape were selected for model construction.

[0055] 1) LPS preparation: LPS was purchased from Sigma-Aldrich's L-2630. It is a powder. Add 25 ml of filtered PBS to the container, mix well, and make up to a volume of 1 mg / ml stock solution. Package into 25 vials. Store at -20℃ for long-term storage. When using, thaw and place at 4℃. Use within one week.

[0056] 2) Take logarithmically grown HK-2 cells and evenly spread them in a six-well plate. Usually, cells from a fully grown T25 culture flask can be divided into six wells. The next morning, change the medium and add 2 ml of complete culture medium to each well. Administer different final concentrations (0, 1, 10, 20, 50, 100 μg / ml) of LPS for 24 hours. Observe the cells and proceed with the next experiment.

[0057] 1.2 Cell counting and viability assessment 1) Prepare HK-2 cell suspensions with different concentrations of LPS intervention according to the digestion steps, and dilute them appropriately (10 cells / ml). 2) Mix trypan blue solution (0.4%) with cell suspension at a ratio of 1:9, take 10 μl and drop it onto a cell counting plate, complete the counting within three minutes, and count live cells and dead cells separately; observe under a light microscope, dead cells appear pale blue, while live cells are unstained. Count quickly to avoid false positives caused by prolonged staining; 3) Calculate cell viability using the formula: viable cell rate (%) = total number of viable cells / (total number of viable cells + total number of dead cells) × 100.

[0058] 1.3 Annexin V-FITC / PI staining combined with flow cytometry to detect apoptosis Staining was performed using an apoptosis kit from Jiangsu Kaiji Biotechnology Co., Ltd., and detection was performed using an Accuri C6 flow cytometer from the Clinical Sample Bank of Zhongda Hospital Affiliated to Southeast University. 1.4 Plasma collection and EV extraction before and after RIPC in healthy volunteers 1.4.1 Plasma collection before and after RIPC in healthy volunteers This embodiment included 9 healthy volunteers. 2ml of plasma was extracted from the plasma of each of the 9 volunteers before and after RIPC and mixed into two separate samples: control plasma (18ml) and plasma after RIPC (18ml). The EVs extraction step was started immediately after extraction.

[0059] 1.4.2 Extraction and Identification of Extracellular Vesicles (EVs) Relatively pure EVs were obtained from human plasma using ultracentrifugation. The steps are as follows: First, perform gradient centrifugation: 2000g (20 minutes, 4℃), 10000g (30 minutes, 4℃). Use a 20ml syringe to draw up the supernatant after centrifugation and filter it through a 0.22μm pore size filter to remove large particles. The filtered sample was placed in an ultracentrifuge tube, balanced to 0.001 g, and centrifuged at 110,000 g (1 hour, 4°C). The precipitate was resuspended in filtered PBS and centrifuged at 110,000 g (1 hour, 4°C). The liquid in the tube was aspirated, and the precipitate adhering to the tube wall was resuspended in 100 μl of filtered PBS, which was mostly pale yellow. During the experiment, it was repeatedly found that no precipitate appeared on the tube wall after ultracentrifugation. Therefore, this invention marks the possible precipitate locations on the tube wall beforehand. After resuspension in PBS, the precipitate was confirmed to be EVs. The extracted EVs samples were stored at -80°C to avoid repeated freeze-thaw cycles. The two samples were labeled Nor-EVs and RIPC-EVs, respectively. The extracted EVs samples were observed for morphology using transmission electron microscopy, and the particle size distribution and concentration were determined by nanoparticle tracking analysis. The expression of EVs marker proteins was detected using Western blotting.

[0060] 1.4.3 Fluorescently labeled EVs The extracted EVs were fluorescently labeled (red light) using the PKH26 kit.

[0061] 1.5 HK-2 cells’ in vitro uptake and co-culture of human plasma-derived EVs HK-2 cells were cultured in T25 flasks. When the cells reached a confluence of more than 80%, they were seeded into six-well plates. The complete culture medium used at this time was prepared by removing exosomes from FBS. Human plasma-derived EVs were labeled with PKH-26 and EVs (20 μg) were added to each well for co-culture.

[0062] 1) The labeling procedure for EVs with PKH26 staining solution is the same as in 1.4.3; 2) The labeled EVs (20 μg) were co-cultured with HK-2 cells for 12 hours. In addition, the HK-2 group with unstained EVs was used as a negative control, and the HK-2 cells labeled with PKH26 were used as a positive control. 3) Digest the cells, adjust the cell count to the order of 1 million, add 400 μl of Binding Buffer and mix well. Analyze the cell fluorescence intensity quantitatively using flow cytometry within 1 hour to determine the cell uptake of EVs. 4) In advance, seed HK-2 cells onto cell slides, repeat steps 1 and 2, and co-culture for 24 hours. Discard the supernatant, wash with PBS, and fix with paraformaldehyde (4%) for 20 minutes. Wash gently with PBS 3 times and blot dry with filter paper. Add nucleoside staining reagent to the slides, cover with coverslips, transport in the dark, and observe under a fluorescence microscope.

[0063] The results of HK-2 cell uptake of human plasma-derived EVs showed that after 12 hours, the vast majority of EVs were taken up by the cells. Therefore, the subsequent co-culture time of HK-2 cells and EVs was chosen to be 12 hours.

[0064] 1.6 Experimental grouping and procedure for the effect of two groups of EVs on LPS intervention in HK-2 cells HK-2 cells were randomly divided into 4 groups (n=3): Sham group: HK-2 cells were cultured alone; LPS treatment group (LPS): 50 μg / ml LPS was applied for 24 hours; LPS + RIPC-derived EVs group (LPS + RIPC-EVs): RIPC-EVs (20 μg) were added 12 hours before LPS addition and incubated, followed by 50 μg / ml LPS treatment for 24 hours; LPS + control group-derived EVs group (LPS + Nor-EVs): Nor-EVs (20 μg) were added 12 hours before LPS addition and incubated, followed by 50 μg / ml LPS treatment for 24 hours. The supernatant of HK-2 cells from the above four groups (2 ml of complete culture medium was added to each group) was collected, and IL-6 and TNF-α in the cell supernatant were measured.

[0065] 1.7 EdU staining fluorescence assay for HK-2 cells 1) Add 2 ml of glycine (2 mg / ml) to the well, incubate on a shaker for 5 minutes to neutralize paraformaldehyde, and discard the solution; 2) Wash with PBS for 5 minutes, then discard the PBS; 3) Add 100 μl of PBS containing 0.5% Triton X and incubate on a shaker for 10 minutes. Repeat step 2. 4) Add 100 μl of 1×Apollo, shake in the dark for 30 minutes, and discard the solution; 5) Wash twice with 100 μl of PBS containing 0.5% Triton X, 10 minutes each time, then discard the solution; 6) Nuclear staining: Wash the slide with PBS for 5 minutes each time, then drop the nuclear staining reagent into the center of the slide and react in the dark for 10 minutes; 7) Mounting: Wash 3 times with PBS, place the cells face down, and fix them on a glass slide using an anti-fluorescence extractant; 8) Observe and photograph under a fluorescence microscope.

[0066] 1.8 Statistical Analysis Data were analyzed using SPSS (22.0) software and plotted using GraphPad Prism 8.0 software. Quantitative data were expressed as mean ± standard deviation (x ± s). Normality was tested using the Shapiro-Wilk test, and paired t-tests were used for pairwise comparisons between groups. All statistical analyses were two-tailed tests, and p < 0.05 was considered statistically significant.

[0067] 2. Results Analysis 2.1 Remote ischemic preconditioning-induced extracellular vesicles reduce lipopolysaccharide-induced renal tubular epithelial cell damage By intervening with LPS in HK-2 cells to simulate the in vivo process of inflammation leading to kidney damage, the optimal concentration of LPS intervention was screened based on the viability and apoptosis level of HK-2 cells after intervention.

[0068] like Figure 10 As shown, HK-2 cells were treated with different concentrations of LPS for 24 hours. Trypan blue staining results showed that LPS concentrations below 20 μg / ml had no significant effect on HK-2 cell viability, while LPS concentrations of 50 μg / ml or higher significantly affected HK-2 cell viability.

[0069] like Figure 11 As shown, flow cytometry apoptosis experiments on HK-2 cells indicated that when the concentration of LPS reached or exceeded 50 μg / ml, the apoptosis rate of cells significantly increased, and the apoptosis rate increased significantly with increasing LPS concentration. Figure 11 A and Figure 11 B). Ultimately, 50 μg / ml was selected as the optimal intervention concentration for LPS.

[0070] 2.2 Extraction and identification of extracellular vesicles from human plasma after remote ischemic preconditioning EVs derived from human plasma were identified in the normal and RIPC groups. Transmission electron microscopy revealed that both groups of EVs exhibited a cup-shaped bilayer membrane structure. Figure 12 A and Figure 12 C). The nanoparticle tracking analyzer (NTA) detected that the vesicle size was approximately normally distributed, ranging from 30 to 300 nm. The peak value for the normal group was 99.1 nm, and the peak value for the RIPC group was 106.9 nm. Figure 12 B and Figure 12 D). Western blot analysis of the pale yellow precipitate obtained by ultracentrifugation revealed positive expression of three EV surface marker proteins: CD81, TSG101, and HSP70. Figure 12 E).

[0071] 2.3 HK-2 cells can take up EVs derived from human plasma. Human plasma-derived EVs labeled with PKH26 were added to HK-2 cells and co-cultured for 12 hours. Fluorescence microscopy revealed the accumulation of red material in the cytoplasm of HK-2 cells. Figure 13 This suggests that HK-2 cells can take up EVs derived from human plasma.

[0072] 2.4 Remote ischemic preconditioning-induced extracellular vesicles reduce lipopolysaccharide-induced renal tubular epithelial cell injury Flow cytometry was used to quantitatively analyze the uptake of HK-2 cells at different time points during co-incubation with EVs. The percentage of positively stained cells was measured at four time points: 3h, 6h, 9h, and 12h to determine the optimal co-incubation time. Figure 14 A and Figure 14 Results B showed that as the co-incubation time increased, the uptake rate of EVs by cells gradually increased, and after 12 hours of co-incubation, the cell uptake rate gradually stabilized at about 87%.

[0073] 2.5 Human plasma-derived EVs in the RIPC group were able to alleviate LPS-induced apoptosis in HK-2 cells. This invention extracts EVs from the plasma of healthy volunteers before and after RIPC using ultracentrifugation, adds them to HK-2 cells and incubates for 12 hours (protein quantification 20 μg), followed by intervention with 50 μg / ml LPS for 24 hours. Finally, cell apoptosis is detected by flow cytometry and Western blotting. Figure 15 A- Figure 15 The results showed that RIPC-induced EVs could reduce the degree of apoptosis in HK-2 cells.

[0074] 2.6 Human plasma-derived EVs in the RIPC group were able to reduce the inflammatory level of HK-2 cells after LPS intervention. EVs from human plasma before and after RIPC were added to HK-2 cells and incubated for 12 hours. Then, LPS was added to a final concentration of 50 μg / ml for 24 hours. Cell supernatants from four groups were collected, and the levels of IL-6 and TNF-α in the supernatant were measured using ELISA. Figure 16 A and Figure 16 Results showed that RIPC-induced EVs could reduce the inflammatory level of HK-2 cells.

[0075] 2.7 Human plasma-derived EVs in the RIPC group promoted the proliferation of HK-2 cells after LPS intervention. Following acute kidney injury, a hallmark of tissue repair is the significant proliferation of surviving HK-2 cells. EdU dye can enter replicating DNA, and the detection of DNA synthesis is one of the most accurate methods for detecting cell proliferation. To investigate the effect of RIPC-EVs on HK-2 cell proliferation, this invention uses EdU labeling to detect the proliferative capacity of HK-2 cells after LPS intervention. Figure 17 A and Figure 17 Results B showed that EVs derived from human plasma in the RIPC group could promote the proliferation of HK-2 cells after LPS intervention.

[0076] In summary, this invention demonstrates through cell experiments that RIPC-induced EVs can be taken up by HK-2 cells and alleviate LPS-induced cell damage. The mechanism may be that RIPC-EVs deliver systemic anti-inflammatory and anti-apoptotic bioactive substances. This invention uses an LPS intervention time of 24 hours, defining the approximate LPS concentration between 1-100 μg / ml. Cell viability was measured using a concentration gradient of 0, 1, 10, 20, 50, and 100 μg / ml, combined with trypan blue staining to determine cell viability and flow cytometry to detect apoptosis levels, to screen for the optimal concentration. Results showed that when the LPS concentration reached or exceeded 50 μg / ml, HK-2 cell viability significantly decreased and apoptosis levels increased with increasing LPS concentration. In subsequent experiments, this invention used 50 μg / ml as the LPS intervention concentration. This invention identified plasma-derived EVs from the RIPC group and the normal group. Transmission electron microscopy showed that both groups of EVs exhibited a cup-shaped bilayer membrane structure. NTA detected a peak particle size of 99.1 nm in the normal group and 106.9 nm in the RIPC group. Western blot analysis revealed the expression of three extracellular vesicle marker proteins, CD81, HSP70, and TSG101, in the precipitates after ultracentrifugation in both groups. This is similar to the particle size comparison of plasma-derived EVs between the rat RIPC group and the normal group in Example 1, where the particle size of human plasma-derived EVs in the RIPC group was also higher than that in the normal group.

[0077] This invention involves drawing blood immediately after intra-arterial plasma co-culture (RIPC) in the upper limb, extracting endothelial cells (EVs) from the plasma, and co-culturing them with HK-2 cells. The protective effect is expected to occur immediately, theoretically due to the delivery of endogenous proteins released after RIPC to target cells, producing anti-inflammatory and anti-apoptotic protective effects. This invention co-incubates human-derived plasma EVs before and after RIPC with HK-2 cells. Initially, it was found that HK-2 cells can take up EVs, and the uptake rate gradually increases with prolonged co-incubation time, gradually slowing down after 12 hours. Compared to EVs from the normal control group, EVs from the RIPC group significantly reduced apoptosis and inflammation levels in LPS-treated HK-2 cells and promoted their proliferation.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of plasma-derived extracellular vesicles after remote ischemic preconditioning in the preparation of drugs for the prevention and / or treatment of kidney injury.

2. The application according to claim 1, characterized in that, The kidney injury referred to was kidney injury induced by unilateral ureteral obstruction combined with intra-renal pelvic lipopolysaccharide injection.

3. The application according to claim 1, characterized in that, The drug is administered via intravenous injection.

4. The application according to claim 3, characterized in that, The effective dose of the drug is 1 μg to 1000 μg of the extracellular vesicles per kilogram of body weight.

5. The application according to claim 1, characterized in that, The drug can inhibit lipopolysaccharide-induced apoptosis in human renal tubular epithelial cells.

6. The application according to claim 5, characterized in that, The effective dose of the drug is 1×10 5 Individual renal tubular epithelial cells were used with 0.1 μg to 50 μg of the extracellular vesicles.

7. A pharmaceutical composition for preventing and / or treating renal injury induced by unilateral ureteral obstruction combined with intraperitoneal lipopolysaccharide injection, characterized in that, The pharmaceutical composition comprises an effective dose of remotely ischemic preconditioning plasma-derived extracellular vesicles and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The extracellular vesicles derived from plasma after remote ischemic pretreatment were obtained by ultra-high speed centrifugation from the plasma of individuals after remote ischemic pretreatment.

9. The pharmaceutical composition according to claim 8, characterized in that, The extracellular vesicles derived from plasma after remote ischemic preconditioning express at least one of the marker proteins CD81, TSG101, and CD63.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that, The peak particle size of the extracellular vesicles derived from plasma after remote ischemic pretreatment is 100 nm to 150 nm.

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