Use of multi-enzyme NANO complex in preparing drug for treating stroke and other cardiovascular diseases caused by abnormal lactic acid metabolism

Abstract

Provided is the use of a multi-enzyme nano complex in preparing a drug for treating stroke and other cardiovascular diseases caused by abnormal lactic acid metabolism. The active component of the multi-enzyme nano complex is a cascade system consisting of a flavin-type oxidase and a peroxidase. Further provided are the use of the multi-enzyme nano complex in preparing a drug for treating stroke and other cardiovascular diseases caused by abnormal lactic acid metabolism, and a pharmaceutical composition comprising the multi-enzyme nano complex. The multi-enzyme nano complex can simultaneously regulate abnormal metabolism of a focus area and scavenge free radicals, and thus has better treatment effects than a mode of simply scavenging free radicals. Stroke, acute liver injury and acute kidney injury animal experiments verify that the multi-enzyme nano complex can reduce lactic acid accumulation, significantly reduce the cerebral infarction volume of mice with stroke, improve behavioral functions, and also have protective effects on acute liver injury and acute kidney injury. Compared with existing therapeutic drug edaravone, simultaneously regulating the abnormal metabolism and scavenging free radicals is a new therapeutic strategy, which has wide application prospects.

Description

Application of multi-enzyme nanocomplexes in the preparation of drugs for treating stroke and other cardiovascular diseases with abnormal lactate metabolism Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a multi-enzyme nanocomplex for treating stroke and other cardiovascular diseases with abnormal lactate metabolism, a preparation method thereof, and use of the multi-enzyme nanocomplex in preparing drugs for treating stroke and other cardiovascular diseases with abnormal lactate metabolism. Background Art

[0002] When localized tissues and organs (commonly the brain, heart, liver, and kidneys) experience ischemia due to various causes, restoration of blood supply (i.e., reperfusion) exacerbates cellular metabolic dysfunction and structural damage, accompanied by lactate accumulation and a respiratory burst. During the ischemic period, insufficient oxygen supply prevents cells from performing normal oxidative phosphorylation, leading to increased anaerobic glycolysis and the production of large amounts of lactate. During reperfusion, restored intracellular oxygen supply increases the production of oxygen free radicals, exacerbating intracellular oxidative stress and causing cell apoptosis and necrosis, further leading to secondary damage to tissues and organs—a condition known as ischemia-reperfusion injury (IRI). IRI can lead to a range of serious clinical problems and is associated with high morbidity and mortality rates for various diseases, such as stroke, myocardial infarction, acute kidney injury, and acute liver injury. Stroke is a major threat to human health. According to the WHO, stroke ranked second among the leading causes of death or long-term, severe neurological illness worldwide over the past decade. It is characterized by high morbidity, recurrence, disability, and mortality, as well as a significant economic burden. Therefore, developing safe and effective treatment strategies has great clinical value and social significance.

[0003] For diseases related to ischemia-reperfusion injury, including stroke and other cardiovascular diseases with abnormal lactate metabolism, the current treatment measures mainly include: (1) improving hypoxic conditions: by increasing oxygen supply, promoting cellular oxidative phosphorylation and reducing lactate production; (2) cytoprotective agents: using free radical scavengers to inhibit oxidative stress response and reduce cell damage; (3) lactate scavenging therapy: using lactate scavengers or dialysis therapy to promote lactate excretion and improve lactic acidosis; (4) anti-inflammatory treatment: using anti-inflammatory drugs or cytokine antagonists to reduce inflammatory response. Among them, cell protection is a hot topic for researchers. At present, a series of drugs or nanomaterials have been developed to responsively downregulate the reactive oxygen species produced during reperfusion, reduce oxidative stress, reduce tissue damage, and achieve the purpose of treatment. For example, the main function of the clinically used drug molecule Edaravone (Edar) is to scavenge oxygen free radicals and reduce tissue oxidative stress damage; in addition, there are various nanomaterials, such as PNzyme / MnO2 nanozymes (Adv.Mater.2023,2210144) with superoxide dismutase (SOD) and catalase (CAT) catalytic activity, and peroxidase-like CeO2@ZIF-8 (Sci.Adv.2020,6,eaay9751), which remove reactive oxygen species in the lesion area through catalytic oxidation and protect cells from oxidative damage.

[0004] Because the microenvironment in the lesion area of ​​stroke and other cardiovascular diseases with abnormal lactate metabolism is very complex, satisfactory therapeutic effects are often not achieved by simply reducing oxidative stress. Normal metabolic activity is crucial for maintaining tissue microenvironmental homeostasis. As mentioned above, during the development of stroke and other cardiovascular diseases with abnormal lactate metabolism, tissues cannot undergo normal aerobic metabolism, resulting in a large accumulation of lactate, which in turn aggravates disease damage. Currently, there are no reports of drugs that can simultaneously degrade accumulated metabolic substrates and scavenge active free radicals. Therefore, designing and developing a multi-target therapeutic drug system that can simultaneously regulate abnormal metabolism and reduce tissue oxidative stress will be an effective means of treating stroke and other cardiovascular diseases with abnormal lactate metabolism, and has important clinical value. Summary of the Invention

[0005] The present invention is directed to the above-mentioned problems and provides a multi-enzyme nanocomplex that has the dual functions of simultaneously regulating abnormal lactate metabolism in diseased tissues and scavenging active free radicals. It also provides a method for preparing the multi-enzyme nanocomplex and its application in the preparation of therapeutic drugs for stroke and other cardiovascular diseases with abnormal lactate metabolism.

[0006] The technical route of the present invention is as follows: first, the synthesis path and catalytic performance of the multi-enzyme nanocomplex are explored and optimized, and then the mouse cerebral ischemia-reperfusion model (tMCAO), acute liver injury (ALI) and acute kidney injury (AKI) are used as examples to verify its in vivo therapeutic effect on stroke and other cardiovascular diseases with abnormal lactate metabolism. The results showed that after intravenous injection of the multi-enzyme complex, the cerebral infarction volume of stroke mice was significantly reduced, the motor behavior was significantly improved, and the mortality rate was also reduced. After intravenous injection of the multi-enzyme complex in mice with acute liver injury and acute kidney injury, the damaged liver tissue and kidney tissue were also significantly improved, indicating that the simultaneous regulation of lactate metabolism and reactive oxygen species through cascade enzyme catalysis has a significant therapeutic effect on cardiovascular diseases with abnormal lactate metabolism represented by stroke, acute liver injury and acute kidney injury.

[0007] Mechanistically, the flavin oxidases in the multi-enzyme complex of the present invention react specifically with metabolic substrates at the lesion site, reducing metabolite accumulation and regulating abnormal metabolism. Peroxidases, on the other hand, convert generated hydrogen peroxide and endogenous excess hydrogen peroxide into water and oxygen, reducing tissue oxidative stress and alleviating tissue damage. This dual-enzyme cascade, through its highly efficient catalytic action, simultaneously regulates abnormal metabolism and free radical scavenging efficiency in damaged tissues. Compared to current single free radical scavenging approaches, this dual regulatory approach offers a more pronounced therapeutic effect.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] In the first aspect, the present invention provides a multi-enzyme nanocomplex, the active components of which are a cascade system consisting of flavin oxidase or its analogs and peroxidase or its analogs, and also includes a carrier coating the active components to ensure the efficient catalytic activity of the cascade system.

[0010] Preferably, the flavin oxidase is selected from any one or more combinations of lactate oxidase, glutamate oxidase and monoamine oxidase; the peroxidase is selected from any one or more combinations of peroxidase and catalase; and the carrier is selected from any one of nanogel materials, albumin and liposomes.

[0011] More preferably, the concentration ratio of flavin oxidase to peroxidase is (20:1-1:100).

[0012] The second aspect of the present invention provides a method for preparing the multi-enzyme nanocomplex, which is as follows:

[0013] (1) When the carrier is a nanogel material, the multi-enzyme nanocomplex is assembled from flavin oxidase, peroxidase, functional monomer and polysaccharide monomer and then prepared by enzymatic free radical polymerization;

[0014] Preferably, the functional monomer is selected from any one of PEGMA, Lys-MA, Arg-MA, and TMAEMA; the polysaccharide monomer is selected from any one or more combinations of ethylene-functionalized gelatin, dextran, chitosan, chondroitin sulfate, and hyaluronic acid; and the initiator of enzymatic free radical polymerization is selected from the catalytic substrate of the corresponding flavin oxidase.

[0015] (2) When the carrier is albumin, the nanocomplex is assembled from flavin oxidase, peroxidase, alkenyl albumin and functional monomers and prepared by free radical polymerization;

[0016] Preferably, the functional monomer is selected from any one or more combinations of PEGMA, Lys-MA, Arg-MA, and TMAEMA.

[0017] (3) When the carrier is a liposome, the nanocomplex is constructed by self-assembly of the liposome, flavin oxidase, and peroxidase.

[0018] In the specific embodiments of the present invention, the multi-enzyme nanocomplexes LCgel and DL-LCgel prepared when the carrier is a nanogel material were characterized by morphology. The results showed that the LCgel and DL-LCgel particles were spherical, monodisperse, and had a particle size of approximately 50-200 nm (Figures 1 and 13); the results of the cytotoxicity experiment showed that when the LCgel concentration range was 0.01-0.5 mg / ml, the survival rate of fibroblasts NIH3T3 reached more than 90%, indicating that the multi-enzyme complex had no obvious toxicity to cells and had high biosafety (Figure 2).

[0019] The results of animal experiments showed that the multi-enzyme complex of the present invention can significantly reduce the volume of cerebral infarction after stroke in tMCAO mice and significantly improve the behavioral function of mice. It can also significantly improve the damaged liver and kidney tissues in mice with acute liver injury (ALI) and acute kidney injury (AKI), indicating that the multi-enzyme complex has a good therapeutic effect on stroke and other cardiovascular diseases with abnormal lactate metabolism.

[0020] Therefore, the third aspect of the present invention provides the use of a multi-enzyme nanocomplex in the preparation of a therapeutic drug for stroke and other cardiovascular diseases with abnormal lactate metabolism.

[0021] In the specific embodiments of the present invention, animal models of stroke, acute liver injury and acute kidney injury were used as test models to verify the therapeutic effects of the multi-enzyme nanocomplex on stroke, acute liver injury and acute kidney injury. Whether it is stroke or other cardiovascular diseases with abnormal lactate metabolism, they are accompanied by abnormal lactate metabolism and changes in reactive oxygen levels, leading to cell death and tissue damage. The flavin oxidase and peroxidase cascade system of the present invention can effectively degrade excess lactate in lesion tissues, downregulate reactive oxygen species, reduce cellular oxidative damage, and have protective and therapeutic effects on tissue damage in stroke and other cardiovascular diseases with abnormal lactate metabolism.

[0022] Preferably, the drug achieves combined treatment by regulating the cascade effect of abnormal metabolism in the lesion site and clearing reactive oxygen species.

[0023] In a fourth aspect, the present invention provides a pharmaceutical composition for treating stroke and other cardiovascular diseases with abnormal lactate metabolism, comprising an active ingredient and a medically acceptable excipient or diluent, wherein the active ingredient is the multi-enzyme nanocomplex as described above.

[0024] Preferably, the pharmaceutical composition is in the form of an injection. The drug can be prepared into any form of preparation that can be injected intravenously, such as an injection solution or sterile powder for injection.

[0025] The fifth aspect of the present invention also provides a composition for preparing an antioxidant health food that promotes the scavenging of lactic acid and free radicals.

[0026] The beneficial protection and effects of the present invention are as follows:

[0027] Compared with the prior art, the multi-enzyme nanocomplex combination of the present invention can simultaneously regulate abnormal lactate metabolism and free radical scavenging of damaged tissues in stroke and other cardiovascular diseases with abnormal lactate metabolism. The dual action has better therapeutic effects than a single free radical scavenging mode, can significantly reduce the volume of cerebral infarction in the ischemia-reperfusion mouse model, reduce tissue damage, improve behavioral function, and have a significant improvement effect on damaged liver and kidney tissues in mice. Compared with existing therapeutic drugs (such as edaravone), the use of biological enzyme cascade catalysis to simultaneously regulate metabolism and scavenge free radicals is a multi-target treatment strategy. In addition, the multi-enzyme of the present invention is a biological protein and has higher safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a TEM image of the multi-enzyme complex LCgel prepared in Example 1;

[0029] FIG2 is a cytotoxicity experiment of the multi-enzyme complex LCgel in Example 1;

[0030] FIG3 shows the in vitro lactic acid degradation (a) and reactive oxygen species scavenging experiments (b) of the multi-enzyme complex LCgel of Example 1;

[0031] FIG4 shows the blood flow monitoring results of the middle cerebral artery surface projection area in different treatment groups of tMCAO mice in Example 3, wherein a is the cerebral blood flow diagram of different treatment groups, and b is the statistical analysis result;

[0032] Figure 5 shows Nissl and HE staining (a) and statistical analysis results (b) of tMCAO mice in different treatment groups in Example 3;

[0033] FIG6 shows the TTC staining analysis results of tMCAO mice in different treatment groups in Example 3, wherein (a) is the brain slice image of the different treatment groups, and (b) is the statistical analysis result;

[0034] Figure 7 shows brain MRI scan images of tMCAO mice in different treatment groups in Example 3, where (a) and (b) are MRI scan images 24 hours and 72 hours after modeling, respectively; (c) is a comparison of infarct volumes in tMCAO mice at 24 hours and 72 hours; and (d) is a comparison of infarct volume changes in different treatment groups.

[0035] FIG8 shows the behavioral experimental results of tMCAO mice in different treatment groups in Example 3;

[0036] FIG9 is an analysis of the survival rates of tMCAO mice in different treatment groups in Example 3;

[0037] Figure 10 is an analysis of lactic acid content in brain tissue of tMCAO mice in different treatment groups in Example 3;

[0038] Figure 11 is an analysis of lactic acid content in liver tissues of ALI mice in different treatment groups in Example 8;

[0039] FIG12 shows the test results of liver function indicators in different treatment groups of ALI mice in Example 8;

[0040] FIG13 is a TEM image of the multi-enzyme complex DL-LCgel prepared in Example 10;

[0041] FIG14 is a cell experiment showing the scavenging of reactive oxygen species by the multi-enzyme complex DL-LCgel prepared in Example 10;

[0042] Figure 15 is an analysis of lactic acid content in kidney tissues of AKI mice in different treatment groups in Example 11;

[0043] FIG16 shows the renal function index test results of different treatment groups of AKI mice in Example 11. DETAILED DESCRIPTION

[0044] The present invention will now be described in detail with reference to the examples and accompanying drawings, but the present invention is not limited thereto. The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. While the examples do not include detailed descriptions of conventional methods, such methods are well known to those skilled in the art and are described in numerous publications.

[0045] Unless otherwise indicated, percentages and parts are calculated by volume. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0046] Example 1 Preparation and characterization of multi-enzyme complex LCgel

[0047] Using nanogels as immobilized carriers, the target multi-enzyme complex was prepared using a combination of lactate oxidase and catalase as an example. Lactate oxidase and catalase were dissolved in HEPES buffer along with 2% vinyl gelatin and PEGMA (0.1-2%). The mixture was passed through an SPG membrane, initiating polymerization. The resulting precipitate was centrifuged at 6000 rpm, washed, and finally dispersed in PBS buffer to produce the target multi-enzyme complex, LCgel.

[0048] Transmission electron microscopy (TEM) analysis of the resulting LCgel revealed spherical, porous, and monodisperse LCgel particles with a particle size of approximately 200 nm. NIH3T3 fibroblasts were incubated with LCgel at varying concentrations. As shown in Figure 2, the survival rate of NIH3T3 cells exceeded 90% within the 1-100 μg / ml concentration range, demonstrating that the multi-enzyme complex exhibits no significant cellular toxicity and exhibits high biosafety.

[0049] Example 2 In vitro lactate metabolism and ROS scavenging experiments of the multi-enzyme complex

[0050] In vitro lactate degradation and ROS scavenging experiments were conducted on the multi-enzyme complex LCgel prepared in Example 1. First, to simulate an ischemia-reperfusion microenvironment, a 50 mM lactic acid solution was prepared in PBS. Subsequently, LCgel at varying concentrations (0.25-2 mg / ml) was added. After a half-hour reaction, the residual lactic acid content in the solution was measured using a lactate detection kit, and the lactate clearance rate of the multi-enzyme complex LCgel was calculated. As shown in Figure 3a, the lactate clearance rate gradually increased with increasing concentration of the added material, demonstrating that the prepared multi-enzyme complex LCgel is capable of clearing excess lactate and can be used to regulate the accumulation of large amounts of lactic acid in tissues. A ROS scavenging experiment was also conducted, simulating an ischemia-reperfusion microenvironment. A 100μM hydrogen peroxide solution was prepared in PBS, and different concentrations of LCgel (0.25-2mg / ml) were added. The mixture was allowed to react for half an hour. The residual H2O2 content in the solution was measured using a H2O2 assay kit, and the ROS scavenging rate of LCgel was calculated. As shown in Figure 3b, the higher the LCgel concentration, the higher the ROS scavenging rate, demonstrating the LCgel's ability to scavenge excess ROS. This indicates that the prepared multi-enzyme complex, LCgel, can simultaneously scavenge excess lactate and reactive oxygen species, and is expected to have a positive therapeutic effect on cardiovascular diseases caused by abnormal lactate metabolism.

[0051] Example 3 Establishment and treatment of mouse ischemia-reperfusion model (tMCAO)

[0052] The animals used were healthy SPF-grade C57BL / 6 (C57) male mice, all purchased from Southern Model Organisms Science Co., Ltd. The mice were about 8 weeks old and weighed between 22 and 25 g.

[0053] Mice were randomly divided into three groups: (1) sham group, (2) saline group, (3) multienzyme complex group (LCgel, prepared in Example 1), and (4) edaravone group. Edar is a neuroprotective agent (free radical scavenger) that can scavenge free radicals and inhibit lipid peroxidation, thereby inhibiting oxidative damage to brain cells, vascular endothelial cells, and nerve cells. It is mainly used clinically to treat diseases such as cerebral infarction, cerebral hemorrhage, acute cerebral infarction, and cerebral arteriosclerosis. In this experiment, we used Edar as a control to explore the therapeutic effect of multienzyme complex LCgel on stroke.

[0054] The experiment simulated cerebral ischemia-reperfusion by 1-hour middle cerebral artery occlusion (tMCAO) followed by 72-hour reperfusion. Eight-week-old mice were induced with a gas mixture containing 2% isoflurane and maintained with 1% isoflurane. The mice were allowed to breathe spontaneously during the procedure. The skin in the middle of the neck was prepared, disinfected, and covered with a drape. A longitudinal incision was made along the midline of the neck in two layers to expose the common carotid artery, internal carotid artery, and external carotid artery. The small branches on the external carotid artery were freed and cut off with an electrocoagulation pen. The distal end of the external carotid artery was ligated with 6-0 surgical thread, and the proximal end of the common carotid artery was ligated with 4-0 surgical thread. A slipknot was tied in the middle with 6-0 surgical thread. A small incision was made above the slipknot, and then a suture was inserted. The suture was inserted from the external carotid artery to the internal carotid artery. It was stopped when resistance was encountered. At this time, the suture was inserted about 1.0-1.2 cm. The incision was covered with cotton soaked in saline. After 1 hour of ischemia, the suture was pulled out, the skin was sutured, disinfected, and lidocaine ointment was applied to the wound to complete postoperative analgesia.

[0055] The mice in the sham and tMCAO groups were intravenously injected with normal saline, the mice in the LCgel treatment group were intravenously injected with LCgel (dose of 2.5 mg / kg) immediately after reperfusion, and the mice in the Edar group were intravenously injected with edaravone (Edar, dose of 3 mg / kg) after reperfusion.

[0056] Example 4 Cerebral infarction volume in tMCAO mice after different treatments

[0057] The cerebral infarction condition of the experimental mice in Example 3 was analyzed.

[0058] Laser Speckle Blood Flow Monitoring: Mice were induced with a gas mixture containing 2% isoflurane and maintained at 1% isoflurane. The mice were allowed to breathe spontaneously during surgery. Before modeling, the black hair on the top of the head was shaved to expose the scalp. After disinfection with iodine, a 1.5 cm midline incision was made to expose the skull. Debris from the skull surface was removed with a wet cotton swab, and the skull was exposed to the monitoring area. Blood flow in the surface projection area of ​​the middle cerebral artery was monitored 10 minutes, 1 hour, 2 hours, 7 hours, 24 hours, 96 hours, and 168 hours after modeling in the sham, saline, LCgel, and Edar groups. As shown in Figure 4, compared with the saline group, both LCgel and Edar treatments significantly restored middle cerebral artery blood flow in mice after tMCAO modeling (Figure 4a), and LCgel treatment significantly improved middle cerebral artery blood flow in mice after tMCAO modeling (Figure 4b).

[0059] Determination of infarct volume by Nissl, hematoxylin and eosin (HE), and thiazolyl coagulase (TTC) staining: Brain tissue was sectioned 7 days (168 hours) after tMCAO induction and analyzed by Nissl, HE, and TTC staining. Nissl and HE staining results are shown in Figure 5. Nissl staining revealed intact neuronal morphology and clear structure in the sham group. Compared with the sham group, the number of Nissl bodies in the saline group was reduced, with very few Nissl bodies in the infarcted area. Compared with the saline group, Nissl bodies were restored in the peri-infarcted brain tissue in the LCgel- and Edar-treated groups (Figure 5a, scale bar: 1.25 mm). Nissl and HE staining demonstrated that both LCgel and Edar-treated groups reduced infarct size and improved pathological structures in mice with cerebral ischemia, with LCgel treatment being more effective (Figure 5b). After TTC staining, images were taken with a digital camera, and the brain slice images were imported into a computer for analysis of infarct volume using Image J software. TTC staining is shown in Figure 6, which is consistent with the results of Nissl and HE staining. Compared with the Saline group, the infarct volume in the LCgel treatment group was significantly reduced, and the improvement effect was better than that in the Edar treatment group, indicating that the prepared LCgel has a significant therapeutic effect on ischemic stroke and the effect is better than that of Edar treatment.

[0060] MRI scan evaluation: MRI scans were performed on mice to evaluate infarct volume at 24 and 72 hours after tMCAO modeling. The results are shown in Figure 7. Compared with the Saline group, the infarct volume (highlighted area) in both the LCgel-treated and Edar-treated groups was reduced, with the reduction in infarct volume in the LCgel-treated group being more significant, once again demonstrating the therapeutic effect of the multienzyme complex on stroke.

[0061] Example 5 Behavioral analysis of tMCAO mice after different treatments

[0062] The experimental mice in Example 3 were subjected to behavioral testing using the rotarod test.

[0063] The rotarod test is used to assess motor coordination and balance. The experiment consists of two parts: a 3-day preoperative training session and formal testing on days 1, 3, 7, 14, 21, and 28 after surgery. Before the start of the rotarod test, mice were trained at a set rotation speed (15 RPM) for 15 minutes. Then, three trials were conducted. In the formal experiment, mice were placed on an accelerating rotarod (accelerating from 4 RPM to 40 RPM within 5 minutes) until they fell. The time the mice stayed on the rotarod was recorded. If they did not fall within 5 minutes, it was counted as 5 minutes. Each animal underwent three trials with a 20-minute interval between experiments, and the average retention time of the three trials was calculated. Animals with ischemic injuries tend to fall off more quickly than normal animals. The time the mice stayed on the rotarod was recorded one day before modeling as a baseline value; using the same conditions, the mice were tested on days 1, 3, 7, 14, 21, and 28 after modeling, and the time the mice stayed on the rotarod was recorded. The experimental results are shown in Figure 8. After treatment with LCgel and Edar, the time mice stayed on the rotarod was close to that of the sham group and significantly higher than that of the Saline group. In addition, the time mice stayed on the rotarod was longer than that of the Edar group, indicating that LCgel treatment can significantly improve the impaired motor function of mice after ischemia-reperfusion injury.

[0064] Example 6 Analysis of survival rate of tMCAO mice after different treatments

[0065] The survival rates of the experimental mice in Example 3 after 14 days of treatment were statistically analyzed. As shown in Figure 9, the mortality rates of mice were reduced after both LCgel and Edar treatments, and the survival rate of mice after LCgel treatment was significantly higher than that of the Edar treatment group, which also proves that LCgel has a better therapeutic effect on stroke.

[0066] Example 7 Analysis of Lactic Acid Content in Brain Tissue of tMCAO Mice after Different Treatments

[0067] Three days after modeling, the lactate content in the penumbra region tissues of the mice in the different treatment groups in Example 3 was tested. The experimental results are shown in Figure 10. The lactate content in the brain tissue increased significantly after modeling, indicating that lactate accumulation occurred in the brain tissue of mice after acute cerebral infarction. After LCgel treatment, the accumulated lactate in the brain tissue was significantly reduced and was significantly less than that in the Edar group, indicating that LCgel has significant lactate metabolism ability, while Edar does not have this function. This result also shows that simultaneously regulating lactate metabolism and scavenging free radicals has a better therapeutic effect on stroke.

[0068] Example 8 Establishment and Treatment of Acute Liver Injury (ALI) Model in Mice

[0069] The animals used were healthy SPF-grade C57BL / 6 (C57) female mice purchased from Southern Model Organisms Science Co., Ltd. The mice were aged to 8 weeks and weighed between 22 and 25 g.

[0070] Mice were randomly divided into three groups: (1) sham group, (2) saline group, and (3) LCgel group. Mice were anesthetized by intraperitoneal injection of 100 mg / kg of ciprofloxacin. After ensuring that the mice were thoroughly anesthetized, the abdominal cavity was opened along the midline of the abdomen to fully expose the abdominal organs. After the anatomical position of the liver was determined, the left and middle lobes of the liver were separated upwards with a cotton swab moistened with saline. The remaining liver was normally in the abdominal cavity, exposing the hidden blood vessels in the middle of the liver. Microvascular clamps were used to clamp the hepatic vein and artery, causing ischemia in the left and middle lobes of the liver, which accounted for 70% of the total liver. At this time, the color of the ischemic liver lobe changed from bright red to khaki. If the liver color remained khaki for 30 to 60 seconds, it indicated that the ischemia was successful. The clamping was continued for 1 hour. During this period, the abdominal cavity was sutured and covered with moist gauze. After the end, the vascular clamp was released and the abdominal cavity was sutured and closed with surgical sutures.

[0071] Mice in the treatment group were immediately injected intravenously with LCgel (2.5 mg / kg) after reperfusion, while mice in the saline group received intravenous saline. They were then placed in an incubator, their vital signs monitored, and allowed to recover. Mice in the sham group did not undergo any surgical intervention. Mice were sacrificed at specific time points after reperfusion, and approximately 1 mL of blood was collected by eye sampling. The blood was allowed to stand at room temperature for 2 hours. After stratification, the blood was centrifuged at 15,000 rpm for 10 minutes. The upper serum layer was collected for measurement of liver function markers such as ALT and AST. Liver samples, approximately 50 mg each, were also collected for determination of tissue lactate content.

[0072] Example 9 Analysis of the Effects of Different Treatments on Mice with Acute Liver Injury (ALI)

[0073] The liver lactate content and liver function of mice with acute liver injury described in Example 8 were analyzed after various treatments to verify the therapeutic efficacy of the multi-enzyme complex LCgel in this mouse model of acute liver injury. Figure 11 shows the lactate content in the liver tissue of mice after various treatments. The lactate content in the saline group was significantly higher than that in the sham group, indicating that lactate accumulates in the liver tissue of mice after acute liver injury. After LCgel treatment (LCgel group), the accumulated lactate was metabolized, reducing its concentration to a level comparable to that in the sham group. This demonstrates that the prepared LCgel is capable of degrading excess lactate in tissues, regulating abnormal lactate metabolism, and improving damaged tissues. The liver function test results of mice in different groups are shown in Figure 12, including clinical biochemical indicators such as ALB (albumin), ALP (alkaline phosphatase), ALT (alanine transaminase), AST (aspartate transaminase), GGT (glutamyl transferase), and TBA (total bile acid). As can be seen from the figure, the ALB concentration in the Saline group was significantly lower than that in the Sham group, and increased to normal levels after LCgel treatment. The ALP, ALT, AST, GGT, and TBA levels in the Saline group were significantly increased compared with the Sham group, indicating that liver function was severely impaired. After LCgel treatment, these indicators decreased, indicating that LCgel can improve liver function damage and protect the liver.

[0074] Example 10 Preparation and Characterization of Multi-enzyme Complex DL-LCgel

[0075] Lactate oxidase and catalase were dissolved in HEPES buffer with 1% vinyl dextran and 1% Lys-MA. The mixture was passed through an SPG membrane to form a colloid. After initiation of polymerization, the mixture was centrifuged at 6000 rpm, the resulting precipitate was washed, and finally dispersed in PBS buffer to obtain the target multi-enzyme complex DL-LCgel. Transmission electron microscopy was used to analyze the morphology of the resulting multi-enzyme complex DL-LCgel. As shown in Figure 13, the DL-LCgel appeared as uniformly dispersed nanospheres with a particle size of approximately 50 nm, demonstrating good dispersibility. Different concentrations of DL-LCgel were co-cultured with HEK293 cells subjected to an oxygen-glucose deprivation (OGD) model. DCFH-DA fluorescence staining revealed strong fluorescence in the OGD cells. As shown in Figure 14, the DCFH-DA fluorescence intensity decreased with increasing DL-LCgel concentration, demonstrating that the multi-enzyme complex DL-LCgel can effectively scavenge reactive oxygen species.

[0076] Example 11 Establishment and treatment of acute kidney injury (AKI) model in mice

[0077] The animals used were healthy SPF-grade C57BL / 6 male mice purchased from Southern Model Organisms Science Co., Ltd. The mice were 8-12 weeks old and weighed between 22-25 g.

[0078] Mice were randomly divided into three groups: (1) sham group, (2) saline group, and (3) multienzyme complex group. A sepsis-induced acute kidney injury (AKI) model was established by intraperitoneal injection of lipopolysaccharide (LPS) (2.0 mg / ml, 10.0 mg / kg). Mice in the treatment group were intravenously injected with DL-LCgel (dose 2.5 mg / kg), the saline group was intravenously injected with saline, and the sham group did not undergo any surgical treatment. Blood and tissue samples were collected 24 and 72 hours after treatment for the determination of relevant indicators.

[0079] Example 12 Analysis of the effects of different treatments on mice with acute kidney injury (AKI)

[0080] The renal tissue lactate content and renal function of the mice with acute kidney injury (AKI) in Example 11 were analyzed after various treatments to verify the therapeutic effect of the multi-enzyme complex DL-LCgel on the mouse model of acute kidney injury (AKI). The lactate content in the renal tissue of mice 72 hours after administration is shown in Figure 15. It can be seen that the lactate content in the saline group was significantly higher than that in the sham group, indicating that the lactate content in the renal tissue of mice increases significantly after acute kidney injury. After treatment with DL-LCgel (DL-LCgel group), the accumulated lactate was consumed by lactate oxidase, and the concentration was significantly reduced, indicating that the prepared DL-LCgel can degrade excess lactate in tissues and regulate abnormal lactate metabolism.

[0081] Serum creatinine (CRE) and blood urea nitrogen (BUN) are the most commonly used indicators for evaluating renal function. Higher CRE and BUN levels indicate poorer renal function. 24 and 72 hours after administration, blood samples were collected and processed to obtain serum. CRE and BUN levels were measured in mice from different treatment groups using an automated biochemical analyzer. As shown in Figure 16, compared with the sham group, CRE and BUN levels were significantly elevated in AKI mice after modeling. After DL-LCgel treatment, CRE and BUN levels were significantly reduced in AKI mice 24 and 72 hours later, indicating that DL-LCgel can improve impaired renal function and has a significant therapeutic effect on acute kidney injury.

[0082] The results of the examples show that the prepared multi-enzyme complex can significantly reduce the volume of cerebral infarction in stroke mice, improve the motor behavior of mice, reduce mortality, and also has a significant improvement effect on acute liver injury and acute kidney injury, and can prevent and protect acute liver injury and kidney injury to a certain extent, indicating that the simultaneous regulation of lactate metabolism and reactive oxygen species by cascade enzyme catalysis has a significant therapeutic effect on stroke, acute liver injury and acute kidney injury. Other cardiovascular diseases with abnormal lactate metabolism are also accompanied by abnormal lactate metabolism and changes in reactive oxygen levels, leading to cell death and tissue damage. Through the flavin oxidase and peroxidase cascade, the abnormal metabolism in the lesion area is effectively regulated and the reactive oxygen species are downregulated, reducing cell death and reducing tissue damage. In theory, it also has a good therapeutic effect on such diseases and has broad application prospects.

[0083] Any undescribed parts of the present invention are the same as or implemented using existing technologies. The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the detailed methods of the present invention, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A multi-enzyme nanocomplex, characterized in that: The active components are a cascade system consisting of flavin oxidase or its analogues and peroxidase or its analogues.

2. The multi-enzyme nanocomplex according to claim 1, characterized in that: The flavin oxidase is selected from any one or more combinations of lactate oxidase, glutamate oxidase and monoamine oxidase; and the peroxidase comprises any one or more combinations of peroxidase and catalase.

3. The multi-enzyme nanocomplex according to claim 1, characterized in that The concentration ratio of the flavin oxidase to the peroxidase is 20:1-1:

100.

4. The multi-enzyme nanocomplex according to claim 1, characterized in that The invention also comprises a carrier for coating the active component, wherein the carrier is selected from any one of nano gel material, albumin and liposome.

5. The multi-enzyme nanocomplex according to claim 4, characterized in that: in, When the carrier is a nanogel material, the multi-enzyme nanocomplex is assembled from flavin oxidase, peroxidase, functional monomers and polysaccharide monomers and then prepared by an enzymatic free radical polymerization method; When the carrier is albumin, the multi-enzyme nanocomplex is assembled from flavin oxidase, peroxidase, alkenyl albumin and functional monomers and then prepared by an enzymatic free radical polymerization method; When the carrier is a liposome, the multi-enzyme nanocomplex is self-assembled by the liposome, flavin oxidase and peroxidase through physical action to form a nanocomplex.

6. The multi-enzyme nanocomplex according to claim 5, characterized in that: in, The functional monomer is selected from any one of ethylene glycol dimethacrylate PEGMA, lysine acrylate Lys-MA, arginine acrylate Arg-MA, and dimethylaminoethyl methacrylate TMAEMA; The polysaccharide monomer is selected from any one or more combinations of ethylene-functionalized gelatin, dextran, chitosan, chondroitin sulfate, and hyaluronic acid; The initiator of the enzymatic free radical polymerization is selected from the catalytic substrate of the corresponding flavin oxidase.

7. Use of the multi-enzyme nanocomplex according to claim 4 or 5 in the preparation of a drug for treating stroke and other cardiovascular diseases caused by abnormal lactate metabolism, characterized in that: The drug achieves combined treatment by regulating the abnormal metabolism of the lesion site and the cascade effect of clearing reactive oxygen species.

8. A pharmaceutical composition for treating stroke and other cardiovascular diseases caused by abnormal lactate metabolism, characterized in that: The pharmaceutical composition comprises an active ingredient and a pharmaceutically acceptable excipient or diluent; Wherein, the active component comprises the multi-enzyme nanocomplex according to any one of claims 4 to 5.

9. The pharmaceutical composition according to claim 8, characterized in that: in, The pharmaceutical composition is in the form of any one or more of tablets, capsules, granules, suspensions, emulsions, solutions, syrups or injections.

10. Use of the multi-enzyme nanocomplex according to claim 4 or 5 in the preparation of antioxidant health foods that promote the scavenging of lactic acid and free radicals.

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