Application of multienzyme nano-complex in preparation of medicine for treating cerebral apoplexy and other cardiovascular diseases caused by abnormal lactic acid metabolism

Abstract

The invention provides an application of a multienzyme nano-complex in preparation of drugs for treating cerebral apoplexy and other cardiovascular diseases caused by abnormal lactic acid metabolism, the active component of the multienzyme nano-complex is a cascade system composed of flavin oxidase and peroxidase, and the multienzyme nano-complex further comprises a carrier coating the active component of the multienzyme nano-complex. The invention further provides application of the multi-enzyme nano-complex in preparation of drugs for treating cerebral apoplexy and other cardiovascular diseases with abnormal lactic acid metabolism and a pharmaceutical composition containing the multi-enzyme nano-complex. The multi-enzyme nano complex can regulate and control abnormal metabolism of a focus area and scavenge free radicals at the same time, and has a better treatment effect compared with a single free radical scavenging mode. Cerebral stroke, acute liver injury and acute kidney injury animal experiments prove that the compound can reduce lactic acid accumulation, significantly reduce the cerebral infarction volume of cerebral stroke mice, improve behavioral functions, and also have a protective effect on acute liver injury and kidney injury. Compared with the existing therapeutic drug edaravone, simultaneous regulation of abnormal metabolism and free radical removal is a brand new therapeutic strategy, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a multi-enzyme nano-complex for treating stroke and other cardiovascular diseases with abnormal lactate metabolism, a preparation method thereof, and an application thereof in the preparation of drugs for treating stroke and other cardiovascular diseases with abnormal lactate metabolism. Background Art

[0002] When local tissues and organs (commonly found in tissues such as the brain, heart, liver, and kidney) are ischemic due to various reasons and then reperfused (i.e., blood supply is restored), cell functional metabolism disorders and structural damage are aggravated, accompanied by lactate accumulation and respiratory burst. During the ischemic period, due to insufficient oxygen supply, cells cannot carry out normal oxidative phosphorylation, resulting in increased anaerobic glycolysis and the production of a large amount of lactate. During the reperfusion process, after the intracellular oxygen supply is restored, the generation of intracellular oxygen free radicals increases, leading to an exacerbation of the intracellular oxidative stress response, causing apoptosis and necrosis of cells, and further causing secondary damage to tissues and organs, that is, ischemia-reperfusion injury. Ischemia-reperfusion injury can lead to a series of serious clinical problems and is related to the high incidence and mortality of various diseases, such as stroke, myocardial infarction, acute kidney injury, acute liver injury, etc. Among them, stroke is a major disease threatening human health. According to the WHO report, among the causes of death or long-term and severe neurological diseases worldwide in the past decade, stroke ranks second, with the characteristics of high incidence, high recurrence rate, high disability rate, high mortality rate, and heavy 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 the hypoxic condition: by increasing oxygen supply, promoting cell oxidative phosphorylation, and reducing lactate production; (2) cell protectants: using free radical scavengers to inhibit the oxidative stress response and reduce cell damage; (3) lactate clearance treatment: using lactate scavengers or dialysis therapy to promote lactate excretion and improve lactic acidosis; (4) anti-inflammatory treatment: applying anti-inflammatory drugs or cytokine antagonists to reduce the inflammatory response. Among them, cell protection is a hot topic of concern to researchers. Currently, a series of drugs or nano-materials have been developed to down-regulate the reactive oxygen species components generated during reperfusion in a responsive manner, reduce oxidative stress, and reduce tissue damage to achieve the treatment purpose. For example, the main function of the drug molecule Edaravone (Edar) used clinically is to scavenge oxygen free radicals and reduce tissue oxidative stress injury; in addition, there are various nano-materials, such as the PNzyme / MnO nanozyme (Adv. Mater. 2023, 2210144) with superoxide dismutase (SOD) and catalase (CAT) catalytic activities, and the peroxidase-like CeO 2 nanozyme (Adv. Mater. 2023, 2210144), the peroxidase-like CeO 2@ZIF-8 (Sci. Adv. 2020, 6, eaay9751) etc. protect cells from oxidative damage by catalytically oxidizing and scavenging reactive oxygen species in the lesion area.

[0004] Due to the extremely complex microenvironment in the lesion areas of stroke and other cardiovascular diseases with abnormal lactate metabolism, simply relying on a single mode of reducing oxidative stress often fails to achieve satisfactory therapeutic effects. Normal metabolic activities are crucial for maintaining tissue microenvironment homeostasis. As mentioned above, during the development of stroke and other cardiovascular diseases with abnormal lactate metabolism, the tissue cannot carry out normal aerobic metabolism, leading to a large accumulation of lactate, which further exacerbates disease damage. Currently, there are no reports on drugs that can simultaneously degrade the accumulated metabolic substrates and scavenge reactive free radicals. Therefore, designing and developing a class of multi-target therapeutic drug systems that can simultaneously regulate abnormal metabolism and reduce tissue oxidative stress will be an effective means for treating stroke and other cardiovascular diseases with abnormal lactate metabolism, and has important clinical value. Summary of the Invention

[0005] The present invention addresses the above problems and provides a multi-enzyme nanocomplex with dual functions of simultaneously regulating abnormal lactate metabolism in diseased tissues and scavenging reactive free radicals. The present invention also provides a preparation method of 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. Subsequently, taking the mouse model of cerebral ischemia-reperfusion (tMCAO), acute liver injury (ALI), and acute kidney injury (AKI) as examples, its in vivo therapeutic effects on stroke and other cardiovascular diseases with abnormal lactate metabolism are verified. The results show that after intravenous injection of the multi-enzyme complex in stroke mice, the infarct volume in the brain is significantly reduced, the motor behavior is significantly improved, and the mortality rate is also decreased. After intravenous injection of the multi-enzyme complex in mice with acute liver injury and acute kidney injury, the damaged liver and kidney tissues are also significantly improved, indicating that simultaneous regulation of lactate metabolism and reactive oxygen species components 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, in the multi-enzyme complex of the present invention, flavin oxidase can specifically react with the metabolic substrates at the lesion site to reduce metabolite accumulation and regulate abnormal metabolism; peroxidase can convert the generated hydrogen peroxide and endogenous excessive hydrogen peroxide into water and oxygen, reducing tissue oxidative stress response and alleviating tissue damage. Through the efficient catalytic action of the dual-enzyme cascade, the abnormal metabolism and free radical scavenging efficiency of damaged tissues are simultaneously and efficiently regulated. Compared with the current single free radical scavenging mode, the therapeutic effect of the dual regulation is more significant.

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

[0009] In the first aspect of the present invention, a multi-enzyme nano-complex is provided, and its active component is a cascade system composed of a flavin oxidase or its analog and a peroxidase or its analog, and further includes a carrier coating the active component to ensure the high catalytic activity of the cascade system.

[0010] Preferably, the flavin oxidase is selected from any one or a combination of lactic acid oxidase, glutamate oxidase, and monoamine oxidase; the peroxidase is selected from any one or a combination of peroxidase and catalase; and the carrier is selected from any one of nano-gel materials, albumin, and liposomes.

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

[0012] In the second aspect of the present invention, a preparation method of the above multi-enzyme nano-complex is provided, which is specifically as follows:

[0013] (1) When the carrier is a nano-gel material, the multi-enzyme nano-complex is prepared by an enzyme-catalyzed radical polymerization method after the assembly of flavin oxidase, peroxidase, a functional monomer, and a polysaccharide monomer.

[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 a combination of vinyl-functionalized gelatin, dextran, chitosan, chondroitin sulfate, and hyaluronic acid; and the initiator for the enzyme-catalyzed radical polymerization is selected from the catalytic substrates corresponding to the flavin oxidase.

[0015] (2) When the carrier is albumin, the nano-complex is prepared by a radical polymerization method after the assembly of flavin oxidase, peroxidase, alkenyl albumin, and a functional monomer.

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

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

[0018] In the specific embodiment part of the present invention, the multi-enzyme nano-complexes LCgel and DL-LCgel prepared when the carrier is a nano-gel material are respectively subjected to morphological characterization. The results show that both LCgel and DL-LCgel particles are spherical, have good monodispersity, and the particle size is about 50 - 200 nm (Figure 1 and Figure 13 ); The results of the cytotoxicity experiment showed that within the concentration range of 0.01 - 0.5 mg / ml, the survival rate of fibroblast NIH3T3 reached over 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 could significantly reduce the cerebral infarction volume after stroke in tMCAO mice, significantly improve the behavioral function of mice, and could 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 had good therapeutic effects on stroke and other cardiovascular diseases with abnormal lactate metabolism.

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

[0021] In the specific implementation part of the present invention, animal models of stroke, acute liver injury, and acute kidney injury were used as detection models to verify the therapeutic effects of the multi-enzyme nano-complex on stroke, acute liver injury, and acute kidney injury. Whether it is stroke or other cardiovascular diseases with abnormal lactate metabolism, they are all accompanied by abnormal lactate metabolism and changes in the level of reactive oxygen species, resulting in cell death and tissue damage. The flavin oxidase and peroxidase cascade system of the present invention can effectively degrade the excessive lactate in the lesion tissue, down-regulate the reactive oxygen species components, reduce cell oxidative damage, and have protective and therapeutic effects on the tissue damage of stroke and other cardiovascular diseases with abnormal lactate metabolism.

[0022] Preferably, the drug achieves combined treatment through the cascade action of regulating abnormal metabolism at the lesion site and scavenging reactive oxygen species.

[0023] The fourth aspect of the present invention provides a pharmaceutical composition for treating stroke and other cardiovascular diseases with abnormal lactate metabolism, including an active component and a pharmaceutically acceptable excipient or diluent, wherein the active component is the multi-enzyme nano-complex as described above.

[0024] Preferably, the preferred pharmaceutical form of the pharmaceutical composition is an injection. The drug can be made into any intravenous injection preparation form such as an injection solution or a sterile injection powder.

[0025] The fifth aspect of the present invention also provides a composition in the preparation of an antioxidant health food for promoting the clearance of lactate and free radicals.

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

[0027] Compared with the prior art, the multi-enzyme nano-complex combination of the present invention can simultaneously regulate the abnormal lactic acid metabolism and free radical scavenging in damaged tissues of stroke and other cardiovascular diseases with abnormal lactic acid metabolism. The dual effect has a better therapeutic effect than a single free radical scavenging mode, can significantly reduce the cerebral infarction volume in an ischemic reperfusion mouse model, alleviate tissue damage, improve behavioral function, and has an obvious improvement effect on the damaged liver and kidney tissues of mice. Compared with existing therapeutic drugs (such as edaravone), using bio-enzyme cascade catalysis to simultaneously regulate metabolism and scavenge free radicals is a multi-target therapeutic strategy. In addition, the multi-enzyme of the present invention is a biological protein with higher safety. Description of the Drawings

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

[0029] Figure 2 Cytotoxicity experiment of the multi-enzyme complex LCgel in Example 1;

[0030] Figure 3 In vitro degradation of lactic acid (a) and reactive oxygen species scavenging experiment (b) of the multi-enzyme complex LCgel in Example 1;

[0031] Figure 4 Blood flow monitoring results of the surface projection area of the middle cerebral artery in different treatment groups of tMCAO mice in Example 3, where (a) is the cerebral blood flow map of different treatment groups and (b) is the statistical analysis result;

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

[0033] Figure 6 TTC staining analysis results of different treatment groups of tMCAO mice in Example 3, where (a) is the brain slice image of different treatment groups and (b) is the statistical analysis result;

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

[0035] Figure 8 Behavioral experiment results of different treatment groups of tMCAO mice in Example 3;

[0036] Figure 9 Survival rate analysis of different treatment groups of tMCAO mice in Example 3;

[0037] Figure 10 Analysis of lactic acid content in the brain tissues of different treatment groups of tMCAO mice in Example 3;

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

[0039] Figure 12 Test results of liver function indexes of different treatment groups of ALI mice in Example 8;

[0040] Figure 13 TEM image of the multi-enzyme complex DL-LCgel prepared in Example 10;

[0041] Figure 14 Cell experiment on the scavenging of reactive oxygen species by the multi-enzyme complex DL-LCgel prepared in Example 10;

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

[0043] Figure 16 Test results of renal function indexes of different treatment groups of AKI mice in Example 11. Detailed implementation manners

[0044] Now, in combination with the embodiments and the drawings, the present invention will be described in detail, but the implementation of the present invention is not limited thereto. The following embodiments and experimental examples further illustrate the present invention and should not be construed as a limitation of the present invention. At the same time, the embodiments do not include a detailed description of traditional methods, such methods being well known to those of ordinary skill in the art and being described in many publications.

[0045] Unless otherwise stated, percentages and parts are calculated by volume. Unless otherwise defined, all professional and scientific terms used herein have the same meaning 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, and the preferred implementation methods and materials described in the detailed implementation manners are only for demonstration purposes.

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

[0047] Using nanogel as a fixed carrier, a target multi-enzyme complex was prepared using the combination of lactate oxidase and catalase as an example. Lactate oxidase and catalase were dissolved in HEPES buffer together with 2% vinyl gelatin and PEGMA (0.1 - 2%). After polymerization was initiated through an SPG membrane, centrifugation was carried out at 6000 rpm, and the obtained precipitate was washed. Finally, it was dispersed in PBS buffer to obtain the target multi-enzyme complex LCgel.

[0048] The morphology of the obtained LCgel was analyzed using a transmission electron microscope, as Figure 1 shown. The LCgel particles were spherical, porous on the surface, had good monodispersity, and the particle size was approximately 200 nm. Different concentrations of LCgel were co-incubated with fibroblast NIH3T3, and the results were as Figure 2 shown. In the concentration range of 1 - 100 μg / ml, the survival rate of fibroblast NIH3T3 reached over 90%, indicating that this multi-enzyme complex had no obvious toxicity to cells and had 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 carried out on the multi-enzyme complex LCgel prepared in Example 1. First, an ischemic reperfusion microenvironment was simulated, and a 50 mM lactate solution was prepared with PBS. Subsequently, different concentrations of LCgel (0.25 - 2 mg / ml) were added. After reacting for half an hour, the residual lactate content in the solution was measured using a lactate detection kit, and the clearance rate of the multi-enzyme complex LCgel for lactate was calculated. As Figure 3 shown in a, as the concentration of the added material increased, the clearance rate of lactate gradually increased, indicating that the prepared multi-enzyme complex LCgel could scavenge excessive lactate and could be used to regulate the large amount of lactate accumulated in tissues. At the same time, an ROS scavenging experiment was carried out. An ischemic reperfusion microenvironment was simulated, and a 100 μM hydrogen peroxide solution was prepared with PBS. Similarly, different concentrations of LCgel (0.25 - 2 mg / ml) were added. After reacting for half an hour, the residual H 2 O 2 content in the solution was measured using an H 2 O 2 content detection kit, and the clearance rate of LCgel for ROS was calculated. As Figure 3 shown in b, the higher the concentration of LCgel, the higher the clearance rate of ROS, indicating the ROS scavenging ability of LCgel. Thus, it can be seen that the prepared multi-enzyme complex LCgel can simultaneously scavenge excessive lactate and reactive oxygen species, and is expected to have a good therapeutic effect on cardiovascular diseases caused by abnormal lactate metabolism.

[0051] Example 3 Establishment and treatment of a mouse ischemic reperfusion model (tMCAO)

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

[0053] The mice were randomly divided into four groups: (1) sham operation group (sham), (2) saline treatment group (Saline), (3) multi-enzyme complex treatment group (LCgel, prepared in Example 1), and (4) edaravone treatment group (Edar). Edar is a neuroprotective agent (free radical scavenger), which can scavenge free radicals and inhibit lipid peroxidation, thereby inhibiting oxidative damage to brain cells, vascular endothelial cells, and nerve cells. Clinically, it is mainly used 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 the multi-enzyme complex LCgel on stroke.

[0054] The experiment simulated the cerebral ischemia-reperfusion process by occluding the middle cerebral artery (tMCAO) for 1 hour and then reperfusing for 72 hours. Eight-week-old mice were anesthetized with a mixed gas containing 2% isoflurane and maintained with 1% isoflurane, allowing the mice to breathe spontaneously during the operation. The skin of the middle neck was prepared, disinfected, and covered with a surgical drape. A longitudinal incision was made along the midline of the neck (in two layers), exposing the common carotid artery, internal carotid artery, and external carotid artery. A small branch on the external carotid artery was isolated, cauterized with an electrocautery pen to cut off the isolated branch, the distal end of the external carotid artery was ligated with a 6-0 surgical suture, the proximal end of the common carotid artery was ligated with a 4-0 surgical suture, and a slipknot was tied in the middle with a 6-0 surgical suture. A small incision was made above the slipknot, and then a suture plug was inserted. The suture plug was inserted from the external carotid artery into the internal carotid artery and stopped when resistance was encountered. At this time, the suture plug was inserted about 1.0 - 1.2 cm. The incision was covered with cotton dipped in saline, and ischemia was induced for 1 hour. Then the suture plug was removed, the skin was sutured, disinfected, and lidocaine ointment was applied to the wound for postoperative pain relief.

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

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

[0057] The experimental mice in Example 3 were analyzed for cerebral infarction.

[0058] Laser speckle blood imaging for blood flow monitoring: Mice were induced with a mixed gas containing 2% isoflurane, maintained with 1% isoflurane, and allowed to breathe spontaneously during the operation. Before modeling, the black hair on the top of the mouse's head was shaved to expose the scalp. After disinfection with iodophor, a 1.5 cm midline incision was made to expose the skull. The broken hair on the skull surface was removed with a wet cotton swab, and the head was exposed in the monitoring area. Blood flow in the surface projection area of the middle cerebral artery of mice in the sham group, Saline group, LCgel treatment group, and Edar treatment group was monitored at 10 min, 1 h, 2 h, 7 h, 24 h, 96 h, and 168 h after modeling. As Figure 4 shown, compared with the Saline group, both LCgel treatment and Edar treatment could significantly restore the blood flow of the middle cerebral artery in mice after tMCAO modeling ( Figure 4 a), and the effect of LCgel treatment on improving the blood flow of the middle cerebral artery in mice after tMCAO modeling was significantly better than that of Edar treatment ( Figure 4 b).

[0059] Measurement of infarct volume by Nissl, HE, and TTC staining: At 7 days (168 h) after tMCAO modeling, brain tissues were taken for sectioning, and Nissl, HE, and TTC staining analyses were performed respectively. The results of Nissl and HE are shown by Figure 5 As shown, Nissl staining showed that the neurons in the sham group had complete morphology and clear structure; compared with the sham group, the number of Nissl bodies in the Saline group decreased, and there were very few Nissl bodies in the infarcted neurons; compared with the Saline group, the neurons of Nissl bodies in the surrounding brain tissues of the LCgel treatment group and the Edar treatment group were restored ( Figure 5 a, scale bar: 1.25 mm). Nissl and HE staining showed that both the LCgel and Edar treatment groups could reduce the infarct area and improve the pathological structure of ischemic mice, and the LCgel treatment had a better effect ( Figure 5 b). After TTC staining, digital camera photos were taken, the brain slice images were imported into the computer, and the infarct volume was analyzed with Image J software. TTC staining is shown by Figure 6 As shown, 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 of the Edar treatment group, indicating that the prepared LCgel had an obvious therapeutic effect on ischemic stroke and the effect was better than that of Edar treatment.

[0060] MRI scan evaluation: At 24 h and 72 h after tMCAO modeling, mice were scanned by MRI to evaluate the infarct volume. The results are shown by Figure 7As shown, compared with the Saline group, the infarct volumes (highlighted areas) in both the LCgel treatment group and the Edar treatment group decreased, and the decrease in the infarct volume in the LCgel treatment group was more significant, once again demonstrating the therapeutic effect of the multi-enzyme 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 by the rotarod test.

[0063] The rotarod test is used to evaluate motor coordination and balance ability. This experiment includes two parts: training for 3 days before surgery and formal testing on the 1st, 3rd, 7th, 14th, 21st, and 28th days after surgery. Before starting the rotarod test, the mice were trained for 15 minutes at a set rotation speed (15 RPM), and then three trials were conducted. In the formal experiment, the mice were placed on a rotarod that accelerated rotation (from 4 RPM to 40 RPM within 5 min) until the mice fell off. The retention time of the mice on the rotarod was recorded. If the mice did not fall off within 5 min, it was calculated as 5 min. Each animal was subjected to three trials, with an experimental interval of 20 min, and the average retention time of the three trials was calculated. Animals with ischemic injury tend to fall off faster than normal animals. The retention time of the mice on the rotarod was recorded 1 day before modeling as the baseline value; using the same conditions, the mice were tested on the 1st, 3rd, 7th, 14th, 21st, and 28th days after modeling, and the retention time of the mice on the rotarod was recorded. The experimental results are as Figure 8 shown. After LCgel treatment and Edar treatment, the retention time of the mice on the rotarod was close to that of the sham group, significantly higher than that of the Saline group, and the retention time of the mice in the LCgel group on the rotarod was longer than that in the Edar treatment group, indicating that LCgel treatment can significantly improve the impaired motor function of mice after ischemic reperfusion injury.

[0064] Example 6 Survival rate analysis of tMCAO mice after different treatments

[0065] The survival rates of the experimental mice in Example 3 were statistically analyzed 14 days after treatment, as Figure 9 shown. After LCgel treatment and Edar treatment, the mortality rates of the mice both decreased, and the survival rate of the 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 diseases.

[0066] Example 7 Analysis of lactic acid content in the brain tissue of tMCAO mice after different treatments

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

[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 Technology Co., Ltd. The mice were ~8 weeks old and weighed between 22 - 25 g.

[0070] The mice were randomly divided into three groups: (1) sham operation group (sham), (2) normal saline treatment group (Saline), and (3) multi-enzyme complex treatment group (LCgel). The mice were anesthetized by intraperitoneal injection of Zoletil (Zoletil, injection dose 100 mg / kg). After confirming that the mice were completely anesthetized, the abdominal cavity was opened along the midline of the abdomen, and the abdominal organs were fully exposed. After clarifying the anatomical position of the liver, the left and middle lobes of the liver were separated upward with a cotton swab moistened with normal saline, and the remaining liver was left in the abdominal cavity as normal, exposing the hidden blood vessels in the middle of the liver. The hepatic vein and hepatic artery were clamped with a microvascular clamp, resulting in ischemia of the left and middle lobes of the liver, which accounted for 70% of the total liver. At this time, it could be seen with the naked eye that the color of the ischemic liver lobe changed, from bright red to yellowish-brown. Observing that the liver color remained yellowish-brown for 30 - 60 s indicated successful ischemia. The clamping was continued for 1 h, during which the abdominal cavity was sutured and covered with a moist gauze. After completion, the vascular clamp was released, and the abdominal cavity was sutured and closed with surgical sutures.

[0071] Immediately after reperfusion, the mice in the treatment group were intravenously injected with LCgel (dose 2.5 mg / kg), and the Saline group was intravenously injected with normal saline, and then placed in an incubator to monitor the vital signs until they woke up. The sham group did not undergo any surgical treatment. The mice were sacrificed at specific time points after reperfusion, and about 1 mL of mouse blood was collected by eye puncture. The blood was allowed to stand at room temperature for 2 hours, centrifuged at 15000 rmp for 10 minutes after blood stratification, and the upper serum was aspirated for measurement of liver function indexes such as ALT and AST. At the same time, liver samples, about 50 mg each, were collected for determination of tissue lactic acid content.

[0072] Example 9 Analysis of different treatment effects on acute liver injury (ALI) mice

[0073] The lactic acid content and liver function of the liver tissues of the mice with acute liver injury in Example 8 after different treatments were analyzed to verify the therapeutic effect of the multi-enzyme complex LCgel on the mouse model of acute liver injury. The lactic acid content in the liver tissues of the mice after different treatments is as Figure 11 shown. It can be seen that the lactic acid content in the Saline group is significantly higher than that in the Sham group, indicating that lactic acid accumulation occurs in the liver tissues of the mice after acute liver injury. After treatment with LCgel (LCgel group), the accumulated lactic acid is metabolized and the concentration is reduced to the same level as that in the Sham group, indicating that the prepared LCgel can degrade the excessive lactic acid in the tissues, regulate abnormal lactic acid metabolism, and improve the damaged tissues. The test results of the liver function of the mice in different groups are as Figure 12 shown, including clinical biochemical indexes such as ALB (albumin), ALP (alkaline phosphatase), ALT (alanine aminotransferase), AST (aspartate aminotransferase), GGT (gamma-glutamyl transferase), TBA (total bile acid), etc. It can be seen from the figure that the ALB concentration in the Saline group is significantly lower than that in the Sham group and increases to the normal level after treatment with LCgel, while the levels of ALP, ALT, AST, GGT, and TBA in the Saline group are significantly higher than those in the Sham group, indicating that the liver function is severely damaged. After treatment with LCgel, these indexes all decrease, indicating that LCgel can improve liver function injury and play a protective role on the liver.

[0074] Example 10 Preparation and characterization of the multi-enzyme complex DL-LCgel

[0075] Lactate oxidase and catalase were dissolved in HEPES buffer with 1% vinyl dextran and 1% Lys-MA, and a colloid was formed through an SPG membrane. After polymerization was initiated, centrifugation was carried out at 6000 rpm, and the obtained 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 obtained multi-enzyme complex DL-LCgel, as Figure 13 shown. DL-LCgel is a uniformly dispersed nano-gel sphere with a particle size of about 50 nm and good dispersibility. Different concentrations of DL-LCgel were co-cultured with HEK293 cells established with an oxygen-glucose deprivation model (OGD), and fluorescence staining was carried out with DCFH-DA, as Figure 14 shown. It can be seen that the fluorescence of the OGD cells is very strong, and as the concentration of DL-LCgel increases, the fluorescence intensity of DCFH-DA weakens, indicating that the multi-enzyme complex DL-LCgel can effectively scavenge reactive oxygen species.

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

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

[0078] The mice were randomly divided into three groups: (1) sham operation group (sham), (2) saline treatment group (Saline), and (3) multi-enzyme complex treatment group (DL-LCgel). An acute kidney injury (AKI) model induced by sepsis was established by intraperitoneal injection of lipopolysaccharide LPS (2.0 mg / ml, 10.0 mg / kg). After modeling, the mice in the treatment group were intravenously injected with DL-LCgel (dose 2.5 mg / kg), the Saline group was intravenously injected with normal saline, and the sham group received no surgical treatment. Blood samples and tissue samples were collected at 24 h and 72 h after treatment for determination of relevant indicators.

[0079] Example 12 Analysis of Different Treatment Effects on Mice with Acute Kidney Injury (AKI)

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

[0081] Serum creatinine (CRE) and blood urea nitrogen (BUN) are the most commonly used indicators to evaluate renal function. The higher the levels of CRE and BUN, the worse the renal function. At 24 h and 72 h after administration, blood samples were collected and processed to obtain serum, and the CRE and BUN levels of the mice in different treatment groups were measured by an automatic biochemical analyzer. As Figure 16 shown, compared with the sham group, the CRE and BUN levels of the AKI mice after modeling were significantly increased. After treatment with DL-LCgel, the CRE and BUN levels of the AKI mice were significantly decreased at 24 h and 72 h, indicating that DL-LCgel could improve the damaged renal function and had an obvious therapeutic effect on acute kidney injury.

[0082] From the results of the examples, it can be seen that the prepared multi-enzyme complex can significantly reduce the cerebral infarction volume of stroke mice, improve the motor behavior of mice, reduce the mortality rate, and also has an obvious improvement effect on acute liver injury and acute kidney injury. It can prevent and protect acute liver injury and kidney injury to a certain extent, indicating that simultaneous regulation of lactate metabolism and reactive oxygen species components through cascade enzyme catalysis has a significant therapeutic effect on stroke, acute liver injury and acute kidney injury. For other cardiovascular diseases with abnormal lactate metabolism, there will also be abnormal lactate metabolism and changes in reactive oxygen species levels, resulting in cell death and tissue damage. Through the cascade of flavin oxidase and peroxidase, the abnormal metabolism in the lesion area can be effectively regulated and the reactive oxygen species components can be down-regulated, reducing cell death and tissue damage. Theoretically, this also has a good therapeutic effect on such diseases and has broad application prospects.

[0083] The parts not described in this invention are the same as or implemented by the prior art. The applicant declares that the detailed method of this invention is illustrated by the above examples, but this invention is not limited to the above detailed method, that is, it does not mean that this invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of this invention, the equivalent substitution of each raw material of the product of this invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

Claims

1. A multi-enzyme nanocomplex, characterized in that: The active components are a cascade system composed 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; 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 monomer and polysaccharide monomer and prepared by an enzymatic free radical polymerization method; When the carrier is albumin, the multi-enzyme nanocomplex is prepared by assembling flavin oxidase, peroxidase, alkenyl albumin and functional monomers through an enzymatic free radical polymerization method; When the carrier is a liposome, the multi-enzyme nanocomplex is self-assembled by physical action of the liposome, flavin oxidase and peroxidase to form the 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 with 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.

8. A pharmaceutical composition for treating stroke and other cardiovascular diseases with 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 food that promotes the scavenging of lactic acid and free radicals.

Images