Polydopamine modified cobalt monatomic nano-enzyme system as well as preparation method and application thereof

Abstract

The invention provides a cobalt monatomic nano-enzyme system modified by polydopamine. The cobalt monatomic nano-enzyme system comprises cobalt monatomic nano-enzyme and a polydopamine coating layer coating the surface of the cobalt monatomic nano-enzyme, the cobalt monatomic nano enzyme is obtained by pyrolyzing and etching a precursor Co (at) MOF (at) SiO2. According to the invention, Co SA has activity similar to catalase and superoxide dismutase, and can accurately regulate and control oxidative stress and inflammatory reaction pathways induced by alcohol; the PDA realizes effective retention of Co SA in the stomach by virtue of biological adhesion, and the application safety is improved by virtue of good biocompatibility. The system can activate an NRF2 signal channel and inhibit an NF-kappa B signal channel to enhance the oxidation resistance of tissues and block an inflammatory cascade reaction, so that gastric mucosal lesion and hepatic lesion caused by alcohol are remarkably relieved, and oxidative stress and inflammatory reaction are improved. The invention provides a direction and an experimental basis for synchronous combined prevention and treatment of alcoholic gastritis and hepatitis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of preparation of drugs for treating liver / gastric diseases, and relates to a polydopamine modified cobalt monatomic nanoszyme system, a preparation method thereof and application thereof in inhibiting alcoholic "gastric-liver" synergistic injury. BACKGROUND

[0002] Alcohol abuse has become a major public health problem worldwide, and the number of deaths caused by alcohol-related diseases has also increased year by year, of which the damage to the digestive system (gastric mucosa lesion and liver injury) accounts for more than 45%. After alcohol intake, about 20% is directly metabolized in the stomach, and 80% is converted by the liver, and its metabolites (such as reactive oxygen species) can simultaneously cause damage to the gastric mucosal barrier and liver cell degeneration, forming a "gastric-liver" synergistic injury effect--this pathological process not only leads to diseases such as acute gastritis and alcoholic fatty liver, but also develops into gastric mucosal atrophy and cirrhosis in the long term, and the existing clinical intervention means is difficult to achieve synchronous protection of multiple organs, highlighting the treatment gap that needs to be solved in this field.

[0003] The core pathological mechanism of alcoholic "gastric-liver" synergistic injury focuses on the vicious cycle of oxidative stress imbalance and excessive activation of inflammatory response. In the stomach, alcohol directly stimulates gastric mucosal epithelial cells, induces mitochondrial dysfunction and releases a large amount of ROS, such as superoxide anion (O2 -), hydrogen peroxide (H2O2); meanwhile, ROS accumulation inhibits endogenous antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase (CAT), leading to gastric mucosal epithelial cell apoptosis and down-regulation of tight junction protein expression, and ultimately damaging the gastric mucosal physical barrier. Alcohol entering the liver further exacerbates ROS generation, causing hepatocyte cytoplasmic loosening and fatty degeneration; more importantly, ROS can activate the nuclear factor κB (NF-κB) pathway as a signaling molecule. When the NF-κB p65 subunit translocates from the cytoplasm to the nucleus, it regulates the transcription of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), forming an amplification effect of "oxidative stress → inflammation activation → further oxidative stress". In addition, inhibition of the endogenous antioxidant pathway nuclear factor erythroid 2-related factor 2 (NRF2) is a key link in exacerbating this pathological process. Under normal physiological conditions, NRF2 is combined with Kelch-like epoxide chloropropane-associated protein 1 (Keap1) and is in an inactive state; when stimulated by ROS, NRF2 dissociates from Keap1 and enters the nucleus, regulating the expression of antioxidant genes such as heme oxygenase-1 (HO-1) and glutathione peroxidase to clear excess ROS. However, under alcohol exposure conditions, nuclear translocation of NRF2 is blocked, and the antioxidant pathway function is inhibited, ultimately failing to antagonize the inflammatory response mediated by NF-κB, exacerbating "gastric-liver" tissue damage. The above mechanism research shows that simultaneous regulation of the "NRF2 antioxidant pathway-NF-κB inflammatory pathway" and clearance of excess ROS is a core target for breaking through the bottleneck of treatment of alcoholic multiple organ damage.

[0004] The current first-line drug for alcoholic gastric mucosal damage in clinical practice is mainly proton pump inhibitors, which indirectly alleviate gastric mucosal irritation by inhibiting H⁺-K⁺-ATPase to reduce gastric acid secretion, but cannot directly clear ROS or inhibit the NF-κB pathway, and has no regulatory effect on the oxidative stress and inflammatory response that has already formed; while the hepatoprotective drugs for alcoholic liver damage can improve the stability of hepatocyte membranes, but are difficult to protect the gastric mucosal barrier simultaneously. Long-term use of some drugs may also cause water and sodium retention, abnormal liver enzymes and other side effects, limiting their long-term clinical application.

[0005] Therefore, how to find a more suitable therapeutic drug to solve the above problems existing in the existing drugs has become one of the focuses of attention of many forward-looking researchers. SUMMARY

[0006] In view of this, the technical problem to be solved by the present invention is to provide a polydopamine-modified cobalt single-atom nanozyme system and its preparation method, and its application in inhibiting alcoholic "gastric-liver" synergistic damage. The CoSA@PDA provided by the present invention can not only effectively alleviate H2O2-induced oxidative damage to human gastric mucosal epithelial GES-1 cells by directly scavenging excess intracellular ROS and indirectly restoring the level of endogenous antioxidant molecules; it can also significantly inhibit the abnormal release of intracellular inflammatory mediators (NO, TNF-α, L-6, and IL-1β) in RAW264.7 cells, exhibiting good in vitro antioxidant and anti-inflammatory synergistic regulatory capabilities, providing reliable experimental evidence for subsequent in vivo studies of acute alcoholic gastric injury. Moreover, the preparation process is simple, the conditions are mild, and the controllability is good, making it more suitable for industrial production and application.

[0007] This invention provides a polydopamine-modified cobalt single-atom nanozyme system, comprising: a cobalt single-atom nanozyme and a polydopamine coating layer on the surface of the cobalt single-atom nanozyme;

[0008] The cobalt single-atom nanozyme was obtained by pyrolysis and etching of the precursor Co@MOF@SiO2.

[0009] Preferably, the particle size of the cobalt single-atom nanozyme is 100~300nm;

[0010] The cobalt single-atom nanozyme has a dodecahedral-like morphology.

[0011] The thickness of the polydopamine coating layer is 30~60 nm;

[0012] The mass ratio of the cobalt single-atom nanozyme to polydopamine is (1~3):1;

[0013] The cobalt single-atom nanozyme contains 1% to 2% Co by mass.

[0014] The polydopamine-modified cobalt single-atom nanozyme system has a core-shell structure.

[0015] Preferably, the precursor Co@MOF@SiO2 has a core-shell structure, including a Co@MOF core and a SiO2 coating layer covering the surface of the Co@MOF;

[0016] The precursor Co@MOF@SiO2 has a particle size of 130~350nm;

[0017] The thickness of the SiO2 coating layer is 5~30nm;

[0018] The mass ratio of Co@MOF to SiO2 is 1:(0.4~1).

[0019] The Co@MOF is specifically a Co-doped ZIF-8;

[0020] The Co@MOF has a rhombic dodecahedral morphology.

[0021] Preferably, the cobalt single-atom nanozyme partially retains the morphology of the precursor Co@MOF@SiO2, and also has some structural collapse;

[0022] The pyrolysis specifically refers to pyrolysis under a protective atmosphere;

[0023] The etching is specifically alkaline etching;

[0024] In the cobalt single-atom nanozyme, Co is dispersed in single-atom form and forms a local coordination structure with N ligands;

[0025] The N includes graphitic nitrogen, pyrrole nitrogen, and pyridine nitrogen;

[0026] The cobalt single-atom nanozyme system is a nanozyme system used to treat one or more of the following: alcoholic gastric injury, alcoholic gastritis, alcoholic liver injury, and alcoholic hepatitis.

[0027] This invention provides a method for preparing a polydopamine-modified cobalt single-atom nanozyme system, comprising the following steps:

[0028] 1) After mixing zinc salt, cobalt salt and organic solvent, 2-methylimidazole organic solution is added and reacted to obtain Co@MOF;

[0029] 2) Mix the Co@MOF obtained in the above steps with alcohol to obtain an alcohol solution, then add ammonia-alcohol solution, then add tetraethyl orthosilicate, and after reaction, obtain Co@MOF@SiO2;

[0030] 3) Under a protective atmosphere, the Co@MOF@SiO2 obtained in the above steps is pyrolyzed and then etched with an alkaline solution to obtain cobalt single-atom nanozyme CoSA;

[0031] 4) After mixing the cobalt single-atom nanozyme CoSA obtained in the above steps with water, the resulting CoSA dispersion is mixed again with dopamine solution. The pH value of the mixture is adjusted, and a light-protected reaction is carried out to obtain the CoSA@PDA composite system.

[0032] Preferably, the zinc salt comprises zinc nitrate;

[0033] The cobalt salt includes cobalt nitrate;

[0034] The organic solvent and the solvent in the 2-methylimidazol organic solution are each independently selected from DMF and methanol;

[0035] The molar ratio of the zinc salt to the cobalt salt is (6~9):1;

[0036] The mass ratio of the cobalt salt to 2-methylimidazole is 1:(7~10).

[0037] The mixing reaction takes 10 to 14 hours.

[0038] Preferably, the alcohol includes ethanol;

[0039] In the ammonia-alcohol solution, the volume ratio of ammonia to alcohol is 1:(15~18).

[0040] The mass ratio of tetraethyl orthosilicate to Co@MOF is (3~4):1;

[0041] In step 2), the reaction time is 1 to 3 hours;

[0042] In step 2), the reaction is followed by a step of letting it stand overnight.

[0043] The pyrolysis temperature is 900~1000℃;

[0044] The pyrolysis time is 3 to 5 hours.

[0045] Preferably, the alkaline solution includes NaOH solution and / or KOH solution;

[0046] The molar concentration of the alkaline solution is 3~5M;

[0047] The etching time is 10-14 hours;

[0048] The dopamine solution includes an aqueous dopamine solution;

[0049] Specifically, adjusting the pH value of the mixing system involves adjusting the pH value to 7-10.

[0050] The light-avoidance reaction takes 1 to 3 hours.

[0051] The present invention also provides the application of the polydopamine-modified cobalt single-atom nanozyme system described in any one of the above technical solutions or the polydopamine-modified cobalt single-atom nanozyme system prepared by the preparation method described in any one of the above technical solutions in the preparation of drugs for treating one or more of alcoholic gastric injury, alcoholic gastritis, alcoholic liver injury and alcoholic hepatitis.

[0052] Preferably, the drug comprises a cobalt single-atom nanozyme system and pharmaceutically acceptable excipients;

[0053] The dosage forms of the drug include oral preparations, injections, suppositories, inhalers, or dosage forms that can be directly applied to the liver and / or stomach;

[0054] In the pharmaceutical formulation, the mass content of the polydopamine-modified cobalt single-atom nanozyme system is 0.5~10 mg / mL;

[0055] The specific applications include the application of cobalt single-atom nanozymes in the cobalt single-atom nanozyme system in achieving hydrogen peroxide decomposition and / or superoxide anion free radical scavenging;

[0056] The specific applications include the use of polydopamine in cobalt single-atom nanozyme systems to enhance bioadhesion and / or protect the catalytic sites of cobalt single-atom nanozymes to maintain stability.

[0057] The applications also include the use of polydopamine-modified cobalt single-atom nanozyme systems in improving the effective retention time in the gastric mucosal injury area and enhancing the sustained antioxidant and anti-inflammatory effects.

[0058] This invention provides a polydopamine-modified cobalt single-atom nanozyme system, comprising: a cobalt single-atom nanozyme and a polydopamine coating layer on the surface of the cobalt single-atom nanozyme; the cobalt single-atom nanozyme is obtained by pyrolysis and etching of the precursor Co@MOF@SiO2. Compared with the prior art, this invention suggests that developing novel intervention systems with both "multi-target regulation (antioxidant + anti-inflammatory) and high biocompatibility" is one of the important research directions for addressing existing alcoholic "gastric-liver" synergistic damage. Nanozymes, as a novel biomaterial combining the characteristics of nanomaterials and enzyme catalytic activity, have shown potential in the intervention of oxidative stress-related diseases due to their ability to mimic the catalytic function of natural enzymes (such as SOD and CAT), high stability, and strong designability. For example, dextran-encapsulated ultrafine cerium oxide single-atom nanozymes possess both SOD and CAT-like activities, effectively scavenging ROS and significantly reducing inflammatory responses in mice with acute urinary tract infections; and single-atom nanozymes with Pt-N6 active sites effectively reduced cartilage damage and inflammatory responses in a rat model of osteoarthritis. The excellent performance of single-atom nanozymes in anti-inflammatory therapy has attracted widespread attention, but their application in alcoholic gastric injury, especially alcohol-induced gastrohepatic cross-injury, has not yet been reported.

[0059] Based on this, the present invention creatively designs a technical solution of "polydopamine (PDA) encapsulated cobalt single-atom nanozyme system (CoSA@PDA)," selecting CoSA (cobalt single-atom nanozyme) as the core functional unit, on the one hand, through SOD-like activity to encapsulate O2 -The CoSA nanozyme system undergoes a two-step cascade reaction, simultaneously scavenging multiple ROS and blocking oxidative stress at its source. This is achieved through disproportionation into H2O2 and O2, and further decomposition of H2O2 into H2O and O2 via CAT-like activity. PDA was chosen as the encapsulation layer due to its dual functional characteristics: PDA's surface is rich in catechol groups, which can bind to proteins and polysaccharides on the surface of gastric mucosal epithelial cells via hydrogen bonds and hydrophobic interactions, prolonging the retention time of CoSA in the stomach and increasing drug concentration at the site of injury. Secondly, PDA can slowly degrade into dopamine monomers under physiological conditions with no significant cytotoxicity and can mask the metallic surface characteristics of CoSA, reducing its recognition by the immune system and minimizing organ accumulation. Furthermore, this CoSA@PDA nanozyme system can exert a synergistic protective effect through pathway regulation: on the one hand, it enhances tissue antioxidant capacity and reduces alcohol-mediated oxidative stress damage by activating the NRF2 signaling pathway; on the other hand, it blocks the inflammatory cascade reaction by inhibiting the NF-κB signaling pathway. This design not only achieves synergistic optimization of the functional properties and biosafety of single-atom nanozymes, but also provides novel nanobiomaterials for the clinical treatment of alcoholic "stomach-liver" synergistic injury, and opens up new research pathways for synergistic intervention of cross-organ injury.

[0060] This invention develops a polydopamine-encapsulated cobalt single-atom nanozyme (CoSA@PDA) for the synergistic treatment of alcohol-induced acute gastritis and chronic liver injury. This system fully utilizes the high catalytic activity of single-atom nanozymes, combined with the excellent adhesion properties of polydopamine, to achieve long-term retention at the site of injury and efficient clearance of reactive oxygen species (ROS), breaking the vicious cycle of oxidative stress and inflammation between the stomach and liver. This strategy provides a novel synergistic therapeutic platform for alcoholic gastrohepatitis. Attached Figure Description

[0061] Figure 1 TEM transmission electron microscope image of the bimetallic imidazole framework precursor Co@MOF prepared in this invention.

[0062] Figure 2 SEM image of the core-shell precursor Co@MOF@SiO2 prepared in this invention;

[0063] Figure 3 Characterization images of CoSA prepared in this invention;

[0064] Figure 4 The X-ray photoelectron spectrum of CoSA prepared in this invention;

[0065] Figure 5 A series of characterization and analysis figures for the CoSA@PDA composite system prepared in this invention;

[0066] Figure 6These are figures illustrating the performance analysis of the CoSA@PDA composite system prepared in this invention.

[0067] Figure 7 This invention illustrates the effect of different concentrations of CoSA@PDA on the viability of GES-1 cells.

[0068] Figure 8 The effect of different concentrations of CoSA@PDA on the viability of RAW264.7 cells in this invention;

[0069] Figure 9 The following figures illustrate the therapeutic effect of CoSA@PDA on alcohol-induced acute gastritis in mice.

[0070] Figure 10 The images show H&E staining of the heart, liver, spleen, lungs, and kidneys of mice in each group during the acute alcoholic gastritis treatment experiment of this invention.

[0071] Figure 11 This is a statistical graph showing the levels of UA, BUN, and CRE in the serum of mice in each group during the acute alcoholic gastritis treatment experiment of this invention.

[0072] Figure 12 This is a series of images showing the therapeutic effect of Co SA@PDA on mice with alcoholic hepatitis in this invention. Detailed Implementation

[0073] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0074] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0075] There are no particular restrictions on the purity of any raw materials used in this invention. However, it is preferred to use analytical grade or materials that meet the relevant standards for drug purity.

[0076] All raw materials of this invention are conventional in the field, and each brand name and abbreviation is clear and distinct in its relevant application. Those skilled in the art can purchase them from the market or prepare them by conventional methods based on the brand name, abbreviation and corresponding application.

[0077] This invention provides a polydopamine-modified cobalt single-atom nanozyme system, comprising: a cobalt single-atom nanozyme and a polydopamine coating layer on the surface of the cobalt single-atom nanozyme;

[0078] The cobalt single-atom nanozyme was obtained by pyrolysis and etching of the precursor Co@MOF@SiO2.

[0079] In this invention, the particle size of the cobalt single-atom nanozyme is preferably 100~300nm, more preferably 140~260nm, and even more preferably 180~220nm.

[0080] In this invention, the cobalt single-atom nanozyme preferably has a dodecahedral or rhombic dodecahedral morphology. The cobalt single-atom nanozyme retains the morphology of the precursor Co@MOF@SiO2, and due to partial structural collapse, it can be regarded as a rhombic or rhombic dodecahedral morphology.

[0081] In this invention, the thickness of the polydopamine coating layer is preferably 30-60 nm, more preferably 35-55 nm, and even more preferably 40-50 nm.

[0082] In this invention, the mass ratio of the cobalt single-atom nanozyme to polydopamine is preferably (1~3):1, more preferably (1.4~2.6):1, and even more preferably (1.8~2.2):1.

[0083] In this invention, the mass content of Co in the cobalt single-atom nanozyme is preferably 1% to 2%, more preferably 1.2% to 1.8%, and even more preferably 1.4% to 1.6%.

[0084] In this invention, the polydopamine-modified cobalt single-atom nanozyme system preferably has a core-shell structure.

[0085] In this invention, the precursor Co@MOF@SiO2 has a core-shell structure, preferably including a Co@MOF core and a SiO2 coating layer covering the surface of the Co@MOF.

[0086] In this invention, the particle size of the precursor Co@MOF@SiO2 is preferably 130~350nm, more preferably 180~300nm, and even more preferably 230~250nm.

[0087] In this invention, the particle size of the Co@MOF core is preferably 100~300nm, more preferably 140~260nm, and even more preferably 180~220nm.

[0088] In this invention, the thickness of the SiO2 coating layer is preferably 5~30nm, more preferably 10~25nm, and even more preferably 15~20nm.

[0089] In this invention, the mass ratio of Co@MOF to SiO2 is preferably 1:(0.4~1), more preferably 1:(0.5~0.9), and even more preferably 1:(0.6~0.8).

[0090] In this invention, the Co@MOF is preferably Co-doped ZIF-8. Zn will volatilize at high temperatures during the subsequent pyrolysis process.

[0091] In this invention, the Co@MOF preferably has a rhombic dodecahedral morphology.

[0092] In this invention, the cobalt single-atom nanozyme preferably retains the morphology of the precursor Co@MOF@SiO2, and also has some structural collapse.

[0093] In this invention, the pyrolysis is preferably performed under a protective atmosphere.

[0094] In this invention, the etching is preferably alkaline etching.

[0095] In this invention, in the cobalt single-atom nanozyme, Co is preferably dispersed in single-atom form and forms a local coordination structure with the N ligand.

[0096] In this invention, the N preferably includes graphitic nitrogen, pyrrole nitrogen, and pyridine nitrogen.

[0097] In this invention, the cobalt single-atom nanozyme system is preferably a nanozyme system used to treat one or more of alcoholic gastric damage, alcoholic gastritis, alcoholic liver damage, and alcoholic hepatitis.

[0098] This invention provides a method for preparing a polydopamine-modified cobalt single-atom nanozyme system, comprising the following steps:

[0099] 1) After mixing zinc salt, cobalt salt and organic solvent, 2-methylimidazole organic solution is added and reacted to obtain Co@MOF;

[0100] 2) Mix the Co@MOF obtained in the above steps with alcohol to obtain an alcohol solution, then add ammonia-alcohol solution, then add tetraethyl orthosilicate, and after reaction, obtain Co@MOF@SiO2;

[0101] 3) Under a protective atmosphere, the Co@MOF@SiO2 obtained in the above steps is pyrolyzed and then etched with an alkaline solution to obtain cobalt single-atom nanozyme CoSA;

[0102] 4) After mixing the cobalt single-atom nanozyme CoSA obtained in the above steps with water, the resulting CoSA dispersion is mixed again with dopamine solution. The pH value of the mixture is adjusted, and a light-protected reaction is carried out to obtain the CoSA@PDA composite system.

[0103] The present invention first mixes zinc salt, cobalt salt and organic solvent, then adds 2-methylimidazole organic solution and reacts to obtain Co@MOF.

[0104] In this invention, the zinc salt preferably includes zinc nitrate.

[0105] In this invention, the cobalt salt preferably includes cobalt nitrate.

[0106] In this invention, the solvents in the organic solvent and the 2-methylimidazole organic solution are preferably each independently selected from DMF and methanol.

[0107] In this invention, the molar ratio of the zinc salt to the cobalt salt is preferably (6~9):1, more preferably (6.5~8.5):1, and even more preferably (7~8):1.

[0108] In this invention, the mass ratio of the cobalt salt to 2-methylimidazole is preferably 1:(7~10), more preferably 1:(7.5~9.5), and even more preferably 1:(8~9).

[0109] In this invention, the mixing reaction time is preferably 10 to 14 hours, more preferably 10.5 to 13.5 hours, more preferably 11 to 13 hours, and even more preferably 11.5 to 12.5 hours.

[0110] In this invention, the Co@MOF obtained in the above steps is mixed with an alcohol to obtain an alcohol solution. Then, an ammonia-alcohol solution is added, followed by the addition of tetraethyl orthosilicate. After the reaction is carried out, Co@MOF@SiO2 is obtained.

[0111] In this invention, the alcohol preferably includes ethanol.

[0112] In this invention, the volume ratio of ammonia to alcohol in the ammonia-alcohol solution is preferably 1:(15~18), more preferably 1:(15.5~17.5), and even more preferably 1:(16~17).

[0113] In this invention, the mass ratio of tetraethyl orthosilicate to Co@MOF is preferably (3~4):1, more preferably (3.2~3.8):1, and even more preferably (3.4~3.6):1.

[0114] In this invention, in step 2), the reaction time is preferably 1 to 3 hours, more preferably 1.4 to 2.6 hours, even more preferably 1.8 to 2.2 hours, and specifically 2 hours.

[0115] In this invention, step 2) preferably includes a step of letting the mixture stand overnight after the reaction.

[0116] The present invention then pyrolyzes the Co@MOF@SiO2 obtained in the above steps under a protective atmosphere, and then etches it with an alkaline solution to obtain cobalt single-atom nanozyme CoSA.

[0117] In this invention, the pyrolysis temperature is preferably 900~1000℃, more preferably 920~980℃, even more preferably 940~960℃, and specifically 950℃.

[0118] In this invention, the pyrolysis time is preferably 3 to 5 hours, more preferably 3.4 to 4.6 hours, even more preferably 3.8 to 4.2 hours, and specifically 4 hours.

[0119] In this invention, the alkaline solution preferably includes NaOH solution and / or KOH solution, more preferably NaOH solution or KOH solution.

[0120] In this invention, the molar concentration of the alkaline solution is preferably 3-5M, more preferably 3.4-4.6M, even more preferably 3.8-4.2M, and specifically 4M.

[0121] In this invention, the etching time is preferably 10 to 14 hours, more preferably 10.5 to 13.5 hours, even more preferably 11 to 13 hours, even more preferably 11.5 to 12.5 hours, and specifically 12 hours.

[0122] Finally, the CoSA nanozyme obtained in the above steps is mixed with water to obtain a CoSA dispersion. This dispersion is then mixed with a dopamine solution, the pH of the mixture is adjusted, and a light-protected reaction is carried out to obtain a CoSA@PDA composite system.

[0123] In this invention, the dopamine solution preferably comprises an aqueous solution of dopamine.

[0124] In this invention, the pH value of the mixture is preferably adjusted to 7-10, more preferably 7.5-9.5, more preferably 8-9, and specifically 8.5.

[0125] In this invention, the light-avoidance reaction time is preferably 1 to 3 hours, more preferably 1.4 to 2.6 hours, even more preferably 1.8 to 2.2 hours, and specifically 2 hours.

[0126] This invention employs an encapsulation-pyrolysis-etching strategy to successfully prepare cobalt single-atom materials (CoSA) using Co@MOF as a template. First, Co... 2+ Bimetallic imidazole framework precursor Co@MOF was prepared by encapsulation in a MOF template. The Co@MOF exhibited a rhombic dodecahedral morphology with uniform particle size. To prevent Co atom aggregation during pyrolysis, the Co@MOF was coated with SiO2 to obtain a core-shell structure precursor Co@MOF@SiO2, which tightly coated the Co@MOF surface, forming a complete core-shell structure. This precursor was pyrolyzed under a N2 atmosphere, followed by etching of the pyrolysis products with sodium hydroxide solution to finally obtain Co SA. Co SA retained some of its original geometry and exhibited porous and layered characteristics due to structural collapse.

[0127] This invention provides the application of the polydopamine-modified cobalt single-atom nanozyme system described in any one of the above technical solutions, or the polydopamine-modified cobalt single-atom nanozyme system prepared by the preparation method described in any one of the above technical solutions, in the preparation of drugs for treating one or more of alcoholic gastric injury, alcoholic gastritis, alcoholic liver injury, and alcoholic hepatitis.

[0128] In this invention, the drug preferably comprises a cobalt single-atom nanozyme system and pharmaceutically acceptable excipients.

[0129] In this invention, the dosage form of the drug preferably includes oral preparations, injections, suppositories, inhalers, or dosage forms that can be directly applied to the liver and / or stomach.

[0130] In this invention, the mass content of the polydopamine-modified cobalt single-atom nanozyme system in the pharmaceutical preparation is preferably 0.5~10 mg / mL, more preferably 2~8 mg / mL, and even more preferably 4~6 mg / mL.

[0131] In this invention, the application preferably includes the application of cobalt single-atom nanozymes in the cobalt single-atom nanozyme system in achieving hydrogen peroxide decomposition and / or superoxide anion free radical scavenging, and more preferably the application of cobalt single-atom nanozymes in the cobalt single-atom nanozyme system in achieving hydrogen peroxide decomposition or superoxide anion free radical scavenging.

[0132] In this invention, the application preferably includes the use of polydopamine in the cobalt single-atom nanozyme system in enhancing bioadhesion and / or protecting the catalytic sites of cobalt single-atom nanozymes to maintain stability.

[0133] In this invention, the application preferably includes the application of polydopamine-modified cobalt single-atom nanozyme system in improving the effective retention time in the gastric mucosal damage area and enhancing the sustained antioxidant and anti-inflammatory effects.

[0134] The Co SA@PDA provided by this invention exhibits superior protective efficacy against alcoholic liver injury. Its advantages likely stem from two synergistic effects: firstly, PDA possesses excellent biocompatibility, providing a fundamental guarantee for its stable function in vivo; secondly, Co SA@PDA, through its superior oxidative stress regulation and anti-inflammatory activity, reduces alcohol-induced oxidative and inflammatory damage to hepatocytes, thereby achieving hepatocyte structural repair and lipid metabolism regulation, ultimately improving liver morphology and metabolic function. These results highlight the comprehensive advantages of Co SA@PDA in the prevention and treatment of alcoholic liver injury, providing crucial experimental support for subsequent intervention studies on alcohol-related liver diseases.

[0135] The present invention provides a polydopamine-modified cobalt single-atom nanozyme system and its preparation method, as well as its application in inhibiting alcoholic "gastric-liver" synergistic damage. The "polydopamine (PDA)-encapsulated cobalt single-atom nanozyme system (CoSA@PDA)" scheme designed in this invention selects CoSA (cobalt single-atom nanozyme) as the core functional unit, which, on the one hand, utilizes SOD-like activity to encapsulate O2... - The CoSA nanozyme system undergoes a two-step cascade reaction, simultaneously scavenging multiple ROS and blocking oxidative stress at its source. This is achieved through disproportionation into H2O2 and O2, and further decomposition of H2O2 into H2O and O2 via CAT-like activity. PDA was chosen as the encapsulation layer due to its dual functional characteristics: PDA's surface is rich in catechol groups, which can bind to proteins and polysaccharides on the surface of gastric mucosal epithelial cells via hydrogen bonds and hydrophobic interactions, prolonging the retention time of CoSA in the stomach and increasing drug concentration at the site of injury. Secondly, PDA can slowly degrade into dopamine monomers under physiological conditions with no significant cytotoxicity and can mask the metallic surface characteristics of CoSA, reducing its recognition by the immune system and minimizing organ accumulation. Furthermore, this CoSA@PDA nanozyme system can exert a synergistic protective effect through pathway regulation: on the one hand, it enhances tissue antioxidant capacity and reduces alcohol-mediated oxidative stress damage by activating the NRF2 signaling pathway; on the other hand, it blocks the inflammatory cascade reaction by inhibiting the NF-κB signaling pathway. This design not only achieves synergistic optimization of the functional properties and biosafety of single-atom nanozymes, but also provides novel nanobiomaterials for the clinical treatment of alcoholic "stomach-liver" synergistic injury, and opens up new research pathways for synergistic intervention of cross-organ injury.

[0136] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes a polydopamine-modified cobalt single-atom nanozyme system and its preparation method, as well as its application in inhibiting alcoholic "gastric-liver" synergistic damage. However, it should be understood that these embodiments are implemented under the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures, only to further illustrate the features and advantages of the present invention, and not to limit the scope of protection of the claims of the present invention. The scope of protection of the present invention is not limited to the following embodiments.

[0137] Main instruments and reagents

[0138] Zinc nitrate, cobalt nitrate, 2-methylimidazole, ammonia, tetraethyl orthosilicate, and DMPO were purchased from Sigma (Germany). Titanium sulfate, sodium hydroxide, omeprazole, DMF, methanol, and ethanol were purchased from Aladdin Biotech (China). 3% hydrogen peroxide solution was purchased from Likang Medical Technology Co., Ltd. (China). Dopamine hydrochloride, NBT, riboflavin, L-methionine, silymarin, and 4% paraformaldehyde were purchased from Yuanye Biotechnology Co., Ltd. (China). Fetal bovine serum was purchased from Ecosei Biotechnology Co., Ltd. (China). DMEM was purchased from Gibco Life Sciences (China). Trypsin and penicillin-streptomycin mixed solution were purchased from Shanghai Xiaopeng Biotechnology Co., Ltd. (China). CCK-8 was purchased from Invigentech (USA). PBS was purchased from Cytiva (UK). Enzyme-linked immunosorbent assay (ELISA) kits (IL-1β, TNF-α, and IL-6) and BCA protein assay kits were purchased from Beijing Solarbio Biotechnology Co., Ltd. (China). NO assay kits were purchased from Beyotime Biotechnology Co., Ltd. (China). MDA assay kit, GSH assay kit, TC / TG assay kit, AKP, ALT, AST, BUN, CRE, and UA assay kit were provided by Nanjing Jiancheng Biotechnology Institute (China). p65, p-p65, IκB, p-IκB, ZO-1, Occludin, Claudin-1, HO-1, and iNOS were purchased from Wuhan Sanying Biotechnology Co., Ltd. (China). NRF2 was purchased from Abmart Pharmaceutical Technology Co., Ltd. (China).

[0139] Example

[0140] Preparation of Co SA

[0141] Weigh 1.07 g of zinc nitrate and 0.135 g of cobalt nitrate, add them to a mixture of 24 mL of DMF and 6 mL of methanol, and stir at room temperature for 30 min. Separately, dissolve 1.16 g of 2-methylimidazole in a mixture of 24 mL of DMF and 6 mL of methanol, add it to the above solution, and stir at room temperature for 12 h to obtain Co@MOF. Take 108 mg of Co@MOF and add it to 108 mL of ethanol, sonicate until completely dispersed. Add 6.48 mL of ammonia to 108 mL of ethanol, add the ammonia-ethanol solution first under vigorous stirring, then add 408 μL of tetraethyl orthosilicate, stir for 2 h, and let stand overnight at room temperature to obtain Co@MOF@SiO2. Place the dried Co@MOF@SiO2 in a tube furnace and react at 950 °C under a nitrogen atmosphere for 4 h. Etch the product with 4 M NaOH solution for 12 h and centrifuge to obtain Co SA.

[0142] Preparation of Co SA@PDA composite system

[0143] Weigh a certain mass of Co SA, add 5 mL of deionized water, and ultrasonically disperse to obtain a Co SA dispersion. Accurately transfer 50 μL of 189.64 mg / mL dopamine solution and add it to the above dispersion. Adjust the pH of the mixture to 8.5 with NaOH solution. Stir the system magnetically in the dark and react for 2 h to obtain the Co SA@PDA composite material.

[0144] See Figure 1 , Figure 1 This is a TEM transmission electron microscope image of the bimetallic imidazole framework precursor Co@MOF prepared in this invention. Figure 1 As can be seen, Co@MOF exhibits a rhombic dodecahedral morphology with uniform particle size and a size of approximately 170 nm.

[0145] See Figure 2 , Figure 2 This is a scanning electron microscope (SEM) image of the core-shell precursor Co@MOF@SiO2 prepared for this invention. The image shows that SiO2 is tightly coated on the Co@MOF surface, forming a complete core-shell structure with a size of approximately 180 nm.

[0146] See Figure 3 , Figure 3The images show the characterization of Co SA prepared in this invention. (A) TEM image of Co SA; (B) AC-STEM image and magnified view of Co SA; (C) Elemental distribution map of Co SA, showing the distribution of Co (yellow), C (red), and N (green); (D) XANES spectra; (E) Fourier transform EXAFS spectra and (F) k-space of CoPc, Co SA, and Co Foil; (G) Extended X-ray absorption fine structure fitting curves of Co SA in R-space and (H) k-space; (IK) WT EXAFS of Co SA, Co Foil, and CoPc, respectively.

[0147] Figure 3 This indicates that Co SA retains some of its original geometry, but some structural collapse occurs due to the carbonization and shrinkage of the Co@MOF framework during pyrolysis. Figure 3 A). The dispersion state of cospecies was analyzed by aberration-corrected scanning transmission electron microscopy (AC-STEM). Figure 3 B), uniformly distributed isolated bright spots (marked with red circles) can be observed in the AC-STEM image of Co SA, confirming that Co is dispersed at the atomic level. Energy dispersive spectroscopy (EDS) elemental mapping results ( Figure 3 C) shows that the three elements Co, C, and N are all uniformly distributed in the material.

[0148] The surface elemental composition of Co SA was analyzed using X-ray photoelectron spectroscopy (XPS). Figure 4As shown in the image, the full spectrum reveals characteristic peaks for Co 2p, N 1s, C 1s, and O 1s, indicating that the material is mainly composed of Co, N, and C elements (where O originates from oxygen adsorbed from the air on the material surface). The high-resolution C 1s spectrum shows three absorption peaks, attributed to C=C / CC (~284.5 eV), C=N (~285.5 eV), and CN (~288 eV), respectively. These carbon species provide the coordinating carbon framework for Co single atoms. The high-resolution N 1s spectrum exhibits three absorption peaks, attributed to pyridine nitrogen (~398.3 eV), pyrrole nitrogen (~400.4 eV), and graphitic nitrogen (~401.9 eV), respectively. These nitrogen species provide a crucial coordination environment for the anchoring of Co single atoms. The high-resolution Co 2p spectrum shows four absorption peaks, attributed to the main peaks of 2p³ / 2 (~780.5 eV) and 2p¹ / ² (~795.8 eV) and satellite peaks (~785.1 eV, ~803.4 eV). The satellite peaks corroborate the oxidation state and coordination environment of Co, indicating that Co exists in a coordinated state rather than a nonmetallic state. See also Figure 4 , Figure 4 The X-ray photoelectron spectrum of CoSA prepared according to this invention.

[0149] Synchrotron radiation further analyzed the chemical state and coordination environment of Co. X-ray absorption near-edge structure (XANES) showed that the absorption edge energy of Co SA was between that of Co foil and Co phthalocyanine (Co Pc), indicating that the valence state of Co was in the range of 0 to +2. Figure 3 D); k 2 -Weighted Fourier transform of the extended X-ray absorption fine structure (EXAFS) analysis showed that Co SA had a distinct main peak at 1.38 Å, which was similar to the Co-N coordination peak (1.47 Å) in Co Pc (attributed to Co-N coordination) and significantly different from the Co-Co bond (2.14 Å) in Co Foil. Figure 3 E), confirming that the Co atom is mainly coordinated with N; the fitting results of the R space show that the coordination number of the Co atom is about 3.97 and the average bond length is about 1.91 Å, which indicates that the Co atom is in a four-coordinate N atom environment ( Figure 3 F, G and Table 1). Table 1 shows the Co K-edge detection data in EXAFS.

[0150] Table 1

[0151]

[0152] Furthermore, wavelet transform analysis of the k²-weighted EXAFS spectra was also conducted to provide the atomic configuration of the Co atom in the CoSA. The results showed that the maximum intensity of the CoSA occurred at approximately 4.0 Å⁻¹, similar to that of Co Pc (4.0 Å⁻¹), but significantly different from that of Co foil (7.0 Å⁻¹). Figure 3 H~ Figure 3 J). This further confirms that Co is dispersed in monatomic form and forms local coordination structures with N ligands.

[0153] H2O2 removal and O2 generation capabilities of Co SA@PDA

[0154] A reaction system was constructed by mixing 10 mM H2O2 solution and Co SA@PDA solution in deionized water to monitor the H2O2 consumption capacity of Co SA@PDA. 532 μL of 24% Ti(SO4)2 solution was added to a mixture of 3.332 mL H2SO4 and 16.136 mL water to prepare a Ti(SO4)2 precursor solution. After a specified time, the Co SA@PDA reaction solution and the Ti(SO4)2 precursor solution were mixed 1:1, and the H2O2 concentration at different concentrations and time points was determined by measuring the absorbance at 405 nm. H2O2 was repeatedly added under the same experimental conditions to detect the repeated H2O2 scavenging capacity of Co SA@PDA. In the dissolved oxygen experiment, the O2 concentration in the Co SA@PDA and H2O2 mixture was monitored every 10 s using an oxygen probe.

[0155] Co SA@PDA's O2 • ⁻Clearing ability

[0156] 130 mM L-methionine, 200 μM riboflavin, and 750 μM NBT were prepared separately; different concentrations of CoSA@PDA solutions (0, 25, 75, 125, 175, and 225 μg / mL) were also prepared for later use. 100 μL of CoSA@PDA solution was added to 300 μL of a mixed reaction system containing L-methionine, riboflavin, and NBT. After mixing, the mixture was irradiated under a 450 nm UV lamp for 1 min, and the absorbance at 560 nm was measured. O2 was detected using electron spin resonance spectroscopy. •For the signal, take 5 μL of 252.4 mM L-methionine, 5 μL of 0.8 mM riboflavin, 20 μL of DMPO, and 5 μL of 3 mg / mL Co SA solution, add them to 465 μL of methanol, mix well, and irradiate under a 450 nm UV lamp for 1 min before detection.

[0157] Cell viability assay

[0158] GES-1 and RAW264.7 cells were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 12 h. Subsequently, different concentrations of CoSA@PDA (2, 4, 6, and 8 μg / mL) were added and co-incubated with the cells for another 12 h. After incubation, the culture medium was discarded and replaced with fresh culture medium containing CCK8 reagent (culture medium: CCK8 = 10:1). 110 μL of reagent was added to each well, and the plate was incubated at 37°C for 45 minutes. The absorbance at 450 nm was measured, and cell viability was calculated.

[0159] GES-1 cells were seeded in 96-well plates and cultured for 12 hours. The cells were then treated with a final concentration of 450 μM H2O2 for 12 hours (the blank control group received an equal volume of culture medium). Subsequently, different concentrations of CoSA@PDA (2, 4, 6, and 8 μg / mL) were added to each well and co-incubated with the cells for 12 hours (the blank control group and model group received an equal volume of culture medium). After incubation, the culture medium was discarded, and the absorbance at 450 nm was read using the same method as described above to calculate cell viability.

[0160] Cellular MDA and GSH detection

[0161] GES-1 cells were seeded in culture dishes and incubated at 37°C in a 5% CO2 incubator for 12 h. The cells were then treated with a final concentration of 450 μM H2O2 for 12 h (the blank control group received an equal volume of basal medium without H2O2 treatment). Subsequently, different concentrations of CoSA@PDA (2, 4, 6, and 8 μg / mL) were added to each well for co-incubation for 12 h (the blank control group and model group received equal volumes of culture medium). After the intervention, cells were collected, resuspended in 0.3 mL of isotonic PBS buffer, and sonicated at 200 W for 5 min to disrupt the cells. The cell suspension was then analyzed using an MDA and GSH assay kit (Nanjing Jiancheng Biotechnology Research Institute) according to the manufacturer's instructions to determine the MDA and GSH content of the samples.

[0162] Intracellular ROS detection

[0163] GES-1 cells (8 × 10⁶ cells per well) 5Cells were seeded in 6-well plates and incubated at 37°C with 5% CO2 for 12 h. Cells were then treated with a final concentration of 450 μM H2O2 for 12 h (the blank control group received an equal volume of culture medium). Subsequently, different concentrations of CoSA@PDA (2, 4, 6, and 8 μg / mL) were added to each well and co-incubated with the cells for 12 h (the blank control group and model group received an equal volume of culture medium). After the intervention, staining was performed using the DCFH-DA detection kit and Hoechst 33342 staining solution according to the manufacturer's instructions. Finally, the cells were observed and images were captured under a fluorescence inverted microscope.

[0164] Intracellular NO and inflammatory factor level detection

[0165] RAW264.7 cells were spaced at 5 × 10⁶ cells per well. 4 Cells were incubated in 96-well plates at 37°C with 5% CO2 for 12 h. After cell attachment, they were treated with lipopolysaccharide (LPS) at a final concentration of 1 μg / mL for 12 h to construct a cell inflammation model (the blank control group received an equal volume of basal medium without LPS treatment). Subsequently, different concentrations of CoSA@PDA (2, 4, 6, and 8 μg / mL) were added to each well and co-incubated with the cells for 12 h (the blank control group and the model group received an equal volume of medium). After the intervention, the cell supernatant was collected for later use. The levels of each indicator in the supernatant were detected using a NO assay kit (Beyotime Biotechnology Co., Ltd.) and TNF-α, IL-6, and IL-1β assay kits (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions.

[0166] Acute alcoholic gastritis experiment in mice

[0167] Forty C57BB / 6 mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. They were acclimatized for 7 days before the experiment and housed in a constant temperature environment of 22±2℃ with a 12-hour light-dark cycle and free access to water. All animal experiments were conducted in accordance with the requirements of the Experimental Animal Ethics Committee of Changchun University of Traditional Chinese Medicine. Mice were randomly divided into 5 groups (n=8): Control group, Model group, Omeprazole positive control group (40 mg / kg), CoSA group (2.5 mg / kg), and CoSA@PDA group (2.5 mg / kg). All mice were acclimatized with normal food and water during the experiment. After the acclimatization period, the drug administration was performed by gavage every other day for a total of 5 times. The Control and Model groups were administered physiological saline by gavage for all 5 times, while the CoSA@PDA and CoSA groups were administered the corresponding drugs by gavage for each of the 5 times. The Omeprazole group was administered physiological saline by gavage for the first 4 times and Omeprazole solution by gavage for the 5th time. All groups (except the Control group) were administered anhydrous ethanol by gavage 2 hours after the last drug administration. During the experiment, changes in body weight were recorded. On the last day of the experiment, mice were euthanized 4 hours after being administered anhydrous ethanol by gavage, and blood, heart, liver, spleen, lungs, kidneys, and stomach were collected. Whole blood was used for routine blood tests; serum was separated for biochemical indicators and ELISA analysis; tissues such as heart, liver, spleen, lungs, kidneys, and stomach were stained with H&E, and a portion of the stomach was used for immunofluorescence analysis.

[0168] Mouse alcoholic hepatitis experiment

[0169] Forty C57BL / 6 mice, after completing the adaptive feeding period, were randomly divided into five groups (n=8 per group): Control group, Model group, Silymarin positive control group (80 mg / kg), CoSA group (2.5 mg / kg), and CoSA@PDA group (2.5 mg / kg). All mice had free access to food and water during the experiment. Each group of mice was administered the corresponding drug via gavage daily, followed by gavage administration of alcohol three hours after drug administration (10% alcohol for days 0-5, 30% alcohol for days 6-12, and 40% alcohol for days 13-24). The Control group was administered an equal volume of physiological saline via gavage. Weight changes were recorded throughout the experiment. On the last day of the experiment, the mice were euthanized, and blood, heart, liver, spleen, lungs, and kidneys were collected. Whole blood was used for complete blood count; serum was separated for biochemical indicators and ELISA analysis; H&E staining was performed on the heart, liver, spleen, lungs, and kidneys; liver tissue was stained with Oil Red O; and the remaining tissue was used for immunofluorescence analysis.

[0170] Blood and serum analysis

[0171] Mice whole blood was analyzed using a fully automated blood typing analyzer. Serum levels of IL-1β, TNF-α, and IL-6 were quantitatively determined according to the standard operating procedure of reagent kits from Beijing Solarbio Biotechnology Co., Ltd.; serum levels of TC, TG, AKP, ALT, AST, BUN, CRE, and UA were quantitatively determined according to the standard operating procedure of reagent kits from Nanjing Jiancheng Bioengineering Institute.

[0172] H&E and Oil Red O staining

[0173] Heart, liver, spleen, lung, kidney, and stomach tissues from mice were fixed with 4% paraformaldehyde. After embedding and paraffin sectioning, the tissues were stained with hematoxylin-eosin and Oil Red O staining solutions. Stained images were obtained under an optical microscope.

[0174] Detection of oxidative stress and lipid metabolism indicators

[0175] Mouse liver and stomach tissues were collected and homogenized into 10% (w / v) tissue homogenates at a ratio of 1:9 (tissue: sterile saline). The supernatant was collected by centrifugation at 4°C and 3000 r / min. The MDA and GSH levels in the samples were determined using an MDA and GSH assay kit (Nanjing Jiancheng Biotechnology Research Institute) according to the manufacturer's instructions. The TC and TG levels in the liver tissue samples were determined using a TC and TG assay kit (Nanjing Jiancheng Biotechnology Research Institute) according to the manufacturer's instructions.

[0176] Immunofluorescence analysis

[0177] Paraffin sections of the tissue to be tested were dewaxed and hydrated before antigen retrieval and blocking. Primary antibodies (p65, p-p65, IκB, p-IκB, iNOS, NRF2, HO-1, Occludin, Claudin-1, and ZO-1) were added sequentially, followed by washing and staining with FITC-labeled goat anti-rabbit IgG secondary antibody. Finally, cell nuclei were counterstained with DAPI, and images were captured under a fluorescence microscope (results showed that DAPI-stained cell nuclei appeared blue, and areas of positive expression of the target protein appeared red).

[0178] Statistical analysis

[0179] Statistical data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 10.0 and IBM SPSS 9.8.0 software. T-tests and one-way ANOVA were used to compare differences between data. For all experimental data (n≥3), p < 0.05 was considered statistically significant.

[0180] Co SA@PDA for H2O2 and O2 • Research on the clearance ability of ⁻

[0181] To address key pathological issues such as excessive oxidative stress and inflammation in acute alcoholic gastritis, this invention constructs a nanozyme system with both antioxidant and anti-inflammatory properties, and characterizes and analyzes its performance. Figure 6 Using cobalt single-atom nanozymes (CoSA) as the core catalytic unit, the decomposition of hydrogen peroxide (H2O2) and superoxide anion radicals (O2) can be efficiently achieved. • (⁻) Clearance provides a basis for combating oxidative stress and inflammatory response; secondly, CoSA is modified with polydopamine (PDA). PDA has excellent biocompatibility, and its hydroxyl and amino groups can enhance bioadhesion and protect the catalytic sites of CoSA to maintain stability. This system can effectively stay in the gastric mucosal damage area of ​​acute alcoholic gastritis, continuously resist oxidation and inflammation, and significantly improve the therapeutic effect.

[0182] To verify the successful encapsulation of Co SA in PDA, a series of characterization analyses were performed on the synthesized Co SA@PDA composite system. See [link to documentation]. Figure 5 , Figure 5 The following are characterization and analysis images of the CoSA@PDA composite system prepared in this invention. (A) TEM image of CoSA@PDA (scale bar: 100 nm); (B) Fourier transform infrared (FT-IR) spectra of PDA, CoSA, and CoSA@PDA; (C) Zeta potential diagrams of CoSA, PDA, and CoSA@PDA; (D) UV-Vis absorption spectra of H2O2 decomposition by CoSA@PDA at different concentrations (same concentration); (E) Time-dependent decomposition curve of H2O2 by CoSA@PDA (100 μg / mL); (F) Repeated catalytic H2O2 consumption performance of CoSA@PDA with repeated addition of H2O2 (10 mM); (G) ΔOxygen concentration change curve during the H2O2 catalytic reaction of CoSA@PDA (100 μg / mL); (H) Effect of different concentrations of CoSA@PDA on O2. • ⁻ Scavenging activity; (I) O2 before and after treatment with Co SA@PDA (100 μg / mL) • ⁻ Comparison of ESR spectra of absorption intensity.

[0183] Transmission electron microscopy (TEM) Figure 5 A) The image shows that CoSA@PDA clearly exhibits core-shell structure characteristics, intuitively indicating that PDA has been successfully encapsulated on the CoSA surface; Fourier transform infrared (FT-IR) spectroscopy (… Figure 5 B) The results showed that CoSA@PDA exhibited characteristic vibrational peaks of PDA, specifically the stretching vibration peak of the -OH / NH group (~3400 cm⁻¹). -1Characteristic absorption peaks of C=C / C=N groups (~1600 cm⁻¹) -1 This further corroborates the presence of PDA in the composite system; in addition, the Zeta potential test results show ( Figure 5 C) The Zeta potential of Co SA is -10.9 mV, that of PDA is -16.8 mV, while the Zeta potential of Co SA@PDA shifts significantly to -20.4 mV. The above morphological, spectral, and surface potential characterization results corroborate each other, all confirming that Co SA and PDA have been successfully recombinated.

[0184] Hydrogen peroxide and superoxide anion radicals are key reactive oxygen species pathologically overproduced in acute alcoholic gastritis. Their abnormal accumulation in the gastric mucosa further exacerbates oxidative damage to gastric mucosal epithelial cells, disrupts the integrity of the gastric mucosal barrier, and thus promotes pathological progression. First, this invention evaluated the performance of CoSA@PDA in catalyzing the decomposition of H2O2. Using the Ti(SO4)2 colorimetric method, under a fixed H2O2 concentration (10 mM), the UV-Vis absorption spectra of H2O2 under the action of different concentrations of CoSA@PDA were detected. Figure 5 D), the results showed that as the concentration of CoSA@PDA increased from 0 to 100 μg / mL, the absorption peak intensity at 405 nm gradually decreased, indicating that it has a concentration-dependent catalytic effect on the decomposition of H2O2; the H2O2 decomposition curves at different times ( Figure 5 E) Further, it was shown that compared with the control group containing only H2O2, under the action of 100 µg / mL Co SA@PDA, more than 90% of H2O2 (10 mM) was decomposed within 60 min, indicating that the material has highly efficient H2O2 catalytic decomposition efficiency. Considering the continuous generation of ROS in the pathological environment of acute alcoholic gastritis, in order to evaluate the performance stability of Co SA@PDA under dynamic damage, an equal amount of H2O2 was repeatedly added to the reaction system every 60 min, and the change in H2O2 concentration was dynamically monitored. Figure 5 F). The results show that after multiple H2O2 replenishments, Co SA@PDA can still rapidly and fully catalyze the decomposition of H2O2 without a significant decrease in catalytic efficiency, confirming that the material possesses excellent repeatable catalytic stability. Furthermore, dissolved oxygen experiments ( Figure 5 G) The results showed that in the Co SA@PDA and H2O2 co-incubation system, the concentration of oxygen in the solution increased significantly over time and gradually reached a plateau, while the H2O2 group alone showed no significant change, directly confirming that Co SA@PDA can efficiently catalyze the decomposition of H2O2 to generate oxygen.

[0185] Besides H2O2, superoxide anion radicals (O2) •⁻) As another key reactive oxygen species, its excessive generation can also exacerbate oxidative stress damage. Therefore, it is necessary to investigate the effect of CoSA@PDA on O2. • The scavenging ability of CoSA@PDA is of great significance. Different concentrations of CoSA@PDA have different effects on O2. • ⁻ Clearance experiment ( Figure 5 H) shows that as the concentration of CoSA@PDA increases, the O2 in the system... • The relative concentration of ⁻ gradually decreased, suggesting that CoSA@PDA has a positive effect on O2. • The scavenging effect of O2 is concentration-dependent; to further verify this effect, electron spin resonance (ESR) technology was used with DMPO as a spin trap to detect O2. • ⁻( Figure 5 I) The results showed that significant O2 was observable in the control group. • The characteristic signal peak was significantly weakened or even disappeared after processing with Co SA@PDA, confirming that Co SA@PDA has the ability to efficiently remove O2. • ⁻ Ability.

[0186] The above results confirm that CoSA@PDA possesses both highly efficient catalase-like and superoxide dismutase-like synergistic catalytic activities, enabling it to jointly eliminate excess H2O2 and O2 generated in the inflammatory areas of the gastric mucosa during acute alcoholic gastritis. • ⁻ and other ROS, thereby significantly reducing local oxidative stress damage.

[0187] See Figure 6 , Figure 6 The following figures illustrate the performance analysis of the CoSA@PDA composite system prepared in this invention. (A) GES-1 cell viability; (B) intracellular malondialdehyde (MDA) content in GES-1 cells; (C) intracellular glutathione (GSH) level in GES-1 cells; (D) fluorescence image of changes in intracellular reactive oxygen species (ROS) levels in GES-1 cells (scale bar: 100µm); (E) quantitative analysis of DCF fluorescence intensity; (F) intracellular NO content in RAW264.7 cells; (GI) intracellular TNF-α, IL-6, and IL-1β levels in RAW264.7 cells (ELISA). Compared with the control group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, and ***p<0.001, analyzed by one-way ANOVA.

[0188] Regulatory Role of CoSA@PDA in Cellular Oxidative Stress and Inflammatory Response

[0189] Acute alcoholic gastric injury is characterized by the superposition and synergistic exacerbation of gastric mucosal damage by inflammatory response and oxidative stress. During this pathological process, gastric tissue abnormally generates large amounts of ROS (mainly hydrogen peroxide) and inflammatory factors. These molecules are core targets for evaluating the effectiveness of interventions. Based on this, this invention uses ROS and inflammatory factors as core detection indicators to systematically explore the in vitro antioxidant and anti-inflammatory activities of CoSA@PDA. To simulate oxidative damage characteristics in vitro, an oxidative damage model was constructed using human gastric mucosal epithelial GES-1 cells as the research subject. H2O2 was selected as the modeling agent, and concentration gradient screening experiments were conducted. The results showed that a H2O2 concentration of 450 μM effectively induced oxidative stress damage in GES-1 cells while avoiding interference from excessive cell death in subsequent detections. Therefore, 450 μM H2O2 was selected as the optimal modeling concentration.

[0190] To clarify the safety of CoSA@PDA, its effect on GES-1 cells was first verified through cytotoxicity experiments (see [link to study]). Figure 7 , Figure 7 (This study investigated the effect of different concentrations of Co SA@PDA on the viability of GES-1 cells. The results showed that when the concentration of Co SA@PDA was in the range of 0~8 μg / mL, the survival rate of GES-1 cells remained above 90%, with no statistically significant difference compared with the control group (p>0.05), confirming that Co SA@PDA had no significant toxicity to GES-1 cells within this concentration range.

[0191] Subsequently, the effect of CoSA@PDA on the survival rate of GES-1 cells after H2O2 damage was investigated. Figure 6 A) The survival rate of cells in the model group treated with H2O2 was significantly reduced (Model vs Control, ###p < 0.001); however, as the concentration of Co SA@PDA increased, the survival rate of damaged cells significantly recovered (Co SA@PDA vs Model, ***p < 0.001), indicating that Co SA@PDA can effectively alleviate H2O2-mediated GES-1 cell damage.

[0192] To further elucidate the antioxidant effect of CoSA@PDA, oxidative stress-related indicators in GES-1 cells after H2O2 damage were detected. Malondialdehyde (MDA), as a characteristic product of lipid peroxidation, directly reflects the degree of oxidative damage. The results showed that the MDA content in GES-1 cells of the model group was significantly increased (…). Figure 6(B) (Model vs Control, p < 0.001); After intervention with different concentrations of CoSA@PDA, the intracellular MDA content gradually decreased, indicating that CoSA@PDA can effectively alleviate H2O2-induced cellular lipid peroxidation damage. Glutathione (GSH) is a key endogenous antioxidant molecule in cells, and its level changes can reflect antioxidant capacity. The results showed that the intracellular GSH level in the model group was significantly lower than that in the control group (B). Figure 6 C) (Model vs Control, ###p < 0.001); however, the intracellular GSH level significantly increased after treatment with Co SA@PDA (Co SA@PDA vs Model, ***p < 0.001), further supporting the evidence that Co SA@PDA can inhibit H2O2-induced oxidative damage in GES-1 cells and thereby regulate the level of endogenous antioxidant molecules.

[0193] Abnormal ROS accumulation is a key inducing factor leading to gastric mucosal cell damage. This invention uses 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence staining to specifically detect the accumulation level of ROS in GES-1 cells after H2O2 damage. Figure 6 D, scale bar: 100 μm). Fluorescence imaging and quantitative analysis of fluorescence intensity ( Figure 6 E) The results showed that the Model group cells exhibited strong green fluorescence (indicating a large accumulation of ROS); after treatment with Co SA@PDA, the fluorescence intensity gradually decreased with increasing drug concentration. This indicates that Co SA@PDA can reduce the level of intracellular ROS in a concentration-dependent manner, confirming that Co SA@PDA has a highly efficient scavenging effect on H2O2-induced intracellular ROS in GES-1 cells.

[0194] Macrophages, as core effector cells in the inflammatory response, play a crucial role in disease progression through activation and the release of inflammatory mediators. Therefore, this invention constructs an inflammatory response model using mouse RAW264.7 macrophages to systematically evaluate the in vitro anti-inflammatory activity of CoSA@PDA. First, to clarify the safety of CoSA@PDA, its effects on RAW264.7 cells were verified through cytotoxicity experiments (see [link to relevant documentation]). Figure 8 , Figure 8 (This study investigated the effect of different concentrations of Co SA@PDA on the viability of RAW264.7 cells. The results showed that when the concentration of Co SA@PDA was in the range of 0~8 μg / mL, the survival rate of RAW264.7 cells remained above 90%, confirming that Co SA@PDA had no significant toxicity to cells within this concentration range and could be used for subsequent detection of inflammatory factors.)

[0195] Nitric oxide (NO) is a key mediator in the inflammatory response, and its secretion level directly reflects the degree of inflammatory activation. This study evaluated the anti-inflammatory effect of CoSA@PDA by detecting NO content. Figure 6 F). The results showed that the intracellular NO content in RAW264.7 cells induced by 1 μg / mL lipopolysaccharide (LPS) was significantly increased (Model vs Control, p < 0.001); after Co SA@PDA intervention, the intracellular NO production decreased in a concentration-dependent manner, suggesting that Co SA@PDA can effectively inhibit the abnormal release of NO in RAW264.7 cells. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) are core pro-inflammatory cytokines mediating the inflammatory cascade, and their secretion levels are core molecular markers of cellular inflammatory activation and the intensity of inflammatory responses. To further elucidate the molecular mechanism by which Co SA@PDA regulates inflammatory responses, this invention uses enzyme-linked immunosorbent assay (ELISA). The levels of the three pro-inflammatory cytokines secreted by RAW264.7 cells were detected using an ELISA method. The results showed that the secretion levels of TNF-α, IL-6, and IL-1β in the RAW264.7 cell model group treated with inflammation induction were significantly lower. Figure 6 The levels of G and I were significantly increased (Model vs Control, ###p < 0.001), indicating that the cells had been successfully induced into an inflammatory activation state; however, after treatment with different concentrations of CoSA@PDA, the secretion of the three pro-inflammatory cytokines was downregulated in a concentration-dependent manner.

[0196] This result further confirms that CoSA@PDA can effectively inhibit the inflammatory activation process of RAW264.7 cells by downregulating the secretion of pro-inflammatory cytokines, providing molecular-level support for its in vitro anti-inflammatory activity.

[0197] The therapeutic effect of Co SA@PDA on mice with acute alcoholic gastritis

[0198] See Figure 9 , Figure 9This is a series of images illustrating the therapeutic effect of Co SA@PDA on alcohol-induced acute gastritis in mice. (A) Schematic diagram of the experimental procedure for the acute alcoholic gastritis animal model; (B) Body weight change curve; (C) Morphological images of gastric tissue damage in each group of mice (scale bar is 1 cm); (D) H&E stained pathological sections of gastric tissue (scale bar: 300 μm); (EF) MDA and GSH content in gastric tissue; (GI) Levels of TNF-α, IL-6, and IL-1β in gastric tissue (ELISA method); (JL) Serum levels of AST, ALT, and AKP. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, analyzed by one-way ANOVA.

[0199] A schematic diagram of the experimental procedure for an animal model of acute alcoholic gastritis is shown in Figure F. Figure 9 As shown in Figure A, C57BL / 6 mice were randomly divided into 5 groups (n=5): Control group, Model group, Omeprazole positive drug group (40 mg / kg), CoSA group (2.5 mg / kg), and CoSA@PDA group (2.5 mg / kg). All mice had normal food and water intake during the experiment. After intervention according to the experimental procedure diagram, the effects were evaluated from the dimensions of weight change, gastric tissue morphology, histopathology (H&E staining), serum inflammatory factors, and biochemical indicators.

[0200] Results of weight change ( Figure 9 (B) showed that there was no significant difference in the overall body weight of mice in each group over time. The pre-modeling fasting procedure resulted in a slight decrease in body weight at the end of the experiment, but the weight change trends in each treatment group and the control group were basically consistent, indicating that CoSA and CoSA@PDA have good biosafety in experimental animals and did not produce obvious toxic reactions. (Gastric tissue morphology observation) Figure 9 C) showed that the gastric mucosa in the Control group was smooth, without pathological changes such as bleeding or erosion; the gastric mucosa in the Model group showed significant erosion and obvious focal bleeding, presenting typical gastric injury. After drug intervention, the degree of gastric mucosal erosion and bleeding was relieved to varying degrees. Among them, the gastric tissue appearance of the CoSA@PDA group was closest to normal, and the improvement effect was better than that of the Omeprazole group and the CoSA group. Gastric tissue H&E staining ( Figure 9D) Further histological verification showed that the gastric mucosal epithelium in the Control group was intact, the glands were arranged in a regular manner, and there was no obvious inflammatory cell infiltration; the gastric mucosal epithelium in the Model group was sloughed off, the glandular structure was disordered, and there was inflammatory cell infiltration. The Co SA@PDA group more effectively restored the integrity of the epithelium, reduced inflammatory cell infiltration, and normalized the glandular structure. Its repair effect on the pathological damage of the gastric mucosa was better than that of the Omeprazole group and the CoSA group.

[0201] Oxidative stress is a key damaging mechanism in the development and progression of acute alcoholic gastritis. Based on this, the levels of malondialdehyde (MDA), an oxidative damage product, and glutathione (GSH), an antioxidant, in gastric tissue were measured. Figure 9 E, Figure 9 F). The results showed that the Model group had significantly increased MDA levels and significantly decreased GSH levels due to alcohol-induced oxidative damage (Model vs Control, ***p < 0.001) (Model vs Control, ***p < 0.001). After intervention, Co SA@PDA significantly reduced MDA levels (Co SA@PDA vs Model, ***p < 0.001) and increased GSH levels in gastric tissue (Co SA@PDA vs Model, ***p < 0.001), and its regulatory effect was superior to that of the Omeprazole group and the Co SA group, suggesting that Co SA@PDA can effectively alleviate alcohol-induced oxidative stress damage.

[0202] Serum inflammatory factor test results ( Figure 9 The G~I study showed that the levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β were significantly upregulated in the Model group (Model vs Control, ***p < 0.001), suggesting that alcohol-induced acute gastric injury is accompanied by severe local and systemic inflammatory response activation. However, after CoSA@PDA intervention, the levels of pro-inflammatory cytokines significantly decreased (CoSA@PDA vs Model, ***p < 0.001), and the anti-inflammatory regulatory effect was optimal, indicating that CoSA@PDA can significantly inhibit the excessive secretion of pro-inflammatory factors and efficiently maintain the homeostasis of the inflammatory microenvironment by regulating inflammatory factors.

[0203] Serum biochemical index test results ( Figure 9The study showed no significant difference in alkaline phosphatase (AKP) levels among the groups, which is speculated to be due to the short duration of alcohol exposure, which has not yet significantly affected AKP-related metabolic pathways. However, the Model group exhibited abnormally elevated levels of key liver function indicators such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), suggesting that alcohol, while inducing gastric mucosal damage, also caused hepatocellular damage and abnormal liver function, forming a synergistic "stomach-liver" damage effect. After Co SA@PDA intervention, the abnormally elevated ALT and AST levels were significantly reduced (Co SA@PDA vs Model, ***p < 0.001), and the improvement was superior to that of the Omeprazole and Co SA groups. This indicates that Co SA@PDA not only effectively alleviates alcohol-induced gastric tissue damage and metabolic disorders but also improves alcohol-induced liver damage, demonstrating a comprehensive advantage in multi-organ protection.

[0204] Results of H&E staining of major organs (see [link]) Figure 10 , Figure 10 In the acute alcoholic gastritis treatment experiment of this invention, H&E staining images of the heart, liver, spleen, lungs, and kidneys of mice in each group showed that the liver tissue of the Model group exhibited mild vacuolar structures and cellular edema-like changes, which highly consistent with the detection results of the above-mentioned serum biochemical indicators (elevated ALT and AST), further confirming the hepatocellular damage induced by alcohol exposure. After treatment with CoSA@PDA, the degree of the above-mentioned pathological damage was significantly reduced, and the cytoplasmic loosening phenomenon was significantly improved, while no obvious pathological damage was observed in other major organs such as the heart, spleen, lungs, and kidneys. The results indicate that under the intervention dose and cycle of this experiment, neither CoSA nor CoSA@PDA produced significant toxic effects on the body.

[0205] In addition, there were no significant differences in renal function indicators such as blood urea nitrogen (BUN), serum creatinine (CRE), and uric acid (UA) between the treatment group and the control group (see [link to relevant documentation]). Figure 11 , Figure 11 This is a statistical chart showing the serum UA, BUN, and CRE levels in mice from each group during the acute alcoholic gastritis treatment experiment of this invention. Blood routine indicators, including lymphocyte count (LYM), intermediate cell count (MID), granulocyte count (GRAN), total white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), red blood cell distribution width coefficient of variation (RDW-CV), and red blood cell distribution width standard deviation (RDW-SD), showed no significant differences between the treatment and control groups (Fig. S10). Combined with changes in body weight and H&E staining results of major organs, multiple dimensions confirmed that CoSA and CoSA@PDA did not produce significant toxic reactions, had no adverse effects on experimental animals, and possessed good biosafety.

[0206] In summary, Co SA@PDA demonstrated outstanding performance in the treatment of alcohol-induced acute gastritis. Its advantage is presumably due to the gastric adhesion properties of its component PDA, which forms a physical protective barrier on the gastric mucosa, directly reducing the direct chemical damage to the mucosa caused by alcohol. Furthermore, through synergistic antioxidant and anti-inflammatory effects, combined with its gastric mucosal repair and liver damage protection efficacy, it exhibits excellent results in improving gastric tissue morphology and pathological damage, regulating oxidative stress balance, modulating pro-inflammatory factors, and repairing liver function. These results fully demonstrate the synergistic and multi-organ protective advantages of Co SA@PDA in the treatment of alcohol-induced acute gastritis, providing experimental support for subsequent intervention studies in related diseases.

[0207] The therapeutic effect of Co SA@PDA on mice with alcoholic hepatitis

[0208] See Figure 12 , Figure 12 This is a series of images illustrating the therapeutic effect of Co SA@PDA on mice with alcoholic hepatitis in this invention. (A) Schematic diagram of the experimental procedure for the animal model of chronic alcoholic liver injury; (B) Liver index histogram; (C) H&E stained pathological section of liver tissue and a magnified view (scale bar: 300 μm); (D) Oil Red O stained pathological section of liver tissue (scale bar: 500 μm); (EF) Serum triglyceride (TG) and total cholesterol (TC) levels; (GI) Serum AST, ALT, and AKP levels; (JK) MDA and GSH levels in liver tissue; (LN) Levels of TNF-α, IL-6, and IL-1β in gastric tissue (ELISA method); *p < 0.05, **p < 0.01, ***p < 0.001, analyzed by one-way ANOVA.

[0209] The CoSA@PDA complex system exhibits a significant protective effect against alcohol-induced gastric injury; it also shows potential protective effects against liver injury associated with alcohol exposure. Based on the above research, we plan to further systematically explore the interventional effect of CoSA@PDA on alcoholic hepatitis. A schematic diagram of the experimental procedure for an animal model of alcoholic liver injury is shown below. Figure 12As shown in Figure A, C57BL / 6 mice were randomly divided into 5 groups (n=8 per group): Control group, Model group, Silymarin positive control group (80 mg / kg), CoSA group (2.5 mg / kg), and CoSA@PDA group (2.5 mg / kg). All mice had free access to food and water during the experiment. After 7 days of acclimatization, each group of mice was intervened daily according to the experimental procedure diagram. The effects of each intervention group were comprehensively evaluated by monitoring changes in mouse body weight, organ indices, histopathological analysis, serum inflammatory factors, and biochemical indicators.

[0210] Weight monitoring results showed that the mice in the Control group maintained a normal physiological growth trend in weight, while the mice in the Model group showed a continuous decline in weight due to the damage of alcohol to the body's metabolism and nutrient absorption. After drug intervention, the weight of mice in each treatment group showed a significant recovery trend, among which the Co SA@PDA group had the best effect and its weight level was closest to that of the Control group.

[0211] Liver index ( Figure 12 B) The results of spleen index testing showed that the liver and spleen indices of mice in the Control group were within the normal physiological range, while the liver and spleen indices of mice in the Model group were significantly elevated (Model vs Control, ***p< 0.001). This may be due to the direct damage of hepatocyte structure by alcohol metabolites, leading to hepatocyte edema and fatty degeneration, resulting in elevated liver indices, as well as alcohol's disruption of the body's immune function, damage to the normal immune structure of the spleen, and promotion of splenomegaly. After drug intervention, the liver and spleen indices of all groups significantly decreased and tended to normal. The CoSA@PDA group showed the best improvement, with its index levels not significantly different from the Control group and superior to the Silymarin and CoSA groups, indicating that CoSA@PDA can repair alcohol-induced liver and spleen damage and restore the normal morphology and function of the organs.

[0212] Liver histological H&E staining results ( Figure 12 C) shows that in the Control group, hepatocytes were arranged in a regular manner without degeneration, necrosis, or structural disorder. In the Model group, hepatocytes showed obvious degeneration, accompanied by the formation of a large number of fatty vacuoles and disordered liver tissue structure, suggesting that alcohol-induced liver damage can cause significant liver damage. After intervention with each drug, the degree of hepatocyte damage in each group of mice was significantly reduced, the liver tissue structure gradually recovered, the hepatocyte arrangement became more regular, and no obvious pathological changes were observed. Among them, the liver tissue morphology of the CoSA@PDA group was closest to that of normal, with only a very small number of hepatocytes showing slight degeneration, further confirming its advantage in repairing alcohol-induced liver tissue structural damage.

[0213] One of the pathological features of alcoholic hepatitis is the abnormal accumulation of large amounts of lipids in the liver. This process is an early marker of alcohol-induced liver damage. To clarify the specific morphology and metabolic characteristics of lipid accumulation in the liver under alcohol exposure, this study used Oil Red O staining to visually observe the distribution and accumulation degree of lipid droplets in liver tissue, while simultaneously detecting the levels of triglycerides (TG) and total cholesterol (TC). Oil Red O staining of liver tissue ( Figure 12 In the D (scale bar: 500 μm) diagram, the red stained areas represent lipid deposition within hepatocytes. In the Control group, only a very small amount of scattered pale red signal was observed in the liver tissue, indicating minimal lipid deposition in hepatocytes under normal physiological conditions. In the Model group, large, dense, bright red areas appeared in the liver tissue, indicating fatty degeneration of hepatocytes after alcohol induction, accompanied by a large accumulation of lipids within the cells. After drug intervention, the red stained areas in hepatocytes of all groups were significantly reduced; the CoSA@PDA group had the smallest red area, with only a small number of scattered lipid droplets. Serum triglycerides (TG) ( Figure 12 E) and total cholesterol (TC) Figure 12 F) Content detection further verified that the Model group had significantly elevated TG and TC levels due to lipid metabolism disorder (Model vs Control, ***p < 0.001); the CoSA@PDA group significantly reduced TG and TC levels (CoSA@PDA vs Model, ***p < 0.001), and the improvement effect was better than that of the Silymarin group and the CoSA group. This fully demonstrates that CoSA@PDA significantly reduces alcohol-induced hepatic lipid deposition and fully reflects its excellent efficacy in improving hepatic steatosis and regulating lipid metabolism.

[0214] AST and ALT are mainly found in hepatocytes, while AKP participates in hepatocyte membrane transport. Elevated levels of all three indicate mitochondrial damage or disruption of cell membrane integrity in hepatocytes, serving as hallmark indicators of liver injury. Therefore, the levels of AST, ALT, and AKP in mouse serum were measured. Figure 12The results showed that all indicators in the Control group were within the normal reference range. In the Model group, due to alcohol-induced hepatocellular damage, AST, ALT, and AKP levels were significantly elevated (Model vs Control, ***p <0.001), indicating alcohol-induced liver damage and abnormal liver function. However, after Co SA@PDA intervention, the abnormally elevated AST, ALT, and AKP levels were significantly downregulated (Co SA@PDA vs Model, ***p < 0.001), and the downregulation was greater than that in the Silymarin and Co SA groups. This indicates that Co SA@PDA can effectively repair alcohol-induced hepatocellular damage, restore normal liver function, and alleviate liver metabolic disorders.

[0215] Oxidative stress is a key pathogenic mechanism of alcoholic liver injury. This study assessed oxidative stress status by detecting the levels of malondialdehyde (MDA), an oxidative damage product, and glutathione (GSH), an antioxidant, in liver tissue. Results showed that MDA and GSH levels in the Control group were within the normal reference range, while those in the Model group were significantly lower. Figure 12 J~K) levels were significantly increased (Model vs Control, ***p < 0.001), while GSH levels (Fig. 5K) were significantly decreased (Model vs Control, ***p < 0.001). After intervention, the Co SA@PDA group significantly reduced MDA levels and increased GSH levels (both Co SA@PDA vs Model, ***p < 0.001), and its regulatory effect was superior to that of the Silymarin group and the Co SA group, suggesting that Co SA@PDA can effectively alleviate alcohol-induced oxidative stress damage.

[0216] Serum inflammatory factor test results ( Figure 12 The results (L~N) showed that in the Control group, the levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β remained at low physiological levels; in the Model group, due to alcohol activation of liver inflammatory signaling pathways, the levels of pro-inflammatory cytokines were significantly upregulated (Model vs Control, ***p < 0.001), suggesting that alcoholic liver injury is accompanied by a severe inflammatory response. After CoSA@PDA intervention, the overexpression of pro-inflammatory cytokines was significantly inhibited (CoSA@PDA vs Model, ***p < 0.001), and its anti-inflammatory regulatory effect was superior to that of the Silymarin group and the CoSA group, demonstrating the intervention effect of CoSA@PDA on inflammation and its ability to effectively maintain the homeostasis of the liver inflammatory microenvironment.

[0217] Furthermore, there were no significant differences in renal function indicators such as blood urea nitrogen (BUN), serum creatinine (CRE), and uric acid (UA) between the treatment and control groups. Blood routine indicators, including LYM, MID, GRAN, WBC, RBC, HGB, RDW-CV, and RDW-SD, also showed no significant differences between the treatment and control groups. H&E staining of major organs showed that CoSA@PDA could alleviate mild splenic inflammation, and no significant pathological damage was observed in other major organs. These findings confirm that CoSA and CoSA@PDA have no adverse effects on experimental animals, exhibit good biosafety, and did not produce significant toxic reactions.

[0218] In summary, Co SA@PDA exhibits the best protective effect against alcoholic liver injury, likely due to two synergistic effects: firstly, PDA's good biocompatibility provides a fundamental guarantee for its stable function in vivo; secondly, Co SA@PDA, through its excellent oxidative stress regulation and anti-inflammatory activity, reduces alcohol-induced oxidative and inflammatory damage to hepatocytes, thereby achieving hepatocyte structural repair and lipid metabolism regulation, ultimately improving liver morphology and metabolic function. These results highlight the comprehensive advantages of Co SA@PDA in the prevention and treatment of alcoholic liver injury, providing crucial experimental support for subsequent intervention studies on alcohol-related liver diseases.

[0219] The foregoing has provided a detailed description of the polydopamine-modified cobalt single-atom nanozyme system and its preparation method, as well as its application in inhibiting alcoholic "gastric-liver" synergistic damage. Specific examples have been used to illustrate the principles and implementation methods of the invention. The descriptions of these embodiments are merely for the purpose of helping to understand the method and core ideas of the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that for those skilled in the art, several improvements and modifications can be made to the invention without departing from the principles of the invention, and these improvements and modifications also fall within the scope of protection of the claims. The scope of protection of this patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements that are not different from the textual description of the claims, or if they include equivalent structural elements that are not substantially different from the textual description of the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A polydopamine modified cobalt monatomic nanoszyme system, characterized in that, The application relates to a cobalt monatomic nanoscale enzyme and a polydopamine coating layer coated on the surface of the cobalt monatomic nanoscale enzyme. The cobalt monatomic nanoscale enzyme is obtained by pyrolysis and etching of a precursor Co@MOF@SiO2. The particle size of the cobalt monatomic nanoscale enzyme is 100-300 nm.

2. The Co-soloatom nanoszyme system according to claim 1, wherein, The cobalt monatomic nanoscale enzyme has a dodecahedron-like morphology. The thickness of the polydopamine coating layer is 30-60 nm. The mass ratio of the cobalt monatomic nanoscale enzyme to polydopamine is (1-3):

1. The mass content of Co in the cobalt monatomic nanoscale enzyme is 1%-2%. The polydopamine-modified cobalt monatomic nanoscale enzyme system has a core-shell structure. The precursor Co@MOF@SiO2 has a core-shell structure and comprises a Co@MOF core and a SiO2 coating layer coated on the surface of the Co@MOF.

3. The Co-soloatom nanoszyme system according to claim 1, wherein, The particle size of the precursor Co@MOF@SiO2 is 130-350 nm. The thickness of the SiO2 coating layer is 5-30 nm. The mass ratio of the Co@MOF to SiO2 is 1:(0.4-1). The Co@MOF is specifically Co-doped ZIF-8. The Co@MOF has a rhombohedral dodecahedron morphology. The cobalt monatomic nanoscale enzyme partially retains the morphology of the precursor Co@MOF@SiO2 and has part of the structure collapsed.

4. The Co-soloatom nanoszyme system according to claim 3, wherein, The pyrolysis is specifically pyrolysis under a protective atmosphere. The etching is specifically alkali etching. In the cobalt monatomic nanoscale enzyme, Co is dispersed in the form of monatomic atoms and forms a local coordination structure with N ligands. The N includes graphite-type nitrogen, pyrrole-type nitrogen and pyridine-type nitrogen. The cobalt monatomic nanoscale enzyme system is a nanoscale enzyme system for treating one or more of alcoholic gastric injury, alcoholic gastritis, alcoholic liver injury and alcoholic hepatitis. The application further relates to a preparation method of the cobalt monatomic nanoscale enzyme system.

5. A preparation method of a polydopamine modified cobalt monatomic nanoszyme system, characterized in that, 1) mixing a zinc salt, a cobalt salt and an organic solvent, then adding an organic 2-methyl imidazole solution to mix and react, and obtaining Co@MOF; 2) mixing the Co@MOF obtained in the above step with alcohol to obtain an alcohol solution, then adding ammonia water-alcohol solution, adding tetraethyl orthosilicate, and reacting to obtain Co@MOF@SiO2; 3) pyrolyzing the Co@MOF@SiO2 obtained in the above step under a protective atmosphere, then etching with an alkali solution to obtain cobalt monatomic nanoscale enzyme Co SA; 4) mixing the cobalt monatomic nanoscale enzyme Co SA obtained in the above step with water to obtain a Co SA dispersion, then mixing with a dopamine solution again, adjusting the pH value of the mixed system, and performing light-shielded reaction to obtain a Co SA@PDA composite system. The zinc salt comprises zinc nitrate.

6. The preparation method according to claim 5, characterized in that, The cobalt salt comprises cobalt nitrate. The solvents in the organic solvent and the organic 2-methyl imidazole solution are each independently selected from DMF and methanol. The molar ratio of the zinc salt to the cobalt salt is (6-9):

1. The mass ratio of the cobalt salt to 2-methyl imidazole is 1:(7-10). The mixing reaction time is 10-14 hours. The alcohol comprises ethanol.

7. The preparation method according to claim 5, characterized in that, In the ammonia water-alcohol solution, the volume ratio of ammonia water to alcohol is 1:(15-18). ​ The mass ratio of the tetraethyl orthosilicate to the Co@MOF is (3-4):1; In the step 2), the reaction time is 1-3 hours; In the step 2), after the reaction, a step of standing overnight is further included; The pyrolysis temperature is 900-1000℃; The pyrolysis time is 3-5 hours.

8. The preparation method according to claim 5, characterized in that, The alkali solution includes a NaOH solution and / or a KOH solution; The molar concentration of the alkali solution is 3-5M; The etching time is 10-14 hours; The dopamine solution includes a dopamine aqueous solution; The adjustment of the pH value of the mixed system specifically is adjusting the pH value to 7-10; The light-avoiding reaction time is 1-3 hours.

9. Use of the polydopamine-modified cobalt single-atom nanoszyme system of any one of claims 1-4 or the polydopamine-modified cobalt single-atom nanoszyme system prepared by the preparation method of any one of claims 5-8 in the preparation of a drug for treating one or more of alcoholic gastric injury, alcoholic gastritis, alcoholic liver injury, and alcoholic hepatitis.

10. Use according to claim 9, characterized in that, The drug includes the cobalt single-atom nanoszyme system and a pharmaceutically acceptable excipient; The preparation form of the drug includes oral preparation, injection, suppository, inhalation, or a form that can be directly applied to the liver and / or stomach; In the preparation of the drug, the mass content of the polydopamine-modified cobalt single-atom nanoszyme system is 0.5-10 mg / mL; The application specifically includes the application of the cobalt single-atom nanoszyme in the cobalt single-atom nanoszyme system in the decomposition of hydrogen peroxide and / or the clearance of superoxide anion radicals; The application specifically includes the application of the polydopamine in the cobalt single-atom nanoszyme system in the enhancement of biological adhesion and / or the protection of the catalytic site of the cobalt single-atom nanoszyme to maintain stability; The application further includes the application of the polydopamine-modified cobalt single-atom nanoszyme system in the improvement of the effective residence time in the gastric mucosa injury area and the improvement of the sustained antioxidant and anti-inflammatory effect.

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