Research On The Antioxidant Damage Effect And Mechanism Of Manganese Based Nanozyme

Nanozymes are nanomaterials with enzyme like activity,which have a wide range of potential applications in biomedical fields such as biomolecular detection,in vivo imaging,biosensing,therapy,drug delivery,and environmental remediation due to their adjustable physicochemical properties and good stability.So far,a large number of nanozymes have been synthesized and applied,but research on the toxicity and biological effects of nanozymes is not yet in-depth,and their biological mechanisms of action have not been fully elucidated.This study synthesized a manganese based nanozymes（MnO₂-BSA NPs）with multiple antioxidant mimetic enzyme activities through BSA biomineralization.The safety of MnO₂-BSA NPs was comprehensively studied using multiple cell and animal models.The biological effects of MnO₂-BSA NPs on antioxidant damage were comprehensively explored in vitro and in vivo,and the mechanism of MnO₂-BSA NPs on antioxidant damage was systematically elucidated,providing scientific basis for the study of the biological effects and mechanisms of nanozymes.PartⅠSynthesis,characterization,and antioxidant activities of manganese based nanozymeObjective:To synthesize manganese based nanozyme（MnO₂-BSA NPs）and characterize its composition,structure,and morphology.To investigate the stability and multi antioxidant mimetic enzyme activities of MnO₂-BSA NPs.Methods:1.MnO₂-BSA NPs was synthesized through BSA biomineralization,the concentrations of reactants KMnO₄and BSA,pH value of the solution,and reaction time were optimized.Characterization of MnO₂-BSA NPs was performed using transmission electron microscopy,marvin particle size analyzer,UV visible spectrophotometer,fourier transform infrared spectrometer,and inductively coupled plasma mass spectrometer.2.The elemental composition,atomic valence states,molecular structure,and chemical bonds of MnO₂-BSA NPs were determined by X-ray diffraction,high-resolution transmission electron microscopy lattice analysis,and X-ray photoelectron spectroscopy.Potassium periodate spectrophotometry combined with ICP-MS was used to determine the manganese content in MnO₂-BSA NPs.The stability of MnO₂-BSA NPs in H₂O,PBS,and DMEM was analyzed by hydration particle size and Zeta potential analysis.3.The H₂O₂scavenging ability of MnO₂-BSA NPs was determined by UV spectroscopy.Terephthalic acid（TA）was used as a fluorescent probe to determine the·OH scavenging activity of MnO₂-BSA NPs.The CAT,SOD,and GPx simulated enzyme activities of MnO₂-BSA NPs were detected using enzyme activity assay kits,and the thermal stability and pH stability of MnO₂-BSA NPs were compared to SOD natural enzyme.The kinetic parameters of MnO₂-BSA NPs were calculated using the Michaelis Menten curve.The POD and OXD activities of MnO₂-BSA NPs were detected under pH=3～8 conditions through TMB colorimetric reaction.Results:1.The optimal reaction conditions for the synthesis of MnO₂-BSA NPs were BSA concentration of 25 mg/mL,KMnO₄concentration of 100 mM,pH=11.23,and reaction time of 3 hours.2.The particle size of MnO₂-BSA NPs was 5.68±0.08 nm,the zeta potential was-12.3±2.7 mV,and the hydrated particle size was 20.52±0.21 nm.The UV visible characteristic peak of MnO₂nanomaterials appeared at 350 nm.3.The XRD results showed that diffraction peaks corresponding to MnO₂（311）and（440）crystal planes（JCPDF-#42-1169）were observed at 2θ=37.06°and65.65°,respectively;A lattice stripe with a spacing of 0.242 nm corresponding to the MnO₂（311）crystal plane could be observed in the HRTEM image.XPS results showed that MnO₂-BSA NPs were composed of elements C,N,O,and Mn;The two strong peaks at 642.4eV and 653.9eV were the characteristic peaks of MnO₂at Mn2p3/2 and Mn 2p1/2,respectively;There were three types of oxygen-containing functional groups in MnO₂-BSA NPs,with Mn-O at 531.4eV,C-O at 533.0eV,and H-O-H at 534.8eV.4.Compared with the H₂O,PBS and DMEM solutions of newly prepared MnO₂-BSA NPs,there was no statistically significant difference in hydration particle size after 24 hours of storage（P>0.05）.5.MnO₂-BSA NPs exhibited multi enzyme activities.MnO₂-BSA NPs exhibited OXD and POD activity under pH=3～6 conditions;Under pH 7.2 conditions,MnO₂-BSA exhibited CAT,SOD,and GPx mimetic enzyme activities,which could eliminate H₂O₂,·OH and O₂^·-;The CAT and GPx mimetic enzyme activities of MnO₂-BSA were dose-dependent,and the thermal and pH stability of MnO₂-BSA was superior to that of natural enzymes;When using GSH as the substrate,the K_Mand V_maxvalues of MnO₂-BSA NPs were 4.03 mM and 0.016 mM min^-1,respectively;When H₂O₂was used as the substrate,the K_Mand V_maxvalues were 0.15 mM and0.004 mM min^-1,respectively.Conclusions:1.The particle size of MnO₂-BSA NPs was uniform and the dispersibility was excellent.MnO₂-BSA NPs exhibited excellent solubility and stability in three media:H₂O,PBS and DMEM.2.MnO₂-BSA NPs exhibited excellent CAT,SOD,and GPx antioxidant mimetic enzyme activities,which could efficiently remove H₂O₂,·OH,and O₂^·-.The thermal stability and pH stability of MnO₂-BSA NPs were superior to those of SOD natural enzymes.PartⅡResearch on the toxicity of manganese based nanozymeObjective:To clarify the biological safety of MnO₂-BSA NPs and explore their cytotoxicity and acute toxicity to mice.Methods:1.The cellular uptake of MnO₂-BSA NPs was observed through biological transmission electron microscopy.The cytotoxicity of MnO₂-BSA NPs on BEAS-2B and HEK293 cells incubated for 12～72 hours was detected using MTT assay.The concentration range of MnO₂-BSA NPs was 0～400μg/mL.2.The safety of intravenous administration of MnO₂-BSA NPs was evaluated through hemolysis tests.3.The toxicity of MnO₂-BSA NPs in vivo was analyzed through acute toxicity testing,20 ICR mice were randomly divided into two groups:the MnO₂-BSA NPs treatment group and the control group,with half male and half female in each group（n=10）.The administration group received three tail vein injections of 320 mg/kg MnO₂-BSA NPs within 24 hours,with a time interval of 8 hours between each injection.The daily status of mice was observed and their weight was recorded.After14 days of feeding,the mice were euthanized and the blood and tissue samples were collected to analyze tissue pathology,whole blood parameters,and blood biochemical indicators.Results:1.The results of biological transmission electron microscopy showed that MnO₂-BSA NPs can be internalized into the cytoplasm of normal somatic cells.2.MnO₂-BSA NPs incubated at a dose of 2.5～10.0μg/mL for 12～72 hours did not affect the viability of BEAS-2B cells,and did not affect the viability of HEK293cells within the concentration range of 0.625～10.0μg/mL,indicating that lower doses of MnO₂-BSA NPs are not toxic to cells（P>0.05）.3.The hemolysis test results showed that MnO₂-BSA NPs within the concentration range of 50-1000μg/mL did not cause hemolysis in red blood cells（P>0.05）.4.The results of the acute toxicity test show that there were no significant changes in survival,body weight,organ index,tissue pathology,whole blood parameters,and liver and kidney function of all mice,indicating that MnO₂-BSA NPs did not have acute toxicity to mice when administered through tail vein injection（P>0.05）.Conclusions:1.MnO₂-BSA NPs below 20μg/mL do not exhibit cytotoxicity.2.MnO₂-BSA NPs do not cause red blood cell hemolysis.MnO₂-BSA NPs at a concentration of 1000 mg/kg/d do not exhibit toxicity in mice.PartⅢ Research on the antioxidant damage effect of manganese based nanozymeObjective:To clarify the protective effects of MnO₂-BSA NPs against oxidative damage in vitro and in vivo,and to explore the effects of MnO₂-BSA NPs on regulating oxidative stress,inhibiting cell apoptosis,and inflammation.Methods:1.The toxic doses of H₂O₂on BEAS-2B and HEK293 cells were determined using the MTT method.2.Cell experiment grouping:For BEAS-2B cells,the experimental groups were control group,injury group,low-dose protection group,medium dose protection group,and high-dose protection group.The experimental groups for HEK293 cells were control group,MnO₂-BSA NPs group,injury group,low-dose protection group,medium dose protection group,and high-dose protection group.3.The cell apoptosis and intracellular ROS levels were detected through flow cytometry and cell fluorescence imaging.Cell cycle and mitochondrial membrane potential detection were performed by flow cytometry.After ultrasound fragmentation,the cells were subjected to protein quantification,and the activities of CAT,SOD,and GPx,as well as the content of GSH and MDA,were measured.4.Establishment of a mouse RM-AKI model,in which female ICR mice（6～8weeks old,18～22 g）were adaptively fed for 5 days and stopped drinking water,but were free to eat for 15 hours.Inject 50%glycerol at a dose of 6 mL/kg into the muscles of both hind limbs of the mice,and then all mice resumed free drinking and eating.5.In vivo experimental grouping,female ICR mice were randomly divided into 5groups（n=6）,each being a blank control group;MnO₂BSA NPs group;AKI group;MnO₂BSA NPs protection group;NAC protection group.6.The levels of BUN and CRE were detected in mouse serum,and the levels of CAT,SOD,GPx,GSH,and MDA in mouse kidney tissue and serum were measured.HO-1,KIM-1,IL-1β,and TNF-αin mouse kidney tissue were determined using ELISA method.Pathological damage to mouse kidney tissue was detected through H&E staining.Results:1.Compared to the BEAS-2B cell injury group,the high-dose protection group with MnO₂-BSA NPs significantly reduced the proportion of cell apoptosis/necrosis,significantly decreased intracellular ROS levels,and significantly increased CAT and GPx activities（P<0.05）.2.Compared to the HEK293 cell injury group,the high-dose protection group with MnO₂-BSA NPs significantly reduced the proportion of cell apoptosis/necrosis,significantly decreased intracellular ROS levels,significantly regulated cell cycle and mitochondrial membrane potential,and significantly increased intracellular CAT,SOD,and GSH levels,while significantly decreased MDA content.The results indicate that MnO₂-BSA NPs have a protective effect against cellular oxidative damage（P<0.05）.3.MnO₂-BSA NPs can restore weight gain in AKI mice.H&E staining results show that MnO₂-BSA NPs can slow down renal tissue lesions in AKI mice and significantly reduce serum BUN and CRE levels.Compared to the AKI group,the levels of CAT,SOD,GPx,and GSH in the kidneys and serum of the MnO₂-BSA NPs protection group were significantly increased,while the MDA content was significantly reduced.The results indicate that MnO₂-BSA NPs can protect the renal function of AKI mice and help regulate the redox balance in the body of AKI mice（P<0.05）.4.ELISA results showed that compared to the AKI group,the KIM-1,TNF-α,and IL-1βin the MnO₂-BSA NPs protected group were significantly reduced,while HO-1 was significantly increased.The results indicate that MnO₂-BSA NPs can alleviate the severity of AKI and inhibit inflammation（P<0.05）.5.MnO₂-BSA NPs can prolong the survival of AKI mice,promote their weight gain and renal function recovery（P<0.05）.Conclusions:1.MnO₂-BSA NPs can exert a protective effect of inhibiting cell apoptosis and clearing excess intracellular ROS in H₂O₂induced oxidative damage in BEAS-2B cells.2.MnO₂-BSA NPs can effectively alleviate oxidative damage in HEK293 cells and have a cellular protective effect by regulating intracellular redox homeostasis.3.MnO₂-BSA NPs can slow down weight loss in AKI mice;Relieve symptoms of renal tissue lesions in AKI mice;Restore mouse kidney function and regulate oxidative stress and inflammation in mice.Part Ⅳ Research on the mechanism of antioxidant damage of manganese based nanozymeObjective:To clarify the mechanism of protective effect of MnO₂-BSA NPs against oxidative damage,analyze the effects of MnO₂-BSA NPs on the expression of Nrf2/HO-1,PI3K/Akt/NF-κB and endogenous mitochondrial cell apoptosis pathway related genes,and elucidate the mechanism of MnO₂-BSA NPs regulating mitochondrial cell apoptosis pathway via Nrf2/HO-1 signaling pathway.Methods:1.The experimental groups of HEK293 cells inhibited by Nrf2 were:control group,MnO₂-BSA NPs group,ML385 group,injury group,MnO₂-BSA NPs protection group,and Nrf2 inhibited MnO₂-BSA NPs protection group.2.The expression levels of endogenous mitochondrial apoptosis pathway related proteins such asγH2AX,Cyt C,Apaf-1,Cleaved caspase-3,Bax,and Bcl-2 in BEAS-2B cells were analyzed using Western blot.The m RNA and protein expression levels ofγH2AX,Bax,Bcl-2,Cyt C,and Caspase-3 genes in HEK293 cells were measured by RT q PCR and Western blot.3.The m RNA and protein expression levels of Nrf2,keap-1,HO-1,SOD2,PI3K,Akt,and NF-κB genes in HEK293 cells were analyzed through RT q PCR and Western blot.4.ML385 was used to specifically inhibit the expression of Nrf2 gene in HEK293 cells,the cell apoptosis/necrosis and ROS levels in each group were analyzed through MTT and cell fluorescence imaging,and the protein expression levels ofγH2AX,Cyt C,Caspase-3,Bax,and Bcl-2 were determined through Western blot.5.Western blot was used to detect the protein expression levels of Bax,Bcl-2,Cyt C,Caspase-3,γH2AX,PI3K,Akt,NF-κB,Nrf2,keap-1,and HO-1 genes in mouse kidneys.Results:1.Compared to the BEAS-2B cell injury group,the high-dose protection group of MnO₂-BSA NPs significantly reduced the expression levels ofγH2AX,Cyt C,Apaf-1,Cleaved caspase-3,and Bax/Bcl-2 proteins in the cells.Compared with the HEK293 cell injury group,the m RNA and protein expression levels ofγH2AX,Bax,Cyt C,and Caspase-3 genes in the high-dose protection group of MnO₂-BSA NPs showed significant changes,indicating that MnO₂-BSA NPs can inhibit oxidative damage by regulating the mitochondrial cell apoptosis pathway（P<0.05）.2.Compared to the AKI group,the expression levels ofγH2AX,Cyt C,Cleaved caspase-3,and Bax/Bcl-2 proteins in the kidneys of MnO₂-BSA NPs protected mice were significantly reduced,indicating that MnO₂-BSA NPs can inhibit oxidative damage in mouse kidneys by regulating the mitochondrial cell apoptosis pathway（P<0.05）.3.Compared to the HEK293 cell injury group,the high-dose protection group of MnO₂-BSA NPs significantly increased the m RNA and protein expression levels of Nrf2 and HO-1 genes,and significantly increased the expression of SOD2 protein.The m RNA expression level of NF-κB gene was significantly reduced,and the expression levels of PI3K,p-Akt,and p-NF-κB proteins were significantly reduced.This indicates that MnO₂-BSA NPs can regulate oxidative stress in vitro through the Nrf2/HO-1 and PI3K/Akt/NF-κB signaling pathways（P<0.05）.4.Compared to the AKI group,the expression level of Nrf2 protein in the kidneys of MnO₂-BSA NPs protected mice was significantly increased,while the expression of keap-1 protein was significantly reduced.The expression of PI3K,p-Akt,and p-NF-κB proteins was significantly decreased,indicating that MnO₂-BSA NPs can alleviate oxidative stress and inflammation by regulating the Nrf2/HO-1 and PI3K/Akt/NF-κB signaling pathways in vivo（P<0.05）.5.Compared to the MnO₂-BSA NPs protection group,the MnO₂-BSA NPs protection group inhibited by Nrf2 showed a significant increase in cell apoptosis rate and ROS level,as well as a significant increase in protein expression levels ofγH2AX,Cyt C,Caspase-3,and Bax/Bcl-2,indicating that the inhibition of Nrf2/HO-1 pathway affects the regulation of MnO₂-BSA NPs on mitochondrial cell apoptosis pathway（P<0.05）.Conclusions:1.MnO₂-BSA NPs resist oxidative damage in HEK293 cells through Nrf2/HO-1,PI3K/Akt/NF-κB,and mitochondrial apoptosis pathways.2.MnO₂-BSA NPs regulate oxidative damage and inflammation via Nrf2/HO-1,PI3K/Akt/NF-κB,and mitochondrial cell apoptosis pathways in vivo.3.MnO₂-BSA NPs regulate mitochondrial cell apoptosis pathways through the Nrf2/HO-1 signaling pathway,thereby resisting oxidative damage.