| Background:the structural development of hippocampal neurons is closely related to brain learning and memory,therefore abnormal structural development of hippocampal neurons may lead to neurodegenerative diseases.AD is the most common neurodegenerative disease in the world.More and more studies have found that during the development of AD,a large number of reactive oxygen species(ROS)will be produced,which will cause oxidative stress,lead to neuronal death and aggravate the pathogenesis of AD.At present,the antioxidant therapy of AD has become a research hotspot,and many nanomaterials have been studied as antioxidants to antagonize oxidation and treat AD.Therefore,we prepared Nb2C MXene nanosheets with high electrical conductivity,strong antioxidant capacity and biodegradability to explore the promoting effect of the nanosheets on the structural development of normal hippocampal neurons and improving the growth of Glu-induced injured hippocampal neurons by antagonizing oxidative stress.Objective:Firstly,Nb2C MXene nanosheets with biodegradability,high conductivity,strong antioxidant stress,safety and low toxicity were prepared by two-step liquid phase stripping method.The prepared Nb2C MXene nanosheets were used as substrates to culture hippocampal neurons to explore the promoting effect of Nb2C MXene nanosheets on the growth and development of hippocampal neurons.Through the injury of hippocampal neurons induced by Glu,the AD cell model was established to explore the repair and protective effect of Nb2C MXene nanosheets on injured hippocampal neurons,so as to provide a new method for the effective treatment of AD.Methods:(1)Phalloidin/β Ⅲ-Tubulin and MAP2/GFAP immunofluorescence staining were used to identify mouse hippocampal neurons cultured in vitro for 1 day and 7 days,respectively.(2)Nb2C MXene nanosheets were prepared by a two-step liquid phase stripping method of HF etching and TPAOH intercalation.The materials were characterized by SEM,TEM,EDS,XRD,DLS and Zeta potentials.At the same time,the total antioxidant capacity test kit was used to detect the antioxidant capacity of Nb2C MXene nanosheets.(3)The CCK-8 kit was used to detect the activity of hippocampal neurons in the control group and nanomaterial treatment group cultured in vitro for 7 days.(4)the hippocampal neurons of the control group and the nanomaterial-treated group were stained with β Ⅲ-Tubulin immunofluorescence for 5 days.The neurons were scanned by the Neurolucida system and their axons and dendrites were measured.(5)on the 6th day,hippocampal neurons were injured by Glu of different concentrations(0,2,6,10,14,18 mM)for 30 minutes,and the AD cell model was established.On the 7th day,the viability of hippocampal neurons was detected by the CCK-8 kit,and an appropriate concentration of Glu was selected to model hippocampal neurons,to provide the AD cell model with appropriate degree of injury for follow-up experiments.(6)the CCK-8 kit was used to detect the cell viability of hippocampal neurons in the control group,the Glu damage group and the Glu+Nb2C material group cultured in vitro for 7 days.(7)ROS activity detection kit was used to detect the ROS activity of hippocampal neurons in the control group,the Glu damage group and the Glu+Nb2C material group cultured in vitro for 7 days.(8)the mitochondrial membrane potential of hippocampal neurons in the control group,the Glu damage group and the Glu+Nb2C material group cultured in vitro for 7 days was detected by the mitochondrial membrane potential kit.(9)the Superoxide dismutase(Superoxide Dismutase,SOD)activity detection kit was used to detect the SOD activity of hippocampal neurons in the control group,the Glu damage group and the Glu+Nb2C material group cultured in vitro for 7 days.(10)the hippocampal neurons of the control group,the Glu damage group and the Glu+Nb2C material group cultured in vitro for 7 days were stained with β Ⅲ-Tubulin immunofluorescence.The neurons were scanned by the Neurolucida system and their axons and dendrites were measured.(11)the expression levels of MAP2 in hippocampal neurons of the control group,the Glu damage group and the Glu+Nb2C material group cultured in vitro for 7 days were measured by the Western Blot method.Results:(1)From the results of Phalloidin/β Ⅲ-Tubulin immunofluorescence staining,we can see that there are more filamentous pseudopodia and flaky pseudopodia in hippocampal neurons cultured in vitro for 1 day.We saw that the hippocampal neurons cultured for 7 days were all MAP2 positive cells by MAP2/GFAP immunofluorescence staining,indicating that the hippocampal neurons cultured in vitro grew well and had high purity.(2)the SEM results showed that the Nb2C MXene etched by HF showed a layered structure;the results of TEM,EDS,XRD,DLS and Zeta potentials showed that Nb2C MXene nanosheetswas a thin lamellar structure with a size of about 50 nm,containing C and Nb elements,and the hydration radius was 150～200 nm,indicating that Nb2C MXene nanosheets had good hydrophilicity,and the Zeta potential was about-22 mV,indicating that Nb2C MXene nanosheets was negatively charged and difficult to enter cells;the results of the total antioxidant capacity detection kit showed that Nb2C MXene nanosheets had a strong antioxidant capacity.(3)CCK-8 results showed that the Nb2C MXene nanosheets had no obvious toxic effect on cells up to the concentration of 200 μg/mL,indicating that the nanosheets had the advantages of safety and low toxicity.(4)the results of βⅢ-Tubulin immunofluorescence staining of normal hippocampal neurons cultured in vitro for 5 days showed that compared with the control group,the length of axons and dendrites of hippocampal neurons treated with Nb2C MXene nanosheets increased significantly,and the number of branch nodes and dendrites also increased,indicating that Nb2C MXene nanosheets can promote the growth and development of hippocampal neurons.(5)CCK-8 results showed that the neuronal viability of 30 min treated with Glu of the concentration of 14 mM was about 66.79±3.44%.In the follow-up experiment,the AD cell model was established with this concentration and time point.(6)CCK-8 results showed that 12～100 μg/mL Nb2C MXene nanosheets could significantly improve the cell viability of injured neurons,and subsequent experiments will be carried out with 12 μg/mL and 25μg/mL Nb2C MXene nanosheets.(7)The results of the ROS activity detection kit showed that the ROS activity of hippocampal neurons in the Glu+Nb2C group was lower than that in the Glu damage group.(8)The results of the mitochondrial membrane potential kit showed that the mitochondrial membrane potential of hippocampal neurons in the Glu+Nb2C group was higher than that in the Glu injury group.(9)The results of the SOD activity detection kit showed that the SOD activity of hippocampal neurons in the Glu+Nb2C group was higher than that in the Glu injury group.(10)The results of βⅢ-Tubulin immunofluorescence staining showed that the length of axons and dendrites of hippocampal neurons and the number of branch nodes and dendrites of hippocampal neurons in the Glu+Nb2C group were significantly longer than those in the Glu injury group.(11)the Western blot results showed that the protein expression levels of MAP2 in hippocampal neurons in the Glu+Nb2C group were higher than those in the Glu damage group.Conclusion:(1)Nb2C MXene nanosheets can promote the growth and development of hippocampal neurons by increasing the length of axons and dendrites and the number of branch nodes and dendrites of normal hippocampal neurons.(2)Nb2C MXene nanosheets can repair and protect the injured hippocampal neurons by increasing the cell viability,decreasing the ROS level,increasing the SOD activity,recovering the mitochondrial membrane potential,lengthening the axons and dendrites,increasing the number of branch nodes and dendrites,and improving the protein expression levels of MAP2. |
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