Effects Of Nasal Delivery Of Selenium Nanoparticles On P-gp Expression And Neuroprotective Effect In Hippocampus Of Temporal Lobe Epilepsy Rats

Part Ⅰ:Construction and characterization of the nasal delivery system of selenium nanoparticles modified by P-gp monoclonal antibodyObjective:To construct a targeted delivery system for selenium nanoparticles(SeNPs)and P-gp monoclonal antibody modified selenium nanoparticles(Pgp@SeNPs),and to preliminically characterize its performance,so as to provide a basis for the following research.Materials and Methods:Selenium nanoparticles(SeNPs)were constructed by mixing quantitative selenite and PEG solution.Then SeNPs was mixed with P-gp monoclonal antibody,lyophilized to obtain the sample,dissolved and washed to obtain P-gp@SeNPs.Sstructure and antibacterial properties of SeNPs and P-gp@SeNPs were determined by ultraviolet coefficient spectroscopy,transmission electron microscopy(TEM)and toxic plate method.Results:In the UV spectrum analysis of SeNPs,there were two characteristic absorption peaks at 210 nm to 259 nm.When the absorption peak of SeNPs was blue shifted at the late stage of P-gp monoclonal antibody modification,the characteristic peak occurred at 6 nm.TEM results showed that the particle properties of SeNPs and Pgp@SeNPs samples showed spherical shape,and the particle size was relatively uniform,and the particle size was about 61.7 nm.The concentration of P-gp monoclonal antibody supported on the surface of selenium nanoparticles was 2.55 mg/mL.Pgp@SeNPs samples have good antibacterial effect on Staphylococcus aureus and Escherichia coli,and the antibacterial rate of both can reach about 100%.Conclusion:P-gp@SeNPs samples with small particle size and uniform dispersion were successfully prepared in this part.The sample stability and antibacterial activity were high,which is expected to become a new high-efficiency substance and provide the basis for the following research.Part Ⅱ:Study on the effect of intranasal delivery of selenium nanoparticles on P-gp expression in hippocampus of rats with temporal lobe epilepsyObjective:To study the construction of P-gp@SeNPs intranasal delivery system and its effect on the expression of P-gp in hippocampus of rats with temporal lobe epilepsy.Materials and Methods:(1)Amygdala kainic acid injection model was established(TLE rats),and P-gp expression levels(WB)in hippocampal tissues of rats were measured on 1d,7d,14d and 30d after injection.Te highest point(30d)was selected as the time of administration for subsequent experiments.Iflour594-labeled P-gp@SeNPs were administered through the intranasal cavity for 6 h and brain sections were taken for microvascular endothelial(GLUT1)specific staining.(2)Sham group(control group),Model group(TLE rats group),SeNPs(oral)group and P-gp@SeNPs(intranasal delivery)group were set up.The behavioral seizures of each group was determined.Western blot and immunohistochemistry were used to detect P-gp protein expression in brain tissue of rats in each group.Results:(1)With the prolongation of postoperative time,the expression of P-gp protein in rat brain tissue was gradually increased(P<0.05).Microvascular endothelial(GLUT1)specific staining showed that P-gp@SeNPs colocalized with microvascular endothelial cells in normal rat hippocampal tissue,suggesting that P-gp@SeNPs can effectively accumulate in microvascular endothelial cells,the main expression region of P-gp,after entering the brain through the intranasal pathway.(2)Compared with Sham group,P-gp protein expression in hippocampus of TLE rats increased significantly,while P-gp protein expression in hippocampus of TLE rats could be down-regulated by SeNPs and P-gp@SeNPs intervention(P<0.05).Conclusion:(1)The expression level of P-gp in the hippocampal tissue at the operative side showed a continuous upward trend within 30 days after the KA injection in amygdaloid.(2)Oral administration of SeNPs and intranasal delivery of P-gp@SeNPs significantly inhibited P-gp overexpression after epileptic seizure.Part Ⅲ:Studies of the neuroprotective effect of intranasal delivery of selenium nanoparticles on temporal lobe epileptic ratsMaterials and Methods:Sham group(control group),Model group(TLE rats group),SeNPs group and P-gp@SeNPs group were set up.The behavioral serzures of each group were determined.The pathological changes of hippocampus were analyzed by Nissl staining and HE staining.The concentrations of inflammatory factors(NF-κB,IL-6,IL-1β,TNF-α),glutathione reductase(GR),glutathione peroxidase(GPx),catalase(CAT)and superoxide dismutase(SOD)in hippocampal tissues of each group were detected by ELISA.TUNEL method was used to detect the apoptosis of hippocampal cells in each group.The expressions of apoptosis-related proteins Bcl-2,Caspase3 and Bax were analyzed by Western blot and RT-qPCR.Results:In the control group,the hippocampal tissue structure and morphology were normal,the hippocampal cells were uniform and arranged neatly,the size of nuclei and nucleolus were normal,and the number of Nissl bodies was large.After the construction of the TLE model,the neurons in rat hippocampal tissue were moderate to varying degrees,the cells showed clear vacuolation,the nucleus and cytoplasm were non-uniformly colored,and the cell structure and shape were disordered.At the same time,the volume of neuron cells gradually shrank,and the phenomenon of hyperchromatism showed a decrease in the number of Nissl bodies.SeNPs can improve the histopathological morphology of rat hippocampus,and the cell morphology and structure are restored compared with model group,while the number of Nissl bodies is increased.After P-gp monoclonal antibody modification,the pathological morphology of hippocampus was further improved,the color of neurons was more uniform,the shape was more regular,and the number of Nissl bodies was further increased.Compared with Sham group,the construction of TLE rat model could increase the expressions of inflammatory factors(NF-κB,IL-6,IL-1β,TNF-α),Caspase3 and Bax in hippocampus,and decrease the levels of GR,CAT,GPx,SOD and Bcl-2(P<0.05).Compared with Model group,SeNPs and P-gp@SeNPs can down-regulate the levels of P-gp protein,inflammatory factors(NF-κB,IL-6,IL-1β,TNF-α)and the expression of Caspase3 and Bax in hippocampus of TLE rats.The levels of GR,CAT,GPx,SOD and Bcl-2 were up-regulated(P<0.05).Conclusion:(1)The delivery of SeNPs in TLE rats inhibited the development of neuroinflammation by alleviating the oxidative stress caused by epileptic seizures,alleviated the apoptosis of neurons in rat hippocampus,thus played a neuroprotective role.(2)Nasal delivery of P-gp@SeNPs further enhances this neuroprotective effect by bypassing theBBB directly into the brain and targeted to areas with high P-gp expression.Part Ⅳ:Study on the neuroprotective mechanism of intranasal delivery of selenium nanoparticlesin rats with temporal lobe epilepsyObjective:To explore the molecular mechanism of neuroprotective effects of SeNPs and P-gp@SeNPs delivery system on TLE rats.Materials and Methods:(1)Rat amygdala kindling Model(TLE rats)was constructed,and Sham group(control group),Model group(TLE rats group),SeNPs group and P-gp@SeNPs group were set up.The expression levels of key proteins of MAPK signaling pathway p-p38,p-JNK and p-ERK1/2 in hippocampal tissues of rats in each group were detected by Western-blot analysis.(2)MAPK signaling pathway inhibitor VX-702 was used to intervene the Model group.Then model group,VX-702 group,SeNPs group,P-GP-@SENps group,and PGP-@SENPS+VX-702 group were set;The expression levels of the key proteins of MAPK signaling pathway p-p38,p-JNK and p-ERK1/2 in hippocampal tissues of rats in each group were detected by Western-blot analysis.To determine the inhibitory effect of P-gp@SeNPs intranasal delivery system on MAPK signaling pathway in rat hippocampal tissue.Results:(1)Compared with Sham group,the protein expressions of p-p38,p-JNK and p-ERK1/2 in hippocampus of TLE rats were increased after the establishment of the model(P<0.05).SeNPs and P-gp@SeNPs delivery could decrease the expression of pp38,p-JNK and p-ERK1/2 proteins(P<0.05).(2)Compared with the Model group,the intervention of MAPK signaling pathway inhibitors can significantly reduce,the expression of p-p38,p-JNK and p-ERK1/2 protein in hippocampus tissues(P<0.05).SeNPs and P-gp@SeNPs delivery could decrease the expression of p-p38,p-JNK and p-ERK1/2 proteins(P<0.05).Pgp@SeNPs combined with VX-702 could further reduce the levels of p-p38,p-JNK and p-ERK1/2(P<0.05).Conclusion:SeNPs and P-gpSeNPs inhibit the activity of MAPK signaling pathway,and then alleviate the pathological progression of TLE,providing a basis for further clinical research.