Neuroprotection And Tissue Distribution Of Selenium-enriched Spirulina Protein After Subarachnoid Hemorrhage

As a necessary trace element in human metabolism,Se（Se）element plays an important role in various physiological activities and biochemical reactions^[1].Selenium compounds exhibit strong antioxidant and anticancer activity,enhanced immunity,and liver protection^[2].Spirulina,as a high quality selenium carrier^[3-5],has been proved to be powerful in scavenging free radicals,antioxidation,anti-tumor and immune regulation^[6].Subarachnoid hemorrhage（SAH）due to abnormal blood vessels at the bottom of the brain and the brain surface rupture rupture caused by various reasons,blood flow out,in and of subarachnoid and cause a syndrome^[7],with great harm after SAH high incidence,high disabling,the more difficult^[6,8],the majority of patients have sequela,seriously affected normal life,family and society bring great burden to patients.The treatment of subarachnoid hemorrhage has been widely concerned,and it has become the focus of attention to find the drug or treatment which has the exact curative effect and has the potential of wide clinical application.This topic with sodium selenite inorganic selenium spirulina,spirulina protein extracted,by Trypsin digestion treatment for pancreatic enzyme treatment of se-enriched spirulina protein（Trypsin-SeSP）,inductively coupled plasma mass spectroscopy（icp-ms）determination of the generation of neurons,selenium content in brain tissue,and the important tissues and organs,detect tissue distribution,and investigate the correlation between the Trypsin-SeSP protective effect and its mechanism of neurons after SAH.Experiment purpose:1.The small molecule polypeptide trypsin-sesp,which was prepared for enzymatic hydrolysis,was used to extract the original neurons in the hippocampus and provide a basis for the follow-up mechanism of tissue distribution and post-sah protection.2.Establish an efficient,accurate and reliable icp-ms method for determining the content of Seelements in cells and tissues;To evaluate the membrane permeability and tissue distribution of trypsin-sesp.3.The effects of trypsin-sesp on free radical production,caspase and mitochondrial membrane potential were clarified through the mechanism of trypsin-sesp through the blood-brain barrier after SAH and the role of neurons in the cell.4.Comprehensive evaluation of the therapeutic effect of trypsin-sesp on SAH in rats.Research Methods:1.To cultivate spirulina with sodium selenite gradually,extract SeSP from selenium-enriched spirulina,and make trypsin-sesp with 0.25%Trypsin digestion.The original neuron cells of the hippocampus were extracted from the rats with SD rats at 24h,and the neuron specific fluorescence probe Tululin was used to identify the original neurons.2.The original neuron cells were divided into 3 groups,which were measured by trypsin-sesp,SeSP and SP,and the contents of Seelements in the neurons were determined by icp-ms after 0h,2h,4h,6h,12h and 24h.SD rats were divided into four groups,to establish the rat model of SAH,give 30 mg/kg/day Trypsin-SeSP,SP,SeSP,PBS treatment 3 days,beheaded kill,brain,liver,lung,kidney,heart tissue,microwave digestion,using icp-ms determination of Secontent in organization change,evaluation of Trypsin-SeSP through the function of cell membrane and distribution in the body.3.The original generation of neurons with 0,2.5,5,10,20,40 mu g/ml concentration gradient of Trypsin,SeSP cultivating,with CCK 8 standard instrument testing kits and enzymes cell activity,evaluation of Trypsin-SeSP’s impact on the original generation of neuron survival time.The original generation of neurons with 0,2.5,5,10,20,40,80 mu g/ml of oxygenated hemoglobin（foxy-Hb）training with CCK 8 standard instrument testing kits and enzymes neuron activity,evaluation foxy-Hb on the toxic effects of neurons;The original generation of neurons is divided into four groups,normal neurons in the control group,foxy-Hb neurons injury group,foxy-Hb neurons after injury to Trypsin-SeSP protection group,foxy-Hb neurons after injury for SeSP protection group,detecting the change of the neuron cell activity,Trypsin-SeSP evaluation under the condition of foxy-Hb protection of neurons.The original generation of neurons is divided into four groups,normal neurons in the control group,foxy-Hb neurons injury group,foxy-Hb neurons after injury to Trypsin-SeSP protection group,Trypsin-SeSP neurons group,four groups of cells with Tubulin,TUNEL-DAPI,the Mito-traker,JC-1,DCFH-DA,DHE specific fluorescent probe for immunofluorescence staining and detection of Caspase 3 activity change,To explore the mechanism of the protective effect of Trypsin-SeSP in oxy-hb.Normal SD rats,SAH model of SD rats and to give 30 mg/kg/day after SAH Trypsin-SeSP treatment 3 days of SD rats,beheaded kill in brain tissue,frozen section,application specific fluorescent probe Neu N,TUNEL DAPI,Acive Caspase 3immunofluorescence staining,observed the fluorescent changes,to explore the level of animal Trypsin-after SAH SeSP give play to the role of neural protection mechanism.4.SD rats in SD rats,SAH model SD rats,and SD rats treated with trypsin-sesp after the SAH model were evaluated for neurological function,and evaluated the improvement of trypsin-sesp on neurofunctional disorders after SAH.Anesthesia,severed head,brain,brain tissue from 50℃constant temperature box is used to detect the wet weight,the determination of brain tissue dry wet weight,evaluation of Trypsin-SeSP’s an adverse effect on cerebral edema.Results:1.The morphological and specific fluorescence characteristics of spirulina proved that the experimental culture was enriched with selenium-rich spirulina;The morphological and specific fluorescence probe Tululin of neuronal cells showed that the extracted cells were the original neurons.2.The content of Seelements in the brain tissues of the SD rats treated with trypsin-sesp treated with neuronal cells and SAH after trypsin-sesp was significantly increased;Trypsin-sesp was found in the liver and kidney of SD rats.3.Oxy-hb processes the protozoa cells of god,and the morphological changes of neuronal cells are the cytoplasmic contractions,the number of cells decreased,the growth of the cells,the vacuolation of the cytoplasm and the reduction of intercellular connections.Foxy to Hb induction of mitochondrial membrane potential lowers,caspase activation,free radical generation,but Trypsin-SeSP inhibits foxy-Hb neurons induced by change of the above,prove that foxy-Hb induced neuronal apoptosis can be effective inhibit Trypsin-SeSP.The results showed that trypsin-sesp inhibited apoptosis of SAH after the SAH model was treated with trypsin-sesp.4.Trypsin-sesp can significantly improve the neurological dysfunction after subarachnoid hemorrhage,and improve the spontaneous activity,responsiveness,sensitivity,and movement range of SD rats after SAH.Trypsin-sesp significantly inhibited the occurrence of cerebral edema in the posterior cerebral tissue of SAH,reducing the water content in the brain tissue surrounding SAH hematoma and inhibiting the occurrence of brain injury.Conclusion:1.The original neuron and selenium-rich spirulina were pure.2.Trypsin-sesp can play a neuroprotective role after SAH through membrane and blood-brain barrier.Trypsin-sesp enters the body and enters the brain tissue to play the neuroprotective role,which is metabolized by the liver and excreted by the kidneys.Icp-ms method for determining Seelements in cells and tissues is reliable.3.Trypsin-sesp can inhibit the apoptosis of neuronal cells induced by oxidative stress and protect the neurons after SAH.4.Trypsin-sesp inhibits nerve function damage after SAH,and reduces the occurrence of cerebral edema with significant therapeutic effect.