Protective Mechanism Of Selenium-Containing Spirulina Protein On Neurons After Cerebral Ischemia By Inhibiting Oxidative Stress

1.Objective1.1 Spirulina,as a traditional Chinese medicine,has been confirmed that has powerful effects such as antioxidation,radiation resistance,and anti-tumor,and spirulina protein is one of its main active components at present.The trace element selenium exists in the human body in the form of selenoprotein and selenopeptide,participating in multiple functions such as redox,reproductive metabolism,and so on.As a suitable carrier of selenium known at present,spirulina platensis can cultivate and obtain selenium containing spirulina protein(Se-SP).However,the protective effect of Se-SP on neurons after cerebral ischemia has not been reported,and the mechanism is unclear.1.2 In this study,the traditional Chinese medicine Spirulina platensis was cultured with inorganic selenium sodium selenite(Na2SeO3),and selenium containing spirulina protein(Se-SP)was isolated,purified,and extracted;Rat hippocampal neurons were cultured in primary culture,and an in vitro model of cerebral ischemia injury was established using oxygen glucose deprivation(OGD);A middle cerebral artery occlusion(MCAO)model was established in adult SD rats using thread occlusion method.The protective effects and mechanisms of Se-SP on neurons after cerebral ischemia were systematically evaluated using multiple techniques such as cellular,molecular,and pathological methods.2.Methods2.1 Spirulina was cultured with sodium selenite and selenium was gradually added.The morphological appearance of selenium containing spirulina was observed using optical and fluorescence microscopes.Separation and purification of selenium containing spirulina protein using ultrasound fragmentation,low-temperature centrifugation,and trypsin digestion;and the protein without selenium(SP)was used as negative control.After excitation at 544 nm,the emission peak at 660 nm was determined.Hippocampal tissues of newborn rats were isolated,and primary hippocampal neurons were cultured and stained by tubulin immunofluorescence to identify the neuron purity.The selenium content of neurons was determined by elemental analysis to examine the selenium cell uptake.The neurons were cultured with glucose-free medium under 95%N2 and 5%CO2 for 6 h to construct OGD injury model.MTT assay was used to detect the activity of neurons.TUNEL staining was used to detect the apoptosis of neurons.JC-1 probe was used to detect the changes of mitochondria membrane potential of neurons.ROS and superoxide anion were detected by DCFH-DA and DHE probes,respectively.Western blott was used to detect protein expression.2.2 Cerebral ischemia(MCAO)model was established in adult SD rats by thread embolization,and Se-SP or SP was administered intravenously for 4 times.After perfusion by paraformaldehyde solution,brain tissues were dehydrated,fixed,paraffin embedded,sectionalized,and Neun-DAPI staining,Nissl staining,Tunel-DAPI staining,PCNA staining,and IB A-1-GFAP staining were used to evaluate the protective mechanism of Se-SP on neurons after cerebral ischemia.3.Results3.1 Selenium-rich Spirulina was successfully cultured.The Spirulina filaments showed obvious spiral structure under the microscope,and showed bright red fluorescence under the fluorescence microscope,which was consistent with the characteristics of Spirulina.Se-SP was successfully separated,and a significant characteristic peak was detected at 660 nm after excitation at 544 nm.The selenium content of neurons was detected by elemental analysis,and the results showed that Se-SP effectively entered neurons.MTT results showed that OGD significantly inhibited the growth of neurons in a time-dependent manner.Se-SP co-treatment significantly attenuated OGD-induced neurotoxicity and improved the morphology of neurons.Se-SP and SP alone showed no significant toxicity to neurons.3.2 In vitro,OGD can induce neuronal apoptosis in a time-dependent manner by activating Caspase-3,Caspase-8 and Caspase-9 in the caspase specific protein(caspase)family;However,Se-SP co-treatment significantly inhibited OGD induced neuronal apoptosis,manifested by a decrease in the number of TUNEL positive cells and a decrease in the activity of Caspase-3 and Caspase-9.JC-1 probe detection and WB detection results showed that Se-SP co treatment significantly improved the reduction of mitochondrial membrane potential(MPP)of neurons after OGD treatment,and improved the expression of B-cell lymphoma-2(Bcl-2)family proteins.The results of DCFH-DA and DHE probe detection showed that Se-SP co treatment significantly inhibited the accumulation of ROS and O2-induced by OGD,weakened DNA damage,manifested as causing OGD induced ataxia telangiectasia mutated kinase/ataxia telangiectasia mutated gene/Ataxia Telangiectasia and Rad3 related kinase(ATM/ATR),histone H2A The phosphorylation level of p53 tumor protein(p53)decreases.In addition,the addition of mitochondrial permeability transition pore(MPTP)inhibitor CsA can significantly inhibit OGD induced reduction of MPP,free radical production,apoptosis,and DNA damage,ultimately improving neuronal activity.3.3 NeuN staining results showed that Se-SP significantly inhibited the neuron loss caused by cerebral ischemia in SD rats;The results of Nissl staining showed that Se-SP obviously increased the number of Nissl corpuscles and improved the morphology of Nissl corpuscles in cortical neurons of rats with cerebral ischemia;Se-SP apparently reduced the expression of PCNA protein,an indicator of oxidative stress injury in vivo;The double staining results of Iba-1 and GFAP confirmed that Se-SP significantly inhibited the expression of Iba-1 and GFAP protein in vivo;TUNEL staining showed that Se-SP significantly attenuated OGD-induced neuronal apoptosis in vivo.4.Conclusion4.1 Se-SP inhibits mitochondrial dysfunction induced by OGD by regulating the expression of Bcl-2 family proteins in vitro.4.2 Se-SP regulates the shutdown of MPTP,inhibits free radical production and DNA damage,improves mitochondrial membrane potential,and inhibits OGD-induced neuronal toxicity and apoptosis in vitro.4.3 Se-SP can protect neurons from damage caused by cerebral ischemia after cerebral thrombosis by inhibiting oxidative stress damage and glial cell activation in vivo.