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Objective To investigate the neuroprotective effect of Ghrelin on traumatic brain injury (TBI) in mice.Methods TBI model of C57BL/6 mice was established by electronic cortical impact instrument (eCCI).According to the random figure table method,twenty-four mice were randomly divided into sham group(Sham group),TBI group and Ghrelin intervention group(Ghrelin group) with 8 mice in each group.The model of TBI was established in TBI group and Ghrelin group.The mice in Ghrelin group was injected intraperitoneally 0.5 g/kg before and 1 h after injury respectively.And the mice Sham group and TBI group were injected with the same amount of normal saline.The changes of cerebral blood perfusion (CBP)were monitored in real time by laser speckle contrast analysis(LSCI),the changes of neuroelectrophysiology were observed by monitoring motor evoked potential (MEP),and the status of neurological deficit was evaluated by modified neurological deficit score (mNSS).Results Compared with Sham group,the mice in TBI group had significantly lower cerebral blood perfusion(CBP) (t=-12.36,P＜0.01),longer latency and lower amplitude of motor evoked potential (MEP) (t=5.03,-11.55,all P＜0.01),and significantly higher mNSS scores (t=9.34,P＜0.01).However,compared with the TBI group,the cerebral blood perfusion(CBP) of Ghrelin group increased significantly at 12 h after TBI((196.87± 17.36) PU/mm2 vs (123.62±8.04)PU/mm2,t=10.45,P＜0.01),while the latency of MEP decreased((5.30±0.33) ms vs (6.80±0.97) ms,t =-5.01,P＜0.01),the amplitude of MEP increased ((2.21 ± 0.16) mV vs (1.27± 0.27) mV,t =9.65,P＜0.01).And compared with the TBI group,the neurological deficit score of Ghrelin group decreased significantly at 24 h after TBI((4.9±1.2) vs (8.4±2.6),t=-3.87,P＜0.01).Conclusion Ghrelin exhibits a significant neuroprotective role by increasing cerebral blood flow perfusion,reducing the degree of neurological deficit and promoting motor function recovery in TBI mice.
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Objective@#To investigate the neuroprotective effect of Ghrelin on traumatic brain injury (TBI) in mice.@*Methods@#TBI model of C57BL / 6 mice was established by electronic cortical impact instrument (eCCI). According to the random figure table method, twenty-four mice were randomly divided into sham group(Sham group), TBI group and Ghrelin intervention group(Ghrelin group) with 8 mice in each group. The model of TBI was established in TBI group and Ghrelin group.The mice in Ghrelin group was injected intraperitoneally 0.5 g/kg before and 1 h after injury respectively. And the mice Sham group and TBI group were injected with the same amount of normal saline. The changes of cerebral blood perfusion (CBP) were monitored in real time by laser speckle contrast analysis(LSCI), the changes of neuroelectrophysiology were observed by monitoring motor evoked potential (MEP), and the status of neurological deficit was evaluated by modified neurological deficit score (mNSS).@*Results@#Compared with Sham group, the mice in TBI group had significantly lower cerebral blood perfusion(CBP) (t=-12.36, P<0.01), longer latency and lower amplitude of motor evoked potential (MEP) (t=5.03, -11.55, all P<0.01), and significantly higher mNSS scores (t=9.34, P<0.01). However, compared with the TBI group, the cerebral blood perfusion(CBP) of Ghrelin group increased significantly at 12 h after TBI((196.87±17.36) PU/mm2 vs (123.62±8.04)PU/mm2, t=10.45, P<0.01), while the latency of MEP decreased((5.30±0.33)ms vs (6.80±0.97)ms, t=-5.01, P<0.01), the amplitude of MEP increased((2.21±0.16)mV vs (1.27±0.27)mV, t=9.65, P<0.01). And compared with the TBI group, the neurological deficit score of Ghrelin group decreased significantly at 24 h after TBI((4.9±1.2) vs (8.4±2.6), t=-3.87, P<0.01).@*Conclusion@#Ghrelin exhibits a significant neuroprotective role by increasing cerebral blood flow perfusion, reducing the degree of neurological deficit and promoting motor function recovery in TBI mice.
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Objective@#To investigate the effects of Casein kinase 2-interacting protein 1 (CKIP-1) gene silencing on the proliferation of glioma cells U87-MG.@*Methods@#The recombinant lentiviral vectors targeting CKIP-1 gene or negative control were constructed and then used to infect glioma U87-MG cell line.The effects of knock-down on the mRNA or protein expression of CKIP-1 were evaluated by real-time qPCR and western blotting.Cell cycle was detected by the flow cytometry assay, and cell proliferation changes were evaluated by cell counting, MTT, and BrdU assay, respectively.Lastly, the colony formation was used to investigate the effect of CKIP-1 knock-down on the clone formation.@*Results@#Compared with the group of Ctrl, CKIP-1 siRNA was observed to significantly inhibit CKIP-1 expression at the mRNA levels (Ctrl (1.01±0.13) vs CKIP-1 siRNA (0.23±0.02), P<0.01) and protein levels in the U87-MG cells.Also, CKIP-1 suppression mediated by RNAi decreased the ratio of G0/G1 phase (Ctrl (69.64±0.79) vs CKIP-1 siRNA(62.64±0.66), P<0.01), increased that of G2/M phase (Ctrl (8.36±0.52) vs CKIP-1 siRNA(13.87±2.90), P<0.05), and significantly inhibited the cell proliferation and clone formation (Colony number: Ctrl (25±2) vs CKIP-1 siRNA(2±1), P<0.05) of transfected U87-MG cells.@*Conclusion@#Knocking down the expression of CKIP-1 significantly inhibit cell proliferation in human U87-MG glioma cells, indicating that CKIP-1 is involved in the development of gliomas and could promote the cell proliferation.
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Objective To screen altered proteins of hippocampus in the stress-induced depression (STRID) rat model, and explore the potential molecular mechanism. Methods Twenty Sprague-Dawley rats were randomly divided into the control group and STRID group, 10 rats in each group. Chronic unpredictable mild stress (CUMS) methods including fasting for solids and liquids, electric foot-shock, reversing day and night, cold water swimming, cage tilt, scare stimulation and tail pinch were conducted on STRID rats with no repeats for 28 days to make up the depression animal model. The control group was normally fed during this period. After the stress stimulation, the hippocampus protein samples were used for two dimensional electrophoresis to screen the differentially expressed protein, and then mass spectrum identification and function analyze were conducted. Results Compared with the control group, 34 proteins were altered in STRID group. Among which, 18 were up-regulated, and 16 were down-regulated. The differentially expressed proteins mainly located in cytoplasm, mitochondrion, extracellular exosome and myelin sheath. The involved signaling pathways included metabolic pathway, oxidative phosphorylation pathway, and Alzheimer's disease, Parkinson's disease and Huntington's disease pathways. Conclusion The altered proteins and dysfunction of nerve signaling, and the excess of oxidative phosphorylation in hippocampus of STRID rats may be one of the pathogenesises.
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The refractory epilepsy refers to the epilepsy whose seizures couldn't be cured after using two kinds of correctly selected antiepileptic drugs which can be tolerated and enough dosage and duration of monotherapy or combination therapy.The pathogenesis of intractable epilepsy is complex,and there is no established theory at home and abroad.Recently,more and more attention has been paid to the correlation between mitochondrial dysfunction caused by oxidative stress and intractable epilepsy.Based on the summary of common treatment of refractory epilepsy and by searching the related literature at home and abroad,further investigation would be made to explore the diagnosis and treatment of intractable epilepsy theory in order to provide new strategies for diagnosis and treatment of intractable epilepsy.
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Objective To explore the effects of different doses of dexmedetomidine on acute brain edema in mice with traumatic brain injury (TBI).Methods A total of 132 male C57BL/6J mice were randomly divided into six groups:control group (group C),sham-operation group (group Sham),traumatic brain injury group (group TBI),Dex 20 μg/kg (group D20),40 μg/kg (group D40),and 60 μg/kg (group D60),n=22 in each group.The TBI animal model was established by electric controlled cortical impactor (eCCI),then intraperitoneal injected by the administration of different doses of dexmedetomidine at 0,2 and 4 h after TBI.Twenty-four hours post-TBI,brain water content was measured by the dry-wet method,histological observation was performed using HE staining,and aquaporin 4 (AQP4) and NF-κB expression were detected using Western blot assay,respectively.Then,the modified neurological scale scores (mNSS) on 1,2,3,and 7 d and Morris water maze (MWM) test on 4,5,6 and 7 d post-TBI were used to evaluate the neurologic deficit of TBI mice.Results After traumatic brain injury,the mNSS scores,the escape latency,the brain water content and the expression of AQP4 and NF-κB increased significantly in group TBI (P＜0.01).Different doses of dexmedetomidine significantly reduced the mNSS scores,the escape latency,the brain water content and the expression of AQP4 and NF-κB (P ＜ 0.05 or P ＜ 0.01).And meanwhile dexrnedetomidine can lessen neuronal degeneration,and inflammation response.Additionally,the effect was remarkably in group D60 compared with group D20 (P ＜ 0.05 or P ＜ 0.01).Conclusion Dexmedetomidine can lessen brain edema and cognition impairment induced with traumatic brain injury,which is a dose-effect relationship within 20-60 μg/kg,and this effect may be related to the downregulation of AQP4 and NF-κB expression.
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Objective To explore the effect and mechanism of necroptosis related proteins in middle cerebral artery occlusion (MCAO) induced brain ischemia/reperfusion injury in mice.Methods C57BL/6 mice were used to establish the brain ischemia/reperfusion injury model induced by MCAO.MCAO mice were treated with z-VAD.fmk (zVAD,1.1 g/kg),GSK'872 (0.7 g/kg) and combined intervention of zVAD and GSK'872,and neurological defect was evaluated by mNSS while brain infarct volume was measured by TTC staining.Western blot and immunofluorescence assay were used to detect protein expression and location of RIP1,RIP3 and MLKL,respectively.Results Neurological defect and brain infarction were caused by MCAO.Compared with MCAO group,zVAD,GSK'872 and the combined intervention alleviated neurological defect and reduced brain infarct volume significantly (P＜0.05 or P＜0.01).The protein levels of RIP3 and RIP1 MLKL were increased in mice of MCAO group,while GSK'872 and the combined intervention obviously downregulated the aforementioned protein expression [RIP1 (GSK'872:0.64± 0.02 vs MCAO:1.28±0.02,P＜0.01);RIP3 (GSK'872:1.08±0.02 vs MCAO:1.45±0.02,P＜0.01);MLKL (GSK'872:0.54±0.01 vs MCAO:1.00±0.01,P＜0.01)].However,zVAD only slightly reduced protein expression of MLKL (P＜0.05) but didn't change the protein expression of RIP1 and RIP3 (P＞0.05).Conclusion RIP1,RIP3 and MLKL are involved in the execution of necroptosis and contribute to the pathological progress of brain ischemia/reperfusion injury.
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Objective To investigate the signaling pathway and the key signal molecules of protein kinase (RIPK)3 in SH-SY5Y cells. Methods SH-SY5Y cells were transfected with RIPK3 expression plasmid vector to upregulate intracellular RIPK3, while the SH-SY5Y cells were transfected with empty vector plasmid, which was considered as control group. Western blot assay was used to check the expression of exogenous RIPK3 in cells. The proliferation rate of SH-SY5Y cells was determined by MTT assay at designated time to detect exogenous RIPK3 activity. Whole transcriptome sequencing (RNAseq) was used to detect the transcription of genes. Whole-transcriptomic gene transcription was measured by following Ingenuity Pathway Analysis (IPA) to obtain downstream signaling pathways and the key molecule, which were partly confirmed by following droplet digital PCR (ddPCR). Results Exogenous RIPK3 showed biological activity in SH-SY5Y, which inhibited the proliferation of cells. IPA showed that znic finger protein 36 (ZFP36) was significantly up-regulated as compared with that of the control group. The tran?scription levels of ZFP36 downstream genes such as tumor necrosis factor (TNF), brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF) and mRNA-decapping enzyme 2 (DCP2) were affected at the same time. Conclusion Within the limitations of this study, it seems that RIPK3 is notable for the development, inflammation and tumorigenesis of the nervous system as an independent regulator of ZFP36 gene and downstream effectors.
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Objective To investigate the effects of synuclein-γ (SNCG) gene silencing on the proliferation and apoptosis of glioma U87-MG cells.Methods Five small hairpin RNA templates targeting SNCG and a negative control were synthesized and cloned into the lentiviral vector system and all the constructs were sequenced.Then the recombinant lentiviral vectors were used to infect U87-MG cells.The lentiviruses which can effectively inhibit protein expression levels of SNCG were selected by RT-PCR for further study.Colony formation and flow cytometry assay were used to investigate the effects of SNCG downregulation by RNA interference on the clony formation,proliferation,and apoptosis of U87-MG cells,respectively.Results The lentiviral vectors carrying 5 shRNAs targeting the SNCG gene were successfully constructed,and SNCG siRNA3 and siRNA5 showed higher interfering efficiency than other vectors.In comparison with the group of negative control,SNCG siRNA3 and siRNA5 were observed to significantly inhibit SNCG expression at the mRNA levels (the relative mRNA levels:siRNA3 (0.17± 0.01)％,siRNA5 (0.13±0.01)％ vs (1.00±0.10)％,P＜0.05).Also,SNCG suppression mediated by RNAi significantly inhibited the clone formation (colony number:siRNA3 (66± 12),siRNA5 (1 ± 1) vs (80± 5),P＜0.05),and the proliferation (ratio of cells in S phase:siRNA3 (41.2±0.7) ％,siRNA5 (39.9±0.5) ％ vs (47.6±2.2) ％,P ＜0.05),but promoted the apoptosis (cell apoptosis:siRNA3 (22.9± 0.4) ％,siRNA5 (28.6± 0.9) ％ vs (1.1 ± 0.1) ％,P＜0.01) of transfected U87-MG cells.Conclusion SNCG suppression at the mRNA level mediated by RNAi can inhibit the proliferation and the clony formation,but induce the apoptosis of glioma U87-MG cells in vitro,suggesting that SNCG suppression mediated by an RNAi strategy may become a novel approach for treating human gliomas.
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The phosphorylation is one of most common protein post-translational modifications. The protein phosphorylation plays important roles in the life through the reversible process of phosphorylation and dephosphorylation by kinases and phosphatases. Systematical analysis of the phosphorylation state of proteins would greatly help to reveal the mystery of the life. Recently, with the development of mass spectrometer, bioinformatics sortwares and enrichment methods of phosphopeptides, phosphorylation stduy of orgnism proteins by mass spectrometer has become mature gradually. Liver is one of the most important metabolic and immune organs. In-depth study of protein phosphorylation in liver is of great importance to reveal its function. And booming phosphoproteomics has been applied into the study of liver, which has deepened the knowledge of molecular mechnism of its physiology and pathology states. Here, we review the recent progress on the research and development of phosphoproteomics and their application in liver proteomics study.
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Humans , Computational Biology , Liver , Metabolism , Pathology , Physiology , Mass Spectrometry , Phosphopeptides , Metabolism , Phosphorylation , Protein Processing, Post-Translational , ProteomicsABSTRACT
The stable isotope labeling by amino acids in culture (SILAC) based quantitative proteomics serves as a gold standard because of the high accuracy and throughput for protein identifications and quantification. In this study, we discussed the application of SILAC technology in mammal model, and developed quantitative internal standard for comparative proteomics of disease model. The C57BL/6J mice fed by special diet containing the 13C6-Lysine and bred F2 generation. We identified and analyzed total proteins of 9 mice tissues of F2 generation, including brain, lung, heart, stomach, intestine, liver, spleen, kidney, and muscle. Quantitative analysis information could evaluate the mice and different tissues' labeling efficiency. Liver was the most efficient, brain the least, and the labeling efficiency were 96.34%±0.90% and 92.62%±1.98% respectively. The average of the labeling efficiency of F2 generation was 95.80%±0.64%, which met the international standard (≥ 95%) for SILAC quantitative proteomics effective study. SILAC technology was successfully extended to mammalian model system, which will provide powerful tools for the mechanism study of the pathophysiology process with mouse model.
