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Epilepsy is a disorder of the brain charac-terized by abnormal neuron excitability.However,the underlying molecular mechanism of neuron excitability modulation remains elusive.With the help of bioinformatic methods,we have identified receptor-type tyrosine-pro-tein phosphatase-like N(PTPRN)as a critical gene dur-ing epileptogenesis.PTPRN recruits NEDD4L ubiquitin E3 ligase to NaV1.2 sodium channels,facilitating NEDD4L-mediated ubiquitination and endocytosis.Knockout of PTPRN endows hippocampal granule cells with augmented depolarization currents and higher intrinsic excitability,which is reflected by increased seizure susceptibility of transgenic mice.On the contrary,reduced neuron excit-ability and decreased seizure susceptibility are observed after PTPRN overexpression.Meanwhile,we find that a 133 aa fragment recaptures modulation effect of PTPRN full-length,and this fragment shows therapeutic potential towards epilepsy caused by NaV1.2 gain of function vari-ants.In brief,our results demonstrate PTPRN playsa criti-calroleinregulatingneuronexcitability,providing a poten-tial therapeutic approach for epilepsy.
RESUMO
AIM:To investigate the effect of tert-butylhydroquinone ( tBHQ) on the replicative senescence of bone marrow mesenchymal stem cells (BMSCs).METHODS: Late stage BMSCs were continuously treated with tBHQ at concentration of 30 μmol/L for 4 weeks and the cells were used for the following assays immediately .The proteasomal ac-tivity was determined by chemiluminescence method .The samples were subjected to CCK-8 assay and BrdU incorporation as well as flow cytometry analysis for analyzing the cell vitality and proliferation .Percentage of senescent cells was detected by senescence-associatedβ-galactosidase ( SA-β-Gal) staining.The expression of P53 was measured by Western blot .RE-SULTS:After the continuous treatment of tBHQ (30 μmol/L) for 4 weeks, the proteasomal activity of late stage BMSCs increased by 21.96%±1.98%(P<0.05).The cell vitality and survival were significantly increased with the increases in tBHQ doses till 40 μmol/L, and no cytotoxicity reaction with the increased dose of tBHQ till 120 μmol/L was observed . BrdU-positive cells, which represented the cell proliferation , were significantly increased (P<0.05).The proliferation in-dex was also significantly increased by flow cytometry analysis (P<0.05).The SA-β-Gal positive cells and the expression of P53 were decreased (P<0.05).CONCLUSION:tBHQ delays the proteasome dysfunction associated senescence pro-gress of BMSCs by increasing the proteasomal activity .
RESUMO
Objective To investigate the role of endothelial progenitor cells ( EPCs ) transplantation in rats with sepsis induced by endotoxin ( lipopolysaccharides, LPS ). Methods Sixty clean grade Sprague-Dawley ( SD ) rats with genetic background were divided into three groups according to random number table method:control group, model group, and EPCs transplantation group, with 20 rats in each group. The sepsis model was reproduced by intravenous delivery of LPS 5 mg/kg. Rats in control group were injected with the same amount of normal saline. EPCs were isolated, and cultured and identified were fluorescently labeled with the green fluorescent protein ( GFP ) adenoviral transfection method. The EPC transplantation group was injected with LPS, then a fluorescently labeled EPCs suspension was injected via the tail vein 1 hour later. The expression of fluorescent markers of EPCs was detected with both small animal in vivo imaging instrument and frozen section. Seven days after transplantation, abdominal aorta blood was collected to determine interleukins ( IL-6 and IL-10 ) in peripheral blood with enzyme linked immunosorbent assay ( ELISA ), and the lung, liver, and kidney tissues were harvested, the wet/dry ratio of the lung ( W/D ) was calculated, and hematoxylin and eosin ( HE ) staining was performed to observe, the change in histopathology. Toll-like receptor 4 ( TLR4 ) mRNA expression in lung, liver, and kidney tissues was determined with real-time reverse transcription-polymerase chain reaction ( RT-PCR ). Results The positive rate of EPCs cells with double marking of CD133 and CD34 was 99.0% at the 5th generation of subculture by using flow cytometry. After the transplantation of EPCs labeled with the green fluorescent protein, the appearance of fluorescence indicated that EPCs were mainly localized in the chest, and a stronger fluorescence was observed near the blood vessels. EPCs transplantation could significantly reduce the inflammatory cell infiltration and cell damage in lung, liver, and kidney tissue in septic rats. Compared with control group, the expression of IL-6 and IL-10 in the peripheral blood, W/D ratio, and TLR4 mRNA in lung, liver, and kidney were increased significantly in the model group. Compared with model group, the expressions of IL-6 and IL-10 in the peripheral blood were significantly reduced after EPCs transplantation [ IL-6 (μg/L ):2.127±0.118 vs. 2.664±0.438, IL-10 ( ng/L ): 24.5±3.9 vs. 31.5±3.8, both P < 0.01 ]. EPCs transplantation reduced the W/D ratio of lung, liver and kidney tissues ( lung: 4.68±0.24 vs. 5.48±0.15, liver: 3.33±0.11 vs. 3.94±0.09, kidney: 4.08±0.20 vs. 4.84±0.21, all P < 0.05 ], and down-regulated the expression of TLR4 mRNA ( ×103, lung: 782±131 vs. 1 136±126, liver: 39.1±14.0 vs. 69.2±8.7, kidney: 52.2±15.2 vs. 83.5±17.1, all P < 0.01 ). Conclusions EPCs can enter the lung, liver and kidney tissues of the rat successfully after transplantation of EPCs via vein. EPCs transplantation can down-regulate pro-inflammatory process, help to recover the balance of pro-and anti-inflammatory processes, alleviate the damage to the lung, liver, and kidney tissue significantly.