Objective To investigate the location of receptor interacting protein 3( RIP3) in Necroptosis and its function in this signal passage, and explore the relationship between receptor interacting protein 1 ( RIP1 ) and RIP3 in nuclear translocation. Methods Primary cerebrocortical neurons were cultured for 12 days,then pre-treated with zVAD-fmk(20μ,mol/L) for half an hour to block apoptosis. ①Extracting nuclear and cytoplasmic protein after neurons were exposed to TNF for different time ,then protein levels of RIP3 were analyzed by western blot and immunofluorescence for qualitative observation;②In the following research,the neurons were treated with Nec-1 and shRlPl ,then the protein level of RIP1 and RIP3 with western blot were analyzed, cell viability were determined by measuring LDH levels. Results ①In signaling pathways of necroptosis, the protein level of RIP3 in cytoplasmic decreased gradually with prolonged TNF exposure, to the corresponding it rolled up in nucleus and a-chieved the peak in 12 hours of TNF treatment ( Cytoplasmic 0. 45 ± 0. 03 ,0. 41 ± 0. 02,0. 73 ± 0. 03 ,0. 90 ± 0.01,1.15 ±0.04,1.30 ±0.02,0.99 ±0.03,0.63 ±0. 03;Nucleus 0. 07 ±0.02,0. 26 ±0.02,0. 57 ±0. 02,0. 68 ± 0.02,0. 80 ± 0.01,0.92 ± 0.02,1.28 ± 0.03,0. 87 ± 0.02) (P < 0.01). ②Blocking the relationship between RIP1 and RIP3 with necrostatin-1 and shRIPl , nuclear translocation of RIP3 decreased and caused a great increase in cell viability( 1.00 ±0.05,0.39 ±0.03,0.50 ±0. 03) (P<0. 01). Conclusion RIP3 mainly locates in cy-tolymph of normal cells,it translocates into nucelus as necroptosis takes place. RIP1 function with RIP3 in nuclear translocation. Block nuclear translocation of RIP3 is a potential way to protect cells.

BACKGROUND:Preliminary data showed that the application of human platelet lysate to human umbilical cord mesenchymal stem cell culture can better maintain the characteristics of stem cells than the application of serum-free medium. However, the serum-free medium can better improve the proliferation of mesenchymal stem cells in vitro than the human platelet lysate.OBJECTIVE:To screen out a better mesenchymal stem cell cultivation system that can greatly maintain the characteristics and proliferation rate of human umbilical cord mesenchymal stem cells.METHODS:Six human umbilical cord specimens were inoculated in six culture systems, and the primary culture of umbilical cord mesenchymal stem cell was performed. These six culture systems were respectively MesenCult serum-free medium with 2% human platelet lysate (group A), StemPro serum-free medium with 2% human platelet lysate (group B), MesenCult serum-free medium (group C), StemPro serum-free medium (group D), low glucose-DMEM with 10% fetal bovine serum (group E), low glucose-DMEM with 10% human platelet lysate (group F). The cells were subcultured at 14 days after inoculation to compare the effects of different culture systems on the morphology, surface markers, differentiation and proliferation of human umbilical cord mesenchymal stem cells.RESULTS AND CONCLUSION:(1) The morphology of passage 3 cells in group D was elongated and uneven in size. The morphology of passage 3 cells was flattened in groups E and F, but the cells in the other groups were spindle-shaped and uniform. There were no significant changes in morphology and size between passage 3 and 5 cells in A and B. In group C, the morphology of passage 5 cells was more flattened and uneven in size compared with passage 3 cells. In group D, the morphology of passage 5 cells was more elongated than that of passage 3 cell. In group E, the morphology of passage 5 cells was more flattened than that of passage 3 cells. There was no significant difference in morphology between passage 3 and 5 cells in group F. (2) The expression rate of cell surface markers had no significant difference at different passages in each group. (3) The adipoinduction and osteoinduction rates were relatively higher in groups A and B compared with groups E and F, and lowest in groups C and D. (4) The cell proliferation rate for each passages in group A was significantly higher than that in group C. The cell proliferation rate for each passage in group B was significantly higher than that in group D. The cell proliferation rate for each passage in groups E and F was significantly lower than that in groups A and B. To conclude, these results suggest that the combination of serum-free medium with human platelet lysate could better maintain the characteristics and the proliferation efficiency of mesenchymal stem cells.

Objective To investigate the effects of glutamine (Gln) on proliferation and survival of small cell lung cancer H446 cells, and further to explore the potential mechanism. Methods The proliferation of H446 cells was detected at different time points (0, 24, 48, 72 and 96 h) by CCK-8 assay in Gln (+) group and Gln (-) group, and an optimal time was selected. Under the optimal time, Annexin V-FITC/PI staining, CellTiter-Glo? assay kit and flow cytometer were used to detect cell survival, cellular adenosine triphosphate (ATP) and reactive oxygen species (ROS) levels. Gln (-) group was used as the control group, under the condition of Gln deficiency, cellular ATP, cell proliferation and survival were detected after adding oxaloacetic acid (OAA) or dimethyl-α-ketoglutarate (DM-αKG). Gln (-) group was used as the control group, cellular ROS, cell proliferation, colony and survival were detected after treated with ROS scavenger N- acetyl cysteine (NAC). With different concentrations (0, 2, 5, 10 μmol/L) of glutaminase inhibitor BPTES, the optimal concentration was selected through the colony assay. The cellular ATP and ROS levels and cell proliferation were detected under the optimal concentration. H446 cells were treated with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES), ROS inducer hydrogen peroxide (H2O2) or the combination of them, and cell survival ratio was compared between two groups. Results The proliferation levels of H446 cells at 24, 48, which were decreased most significantly in 72 h in Gln (-) group. When 72 h was used as the optimal time, the cell survival ratio and ATP level were decreased, and the ROS level was increased, in Gln (-) group compared with those of Gln (+) group (P<0.05). There was a higher survival ratio in H446 cells in Gln (-)+OAA group and Gln (-)+DM-αKG group than that of Gln (-) group (P<0.05), but there were no significant differences in cell proliferation and ATP levels between Gln (-) group, Gln (-)+OAA group and Gln (-)+DM-αKG group. The ROS level was reduced, the cell proliferation, colony level and survival ratio were increased in Gln (-)+NAC group compared with those of Gln (-) group (P<0.05). Cloning assay showed that 10μmol/L was the optional concentration. Under this concentration, the proliferation and ATP level were decreased in Gln(+)+BPTES group (P<0.05), and cellular ROS level was up-regulated compared with Gln(+) group. The survival ratio was significantly lower in BPTES+H 2O2 group compared with BPTES (+) group or H2O2 (+) group. Conclusion Glutamine deficiency inhibits the proliferation and survival ratio of H446 cells through enhancing ROS level. BPTES and H2O2 show synergistically inhibitory effect on the survival of H446 cells.

Objective To explore whether hypoxia-inducible factor-1α( HIF-1α )is involved in oxygenglucose deprivation (OGD)induced caspase-independent cell death in primary cortical neurons and whether their expression is infected by necrostatin-1 ( Nec-1 ).Methods ( 1 ) Primary cerebrocortical neurons were cultured for 14 days.Pretred z-VAD.Fmk (z-VAD)and Nec-1 with 0.1,1,5,10,25 and 50 μ mol/L before the neurons were exposed to OGD for 2 hours and reoxygenated for 12 hours,then cell viability was determined by measure LDH level.(2)Pretred z-VAD before the neurons were exposed to OGD for 2 hours,then reoxygenated for 0,2,6,12,24and 48 hours.Then western blot analysis protein level of HIF-1 α ;rt-PCR check its RNA level.(3)Pretred z-VAD and Nec-1 with 25μ mol/L before the neurons were exposed to OGD for 2 hours and reoxygenated for 12 hours.Then western blot analysis protein level of HIF-1 α; rt-PCR check its RNA level.Result ( 1 ) When cells were pretread Nec-1 with 5 μ mol/L(6.97 ± 0.06),the level of LDH was lower than cells untreated( 14.23 ± 0.08 ) (P＜ 0.05);At 25 μmol/L( 2.21 ± 0.05),the level of LDH was essentially the same as that of the control( 1.03 ±0.03 ) (P＞0.05).(2)The protein level of HIF-1 αwas different from normal (0.24 ±0.01 ) when exposed to OGD for 2 hours and reoxygenated for 2 hours (0.57 ± 0.09) and was highest after cells were exposed to OGD for 2 hours and reoxygenated for 12 hours(0.91 ± 0.08 ) (P＜ 0.05 ).The RNA level of HIF-1 α when cells were exposed to OGD was not deferent from normal (P ＞ 0.05 ).( 3 ) When cells were pretread with Nec-1 (0.32 ± 0.04 ),the protein level of HIF-1α were lower than untreated(0.83 ±0.03) (P＜0.05),but the RNA level of HIF-1α had no deference(P ＞ 0.05).Conclusion HIF-1α was involved in cell' s caspase-independent cell death;Nec-1 can protect neurons through inhibiting the expression of HIF-1α.

Considerable attention has been directed toward studying the impact of diabetes on the central nervous system. The current study investigates the biochemical changes in the brain tissue of streptozotocin (STZ)-induced diabetic rat using 31P magnetic resonance spectroscopy (31P MRS). The 31P NMR spectra of the whole brain show no significant changes of phosphomonoesters and phosphodiesters levels one week after STZ induction, suggesting no apparent structural changes in cell membranes. The results identifies the increased level of adenosine diphosphate, negligible changes of phosphocreatine ( PCr ) and adenosine triphosphate ( ATP) , but the decreased ratio of PCr/ATP, indicating that PCr plays a role of balancing the energy. Moreover, the decreased pH value indicates the changes of the intracellular environment in STZ-diabetic brains in rats. After 15 weeks of STZ injection, the metabolism of phospholipid membrane and brain energy metabolism has been obviously disturbed. Our study successfully shows that 31 P MRS can not only study phospholipid and energy metabolism non-invasively, but also measure intracellular pH and other important biochemical information. All of these spectroscopic characterizations contribute significantly to the understanding of pathogenesis and evolution of diabetes, and provide theoretical basis for early diagnosis and clinical treatment in diabetes.

Objective To investigate whether chlorophyllin could protect human umbilical vein endothelial cell (HUVEC) against oxidative damage by inducing the expression of heme oxygenase-1 (HO-1) and to explore the underlying mechanism.Methods The cellular protection of chlorophyllin against oxidative damage was detected by cell-survival assay with flow cytometry.The level of free radicals was detected directly by electron spin resonance spectra.The induced expression of HO-1 was shown by RT-PCR,Western blot,immunofluorescence confocal laser microscopy and enzymatic activity test.Whether the activation of PI3K/Akt pathway was involved was detected by Western blot.Results Chlorophyllin could protect HUVEC against oxidative damage caused by H2O2 via scavenging the excessive free radicals.Chlorophyllin treatment could induce expression of HO-1 in a dose- and time-dependent manner.The activation of PI3K/Akt pathway was required in the induction of HO-1.LY294002,the specific inhibitor of PI3K,could suppress the activation of PI3K/Akt and the induced expression of HO-1 in a dose-dependent manner.Conclusions Chlorophyllin shows cellular protection against oxidative damage by counteracting the excessive free radicals.Up-regulation of HO-1 expression plays a pivotal role in the protection of chlorophyllin,while the activation of PI3K/Akt signaling pathway is required in the induction of HO-1.

Objective:To examine the differences of local functional connectivity in male and female children with autism spectrum disorders (ASD), and analyze the correlation of local functional connectivity with severity of clinical symptoms in ASD children.Methods:In the International Autism Brain Imaging Exchange Database, resting-state functional MR imaging (rs-fMRI) and T1WI data were extracted from 72 children with ASD (36 male and 36 female) and 72 normal controls (36 male and 36 female) who met the inclusion criteria at 5 sites. Regional homogeneity (ReHo) method was used to evaluate the local functional connectivity of the 4 groups. A covariance analysis (gender and diagnosis as factors; age, total intelligence quotient, and head movement parameters as concomitant variables) was used to find out the differences of ReHo in the 4 groups. Pearson correlation analysis was used to examine the correlation between ReHo values with significant differences in covariance analysis and revised autism diagnostic interview (ADI-R) scale scores in children with ASD.Results:(1) Regarding the main effect of diagnosis, significantly decreased ReHo values in the right posterior cerebellar lobe, and significantly increased ReHo values in the left anterior central gyrus and the left precuneus of the ASD children were noted as compared with those in the normal controls (GRF calibration, voxel level: P<0.01, cluster level: P<0.05); regarding the main effect of gender, the male subjects had significantly higher ReHo value in the right middle temporal gyrus, and significantly lower ReHo values in the left inferior frontal gyrus, the left precuneus, the right angular gyrus, and the right supramarginal gyrus than the female subjects (GRF calibration, voxel level: P<0.01, cluster level: P<0.05); interaction effect (diagnosis×gender) results showed that there was significant difference in ReHo values of the left middle frontal gyrus among the 4 groups (GRF calibration, voxel level: P<0.01, cluster level: P<0.05). (2) The results of Pearson correlation analysis showed that the ReHo value in the right middle temporal gyrus was positively correlated with the verbal performance scores measured by ADI-R scale ( r=0.387, P=0.026), and the ReHo value in the right posterior cerebellar lobe was positively correlated with the social dimensionality scores ( r=0.364, P=0.037) in male patients with ASD; the ReHo value in the right supramarginal gyrus was negatively associated with the social dimensionality scores ( r=-0.395, P=0.031), and the ReHo value in the left middle frontal gyrus was negatively associated with the repetitive behavior scores in female patients with ASD ( r=-0.398, P=0.029). Conclusion:There are brain regions with abnormal local functional connectivity in ASD children, and there are differences in local functional connectivity between male and female patients with ASD, which may be the brain functional basis of the differences in the clinical manifestations of male and female patients with ASD, and local functional connectivity in some brain regions is related to the severity of clinical symptoms of the children with ASD.

Objective To study the impact of earthquake disasters on the mental health of military medical personnel during emergency rescue. Methods Three hundred military medical personnel who participated in the emergency rescue of earthquake disasters in five military hospitals in Shaanxi Province were selected as the study subjects, and their mental health levels were analyzed using the Mental Symptom Checklist 90 (SCL-90). Results In this survey, a total of 300 SCL-90 questionnaires were issued, 300 were recovered, and the effective recovery rate was 100%. Only 30.67%(92/300) of people participated in emergency disaster rescue during investigation, 31.33% (94/300) had psychological problemsafter arriving in the earthquake-stricken area after one week, such as somatization, obsessive-compulsive disorder, anxiety, and horror. SCL-90 scores were 0.45±0.03, 1.13±0.13, 0.79 ±0.08, 1.07±0.09, 1.01±0.14. The psychological stress response rates of the emergency response of military medical staff of different characteristics were different. Among them, the stress response rates of military personnel with different degrees (specialty, bachelor or above) and whether they have participated in rescue work were 74.07% (40/54), 21.95% (54/246), 15.22% (14/92), 38.46% (80/208).The difference was statistically significant (χ2=55.913,16.018, P<0.01). Conclusions The medical staff of the army participating in the earthquake disaster rescue has psychological pressure due to many injuries, environmental risks, lack of supplies, etc. The medical unit should provide adequate training in theory and practice to its personnel and focus on the mental health of rescue workers, give timely psychological counseling to ensure the successful completion of the rescue.

Objective:To explore the influencing factors of intracranial hemorrhage in elderly patients with acute occlusion of intracranial arteries after treatment and reperfusion.Methods:A retrospective selection was conducted on elderly patients with acute occlusion of the intracranial artery who were treated at the Hebei Petro China Central Hospital from February 2019 to May 2021. Sixty patients who received mechanical thrombectomy treatment were selected as the observation group, and 60 patients who received combined arterial and venous thrombolysis treatment were selected as the control group. The vascular reperfusion rate and incidence of intracranial hemorrhage were observed and compared between the two groups. Meanwhile, multiple logistic regression analysis was used to identify the influencing factors of intracranial hemorrhage.Results:The reperfusion rate of the observation group′s blood vessels was 85.00%(51/60), significantly higher than the control group′s 68.33%(41/60), and the difference was statistically significant (χ 2=4.658, P=0.031). The National Institutes of Health Neurological Deficit Score (NIHSS) of the observation group after treatment was (10.57±2.23), significantly lower than that of the control group (14.73±2.84), and the difference was statistically significant ( P<0.05). The reperfusion rate of blood vessels in patients under 80 years old in the observation group was significantly higher than that in patients ≥80 years old ( P<0.05). The results of univariate analysis showed that there were statistically significant differences in age, NIHSS at admission, Alberta Stroke Program Early CT Score (ASPECTS), and neutrophil absolute value/lymphocyte ratio (NLR) between patients with and without intracranial hemorrhage (all P<0.05); Multivariate logistic regression analysis showed that age ( OR=1.756, 95% CI: 1.184-2.604) and NIHSS at admission ( OR=2.392, 95% CI: 1.401-4.084) were risk factors for postoperative intracranial hemorrhage in elderly patients with acute occlusion of the large intracranial artery, while ASPECTS ( OR=0.364, 95% CI: 0.190-0.697) was a protective factor. Conclusions:Mechanical thrombectomy has good clinical efficacy in the treatment of elderly patients with acute occlusion of intracranial arteries, and is worthy of clinical use; The intracranial hemorrhage after reperfusion is mainly influenced by the patient′s age, NIHSS at admission, and ASPECTS.