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Objective:To evaluate the effect of hypothermia on the polarization of microglia and TLR4/NF-κB signaling pathway during oxygen-glucose deprivation/restoration (OGD/R).Methods:BV2 microglia were cultured in vitro and divided into 3 groups ( n=18 each) using the random number table method: control group (group C), group OGD/R and OGD/R plus hypothermia group (group OGD/R+ HT). Group C was cultured normally for 24 h. In group OGD/R, the cells were exposed to 5% CO 2-1% O 2 at 37 ℃ for 2 h in a glucose-free medium, followed by restoration of glucose and oxygen for 24 h. In group OGD/R+ HT, the high-glucose medium was replaced with a glucose-free medium, the cells were exposed to 5% CO 2-1% O 2 for 2 h in a 33 ℃ cryostat, followed by restoration of glucose and oxygen for 24 h. The cell survival rate was measured by CCK-8 assay.The expression of M1 microglia markers CD32 and iNOS protein and mRNA and M2 microglia markers CD206 and Arg-1 and mRNA was detected by immunofluorescence and real-time quantitative polymerase chain reaction.The expression of TLR-4 and NF-κB in cells was detected by Western blot, and the expression of TLR4 and NF-κB mRNA in cells was detected by real-time quantitative polymerase chain reaction.The concentrations of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and interleukin 10 (IL-10) in the cell supernatant were detected by enzyme-linked immunosorbent assay. Results:Compared with group C, the cell survival rate was significantly decreased, the expression of CD32, iNOS, CD206, Arg-1, TLR4 and NF-κB protein and mRNA was up-regulated, and the concentrations of TNF-α, IL-1β and IL-10 in supernatant were increased in OGD/R and OGD/R+ HT groups ( P<0.05). Compared with group OGD/R, the cell survival rate was significantly increased, the expression of CD32, iNOS, TLR4 and NF-κB protein and mRNA was down-regulated, the expression of CD206 and Arg-1 protein and mRNA was up-regulated, the concentrations of TNF-α and IL-1β in supernatant were decreased, and the concentration of IL-10 was increased in group OGD/R+ HT ( P<0.05). Conclusions:Hypothermia can significantly inhibit microglia polarization toward M1 phenotype, increase microglia polarization toward M2 phenotype and inhibit the development of inflammatory responses during OGD/R, and the mechanism may be related to inhibition of TLR4/NF-κB signaling pathway.
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Objective:To evaluate the role of exosomes in M2 microglia-induced reduction of oxygen-glucose deprivation and restoration (OGD/R) injury to astrocytes.Methods:The primary astrocytes were cultured in vitro to the logarithmic growth phase and divided into 5 groups ( n=14 each) using a random number table method: control group (group C), OGD/R group (group O), OGD/R+ M2 microglia group (O+ M2 group), OGD/R+ M2 microglia+ GW4869 group (O+ M2+ G group) and OGD/R+ M2 microglia-derived exosome group (O+ M2-E group). Cells in group C were cultured routinely.Cells in group O were subjected to 4 h of oxygen-glucose deprivation (OGD) and 24 h of restoration of O 2-glucose supply.In group O+ M2, cells were subjected to 4 h of OGD, and the supernatant of M2 microglia 2 ml was added to the medium during restoration of O 2-glucose supply, and the cells were cultured for 24 h. In group O+ M2+ G, cells were subjected to 4 h of OGD, and the supernatant of M2 microglia 2 ml treated with the exosome inhibitor GW4869 10 μmol/L was added to the medium during restoration of O 2-glucose supply, and the cells were cultured for 24 h. In group O+ M2-E, cells were subjected to 4 h of OGD, and the M2 microglia-derived exosome 10 μg/ml was added to the medium during restoration of O 2-glucose supply, and the cells were cultured for 24 h. The morphological changes of cells were observed with a light microscope, the cell viability was detected by CCK-8 assay, the expression of aquaporin 4 (AQP4) mRNA was detected by quantitative real-time polymerase chain reaction, and the expression of AQP4 and porimin was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the expression of AQP4 protein and mRNA and porimin was up-regulated ( P<0.05), and cell swelling occurred in the other four groups.Compared with group O, the cell viability was significantly increased, and the expression of AQP4 protein and mRNA and porimin was down-regulated in O+ M2 and O+ M2-E groups ( P<0.05), and no significant change was found in the parameters mentioned above ( P>0.05), and the cell viability was significantly attenuated in group O+ M2+ G.Compared with group O+ M2, the cell viability was significantly decreased, and the expression of AQP4 protein and mRNA and porimin was up-regulated in group O+ M2+ G ( P<0.05), and no significant change was found in the parameters mentioned above ( P>0.05), and the degree of cell swelling was increased in group O+ M2-E. Conclusions:M2 microglia can mitigate OGD/R injury to astrocytes through exosomes.
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Objective:To evaluate the role of miR-20a-5p in M1 microglia aggravating oxygen-glucose deprivation and restoration (OGD/R)-induced injury to neurons and the relationship with mitofusin2 (MFN2).Methods:The well-growing BV2 microglia (M0 type) were polarized into M1 phenotype by lipopolysaccharide (100 ng/ml) and IFN-γ (20 ng/ml) and identified by quantitative real-time polymerase chain reaction and immunofluorescence.The well-growing N2a cells were divided into 6 groups ( n=6 each) by the random number table method: control group (group C), OGD/R group, M0 microglia co-culture group (group M0), M1 microglia co-culture group (group M1), miR-20a-5p inhibitor transfection group (group I) and negative control group (group NC). The cells were routinely cultured in group C, and the cells were subjected to OGD for 3 h followed by restoration of oxygen-glucose supply to develop the model of OGD/R injury in group OGD/R.The cells were subjected to OGD for 3 h and were co-cultured with M0 microglia for 24 h during restoration of oxygen-glucose supply in group M0.The cells were subjected to OGD for 3 h and were co-cultured with M1 microglia for 24 h during restoration of oxygen-glucose supply in group M1.In group I and group NC, cells were transfected with miR-20a-5p inhibitor and negative control miRNA into M1 microglia, respectively, and N2a cells were subjected to OGD for 3 h and co-cultured with M1 microglia for 24 h during restoration of oxygen-glucose supply.The cell viability was determined by cell counting kit-8 assay, amount of lactate dehydrogenase (LDH) released was determined, the expression of miR-20a-5p and MFN2 mRNA was detected by quantitative real-time polymerase chain reaction, and MFN2 expression was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the amount of LDH released was increased, and the expression of MFN2 protein and mRNA was down-regulated in the other five groups, miR-20a-5p expression was significantly up-regulated in OGD/R, M0 and M1 groups, and miR-20a-5p expression was significantly down-regulated in group I ( P<0.05). There were no significant differences in the cell viability, amount of LDH released, and expression of miR-20a-5p, MFN2 protein and mRNA between group OGD/R and group M0 ( P>0.05). Compared with group OGD/R and group M0, the cell viability was significantly decreased, the amount of LDH released was increased, and the expression of MFN2 protein and mRNA was down-regulated, and miR-20a-5p expression was up-regulated in group M1 ( P<0.05). Compared with group M1, the cell viability was significantly increased, the amount of LDH released was decreased, the expression of MFN2 protein and mRNA was up-regulated, and miR-20a-5p expression was down-regulated in group I ( P<0.05). Conclusions:The mechanism by which M1 microglia aggravates OGD/R-induced damage to N2a cells may be related to the up-regulation of miR-20a-5p expression in M1 microglia and the inhibition of MFN2 expression in N2a cells.
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Objective:To evaluate the role of miR-205-3p in oncosis in astrocytes subjected to oxygen-glucose deprivation and restoration (OGD/R) and the relationship with aquaporin4 (AQP4).Methods:Primary astrocytes were cultured in vitro to the logarithmic growth phase and divided into 5 groups ( n=16 each) using a random number table method: control group (C group), OGD/R group (O group), OGD/R+ miR-205-3p mimic group (M group), OGD/R+ miR-205-3p inhibitor group (I group), and OGD/R+ negative control group (NC group). Cells were cultured routinely in C group.Cells were subjected to 4 h of oxygen-glucose deprivation in a 37℃ anaerobic incubator (containing 94% N 2, 1% O 2 and 5% CO 2) followed by restoration of O 2-glucose supply for 24 h in O group.Cells in M, I and NC groups were transfected with miR-205-3p mimic, miR-205-3p inhibitor and miR-205-3p negative control for 48 h, respectively, and then cells were subjected to 4 h of oxygen-glucose deprivation followed by restoration of O 2-glucose supply for 24 h. The cell viability was evaluated by CCK-8 assay, the cell injury and oncosis were analyzed by flow cytometry, the expression of AQP4 mRNA was detected by quantitative reverse transcription-polymerase chain reaction, and the expression of AQP4 and porimin was detected by Western blot. Results:Compared with C group, the expression of miR-205-3p was significantly down-regulated, the cell viability was decreased, the rates of cell injury and oncosis were increased, and the expression of AQP4 protein and mRNA and porimin was up-regulated in O group ( P<0.05). Compared with O group, the expression of miR-205-3p was significantly up-regulated, the cell viability was increased, the rates of cell injury and oncosis were decreased, and the expression of AQP4 protein and mRNA and porimin was down-regulated in M group, the expression of miR-205-3p was significantly down-regulated, the cell viability was decreased, the rates of cell injury and oncosis were increased, and the expression of AQP4 protein and mRNA and porimin was up-regulated in I group ( P<0.05), and no significant changes were found in NC group( P>0.05). Conclusions:miR-205-3p is involved in oncosis in astrocytes subjected to OGD/R, which is associated with regulation of AQP4 expression.
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Objective:To evaluate the role of exosomes in neuronal injury induced by M1 microglia.Methods:Liposolysaccharide 100 ng/ml and interferon-γ (IFN-γ)20 ng/ml were added to well-growing BV2 microglia to induce the polarization of microglia into M1 phenotype.Cell supernatant of M1 microglia was collected and M1 microglia exosomes (M1-exo) were extracted with exosome kit.The well-growing N2a cells were divided into 4 groups ( n=24 each) using a random number table method: control group (group C), M1 microglia group (group M), exosome group (group E), and exosome inhibitor+ M1 microglia group (group G+ M). The cells in group C were conventionally cultured, the cells in group M were cultured with the supernatant of M1 microglia for 24 h, and the cells in group E were cultured with M1 microglia-derived exosomes for 24 h. In G+ M group, exosome inhibitor GW4869 was added, M1 microglia were incubated for 24 h, then the supernatant was collected and added to N2a cells, and the cells were incubated for 24 h. Cell viability of N2a cells was measured by the cell counting kit 8 assay, cell apoptosis rate was determined by flow cytometry.The expression of apoptosis-related genes Bcl-2 and Bax mRNA was detected by quantitative real-time-polymerase chain reaction, and the expression of apoptosis-related genes Bcl-2 and Bax protein was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the apoptosis rate was increased, the expression of Bcl-2 protein and mRNA was down-regulated, and the expression of Bax protein and mRNA was up-regulated in the other three groups ( P<0.05). Compared with group M, the cell viability was significantly increased, the apoptosis rate was decreased, the expression of Bcl-2 protein and mRNA was up-regulated, and the expression of Bax protein and mRNA was down-regulated in group G+ M ( P<0.05). There was no significant difference in the above indexes between group E and group M ( P>0.05). Conclusions:M1 microglia can mediate neuronal injury via exosomes.
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Objective:To investigate the effect of M1 microglia-derived exosomes (M1-exo) on neuronal injury after oxygen-glucose deprivation and restoration, and to explore its mechanism.Methods:The mouse microglia BV2 cells grown in logarithmic growth phase were added with 100 μg/L liposolysaccharide (LPS) and 20 μg/L interferon-γ (IFN-γ) to induce the polarization of microglia into M1 phenotype. M1 microglia were identified by Western blotting, quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence. The supernatant of M1 microglia was collected, and exosomes were extracted by ExoQuick-TC TM kit. The morphology of exosomes were observed by transmission electron microscope and nanoparticle tracking analysis (NTA), and the expression of characteristic proteins CD9 and CD63 of exosomes were detected by Western blotting. The well-growing mouse neuroblastoma N2a cells were divided into six groups: the cells in group C were conventionally-cultured; and the cells in group O were subjected to oxygen-glucose deprivation for 3 hours followed by restoration of oxygen-glucose supply 24 hours to establish the model of oxygen-glucose deprivation and restoration injury; and the N2a cells in group E were co-cultured with M1-exo 24 hours after oxygen-glucose deprivation 3 hours; NC group, M group and I group constructed negative control, overexpression and knockdown of microRNA-20a-5p (miR-20a-5p) M1-exo, respectively. The succession of transfection was detected by qPCR and N2a cells in group NC, group M and group I were co-cultured with such transfected M1-exo for 24 hours after oxygen-glucose deprivation 3 hours. Cell viability were detected by cell counting kit-8 (CCK-8) assay, cell apoptosis were detected by flow cytometry, and the expression of miR-20a-5p were detected by qPCR. Results:Compared with M0 microglia, the fluorescence intensity and mRNA and protein expressions of CD32 and inducible nitric oxide synthase (iNOS), specific markers of M1 microglia, were increased [CD32 (fluorescence intensity): 36.919±1.541 vs. 3.533±0.351, CD32 mRNA (2 -ΔΔCt): 4.887±0.031 vs. 1.003±0.012, CD32/β-actin: 2.663±0.219 vs. 1.000±0.028; iNOS (fluorescence intensity): 29.513±1.197 vs. 7.933±0.378, iNOS mRNA (2 -ΔΔCt): 4.829±0.177 vs. 1.000±0.016, iNOS/β-actin: 1.991±0.035 vs. 1.000±0.045; all P < 0.01], indicating M1 microglia were successfully activated. Under electron microscopy, M1-exo had round or oval vesicular bodies with obvious membranous structures, with diameters ranging from 100 nm. Western blotting showed that the exosomes expressed specific CD63 and CD9 proteins. Compared with group C, the cell viability was decreased, the apoptosis rate and the expression of miR-20a-5p were significantly increased in group O [cell viability ( A value): 0.540±0.032 vs. 1.001±0.014, apoptosis rate: (19.857±0.910)% vs. (13.508±0.460)%, miR-20a-5p (2 -ΔΔCt): 5.508±0.291 vs. 1.033±0.101, all P < 0.01]. Compared with O group, cell viability was decreased, apoptosis rate and the expression of miR-20a-5p were increased in group E [cell viability ( A value): 0.412±0.029 vs. 0.540±0.032, apoptosis rate: (31.802±0.647)% vs. (19.857±0.910)%, miR-20a-5p (2 -ΔΔCt): 8.912±0.183 vs. 5.508±0.291, all P < 0.01], indicating that M1 microglia-derived exosomes further aggravated the damage of N2a cells after oxygen-glucose deprivation and restoration. Compared with group E, cell viability was decreased, apoptosis rate and the expression of miR-20a-5p were increased in group M [cell viability ( A value): 0.311±0.028 vs. 0.412±0.029, apoptosis rate: (36.343±0.761)% vs. (31.802±0.647)%, miR-20a-5p (2 -ΔΔCt): 32.348±0.348 vs. 8.912±0.183, all P < 0.01]; and the cell viability was increased, apoptosis rate and the expression of miR-20a-5p were decreased in group I [cell viability ( A value): 0.498±0.017 vs. 0.412±0.029, apoptosis rate: (26.437±0.793)% vs. (31.802±0.647)%, miR-20a-5p (2 -ΔΔCt): 6.875±0.219 vs. 8.912±0.183, all P < 0.01]. There was no significant difference in cell viability, apoptosis rate and the expression of miR-20a-5p between group E and group NC. Conclusion:M1 microglia-derived exosomes aggravate the injury of neurons after oxygen and glucose deprivation and reoxygenation, which may be related to miR-20a-5p carried by M1-exo.
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OBJECTIVE To optimize the extraction technology of modified Tabusen- 2（MT-2），and to investigate inhibitory effects of the extract obtained by the optimal technology on osteoclast differentiation. METHODS The index components of MT- 2 process optimization were selected by using network pharmacology. Based on single factor tests ，the extraction technology of MT- 2 was optimized by Box-Behnken design-response surface methodology according to the comprehensive score of contents of above index components ，and then validated. RAW 264.7 cells were induced by receptor activator of nuclear factor-κB ligand（100 ng/mL） to prepare osteoclast differentiation model. Inhibitory effects of MT- 2 extract（18.6，37.2，74.4 ng/mL）obtained by the optimal technology on osteoclast differentiation were investigated. RESULTS The index components screened by network pharmacology included chlorogenic acid ，terpineol diglucoside ，isochlorogenic acid A ，1，5-dicaffeoylquinic acid ，hydroxysafflower yellow A ， ginsenoside Rg 1 and ginsenoside Rb 1. The optimal extraction technology of MT- 2 was ethanol volume fraction of 60％ ，the solid-liquid ratio of 1 ∶ 14（g/mL），extraction time of 94 min and extraction times of twice. The average comprehensive score obtained by the three validation experiments was 95.50，and the relative error with the predicted value （95.75）was －0.26％. Compared with osteoclastic differentiation model cells ，the cells treated with MT- 2 extract prepared by the optimal technology were mostly mononuclear round cells ，and the number of osteoclasts decreased significantly （P＜0.05），its inhibitory effects tended to strengthen with the increase of drug concentration. CONCLUSIONS The optimal extraction technology of MT- 2 is stable and feasible. Obtained extract can inhibit osteoclast differentiation.
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OBJECTIVE@#To assess the value of m7G-lncRNAs in predicting the prognosis and microenvironment of colorectal cancer (CRC).@*METHODS@#We screened m7G-lncRNAs from TCGA to construct an m7G-lncRNAs risk model using multivariate Cox analysis, which was validated using ROC and C-index curves. Calibration and nomogram were used to predict the prognosis of CRC patients. Point-bar charts and K-M survival curves were used to assess the correlation of risk scores with the patients' clinical staging and prognosis. CIBERSORT and ESTIMATE were used to explore the association between the tumor microenvironment and immune cell infiltration in patients in high and low risk groups and the correlation of risk scores with microsatellite instability, stem cell index and immune checkpoint expression. A protein-protein interaction network was constructed, and the key targets regulated by m7G-lncRNAs were identified and validated in paired samples of CRC and adjacent tissues by immunoblotting.@*RESULTS@#We identified a total of 1722 m7G-lncRNAs from TCGA database, from which 12 lncRNAs were screened to construct the risk model. The AUCs of the risk model for predicting survival outcomes at 1, 3 and 5 years were 0.727, 0.747 and 0.794, respectively. The AUC of the nomogram for predicting prognosis was 0.794, and the predicted results were consistent with actual survival outcomes of the patients. The patients in the high-risk group showed more advanced tumor stages and a greater likelihood of high microsatellite instability than those in the low-risk group (P < 0.05). The tumor stemness index was negatively correlated with the risk score (r=-0.19; P=7.3e-05). Patients in the high-risk group had higher stromal cell scores (P=0.0028) and higher total scores (P=0.007) with lowered expressions of activated mast cells (r=-0.11; P=0.045) and resting CD4+ T cells (r=-0.14; P=0.01) and increased expressions of most immune checkpoints (P < 0.05). ATXN2 (P= 0.006) and G3BP1 (P=0.007) were identified as the key targets regulated by m7G-lncRNAs, and their expressions were both higher in CRC than in adjacent tissues.@*CONCLUSION@#The risk model based on 12 m7G-lncRNAs has important prognostic value for CRC and can reflect the microenvironment and the efficacy of immunotherapy in the patients.
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Humans , Biomarkers, Tumor/metabolism , Colonic Neoplasms , DNA Helicases/metabolism , Gene Expression Regulation, Neoplastic , Microsatellite Instability , Poly-ADP-Ribose Binding Proteins/metabolism , Prognosis , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , RNA, Long Noncoding/metabolism , Tumor MicroenvironmentABSTRACT
OBJECTIVE@#To investigate the protective effect against intestinal mucosal injury in rats following traumatic brain injury (TBI) and explore the underlying mechanism.@*METHODS@#SD rat models of TBI were established by fluid percussion injury (FPI), and the specimens were collected at 12, 24, 48, and 72 h after TBI. Another 15 rats were randomly divided into shamoperated group (n=5), TBI with saline treatment (TBI+NS) group (n=5), and TBI with PD treatment (TBI+PD) group (treated with 30 mg/kg PD after TBI; n=5). Body weight gain and fecal water content of the rats were recorded, and after the treatments, the histopathology of the jejunum was observed, and the levels of D-lactic acid (D-LAC), diamine oxidase (DAO), ZO-1, claudin-5, and reactive oxygen species (ROS) were detected. Lipid peroxide (LPO) and superoxide dismutase (SOD) 2 content, jejunal pro-inflammatory factors (IL-6, IL-1β, and TNF- α), Sirt1 activity, SOD2 and HMGB1 acetylation level were also determined after the treatments.@*RESULTS@#The rats showed significantly decreased body weight and fecal water content and progressively increased serum levels of D-LAC and DAO after TBI (P < 0.05) with obvious jejunal injury, significantly decreased expression levels of ZO-1 and claudin-5, lowered SOD2 and Sirt1 activity (P < 0.05), increased expression levels of LPO, ROS, and pro-inflammatory cytokines, and enhanced SOD2 and HMGB1 acetylation levels (P < 0.05). Compared with TBI+NS group, the rats in TBI+PD group showed obvious body weight regain, increased fecal water content, reduced jejunal pathologies, decreased D-LAC and DAO levels (P < 0.05), increased ZO-1, claudin-5, SOD2 expression levels and Sirt1 activity, and significantly decreased ROS, LPO, pro-inflammatory cytokines, and acetylation levels of SOD2 and HMGB1 (P < 0.05).@*CONCLUSION@#PD alleviates oxidative stress and inflammatory response by activating Sirt1-mediated deacetylation of SOD2 and HMGB1 to improve intestinal mucosal injury in TBI rats.
Subject(s)
Animals , Rats , Brain Injuries, Traumatic , Glucosides/pharmacology , HMGB1 Protein/metabolism , Oxidative Stress , Rats, Sprague-Dawley , Sirtuin 1/metabolism , Stilbenes/pharmacology , Superoxide Dismutase/metabolismABSTRACT
Objective@#To understand sleep behavior among primary and middle school students and its impact on overweight and obesity changes, to provide evidence for developing obesity prevention and controlling strategies in children and adolescents.@*Methods@#Primary and middle school students from three cities in Zhejiang Province who participated in questionnaire surveys and physical measurements in both 2017 and 2019 were selected. A follow up dataset of 605 students was developed and the relationship between sleep duration and body mass index was analyzed.@*Results@#From 2017 to 2019, BMI Z scores for male and female participants increased by 0.24 and 0.13, respectively. BMI Z scores increased by 0.29 in students of 9-12 years old and increased by 0.11 and 0.25 in urban and rural students, respectively ( P <0.05). The prevalence of insufficient sleep duration increased from 37.0 % to 41.8% simultaneously ( χ 2=3.68, P =0.06). After adjusting for confounding factors, the BMI Z score of students with insufficient sleep was 0.20 higher than those with sufficient sleep duration ( P <0.01). Compared with participants who had sufficient sleep duration from 2017 to 2019, participants whose sleep duration changed from sufficient to insufficient, and those who always had insufficient sleep duration increased by 0.23, respectively ( P <0.05).@*Conclusion@#Insufficient sleep duration is a risk factor for obesity. Shortened sleep duration is related to weight gain, and maintaining sufficient sleep duration may reduce the risk of obesity in children and adolescents.
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Objective:To evaluate the effect of electrical stimulation on lipopolysaccharide (LPS)-induced activation of M1 microglia.Methods:The well-growing BV2 microglia cells were divided into 3 groups ( n=18 each) using a random number table method: control group (group C), group LPS, LPS and electrical stimulation group (group LE). The cells were cultured for 24 h in normal culture atmosphere in group C. In group LPS and group LE, the LPS medium culture 100 ng/ml was added, and the cells were cultured for 24 h. In group LE, cells were stimulated with 100 mV/mm direct current for 4 h before LPS incubation.The levels of tumor necrosis factor-α (TNF-α) and leukocyte interleukin-1β (IL-1β) were determined by enzyme-linked immunosorbent assay.The expression of the M1 microglia surface markers CD32 and inducible nitric oxide synase (iNOS) was detected using immunofluorescent staining.The expression of CD32 and iNOS mRNA was detected using quantitative real-time polymerase chain reaction. Results:Compared with group C, the concentrations of TNF-α and IL-1β were significantly increased, and the expression of CD32 and iNOS protein and mRNA was up-regulated in LPS and LE groups ( P<0.05). Compared with group LPS, the concentrations of TNF-α and IL-1β were significantly decreased, and the expression of CD32 and iNOS protein and mRNA was down-regulated in group LE ( P<0.05). Conclusions:Electrical stimulation can inhibit LPS-induced activation of M1 microglia and thus alleviate the inflammatory responses.
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Objective:To evaluate the effect of long-term intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) on the activation of hippocampal microglia in a mouse model of postoperative cognitive dysfunction (POCD).Methods:Ninety-six clean-grade healthy male C57BL/6 mice, aged 8 weeks, weighing 18-24 g, were stratified according to body weight and divided into 4 groups ( n=24 each) by a random number table method: control diet group (group C), ω-3 PUFAs group (group ω), control diet plus POCD group (group C+ P) and ω-3 PUFAs plus POCD group (group ω+ P). Mice were fed a special ω-3 PUFAs diet (DHA 0.14 g/100 g, EPA 0.03 g/100 g) for 12 weeks in group ω and group ω+ P, while mice were fed with a control diet for 12 weeks in group C and group C+ P.Tibial fracture procedures were performed under isoflurane anesthesia to develop the POCD model after 12 weeks of feeding.The fear conditioning test and Y maze test were performed on 1st and 3rd days after developing the model.The mice were sacrificed after behavioral tests, and the hippocampal tissues were removed for determination of the contents of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (by gas chromatography-mass spectroscopy), density of Iba-1 positive microglia (by immunofluorescence staining), and expression of mature brain-derived neurotrophic factor (mBDNF) and precursor brain-derived neurotrophic factor (pro-BDNF) (by Western blot), and contents of interleukin-1beta (IL-1β) and interleukin-6 (IL-6) (by enzyme-linked immunosorbent assay). Results:Compared with group C, the contents of DHA and EPA were significantly increased, the percentage of freezing time in the contextual test was increased, mBDNF/pro-BDNF ratio was increased ( P<0.05), no significant change was found in the rotation accuracy in Y maze test, density of Iba-1 positive microglia and contents of IL-1β and IL-6 in hippocampus ( P>0.05) in group ω ( P<0.05), and no significant change was found in the contents of DHA and EPA ( P>0.05), the percentage of freezing time in the contextual test and accuracy of rotation in Y maze test were decreased on 1st and 3rd days after operation, the density of Iba-1 positive microglia and contents of IL-1β and IL-6 were increased, and mBDNF/pro-BDNF ratio was decreased in group C+ P ( P<0.05). Compared with group C+ P, the contents of DHA and EPA were significantly increased, the percentage of freezing time in the contextual test and accuracy of rotation in Y maze test were increased on 1st and 3rd days after operation, the density of Iba-1 positive microglia and contents of IL-1β and IL-6 were decreased, and mBDNF/pro-BDNF ratio was increased in group ω+ P ( P<0.05). Conclusions:Long-term intake of ω-3 PUFAs can improve cognitive function in a mouse model of POCD, and the mechanism may be related to inhibition of activation of hippocampal microglia, reduction of inflammatory responses, and thus increasing the mBDNF/Pro-BDNF ratio.
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Objective:To establish the risk prediction models for postoperative delirium (POD) in elderly patients undergoing non-cardiac surgery and to evaluate the predictive efficacy.Methods:A total of 685 patients of both sexes, aged 65-90 yr, of American Society of Anesthesiologists (ASA) physical status Ⅰ-Ⅳ, who underwent non-cardiac elective surgery requiring tracheal intubation during general anesthesia in general surgery, orthopedics, urology, hepatobiliary and pancreatic surgery in our hospital from January 2020 to December 2020, were selected.Patients were assigned to the training set and validation set at a ratio of 7∶3 using a simple random sampling method.The clinical data of patients in the perioperative period were collected, and the patients were followed up within 1-7 days after operation (or before discharge), and the occurrence of POD was recorded.Univariate and multivariate logistic regression analysis was used to identify the independent risk factors for POD.The risk prediction model for POD was established based on the results of multivariate logistic regression analysis of the training set, a nomogram and receiver operating characteristic (ROC) curve were drawn, and the area under the curve (AUC) was calculated.The validation set was used to verify the prediction model and assess the efficacy of the risk prediction model for POD.Results:A total of 653 patients were enrolled in this study, 139 patients developed POD, and the incidence was 21.3%.The results of multivariate logistic regression analysis showed that advanced age, high ASA physical status classification, low preoperative Mini-Mental State Examination score, complication with diabetes mellitus, low years of education, high preoperative Pittsburgh Sleep Quality Index scale score, long anesthesia time and high numerical rating scale score after operation were independent risk factors for POD in elderly patients undergoing non-cardiac surgery.The risk prediction model for POD was established based on the independent risk factors mentioned above.The AUC of the training set was 0.981, the Youden index was 0.881, the sensitivity was 95.95%, and the specificity was 92.92%; the AUC of the validation set was 0.939, the Youden index was 0.795, the sensitivity was 94.44%, and the specificity was 85.09%.Conclusion:The risk prediction model for POD established based on age, ASA physical status classification, history of diabetes melittus, years of education, preoperative Mini-Mental State Examination score, preoperative Pittsburgh sleep quality index scale score, anesthesia time and postoperative numerical rating scale score has good predictive efficacy in elderly patients undergoing non-cardiac surgery.
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Objective:To evaluate the changes in proteome in hippocampus and bioinformatics analysis in mice with perioperative neurocognitive disorders (PND).Methods:Clean-grade healthy male C57BL/6 mice, aged 15 months, weighing 30-35 g, were divided into 2 groups ( n=9 each) using a random number table method: control group (group C) and group PND.The model of PND was established by performing open tibial fracture with intramedullary fixation under isoflurane anesthesia in anesthetized mice.The Morris water maze test, open field test and fear conditioning test were performed at 1 day before operation and at 1, 3 and 7 days after operation.At 1, 3 and 7 days after operation, 3 mice with worst cognitive performance in each cognitive function assessments were sacrificed in group P, and three mice were randomly sacrificed in group C. The hippocampal tissues were then obtained, the expression of differentially expressed proteins was identified by high-performance liquid chromatography-mass spectrometry, and Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to analyze the differentially expressed proteins. Results:Compared with group C, the escape latency at different time points was significantly prolonged, and the percentage of time spend on target quadrant and the percentage of freezing time in fear conditioning test were decreased in group P ( P<0.05). There were 21 differentially expressed proteins, of which 12 proteins showed up-regulated expression and 9 proteins showed down-regulated expression.The GO functional analysis showed that the differentially expressed proteins were involved in the process such as the metabolism, signal transmission, regulation of biological processes, formed cell components such as synapses and organelles, and were related to molecular function such as binding and transportation.KEGG signaling pathway analysis showed that there were also differences in MAPK signaling pathway, ErbB signaling pathway, AMPK signaling pathway and the transport of SNARE protein in vesicle and etc. Conclusion:There are 21 differentially expressed proteins in the hippocampus of PND mice, and these proteins are involved in the pathophysiological process probably related to PND such as neuroinflammatory responses, abnormal synaptic structure, mitochondrial dysfunction and decreased autophagy.
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Objective:To evaluate the relationship between preoperative cerebrospinal fluid/serum albumin ratio (Q-alb) and postoperative delirium (POD) in patients undergoing neuraxial anesthesia.Methods:The patients, aged 40-90 yr, of American Society of Anesthesiologists physical status Ⅰ or Ⅱ, underwent total knee/hip replacement under combined spinal-epidural block in our hospital from January 2018 to December 2020, were collected.After admission to the operating room, venous blood and cerebrospinal fluid samples were collected for determination of cerebrospinal fluid albumin, β-amyloid (Aβ) 1-42, Aβ 1-40, total tau protein (t-Tau), phosphorylated tau protein (p-Tau) and serum albumin levels (by enzyme-linked immunosorbent assay) and for calculation of Q-alb.When Q-alb was more than 10.2, the patient was considered to have blood-brain barrier disruption.Mini-Mental State Examination scale was used to evaluate the cognitive level on 1 day before surgery. The development of POD was evaluated using Confusion Assessment Method Chinese Reversion and Memorial Delirium Assessment Scale at 1-7 days after surgery.The patients were divided into POD group (P group) and non-POD (NP group) according to whether POD occurred.The receiver operating characteristic (ROC) curve was used to analyze the accuracy of Q-alb in predicting POD. Results:There were 49 cases in each group.Compared with group NP, concentrations of Aβ 1-42 and Aβ 1-40 were significantly decreased, concentrations of t-Tau and p-Tau albumin were increased, the ratio of Q-alb and blood-brain barrier disruption was increased in group P ( P<0.05). Before and after adjusting for confounding factors, Q-alb, cerebrospinal fluid Aβ 1-42, Aβ 1-40, t-Tau and p-Tau levels were risk factors for POD ( P<0.05). There was a positive linear regression relationship between Q-alb and levels of t-Tau and p-Tauin cerebrospinal fluid (t-Tau: β=0.587, P<0.001; p-Tau: β=0.427, P<0.001), and there was a negative linear regression relationship between Q-alb and levels of Aβ 1-42 and Aβ 1-40 in cerebrospinal fluid (Aβ 1-42: β=-0.762, P<0.001; Aβ 1-40: β=-0.531, P<0.001). There was no linear regression relationship between Q-alb and level of p-Tau in group P ( P=0.121). There was no linear regression relationship between Q-alb and level of Aβ 1-40 in group NP ( P=0.467). The results of ROC curve analysis showed that the area under the curve for Q-alb in predicting POD (95% confidence interval) was 0.827 (0.738-0.896). Conclusion:Preoperative higher Q-alb is the risk factor for POD in patients undergoing neuraxial anesthesia, and is more accurate in predicting POD.
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Objective:To investigate the relationship between preoperative subjective cognitive decline (SCD) and postoperative delirium (POD) in elderly patients.Methods:A total of 292 elderly patients of both sexes, aged 65-90 yr, weighing 50-90 kg, of American Society of Anesthesiologists physical status Ⅰ-Ⅱ, with Mini-Mental State Examination (MMSE) score>23 and Montreal Cognitive Assessment (MoCA) score > 26 at 1 day before operation, underwent total knee/hip arthroplasty under combined spinal-epidural block in our hospital from January to December 2020, were collected.The development of SCD was evaluated using subjective cognitive decline scale at 1 day before operation.Cerebrospinal fluid (CSF) was extracted after successful spinal-epidural anesthesia puncture, the concentrations of β-amyloid protein 40 (Aβ 40), Aβ 42, total tau (t-tau) and phosphorylated tau (p-tau) were determined by enzyme-linked immunosorbent assay.The incidence of POD was evaluated using confusion assessment method during post-anesthesia care unit and at 1-7 days after operation (or before discharge). Patients were divided into POD group and non-POD group according to whether POD occurred within 7 days after operation.The risk factors of which P values were less than 0.05 would enter the logistic regression analysis to stratify the risk factor for incidence of POD. Results:A total of 205 patients were enrolled and 53 patients developed POD (25.8%). The results of logistic regression analysis showed that preoperative SCD, and increased CSF p-tau and t-tau concentrations were risk factors for POD of elderly patients, and increased CSF Aβ 42 concentration and Aβ 40/p-tau, Aβ 40/t-tau, Aβ 42/p-tau and Aβ 42/t-tau were the protective factors for POD in elderly patients ( P<0.05). After correction of the confounding factors such as age, sex, body weight, education, the history of smoking and drinking, hypertension, diabetes and coronary heart disease, family history of dementia, Pittsburgh sleep quality index (PSQI), MMSE and MoCA score at 1 day before operation, duration of surgery, duration of anesthesia, intraoperative volume of infusion and blood loss and postoperative pain score, SCD, and increased CSF p-tau and t-tau concentrations were still the risk factors for POD in elderly patients, and increased CSF Aβ 42 concentration and Aβ 40/p-tau, Aβ 40/t-tau, Aβ 42/p-tau and Aβ 42/t-tau were still the protective factors for POD in elderly patients ( P<0.05). Conclusion:Preoperative SCD is the risk factor for POD in elderly patients.
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Objective:To evaluate the value of cerebrospinal fluid (CSF) nerve injury-related proteins levels in predicting postoperative delirium (POD) in patients.Methods:A total of 1 000 patients of both sexes, aged 40-90 yr, of American Society of Anesthesiologists physical status Ⅰ or Ⅱ, with Mini-Mental State Examination score>24 at 1 day before operation, undergoing elective knee/hip arthroplasty under spinal-epidural anesthesia , were enrolled in this study.Cubital venous blood samples were drawn before anesthesia for detection of the concentrations of plasma total cholesterol, high-density lipoprotein, low-density lipoprotein and triglyceride.CSF 2ml was extracted after successful spinal-epidural anesthesia puncture for measurement of concentrations of α-synuclein (α-syn), β-amyloid protein 1-40 (Aβ 1-40), Aβ 1-42, total-Tau (t-Tau), phosphorylated Tau (p-Tau), progranulin (PGRN) and soluble myeloid cell triggering receptor 2 (sTREM2) (by enzyme-linked immunosorbent assay). The Confusion Assessment Method was used at 1, 3 and 7 days after surgery to evaluate the occurrence of POD.The patients were divided into POD group and non-POD group according to whether POD occurred after operation.Logistic regression analysis was used to analyze the variables of which P values were less than 0.05 to analyze the risk factors for POD.The receiver operating characteristic (ROC) curve was drawn and area (AUC) under the curve was calculated to evaluate the accuracy of the related risk factors in predicting POD. Results:A total of 964 patients were enrolled in the study, and 108 patients were diagnosed with POD, and the incidence was 11.2%.The results of logistic regression analysis found that age and and increased α-syn in CSF concentration were risk factors for POD, and decreased PGRN in CSF concentration and Aβ 1-42/p-Tau in CSF were the protective factors for POD ( P<0.05). ROC curve analysis showed that α-syn (AUC 0.69, 95% confidence interval (CI) 0.634-0.748, sensitivity 57.41%, specificity 82.10%, Youden Index 0.3951), PGRN in CSF concentration (AUC 0.695, 95%CI 0.637-0.750, sensitivity 59.26%, specificity 80.86%, Youden Index 0.4012) and Aβ 1-42/p-Tau in CSF (AUC 0.635, 95%CI 0.574-0.692, sensitivity 93.52%, specificity 30.25%, Youden Index 0.2377) could predict the occurrence of POD. Conclusion:PGRN, α-syn concentration and Aβ 1-42/p-Tau in CSF can predict the occurrence of POD in patients.
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Objective:To investigate the value of α-synuclein (α-syn) concentration in cerebrospinal fluid (CSF) in predicting postoperative delirium (POD).Methods:One thousand patients underwent elective surgery with combined epidural-spinal anesthesia in our hospital from January 2018 to September 2020 were selected.The epidural puncture was performed at L 3, 4 interspace, and 2 ml of CSF was collected after the needle reaching the subarachnoid space.The concentrations of α-syn, β-amyloid (Aβ)40, Aβ42, total tau protein (T-tau), and phosphorylated tau protein (P-tau) in CSF were determined by enzyme-linked immunosorbent assay.The concentrations of α-syn in CSF and occurrence of POD in patients of different ages were recorded.Patients were divided into POD group and non-POD group according to whether POD occurred, and frequency matching (1∶1) was performed based on five matching variables of age, ASA physical status, education level, duration of operation, and intraoperative blood loss. Results:Eight hundred and forty-one patients were finally included in the study, and the incidence of POD was 15.0%. There were 126 cases in POD group and 126 cases in non-POD group after matching. The concentrations of α-syn in CSF and incidence of POD were gradually increased with age ( P<0.05). Compared with non-POD group, the concentrations of α-syn, T-tau and P-tau in CSF were significantly increased, the concentrations of Aβ40 and Aβ42 were decreased, Aβ40/P-tau, Aβ42/P-tau, Aβ42/Aβ40 and P-tau/T-tau were decreased in POD group ( P<0.05). After confounding factors were corrected by logistic regression analysis, increased concentrations of α-syn, p-tau, and T-tau in CSF were risk factors for POD ( P<0.05). Increased concentrations of Aβ40 and Aβ42 in CSF and increased Aβ40/P-tau and Aβ42/P-tau were protective factors for POD ( P<0.05). Multiple linear regression analysis showed that the concentration of α-syn in CSF was negatively correlated with Aβ40 and Aβ42 concentrations and positively correlated with P-tau and T-tau concentrations ( P<0.05). The area under the receiver operating characteristic curve of concentrations of α-syn in CSF predicting POD was 0.895, Youden index was 0.664, sensitivity was 80.00%, and specificity was 86.36% ( P<0.001). Conclusion:The concentration of α-syn in CSF is related to the occurrence of POD, and it provides higher accuracy in predicting POD.
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Objective:To evaluate collared and bipolar hemiarthroplasty combined with medial femoral calcar reconstruction in the treatment of senile patients with osteoporotic unstable intertrochanteric fracture.Methods:The data of 28 senile patients with unstable femoral intertrochanteric fracture were retrospectively analyzed who had been admitted to Department of Orthopedics, The First Affiliated Hospital to Zhejiang University of Chinese Medicine from March 2014 to February 2020. They were 8 males and 20 females, aged from 75 to 99 years (average, 81.5 years). All the fractures were low violence injuries due to falls. By the Evans-Jensen classification, there were 2 cases of type Ⅲ, 5 cases of type Ⅳ and 21 cases of type Ⅴ. All patients were osteoporotic, with a BMD T-score ranging from -4.5 to -2.0. Surgery was performed 2 to 6 days after injury (3.8 days on average). Corail collared femoral stems and bipolar ball heads produced by DePuy company were selected for implantation during surgery. Their fractures were reduced and fixated by titanium wire bundling system or bifilar winding wire bundles. The femoral calcar reconstruction was accomplished by inserting the beak-shaped distal part of the head-neck fracture fragment into the femoral medullary cavity together with the medial side of the stem just under the collar and impacting it to a tight position.Results:The average operation time for this group of patients was 62 min (from 50 to 85 min) and the average intraoperative blood loss 170 mL (from 110 to 320 mL). All the 28 patients were followed up for 10 to 71 months (average, 46 months). Their Harris hip scores averaged 92 points (from 89 to 96 points) at 6 months after operation. Two patients developed intermuscular venous thrombosis in the calf after operation. During follow-up, none of the patients had such complications as deep iliac femoral vein thrombosis, pulmonary embolism, incision infection, or deep prosthesis infection. At the last follow-up, their Harris hip scores averaged 88 points (from 82 to 96 points).Conclusion:For some senile patients with osteoporotic unstable intertrochanteric fracture, collared and bipolar hemiarthroplasty combined with medial femoral calcar reconstruction can achieve fine therapeutic efficacy.
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Objective:To evaluate the prognostic value of extravascular lung water index (EVLWI) , soluble intercellular adhesion molecule-1(sICAM-1) and Krebs yon den lungen-6 (KL-6) in severe pneumonia patients with Severe Acute Respiratory Syndrome (ARDS).Methods:A prospective study was conducted in Respiratory Intensive Care Unit of the Affiliated Zhengzhou Central Hospital of Zhengzhou University from October 2017 to February 2020. The study included 65 severe pneumonia patients with ARDS, who was performed by measurement of pulse index continuous cardiac output and survived more than 3days after admission. The Extravascular Lung Water Index (EVLWI) , sICAM-1, KL-6 and Oxygenation Index(OI) on 1st, 3rd and 5th day were detected. APACHEⅡ score, patient survival events (days) and survival outcome were recorded. Correlation analysis between EVLWI, sICAM-1, KL-6 and OI was performed on the 1st, 3rd and 5th day after admission. Independent risk factors of mortality in severe pneumonia patients with ARDS were analyzed by multiple logistic regression. Receiver operating characteristic curve was drawn, and the prognostic value of each parameter was assessed finally.Results:The PCT, EVLWI, sICAM-1, KL-6 and APACHEⅡ score in the death group were significantly higher than those in the survival group ( P<0.05) at RICU admission, and the length of RICU stay was significantly shorter than that in the survival group ( P<0.05), while differences in other clinical characteristics between two groups were not statistically significant ( P>0.05) . These parameters including levels of EVLWI, sICAM-1, KL-6, Procalcitonin and APACHE Ⅱscore in the death group were significantly higher than those in the survival group on the 1st, 3rd and 5th day ( P<0.05), whereas the OI was significantly lower than that of the survival group on the 3rd and 5th day ( P<0.05). Logistic regression analysis showed that EVLWI, sICAM-1, KL-6 level were significantly related with the mortality of these patients. The levels of sICAM-1, kl-6 and EVLWI on 1st, 3rd and 5th day after RICU admission showed a significant negative correlation with OI ( P<0.001). Whereas, The levels of sICAM-1, kL-6 on 1st, 3rd and 5th day showed a significant positive correlation with EVLWI ( P<0.001). The sensitivity and specificity of sICAM-1, KL-6 combined with EVLWI in prognosis evaluation on 1st, 3rd and 5th day were 75.0%, 84.4%, 85.0%, 66.7%, 80.0%, 86.7%, respectively. The AUC was 0.864, 0.881, 0.892 on 1st, 3rd and 5th day, respectively ( P<0.001), which had a better prognostic value than each of them. Conclusions:EVLWI, sICAM-1 and KL-6 were independent risk factors for the prognosis of severe pneumonia patients with ARDS. The combination of EVLWI, sICAM-1 and KL-6 might be important in early predicting the prognosis of the 28d mortality.