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Objective:To evaluate the relationship between M2-type microglia-derived exosomes (M2-exo) and neuronal oxygen-glucose deprivation and restoration (OGD/R) injury in mice.Methods:Mouse neuroblastoma cells (N2a cells) and BV2 microglia were cultured in vitro, and BV2 microglia were activated to M2 type using 20 ng/ml IL-4, and M0-type microglia-derived exosomes (M0-exo) and M2-exo were extracted.N2a cells were divided into 4 groups ( n=23 each) using the random number table method: control+ M0-exo group (C+ M0 group), control+ M2-exo group (C+ M2 group), OGD/R+ M0-exo group (O+ M0 group) and OGD/R+ M2-exo group (O+ M2 group).M0-exo and M2-exo (final concentration 100 μg/ml) were added in C+ M0 and C+ M2 groups, respectively, and the cells were incubated for 24 h. M0-exo and M2-exo (final concentration 100 μg/ml) were added at 3 h after oxygen and glucose deprivation, and then the cells were incubated for 24 h in O+ M0 and O+ M2 groups, respectively.N2a cell viability was measured by the CCK-8 method, and the severity of cell damage was assessed using the lactic dehydrogenase (LDH) release rate.The expression of Bax and Bcl-2 protein and mRNA was detected by quantitative real-time polymerase chain reaction and Western blot. Results:Compared with C+ M0 group, no significant changes were found in N2a cell viability, LDH release rate, Bax/Bcl-2 ratio and Bax mRNA/Bcl-2 mRNA ratio in C+ M2 group ( P>0.05), and N2a cell viability was significantly decreased, and the LDH release rate, Bax/Bcl-2 ratio and Bax mRNA/Bcl-2 mRNA ratio were increased in O+ M0 group ( P<0.05).Compared with C+ M2 group, the N2a cell viability was significantly decreased, and the LDH release rate, Bax/Bcl-2 ratio and Bax mRNA/Bcl-2 mRNA ratio were increased in O+ M2 group ( P<0.05).Compared with O+ M0 group, N2a cell viability was significantly increased, and LDH release rate, Bax/Bcl-2 ratio, and Bax mRNA/Bcl-2 mRNA ratio were decreased in O+ M2 group ( P<0.05). Conclusions:M2-exo exerts an endogenous protective effect during OGD/R in mouse neurons, which may be related to the inhibition of cell apoptosis.
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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 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 clinical and electrophysiological features of patients with non-inflammatory myopathy with neurogenic lesions. Method The clinical and electromyography data was retrospectively collected from 110 patients who were diagnosed with myopathy and completed routine electromyography examination from 2015 to 2017. A retrospective analysis of clinical and electrophysiological features was conducted on 4 patients with non-inflammatory myopathy with neurogenic lesions. Result Of the 110 patients, 10 patients with neurogenic lesions and 4 of them were diagnosed to have non-inflammatory myopathy. These 4 patients had limb and trunk weakness with muscle atrophy and the electromyography showed neurogenic lesion with or without peripheral nerve lesion. Conclusion This study has revealed neurogenic lesions in a small number of non-inflammatory myopathy on the electromyography, suggesting that the electromyography alone may not be sufficient for diagnosis of myopathy.
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Objective To study the effect and safety of compound Biejiaruangan tablets combined with entecavir on the treatment of viral hepatitis B cirrhosis.Methods100 cases with hepatitis B cirrhosis in Zhejiang Taizhou hospital from June 2014 to December 2016 were selected as the research object in this study which were randomly divided into the control group and the experimental group 50 cases in each group.The control group were given entecavir, the experimental group were given compound Biejiaruangan tablets combined with entecavir.The clinical effect and the adverse reaction in the two groups were compared.ResultsThe effective rate was 90.0% in the experimental group, which was significantly higher than 72.0% in the control group(P<0.05).There were 2 patients vomiting in the experimental group, the adverse reaction rate was 4.0%.There were 9 cases of vomiting and headache in the control group, the adverse reaction rate was 18.0%.T,he adverse reaction rate(in the experimental group was significantly lower than that in the control group(P<0.05).ConclusionThe clinical effect of compound Biejiaruangan tablets combined with entecavir on the treatment of viral hepatitis B cirrhosis is better, which can reduce the rate of adverse reactions in a certain extent, is high safety, and worth popularizing.
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Objective:To clarify the significance of the fractional anisotropy(FA) value and apparent diffusion coefficient(ADC) value of diffusion tensor imaging(DTI) applied in the glioma grading with Meta-analysis, and to provide a reference for selecting the treatment program and judging the prognosis of glioma.Methods:Computer search was performed in PubMed database, EMBASE database,CBM, CNKI, Wanfang database, and VIP database.The relevant neurosurgery magazines and neuroimaging magazines, the proceedings of academic meeting were retrieved by hand and the references of related reviews were retrospectively retrieved.Then the case-series studies according to inclusion and exclusion criteria were screened,and the qualities of included studies were evaluated.Meta-analysis was carried out by Review Manager 5.2 software.Results:Nine studies were included.The FA value of low-grade glioma(LGG) in tumor centre was lower than that of high-grade glioma(HGG) (χ2=11.94,df=8,P=0.15;I2=33%,Z=2.78,P=0.005;WMDFA=-0.02,95%CI:-0.03--0.01),and the ADC value of LGG in tumor centre was higher than that of HGG (χ2=5.26,df=4,P=0.26;I2=24%,Z=2.19,P=0.03;WMDADC=0.15,95%CI:0.02-0.28).Conclusion:When the FA value in tumor centre of the glioma is increasd,the ADC value is decreased,and the grade of glioma is increasd.The FA value and ADC value of DTI can help determine the grade of gliomas.
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Objective To compare the effect of oxycodone or sufentanil in preventing post-operative hyperalgesia induced by remifentanil.Methods One hundred and twenty patients scheduled for radical operation for carcinoma of stomach undergoing general anesthesia, 62 males and 58 females, aged 18-65 years, ASA physical status Ⅰ or Ⅱ, were randomly divided into 3 groups.Thirty minutes before the end of operation, oxycodone hydrochloride 10 mg was administered intravenously in oxycodone hydrochloride group (group O, n=42) while sufentanil 10 μg was injected in group S (group S, n=40) and equal normal saline was injected in control group (group C, n=38).The VAS score after waking up was recorded.VAS score>3 was defined as hyperalgesia, and sufentanil was injected when VAS score≥6.The BCS scores were recorded immediately after extubation (T1), 30 min (T1), 1 h (T3), 2 h (T4) and 4 h (T5) after extubation.Awakening time and extubation time and the incidence of nausea and vomiting 4 h after extubation were compared.Results At T1-T5, the BCS scores in group O were significantly higher than those in groups S and C (P<0.05).Group S has a higher adverse reaction (P<0.05).There was no significant differences of awakening time and extubation time in 3 groups.Conclusion Prophylactic injection of oxycodone 10 mg can reduce incidence rate of post-operative hyperalgesia, and increase BCS score with little side effects.
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Objective To investigate the loss of motor unit and it's influencing factors in the lower motor neurons after middle cerebral artery infarction. Method Forty patients with first onset and unilateral middle cerebral artery infarction were divided into cortical-basal ganglia(26)and basal ganglia(14)groups and 10 healthy controls were served as control group.All included patients were scored by National Institute of Health stroke scale(NIHSS),modified Rankin scale (mRS), Fugl-Meyer Assessment (FMA) at 48 hours of admission. Nerve conduction study on the limb and motor unit number estimation (MUNE) on abductor pollicis brevis were performed at 2-4 weeks after onset, and the data of single motor action potential (SMUAP) were collected. SPSS 20.0 software was used to statistical analysis. Result The MUNE on were significantly lower and the amplitude and area of SMUAP were significantly increased in ipsilateral than contralateral sides (cortical-basal ganglia group:95.85±26.82 vs. 143.65±38.86, P<0.001; basal ganglia group: 126.71± 44.13 vs. 157.36±56.72, P=0.001). The affected MUNE was significantly decreased in the cortex-basal ganglia than in basal ganglia groups (95.85±26.82 vs.161.40±48.90,P=0.027). The MUNE was negatively correlated with NIHSS score (r=-0.362,P=0.022)and mRS score(r=-0.339,P=0.032).NIHSS score(β=-1.603,P=0.032,95%CI:-3.064~-0.142)and mRS score(OR=2.885,P=0.025,95%CI:1.139~7.158)on admission could predict the loss of MUNE on the affected side. Conclusion This study reveals the loss of motor unit and the compensation of remained motor unit on the affected side after middle cerebral artery infarction,NIHSS score and mRS score on admission may predict the loss of MUNE after stroke.
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This paper introduces and analyzes the changes of scientific understanding in the linkage of global envi-ronment and health from historical and environment perspective, emphasizing on the fact that:the environmental issue im-pacts on human health are attracting an increased attention and emphasis from the international community;the existing re-search has fully proved a clear causal relationship between environmental issues which constitute one of the major factors that lead to a variety of human diseases and human health risks;the environmental issues effects on health quantified at the global level are the highlights and difficulties in the related research, differences in the current research methods and con-clusions exist internationally, but quantitative research is popular; the integration perspective of the existing scientific knowledge and global policy is a huge challenge placed in front of the global environment and health governance.
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China faces opportunities and challenges as well when it participates in global environment and health governance. Major opportunities are as follows:(1) The convergence between global environment and health governance’s future trend and China’s environment and health strategies is a strongest incentive to push China to par-ticipate in global environment and health governance;(2) China can benefit from this participation in term of more access to international resources; ( 3 ) Global environmental and health issues are the areas that can best illustrate China’s international image as a responsible member; ( 4 ) Since global environmental and health governance is still young, early participation will give China a louder voice in its decision-making. The challenges that China faces are as follows:(1) Huge pressure from domestic environment and health challenges forces China to be primarily inward-looking and makes it very hard for the country to keep a high profile in global environment and health governance. China also faces the increasing international pressure. ( 2 ) The lack of global environment and health governance strategy prevents China from effectively participating in global environment and health governance. (3)“The Grand Health Outlook”hasn’t been fully established yet in China, leaving much room for improvement. (4) Intellectual sup-port is inadequate. (5) Some countries have too high expectations of China in terms of its contribution to global envi-ronmental and health governance. In order to effectively participate in this field, China should undertake coordinated planning on both the international and domestic scenes, and take some specific policies both at national and interna-tional level.
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In order to strengthen the education for university students’all-round devel-opment,and to improve the quality of teaching,Shantou University,by making good use of higher education resources,built a digital campus consisting of virtual classrooms based on advantage of computer and Internet network environment,and plenty of general education programs were carried out.The teaching practice showed that virtual classroom,as a network platform,played an important role in general education programs.