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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 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 role of has_circ_0008039 and miR-484 in oxygen-glucose deprivation/reoxygenation (OGD/R) injury in SK-N-SH cells and the relationship with Fis1.Methods:SK-N-SH cells were cultured in vitro to logarithmic growth stage and divided into 5 groups ( n=25 each) according to the random number table method: control group (group C), OGD/R group, has_circ_0008039 siRNA group (group S), hsa_circ_0008039 over-expression group (group E) and has_circ_0008039 siRNA plus miR-484 inhibitor group (group S+ I). Cells were cultured in normal condition in group C. In S, E and S+ I groups, after the cells were transfected with hsa_circ_0008039 siRNA, has_circ_0008039 over-expression vector, hsa_circ_0008039 siRNA and miR-484 inhibitor, the cells were subjected to oxygen-glucose deprivation for 12 h followed by 24 h restoration of O 2-glucose supply to develop the OGD/R model.At 24 h of restoration of O 2-glucose supply, the cell viability and amount of lactic dehydrogenase (LDH) released were measured using CCK-8 assay, the expression of hsa_circ_0008039, miR-484 and Fis1 mRNA was detected using real-time polymerase chain reaction, and the expression of Fis1 was detected by Western blot.A dual-fluorescein experimental report was used to verify the targeting relationship between hsa_circ_0008039 and miR-484. Results:Compared with group C, the cell viability was significantly decreased, and the amount of LDH released was increased in the other 4 groups, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in OGD/R and E groups, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group OGD/R, the cell viability was significantly decreased, and the amount of LDH released was increased in E and S+ I groups, the cell viability was significantly increased, and the amount of LDH released was decreased in group S, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in group E, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and the expression of miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group S, the cell viability was significantly decreased, the amount of LDH released was increased, the expression of miR-484 was down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.01). The dual-fluorescein experimental report verified that miR-484 was the target of hsa_circ_0008039 which binded to miR-484 specifically. Conclusions:has_circ_0008039 is involved in OGD/R injury in SK-N-SH cells by targetedly binding to miR-484, which is associated with up-regulation of Fis1 expression.
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Three children with Skene duct cyst were presented in this article. By reviewing literature, in pediatric population, Skene duct cycts mostly occur in newborns and conservative therapy is the first choice in this group.In contrast, it is extremely rare between the ages of 1 and 12, and surgical excised is the preferred therapy because of having a similar pathogenesis to adults.
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Objective:To explore the clinical features and treatment strategy of congenital anterior urethrocutaneous fistula.Methods:A total of 7 cases with congenital anterior urethrocutaneous fistula were repaired by surgery between January 2006 and February 2019 in Affiliated Children’s Hospital of Xi’an Jiaotong University. The median age was 30 (18-92) months. All of cases had a intact prepucs and a normal external urethal meatus located at the tip of glans. Fistula located at subcoronal culus in 2 cases, midshaft in 3 cases, penioscrotal region in 1 case, scrotum in 1 case, respectively.Defect longitudinal diameter was 0.5-1.5cm. Associated anomalies including division of scrotum in 3 cases, penile chordee in 2 cases, urethral meatus stenosis in 1 case, right hydrocele in 1 case. Six cases had underwent one-stage fistula repair incluing Duplay procedure in 4 cases(case 1, 2, 4 and 6), Onlay preputial flap in 1 case(case 3), TIP repair with dorsal plication for straightening and urethrotomy in 1 case(case 5). Case 7 had underwent a two-stage repair, which received Duckett flap repair with urethrostomy simultaneously at the base of the penis, and the defect was closed in second procedure. All of neourethras were reinforced by soft tissues from different places.Results:Of 6 cases with one-stage repair, the catheter was removed 10-14 days after surgery in 5 cases. Removal of the catheter was delayed until 3 weeks in case 3 because of poor wound healing. Case 7 received Duckett flap repair with urethrostomy in the initial surgery, who recovered uneventfully and was resolved during the second operation. No recurrence, urethral stricture or chordee occurence were noted in all after a 1-8 years followup period.Conclusions:Congenital anterior urethrocutaneous fistula have a high overall success rate.Duplay could be applied to cases without penile curvature, and well-developed urethral plate. Onlay or TIP is more suitable for cases with narrow urethral plate. The principle of hypospadias repair should be followed for those cases with severe penile curvature.
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Objective:To evaluate the role of miR-188-5p in oxygen-glucose deprivation and restoration (OGD/R) injury to mouse neuroblastoma (N2a) cells and its relationship with small ubiquitin-like modifier-specific proteases 3 (SENP3).Methods:N2a cells were cultured and divided into 5 groups ( n=23 each) using a random number table method: control group (group C), OGD/R group, group NC, transfection of mir-188-5p agonist group (group M) and transfection of mir-188-5p inhibitor group group (group I). Cells in group C were cultured routinely.Cells in group NC, group M and group I were transfected with mir-188-5p negative control miRNA, agonist and inhibitor, respectively.N2a cells were subjected to OGD for 3 h followed by restoration of oxygen-glucose supply to establish the model of OGD/R injury.At 24 h of oxygen-glucose restoration, the cell viability was recorded by the cell counting kit-8 assay, the amount of lactic dehydrogenase (LDH) released was detected, the expression of miR-188-5p and SENP3 mRNA was detected by quantitative real-time polymerase chain reaction, and SENP3 expression was determined by Western blot.The targeting relationship between miR-188-5p and SENP3 mRNA was detected using dual luciferase reporter assay. Results:Compared with group C, the cell viability was significantly decreased, amount of LDH released was increased, and expression of SENP3 and its mRNA was up-regulated in the other 4 groups, miR-188-5p expression was down-regulated in OGD/R and I groups, and miR-188-5p expression was up-regulated in group M ( P<0.05 or 0.01). Compared with group OGD/R, the cell viability was significantly decreased, amount of LDH released was increased, and expression of SENP3 and its mRNA was up-regulated, and miR-188-5p expression was down-regulated in group I, and the cell viability was increased, amount of LDH released was decreased, expression of SENP3 and its mRNA was down-regulated, and miR-188-5p expression was up-regulated in group M ( P<0.05 or 0.01). The dual luciferase reporter assay showed that miR-188-5p could act directly on SENP3. Conclusion:miR-188-5p is involved in OGD/R injury, which is associated with targeted down-regulation of SENP3 expression in N2a cells.
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Objective:To evaluate the gastric emptying of orally administered enzyme-hydrolyzed rice flour solution before surgery in the patients undergoing laparoscopic cholecystectomy and effect on insulin resistance.Methods:One hundred patients, of American Society of Anesthesiologists physical status Ⅰ or Ⅱ, aged 18-64 yr, with body mass index of 19-30 kg/m 2, scheduled for elective laparoscopic cholecystectomy under general anesthesia, were divided into 2 groups ( n=50 each) using a random number table method: water group (group C) and enzyme-hydrolyzed rice flour group (group M). Routine fasting and water deprivation were executed at 1 day before operation in two groups, and 300 ml water in group C or 300 ml enzyme-hydrolyzed rice flour solution in group M were taken orally at 2-3 h before induction on the day of surgery.Bedside antrum ultrasonography was used to calculate the gastric volume (GV) before oral administration (V 0), immediately after oral administration (V 1), and before induction (V 2), and then the ΔGV (GV 1-GV 0) was calculated.Fasting plasma glucose and insulin CONCENTRATIONS were measured on admission to hospital (T 1) and on an empty stomach on 1st morning after surgery (T 2), and then the homeostasis model assessment of insulin resistance (HOMA-IR) was calculated according to HOMA steady-state model formula.Visual analog scale (VAS) scores for subjective comfort (thirst, hunger, fatigue and anxiety) and grip strength were assessed before anesthesia (T 3) and before leaving PACU (T 4). Reflux and aspiration during induction, nausea and vomiting within 24 h after surgery, and anal exhaust time after surgery were recorded. Results:There was no significant difference in GV at V 0, V 1 and V 2 between the two groups ( P>0.05). Compared with the baseline at V 0, no significant was found in the GV at V 2 in both groups ( P>0.05). The fasting plasma glucose and insulin concentrations and HOMA-IR were significantly increased at T 2 than at T 1 in both groups ( P<0.05 or 0.01). Compared with group C, the fasting plasma glucose and insulin concentrations and HOMA-IR were significantly decreased at T 2, VAS scores for hunger, fatigue and anxiety were decreased at T 3, 4, grip strength was increased at T 3, 4, the postoperative anal exhaust time was shortened, and the incidence of nausea was reduced in group M ( P<0.05). No reflux and aspiration happened during induction in either group. Conclusion:The gastric emptying of 300 ml enzyme-hydrolyzed rice flour solution orally administered at 2 h before surgery is normal in the patients undergoing laparoscopic cholecystectomy, which does not increase the risk of reflux and aspiration during anesthesia induction, reduces postoperative insulin resistance, and increases patient′s subjective comfort, and enhances the postoperative recovery of intestinal function.
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Objective:To investigate the effect of irisin on pyroptosis in rats with ventilator-induced lung injury.Methods:Thirty-six healthy clean-grade male Sprague-Dawley rats, weighing 200-250 g, aged 6-8 weeks, were divided into 3 groups ( n=12 each) using a random number table method: control group (group C), ventilator-induced lung injury group (group V) and ventilator-induced lung injury plus irisin group (group V+ I). In group V+ I, irisin 1 μg/kg was injected via the tail vein before mechanical ventilation.The animals were mechanically ventilated (tidal volume of 40 ml/kg, respiratory rate 60 breaths/min, inspiratory/expiratory ratio 1∶2, positive end expiratory pressure 0 and inspired oxygen fraction ratio 21%.Blood samples were then taken from the femoral artery for blood gas analysis, and PaO 2 was recorded.Bronchoalveolar lavage fluid (BALF) was collected, the total protein concentrations in BALF were measured, and the concentrations of BALF and serum interleukin-1β (IL-1β) and IL-18 were measure by enzyme-linked immunosorbent assay.The lung tissues were obtained for determination of the pathological changes after HE staining which were scored, wet to dry weight (W/D) ratio, expression of pyroptosis-related proteins N-terminal gasdermin D (GSDMD-N) and caspase-1 protein and mRNA (by Western blot or using real-time polymerase chain reaction). Results:Compared with group C, the lung injury score and W/D ratio were significantly increased, PaO 2 and OI were decreased, the total protein concentrations in BALF, concentrations of IL-1β and IL-18 in BALF and serum were increased, and the expression of caspase-1 and GSDMD-N protein and mRNA was up-regulated in group V ( P<0.01). Compared with group V, the lung injury score and W/D ratio were significantly decreased, PaO 2 and OI were increased, the total protein concentrations in BALF, concentrations of serum IL-1β and IL-18 in BALF and serum were decreased, and the expression of caspase-1 and GSDMD-N protein and mRNA was down-regulated in group V+ I ( P<0.01). Conclusion:The mechanism by which irisin reduces ventilator-induced lung injury is probably related to inhibiting pyroptosis in rats.
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Objective:To evaluate the role of hsa_circ_0025853 in hypothermia-induced reduction of oxygen-glucose deprivation and restoration (OGD/R) injury to neuroblastoma cells (SK-N-SH cells). Methods:SK-N-SH cells were cultured in vitro to the logarithmic phase and divided into 4 groups ( n=20 each) using a random number table method: control group (group C), group OGD/R, hypothermia group (group H) and hsa_circ_0025853 small interfering RNA (siRNA) plus hypothermia group (group S+ H). The cells were cultured in normal culture atmosphere in group C. The cells were subjected to OGD for 3 h followed by restoration of oxygen-glucose supply for 24 h in group OGD/R.The cells were subjected to OGD for 3 h followed by restoration of oxygen-glucose supply for 24 h at 32 ℃ in group H. The cells were transfected with siRNA at 48 h before establishing OGD/R model, and the other treatments were similar to those previously described in group H. At 24 h of restoration of oxygen-glucose, cell viability was measured by Cell Counting Kit-8, apoptosis rate were measured by flow cytometry.the expression of dynamin-related protein 1 (Drp1) mRNA and hsa_circ_0025853 was detected by quantitative real-time-polymerase chain reaction, and the expression of Drp1 protein was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the apoptosis rate of cells was increased, the expression of hsa_circ_0025853 was down-regulated, and the expression of Drp1 protein and mRNA was up-regulated in the other 3 groups ( P<0.05). Compared with group OGD/R, the cell viability was significantly increased, the apoptosis rate of cells was decreased, the expression of hsa_circ_0025853 was up-regulated, and the expression of Drp1 protein and mRNA was down-regulated in H and S+ H groups ( P<0.05). Compared with group H, the cell viability was significantly decreased, the apoptosis rate of cells was increased, the expression of hsa_circ_0025853 was down-regulated, and the expression of Drp1 protein and mRNA was up-regulated in group S+ H ( P<0.05). Conclusion:The mechanism by which hypothermia alleviates the OGD/R injury to SK-N-SH cells is related to the up-regulation of hsa_circ_0025853 expression, thus reducing the expression level of Drp1.
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Objective:To evaluate the effect of mild hypothermia on inositol requiring enzyme 1-X-box binding protein 1 (IRE1-XBP1) signaling pathway in endoplasmic reticulum in cortex in a rat model of focal cerebral ischemia-reperfusion (I/R).Methods:Fifty-four clean-grade healthy male Sprague-Dawley rats, aged 8-10 weeks, weighing 200-230 g, were divided into 3 groups ( n=18 each) using a random number table method: sham operation group (group S), cerebral I/R group (group I) and mild hypothermia group (group T). Cerebral I/R was induced by inserting a nylon thread with rounded tip into the internal carotid artery which was occluded for 2 h and then released for reperfusion.The surface cooling was started immediately after reperfusion, and the rectal temperature was maintained at 32-34 ℃ for 3 h in group T. Blood vessels were only exposed, without occlusion in group S. The neurologic deficit was assessed and scored at 24 h of reperfusion.The animals were then sacrificed and the ischemic area of the cerebral cortex was removed for examination of the ultrastructure of the cells (with a transmission electron microscope), for determination of nerve cell apoptosis (using TUNEL), for detection of the expression of IRE1 and XBP1 (by Western blot) and for determination of the expression of IRE1 and XBP1 protein mRNA (using quantitative real-time polymerase chain reaction). Results:Compared with group S, the neurologic deficit scores were significantly increased, nerve cell apoptosis in the ischemic area of the cerebral cortex was increased, the expression of IRE1, XBP1 protein and mRNA was up-regulated ( P<0.05), the neuronal nuclei was degenerated and swollen, the nuclear membrane was fragmented and defective, the chromatin was pyknotic and marginalized, and the endoplasmic reticulum was dilated and cisternal in group I and group T. Compared with group I, the neurologic deficit scores were significantly decreased, nerve cell apoptosis in the ischemic area of the cerebral cortex was decreased, the expression of IRE1, XBP1 protein and mRNA was up-regulated ( P<0.05), and the damage to the ultrastructure of nerve cells was reduced in group T. Conclusion:The mechanism by which mild hypothermia alleviates focal cerebral I/R injury is associated with further activation of neuronal IRE1-XBP1 signaling pathway and alleviation of endoplasmic reticulum stress response in rats.
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Objective:To evaluate the effect of electroacupuncture (EA) preconditioning on expression of cortical Ubc9 during cerebral ischemia-reperfusion (I/R) in rats.Methods:A total of 80 healthy clean-grade male Sprague-Dawley rats, aged 8-12 weeks, weighing 200-250 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (group S), group I/R, EA preconditioning group (group E) and sham EA group (group SE). Blood vessels were only exposed, without occlusion in group S. In the other three groups, the cerebral I/R model was established by middle cerebral artery occlusion using suture-occluded method, and the suture was removed after 2-h occlusion to restore the perfusion in anesthetized rats.EA was performed at 5 days before establishing the model in group E and group SE.Baihui acupoints were stimulated with an electric stimulator (2/12 Hz disperse-dense waves, intensity 1 mA) for 30 min once a day for 5 consecutive days, and the model was established at 24 h after the last stimulation.EA was performed at the points 1 cm lateral to the acupoints of Baihui, and the other operating parameters were the same as those previously described in group E. Neurological deficit scores (NDSs) were evaluated at 24 and 48 h of reperfusion.Then the rats were sacrificed, and tissues in the ischemic penumbra of cerebral cortex were obtained for determination of cell apoptosis (by TUNEL) and expression of Ubc9 and conjugated small ubiquitin-like modifier (SUMO) 2/3 (by Western blot). The apoptosis rate was calculated. Results:Compared with group S, NDSs at 24 and 48 h of reperfusion and apoptosis rate were significantly increased, and the expression of Ubc9 and conjugated SUMO2/3 was up-regulated in the other three groups ( P<0.05). Compared with group I/R and group SE, NDSs at 24 and 48 h of reperfusion and apoptosis rate were significantly decreased, and the expression of Ubc9 and conjugated SUMO2/3 was up-regulated in group E( P<0.05). Conclusion:The mechanism by which EA preconditioning reduces cerebral I/R injury is related to up-regulating Ubc9 expression and thus enhancing SUMO2/3ylation in rats.
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Objective:To investigate the rate of compliance with sepsis Bundle and outcomes in patients with septic shock in intensive care units (ICU) in Changshu area.Methods:A multi-center retrospective study was conducted on patients who were diagnosed as septic shock in ICU of three hospitals in Changshu area from January 2014 to October 2017. Patients who were diagnosed as septic shock meeting the 2016 diagnostic criteria were enrolled. The exclusion criteria were paients younger than 18 years, pregnancy, death within 6 h of admission, halfway abandoned treatment, and those who had entered other studies on septic shock intervention. Clinical characteristics, past history, situation on admission, Bundle completion at 1 h, 3 h and 6 h, and prognosis were recorded. Patients were divided into the survival group and death groups according to whether they survived or not. The differences of completion of Bundle indicators between the two groups were compared. The independent risk factors of prognosis were analyzed by Logistic regression analysis, and the survival curve was plotted by Kaplan-Meier method.Results:Totally 207 patients from 3 hospitals were enrolled in this study. The complition rate of 1 h Bundle was 27.1%, of which the completion rate of serum lactate determination was 81.2%, the completion rate of blood culture before antibiotic administration was 72.5%, the completion rate of broad-spectrum antibiotic administration was 48.4%, the completion rate of fluid resuscitation was 74.4%, and the completion rate of vasopressors to maintain MAP≥65 mmHg was 86.0%; the completion rate of 3 h Bundle was 67.6%, of which the completion rate of serum lactate determination was 97.1%, the completion rate of blood culture before antibiotic administration was 84.5%, the completion rate of broad-spectrum antibiotic administration was 97.1%, and the completion rate of fluid resuscitation was 76.8%; the completion rate of 6 h Bundle was 30.4%, of which the completion rate of vasopressors to maintain MAP≥65 mmHg was 98.1%, the completion rate of reassessed volume stasis (at least two indexes) was 48.3%, the completion rate of central venous pressure (CVP) was 42.5%, the completion rate of ScvO 2 was 10.6%, the completion rate of bedside ultrasound was 48.8%, the completion rate of passive leg raising and rehydration therapy was 42.5%, and the completion rate of re-evaluation of lactate was 55.1%. The total Bundle completion rate at 3 h and 6 h was 27.1%. There was no significant difference in the completion of the three hospitals. The mortality of patients with septic shock was 58.9%, length of stay in the ICU was 10 d (5, 23) d, and length of hospital stay was 14 d (6, 26) d. Univariate analysis showed that antibiotic use time, CVP, bedside ultrasound, passive leg raising and rehydration experiments, re-evaluation after elevated lactate, 6 h Bundle completion, total Bundle completion at 3 and 6 h, and APACHE Ⅱ score were associated with the prognosis (all P<0.01). Logistic regression analysis showed that CVP ( OR=23.236, P=0.001), passive leg raising and rehydration experiments ( OR=0.102, P=0.012), re-evaluation after elevated lactate ( OR=0.197, P=0.001) and APACHEⅡ score ( OR=1.103, P<0.01) were risk factors of the prognosis. Kaplan-Meier analysis showed that the 28 d survival rate was significantly higher if 6 h Bundle was completed (Log Rank χ 2=8.944, P=0.003). Conclusions:The compliance with sepsis Bundle is not high in Changshu area, and the compliance is closely related to the prognosis, so it needs to improve compliance with the guidelines.
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Objective To evaluate the effects of selective brain hypothermia on the expression of miR-484 during cerebral ischemia-reperfusion ( I/R) in rats. Methods A total of 120 clean-grade healthy male Sprague-Dawley rats, aged 8-10 weeks, weighing 250-300 g, were divided into 4 groups ( n=30 each) using a random number table method: sham operation group ( group S) , cerebral I/R group ( group I/R) , hypothermia group ( group H) and normothermia group ( group N) . Blood vessels were only separa-ted and ligated without blockade in group S. In the other three groups, cerebral I/R was induced by inser-ting a nylon thread with rounded tip into the right internal carotid artery, and the middle cerebral artery was occluded for 2 h followed by reperfusion. In group H, 4℃ normal saline was infused for 15 min at a rate of 80 ml·kg-1 ·h-1 through the internal carotid artery immediately after removing the nylon thread. In group N, 37 ℃ normal saline was infused in the same way. Neurological deficit scores were evaluated at 6, 24 and 48 h after reperfusion. Animals were then sacrificed, the cerebral cortex of the ischemic penumbra was removed for determination of nerve cell apoptosis ( by TUNEL method) , expression of mitochondrial fission protein 1 ( Fis1) ( by Western blot) and expression of miR-484 ( quantitative real-time polymerase chain re-action) . The apoptotic rate was calculated. Results Compared with group S, the neurological deficit score and apoptotic rate of nerve cells were significantly increased, and the expression of Fis1 was up-regulated at each time point in the other three groups, the expression of miR-484 was significantly down-regulated in I/R and N groups, and the expression of miR-484 was significantly up-regulated in group H ( P＜0. 05 or 0. 01) . Compared with group I/R and group N, the neurological deficit score and apoptotic rate of nerve cells were significantly decreased, and the expression of Fis1 was down-regulated, and the expression of miR-484 was up-regulated at each time point in group H ( P＜0. 05 or 0. 01) . Conclusion The mechanism by which se-lective cerebral hypothermia attenuates cerebral I/R injury is associated with up-regulating miR-484 expres-sion and thus down-regulating Fis1 expression in rats.
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Objective To study the effect of laparoscopic hepatectomy resection on immune function,inflammatory factors and prognosis of patients??Methods One hundred and four patients with hepatocellular carcinoma admitted Zigong Fourth People′s Hospital from July 2015 to December 2016 were selected as the study subjects??According to the different surgical methods,they were randomly divided into observation group and control group with 52 cases in each group??The control group received open hepatectomy, while the observation group received laparoscopic hepatectomy??The immune function, inflammatory factors,and surgery related indicators were compared between the two groups??Results The observation time of the observation group was significantly longer than that in the control group??The blood loss was significantly less than the control group??Gastrointestinal function recovery time and postoperative hospital stay were significantly shorter than control group (t＝7??553,13??855,15??100,16??873,all P＜0??01), three days after surgery,the serum IgA,IgG,and IgM levels were significantly higher than control group ( t＝16??009,10??076,14??138,all P＜0??01)??The levels of IL?6,IL?10 and TNF?α were significantly lower than the control group (t＝8??538,6??851,11??103,P＜0??05,all P＜0??01)??Conclusion Laparoscopic hepatectomy can promote the recovery of patients after surgery,which may be related to the reduction of immune function inhibition,inflammatory response and other factors??
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Objective To evaluate the effect of electro-acupuncture preconditioning on the expression of protein kinase R-like endoplasmic reticulum kinase (PERK) in the hippocampus of mice following cerebral ischemia-reperfusion (I/R).Methods A total of 120 healthy male C56BL/6 mice,aged 7 weeks,weighing 20-30 g,were divided into 3 groups (n=40 each) using a random number table method:sham operation group (group S),cerebral I/R group (group I/R) and electro-acupuncture preconditioning group (group EA).Cerebral I/R was induced by clamping bilateral common carotid arteries for 15min followed by restoring cerebral blood flow in anesthetized mice.Dazhui and Baihui acupoints were stimulated with disperse-dense waves of 1 mA for 30 min once a day,lasting for 5 consecutive days,and cerebral I/R model was established at 24 h after the last stimulation in group EA.Animals were sacrificed at 24and 48 h of reperfusion,brains were removed,and hippocampi were isolated for determination of apoptotic neurons in hippocampal CAl region (by TUNEL) and expression of PERK (by Western blot).Results Compared with group S,the neurological deficit score and the number of apoptotic neurons in hippocampal CAl region were significantly increased at each time point of reperfusion in I/R and EA groups (P＜0.05).Compared with group I/R,the neurological deficit score and the number of apoptotic neurons in hippocampal CAl region were significantly decreased at each time point of reperfusion in group EA (P＜0.05).Conclusion The mechanism by which electro-acupuncture preconditioning mitigates endoplasmic reticulum stress during cerebral I/R may be related to inhibiting PERK activity in mice.