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Objective:To evaluate the role of miR-124-3p in reduction of oxygen-glucose deprivation and restoration (OGD/R) injury by electrostimulation preconditioning in microglia and its relationship with microglial polarization.Methods:The well-growing BV2 cells were divided into 4 groups ( n=30 each) by the random number table method: control group (group C), OGD/R group, electrostimulation preconditioning group (group E) and miR-124-3p inhibitor group (group I). Group C was cultured under normal conditions, and group OGD/R was deprived of oxygen and glucose for 2 h followed by restoration of oxygen and glucose supply for 24 h to develop the OGD/R injury model. In group E, cells were stimulated with 100 mV/mm direct current for 4 h before oxygen-glucose deprivation, and the other treatments were similar to those previously described in group OGD/R. Group I was transfected with micrOFF? mmu-miR-124-3p inhibitor at 48 h before oxygen-glucose deprivation, and the other treatments were similar to those previously described in group E. The cell survival rate was determined by CCK-8 assay, the concentrations of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and IL-10 in the cell supernatant were measured by enzyme-linked immunosorbent assay. The expression of a surface marker of M1 microglia inducible nitric oxide synthase (iNOS) and a surface marker of M2 microglia arginase 1 (Arg-1) was detected by immunofluorescence and Western blot, respectively. The expression of iNOS and Arg-1 mRNA and miR-124-3p was detected by quantitative polymerase chain reaction. Results:Compared with group C, the cell survival rate was significantly decreased, the concentrations of TNF-α, IL-1β and IL-10 in the supernatant were increased, and the expression of iNOS and Arg-1 protein and mRNA and miR-124-3p was up-regulated in the remaining three groups ( P<0.05). Compared with group OGD/R, the cell survival rate was significantly increased, the concentrations of TNF-α and IL-1β in the supernatant were decreased, the IL-10 concentration was increased, the expression of iNOS protein and mRNA was down-regulated, and the expression of Arg-1 protein and mRNA and miR-124-3p was up-regulated in E and I groups ( P<0.05). Compared with group E, the cell survival rate was significantly decreased, the concentrations of TNF-α and IL-1β in the supernatant were increased, the IL-10 concentration was decreased, the expression of iNOS protein and mRNA was up-regulated, and the expression of Arg-1 protein and mRNA and miR-124-3p was down-regulated in group I ( P<0.05). Conclusions:The mechanism by which electrostimulation preconditioning reduces OGD/R injury in microglia is related to up-regulation of the expression of miR-124-3p, promotion of M2 microglia polarization, inhibition of M1 microglia polarization, and thus inhibiting the inflammatory responses.
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Objective:To evaluate the effect of sodium bicarbonate Ringer′s solution on acute kidney injury(AKI) following laparoscopic hepatectomy in elderly patients.Methods:A total of 362 American Society of Anesthesiologists Physical Status classification Ⅱ or Ⅲ elderly patients, aged 65-79 yr, with body mass index of 18-28 kg/m 2, scheduled for elective laparoscopic hepatectomy, were divided into 2 groups( n=181 each) using a random number table method: bicarbonate Ringer′s solution group(BR group) and lactated Ringer′s solution group(LR group). Bicarbonate Ringer′s solution and lactated Ringer′s solution were intravenously infused in BR group and LR group, respectively. All operations were performed under general anesthesia combined with abdominal fascia block, and the methods of controlled low central venous pressure and intermittent hepatic inflow occlusion were applied to reduce intraoperative bleeding. Radial artery blood samples were collected for blood gas analysis at 5 min before anesthesia induction(T 0), 20 min after occluding liver hilus(T 1), 10 min after hepatectomy and hemostasis(T 2), at the end of surgery(T 3) and at postanesthesia care unit discharge(T 4), and lactate value(Lac) was recorded. Blood samples from cubital vein were collected on admission to hospital(T A) and at 24(T 24) and 48 h after operation(T 48) for determination of serum creatinine(Cr) concentrations. Doppler-based renal resistive index(RRI) was measured at T A, T 4, T 24 and T 48. The incidence of AKI was calculated within 48 h after operation according to the Kidney Disease: Improving Global Outcomes criteria in 2012 for Cr concentration. Adverse reactions(such as nausea and vomiting) and complications(such as incision infection) within 48 h after operation were recorded. Results:Compared with the baseline at T 0, Lac concentrations were significantly increased at T 1-4 in both groups( P<0.01). Cr concentrations were significantly increased at T 24 and T 48, and RRI was increased at T 4, T 24 and T 48 than at T A in both groups( P<0.01). Compared with group LR, the incidence of AKI within 48 h after operation, Lac concentrations at T 3, 4, Cr concentrations at T 24 and T 48, and RRI at T 4, T 24 and T 48 were significantly decreased in group BR( P<0.05 or 0.01). There was no significant difference in the incidence of nausea, vomiting, incision infection, delirium, bile leakage and pulmonary infection within 48 h after operation among the two groups( P>0.05). Conclusions:Sodium bicarbonate Ringer′s solution can decrease the development of AKI following laparoscopic hepatectomy in elderly patients.
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Objective:To evaluate the effect of selective cerebral mild hypothermia on small ubiquitin-like modifier 2/3 (SUMO2/3) modification of dynamin-related protein 1 (Drp1) in a rat model of cerebral ischemia-reperfusion (I/R).Methods:Sixty clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 240-260 g, were divided into 4 groups ( n=15 each) using a random number table method: sham operation group (S group), cerebral I/R group (I/R group), selective cerebral mild hypothermia group (HT group) and normal temperature group (NT group). The operation was performed under the monitoring of cerebral temperature and rectal temperature.Only the cervical blood vessels were exposed in S group, while focal cerebral I/R was induced by 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion in anesthetized animals in the other three groups.In HT group and NT group, 4 and 37 ℃ normal saline was perfused through the left internal carotid artery at a rate of 80 ml·kg -1·h -1 for 15 min, respectively. Modified neurological severity score (mNSS) was assessed at 24 h of reperfusion. Then the rats were sacrificed under deep anesthesia, brains were removed, brain tissues were obtained for determination of the percentage of cerebral infarct size (by TTC staining), and the ischemic penumbra tissues in the cerebral cortex were removed for examination of the ultra-structural changes of mitochondria (with a transmission electron microscope) and for determination of the SUMO2/3 modification of Drp1 (by CO-IP), expression of total Drp1 (T-Drp1) and total cytochrome c (T-Cytc) (by Western blot), and expression of mitochondrial outer membrane Drp1 (M-Drp1) and cytoplasmic Cytc (C-Cytc) (by Western blot) after isolation of mitochondria and cytoplasm. Results:Compared with S group, the mNSS and percentage of cerebral infarct size were significantly increased, the expression of M-Drp1, T-Drp1, C-Cytc and T-Cytc was up-regulated, and SUMO2/3 modification of Drp1 in ischemic penumbra area was increased ( P<0.05), the fragmentation of mitochondria was aggravated, and cristae rupture and vacuolation were obvious in the other three groups. Compared with I/R group, the mNSS and percentage of cerebral infarct size were significantly decreased, the expression of M-Drp1, T-Drp1, C-Cytc and T-Cytc was down-regulated, SUMO2/3 modification of Drp1 was increased ( P<0.05), the fragmentation of mitochondria was significantly attenuated, and cristae rupture and vacuolation were weakened in HT group. There were no significant differences in these detection parameters between NT group and I/R group ( P>0.05). Conclusions:The mechanism by which selective cerebral mild hypothermia alleviates the cerebral I/R injury is related to increased SUMO2/3 modification of Drp1, decreased binding of Drp1 to mitochondrial outer membrane, and reduced mitochondrial excessive fission in rats.
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Objective:To investigate the effects of mild hypothermia on microglia polarization and janus kinase 2/signal transduction and transcriptional activation factor 3 (JAK2/STAT3) signaling pathway during cerebral ischemia-reperfusion (I/R) in rats.Methods:Forty-five clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 260-280 g, were divided into 3 groups ( n=15 each) by the random number table method: sham operation group (S group), cerebral I/R group (I/R) and mild hypothermia group (H group). In I/R group and H group, cerebral I/R was induced by middle cerebral artery occlusion using a nylon thread in anesthetized animals, the nylon thread was removed to restore the perfusion after 2 h of occlusion, and the rectal temperature was maintained at 36-37 ℃ during the period. Group H was wiped with 75% alcohol for 3 h starting from the time point immediately after reperfusion, and the rectal temperature was maintained at 32-33℃. Modified neurological severity score (mNSS) was evaluated at 24 h of reperfusion. Animals were then sacrificed for determination of the cerebral infarct size (using TTC staining), expression of M1 marker inducible nitric oxide synthase (iNOS), M2 marker arginase 1(Arg-1), phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)(by Western blot), expression of iNOS mRNA and Arg-1 mRNA (by quantitative polymerase chain reaction), and contents of interleukin-6 (IL-6) and IL-10 (by enzyme-linked immunosorbent assay). Results:Compared with group S, mNSS and cerebral infarct size were significantly increased, the expression of iNOS, Arg-1 protein and mRNA in cerebral ischemic penumbral zone was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio, and contents of IL-6 and IL-10 were increased in the other two groups ( P<0.05). Compared with I/R group, mNSS and cerebral infarct size were significantly decreased, the expression of iNOS protein and mRNA in cerebral ischemic penumbral zone was down-regulated, the expression of Arg-1 and mRNA was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio and IL-6 content were decreased, and the IL-10 content was increased in group H ( P<0.05). Conclusions:Mild hypothermia can promote the polarization shift of microglia from M1 to M2 phenotype during cerebral I/R and inhibit the central inflammatory responses, and the mechanism may be related to inhibition of JAK2/STAT3 signaling pathway in rats.
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Objective:To evaluate the relationship between hippocampal miR-3065-5p and insulin-like growth factor-1/phosphatidylinositol 3-kinase/protein kinase B(IGF-1/PI3K/Akt)signaling pathway in a mouse model of perioperative neurocognitive disorder (PND).Methods:Eighty clean-grade healthy male C75BL/6 mice, aged 12-14 weeks, weighing 20-30 g, were divided them into 4 groups ( n=20 each) using the random number table method: control group (C group), PND group, miR-3065-5p agonist group (Ag group) and miR-3065-5p agonist negative control group (Ag-NC group). PND model was prepared by internal fixation of tibial fracture under anesthesia with 1.5% isoflurane. Two days before developing the model, miR-3065-5p agomir 2 μl was injected into the lateral ventricle in Ag group, miR-3065-5p agomir negative control 2 μl was injected into the lateral ventricle in Ag-NC group. Morris water maze test and open field test were performed at 7 days after surgery. The mice were sacrificed after the end of test, and hippocampal tissues were obtained for determination of the expression of miR-3065-5p, IGF-1 mRNA and Bcl-2 mRNA (by quantitative real-time polymerase chain reaction) and expression of IGF-1, phosphorylated Akt (p-Akt), phosphorylated glycogen synthase kinase-3β (p-GSK3β) and Bcl-2 (by Western blot). Results:There was no significant difference in each parameter in the open field test among the four groups ( P>0.05). Compared with group C, the postoperative escape latency was significantly prolonged, the percentage of time of stay at the target quadrant was decreased, the number of crossing the original platform was reduced, the expression of miR-3065-5p was up-regulated, and the expression of IGF-1 mRNA, Bcl-2 mRNA, IGF-1, p-Akt, p-GSK3β and Bcl-2 was down-regulated in the other three groups ( P<0.05). Compared with PND group and Ag-NC group, the postoperative escape latency was significantly prolonged, the percentage of time of stay at the target quadrant was decreased, the number of crossing the original platform was reduced, the expression of miR-3065-5p was up-regulated, and the expression of IGF-1 mRNA, Bcl-2 mRNA, IGF-1, p-Akt, p-GSK3β and Bcl-2 was down-regulated in Ag group ( P<0.05). Conclusions:Up-regulation of miR-3065-5p can inhibit the activation of IGF-1/PI3K/Akt signaling pathway, which might be one of the mechanisms of PND developed in mice.
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Objective:To evaluate the role of Sirtuin 1/nuclear factor κB (SIRT1/NF-κB) signaling pathway in mild hypothermia-induced promotion of microglial polarization during oxygen-glucose deprivation and restoration (OGD/R).Methods:The well-grown BV2 microglia were divided into 4 groups ( n=36 each) using the random number table method: control group (group C), OGD/R group (group O), mild hypothermia group (group M), and mild hypothermia+ SIRT1 specific inhibitor EX527 group (group ME). Cells in group C were commonly cultured without any treatment. Cells in group O were subjected to 3 h of OGD followed by 21 h of restoration of O 2-glucose supply at 37 ℃. Cells in group M were subjected to 3 h of OGD followed by 21 h of restoration of O 2-glucose supply at 33 ℃. Cells in group ME were co-cultured with inhibitor EX527 (final concentration 5 nmol/L) for 12 h in the medium before OGD/R, and the other procedures were conducted as previously described in group M. The cell survival rate was detected by CCK-8 assay. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and interleukin-10 (IL-10) in supernatant were detected by enzyme-linked immunosorbent assay. The expression of CD206, CD32, inducible nitric oxide synthase (iNOS) and arginine synthase 1 (Arg-1) mRNA was detected by quantitative real-time polymerase chain reaction. The expression of CD206 and CD32 was detected by immunofluorescent staining. The expression of iNOS, Arg-1, SIRT1, NF-κB p65 (p65) and acetylated NF-κB (Ac-p65) was detected by Western blot. Results:Compared with group C, the cell survival rate was significantly decreased, the concentrations of TNF-α, IL-6 and IL-10 in the supernatant were increased, the expression of CD206, Arg-1, CD32 and iNOS was up-regulated, the expression of SIRT1 was down-regulated, and the Ac-p65/p65 ratio was increased in group O ( P<0.05). Compared with group O, the cell survival rate was significantly increased, the concentrations of TNF-α and IL-6 in the supernatant were decreased, the concentration of IL-10 was increased, the expression of CD206, Arg-1 and SIRT1 was up-regulated, the expression of CD32 and iNOS was down-regulated, and the Ac-p65/p65 ratio was decreased in group M ( P<0.05). Compared with group M, the cell survival rate was significantly decreased, the concentrations of TNF-α and IL-6 in the supernatant were increased, the concentration of IL-10 was decreased, the expression of CD206, Arg-1 and SIRT1 was down-regulated, the expression of CD32 and iNOS was up-regulated, and the Ac-p65/p65 ratio was increased in group ME ( P<0.05). Conclusions:SIRT1/NF-κB signaling pathway is involved in mild hypothermia-induced promotion of microglial polarization during OGD/R.
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Objective:To evaluate the effects of remimazolam on gastrointestinal motor function in the patients undergoing gastrointestinal endoscopy.Methods:A total of 262 American Society of Anesthesiologists Physical Status classification Ⅰ or Ⅱ patients, aged 18-64 yr, with body mass index of 18-28 kg/m 2, scheduled for elective gastrointestinal endoscopy from May 2022 to August 2022, were divided into 2 groups ( n=131 each) using a random number table method: remimazolam group (group R) and propofol group (group P). The patients in group R received intravenous remimazolam 0.20-0.25 mg/kg, and patients in group P received intravenous propofol 1.5-2.0 mg/kg. The gastrointestinal endoscopy was performed when the patients′ Modified Observer′s Assessment of Alertness/Sedation scores ≤3. During fasting before gastrointestinal preparation, before gastrointestinal endoscopy and while leaving the post-anesthesia care unit (PACU), the concentrations of serum motilin and gastrin were measured by enzyme-linked immunosorbent assay, the intestinal peristalsis rating assessed by the endoscopist during the examination was recorded, the occurrence of hypotension and hypoxemia during the examination and occurrence of abdominal distension, abdominal pain, and nausea and vomiting during stay in PACU were recorded. Results:Compared with group P, the intestinal peristalsis rating was significantly increased, the serum motilin and gastrin concentrations were increased while leaving PACU, the incidence of hypotension and hypoxemia was decreased during the examination, and the incidence of abdominal distention was decreased during stay in PACU in group R ( P<0.05). Conclusions:Remimazolam has a milder inhibitory effect on secretion of gastrointestinal hormones than propofol in the patients undergoing gastrointestinal endoscopy and is helpful for the recovery of gastrointestinal motility.
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Microglia are widely present in the central nervous system and participate in various pathophysiological processes.They play an important role in degenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.In recent years, the study of exosomes produced by microglia activation involved in the pathophysiological processes of various diseases has attracted extensive attention, but the role of exosomes has not been fully clarified.This article reviewed the characteristics and functions of microglia, the characteristics and functions of microglia-derived exosomes and their roles in central neurodegenerative diseases.
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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 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 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 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 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.