Objective:To evaluate the role of cancerous inhibitor of protein phosphatase 2A (CIP2A) in preoperative sleep deprivation (PSD)-induced aggravation of postoperative cognitive dysfunction (POCD) in aged mice.Methods:One hundred and ten healthy C57BL/6J mice of either sex, aged 18-20 months, weighing 29-35 g, were divided into 5 groups ( n=22 each) using a random number table method: sham operation group (S group), abdominal surgery group (O group), PSD + abdominal surgery group (D+ O group), CIP2A shRNA + abdominal surgery group (CS+ O group), and CIP2A shRNA+ PSD+ abdominal surgery group (CS+ D+ O group). At 14 days before surgery, control shRNA lentivirus was injected into the hippocampus in S, O and CS+ O groups, and CIP2A shRNA was injected into the hippocampus in D+ O and CS+ D+ O groups. PSD was carried out for 3 consecutive days prior to surgery. Cognitive function was assessed using the Morris water maze test at days 7-11 after surgery. The mice were sacrificed under deep anesthesia at day 3 after surgery, and hippocampal tissues were obtained to determine the expression of CIP2A, high mobility group box 1 (HMGB1), ionized calcium-binding adapter molecule 1 (Iba-1), alpha subunit of protein phosphatase 2A (PP2Aa), catalytic subunit of protein phosphatase 2A (PP2Ac), phosphorylated tau protein (p-tau) (S396), and p-tau (S404) (by Western blot), levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD), and count of Iba-1 positive cells in the hippocampal CA1 region (using immunofluorescence staining). Results:Compared with S group, the escape latency was significantly prolonged, the frequency of crossing the platform was reduced, duration of stay in the target quadrant was shortened, the expression of CIP2A, Iba-1 and HMGB1 was up-regulated, PP2Ac expression was down-regulated, levels of ROS and MDA and count of Iba-1 positive cells were increased, and the activity of SOD was decreased in O group ( P<0.05). Compared with O group, the escape latency was significantly prolonged, the frequency of crossing the platform was reduced, duration of stay in the target quadrant was shortened, the expression of CIP2A, Iba-1 and HMGB1 was up-regulated, PP2Ac expression was down-regulated, levels of ROS and MDA and count of Iba-1 positive cells were increased, and the activity of SOD was decreased in D+ O group, and the escape latency was significantly shortened, the frequency of crossing the platform was increased, duration of stay in the target quadrant was prolonged, the expression of CIP2A, Iba-1 and HMGB1 was down-regulated, PP2Ac expression was up-regulated, levels of ROS and MDA and count of Iba-1 positive cells were decreased, and the activity of SOD was increased in CS+ O group ( P<0.05). Compared with D+ O group, the escape latency was significantly shortened, the frequency of crossing the platform was increased, duration of stay in the target quadrant was prolonged, the expression of CIP2A, Iba-1 and HMGB1 was down-regulated, PP2Ac expression was up-regulated, levels of ROS and MDA and count of Iba-1 positive cells were decreased, and the activity of SOD was increased in CS+ D+ O group ( P<0.05). There was no significant difference in PP2Aa expression among the five groups ( P>0.05). Conclusions:The mechanism by which PSD aggravates POCD is related to up-regulating the expression of CIP2A and promoting oxidative stress responses, neuroinflammatory responses and phosphorylation of tau protein in aged mice.

Objective To explore the optimal phases of reconstructed CT images under different heart rates based on dynamic phantom system simulating the motion of left ventricle and coronary arteries.Methods A dynamic phantom system which could simulate the periodic movements of the heart at 50-120 beats per minute(bpm)and the output of electrocardiogram signals was constructed.CT scanning were performed in the simulated R-R interval,and images were reconstructed with every 10％interval between 0 to 90％phases.Then subjective image quality scoring was performed,and inter-observer consistency of image quality scores was assessed.Finally the qualities of reconstructed images were compared among different phases under different heart rates.Results The inter-observer consistency of subjective imaging quality scores was high(Kendall W=0.83,P＜0.05).Under 50-60 bpm simulated heart rates,good reconstructed image qualities were obtained at most phases,especially at 30％,70％and 80％(all P＜0.05).When simulated heart rates were set as 65-75 bpm,the best reconstructing phases included 40％,70％and 80％(all P＜0.05),and images obtained in diastolic phase were better.Under 80-95 bpm,the best reconstructing phase was 30％(all P＜0.05).When the simulated heart rate reached 100 bpm and above,the reconstructed image qualities were poor at all phases.Conclusion The optimal reconstructed phases were different under different heart rates based on dynamic phantom system simulating the motion of left ventricle and coronary arteries.When the simulated heart rate reached 100 bpm and above,the qualities of reconstructed images were poor under all phases.

Objective:To evaluate the effect of selective cerebral mild hypothermia on the expression of histone deacetylase 1-3 (HDAC1-3) during focal cerebral ischemia-reperfusion (I/R) in rats.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), focal cerebral I/R group (I/R group), selective cerebral mild hypothermia group (SCH group), and normothermia group (N group). Only the cervical vessels were isolated in S group. In the other three groups, sutures were inserted into the internal carotid artery to block the middle cerebral artery for 2 h, and then the sutures were pulled out to restore perfusion for 24 h. A focal cerebral I/R model was prepared. Normal saline at 20 ℃ and 37 ℃ was infused into the internal carotid artery at a rate of 0.6 ml/min for 10 min starting from the time point immediately after removal of the sutures in SCH group and N group respectively. Cerebral temperature and rectal temperature were continuously monitored during the operation. The modified neurological severity score (mNSS) was assessed at 24 h of reperfusion. The rats were then sacrificed under deep anesthesia and brains were obtained for determination of cerebral infarct size (by TTC staining). The tissues of the cerebral ischemic penumbra were taken for determination of the apoptosis rate of neurons (by TUNEL method) and lactylation modification and expression of HDAC1-3 (by Western blot) and for observation of the morphology of neurons (by HE staining). Results:Compared with S group, the mNSS, cerebral infarct size and apoptosis rate of neurons were significantly increased, HDAC1-3 expression was down-regulated, and the lactylation modification was increased in the other three groups ( P<0.05). Compared with I/R and N groups, the mNSS, cerebral infarct size and apoptosis rate of neurons were significantly decreased, HDAC1-3 expression was up-regulated, and the lactylation modification was decreased in SCH group ( P<0.05). There was no statistically significant difference in the aforementioned parameters between I/R group and N group ( P>0.05). HE staining showed that the morphology of neurons was intact and well-defined in S group, a large number of cells with edema and irregularly solidified nuclei were found in I/R group and N group, and the nuclear shrinkage and morphological changes of neurons were alleviated in SCH group. Conclusions:The mechanism by which selective cerebral mild hypothermia alleviates cerebral I/R injury may be related to up-regulation of HDAC1-3 expression in rats.

Objective:To evaluate the role of glutathione S-transferase μ1 (GSTM1) expression in mild hypothermia-induced mitigation of cerebral ischemia-reperfusion (I/R) injury and the relationship with microglial polarization.Methods:Eighty clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 260-280 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (S group), cerebral I/R group (I/R group), mild hypothermia group (H group), and GSTM1 inhibitor + mild hypothermia group (IH group). The rat model of cerebral I/R injury was prepared using the filament occlusion method. The filament was removed to restore blood flow after the left middle cerebral artery was blocked for 2 h, and the rats′ brain and rectal temperature were maintained at 36-37 ℃ during the period. The vessels were only isolated and ligated without occlusion in S group. In H group, the entire body was wiped with 75% ethanol immediately after removing the filament, and the brain and rectal temperatures were maintained at 32-33 ℃ for 3 h, and the other procedures were the same as those previously described in I/R group. In IH group, GSTM1 inhibitor itaconic acid 8.6 mg/kg was intraperitoneally injected at 24 and 1 h before developing the model, and the other procedures were the same as those previously described in H group. Neurological deficits were evaluated using a modified neurological severity score (mNSS) at 24 h of reperfusion, and then the animals were sacrificed and the brains were removed for observation of cerebral infarction (by TTC staining) and for determination of the expression of GSTM1, M1-type microglial marker inducible nitric oxide synthase (iNOS), and M2-type microglial marker arginase-1 (Arg-1) (by Western blot), expression of GSTM1, iNOS and Arg-1 mRNA (quantitative real-time polymerase chain reaction) and contents of interleukin-6 (IL-6), IL-10, tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) (by enzyme-linked immunosorbent assay). Results:Compared with S group, the mNSS and percentage of cerebral infarct size were significantly increased, and the expression of iNOS and Arg-1 protein and mRNA was up-regulated, the expression of GSTM1 and mRNA was down-regulated, and the contents of IL-6, TNF-α, IL-10 and TGF-β were increased in the other three groups ( P<0.05). Compared with I/R group and IH group, the mNSS and percentage of cerebral infarct size were significantly decreased, and the expression of iNOS protein and mRNA was down-regulated, the expression of Arg-1 protein and mRNA and GSTM1 was up-regulated, the contents of TNF-α and IL-6 were decreased, and the contents of TGF-β and IL-10 were increased in H group ( P<0.05). Conclusions:Up-regulated expression of GSTM1 is involved in mild hypothermia-induced mitigation of cerebral I/R injury, which is associated with inhibition of microglial polarization toward the M1 phenotype and promotion of polarization toward the M2 phenotype.

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.

Objective:To evaluate the effect of irisin on the alveolar macrophage polarization in a rat model of ventilator-induced lung injury (VILI).Methods:Thirty SPF healthy adult male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-250 g, were divided into 3 groups ( n=10 each) using a random number table method: control group (group C), VILI group (group V) and irisin group (group I). The rats were mechanically ventilation (tidal volume 20 ml/kg, respiratory rate 80 times/min, inhaled oxygen concentration 21%, inspiratory/expiratory ratio 1∶2, positive end-expiratory pressure 0) for 4 h to develop VILI model.Group C kept spontaneous breathing for 4 h. Irisin 1 μg/kg was injected via the tail vein at 30 min before tracheal intubation in group I, while the equal volume of normal saline was given instead in the other groups.The rats were sacrificed at 4 h of mechanical ventilation, the lung tissues were removed for examination of pathological changes which were scored and for determination of wet to dry weight ratio (W/D ratio), and bronchoalveolar lavage fluid (BALF) was collected for determination of concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and IL-10 (by enzyme-linked immunosorbent assay), expression of inducible nitric oxide synthase (iNOS), argininase 1 (Arg-1), and phosphorylated nuclear factor kappa B (p-NF-κB) p65 and p-NF-κB p50 in alveolar macrophages (by Western blot), and percentage of M1 and M2 alveolar macrophages and M1/M2 ratio (by flow cytometry). Results:Compared with group C, the W/D ratio, lung injury score, and concentrations of IL-6, TNF-α and IL-10 in BALF were significantly increased, the expression of iNOS, Arg-1, p-NF-κB p65 and p-NF-κB p50 was up-regulated, and the percentage of M1 and M2 alveolar macrophages and M1/M2 ratio were increased in group V and group I ( P<0.05). Compared with group V, the W/D ratio, lung injury score, and concentrations of IL-6 and TNF-α in BALF were significantly decreased, the expression of iNOS and p-NF-κB p65 was down-regulated, the percentage of M1 alveolar macrophages and M1/M2 ratio were decreased ( P<0.05), and no significant change was found in levels of IL-10 and Arg-1 in BALF, percentage of M2 alveolar macrophages and expression of p-NF-κB p50 in group I ( P>0.05). Conclusions:The mechanism by which irisin reduces VILI may be related to inhibition of NF-κB signaling pathway activation and reduction of alveolar macrophage polarization to M1 phenotype in rats.

Objective:To analyze the phenotype and genotype of two propositi with inherited hypodysfibrinogenaemia caused by compound heterozygous mutations, and investigate the molecular mechanism.Metheds:Two propositi and their family members(7 person in 3 generations and 10 person in 3 generations,respectively) were investigated. The activity of plasma fibrinogen (Fg:C) and thrombin time (TT) were analyzed by coagulation method, the antigen of plasma fibrinogen (Fg:Ag) was detected by immunoturbidimetry. All of the exons and flanking sequences of FGA,FGB,FGG of two propositi were amplified by PCR, followed by direct sequencing. The ClustalX-2, 1-win software was used to analyze the conservatism of mutated gene locus. PROVEAN and Mutation Taster were applied to analyze the pathogenicity of mutated amino acid. The changes of the protein spatial structure and intermolecular interaction were analyzed by Pymol.Results:Fg:C and Fg:Ag of proposita A and B were both significantly decreased (0.74 and 0.78 g/L, 0.96 and 0.94 g/L, respectively). Gene analysis revealed that proposita A and B both carried a heterozygous mutation c.2185G>A(p.AαGlu710Lys) in exon 6 of FGA. Furthermore, proposita A also carried a heterozygous mutation c.701G>T(p.γTrp208Leu) in exon 7 of FGG, and proposita B carried a heterozygous mutation c.1015A>C(p.γSer313Arg) in exon 8 of FGG. Phylogenetic analysis suggested that p.AαGlu710,p.γTrp208 and p.γSer313 were highly conserved among homologous species. All variants were predicted to be deleterious by two online bioinformatic softwares. The protein model analysis indicated that protein spatial structure and intermolecular hydrogen bonds were changed by these variants, which destroyed the stability of Fg.Conclusion:The compound heterozygous mutations of p.AαGlu710Lys and p.γTrp208Leu,p.AαGlu710Lys and p.γSer313Arg might account for the hypodysfibrinogenemia in two propositi.

Objective:To explore the genetic basis for a patient with factor XIII (FXIII) deficiency.Methods:All exons of the F13A1 and F13B genes were amplified by PCR and sequenced directly. The sequencing was performed with a reverse primer if a variant was found. Conservation of variant site was analyzed by the ClustalX software. Four online bioinformatic software including MutationTaster, PolyPhen-2, PROVEAN and SIFT were used to predict the function of the mutation site. The Swiss-PdbViewer software was applied to analyze the changes in the protein model and intermolecular force. Results:The proband was found to harbor a novel c. 515G>C (p.Arg171Pro) variant of the F13A1 gene. The corresponding amino acid Arg171 is conserved among homologous species. Bioinformatic analysis indicated that Arg171Pro variant may affect the protein function. Protein model analysis showed that in the wild-type, there is one hydrogen bond between Arg171 and Pro27; one hydrogen bond between Arg171 and Thr28; two hydrogen bonds between Arg171 and Glu102. When Arg171 was mutated to Pro171, the three hydrogen bonds between Arg171 and Pro27, Glu102 are all disappeared and formed a new benzene ring which might affect the stability of the protein structure. No variant was found in the F13B gene. Conclusion:The Arg171Pro variant may account for the decreased FXIII level. Above finding has enriched the spectrum of F13A1 gene variants.

Objective:To evaluate the effect of irisin on ventilator-induced lung injury (VILI) in rats and the relationship with expression of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasomes.Methods:Thirty-six SPF-grade healthy adult male Sprague-Dawley rats, aged 6-8 weeks, weighing 220-300 g, were divided into 3 groups ( n=12 each) using a random number table method: control group (group C), group VILI and irisin group (group I). All the groups underwent tracheotomy and intubation, group C kept spontaneous breathing for 4 h, and the animals were mechanically ventilated for 4 h in VILI and I groups.Irisin 1 μg/kg was injected via the tail vein at 30 min before tracheal intubation in group I, and the equal volume of normal saline mixture (normal saline∶phosphate buffer solution containing 5% trehalose=1∶9) were given in the other 2 groups via the tail vein.The rats were mechanically ventilated with the tidal volume of 20 ml/kg, respiratory rate 80 breaths/min, inspiratory/expiratory ratio 1∶1, inspired oxygen fraction ratio 21% and positive end-expiratory pressure 0.Blood samples from left femoral artery were collected before tracheal intubation and at the end of mechanical ventilation for detection of PaO 2.The animals were sacrificed and the lung tissue samples and bronchoalveolar lavage fluid (BALF) were then collected for examination of the pathological changes (under the light microscope), and for determination of wet to dry weight (W/D) ratio and the concentrations of total protein in BALF and interleukin-1β (IL-1β) and IL-18 in BALF and serum (by enzyme-linked immunosorbent assay), level of reactive oxygen species (ROS) in alveolar macrophages in BALF (by DCFH-DA) and the expression of NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1 protein and mRNA in lung tissues (by Western blot and by quantitative reverse transcription polymerase chain reaction). The pathological changes of the lung were scored. Results:Compared with group C, PaO 2 was significantly decreased at the end of mechanical ventilation, lung injury score and W/D ratio were increased, concentration of total protein and ROS level in alveolar macrophages in BALF and concentrations of BALF, IL-1β and IL-18 in serum were increased, and the expression of NLRP3, ASC and caspase-1 protein and mRNA in lung tissues was up-regulated in group VILI and group I ( P<0.01). Compared with group VILI, PaO 2 was significantly increased at the end of mechanical ventilation, lung injury score and W/D ratio were decreased, concentration of total protein and ROS level in alveolar macrophages in BALF and concentrations of BALF, IL-1β and IL-18 in serum were decreased, and the expression of NLRP3, ASC and caspase-1 protein and mRNA in lung tissues was down-regulated in group I ( P<0.05). Conclusion:Irisin can reduce VILI, and the mechanism is related to inhibiting activation of NLRP3 inflammasome and reducing inflammatory response in rats.

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.