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.

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.

OBJECTIVE: To explore the molecular pathogenesis of a Chinese pedigree affected with Hereditary coagulation factor Ⅺ (FⅪ) deficiency due to variants of the F11 gene. METHODS: A male proband with Hereditary coagulation factor Ⅺ deficiency who was admitted to the First Affiliated Hospital of Wenzhou Medical University due to urinary calculi on November 30, 2020 and his family members (7 individuals from 3 generations in total) were selected as the study subjects. Clinical data of the proband were collected, and relevant coagulation indices of the proband and his family members were determined. Genomic DNA of peripheral blood samples was extracted for PCR amplification. All exons, flanking sequences, and 5' and 3' untranslated regions of the F11 gene of the proband were analyzed by direct sequencing. And the corresponding sites were subjected to sequencing in other family members. The conservation of amino acid variation sites was analyzed by bioinformatic software, and the effect of the variant on the protein function was analyzed. Variants were graded based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). RESULTS: The proband was a 36-year-old male. His activated partial thromboplastin time (APTT) was 89.2s, which was significantly prolonged. The FⅪ activity (FⅪ:C) and FⅪ antigen (FⅪ:Ag) were 2.0% and 3.5%, respectively, which were extremely reduced. Both the proband and his sister were found to harbor compound heterozygous variants of the F11 gene, including a c.689G>T (p.Cys230Phe) missense variant in exon 7 from their father and a c.1556G>A (p.Trp519*) nonsense variant in exon 13 from their mother. Conservation analysis indicated the Cys230 site to be highly conserved. The c.1556G>A (p.Trp519*) variant was known to be pathogenic, whilst the c.689G>T variant was classified as likely pathogenic (PM2+PM5+PP1+PP3+PP4) based on the ACMG guidelines. CONCLUSION The c.689G>T and c.1556G>A compound heterozygous variants of the F11 gene probably underlay the pathogenesis of FⅪ deficiency in this pedigree.
Adult ; Humans ; Male ; 3' Untranslated Regions ; East Asian People ; Factor XI/genetics* ; Factor XI Deficiency/genetics* ; Partial Thromboplastin Time ; Pedigree

Objective:To investigate the molecular pathogenesis of a newly discovered gene mutation in a family with hereditary coagulation factor Ⅺ(FⅪ) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in September 2021 due to "calculus of intrahepatic duct". The patient had no symptoms of spontaneous bleeding.The clinical data and blood samples of the proband and her family members (10 persons in 3 generations) were collected.The activated partial thromboplastin time (APTT) and FⅪ activity (FⅪ:C) were performed by the one-stage clotting assay. FⅪ antigen (FⅪ:Ag) were detected by enzyme linked immunosorbent assay (ELISA). Genomic DNA extracted from peripheral blood cells of subjects was used as template to analyze F11 gene mutation by DNA direct sequencing. Bioinformatics software was used to analyze the effects of mutations on protein structure and function. Wild-type and mutant FⅪ protein expression vectors were constructed and transient transfected into HEK293T cells. The total RNA was extracted from positive transfected cells and then reversely transcribed into cDNA. The mRNA expression level of F11 gene in transfected cells was detected by real-time fluorescence quantitative PCR (qRT-PCR). The content of FⅪ:Ag and the expression of FⅪ protein in transfected cell lysates and culture supernatant were detected by ELISA and western blot.Results:The APTT of the proband was significantly prolonged to 107.9s (reference range 29.0-43.0s), while FⅪ:C and FⅪ:Ag were significantly decreased to 2% (reference range 84%-122%) and 5% (reference range 76%-127%), respectively. Gene sequencing analysis indicated that the proband had c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation in exon 6 and 13 of the F11 gene, respectively. Bioinformatics analysis showed that the amino acids at site 161 of FⅪ protein were threonine (Thr) in the matrix composed of five different species, indicating that Thr161 site was highly conserved among homologous genes in different species. p.Thr161Met heterozygous mutation affected the stability of local intermolecular structure of FⅪ protein. In vitro expression experiments of p.Thr161Met mutation showed that FⅪ protein had a normal synthesis in the cells but secretion dysfunction.Conclusions:c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation were mainly responsible for the decrease of FⅪ in this family. p.Thr161Met mutation was first reported in the world and did not affect the normal synthesis of FⅪ protein, but caused secretion dysfunction.

Objective:To explore the molecular pathogenesis of hereditary protein C (PC) deficiency due to a p. Gly86Asp variant of the PROC gene through in vitro expression experiment.Methods:Wild type and Gly86Asp mutant expression plasmids of PC were constructed and respectively transfected into HEK 293FT cells. Total RNA was extracted from the transfected cells, and the expression of PROC gene was determined by quantitative real-time PCR (qRT-PCR). PC antigen (PC: Ag) in the supernatant of cell culture and cell lysate was determined by enzyme-linked immunosorbent assay (ELISA), and the level of PC protein was detected by Western blotting. Results:qRT-PCR has detected no significant difference in the transcription level of wild-type and mutant-type PC. Compared with the wild type, the level of mutant PC: Ag in the supernatant and cell lysate were 81.3%±2.6% and 110.0%±2.8%, respectively. No difference was detected in the molecular weight between the wild-type and mutant-type PC by Western blotting. The PC content of mutant type was higher than wild-type in cell lysate, while the opposite was found with the cell culture supernatant.Conclusion:The impaired secretion by mutant PC may be the molecular mechanism of PC deficiency caused by the p.

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 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.

Objective To evaluate the effect of selective brain hypothermia on small ubiquitin-like modifier (SUMO)ylation of cerebral cortex during cerebral ischemia-reperfusion (I/R) in rats.Methods A total of 120 healthy clean-grade male Sprague-Dawley rats,aged 8 weeks,weighing 200-250 g,were divided into 4 groups (n=30 each) by a random number table method:sham operation group (group S),group I/R,selective brain hypothermia group (group H) and 37 ℃ normal saline group (group N).Rats were anesthetized with 10％ chloral hydrate 3 ml/kg.Cerebral ischemia was induced by middle cerebral artery occlusion using a nylon thread with rounded tip inserted into the right internal carotid artery and advanced cranially until resistance was met.The right middle cerebral artery was occluded for 2 h followed by reperfusion in group I/R.10-13 ℃ normal saline was infused at the rate of 100 ml · kg-1 · h-1 for 20 min starting from the time point immediately after removing the nylon thread in group H.37 ℃ normal saline was infused instead at the same rate for 20 min in N group.The neurological deficit were assessed and scored at 6,24 and 48 h after reperfusion.The cerebral cortex on ischemic side was then removed for determination of cell apoptosis (by TUNEL) and expression of SUMO sepcific proteases 3 (SENP3) and SUMO2/3-conjugated protein (by Western blot).The apoptotic rate was calculated.Results Compared with group S,the neurological deficit score and apoptosis rate were significantly increased,and the expression of SUMO2/3-conjugated protein was up-regulated at each time point after reperfusion in the other three groups (P＜0.05),and the expression of SENP3 was significantly up-regulated in I/R and N groups and down-regulated in group H (P＜0.01).Compared with group I/R,the neurological deficit score and apoptosis rate were significantly decreased,the expression of SUMO2/3-conjugated protein was up-regulated,and the expression of SENP3 was down-regulated in group H (P＜0.05).Conclusion The mechanism by which selective brain hypothermia reduces cerebral I/R injury is related to enhancing SUMOylation of cerebral cortex in rats.

Objective To investigate the risk factors of thrombopenia(TP)in septic patients complicated with acute kidney injury (AKI).Methods Two hundred and sixty five septic patients complicated with AKI admitted in Intensive Care Unit ICU of Zhejiang Provincial People''s Hospital during January 2012 and December 2016 were enrolled in the study.The clinical data, results of laboratory tests, Acute Physiology and Chronic Health Evaluation (APACHEII) scores, Sequential Organ Failure Assessment (SOFA) scores, therapeutic intervention, and 28-day mortality were documented.Among 265 patients, TP occurred within 7 days in 112 cases (TP group) and did not occur in 153 cases (non-TP group).Multivariable Logistic regression analysis was performed to analyze the risk factors of TP.Results The 28-day mortality rate in TP group was higher in TP group than that in non-TP group (47.3% vs.33.3%, χ2=5.307,P<0.05).Univariate analysis showed that age, C-reactive protein (CRP), procalcitonin (PCT) and APACHEII score, SOFA score, continuous renal replacement therapy (CRRT), heparin anticoagulation, shock, usage of linezolid and bloodstream infections were associated with TP in septic patients with AKI(all P<0.05).Multivariable Logistic regression analysis showed that age≥65 (OR=4.53, 95%CI 1.23-9.24,P<0.05), CRRT(OR=5.24,95%CI 2.14-14.56,P<0.01), heparin anticoagulation(OR=4.56,95%CI 2.13-8.46,P<0.01), usage of linezolid(OR=2.35,95%CI 1.25-5.24,P<0.01), shock(OR=2.15,95%CI 1.03-4.96,P<0.01)and bloodstream infections(OR=4.26,95%CI 1.36-12.48,P<0.01)were independent risk factors for septic patients with TP.Conclusion For septic patients with AKI having these risk factors, the platelet counts should be closely monitored, and intervention measures should be given to reduce the occurrence of TP.

Objective To explore the influence of citalopram on early recovery of motor function in patients with acute cortical infarction.Methods A total of 276 patients with acute cerebral infarction were randomly divided into observation group and control group,138 cases in each group.Patients in control group were treated with anti-platelet,blood lipid,protective nerve and rehabilitation therapy.The observation group was given citalopram hydrobromide tablets on the basis of control group.Results The incidence rate of depression in the observation group was 18.1％,which was significantly lower than 42.7％ in the control group (P ＜ 0.05).After treatment,HDRS score and NIHSS score in the observation group were significantly lower than the control group (P ＜ 0.05).In the observation group,after treatment of 30 days and 90 days,the increasing scores of hand motor function of FMA were 4.1 ± 1.0 points and 6.5 ± 2.5 points,which were significantly higher than 1.4 ± 0.7 points and 2.5 ± 1.5 points (P ＜ 0.05).After treatment of 30 days,the BI score of observation group was significantly higher than control group (P ＜ 0.05).Conclusion Early application of citalopram can effectively reduce the incidence rate of post-stroke depression in patients with acute cerebral infarction,promote the recovery of motor function and improve the quality of prognosis.