Objective To investigate the effects of the tumor suppressor gene PTEN in growing inhibition and down-regulating mTOR in cholangiocarcinoma QBC939 cells in vitro.Methods QBC939 cells were transfected with plasmids wild-type PTEN and C124S-PTEN in vitro.After transfection,the expression of the PTEN and phosphorylation of AKT and mTOR was detected by Western blot.Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells.Results Compared with the control,the expression of phosphorylation AKT was decreased and mTOR were down-regulated respectively when transfected with the wild-type PTEN.However,after transfection with mutation-type PTEN,the level of PTEN in the cells by increased,but phosphorylation AKT level and mTOR expression had no significant change.Conclusions PTEN can be actived by phosphorylated AKT.Actived AKT decreased the mTOR which led to tumor cells apoptosis and regulation of the tumor cell cycle.In the pathway of signal transmission of PI3K/AKT/PTEN/mTOR,PTEN and mTOR are closely related through phosphorylation of AKT.

Objective To investigate the association between RNF213 rs6565666 polymorphisms and intracranial cystic aneurysms in patients from Guangdong province. Methods Two hundred and fifty patients with intracranial cystic aneurysms, admitted to and conformed by digital substraction angiography (DSA) in our hospital from February 2016 to October 2018, were selected as experimental group; and 250 patients without intracranial aneurysms conformed by DSA, CT angiography or magnetic resonance angiography at the same time period were used as control group. The genotypes of rs6565666 locus of RNF213 gene were detected by polymerase chain reaction-ligase detection reaction (PCR-LDR). Results As compared with those in the control group, percentages of AG and AA genotypes were significantly higher and percentage of GG genotype was statistically higher at rs6565666 locus of patients from the experimental group (P<0.05). The proportion of allele A at rs6565666 locus in the experimental group was statistically higher as compared with that in the control group (P<0.05). In the experimental group, 112 patients had ruptured aneurysms and 138 patients did not have ruptured aneurysms; there was no statistically significant difference in the genotype distribution of rs6565666 between the ruptured group and the non-ruptured group (P>0.05). Conclusion RNF213 gene rs6565666 polymorphism is associated with intracranial cystic aneurysms in patients from Guangdong province.

Objective To evaluate the effect of cerebral microbleeds (CMBs) on intracranial aneurysm rupture and hemorrhage. Methods A total of 2023 patients with intracranial single aneurysms (944 un-ruptured aneurysms and 1029 ruptured aneurysms) were enrolled in our study. The 3D-DSA was applied to all patients to evaluate the aneurysm sizes, locations, and morphous features, and to confirm the presence of A1 dominance and variations of Circle of Willis; moreover, aspect ratio (AR), size ratio (SR) and aneurismal inclination angle were measured. The presence of CMBs identified by T2-weighted gradient-recalled-echo sequence on magnetic resonance imaging (MRI) was evaluated; the clinical data of combined with/without CMBs patients were compared. Ninety-two untreated intracranial aneurysms patients combined with CMBs were followed up to evaluate the incubation periods of CMBs-related intracranial aneurysm rupture and hemorrhage; the clinical data of patients with un-ruptured aneurysms and ruptured aneurysms were compared; the risk factors of intracranial aneurysm rupture and hemorrhage were analyzed by univariate and multivariate Logistic regression analyses. Results CMBs confirmed by MR imaging were presented in 158 patients, with 7.81％ incidence rate (158/2023). Age, proportion of smokers, aneurysm inclination Angle, SR, narrow neck, irregular aneurysm shape, proportion of rupture and hemorrhage, aneurysm sites, and hypertension showed significant differences between patients without CMBs and patients with CMBs (P<0.05). In the untreated intracranial aneurysms patients combined with CMBs, 27 had intracranial aneurysm rupture and 65 did not appear intracranial aneurysm rupture; the rupture time was 3-46 months, with an average of (15.07± 10.76) months. As compared with the un-ruptured group, the ruptured group had a statistically higher proportion of patients with irregular aneurysm morphology (P<0.05). Univariate analysis showed that CMBs, female, age, aneurysm size, aneurysm morphology, ICA and ACA aneurysms, AR, variations of Circle of Willis, hypertension grading II and III, diabetes mellitus with fasting blood glucose≤6.0 mmol/L, hyperlipidemia, coronary heart disease, and drinking alcohol were important factors affecting intracranial aneurysm rupture, and the differences were statistically significant (P<0.05). Multivariate Logistic regression analysis showed that CMB was an independent risk factor for intracranial aneurysm rupture and hemorrhage; as compared with patients without CMBs, patients with CMBs had a 1.75 fold increased risk of aneurysm rupture. Conclusions Patients with intracranial aneurysms with older age, smaller aneurysm inclination Angle and larger SR are more likely to be associated with CMBs. Intracranial aneurysms with CMBs patients with irregular morphology are prone to have rupture and hemorrhage. CMBs is an independent risk factor for intracranial aneurysm rupture and hemorrhage.

Objective To investigate the role of hesperitin in regulating inflammatory response in early brain injury after subarachnoid hemorrhage (SAH). Methods A total of 96 adult male SD rats were divided into sham-operated group, SAH group, solvent group and intervention group (n=24) by random number table method. The SAH rat models in the latter three groups were prepared by carotid artery puncture method; the rats in the intervention group were given oral administration of hesperidin solution, which was dissolved in 5％ dimethyl sulfolide (DMSO), with a concentration of 1 mg/100 μL and a dosage of 40 mg/kg within 30 min after operation; rats in the solvent group were given oral administration of an equal volume of 5％ DMSO solution. Modified Garcia behavioral scale was used to evaluate the neurobehavior of rats, and the wet/dry weight method was used to measure the water content in the brain tissues of the left and right hemispheres of the rats 24 h after SAH. Immunofluorescence staining was used to detect the microglia activation, Fluoro-dyed Jade C staining was used to assess the brain neuron degeneration, enzyme-linked immunosorbent assay (ELISA) was employed to detect the inflammatory factors interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) content in the brain tissues, and Western blotting was used to detect the nuclear factor-κB (NF-κB) and phosphorylated (p) -NF-κB protein expressions. Results As compared with the solvent group, intervention group had significantly increased improved modified Garcia behavioral scale scores (10.08±1.73 vs. 13.83±1.70), and significantly decreased water content of brain tissues in the left and right hemispheres ([81.44 ± 1.05]％ vs. ([79.14±0.82]％; [80.55±1.55]％ vs. [78.79±1.02]％), significantly smaller number of CD68+ and Iba1+ microglias (30.17±1.04 vs. 10.67±0.75; 29.33±1.16 vs. 12.00±0.41), significantly smaller number of degenerate neurons (53.21±0.94 vs. 31.33±0.28), significantly reduced levels of inflammatory cytokines IL-1β, IL-6 and TNF-α ([429.88±106.32] pg/mL vs. [221.50±48.80] pg/mL; [1015.50±221.80] pg/mL vs. [448.11±93.40] pg/mL; [1021.75±149.17] pg/mL vs. [595.71±190.81] pg/mL), and significantly lower p-NF-κB/NF-κB ratio (1.13±0.07 vs. 0.71±0.02, P<0.05). Conclusion Hesperitin may reduce the inflammatory response mediated by microglia after subarachnoid hemorrhage by inhibiting NF-κB pathway, thereby improving the neurological dysfunction of rats.

Objective To explore the effect of Willis circle variation and hemodynamic forces alteration arised from vascular structural abnormity on intracranial aneurysm (IA) rupture using 3D-digital subtraction angiography (DSA) and transcranial color Doppler (TCCD) detection.Methods Two hundred and twenty-three patients with IA,admitted to and conformed by DSA in our hospital from November 2010 to November 2011,were divided into ruptured IA group (n=182) and un-ruptured IA group (n=41).The 3D-DSA was applied in all patients to carefully evaluate the aneurysm sizes,locations,and morphous features,and to confirm the presence of A1 dominance and Willis circle variation.Moreover,aneurysmal neck area,diameter of parent artery,angle between A2 segments of bilateral anterior cerebral artery,angle between aneurysmal longitudinal axis and parent artery,aortic diameter (AD) and aspect ratio (AR) were measured with assistance of 3D-DSA images.Besides,TCCD was applied to all patients,and the hemodynamic parameters were recorded to calculate wall shear stress (WSS) and mechanical stretch.The risk factors of IA rupture were analyzed by receiver operating characteristic (ROC) curve and multivariate Logistic regression with emphasis on Willis circle variation and hemodynamic forces alteration.Results Whether it was in ruptured IA group or in un-ruptured IA group,the incidence rate of variation of anterior Willis circle was higher than that of variation of posterior Willis circle.A1 dominance on the left side was the most common asymmetry.As compared with those in un-ruptured IA group,statistically elder age,smaller AD,larger angle between aneurysmal longitudinal axis and parent artery,decreased WSS and increased mechanical stretch in the ruptured IA group were noted (P＜0.05).ROC curve indicated that angle between aneurysmal longitudinal axis and parent artery,AD,WSS and mechanical stretch could be used to evaluate IA rupture (area under the curve:0.606、0.618、0.396、0.637).Age (OR=8.618,95％CI:2.866-25.917,P=0.000),hypertension (grade Ⅲ OR=16.320,95％CI:1.628-163.556,P=0.018),angle between aneurysmal longitudinal axis and parent artery (OR=3.053,95％CI:1.131-8.242,P=0.028),AD (OR=5.638,95％CI:1.507-20.251,P=0.008) and mechanical stretch (OR=4.230,95％CI:1.554-11.516,P=0.000) were risk factors of IA rupture.A1 dominance (OR=0.242,95％CI:0.074-0.785,P=0.018),small aneurysms (2-5 mm,OR=0.207,95％CI:0.054-0.788,P=0.002) and WSS (OR=0.021,95％CI:0.060-0.672,P=0.009) were identified as protective factors.Conclusions Willis circle variation exists in IA patients.Age,hypertension (grade ⅢD,angle between aneurysmal longitudinal axis and parent artery,AD and mechanical stretch are risk factors of IA rupture,while A1 dominance,small aneurysm (2-5 mm) and WSS are identified as protective factors.Accurate assessment of these factors is of great clinical significance for the prevention and treatment of IA in the future.

Objective:To investigate the effect of neutrophils on cell pyroptosis and its mechanisms in mice with early brain injury (EBI) following subarachnoid hemorrhage (SAH).Methods:Seventy six male C57BL/6J mice were randomly divided into sham-operated group, SAH group, SAH+vehicle group, and SAH+anti-ly6G group ( n=19). SAH models in the latter 3 groups were established by modified endovascular perforation. Mice in the SAH+vehicle group and SAH+anti-ly6G group received intravenous injection of equal normal saline or anti-ly6G antibody (4 mg/kg) 24 h before SAH. At 24 h after SAH, immunofluorescent staining was used to detect the locations/expressions of neutrophils, S100 calcium binding protein A8 (S100A8) and gasdermin D (GSDMD); FJC staining was performed to assess the neuronal injury; modified Garcia test and rotarod test were used to evaluate the neurological functions, and brain water content test was applied to evaluate the brain edema; Western blotting was used to detect the expressions of S100A8, Toll-like receptor 4 (TLR4), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved cysteinyl aspartate specific proteinase-1 (cleaved-caspase1), and cleaved N-terminal gasdermin D (GSDMD-N). Results:(1) Compared with those in the sham-operated group, neutrophil infiltration at the damaged cortex with highly expressed S100A8 in neutrophils was observed in the SAH group, and increased GSDMD expression at the damaged cortex and GSDMD co-localization in astrocytes, microglia and neurons were observed in the SAH group. (2) Compared with the sham-operated group, the SAH group and SAH+vehicle group had significantly increased numbers of infiltrated neutrophils and FJC-positive neurons, significantly decreased falling latency in the modified Garcia score and rotarod test, significantly increased brain water content, and significantly elevated expressions of S100A8, TLR4, NLRP3, cleaved-caspase1 and GSDMD-N ( P<0.05); the SAH+anti-ly6G group had statistically decreased numbers of infiltrated neutrophils and FJC-positive neurons, statistically increased falling latency in the modified Garcia score and rotarod test, statistically decreased brain water content, and statistically decreased expressions of S100A8, TLR4, NLRP3, cleaved-caspase1 and GSDMD-N compared with the SAH group and SAH+vehicle group ( P<0.05). Conclusion:Inhibition of neutrophils can down-regulate the S100A8 expression after SAH and attenuate TLR4/NLRP3 activation-mediated cell pyroptosis, thereby improving EBI.