Objective To investigate the cross-talk between extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) signal transduction pathways in A375 human melanoma cells.Methods Cultured A375 cells were randomly divided into 5 groups:control group receiving no treatment,two U0126 (a selective inhibitor of the ERK signaling pathway) groups treated with U0126 of 10 and 5 μmol/L,and two BMS-345541 groups treated with BMS-345541 of 10 and 5 μmol/L.After 24-hour treatment,Western blot and reverse transcription PCR were performed to measure the protein expressions of NF-κB P65,phosphorylated IκBα (p-IκBα),ERK1/2,as well as p-ERK1/2,and the mRNA expressions of NF-κB P65 and ERK1,respectively.One-way analysis of variance and least significant difference (LSD)-t test were carried out for statistical analysis.Results After 24 hours of treatment with U0126 of 10 and 5 μmol/L,a significant decrease was noted in the relative expression level of NF-κB p65 protein (0.60 ± 0.04 and 0.56 ± 0.06 vs.1.54 ± 0.15,both P＜ 0.01) and mRNA (0.79 ± 0.05 and 0.75 ± 0.04 vs.0.86 ± 0.05,both P ＜ 0.01),but a statistical increase in that of p-IκBα protein (0.90 ± 0.05 and 0.70 ± 0.02 vs.0.61 ± 0.03,both P ＜ 0.01) in the two U0126 groups compared with the control group; significant differences were observed in the expression level of p-IκBo protein (P ＜ 0.01) but not in that of NF-κB p65 protein (P ＞ 0.01) between the two U0126 groups.The relative expression levels of ERK1/2 and p-ERK1/2 proteins as well as ERK1 mRNA were significantly higher in the control A375 cells than those in the cells treated with BMS-345541 of 10 μmol/L (0.73 ± 0.07,0.75 ± 0.09,1.51 ± 0.02,all P ＜ 0.01),but similar to those treated with BMS-345541 of 5 μmol/L (0.94 ± 0.11,0.99 ± 0.04,1.62 ± 0.03,all P ＞ 0.05).Conclusion There is a cross-talk between ERK and NF-κB signal transduction pathways in A375 melanoma cells.

Group B Streptococcus ( GBS) is a leading infectious cause of adverse pregnancy out-comes such as preterm birth. GBS colonizes the vagina during pregnancy and can ascend into the uterus and then infect the fetus. It encodes a series of virulence factors such as adhesion and invasion factors, hemolytic pigments and hyaluronidase, which are important to vaginal colonization and immune evasion. Immune re-sponses to GBS cause the release of a multiple of inflammatory mediators, leading to the premature rupture of membranes, preterm birth and fetal injury. This paper reviews the pathogenesis of GBS vaginal colonization and ascending infection causing adverse pregnancy outcomes.

Objective:To investigate the role of receptor-interacting protein kinse3 (RIPK3)-mediated necroptosis in diabetic mellitus-caused abolition of cardioprotection induced by sevoflurane postconditioning in rats.Methods:Eighty rats with diabetes mellitus, aged 4-5 weeks, weighing 90-100 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (group Sham), myocardial ischemia-reperfusion (I/R) group (group I/R), sevoflurane postconditioning group (group SP) and sevoflurane postconditiong plus RIPK3 inhibitor GSK-872 group (group GSK). Myocardial I/R was induced by 40 min occlusion of the left anterior descending branch of the coronary artery followed by 120 min reperfusion.In group SP, 2.4% sevoflurane was inhaled for 15 min at the beginning of reperfusion.In group GSK, GSK-872 3.3 mg/kg (dissolved in normal saline) was intraperitoneally injected at 24 and 2 h before surgery, and the other treatments were similar to those previously described in group SP.After 120 min of reperfusion, blood samples from the abdominal aorta were collected for determination of concentrations of serum lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB). Myocardial tissues were taken for determination of percentage of myocardial infarct size (by TTC staining) and expression of RIPK3, phospho-Ca 2+ -calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) and phospho-mixed lineage kinase domain-like protein (p-MLKL) (by Western blot), and the ultrastructure of myocardium was observed by transmission electron microscopy. Results:Compared with group Sham, the serum LDH and CK-MB concentrations and percentage of myocardial infarct size were significantly increased, the expression of RIPK3, p-MLKL and p-CaMKⅡ in myocardial tissues was up-regulated ( P<0.05), and the damage to cardiomyocytes was severe in group I/R.Compared with group I/R, no significant change was found in the parameters mentioned above in group SP ( P>0.05). Compared with group SP, the serum LDH and CK-MB concentrations and percentage of myocardial infarct size were significantly decreased, the expression of RIPK3, p-MLKL and p-CaMKⅡ in myocardial tissues was down-regulated ( P<0.05), and the damage to cardiomyocytes was reduced in group GSK. Conclusion:The mechanism of diabetic mellitus-caused abolition of cardioprotection induced by sevoflurane postconditioning is related to excessive activation of RIPK3-mediated necroptosis in rats.

Anisodamine, which is originally extracted from scopolia tangutica and is currently produced in China, is a tropane alkaloid and a muscarinic cholinoceptor blocker. Our previous study found that anisodamine did not alter high K(+)-evoked contraction of rabbit aortic rings using isometric tension recording methods, but could attenuate noradrenaline (NA)-, histamine- or 5-hydroxytryptamine-induced contraction in an endothelium-independent manner. Since the high K(+)-elicited depolarization non-selectively inhibits potassium channels in vascular smooth muscle cell (VSMC) membrane, the vasodilation effect of some potassium channel activators may be inhibited or abolished in high K(+) solution. We hypothesized that some potassium channels in VSMC membrane might play a role in the anisodamine-induced relaxation of blood vessels. The present experiment was designed to investigate whether potassium channel blockers inhibit anisodamine-induced relaxation of the rabbit isolated aortic rings. In a 8-min period, 1, 3 and 10 micromol/L of anisodamine, significantly relaxed the 0.01 micromol/L NA precontracted aortic ring by (19.1+/-3.1)%, (30.1+/-3.8)% and (38.3+/-4.2)%, respectively, compared with the controls [by (4.8+/-2.4)%, (5.1+/-1.8)% and (5.6+/-2.5)%, respectively] (P<0.01). 10 mmol/L of CsCl (a non-selective potassium channel blocker), 1 mmol/L of 4-aminopyridine [a selective voltage-activated potassium channel (K(V)) blocker], 10 mumol/L BaCl2 (a selective inwardly-rectifying potassium channel blocker), 10 micromol/L of glibenclamide (a selective ATP-sensitive potassium channel blocker), 3 micromol/L of charybdotoxin (a large- and intermediate-conductance Ca(2+)-activated potassium channels blocker) and 3 micromol/L of apamin (a selective small conductance Ca(2+)-activated potassium channel blocker) significantly increased the NA-induced contraction by (14.4+/-3.2)%, (16.3+/-5.8)%, (12.7+/-4.2)%, (13.6+/-2.0)%, (11.1+/-5.5)% and (13.4+/-4.3)%, respectively, compared with the control [by (5.6 +/-1.2)%] (P<0.01). In the presence of 10 and 30 mmol/L CsCl or 1 and 3 mmol/L 4-aminopyridine, anisodamine-induced relaxation of the 0.01 micromol/L NA contracted rabbit aortic rings [(28.8+/-3.0)% and (15.9+/-3.7)% or (29.7+/-3.9)% and (19.0+/-5.0)%] significantly deceased, compared with that in the absence of any potassium channel blocker [(38.3+/-4.2)% (P<0.01)] in a 8-min period. However, in the presence of 10, 30 micromol/L of BaCl2, 10, 30 micromol/L of glibenclamide, 3 micromol/L of charybdotoxin, or 3 micromol/L apamin, 10 micromol/L anisodamine-induced relaxation [(37.1+/-3.8)%, (36.2+/-4.7)%, (36.1+/-2.7)%, (35.6+/-3.3)%, (37.8+/-2.0)% and (39.3 +/-4.7) %, respectively] did not decrease, compared with the control [(38.3+/-4.2)%] (P>0.05). This study suggests that K(V) blockers inhibit anisodamine-induced relaxation of the rabbit aortic smooth muscle precontracted with NA and implies that the K(V) in VSMC membrane plays a role in anisodamine-induced relaxation of blood vessels.
Animals ; Aorta ; cytology ; Female ; Male ; Muscle Contraction ; drug effects ; Muscle Relaxation ; drug effects ; Muscle, Smooth, Vascular ; physiology ; Norepinephrine ; antagonists & inhibitors ; Potassium Channel Blockers ; pharmacology ; Potassium Channels, Voltage-Gated ; Rabbits ; Solanaceous Alkaloids ; pharmacology

OBJECTIVETo investigate the general pattern of cholinergic nerve distribution and M(2) receptors in adult rat heart.

METHODSKarnovsky-Roots histochemical staining combining point counting method and immunochemical SABC method with image analysis were used to identify the cholinergic nerves and M(2) receptors, respectively, in adult rat heart.

RESULTSPositive staining of cholinergic nerves and M(2) receptors was found in all regions of the rat heart, and the point count of cholinergic nerves in the atria was 4.6 times as much as that in ventricles, and the area of immunoreactive substance for M(2) receptors two-fold higher in the atria than in the ventricles. The point counts of the cholinergic nerves in the medial-layer myocardium were fewer than that in subepicardial and endocardial tissues of the left ventricular free wall. However, M(2) receptors were comparable among the 3 layers of the left free ventricular wall.

CONCLUSIONCholinergic nerves and M(2) receptors are located in both rat atria and ventricles, but their density is much higher in the atria than in the ventricles. Transmural heterogeneity characterizes cholinergic nerve innervation in the left ventricular free wall without significant differences in M(2) receptor density.

Animals ; Cholinergic Fibers ; metabolism ; Female ; Heart ; innervation ; Heart Atria ; innervation ; metabolism ; Heart Ventricles ; innervation ; metabolism ; Immunohistochemistry ; Male ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Muscarinic M2 ; analysis

OBJECTIVETo investigate JAK2 exon 12 mutations in patients with Philadelphia (Ph) chromosome-negative myeloproliferative neoplasms (MPN) and the clinical characteristics of patients with JAK2 exon 12 mutants.

METHODSAllele-specific PCR (AS-PCR) was applied to identify JAK2 V617F mutation. Genomic DNA corresponding to exon 12 of JAK2 gene and epigenetic regulator gene (TET2, ASXL1, EZH2) were amplified by polymerase chain reaction (PCR). Identification of mutants was by direct sequencing and classification of mutation types by sequencing followed by plasmid cloning. SNP genotyping of two 46/1 tag SNPs, rs12340895 and rs10974944, was analyzed using commercially available Taqman assays on the 7500HT real-time PCR instrument according to standard protocols.

RESULTSNo JAK2 exon 12 mutation was detected in patients with ET, PMF or JAK2 V617F positive PV. Among 13 JAK2 V617F negative PV patients, JAK2 exon 12 mutation was detected as N542-E543del in 2(15.4%) patients who presented with a phenotype of predominant erythrocytosis and erythroid colonic grown from their bone marrow samples in the absence of exogenous EPO, reduced serum erythropoietin (EPO) level, and no mutations in TET2, ASXL1 or EZH2 genes. One of the affected patients was heterozygous for 46/1 but the second was negative for this haplotype.

CONCLUSIONThere was no need to detect JAK2 exon 12 mutation in ET, PMF or MPN-U patients without JAK2 V67F mutation. Ph negative MPN patients with JAK2 exon 12 mutations had somewhat unique clinical and laboratory features.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow Neoplasms ; genetics ; DNA Mutational Analysis ; Exons ; Female ; Genotype ; Humans ; Janus Kinase 2 ; genetics ; Male ; Middle Aged ; Myeloproliferative Disorders ; genetics ; Philadelphia Chromosome ; Young Adult

OBJECTIVETo observe the effect of recombinant human erythropoietin (rhuEPO) on p-Akt and caspase-9 expressions in the hippocampus of rats with status epilepticus (SE) and explore the neuroprotective mechanism of rhuEPO.

METHODSAdult male SD rats were randomized into control, PTZ, rHuEPO, LY294002 group, and DMSO groups and treated with normal saline (NS), PTZ, PTZ+rHuEPO, PTZ+LY294002+rHuEPO, and PTZ+DMSO+rHuEPO, respectively. The behavioral and electroencephalogram (EEG) changes of the rats were recorded, and the expressions of p-Akt and caspase-9 were detected using immunohistochemistry. The hippocampal expression of caspase-9 mRNA was detected using RT-PCR, and the expressions of Akt and p-Akt proteins were determined with Western blotting.

RESULTSThe p-Akt-positive cell and p-Akt protein expression increased significantly while the caspase-9-positive cell and caspase-9 mRNA expression decreased in rHuEPO group as compared with those in PTZ group (P<0.05). LY294002 treatment prior to rHuEPO injection significantly abolished the effects of rHuEPO on caspase-9 and p-Akt immunohistochemical positivity and caspase-9 mRNA and p-Akt protein expressions (P<0.05).

CONCLUSIONAdministration of rHuEPO activates the PI3K/Akt signaling pathway in SE rats and increases the expression of p-Akt protein to regulate the expression of caspase-9, a regulatory factor of the mitochondrial-dependent apoptotic pathway, and therefore provides anti-apoptotic and neuroprotective effects.

Animals ; Caspase 9 ; genetics ; metabolism ; Erythropoietin ; therapeutic use ; Hippocampus ; metabolism ; Male ; Neuroprotective Agents ; therapeutic use ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; Status Epilepticus ; drug therapy ; metabolism

BACKGROUND:The connection between high-level central nervous system and spinal cord and peripheral nerve under injury level is blocked by traumatic spinal cord injury,and the whole function of the body is thereby influenced.The loss of motor function and feeling both severely affect the patient's life quality in views of physiology,psychology,function and social economics.RNA interference is an effective method to silence target genes,which provides a new treatment strategy for spinal cord injury.OBJECTIVE:To explore the application status of RNA interference in spinal cord injury.METHODS:PubMed and CNKI databases were retrieved using the keywords of "RNA interference,RNAi,spinal cord injury" in English and Chinese,respectively.The articles addressing the application of RNA interference in spinal cord injury were collected and reviewed.RESULTS AND CONCLUSION:Totally 44 articles were enrolled.It is urgent to find a new way to treat spinal cord injury.Compared with the traditional gene silencing technology,RNA interference owing to high specificity,high efficiency,high stability and high penetrability is considered to be a new direction for the studies on the pathogenesis and treatment of spinal cord injury.

OBJECTIVETo investigate the correlation between smoking status and carotid plaque in rural population residing in Eastern part of China.

METHODSBetween July and September of 2013, an epidemiological survey was carried out in residents aged 40 or above men who were enrolled randomly in rural areas of Anqing, Anhui province and Lianyungang, Jiangsu province. The data on epidemiological characteristics including smoking status, physical examination were collected using standardized protocol, and carotid ultrasonography was applied to examine the incidence of carotid plaque among never smokers, former smokers and current smokers. Logistic regress analysis was performed to determine the effect of smoking on carotid plaque.

RESULTSIn the study, a total of 625 male participants were included in the study. 51.4% (321 cases) were current smokers, 21.3% (133 cases) were former smokers, and 27.4% (171 cases) were never smokers. 32.0% (200/625) had carotid plaque. The incidence of carotid plaques was significantly higher in current smokers (35.2%, 113/321) than that in never smokers(23.4%,40/171) (χ(2) = 7.26, P = 0.007) and the incidence in former smokers (35.3%, 47/133) was also higher than that in never smokers (23.4%, 40/171) (χ(2) = 5.23, P = 0.022). Multiple logistic regression analysis showed that current cigarette smoking is significantly associated with the increased risk of carotid plaque (OR = 1.84, 95% CI: 1.13-2.98, P = 0.014) in comparison with never smokers, and there was an interaction between current smoking and age in association with carotid plaque. Compared with the young (≤60 years old) and never smoking group (8%, 3/40), prevalence of carotid plaque among the elderly (>70 years old) and smoking group (55%, 31/56) was significantly higher (OR = 8.06, 95% CI: 2.07-31.45) after adjusting for age, systolic blood pressure, diastolic blood pressure, blood glucose, total cholesterol, triglyceride high-density lipoprotein, body mass index, drinking and regional differences.

CONCLUSIONIt found that cigarette smoking was associated with increased risk of carotid plaque in rural elderly population residing in Eastern part of China.

Aged ; Alcohol Drinking ; Blood Glucose ; Blood Pressure ; Body Mass Index ; Carotid Stenosis ; China ; Cholesterol ; Humans ; Lipoproteins, HDL ; Male ; Middle Aged ; Prevalence ; Risk Factors ; Rural Population ; Smoking ; Triglycerides

Objective To explore the risk factors for intracranial infection in patients after neurosurgery,con-struct and validate a Nomogram prediction model.Methods Data of 978 patients who underwent neurosurgery in a hospital in Nanjing from January 1,2019 to December 31,2022 were retrospectively analyzed.Independent risk fac-tors were screened through logistic univariate and multivariate analyses.Modeling variables were screened through Lasso regression.A Nomogram model was constructed and internally validated by logistic regression.Effectiveness of the model was evaluated with receiver operating characteristic(ROC)curve,calibration curve and decision curve.Results Among 978 patients underwent neurosurgery,293 had postoperative intracranial infection,with an inci-dence of healthcare-associated infection of 29.96％.There was no significant difference in age,gender,proportion of coronary heart disease,cerebral infarction,diabetes and hypertension between the infected group and the non-in-fected group(all P＞0.05).Multivariate logistic analysis showed that postoperative intracranial hypertension,fe-ver,increased neutrophil percentage in blood routine examination,turbid cerebrospinal fluid,positive Pan's test,decreased glucose concentration,abnormal ratio of cerebrospinal fluid/serum glucose,positive microbial culture,absence of indwelling external ventricular drainage tubes,presence of indwelling lumbar cistern drainage tubes,use of immunosuppressive agents,and long duration of surgery were independent risk factors for postoperative intracra-nial infection in patients who underwent neurosurgery(all P＜0.05).Fifteen variables were screened out through Lasso regression.Fourteen variables were finally included for modeling after collinear screening,missing data impu-tation(random forest method)and checking pairwise interaction items.A Nomogram prediction model was con-structed,with the area under ROC curve,sensitivity,specificity,and accuracy of 0.885,0.578,0.896,and 0.704,respectively.Internal validation of the model was conducted.The modeling and validation groups presented similar effects.The calibration curve and decision curve also indicated that the model had good predictive efficacy.Conclusion The constructed Nomogram prediction model for postoperative intracranial infection after neurosurgery is scientific,and the prediction indicators are easy to obtain.The model presents with high stability,reliability,and application value,thus can provide reference for the assessment of postoperative intracranial infection after neuro-surgery.