p53 siRNA promotes autophagy of U2OS cells through its target gene Rap2B

The present study aims to explore the effects of p53 and its target gene Rap2B on the autophagy of U2OS cells.U2OS cells were treated with siRNA against p53,Rap2B,and PLCε.Relative expressions of p53,Rap2B,and PLCεwere determined using quantitative polymerase chain reaction(qPCR)and Western blotting,respectively.Levels of IP3 in the cells were determined using Enzyme-linked Immunosorbent Assay(ELISA).Levels of Ca^(2+) were detected using Flow cytometry.Fluorescence microscopy was used to observe the autophagy of cells.Knockdown of p53 significantly decreased the expressions of Rap2B protein.Additionally,knockdown of p53 significantly decreased the mRNA levels of PLCε.The knockdown of p53,Rap2B,and PLCεsignificantly decreased the levels of intracellular IP3 and Ca^(2+) and promoted autophagy of U2OS cells.Our results demonstrated that p53-Rap2B-PLCε-IP3 signaling pathway regulated autophagy of U2OS cells.

Electroacupuncture Attenuates Visceral Pain and Reverses Upregulation of TRPV1 Expression in Adult Rats with Neonatal Maternal Deprivation

Irritable bowel syndrome (IBS) is characterized by chronic visceral hypersensitivity that companied by altered bowel movement. However, the treatment options are very limited. The aim of this study was to investigate effects of electroacupuncture (EA) on visceral hypersensitivity in a rat model of IBS and to explore the underlying mechanisms of EA effects. Visceral hypersensitivity was established by neonatal maternal deprivation (NMD) in male rats on postnatal days 2 - 15. Behavioral experiments were conducted at the age of 7 weeks. Treatment with EA at Zusanli (stomach-36, ST-36) significantly reduced abdominal withdrawal reflex (AWR) scores in NMD rats but not in age-matched healthy control rats. In addition, EA treatment hyperpolarized resting membrane potentials, increased the rheobase and reduced the numbers of action potentials evoked by 2 and 3 times rheobase current stimulation of dorsal root ganglion (DRG) neurons innervating the colon. NMD markedly enhanced expression of TRPV1 in colon related DRGs while EA treatment drastically suppressed the expression of TRPV1 in DRGs of NMD rats. These data suggest that EA treatment produced an analgesic effect, which might be mediated at least in a part by suppression of TRPV1 expression and by inhibition of neuronal excitability of primary sensory neurons in rats with chronic visceral pain.

NIMA related kinase 2 promotes gastric cancer cell proliferation via ERK/MAPK signaling

BACKGROUND NIMA related kinase 2(NEK2) is closely related to mitosis, and it is currently considered to be over-expressed frequently in many poorly prognostic cancers.However, the effect of the up-regulated NEK2 on cellular signaling in tumors,such as gastric cancer(GC), is con-fusing.AIM To determine the role of the up-regulation of NEK2 in GC.METHODS To investigate the pathological significance of NEK2 in GC, the expression pattern of NEK2 in GC was investigated based on the 'Oncomain' database and compared between 30 pairs of cancer samples and adjacent tissues. The coexpression of NEK2 and ERK in GC was analyzed using The Cancer Genome Atlas(TCGA) database and confirmed in clinical samples by quantitative realtime PCR(qRT-PCR), and the survival curve was also plotted. Western blot or qRT-PCR was used to analyze the effect of NEK2 on the phosphorylation levels of ERK and c-JUN in two GC cell lines(BGC823 and SGC7901) with NEK2 overexpression, and the expression of the downstream effector cyclin D1.Furthermore, CCK8, EdU incorporation assay, and flow cytometry were used to detect the proliferative ability of BGC823 and SGC7901 cells with stably silenced ERK.RESULTS NEK2 was significantly up-regulated in human GC tissues. ERK was significantly associated with NEK2 expression in human clinical specimens, and combined overexpression of NEK2 and ERK potentially forecasted a poor prognosis andsurvival in GC patients. NEK2 knockdown in GC cells inhibited ERK and c-JUN phosphory-lation and reduced the transcription of cyclin D1. More interestingly,NEK2 can rescue the inhibition of cellular viability, proliferation, and cell cycle progression due to ERK knockdown.CONCLUSION Our results indicate that NEK2 plays a carcinogenic role in the malignant proliferation of GC cells via the ERK/MAPK signaling, which may be important for treatment and improving patient survival.

TNF-αTNFR1 Signaling is Required for the Full Expression of Acute and Chronic Itch in Mice via Peripheral and Central Mechanisms

Increasing evidence suggests that cytokines and chemokines play crucial roles in chronic itch. In the present study, we evaluated the roles of tumor necrosis factor-alpha （TNF-c0 and its receptors TNF receptor subtype-I （TNFR1） and TNFR2 in acute and chronic itch in mice. Compared to wild-type （WT） mice, TNFRl-knockout （TNFR1-KO） and TNFR1/R2 double-KO （DKO）, but not TNFR2-KO mice, exhibited reduced acute itch induced by compound 48/80 and chloroquine （CQ）. Application of the TNF-synthesis inhibitor thalidomide and the TNF-at antagonist etanercept dose-dependently suppressed acute itch. Intradermal injection of TNF-α was not sufficient to evoke scratching, but potentiated itch induced by compound 48/80, but not CQ. In addition, compound 48/80 induced TNF-α mRNA expression in the skin, while CQ induced its expression in the dorsal root ganglia （DRG） and spinal cord. Furthermore, chronic itch induced by dry skin was reduced by administration of thalidomide and etaner- cept and in TNFR1/R2 DKO mice. Dry skin induced TNF- expression in the skin, DRG, and spinal cord and TNFR1 expression only in the spinal cord. Thus, our findings suggest that TNF-c^-fNFR1 signaling is required for the full expression of acute and chronic itch via peripheral and central mechanisms, and targeting TNFR1 may be benefi- cial for chronic itch treatment.

Cardiac electrical and mechanical synchrony of super-responders to cardiac resynchronization therapy

Background:Super-responders(SRs)to cardiac resynchronization therapy(CRT)regain near-normal or normal cardiac function.The extent of cardiac synchrony of SRs and whether continuous biventricular(BIV)pacing is needed remain unknown.The aim of this study was to evaluate the cardiac electrical and mechanical synchrony of SRs.Methods:We retrospectively analyzed CRT recipients between 2008 and 2016 in 2 centers to identify SRs,whose left ventricular(LV)ejection fraction was increased to≥50%at follow-up.Cardiac synchrony was evaluated in intrinsic and BIV-paced rhythms.Electrical synchrony was estimated by QRS duration and LV mechanical synchrony by single-photon emission computed tomography myocardial perfusion imaging.Results:Seventeen SRs were included with LV ejection fraction increased from 33.0±4.6%to 59.3±6.3%.The intrinsic QRS duration after super-response was 148.8±30.0 ms,significantly shorter than baseline(174.8±11.9 ms,P=0.004,t=3.379)but longer than BIV-paced level(135.5±16.7 ms,P=0.042,t=2.211).Intrinsic LV mechanical synchrony significantly improved after super-response(phase standard deviation[PSD],51.1±16.5°vs.19.8±8.1°,P<0.001,t=5.726;phase histogram bandwidth(PHB),171.7±64.2°vs.60.5±22.9°,P<0.001,t=5.376)but was inferior to BIV-paced synchrony(PSD,19.8±8.1°vs.15.2±6.4°,P=0.005,t=3.414;PHB,60.5±22.9°vs.46.0±16.3°,P=0.009,t=3.136).Conclusions:SRs had significant improvements in cardiac electrical and LV mechanical synchrony.Since intrinsic synchrony of SRs was still inferior to BIV-paced rhythm,continued BIV pacing is needed to maintain longstanding and synchronized contraction.

Overexpression of Purinergic P2X4 Receptors in Hippocampus Rescues Memory Impairment in Rats with Type 2 Diabetes

Purinergic receptors have been reported to be involved in brain disorders.In this study,we explored their roles and mechanisms underlying the memory impairment in rats with type 2 diabetes mellitus(T2 DM).T2 DM rats exhibited a worse performance in the T-maze and Morris water maze(MWM) than controls.Microglia positive for P2 X purinoceptor 4(P2 X4 R) in the hippocampus were reduced and activated microglia were increased in T2 DM rats.Long Amplicon PCR(LA-PCR) showed that DNA amplification of the p2 x4 r gene in the hippocampus was lower in T2 DM rats.Minocycline significantly reduced the number of activated microglia and the mean distance traveled by T2 DM rats in the MWM.Most importantly,P2 X4 R overexpression suppressed the activated microglia and rescued the memory impairment of T2 DM rats.Overall,T2 DM led to excessive activation of microglia in the hippocampus,partly through the DNA damagemediated downregulation of P2 X4 Rs,thus contributing to memory impairment.

More than Scratching the Surface:Recent Progress in Brain Mechanisms Underlying Itch and Scratch

Itch(also called pruritus)is an unpleasant somatosensation that evokes a desire or reflex to scratch in humans and other mammals[1],Itch is now considered to be a unique sensory modality that is encoded by a labeled line that has genetically distinguishable neurons in the peripheral and central nervous systems[1],Acute itch evokes scratching that may help to remove potentially harmful irritants from the skin,which is believed to be evolutionarily important for survival.Pathological chronic itch often occurs in patients with inflammatory skin diseases,systemic diseases,and neurological conditions.Importantly,chronic itch causes uncontrollable itch-scratch cycles that induce skin damage,affect sleep,and seriously reduce the quality of life[2].At present,effective treatment for chronic itch is still lacking,possibly due to limited understanding of the mechanism of itch information-processing in the nervous system.Therefore,elucidation of the molecular,cellular,and circuitry mechanisms of itch will eventually help to develop new effective strategies for the management of chronic itch.

Targeting GATA1 and p2x7r Locus Binding in Spinal Astrocytes Suppresses Chronic Visceral Pain by Promoting DNA Demethylation

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread,chronic abdominal pain associated with altered bowel movements.Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases.In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1(GATA1)in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation(NCI).The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay,chromatin immunoprecipitation,patch clamp,and interference in vitro and in vivo.In addition,a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island.We showed that NCI caused the induction of GATA1,Ten-eleven translocation 3(TET3),and purinergic receptors(P2X7Rs)in astrocytes of the spinal dorsal horn,and demonstrated that inhibiting these molecules markedly increased the pain threshold,inhibited the activation of astrocytes,and decreased the spinal sEPSC frequency.NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1–TET3 physical interaction and GATA1 binding at the p2x7r promoter.Importantly,we showed that demethylation of the p2x7r locus(and the attendant increase in P2X7R expression)was reversed upon knockdown of GATA1 or TET3 expression,and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter.These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes,and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.