Role of tumor necrosis factor-alpha in zebrafish retinal neurogenesis and myelination

AIM： To investigate the role of tumor necrosis factoralpha （TNF-α） in zebrafish retinal development and myelination. METHODS： Morpholino oligonucleotides （MO）, which are complementary to the translation start site of the wild-type embryonic zebrafish TNF-α mRNA sequence, were synthesized and injected into one to four-cell embryos. The translation blocking specificity was verified by Western blotting using an anti-TNF-α antibody, whole-mount in sltuhybridization using a hepatocytespecific mRNA probe ceruloplasmin （cp）, and coinjection of TNF-α MO and TNF-α mRNA. An atonel homolog 7 （atoh7） mRNA probe was used to detect neurogenesis onset. The retinal neurodifferentiation was analyzed by immunohistochemistry using antibodies Zn12, Zprl, and Zpr3 to label ganglion cells, cones, and rods, respectively. Myelin basic protein （mbp）was used as a marker to track and observe the myelination using whole-mount in situ hybridization. RESULTS： Targeted knockdown of TNF-α resulted in specific suppression of TNF-α expression and a severely underdeveloped liver. The co-injection of TNF-α MO and mRNA rescued the liver development. Retinal neurogenesis in TNF-cc morphants was initiated on time. The retina was fully laminated, while ganglion cells, cones, and rods were well differentiated at 72 hours post-fertilization （hpf）. mbp was expressed in Schwann cells in the lateral line nerves and cranial nerves from 3 days post -fertilization （dpf） as well as in oligodendrocytes linearly along the hindbrain bundles and the spinal cord from 4 dpf, which closely resembled its endogenous profile. CONCLUSION： TNF-α is not an essential regulator for retinal neurogenesis and optic myelination.

Hypoxia inducible factor 1α promotes interleukin-1 receptor antagonist expression during hepatic ischemia-reperfusion injury

BACKGROUND Ischemia-reperfusion injury(IRI) is a major risk associated with liver surgery and transplantation,and its pathological mechanism is complex.Interleukin-1 receptor antagonist(IL-1ra) can protect the liver from IRI.However,the regulatory mechanism of IL-1ra expression is still unclear.AIM To identify the mechanism that could protect the liver in the early stage of IRI.METHODS To screen the key genes in hepatic IRI,we performed RNA sequencing and gene enrichment analysis on liver tissue from mice with hepatic IRI.Subsequently,we verified the expression and effect of IL-1ra in hepatic IRI.We also used promoter mutagenesis and chromatin immunoprecipitation assay to search for the transcriptional regulatory sites of hypoxia-inducible factor(HIF)-1α.Finally,to explore the protective mechanism of ischemic preconditioning(IP),we examined the expression of HIF-1α and IL-1ra after IP.RESULTS We identified IL-1ra as a key regulator in hepatic IRI.The expression of IL-1ra was significantly upregulated after hepatic IRI both in vivo and in vitro.Furthermore,we found that HIF-1αregulated Il-1ra transcription in response to hypoxia.Increased HIF-1α accumulation promoted IL-1ra expression,whereas inhibition of HIF-1α exhibited the opposite effect.We also confirmed a predominant role for hypoxia response element in the regulation of Il1ra transcription by HIF-1αactivation.Of note,we demonstrated that IP protects against hepatic IRI by inducing IL-1ra expression,which is mediated through HIF-1α.CONCLUSION We demonstrated that ischemia or hypoxia leads to increased expression of IL-1ra through HIF-1α.Importantly,IP protects the liver from IRI via the HIF-1α–IL-1ra pathway.

Rongchang and Xifeng capsules suppress pentylenetetrazole-induced seizures and change rest/wake behavior in zebrafish larvae

OBJECTIVE:To evaluate the effects of Rongchang capsule and Xifeng capsule on pentylenetetrazoleinduced epilepsy in zebrafish larvae and to explore the possible mechanisms behind their actions.METHODS:We utilized a trajectory tracking system to monitor seizures in zebrafish larva to confirm that certain concentrations of Rongchang capsule and Xifeng capsule produce antiepileptic effects.c-fos expression was assessed by quantitative reverse transcription-polymerase chain reaction to validate the efficacy of the capsules.Rest/wake behavior and correlation analysis predicted the targets of Rongchang capsule and Xifeng capsule.RESULTS:Larval movement times and total distances traveled by zebrafish larvae experiencing pentylenetetrazole(PTZ)-induced seizures were decreasedbyvalproatetreatment.Rongchang(500μg/m L)and Xifeng(200μg/m L)rescued the epileptic behaviors and down-regulated c-fos expression in the brains of larvae,which indicated antiepileptic effects.The rest/wake behavioral profiles showed that Rongchang and Xifeng differentially decreased rest time at night and increased larval locomotor activities during the day.Based on correlation between the actions of the two capsules and known compounds,we predicted that they might change rest/wake behaviors by affecting serotonin,GABAergic and histamine signaling pathways.CONCLUSION:The efficacy of Rongchang capsule and Xifeng capsule in alleviating epilepsy-like behaviors and molecular responses was confirmed.Our study provides insight into the capsules'effect on epilepsy.

USP51/PD-L1/ITGB1-deployed juxtacrine interaction plays a cell-intrinsic role in promoting chemoresistant phenotypes in non-small cell lung cancer

Background:Programmed death ligand 1(PD-L1)has been demonstrated to facilitate tumor progression and therapeutic resistance in an immuneindependent manner.Nevertheless,the function and underlying signaling network(s)of cancer cell-intrinsic PD-L1 action remain largely unknown.Herein,we sought to better understand how ubiquitin-specific peptidase 51(USP51)/PD-L1/integrin beta-1(ITGB1)signaling performs a cell-intrinsic role in mediating chemotherapeutic resistance in non-small cell lung cancer(NSCLC).Methods:Western blotting and flow cytometry were employed for PD-L1 detection in NSCLC cell lines.Coimmunoprecipitation and pulldown analyses,protein deubiquitination assay,tissue microarray,bioinformatic analysis and molecular biology methods were then used to determine the significance of PD-L1 in NSCLC chemoresistance and associated signaling pathways in several different cell lines,mouse models and patient tissue samples.Ubiquitin-7-amido-4-methylcoumarin(Ub-AMC)-based deubiquitinase activity,cellular thermal shift and surface plasmon resonance(SPR)analyses were performed to investigate the activity of USP51 inhibitors.Results:We provided evidence that cancer cell-intrinsic PD-L1 conferred the development of chemoresistance by directly binding to its membrane-bound receptor ITGB1 in NSCLC.At the molecular level,PD-L1/ITGB1 interaction subsequently activated the nuclear factor-kappa B(NF-κB)axis to elicit poor response to chemotherapy.We further determined USP51 as a bona fide deubiquitinase that targeted the deubiquitination and stabilization of the PD-L1 protein in chemoresistant NSCLC cells.Clinically,we found a significant direct relationship between the USP51,PD-L1 and ITGB1 contents in NSCLC patients with chemoresistant potency.The elevated USP51,PD-L1 and ITGB1 levels were strongly associated with worse patient prognosis.Of note,we identified that a flavonoid compound dihydromyricetin(DHM)acted as a potential USP51 inhibitor and rendered NSCLC cells more sensitive to chemotherapy by targeting USP51-dependent PD-L1 ubiquitination and degradation in vitro and in vivo.Conclusions:Together,our results demonstrated that the USP51/PD-L1/ITGB1 network potentially contributes to the malignant progression and therapeutic resistance in NSCLC.This knowledge is beneficial to the future design of advanced cancer therapy.

CDK4/6 inhibition blocks cancer metastasis through a USP51-ZEB1-dependent deubiquitination mechanism

Tumor metastasis is the most common cause of cancer-related deaths,yet it remains poorly understood.The transcription factor zinc-finger E-box binding homeobox 1(ZEB1)is involved in the epithelial-to-mesenchymal transition(EMT)and plays a pivotal role in tumor metastasis.However,the underlying mechanisms of the posttranslational modification of ZEB1 remain largely unknown.Herein,we demonstrated that specific inhibition of CDK4/6 was able to block tumor metastasis of breast cancer by destabilizing the ZEB1 protein in vitro and in vivo.Mechanistically,we determined that the deubiquitinase USP51 is a bona fide target of CDK4/6.The phosphorylation and activation of USP51 by CDK4/6 is necessary to deubiquitinate and stabilize ZEB1.Moreover,we found a strong positive correlation between the expression of p-RB(an indicator of CDK4/6 activity),p-USP51 and ZEB1 in metastatic human breast cancer samples.Notably,the high expression of p-RB,p-USP51,and ZEB1 was significantly correlated with a poor clinical outcome.Taken together,our results provide evidence that the CDK4/6-USP51-ZEB1 axis plays a key role in breast cancer metastasis and could be a viable therapeutic target for the treatment of advanced human cancers.