METTL5 promotes cell proliferation,invasion,and migration by up-regulating Toll-like receptor 8 expression in colorectal cancer

BACKGROUND N6-methyladenosine(m6A)modification represents the predominant alteration found in eukaryotic messenger RNA and plays a crucial role in the progression of various tumors.However,despite its significance,the comprehensive investigation of METTL5,a key m6A methyltransferase,in colorectal cancer(CRC)remains limited.AIM To investigate the role of METTL5 in CRC.METHODS We assessed METTL5 expression levels in clinical samples obtained from CRC patients as well as in CRC cell lines.To elucidate the downstream targets of METTL5,we performed RNA-sequencing analysis coupled with correlation analysis,leading us to identify Toll-like receptor 8(TLR8)as a potential downstream target.In vitro functional assessments of METTL5 and TLR8 were conducted using CCK-8 assays,scratch assays,as well as assays measuring cell migration and invasion.RESULTS Our findings reveal a pronounced upregulation of METTL5 expression in both CRC cells and tissues,which correlated significantly with an unfavorable prognosis.In vitro experiments unequivocally demonstrated the oncogenic role of METTL5,as evidenced by its promotion of CRC cell proliferation,invasion,and migration.Notably,we identified TLR8 as a downstream target of METTL5,and subsequent down-regulation of TLR8 led to a significant inhibition of CRC cell proliferation,invasion,and tumor growth.CONCLUSION The heightened expression of METTL5 in CRC is strongly associated with clinicopathological features and a poor prognosis,thereby underscoring its potential utility as a critical marker for facilitating early diagnosis and prognostication in CRC.

Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

The noble gas argon has the potential to protect neuronal cells from cell death.So far,this effect has been studied in treatment after acute damage.Preconditioning using argon has not yet been investigated.In this study,human neuroblastoma SH-SY5Y cells were treated with different concentrations of argon(25%,50%,and 74%;21%O_(2),5%CO_(2),balance nitrogen)at different time intervals before inflicting damage with rotenone(20μM,4 hours).Apoptosis was determined by flow cytometry after annexin V and propidium iodide staining.Surface expressions of Toll-like receptors 2 and 4 were also examined.Cells were also processed for analysis by western blot and qPCR to determine the expression of apoptotic and inflammatory proteins,such as extracellular-signal regulated kinase(ERK1/2),nuclear transcription factor-κB(NF-κB),protein kinase B(Akt),caspase-3,Bax,Bcl-2,interleukin-8,and heat shock proteins.Immunohistochemical staining was performed for TLR2 and 4 and interleukin-8.Cells were also pretreated with OxPAPC,an antagonist of TLR2 and 4 to elucidate the molecular mechanism.Results showed that argon preconditioning before rotenone application caused a dose-dependent but not a time-dependent reduction in the number of apoptotic cells.Preconditioning with 74%argon for 2 hours was used for further experiments showing the most promising results.Argon decreased the surface expression of TLR2 and 4,whereas OxPAPC treatment partially abolished the protective effect of argon.Argon increased phosphorylation of ERK1/2 but decreased NF-κB and Akt.Preconditioning inhibited mitochondrial apoptosis and the heat shock response.Argon also suppressed the expression of the pro-inflammatory cytokine interleukin-8.Immunohistochemistry confirmed the alteration of TLRs and interleukin-8.OxPAPC reversed the argon effect on ERK1/2,Bax,Bcl-2,caspase-3,and interleukin-8 expression,but not on NF-κB and the heat shock proteins.Taken together,argon preconditioning protects against apoptosis of neuronal cells and mediates its action via Toll-like receptors.Argon may represent a promising therapeutic alternative in various clinical settings,such as the treatment of stroke.

TLR7/8 signalling affects X-sperm motility via the GSK3α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos

Background:Goat milk is very similar to human milk in terms of its abundant nutrients and ease of digestion.To derive greater economic benefit,farmers require more female offspring(does);however,the buck-to-doe offspring sex ratio is approximately 50%.At present,artificial insemination after the separation of X/Y sperm using flow cytometry is the primary means of controlling the sex of livestock offspring.However,flow cytometry has not been successfully utilised for the separation of X/Y sperm aimed at sexing control in dairy goats.Results:In this study,a novel,simple goat sperm sexing technology that activates the toll-like receptor 7/8(TLR7/8),thereby inhibiting X-sperm motility,was investigated.Our results showed that the TLR7/8 coding goat Xchromosome was expressed in approximately 50%of round spermatids in the testis and sperm,as measured from cross-sections of the epididymis and ejaculate,respectively.Importantly,TLR7/8 was located at the tail of the Xsperm.Upon TLR7/8 activation,phosphorylated forms of glycogen synthase kinaseα/β(GSK3α/β)and nuclear factor kappa-B(NF-κB)were detected in the X-sperm,causing reduced mitochondrial activity,ATP levels,and sperm motility.High-motility Y-sperm segregated to the upper layer and the low-motility X-sperm,to the lower layer.Following in vitro fertilisation using the TLR7/8-activated sperm from the lower layer,80.52±6.75%of the embryos were XX females.The TLR7/8-activated sperm were subsequently used for in vivo embryo production via the superovulatory response;nine embryos were collected from the uterus of two does that conceived.Eight of these were XX embryos,and one was an XY embryo.Conclusions:Our study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3α/β-hexokinase pathway;this technique could be used to facilitate the efficient production of sexed dairy goat embryos.

Transforming growth factor-β and toll-like receptor-4 polymorphisms are not associated with fibrosis in haemochromatosis

AIM:To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis.METHODS:A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was studied,with all subjects having liver biopsy data and DNA available for testing.This study assessed the association of eight single nucleotide polymorphisms(SNPs)in a total of six genes including toll-like receptor 4(TLR4),transforming growth factor-beta(TGF-β),oxoguanine DNA glycosylase,monocyte chemoattractant protein 1,chemokine C-C motif receptor 2 and interleukin-10 with liver disease severity.Genotyping was performed using high resolution melt analysis and sequencing.The results were analysed in relation to the stage of hepatic fibrosis in multivariate analysis incorporating other cofactors including alcohol consumption and hepatic iron concentration.RESULTS:There were significant associations between the cofactors of male gender(P=0.0001),increasing age(P=0.006),alcohol consumption(P=0.0001),steatosis(P=0.03),hepatic iron concentration(P<0.0001)and the presence of hepatic fibrosis.Of the candidate gene polymorphisms studied,none showed a significant association with hepatic fibrosis in univariate or multivariate analysis incorporating cofactors.We also specifically studied patients with hepatic iron loading above threshold levels for cirrhosis and compared the genetic polymorphisms between those with no fibrosis vs cirrhosis however there was no significant effect from any of the candidate genes studied.Importantly,in this large,well characterised cohort of patients there was no association between SNPs for TGF-βor TLR4and the presence of fibrosis,cirrhosis or increasing fibrosis stage in multivariate analysis.CONCLUSION:In our large,well characterised group of haemochromatosis subjects we did not demonstrate any relationship between candidate gene polymorphisms and hepatic fibrosis or cirrhosis.

Recombinant E.coli LLO/OVA Induces Murine BMDCs Maturation via TLR4 and NOD1 Receptor and Promotes Specific Cytotoxic T Cell Immunity

Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells （BMDCs） and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their Toll-like receptor （TLR） and nucleotide-binding oligomerization domain （NOD） receptor signalling pathway were examined by superarray hybridization;and the priming effect of the vaccine activated BMDCs on CD4＋T and CD8＋T was determined by [3H]thymidine uptake and ELISA,the tumor cytotoxic effect of activated CD8＋T cells was determined by cytotoxic assay.Results After BMDCs were activated by E.coli LLO/OVA via TLR4,NOD1 receptor and NF-κB signalling pathway,the expression of their surface molecules including MHC class Ⅰ,MHC class Ⅱ,CD40,CD80 and CD86 significantly up-regulated;the secretion of IL-12 and IFN-？ increased also.The mature BMDCs stimulated the allergic CD4＋T and CD8＋T cells proliferation and their IL-2 and IFN-γ secretion,and the activated CD8＋T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.Conclusion E.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.

Upregulated expression of S100A8 in mice brain after focal cerebral ischemia reperfusion

BACKGROUND:Recent studies have showed that S100A8 has been implicated in the pathobiology of inflammatory disorders,and that cerebral ischemia reperfusion(l/R) rapidly activates inflammation responses via Toll-like receptor 4(TLR4).This study aimed to explore the expression of S100A8 and the relationship between S100A8 and TLR4 in focal cerebral ischemia reperfusion injury.METHODS:C3H/HeJ mice(n=30) and C3H/HeN mice(n=30) were divided randomly into a C3H/HeJ model group(n=18),a C3H/HeJ control group(n=12),a C3H/HeN model group(n=18),and a C3H/HeN control group(n=12).Middle cerebral artery l/R model in mice was produced using a thread embolism method.The brains of the mice were collected after ischemia for 1 hour and reperfusion for 12 hours.Stroke outcome was evaluated by determination of infarct volume and assessment of neurological impairment scores.Brain injury after cerebral l/R was observed by an optical microscope after TTC and HE dyeing.The immunofluorescence technique and real time PCR were used to test the expression level of S100A8 in brain damage.RESULTS:Compared with C3H/HeN mice,TLR4-deficient mice(C3H/HeJ) had lower infarct volumes and better outcomes in neurological tests.The levels of S100A8 increased sharply in the brains of mice after l/R injury.In addition,mice that lacked TLR4(C3H/HeJ) had lower expression of l/R-induced S100A8 than C3H/HeN mice in the model group,indicating that a close relationship might exist between the levels of S100A8 and TLR4.CONCLUSION:S100A8 interaction with TLR4 might be involved in brain damage and in inflammation triggered by l/R injury.

The IL-1R/TLR signaling pathway is essential for efficient CD8+ T-cell responses against hepatitis B virus in the hydrodynamic injection mouse model

The outcome of hepatitis B viral(HBV)infection is determined by the complex interactions between replicating HBV and the immune system.While the role of the adaptive immune system in the resolution of HBV infection has been studied extensively,the contribution of innate immune mechanisms remains to be defined.Here we examined the role of the interleukin-1 receptor/Toll-like receptor(IL-1R/TLR)signaling pathway in adaptive immune responses and viral clearance by exploring the HBV mouse model.Hydrodynamic injection with a replication-competent HBV genome was performed in wild-type mice(WT)and a panel of mouse strains lacking specific innate immunity component expression.We found higher levels of HBV protein production and replication in Tlr2^(−/−),Tlr23479^(−/−),3d/Tlr24^(−/−),Myd88/Trif^(−/−)and Irak4^(−/−)mice,which was associated with reduced HBV-specific CD8+T-cell responses in these mice.Importantly,HBV clearance was delayed for more than 2 weeks in 3d/Tlr24^(−/−),Myd88/Trif^(−/−)and Irak4^(−/−)mice compared to WT mice.HBV-specific CD8+T-cell responses were functionally impaired for producing the cytokines IFN-γ,TNF-αand IL-2 in TLR signaling-deficient mice compared to WT mice.In conclusion,the IL-1R/TLR signaling pathway might contribute to controlling HBV infection by augmenting HBV-specific CD8+T-cell responses.

Total glucosides of paeony improve complete freund’s adjuvant-induced rheumatoid arthritis in rats by inhibiting toll-like receptor 2-mediated tumor necrosis factor receptor-associated factor 6/nuclear factor-kappa B pathway activation

OBJECTIVE: To investigate the mechanism underlying anti-inflammatory and immunoregulatory effect of total glucosides of paeony(TGP) based on toll-like receptor 2(TLR2) mediated tumor necrosis factor(TNF) receptor-associated factor 6(TRAF6)/nuclear factor-kappa B(NF-κB) pathway activation in rats with rheumatoid arthritis.METHODS: Adjuvant arthritis(AA) model was developed by complete freund’s adjuvant(CFA) immunization. TGP(100, 50, 25 mg/kg) and celecoxib(2.8 mg/kg) were administered by intragastric administration for 21 d. Right hind paw swelling was assessed every 2 d. After 21 d, synovial changes of the ankle were detected by histopathology. CD4+and CD8+ T cell amounts in peripheral blood were measured by flow-cytometrically. Gene and protein levels of toll-like receptor(TLR)2, TRAF6, tumor necrosis factor ligand superfamily member 6(FASLG)in the spleen were assessed by RT-qPCR and Western Bolt, respectively. Nuclear expression of NF-κB p65 was detected by NF-κB p65 Assay Kit.RESULTS: Paw swelling and synovium lesions were obviously aggravated in AA rats. These symptoms were significantly relieved by TGP.The ratio of CD4+/CD8+ T cell was increased in AA rats, while TGP reduced this increased ratio.Gene and protein levels of splenic TLR2, TFAR6 and FASLG, and nuclear NF-κB p65 in AA rats were significantly increased, but overtly inhibited by TGP.CONCLUSION: These findings suggest that TGP’s anti-inflammatory effect onRA in rats with CFA may be related to the downregulation of TLR2/TRAF6/NF-κB pathway and the regulation of T cell subsets.

Intervention effects and related mechanisms of glycyrrhizic acid on zebrafish with Hirschsprung-associated enterocolitis

BACKGROUND The prevention and treatment of Hirschsprung-associated enterocolitis(HAEC)is a serious challenge in pediatric surgery.Exploring the mechanism of HAEC is conducive to the prevention of this disease.AIM To explore the possible mechanism of glycyrrhizic acid(GA)and its therapeutic effect on HAEC.METHODS We developed a model of enteritis induced by trinitrobenzenesulfonic acid(TNBS)in zebrafish,and treated it with different concentrations of GA.We analyzed the effect of GA on the phenotype and inflammation of zebrafish.RESULTS After treatment with TNBS,the area of the intestinal lumen in zebrafish was significantly increased,but the number of goblet cells in the intestinal lumen was significantly reduced,but these did not increase the mortality of zebrafish,indicating that the zebrafish enteritis model was successfully developed.Different concentrations of GA protected zebrafish with enteritis.In particular,high concentrations of GA were important for the prevention and control of HAEC because it significantly reduced the intestinal luminal area,increased the number of goblet cells in the intestinal lumen,and reduced the levels of interleukin(IL)-1βand IL-8.CONCLUSION GA significantly reduced the intestinal luminal area,increased the number of intestinal goblet cells,and decreased IL-1βand IL-8 in zebrafish,and is important for prevention and control of HAEC.

STING and TLR7/8 agonists-based nanovaccines for synergistic antitumor immune activation

Immunostimulatory therapies based on pattern recognition receptors(PRRs)have emerged as an effective approach in the fight against cancer,with the ability to recruit tumor-specific lymphocytes in a low-immunogenicity tumor environment.The agonist cyclic dinucleotides(CDNs)of the stimulator of interferon gene(STING)are a group of very promising anticancer molecules that increase tumor immunogenicity by activating innate immunity.However,the tumor immune efficacy of CDNs is limited by several factors,including relatively narrow cytokine production,inefficient delivery to STING,and rapid clearance.In addition,a single adjuvant molecule is unable to elicit a broad cytokine response and thus cannot further amplify the anticancer effect.To address this problem,two or more agonist molecules are often used together to synergistically enhance immune efficacy.In this work,we found that a combination of the STING agonist CDGSF and the Toll-like receptor 7/8(TLR7/8)agonist 522 produced a broader cytokine response.Subsequently,we developed multicomponent nanovaccines(MCNVs)consisting of a PC7A polymer as a nanocarrier encapsulating the antigen OVA and adjuvant molecules.These MCNVs activate bone marrow-derived dendritic cells(BMDCs)to produce multiple proinflammatory factors that promote antigen cross-presentation to stimulate specific antitumor Tcell responses.In in vivo experiments,we observed that MCNVs triggered a strong T-cell response in tumor-infiltrating lymphocytes,resulting in significant tumor regression and,notably,a 100%survival rate in mice through 25 days without other partnering therapies.These data suggest that our nanovaccines have great potential to advance cancer immunotherapy with increased durability and potency.

Toll样受体在U937细胞的表达及其作用研究

本研究探讨急性髓系白血病的免疫治疗中以Toll样受体(TLRs)为靶点的可能性,研究人急性髓系白血病U937细胞TLR的表达及TLR8受体激动剂ssRNA40/LyoVec对其增殖、凋亡和细胞周期的影响。运用逆转录-聚合酶链反应(RT-PCR)检测U937细胞TLR1-9mRNA的表达,用流式细胞术检测TLR8的表达。用不同浓度的TLR8激动剂ssRNA40/LyoVec作用于体外培养的U937细胞后,采用CCK-8法检测细胞生长抑制率,流式细胞术检测细胞凋亡和细胞周期。结果表明:U937细胞表达TLR1-9,TLR8激动剂ssRNA40/LyoVec作用于U937细胞明显地抑制了U937细胞生长,抑制率可达70%,且呈明显的量效关系;作用后处于G0/G1期细胞比例由(44.67±1.05)%增高到(54.08±1.19)%,但凋亡细胞的比例无明显变化。结论:TLR1-9可在U937细胞中表达,TLR8激动剂ssRNA40/LyoVec具有抗肿瘤细胞增殖的作用,使细胞阻滞在G0/G1期,但无明显的促凋亡作用。

内毒素作用中的蛋白质和信号通路

综述内毒素作用中有关的蛋白及其传导通路。脂多糖结合蛋白 (LBP)可以结合内毒素并把它传递给受体 ,如CD14和CD11/CD18。Toll样受体 (TLRs)的功能与信号向胞内传递有密切的关系 ,清道夫受体则与肝脏清除内毒素的功能有关。胞内信号转导涉及多种途径 。

Carcinogenesis of nasopharyngeal carcinoma:an alternate hypothetical mechanism

Current proposed mechanisms implicate both early and latent Epstein-Barr virus(EBV)infection in the carcinogenic cascade,whereas epidemiological studies have always associated nasopharyngeal carcinoma(NPC)with early childhood EBV infection and with chronic ear,nose,and sinus conditions.Moreover,most patients with NPC present with IgA antibody titers to EBV capsid antigen(VCA-lgA),which can precede actual tumor presentation by several years.If early childhood EBV infection indeed constitutes a key event in NPC carcinogenesis,one would have to explain the inability to detect the virus in normal nasopharyngeal epithelium of patients at a high risk for EBV infection.It is perhaps possible that EBV resides within the salivary glands,instead of the epithelium,during latency.This claim is indirectly supported by observations that the East Asian phenotype shares the characteristics of an increased susceptibility to NPC and immature salivary gland morphogenesis,the latter of which is influenced by the association of salivary gland morphogenesis with an evolutionary variant of the human ectodysplasin receptor gene(EDAR),EDARV370A.Whether the immature salivary gland represents a more favorable nidus for EBV is uncertain,but in patients with infectious mononucleosis,EBV has been isolated in this anatomical organ.The presence of EBV-induced lymphoepitheliomas in the salivary glands and lungs further addresses the possibility of submucosal spread of the virus.Adding to the fact that the fossa of Rosen Muller contains a transformative zone active only in the first decade of life,one might be tempted to speculate the possibility of an alternative carcinogenic cascade for NPC that is perhaps not dissimilar to the model of human papillomavirus and cervical cancer.

Therapeutic effect of Qinghuayin(清化饮)against chronic atrophic gastritis through the inhibition of toll or interleukin-1 receptor domain-containing adaptor inducing interferon-β signaling pathway

OBJECTIVE:To examine the efficacy of Qinghuayin(清化饮,QHY)in rat chronic atrophic gastritis(CAG)models and explored the molecular mechanism of QHY in treating CAG.METHODS:In total,65 Wistar rats were randomly divided into the control(n=10)and CAG groups(n=55).CAG model rats were further divided into five groups:model(n=10),vitacoenzyme(n=10),low-dose QHY(n=10),medium-dose QHY(n=10),and high-dose QHY groups(n=10).We analyzed histopathological changes using hematoxylin and eosin staining and measured interleukin(IL)-6 and IL-8 levels in serum using enzyme-linked immunosorbent assay(ELISA)(Boster Bio,Pleasanton,USA).In addition,gastrin(GAS),pepsinogen I(PGI),and PGII expressions were evaluated using ELISA.The protein and m RNA expression of toll-like receptor 4(TLR4)and toll or interleukin-1 receptor domaincontaining adaptor inducing interferon-β(TRIF)was detected by Western blotting and quantitative reverse transcription-polymerase chain reaction,respectively.RESULTS:Our results revealed that histopathological changes in CAG model rates could be restored by low-,medium-,and high-dose QHY.The changes in GAS and PGI/II expression demonstrated that QHY improved CAG.Serum IL-6 and IL-levels were decreased by QHY administration.TLR4 and TRIF were upregulated at the m RNA and protein levels in the model group but downregulated by QHY administration.CONCLUSION:We concluded that QHY could effectively improve the histopathological changes of the gastric mucosa induced by CAG in rats.The therapeutic mechanism of QHY may be related to inhibition of the inflammatory factors IL-6 and IL-8 and suppression of TLR4/TRIF m RNA and protein expression.