MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4

BACKGROUND Diabetic kidney disease(DKD)is a major complication of diabetes mellitus.Renal tubular epithelial cell(TEC)damage,which is strongly associated with the inflammatory response and mesenchymal trans-differentiation,plays a significant role in DKD;However,the precise molecular mechanism is unknown.The recently identified microRNA-630(miR-630)has been hypothesized to be closely associated with cell migration,apoptosis,and autophagy.However,the association between miR-630 and DKD and the underlying mechanism remain unknown.AIM To investigate how miR-630 affects TEC injury and the inflammatory response in DKD rats.METHODS Streptozotocin was administered to six-week-old male rats to create a hypergly cemic diabetic model.In the second week of modeling,the rats were divided into control,DKD,negative control of lentivirus,and miR-630 overexpression groups.After 8 wk,urine and blood samples were collected for the kidney injury assays,and renal tissues were removed for further molecular assays.The target gene for miR-630 was predicted using bioinformatics,and the association between miR-630 and toll-like receptor 4(TLR4)was confirmed using in vitro investigations and double luciferase reporter gene assays.Overexpression of miR-630 in DKD rats led to changes in body weight,renal weight index,basic blood parameters and histopathological changes.RESULTS The expression level of miR-630 was reduced in the kidney tissue of rats with DKD(P<0.05).The miR-630 and TLR4 expressions in rat renal TECs(NRK-52E)were measured using quantitative reverse transcription polymerase chain reaction.The mRNA expression level of miR-630 was significantly lower in the high-glucose(HG)and HG+mimic negative control(NC)groups than in the normal glucose(NG)group(P<0.05).In contrast,the mRNA expression level of TLR4 was significantly higher in these groups(P<0.05).However,miR-630 mRNA expression increased and TLR4 mRNA expression significantly decreased in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Furthermore,the levels of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β),and IL-6 were significantly higher in the HG and HG+mimic NC groups than in NG group(P<0.05).However,the levels of these cytokines were significantly lower in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Notably,changes in protein expression were observed.The HG and HG+mimic NC groups showed a significant decrease in E-cadherin protein expression,whereas TLR4,α-smooth muscle actin(SMA),and collagen IV protein expression increased(P<0.05).Conversely,the HG+miR-630 mimic group exhibited a significant increase in E-cadherin protein expression and a notable decrease in TLR4,α-SMA,and collagen IV protein expression than in the HG+mimic NC group(P<0.05).The miR-630 targets TLR4 gene expression.In vivo experiments demonstrated that DKD rats treated with miR-630 agomir exhibited significantly higher miR-630 mRNA expression than DKD rats injected with agomir NC.Additionally,rats treated with miR-630 agomir showed significant reductions in urinary albumin,blood glucose,TLR4,and proinflammatory markers(TNF-α,IL-1β,and IL-6)expression levels(P<0.05).Moreover,these rats exhibited fewer kidney lesions and reduced infiltration of inflammatory cells.CONCLUSION MiR-630 may inhibit the inflammatory reaction of DKD by targeting TLR4,and has a protective effect on DKD.

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.

Toll-like receptors 2 polymorphism is associated with psoriasis: A case-control study in the northern Chinese population

Background:Psoriasis is a disease caused by genetics and immune system dysfunction,affecting the skin and joints.Toll-like receptors(TLRs)play an important role in triggering the innate immune response and controlling adaptive immunity.The role of TLR2 in the progression of psoriasis is not well understood.Methods:A case-control study was conducted on a northern Chinese Han population,consisting of psoriasis patients and healthy control subjects.Genotyping was performed using the tetra-primer amplification refractory mutation system-polymerase chain reaction(ARMS-PCR),and allele and genotype frequencies of four SNPs in TLR2 were analyzed in 270 psoriasis patients and 246 healthy controls.Results:Four TLR2 SNPs(rs11938228,rs4696480,rs3804099,rs5743699)were genotyped and found to be in linkage disequilibrium.The genotype distributions of rs11938228 and rs4696480 in two groups were in Hardy-Weinberg equilibrium and statistically significant except for the overdominance model.The haplotypes ATTC and ATCC were found to be protective against psoriasis.Conclusion:Our study found a correlation between TLR2 genetic variations and the likelihood of psoriasis in northern China.

On the Impairment of Stress-Induced Changes in Triglyceride Levels via a Sub-Toxic Dose of Unmethylated Cytidine Phosphate Guanosine Oligodinucleotide (a Toll-Like Receptor 9 Ligand)

Changes in lipid metabolism have been implicated in protection against infectious diseases. In the first experiment of this study, we measured clinical lipid parameters in a murine model where the unmethylated cytidine phosphate guanosine (CpG) oligodinucleotide (ODN1826), a Toll-like receptor 9 (TLR9) agonist was administered in combination with D-galactosamine (GalN) that caused relatively liver-specific inflammation and toxicity. In the control mice group injected with phosphate-buffered saline (PBS) (acute psychological stress model associated with blood sampling), the serum triglyceride (TG) levels showed a rapid decrease followed by a rebound at 24 h as we have recently reported. However, such a TG rebound was impaired in the CpG/GalN- and solely CpG-treated groups of mice despite an absence of liver injury based on serum alanine aminotransferase levels in the latter group. Thus, the stress-associated serum TG rebound was abrogated by the injection of a sub-hepatotoxic CpG dose. In the second experiment, we simply measured the hepatic CD36 and SACRB1 (the gene for scavenger receptor B1 (SR-B1)) transcripts after the i.p. administration of PBS, CpG or CpG/GalN. There was a remarkable elevation of hepatic CD36 transcript expression in both the CpG- and CpG/GalN-treated mice at 8 h post-CpG injection whereas the increase in the PBS-treated mice was slower than the former two groups, suggesting that hepatic CD36 transcript expression is more pronounced in the combined stress models than under psychological stress alone. The individual mice data showed that the increase in CD36 expression was accompanied by a reduction in SCARB1 mRNA, showing reciprocal regulation between these two genes. Together with our previously reported findings, these data suggest that, in a murine model combining psychological stress with TLR-triggered hepatic inflammation, the psychological stress facilitates liver uptake of plasma TG (and its components fatty acids), but the subsequent re-esterification and/or release of TG-rich lipoproteins from the liver is impaired due to the concomitant TLR-signaling. We hypothesize that lipid metabolism during acute stress shifts toward an elevated hepatic uptake of lipids due to concomitant TLR signaling, facilitating the clearance of bacterial lipids by the liver.

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.

Melanocortin 3,5 receptors immunohistochemical expression in colonic mucosa of inflammatory bowel disease patients:A matter of disease activity?

BACKGROUND Melanocortin 3 and 5 receptors(i.e.,MC3R and MC5R)belong to the melanocortin family.However,data regarding their role in inflammatory bowel diseases(IBD)are currently unavailable.AIM This study aims to ascertain their expression profiles in the colonic mucosa of Crohn’s disease(CD)and ulcerative colitis(UC),aligning them with IBD disease endoscopic and histologic activity.METHODS Colonic mucosal biopsies from CD/UC patients were sampled,and immunohisto-chemical analyses were conducted to evaluate the expression of MC3R and MC5R.Colonic sampling was performed on both traits with endoscopic scores(Mayo endoscopic score and CD endoscopic index of severity)consistent with inflamed mucosa and not consistent with disease activity(i.e.,normal appearing mucosa).RESULTS In both CD and UC inflamed mucosa,MC3R(CD:+7.7 fold vs normal mucosa,P<0.01;UC:+12 fold vs normal mucosa,P<0.01)and MC5R(CD:+5.5 fold vs normal mucosa,P<0.01;UC:+8.1 fold vs normal mucosa,P<0.01)were significantly more expressed compared to normal mucosa.CONCLUSION MC3R and MC5R are expressed in the colon of IBD patients.Furthermore,expression may differ according to disease endoscopic activity,with a higher degree of expression in the traits affected by disease activity in both CD and UC,suggesting a potential use of these receptors in IBD pharmacology.

STAT3-Dependent Effects of Polymeric Immunoglobulin Receptor in Regulating Interleukin-17 Signaling and Preventing Autoimmune Hepatitis

One-third of patients with autoimmune hepatitis(AIH)have cirrhosis at the time of diagnosis.The relevance of these variables,although unknown,is believed to be critical in AIH because of suspected interactions between the gut microbiome and genetic factors.Dysbiosis of the gut flora and elevated polymeric immunoglobulin receptor(pIgR)levels have been observed in both patients and mouse models.Moreover,there is a direct relationship between pIgR expression and transaminase levels in patients with AIH.In this study,we aimed to explore how pIgR influences the secretion of regenerating islet-derived 3 beta(Reg3b)and the flora composition in AIH using in vivo experiments involving patients with AIH and a concanavalin A-induced mouse model of AIH.Reg3b expression was reduced in pIgR gene(Pigr)-knockout mice compared to that in wild-type mice,leading to increased microbiota disruption.Conversely,exogenous pIgR supplementation increased Reg3b expression and maintained microbiota homeostasis.RNA sequencing revealed the participation of the interleukin(IL)-17 signaling pathway in the regulation of Reg3b through pIgR.Furthermore,the introduction of external pIgR could not restore the imbalance in gut microbiota in AIH,and the decrease in Reg3b expression was not apparent following the inhibition of signal transducer and activator of transcription 3(STAT3).In this study,pIgR facilitated the upregulation of Reg3b via the STAT3 pathway,which plays a crucial role in preserving the balance of the intestinal microbiota in AIH.Through this research,we discovered new molecular targets that can be used for the diagnosis and treatment of AIH.

Overexpression of low-density lipoprotein receptor prevents neurotoxic polarization of astrocytes via inhibiting NLRP3 inflammasome activation in experimental ischemic stroke

Neurotoxic astrocytes are a promising therapeutic target for the attenuation of cerebral ischemia/reperfusion injury.Low-density lipoprotein receptor,a classic cholesterol regulatory receptor,has been found to inhibit NLR family pyrin domain containing protein 3(NLRP3)inflammasome activation in neurons following ischemic stroke and to suppress the activation of microglia and astrocytes in individuals with Alzheimer’s disease.However,little is known about the effects of low-density lipoprotein receptor on astrocytic activation in ischemic stroke.To address this issue in the present study,we examined the mechanisms by which low-density lipoprotein receptor regulates astrocytic polarization in ischemic stroke models.First,we examined low-density lipoprotein receptor expression in astrocytes via immunofluorescence staining and western blotting analysis.We observed significant downregulation of low-density lipoprotein receptor following middle cerebral artery occlusion reperfusion and oxygen-glucose deprivation/reoxygenation.Second,we induced the astrocyte-specific overexpression of low-density lipoprotein receptor using astrocyte-specific adeno-associated virus.Low-density lipoprotein receptor overexpression in astrocytes improved neurological outcomes in middle cerebral artery occlusion mice and reversed neurotoxic astrocytes to create a neuroprotective phenotype.Finally,we found that the overexpression of low-density lipoprotein receptor inhibited NLRP3 inflammasome activation in oxygen-glucose deprivation/reoxygenation injured astrocytes and that the addition of nigericin,an NLRP3 agonist,restored the neurotoxic astrocyte phenotype.These findings suggest that low-density lipoprotein receptor could inhibit the NLRP3-meidiated neurotoxic polarization of astrocytes and that increasing low-density lipoprotein receptor in astrocytes might represent a novel strategy for treating cerebral ischemic stroke.

Diabetic cardiomyopathy:Importance of direct evidence to support the roles of NOD-like receptor protein 3 inflammasome and pyroptosis

Recently,the roles of pyroptosis,a form of cell death induced by activated NODlike receptor protein 3(NLRP3)inflammasome,in the pathogenesis of diabetic cardiomyopathy(DCM)have been extensively investigated.However,most studies have focused mainly on whether diabetes increases the NLRP3 inflammasome and associated pyroptosis in the heart of type 1 or type 2 diabetic rodent models,and whether various medications and natural products prevent the development of DCM,associated with decreased levels of cardiac NLRP3 inflammasome and pyroptosis.The direct link of NLRP3 inflammasome and associated pyroptosis to the pathogenesis of DCM remains unclear based on the limited evidence derived from the available studies,with the approaches of NLRP3 gene silencing or pharmaceutical application of NLRP3 specific inhibitors.We thus emphasize the requirement for more systematic studies that are designed to provide direct evidence to support the link,given that several studies have provided both direct and indirect evidence under specific conditions.This editorial emphasizes that the current investigation should be circumspect in its conclusion,i.e.,not overemphasizing its role in the pathogenesis of DCM with the fact of only significantly increased expression or activation of NLRP3 inflammasome and pyroptosis in the heart of diabetic rodent models.Only clear-cut evidence-based causative roles of NLRP3 inflammasome and pyroptosis in the pathogenesis of DCM can help to develop effective and safe medications for the clinical management of DCM,targeting these biomarkers.

Cortico-striatal gamma oscillations are modulated by dopamine D3 receptors in dyskinetic rats

Long-term levodopa administration can lead to the development of levodopa-induced dyskinesia.Gamma oscillations are a widely recognized hallmark of abnormal neural electrical activity in levodopa-induced dyskinesia.Currently,studies have reported increased oscillation power in cases of levodopa-induced dyskinesia.However,little is known about how the other electrophysiological parameters of gamma oscillations are altered in levodopa-induced dyskinesia.Furthermore,the role of the dopamine D3 receptor,which is implicated in levodopa-induced dyskinesia,in movement disorder-related changes in neural oscillations is unclear.We found that the cortico-striatal functional connectivity of beta oscillations was enhanced in a model of Parkinson’s disease.Furthermore,levodopa application enhanced cortical gamma oscillations in cortico-striatal projections and cortical gamma aperiodic components,as well as bidirectional primary motor cortex(M1)↔dorsolateral striatum gamma flow.Administration of PD128907(a selective dopamine D3 receptor agonist)induced dyskinesia and excessive gamma oscillations with a bidirectional M1↔dorsolateral striatum flow.However,administration of PG01037(a selective dopamine D3 receptor antagonist)attenuated dyskinesia,suppressed gamma oscillations and cortical gamma aperiodic components,and decreased gamma causality in the M1→dorsolateral striatum direction.These findings suggest that the dopamine D3 receptor plays a role in dyskinesia-related oscillatory activity,and that it has potential as a therapeutic target for levodopa-induced dyskinesia.

Correlation between Toll-like Receptor Gene Polymorphisms and Idiopathic Nephrotic Syndrome in Chinese Children

Objective Idiopathic nephrotic syndrome(INS)is the most common glomerular disease in children.Toll-like receptors(TLRs)have been reported to be associated with response to steroid treatment in children with INS.Nevertheless,the correlation between TLR genes and the progression of INS has not yet been clarified.The present study aimed to investigate the association of single-nucleotide polymorphisms(SNPs)in TLR2,TLR4,and TLR9 with susceptibility to INS as well as the clinical phenotyping of steroid responsiveness in Chinese children with INS.Methods A total of 183 pediatric inpatients with INS were included and given standard steroid therapy.Based on their clinical response to steroids,the patients were classified into three groups:steroid-sensitive nephrotic syndrome(SSNS),steroid-dependent nephrotic syndrome(SDNS),and steroid-resistant nephrotic syndrome(SRNS).A total of 100 healthy children were employed as controls.The blood genome DNA was extracted from each participant.Six SNPs(rs11536889,rs1927914,rs7869402,rs11536891,rs352140,and rs3804099)in TLR2,TLR4,and TLR9 were selected and detected by multiplex polymerase chain reaction with next-generation sequencing to assess TLR gene polymorphisms.Results Among the 183 patients with INS,89(48.6%)had SSNS,73(39.9%)had SDNS,and 21(11.5%)had SRNS.No significant difference was found in the genotype distribution between healthy children and patients with INS.However,the genotype and allele frequencies of TLR4 rs7869402 were significantly different between SRNS and SSNS.Compared with patients with the C allele and CC genotype,patients with the T allele and CT genotype had an increased risk of SRNS.Conclusion TLR4 rs7869402 affected the steroid response in Chinese children with INS.It might be a predictor for the early detection of SRNS in this population.

Fecal microbiota transplantation alleviates experimental colitis through the Toll-like receptor 4 signaling pathway

BACKGROUND Fecal microbiota transplantation(FMT)has shown promising therapeutic effects on mice with experimental colitis and patients with ulcerative colitis(UC).FMT modulates the Toll-like receptor 4(TLR4)signaling pathway to treat some other diseases.However,it remains unknown whether this modulation is also involved in the treatment of UC.AIM To clarify the necessity of TLR4 signaling pathway in FMT on dextran sodium sulphate(DSS)-induced mice and explain the mechanism of FMT on UC,through association analysis of gut microbiota with colon transcriptome in mice.METHODS A mouse colitis model was constructed with wild-type(WT)and TLR4-knockout(KO)mice.Fecal microbiota was transplanted by gavage.Colon inflammation severity was measured by disease activity index(DAI)scoring and hematoxylin and eosin staining.Gut microbiota structure was analyzed through 16S ribosomal RNA sequencing.Gene expression in the mouse colon was obtained by transcriptome sequencing.RESULTS The KO(DSS+Water)and KO(DSS+FMT)groups displayed indistinguishable body weight loss,colon length,DAI score,and histology score,which showed that FMT could not inhibit the disease in KO mice.In mice treated with FMT,the relative abundance of Akkermansia decreased,and Lactobacillus became dominant.In particular,compared with those in WT mice,the scores of DAI and colon histology were clearly decreased in the KO-DSS group.Microbiota structure showed a significant difference between KO and WT mice.Akkermansia were the dominant genus in healthy KO mice.The ineffectiveness of FMT in KO mice was related to the decreased abundance of Akkermansia.Gene Ontology enrichment analysis showed that differentially expressed genes between each group were mainly involved in cytoplasmic translation and cellular response to DNA damage stimulus.The top nine genes correlating with Akkermansia included Aqp4,Clca4a,Dpm3,Fau,Mcrip1,Meis3,Nupr1 L,Pank3,and Rps13(|R|>0.9,P<0.01).CONCLUSION FMT may ameliorate DSS-induced colitis by regulating the TLR4 signaling pathway.TLR4 modulates the composition of gut microbiota and the expression of related genes to ameliorate colitis and maintain the stability of the intestinal environment.Akkermansia bear great therapeutic potential for colitis.

Relationship of Toll-Like Receptors 2 and 4 Gene Polymorphisms with Essential Hypertension in Chinese Han Population

Objective: There are numerous studies suggesting that genetic polymor-phisms of inflammation factors Toll-like receptors 2 and 4 (TLR2, TLR4) might play a role in the pathophysiological process of hypertension. In this study, we evaluated the association in a sample of members of the Chinese Han population. Method: We selected four single nucleotide polymor-phisms (SNP) of TLR2 (rs3804099, rs3804100, rs7656411) and TLR4 (rs1927906) genes, and measured the distributions of genotypic and allelic frequencies in 1063 participants, including 391 essential hypertension pa-tients and 672 controls. Result: No significant differences in the genotypic and allelic frequencies of the four SNPs were detected between cases and controls. However, three haplotypes, CCG, TTG and TTT of TLR2, were significantly associated with a decrease in the risk of essential hyperten-sion (OR: 0.512, 95% CI: 0.397 - 0.660, P P = 0.0038;OR: 0.797, 95% CI: 0.667 - 0.952, P = 0.0122, respectively). Inversely, the risk of essential hypertension increased sig-nificantly in patients with the CTG, TCG or TCT haplotypes (OR: 2.924, 95% CI: 2.157 - 3.963, P P P Conclusion: Our study suggested that haplotypes (CCG, TTG, TTT, CTG, TCG and TCT) of TLR2 might have profound effects on the development of essential hypertension in the Chinese Han population.

Oxygen-glucose deprivation of neurons transfected with toll-like receptor 3-siRNA Determination of an optimal transfection sequence

Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA （siRNA）-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis

AIM To determine the effects of ω-3 fatty acids(ω-3FA) on the toll-like receptor 4(TLR4)/nuclear factor κB p56(NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis(SAP).METHODS A total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay. RESULTS The expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group(P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h(P < 0.05).CONCLUSION During the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

Effect of ω-3 Polyunsaturated Fatty Acid on Toll-like Receptors in Patients with Severe Multiple Trauma

This study examined the effects of ω-3 polyunsaturated fatty acid（ω-3PUFA） on the expression of toll-like receptor 2（TLR2）,toll-like receptor 4（TLR4） and some related inflammatory factors in peripheral blood mononuclear cells（PBMCs） of patients with early-stage severe multiple trauma.Thirty-two patients who were admitted to the Department of Traumatic Surgery,Tongji Hospital（Wuhan,China） between May 2010 and November 2010,and diagnosed as having severe multiple trauma with a injury severity score（ISS） no less than 16,were enrolled in the study and divided into two groups at random（n=16 in each）：ω-3PUFA group and control group in which routine parenteral nutrition supplemented with ω-3PUFA or not was administered to the patients in two groups for consecutive 7 days.Peripheral blood from these patients was collected within 2 h of admission（day 0）,and 1,3,5 and 7 days after the nutritional support.PBMCs were isolated and used for detection of the mRNA and protein expression of TLR2 and TLR4 by using real-time PCR and flow cytometry respectively,the levels of NF-κB by quantum dots-based immunofluorescence assay,the levels of TNF-α,IL-2,IL-6 and COX-2 by ELISA,respectively.The results showed that the mRNA and protein expression of TLR2 and TLR4 in PBMCs was significantly lower in ω-3PUFA group than in control group 5 and 7 days after nutrition support（both P0.05）.The levels of TNF-α,IL-2,IL-6 and COX-2 were found to be substantially decreased in PBMCs in ω-3PUFA group as compared with control group at 5th and 7th day（P0.05 for all）.It was concluded that ω-3PUFA can remarkably decrease the expression of TLR2,TLR4 and some related inflammatory factors in NF-κB signaling pathway in PBMCs of patients with severe multiple trauma,which suggests that ω-3PUFA may suppress the excessive inflammatory response meditated by the TLRs/NF-κB signaling pathway.

Effect of Ligands to Toll-Like Receptors (TLR) 3, 7 and 9 on Mice Infected with Mouse Hepatitis Virus A59

Mice infected with mouse hepatitis virus A59 (MHV-A59), an enveloped, positive-strand RNA Co-ronavirus, induce hepatitis, thymus involution, IgG2a-restricted hypergammaglobulinaemia, transaminase release and autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hy-drolase (FAH). Since Toll-like receptors (TLR) play a central role in innate immunity, we explored the effects of TLR3, 7 and 9 stimulation on MHV mouse infection. Thus, the animals were treated with Poly (I:C), Loxoribine and CpG, the respective TLR ligands. MHV-infected mice inoculated with Poly (I:C) had significant lower levels of plasma transaminases and Ig, anti-MHV Ab, and uric acid than MHV-infected animals, whereas autoAb to kidney tissue were observed. Loxoribine only produced a slight decrease of uric acid levels and serum Ig. CpG showed deleterious effects on MHV-infected mice, since survival of animals dramatically dropped to about 10%. AutoAb to murine tissues and uric acid release were not affected, whereas transaminases and anti-MHV Ab were slightly elevated. Besides, CpG administration produced a decrease of the high levels of serum Ig induced by the virus. Therefore, results indicated that TLR3 stimulation appeared to protect the animals against the viral infection, whereas CpG aggravated its signs. Loxoribine, the TLR7 ligand, did not show major effects.

Correlation analysis of Toll-like receptor 3 content in peripheral blood with electroencephalogram parameters and neurotransmitter content in children with intractable epilepsy

Objective:To detect the content of Toll-like receptor 3 (TLR3) in peripheral blood of children with intractable epilepsy, explore the correlation between TLR3 content and EEG parameters, neurotransmitter contents.Methods:37 cases of Intractable epilepsy children in our hospital during September 2016to June 2018 were chosen as Intractable epilepsy group, 30 cases of healthy volunteers who underwent physical examination in our hospital were treated as Normal control group. The levels of TLR3, neurotransmitter [5-hydroxytryptamine (5-HT), dopamine (DA), epinephrine (E), norepinephrine (NE)] and electroencephalogram parameters [alpha, beta, delta, theta] in peripheral blood of two groups were compared. Pearson test was used to evaluate the correlation of TLR3 content in peripheral blood with EEG parameters and neurotransmitter content in children with intractable epilepsy.Results: Content of TLR3 in peripheral blood of Intractable epilepsy group was significantly higher than that of Normal control group;the alpha power and theta power of EEG parameters were lower than those of Normal control group;contents of neurotransmitters such as 5-HT, DA, E and NE were significantly lower than those of Normal control group (P<0.05). The correlation analysis showed that content of TLR3 in peripheral blood of children with intractable epilepsy was negatively correlated with levels of alpha and theta power of EEG, positively correlated with content of neurotransmitters such as 5-HT, DA, E and NE (P<0.05), but had no significant correlation was found with level of beta and delta power (P>0.05).Conclusion: The abnormal increase of TLR3 in peripheral blood of children with intractable epilepsy may be one of the direct causes of neurological impairment in children with intractable epilepsy.

人Toll-like receptor 2胞外段的克隆和表达

目的 克隆、表达人Toll-like receptor2(TLR2)胞外段(A26-T588)基因,获得人TLR2胞外段蛋白。方法 RT-PCR扩增TLR2胞外段基因,以pcDNA3.1+质粒为载体在HEK293细胞中表达TLR2胞外段蛋白,同时以pcDNA3.1+/TLR2(A26-T588)重组质粒免疫昆明鼠,制备抗TLR2胞外段蛋白多抗。结果 PCR扩增及重组质粒测序结果表明成功地构建了pcDNA3.1+/TLR2(A26-T588)真核表达质粒,SDS-PAGE分析纯化产物在M_r为68000处出现明显蛋白条带。重组质粒DNA免疫小鼠3次后,血清抗体滴度可达1∶250。TLR2胞外段蛋白可与LPS结合,并在一定的质量浓度范围内呈剂量依赖性。结论 构建的pcDNA3.1+/TLR2(A26-T588)真核表达质粒可在哺乳动物细胞中表达TLR2胞外段蛋白,并与重组质粒DNA免疫小鼠抗血清及TLR2单克隆抗体TL2.1特异性反应,由此证明其表达正确。

广谱模式识别分子Toll-like receptor 2的研究进展

TLR-2（Toll-like receptor 2,TLR-2）是哺乳动物TLRs（Toll-like receptors,TLRs）家族的一员,作为细胞表面的天然受体蛋白,主要参与病原微生物产物的识别及炎症信号传导,介导天然抗感染兔疫;最近又发现其参与机体对非感染因子所致炎性组织损伤的识别。通过对TLR-2参与的识别和细胞内信号传导机制的研究,可为深入探讨抵御微生物感染的机制、对自身正常与非正常组织的识别提供新的思路。