Effect of Celastrol Based on IRAK4/ERK/p38 Signaling Pathway on Proliferation and Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of celastrol on the proliferation and apoptosis of human multiple myeloma (MM) cell lines, reveal the relationship between IRAK4/ERK/p38 signaling pathway and celastrol regulating the proliferation and apoptosis of H929 and ARP-1 cells, and explore whether celastrol combined with bortezomib has synergistic effect. @*METHODS@#CCK-8 method was used to detect the viability of MM cell lines H929 and ARP-1 treated by different concentrations of celastrol, bortezomib, and their combination, and the synergistic effect was determined by Kim's formula. The apoptosis rate of H929 cells and necrosis rate of ARP-1 were detected by Annexin V/PI method. The expression of key proteins and apoptosis proteins in IRAK4/ERK/p38 signaling pathway were detected by Western blot. @*RESULTS@#Celastrol could significantly inhibit the proliferation of H929 and ARP-1 cells (r=0.9018, r=0.9244) and induce apoptosis in a time-dependent manner. Compared with the control group, celastrol could significantly up-regulate the expression of PARP and cleaved caspase-3 while down-regulate the expression of p-IRAK4, p-ERK, and p-p38 in H929 and ARP-1 cells. Celastrol and bortezomib alone inhibited the proliferation of H929 and ARP-1 cells. Compared with celastrol and bortezomib alone, their combination had lower cell survival rate and higher apoptosis rate (P<0.05). @*CONCLUSION@#Celastrol can inhibit the proliferation and promote the apoptosis of H929 and ARP-1 cells, which may be related to inhibiting the phosphorylation of IRAK4 and blocking the activation of IRAK4/ERK/p38 signaling pathway. Celastrol combined with bortezomib has synergistic effect, which can more effectively inhibit the proliferation and induce apoptosis of H929 and ARP-1 cells.

Celastrol targets IRAKs to block Toll-like receptor 4-mediated nuclear factor-κB activation / 中西医结合学报

<p><b>OBJECTIVE</b>Celastrol has been established as a nuclear factor-κB (NF-κB) activation inhibitor; however, the exact mechanism behind this action is still unknown. Using text-mining technology, the authors predicted that interleukin-1 receptor-associated kinases (IRAKs) are potential celastrol targets, and hypothesized that targeting IRAKs might be one way that celastrol inhibits NF-κB. This is because IRAKs are key molecules for some crucial pathways to activate NF-κB (e.g., the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily).</p><p><b>METHODS</b>The human hepatocellular cell line (HepG2) treated with palmitic acid (PA) was used as a model for stimulating TLR4/NF-κB activation, in order to observe the potential effects of celastrol in IRAK regulation and NF-κB inhibition. The transfection of small interfering RNA was used for down-regulating TLR4, IRAK1 and IRAK4, and the Western blot method was used to detect changes in the protein expressions.</p><p><b>RESULTS</b>The results showed that celastrol could effectively inhibit PA-caused TLR4-dependent NF-κB activation in the HepG2 cells; PA also activated IRAKs, which were inhibited by celastrol. Knocking down IRAKs abolished PA-caused NF-κB activation.</p><p><b>CONCLUSION</b>The results for the first time show that targeting IRAKs is one way in which celastrol inhibits NF-κB activation.</p>

Effect of High MiR-146a Expression on the Inflammatory Reaction in BV2 Cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the effect of MiR-146a regulator function on the inflammatory response in neuroglia cell (microglia).</p><p><b>METHODS</b>BV2 cells were transfected by MiR-146a mimics,and then stimulated by lipopolysaccharide (LPS). MiR-146a expression was measured by real-time polymerase chain reaction (real-time PCR). Interleukin (IL)-6 and tumor necrosis factor α (TNFα) were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) were detected by PCR and Western blotting.</p><p><b>RESULTS</b>Compared to the normal control group, MiR-146a expression was significantly elevated by transfection with MiR-146a mimics (t=5.846, P=0.0021). The expression levels of IRAK1, TRAF6, TNFα, and IL-6 significantly increased in the LPS-stimulated BV2 cells compared to the non-stimulated BV2. The enhancement of MiR-146a resulted in significantly decreased IL-6 (t=5.200, P=0.0003) and TNFα (t=9.812, P<0.0001) secretion. The mRNA (t=5.353, P=0.0007) and protein (t=6.980, P=0.0009) levels of TRAF6, but not IRAK1, also significantly decreased.</p><p><b>CONCLUSION</b>MiR-146a may negatively suppress the inflammatory response of BV2 cells by regulating the expression of IRAF6 molecules in the TLR4 signaling pathway.</p>

Distrofia muscular facioescapulohumeral en Chile: presentación de serie en hospital de referencia terciario / Facioscapulohumeral muscular dystrophy: Report of seven patients

Background: Facioscapulohumeral muscular dystrophy is the third most common muscular dystrophy with an estimated prevalence of 1 per 20.000 and a normal life expectancy in the majority of patients. However, approximately 15% of patients become wheelchair bound in the course of their life. It is a hereditary autosomal dominant disease with high (95%) penetrance by the age of 20, but with variable degree of phenotypic expression even in the same family group. Symptoms frequently start in the second decade of life, with facial and scapular weakness. Aim: To report the clinical features of seven patients with the disease, seen at a public hospital. Material and Methods: Analysis of seven patients with genetic study seen in a public Hospital in Santiago. Results: The age of patients fluctuated from 18 to 61 years and four were females. The mean age at onset of symptoms was 29 years and four had a family history of the disease. The usual presenting complaint was arm or shoulder asymmetric weakness. Four patients had bone pain. Facial involvement was present in four. A genetic study was done in five patients, the other two patients were relatives, confirming the contraction or lower number of repetitions in D4Z4 region. After 12 years of follow up only 2 patients older than 60 years cannot work and one female patients is in a semi dependent state at the age of 30. Conclusions: The clinical workup in the diagnosis and the timely indication of genetic studies are highlighted, to avoid unnecessary and invasive procedures. The variability in the phenotypic expression in a similar genetic defect is discussed and the genetic or epigenetic mechanisms of this muscular dystrophy are described.

Study on effect and mechanism of volatile oil of schizonepetae herba and its essential components against influenza virus / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effect of volatile oil of Schizonepetae Herba (VOSH), and its essential components-menthone and pulegone against anti-influenza virus A/PR/8/34 (H1N1) in vivo and in vitro, as well as the signaling mechanism of its toll-like receptor/interferon (TLR/IFN).</p><p><b>METHOD</b>The lung-adapted PR-8 virus model was prepared in mice. They were administered with preventive and therapeutic drugs, and the hemagglutination titer of model animals was determined to evaluate in vivo effect against H1N1. ELISA test was conducted to observe the effect on IFN-alpha, IFN-beta, IL-2, IL-6 and TNF-alpha in serum, as well as IFN-beta secretion in H1N1 infected MDCK supernatant. Real-time RT-PCR was employed to observe the expression levels of IRAK4 and TLR3 mRNA.</p><p><b>RESULT</b>The in vivo experiment shows that the hemagglutination titer was significantly decreased when the mice were treated with VOSH (0.266 mg x kg(-1)), menthone(0.5 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) in therapeutic way; VOSH (0.226 mg x kg(-1)) had a significant effect on increasing serum levels of IFN-alpha, IL-2; Methone (0.5 mg x kg(-1)) had a significant effect on increasing serum levels of IFN-beta; Methone (0.5 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) had a significant effect on decreasing serum levels of IL-6; VOSH (0.452, 0.226 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) had a significant effect on decreasing serum levels TNF-alpha. The in vitro experiment showed that the expression levels of IRAK4 mRNA and IFN-beta were significantly increased in VOHS (0.1 g x L(-1)) and pulegone (0.1 g x L(-1)) groups; and the menthone (0.25 g x L(-1)) group showed a significant rise in the expression levels of IRAK4 mRNA, but a notable decline in TLR3 mRNA.</p><p><b>CONCLUSION</b>The administration with VOSH, methone and pulegone in therapeutic way can significantly decrease the hemagglutination titer, which demonstrates the anti-virus effect of the administration in therapeutic way, but no notable efficacy of the administration in preventive way. The in vivo anti-virus mechanism is related to regulation of IFN-alpha, IFN-beta and IL-2.</p>

Association between single-nucleotide polymorphisms in the IRAK-4 gene and allergic rhinitis / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the genetic association pattern between single-nucleotide polymorphisms (SNP) in the interleukin-1 receptor-associated kinase 4 (IRAK-4) gene and allergic rhinitis (AR).</p><p><b>METHODS</b>A population of 379 patients with the diagnosis of AR and 333 healthy controls who lived in Beijing region was recruited. A total of 8 reprehensive marker SNP which were in IRAK-4 gene region were selected according to the Beijing people database from Hapmap website. The individual genotyping was performed by MassARRAY platform. SPSS 13.0 software was used for statistic analysis.</p><p><b>RESULTS</b>Subgroup analysis for the presence of different allergen sensitivities displayed associations only in the house dust mite-allergic cohorts (rs3794262: P = 0.0034, OR = 1.7388; rs4251481: P = 0.0023, OR = 2.6593), but not in subjects who were allergic to pollens as well as mix allergens.</p><p><b>CONCLUSION</b>The potential genetic contribution of the IRAK-4 gene to AR demonstrated an allergen-dependant association pattern in Chinese population.</p>

Functional polymorphism in exon 5 and variant haplotype of the interleukin-1 receptor-associated kinase 1 gene are associated with susceptibility to and severity of sepsis in the Chinese population / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) is believed to play an important role in the pathogenesis of sepsis. Recent studies have suggested that the IRAK1 functional genetic variant could affect the severity of sepsis in Caucasians. In this report, we have investigated whether polymorphisms at the IRAK1 gene are associated with the susceptibility to and severity of sepsis among the Chinese population.</p><p><b>METHODS</b>Haplotype-tagging single nucleotide polymorphisms (htSNPs) were selected from the HapMap database. They were genotyped in 255 patients with sepsis and 260 control subjects by PCR/restriction fragment length polymorphism (RFLP) analysis. The association between the selected htSNPs and the susceptibility to and severity of sepsis were estimated by Logistic regression with adjustments for age, sex, smoking, drinking, chronic disease status, Acute Physiology and Chronic Health Evaluation (APACHE) II score and primary diseases.</p><p><b>RESULTS</b>rs1059702 was selected to represent the six linked htSNPs for IRAK1. Genotype frequencies of the htSNPs were in Hardy-Weinberg equilibrium for females, as were allele frequencies for both sex groups. Associations were observed in females between the htSNPs C/C genotype and increased susceptibility to sepsis (odds ratio (OR), 5.46; 95% confidence interval (CI), 1.12 - 26.67; P = 0.018), and such associations were also observed between the IRAK1 variant haplotype (CC/C-allele) and increased susceptibility to sepsis (OR, 1.68; 95% CI, 1.05 - 2.70; P = 0.031) when compared with the T/T + T/C genotype and the wild-type haplotype (TC + TT/T-allele). In the multiple organ dysfunction syndrome (MODS) subgroup, the variant haplotype was also associated with increased severity of sepsis (OR, 2.37; 95% CI, 1.13 - 4.94; P = 0.02) when compared with the wild haplotype. This association was not significant in male patients.</p><p><b>CONCLUSIONS</b>The functional polymorphism in exon 5 and the variant haplotype of IRAK1 gene mediate susceptibility to and severity of sepsis. IRAK1 might be a genetic risk factor for the occurrence and development of sepsis in the Chinese population.</p>

The Effect of IGFBP-3 on Adipokines and Gene Expression in Differentiated 3T3-L1 Adipocytes / 대한소아내분비학회지

PURPOSE: IGFBP-3 leads to the induction of insulin resistance in 3T3-L1 adipocytes. We carried out a series of experiments to elucidate the effects of IGFBP-3 on adipokines and gene expressions. METHODS: We treated fully-differentiated 3T3-L1 adipocytes with IGFBP-3 (0.5, 1, and 2 microg/mL) for one day and measured the mRNA levels of adiponectin, leptin, resistin, and TNF-alpha by RT-PCR, and adiponectin, leptin, resistin, and IL-6 protein levels in the culture supernatant were measured using multiplex adipokine assay ELISA Kits (Linco Research, St. Charles, Missouri). Gene expression in 3T3-L1 adipocyte cells using a microarray method was performed. RESULTS: IGFBP-3 inhibited the expression of adiponectin, leptin, resistin, and TNF-alpha mRNA. IGFBP-3 at 0.5 and 1 micro/mL decreased adiponectin release, but IL-6 release was increased at 2 micro/mL IGFBP-3. A dose-dependent inhibition of leptin was released by IGFBP-3 at 50%. Resistin release was decreased by 40%. The effect of IGFBP-3 on the gene expression in 3T3-L1 adipocyte cells using a microarray assay related to an increase of agouti-realted proteins (Agrp) and Janus kinase 2 (JAK2), and a decrease of the ras homolog gene family (Rhoq), acyl-CoA synthetase long-chain family member 6 (Acsl6), and the interleukin-1 receptor-associated kinase 1 (Irak1). CONCLUSION: IGFBP-3 regulates several adipokines gene expressions that are known to modulate insulin sensitivity, and this regulation may be attributable to the insulin resistance effect of IGFBP-3 on adipocytes.

Effect of Dureping injection on TIR signal pathway on Ana-1 cells / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the influence of Dureping injection to the murinal celiac macrophage Ana-1 on TIR signal pathway.</p><p><b>METHOD</b>Ana-1 cell line was infected by influenza virus FM1 strain and treated with the Dureping injection in different concentrations (10.1 mg x L(-1) group) for 12 h and 24 h. Then we collected the cells, extracted mRNA and measured the expressions of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 respectively by RT-PCR.</p><p><b>RESULT</b>Dureping injection down-regulated the expression of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 mRNA in Ana-1 cell line infected by influenza virus, in a dose-dependent manner significantly.</p><p><b>CONCLUSION</b>Dureping injection has an obvious effect against influenza virus FM1 strain by regulating the TIR signal pathway.</p>

Influence of continuous high-volume hemofiltration on IRAK-4 protein expression in severe acute pancreatitis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the influence of continuous high-volume hemofiltration (CHVHF) on interleukin 1 receptor-associated kinase-4 (IRAK-4) and tumor necrosis factor-alpha (TNF-alpha) levels in patients with severe acute pancreatitis (SAP).</p><p><b>METHODS</b>Forty-one patients with SAP were randomly divided into two groups to receive treatment with CHVHF plus conventional therapy (21 patients) and conventional therapy only (20 patients). Venous blood samples were taken before and 12, 24, and 72 h after the treatment for evaluation of APACHE II scores. The mRNA and protein levels of IRAK-4 in the monocytes were determined by real-time PCR and Western blotting, respectively, and serum TNF-alpha levels was detected using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Among the 21 patients receiving CHVHF, 18 survived and 3 died, and in the conventional therapy group, death occurred in 5 cases. In the surviving patients of CHVHF group, the APACHE II scores, IRAK-4 mRNA and protein levels and TNF-alpha levels were all significantly lowered after the treatment, and these indices were also significantly lower than those in the conventional group after treatment (P<0.05).</p><p><b>CONCLUSION</b>CHVHF is effective in reducing monocyte IRAK-4 levels and serum TNF-alpha level in SPA patients, and thus alleviates the symptoms and improves the prognosis of SAP, possibly by reducing the level of the activators that induce monocyte activation via the Toll-like receptor.</p>

A differential gene expression profiles by cDNA microarrays in endometrioid endometrial carcinoma: a preliminary study / 부인종양

OBJECTIVE: Endometrial carcinoma is the most common gynecological malignant disease in industrialized countries. However, the molecular bases for endometrial tumoriogenesis are not clearly elucidated. Our hypothesis is that there may be some difference in gene expression patterns between normal endometrium and endometrial cancer lesion. In this study, we analyzed the difference of gene expression profile with cDNA microarray. METHODS: Normal endometrial tissues and cancer lesions were gathered from three patient with endometrioid endometrial cancer. cDNA microarray technique (KNU 4.8K chip) was applied to screen the different gene expression profiles. RESULTS: Many genes such as interleukin-1 receptor-associated kinase 1 (IRAK1), bifunctional apoptosis regulator (BFAR), paraneoplastic antigen MA2 (PNMA2), zinc finger protein 257 (ZNF257), ras homolog gene family, member F (in filopodia) (ARHF), cell division cycle 27 (CDC27) were over-expressed in the endometrial cancer tissue. The genes were down-regulated in the endometrial cancer samples included fibronectin 1 (FN1), meiotic checkpoint regulator (MCPR), transforming growth factor beta-stimulated protein TSC-22 (TSC22), programmed cell death 4 (neoplastic transformation inhibitor) (PDCD4), transcript variant 2, matrix metalloproteinase 2 (MMP2), insulin-like growth factor binding protein 4 (IGFBP4), retinoblastoma binding protein 7 (RBBP7), insulin-like growth factor binding protein 3 (IGFBP3), downregulated in ovarian cancer 1 (DOC1). CONCLUSION: The result of this analysis supports the hypothesis that the endometrial cancer tissue has distinct gene expression profile from normal endometium. But, the vaildation of gene expression with RT-PCR and the further study are needed.

Expression changes of interleukin-1 receptor associated kinase-4 during endotoxin tolerance development in kupffer cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the mechanism of endotoxin tolerance (ET) through observing the expression of interleukin 1 receptor associated kinase-4 (IRAK-4) during endotoxin tolerance development in Kupffer cells (KCs).</p><p><b>METHODS</b>Isolated KCs of Balb/c mouse were divided into two groups: the non-endotoxin tolerance (NET) group and the endotoxin tolerance (ET) group, which were pretreated with 10 ng/ml lipopolysaccharide (LPS) for 24 h. Then, the two groups were treated with 100 ng/ml LPS. The expressions of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The activities of NF-kappaB of KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h after LPS stimulation.</p><p><b>RESULTS</b>The ultimate level of IRAK-4, the activities of NF-kappaB and the TNFalpha level were evidently lower in the ET group than those in the NET group (t = 12.4, 17.4 and 138.9 respectively, P<0.01).</p><p><b>CONCLUSIONS</b>Pretreatment with LPS on KCs could induce endotoxin tolerance of KCs and inhibition of IRAK-4 expression may be one of the reasons for its development.</p>

The mechanism and treatment phases chosen of glycine for inhibition lipopolysaccharide induced Kupffer cells activation / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the possible mechanism and optimal treatment phase of glycine for inhibition lipopolysaccharide (LPS) induced Kupffer cells (KCs) activation.</p><p><b>METHODS</b>The KCs were isolated from 40 BALB/c mice and divided into four groups: the endotoxin group, the prevention group, the early treatment group and the later treatment group (n = 10). The endotoxin group was treated with 10 mg/L LPS, and in the other three groups, glycine (1 mmol/L) was given 24 h before, or at 0 h or 4 h respectively after LPS stimulation. At 0 h, 1 h, 2 h, 6 h and 12 h after LPS stimulation, the mRNA levels and protein expression of interleukin-1 receptor associated kinase-4 (IRAK-4) were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively, and nuclear factor-kappaB (NF-kappaB) activities as well as tumor necrosis factor alpha (TNF-alpha) levels were also detected by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The climax values of IRAK-4, NF-kappaB and TNF-alpha were significantly higher in the endotoxin group and the later treatment group than that in the other two groups (t = 3.17, 4.33, 2.47, 126.73, P < 0.01).</p><p><b>CONCLUSION</b>The results indicated that prophylactic or simultaneous treatment with glycine could effectively inhibit LPS-induced KCs activation by inhibiting IRAK-4 expression.</p>

An experimental study of the inhibitory effects on the activation of endotoxin-induced Kupffer cells through short hairpin RNA targeting interleukin-1 receptor associated kinase-4 gene / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the inhibitory effects on the activation of endotoxin-induced Kupffer cells (KCs) through short hairpin RNA (shRNA) targeting interleukin-1 receptor associated kinase-4 (IRAK-4) gene.</p><p><b>METHODS</b>Two effective transfection shRNA plasmid (pSIIRAK-4-A, pSIIRAK-4-B) and one invalidated plasmids (pSIIRAK-4-C) targeting IRAK-4 gene were constructed. The isolated mouse KCs were divided into three groups: the normal control group, the RNAi control group (pSIIRAK-4-C) and the RNAi effective group (pSIIRAK-4-A, pSIIRAK-4-B). Then KCs were stimulated with 0.1 microg/ml lipopolysaccharide (LPS) after 24 h transfection with the constructed plasmid. The expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot at 6 h after LPS stimulation, and the activities of NF-kappaB in KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h.</p><p><b>RESULTS</b>The level of IRAK-4, the activities of NF-kappaB and the TNF-alpha level in the RNAi effective group were evidently lower than those in normal and RNAi control groups (P < 0.01) at 1 h, 3 h, and 6 h. Especially, the pSIIRAK-4-A group in which the changes of the above indices were of no difference (P > 0.05), had better inhibited effects than that of the pSIIRAK-4-B group (P < 0.01).</p><p><b>CONCLUSION</b>The shRNA targeting IRAK-4 gene could effectively inhibit the activation of endotoxin-induced KCs.</p>