Myeloid Differentiation Factor 88 mRNA Research Articles

LXR agonist inhibits inflammation through regulating MyD88 mRNA alternative splicing.

Liver X receptors (LXRs) are important regulators of cholesterol metabolism and inflammatory responses. LXR agonists exhibit potently anti-inflammatory effects in macrophages, which make them beneficial to anti-atherogenic therapy. In addition to transrepressive regulation by SUMOylation, LXRs can inhibit inflammation by various mechanisms through affecting multiple targets. In this study, we found that the classic LXR agonist T0901317 mediated numerous genes containing alternative splice sites, including myeloid differentiation factor 88 (MyD88), that contribute to inflammatory inhibition in RAW264.7 macrophages. Furthermore, T0901317 increased level of alternative splice short form of MyD88 mRNA by down-regulating expression of splicing factor SF3A1, leading to nuclear factor κB-mediated inhibition of inflammation. In conclusion, our results suggest for the first time that the LXR agonist T0901317 inhibits lipopolysaccharide-induced inflammation through regulating MyD88 mRNA alternative splicing involved in TLR4 signaling pathway.

Expression of lnc-MyD88 and its relationship with the prognosis of patients with epithelial ovarian cancer

Objective: To explore the expression of long non-coding RNA-myeloid differentiation factor 88 (lnc-MyD88) and its relationship with the prognosis of patients with epithelial ovarian cancer (EOC). Methods: A total of 70 EOC patients who underwent initial cytoreductive surgery and platinum-based drugs combined with paclitaxel for 6 to 8 courses were selected at Sichuan Cancer Hospital from January 2016 to January 2019. The fresh cancer tissue specimens were collected. In addition, 28 fresh normal ovarian tissues from patients who underwent surgery for benign gynecological diseases during the same period were collected as control group. Reverse transcription (RT) and real-time quantitative polymerase chain reaction (qPCR) were used to detect the expression of lnc-MyD88 and myeloid differentiation factor 88 (MyD88) mRNA in EOC tissues and normal ovarian tissues. The correlation between the expression of lnc-MyD88 and MyD88 mRNA in EOC was analyzed by Pearson's correlation coefficient. The relationship between lnc-MyD88 expression and clinicopathological characteristics of patients with EOC was analyzed. Kaplan-Meier method was used to calculate the survival rate of patients. The log-rank test was used for univariate survival analysis, and Cox proportional hazard model was used for multivariate survival analysis. Results: (1) RT-qPCR showed that the relative expression level of lnc-MyD88 and MyD88 mRNA in EOC were 0.009 (0.000-0.049) and 0.001 (0.000-0.006), respectively, which were significantly higher than those of normal ovarian tissues (all P<0.01); Pearson's correlation coefficient showed that the expression of lnc-MyD88 and MyD88 mRNA in EOC was positively correlated (r2=0.610, P<0.01). (2) The high expression rate of lnc-MyD88 in EOC patients with lymph node metastasis, distant metastasis and chemotherapy resistance (71%, 64% and 70%, respectively) were significantly higher than the patients in control group (41%, 40% and 35%, respectively; all P<0.05). There were no statistically significant in the high expression rate of lnc-MyD88 in EOC patients with different ages, pathological types, pathological grades, surgical pathological stages, postoperative residual lesion size, and ascites cancer cells (all P>0.05). (3) Univariate analysis showed that surgical pathological staging, lymph node metastasis, distant metastasis, postoperative residual tumor size, and high expression of lnc-MyD88 and MyD88 mRNA significantly affected the progression-free survival (PFS) and overall survival (OS) of EOC patients (all P<0.05), ascites cancer cells were the risk factors that significantly affected PFS in EOC patients (P=0.040); multivariate analysis showed that surgical pathological staging and high expression of lnc-MyD88 and MyD88 mRNA were independent factors affecting PFS and OS in EOC patients (all P<0.05), the size of residual lesions after surgery was an independent factor affecting PFS in EOC patients (P=0.001). Conclusions: The level of lnc-MyD88 expression in ovarian cancer tissues was significantly increased. Lnc-MyD88, as a molecular marker for the poor prognosis of EOC, is related to the expression of MyD88 in EOC, and may be involved in its expression regulation, thereby affecting the survival and prognosis of EOC patients.

Antiatherosclerotic effects of corilagin via suppression of the LOX-1/MyD88/NF-κB signaling pathway in vivo and in vitro.

Corilagin, a natural polyphenol compound isolated from Phyllanthus urinaria L., exerts various pharmacological effects, such as antihyperglycemic, antitumor, and antioxidative stress properties, but the mechanisms underlying the antiatherosclerotic effects of corilagin have not been entirely elucidated. In the present study, we investigated the antiatherosclerotic effects of corilagin using a high-fat diet (HFD)-induced atherosclerotic rabbit model and ox-LDL-induced vascular smooth muscle cells (VSMCs) and explored the underlying molecular mechanisms. The serum lipid levels were measured through an enzymatic colorimetric assay. A histological analysis of rabbit aortas was performed after hematoxylin-eosin and oil red O staining. The proliferation of ox-LDL-induced VSMCs was detected using MTT assays, and the migration of cells was determined by wound scratch assays. In addition, the mRNA and protein expression levels of lectin-like ox-LDL receptor-1 (LOX-1), myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assays. Our results indicate that corilagin significantly reduced the serum levels of TC, TG and LDL-C, increased the HDL-C levels, decreased the intimal thickening in the thoracic aorta, and reduced the formation of foam cells in an HFD-induced rabbit atherosclerosis model. Moreover, corilagin suppressed the proliferation and migration of ox-LDL-induced VSMCs and reduced LOX-1, MyD88, NF-κB, MCP-1, and TNF-α mRNA and protein expression in vivo and in vitro. These data demonstrate that corilagin exerts antiatherosclerotic effects in vivo and in vitro and that the mechanisms may be closely associated with downregulation of the LOX-1/MyD88/NF-κB pathway.

Time-serial expression of toll-like receptor 4 signaling during polymicrobial sepsis in rats.

Sepsis caused by aggressive infection is a severe clinical problem with an increasing incidence worldwide. Toll-like receptors and their common adapter myeloid differentiation factor 88 (MyD88) can activate immune responses by recognizing a foreign microbe’s product. This study aimed to identify the different time expression of TLR four signaling pathway in an experimental rodent model of polymicrobial sepsis. A randomized animal study was investigated in rats with septic peritonitis induced by cecal ligation and puncture (CLP). The expressions of MyD88-dependent pathway biomarkers, including MyD88, nuclear factor-κB (NF-κB), and serum tumor necrosis factor-α (TNF-α), were analyzed and compared to the sham controls at the different time points after CLP surgery. CLP-induced sepsis increased liver MyD88 mRNA expression and protein expression compared to the control groups at 2 h after surgery. The MyD88 mRNA and protein expressions in rats with CLP-induced sepsis marked increased at 4 and 6 h, and their NF-κB activities and serum TNF-α levels also increased at 4 h after CLP surgery (both p < .05). The different serial expression of MyD88-ependent pathway during sepsis may be used as biomarkers during sepsis. These results may provide further helpful information for using pro-inflammatory biomarkers of innate immunity such as MyD88 and TNF-α in clinical sepsis or related abdominal surgical emergency in the future.

Effect of moxibustion at "Zusanli"(ST36) and "Shenshu"(BL23) on miR-155-mediated TLR4/NF-κB signaling involving amelioration of synovitis in rheumatoid arthritis rats

To explore the effect of moxibustion at "Zusanli"(ST36) and "Shenshu"(BL23) on synovitis, and expressions of miR-155, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), interlukine（IL-1） receptor-associated kinase (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), nuclear factor κB (NF-κB), IL-1β, tumor necrosis factor receptor (TNF)-α and IL-6 mRNA and protein of synovial membrane in rheumatoid arthritis (RA) rats, so as to explore its mechanism underlying improvement of RA. A total of 48 male Wistar rats were randomly divided into normal control, model, moxibustion and antagonist groups (n=12 rats in each group). The RA model was replicated by placing the rats in a wind, cold and wet environment and injection of Freund's complete adjuvant (CFA, 0.5 mL) into the right hindlimb foot plantar. Moxibustion was applied to bilateral ST36 and BL23 for 30 min, once daily for 21 consecutive days. Rats of the antagonist group was treated by injection of TLR4 antagonist (TAK-242, 1 mg/mL, 0.1 mg/kg) via tail vein, once per day for consecutive 21 d. The joint swelling degree (JSD) and arthritis index (AI, red swelling scale) were determined, and the expression levels of various indicators of miR-155, and TLR4, myeloid MyD88, IRAK1, TRAF6, NF-κB, IL-1β, TNF-α and IL-6 mRNA and protein were assayed by quantitative real time-PCR and Western blot, separately. Compared with the normal control group, the JSD and AI, and the expression levels of synovial miR-155, TLR4, MyD88, IRAK1, TRAF6, NF-κB, IL-1β, TNF-α and IL-6 mRNA and protein were significantly increased in the model group (P<0.01). Compared with the model group, the increased levels of JSD and AI, and the expression levels of synovial miR-155, TLR4, MyD88, IRAK1, TRAF6, NF-κB, IL-1β, TNF-α and IL-6 mRNA and protein were notably down-regulated in both moxibustion and antagonist groups (P<0.01). The effects of moxibustion were evidently superior to the antagonist in down-regulating the abovementioned indexes (P<0.01), except TLR4 mRNA and protein. Moxibustion at ST36 and BL23 can reduce the synovitis of RA rats, which is related to its effects in suppressing the expressions of miR-155, TLR4, MyD88, IRAK1, TRAF6, NF-κB, IL-1β, TNF-α and IL-6 mRNA and protein (i.e., inhibition of miR-155/TLR4/NF-κB signaling).

The effects and combinational effects of Bacillus subtilis and montmorillonite on the intestinal health status in laying hens

This study was conducted to evaluate the effects and combinational effects of Bacillus subtilis (BS) and montmorillonite (MMT) on laying performance, gut mucosal oxidation status, and intestinal immunological and physical barrier functions of laying hens. Three hundred sixty laying hens (29-week-old) were randomly assigned to a 2 × 2 factorial arrangement of treatments (n = 6) for 10 wk as follows: (1) basal diet; (2) the basal diet plus 5 × 108 cfu BS/kg; (3) the basal diet plus 0.5 g MMT/kg; and (4) the basal diet plus 5 × 108 cfu BS/kg and 0.5 g MMT/kg. Dietary supplementation with BS increased egg production and egg mass, the activities of catalase (CAT) and total superoxide dismutase in the intestinal mucosa, and villus height and villus height-to-crypt depth ratio of the jejunum (P < 0.05) but downregulated the mRNA expression levels of toll-like receptor 4 and myeloid differentiation factor 88 (MyD88) in the duodenum and jejunum, interleukin 1 beta in the duodenum, and nuclear factor kappa B P65 (NF-κB P65) and tumor necrosis factor alpha in the jejunum (P < 0.05). Dietary supplementation with MMT increased egg production and egg mass, the concentration of secretory immunoglobulin A in the duodenum, and the occludin mRNA expression level in the jejunum (P < 0.05) but reduced feed conversion ratio, malondialdehyde concentration in the duodenum and jejunum, and the mRNA expression level of MyD88 in the jejunum (P < 0.05). In addition, there was an interaction effect between BS and MMT supplementation on the CAT activity and the MyD88 mRNA expression level in the duodenum and the mRNA expression level of occludin in the jejunum (P < 0.05). In conclusion, dietary BS and MMT and their combination may improve the intestinal health status of laying hens, which may contribute to the increase in hens' laying performance.

Yeast cultures alleviate gossypol induced inflammatory response in liver tissue of Ussuri catfish (Pseudobagrus ussuriensis)

The present study explored the mechanisms of gossypol induced inflammatory response in liver tissue of Ussuri catfish (Pseudobagrus ussuriensis), and investigated whether the yeast culture could relieve the inflammatory response of fish. For this purpose, Ussuri catfish were fed two diets with or without yeast culture for 8 weeks, and then fish were treated with 300 mg gossypol/kg body weight by intragastric administration for 15 days. Fish pre-fed diet without yeast culture supplementation and treated with gossypol oral administration (Gos group) had higher interleukin-1β (IL-1β) and interleukin 8 (IL-8) mRNA levels compared with fish pre-fed diet without yeast culture supplementation and treated with sodium carboxymethylcellulose solution oral administration (control group) and pre-fed yeast culture diet and treated with gossypol oral administration (Gos/YC group), but had lower interleukin 10 mRNA level than the control group (P < .05). Simultaneously, hepatic toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88) and nuclear transcription factor kappa B p65 (NF-κB p65) mRNA levels of fish in Gos group were also up-regulated. However, dietary yeast culture supplementation remarkably prevented the increase of IL-1β and IL-8 mRNA levels and the decrease of IL-10 mRNA level by inhibiting the excessive activation of TLR2-MyD88-NF-κB signaling pathway through down-regulating TLR2, NF-κB p65 (P < .05) and MyD88 (P > .05) mRNA levels. Besides, the lowest serum lysozyme and alkaline phosphatase activities were observed in fish of Gos group. There was an increased trend in immunoglobulin M content among the groups. Fish in Gos group had significantly lower hepatic catalase, superoxide dismutase and total antioxidant capacity compared with those fish in the control group (P < .05). Higher occurrences of the dilatation in sinusoids, karyolysis, hydropic degenerations and nuclei shifting to the cellular periphery were detected in Gos group compared with those in the control group, coupled with increased serum aspartate aminotransferase activity. Lower occurrences of these histopathological symptoms were observed in the Gos/YC group compared with those of fish in the Gos group. Overall, the results mentioned above indicated that gossypol could result in immune and oxidative damage, liver injury and inflammatory response and dietary yeast culture supplementation could alleviate these negative responses for Ussuri catfish.

Effects of dietary n-3 highly unsaturated fatty acids on growth, non-specific immunity, expression of some immune-related genes and resistance to Vibrio harveyi in hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatu)

This study was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acids (n-3 HUFA) on growth performance, non-specific immunity, expression of some immune-related genes and resistance to Vibrio harveyi in juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatu). Six isoproteic and isolipidic experimental diets were formulated with graded levels of n-3 HUFA (0.65, 1.00, 1.35, 1.70, 2.05 and 2.40% of dry matter, respectively), and the 0.65% group was used as control group. Each diet was randomly allocated to triplicate groups of fish in 1000 L fiberglass tank, and each tank was stocked with 40 fish (initial weight 12.06 ± 0.01 g) for 8 weeks. Results showed that feed conversion ratio (FCR), survival rate (SR), hepatosomatic index (HSI) and condition factor (CF) were all not significantly affected by dietary n-3 HUFA levels (P > 0.05). Weight gain (WG) and specific growth rate (SGR) in 1.35% group were significantly higher than those in 2.40% group (P < 0.05). Crude lipid of body in 1.00% group was significantly lower than that in 1.70% and 2.40% groups (P < 0.05). Liver and muscle fatty acid profiles reflected that of diets. Before challenge with Vibrio harveyi, the activity of serum superoxide dismutase (SOD), catalase (CAT) and content of complement 3 (C3) in 1.35% and 1.70% groups significantly higher than those of control group (P < 0.05). After challenge with Vibrio harveyi, serum CAT, glutathione peroxidase (GSH-PX), lysozyme (LZM) and C3 all increased sharply, while SOD showed the opposite trend. Before challenge with Vibrio harveyi, the expression levels of intestine toll-like receptor 22 (TLR22) and myeloid differentiation factor 88 (MyD88) mRNA in 2.40% group were significantly increased, and the expression levels of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) mRNA in 2.05% group were significantly higher than those in 1.00% and 1.35% groups (P < 0.05). In addition, the TLR22 and IL-1β mRNA levels in kidney of 1.70% group were significantly lower than those in control group (P < 0.05). After challenge with Vibrio harveyi, the expression level of MyD88 mRNA in intestine of 1.35% group was significantly higher than that in 1.00% group and from 1.70% to 2.40% groups (P < 0.05), while TNF-α and IL-1β obtained minimum values in 1.70% group. In the kidney, the interleukin 10 (IL10) mRNA expression was significantly higher in 1.70% group than that in other groups, while the IL-1β expression in 1.70% group was on the contrary and significantly lower than that in 2.40% group (P < 0.05). Results of this study suggested that moderate dietary n-3 HUFA (1.47%–1.70% HUFA) could improve the growth performance, non-specific immunity and inhibit the inflammatory response of hybrid grouper.

The effects of silica dust on the expression of MyD88 and TRAF6 mRNA of lung macrophages in rats

Objective: To investigate the effects of silica dust on the expression of Myeloid differentiation factor 88 (MyD88) mRNA and tumor necrosis factor receptor-associated factor (TRAF6) mRNA of lung macrophages in rats. Methods: Selecting 40 SPF-class Wistar rats with average weight (200±20) g randomly divided into control group and 30 d, 60 d, 120 d experimental groups with 10 rats in each group according to body weight. The experimental groups rats were injected with 1 ml of SiO(2) (100 mg/ml) suspension through the trachea into lung only once, then they were respectively killed after 30, 60, 120 days. The control group rats were injected with 1 ml of saline into lung, and killed after 120 days. The lungs of the rats were taken for pathological observation. Lung macrophages were extracted and counted, and their activity was detected by MTT. RT-qPCR was used to assess the relative contents of MyD88 mRNA and TRAF6 mRNA. Results: Silica dust inhalation led to infiltration of lung tissue cells, thickening the alveolar wall and destruction of alveolar structure. The longer the exposure to dust, the more obvious the results were. The number of macrophages in all experimental groups and activity in the 30 d, 60 d groups were significantly higher than that in the control group (P<0.05) . Among them, 30 d group had the largest number and the highest activity. Compared with the control group, the expression of MyD88 mRNA and TRAF6 mRNA of lung macrophages in rats increased in the experimental groups (P<0.05) , especially in the 60 d group. Conclusion: Silica dust inhalation can increase the expression of MyD88 and TRAF6 in macrophages, suggesting that silica dust can induce silicosis fibrosis by activating TLR/NF-κB signal pathway.

Ulinastatin protects the lungs of COPD rats through the HMGB1/TLR4 signaling pathway.

The present study aimed to investigate the protective mechanism of ulinastatin against lung injury. Rat models with chronic obstructive pulmonary disease (COPD) were used to provide guidance for the medical treatment of this disease. The rats were divided into three groups: A control group, a model group and an experimental group (each, n=10). With the exception of the control group, all of the rats were prepared as models of COPD, using the composite molding method of smoking and intratracheal instillation of lipopolysaccharide. The rats in the model group all received a conventional treatment, while the rats in the experimental group received ulinastatin. A small animal lung function detector was used to examine lung function. The forced expiratory volume/sec (FEV) was negatively correlated with the protein expression levels of Toll-like receptor 4 (TLR4) and high mobility group box protein 1 (HMGB1). Real-time fluorescence quantitative polymerase chain reaction and western blot analyses were used to detect TLR4, MyD88 (myeloid differentiation factor 88), TRAF-6 (TNF receptor-associated factor 6), LOX-1 (lectin-type oxidized LDL receptor 1) and HMGB1 mRNA, along with their protein expression levels. The lung function of rats in the model group was significantly decreased compared with in the control group (P<0.05). In the experimental group the lung function was significantly greater, when compared with in the model group; however, it remained lower than in the control group. The mRNA and protein expression levels of TLR4, MyD88, TRAF-6, LOX-1 and HMGB1 were significantly higher in the model group than in the control and experimental groups; however, levels in the experimental group were significantly higher when compared with in the control group (P<0.05). The TLR4 and HMGB1 expression levels were positively correlated in all groups, which indicated involvement of the HMGB1/TLR4 signaling pathway. The FEV was negatively correlated with the protein expression levels of TLR4 and HMGB1. Thus, the protective effect of ulinastatin in the lungs of rats with COPD is associated with changes in the HMGB1/TLR4 signaling pathway.

Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways.

The polymorphic membrane protein D (Pmp18D) is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1) as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s) involved in rPmp18.1-induced IL-1β secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1β cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs) were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs) was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88), nuclear factor kappa beta (NF-κB p50/p65), and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly (p ≤ 0.001) enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1β cytokine secretion, strongly suggesting their involvement in the rPmp18.1-induced IL-1β cytokine secretion. Taken together, these results indicate that C. abortus Pmp18.1 induces IL-1β secretion by TLR4 activation through the MyD88, NF-κB as well as the Caspase-1 signaling pathways and may be a potential C. abortus vaccine candidate. The vaccine potential of Pmp18.1 will subsequently be evaluated in an appropriate animal model, using VCG as an immunomodulator, following immunization and challenge.

Effect of Bacillus subtilis on Aeromonas hydrophila-induced intestinal mucosal barrier function damage and inflammation in grass carp (Ctenopharyngodon idella)

Our study explored the effect of oral intubation of Bacillus subtilis on Aeromonas hydrophila-induced intestinal mucosal barrier function damage and inflammation in grass carp. The mid-intestine mucosal tissue was collected for ATPase activity measurement. Intestinal mucosa was also ultrastructurally examined with transmission electron microscope (TEM), and its permeability was determined using Evans blue (EB) and D-lactic acid. The mid-intestine pro-inflammation cytokine, MyD88 and tight junction (TJ) protein mRNA expression levels were measured using real-time quantitative PCR. The results revealed that B. subtilis was found to prevent the decrease in the activity of Na+, K+-ATPase and Ca2+, Mg2+-ATPase, as well as the increase in EB and D-lactic acid concentration and inflammation induced by A. hydrophila in grass carp. Compared with A. hydrophila groups, B. subtilis safeguarded the integrity of intestinal villi and tight junction structure and restrained A. hydrophila-induced down-regulation of TJ proteins zonula occludens-1 (ZO-1) and occludin. B. subtilis also restrained up-regulation of TJ protein claudin b, pro-inflammation cytokine tumour necrosis factor α (TNF-α), cytokine interleukin 8 (IL-8), IL-1β, and adaptor protein myeloid differentiation factor 88 (MyD88) mRNA levels. Thus, oral intubation of B. subtilis could reduce A. hydrophila-induced intestinal mucosal barrier function damage and inflammation.

Clinical and pathological significance of myeloid differentiation factor 88 expression in human hepatocellular carcinoma tissues.

The innate immune system, which includes toll-like receptor (TLR) signaling, plays an important role in inflammation and oncogenesis. Although TLR common adaptor myeloid differentiation factor 88 (MyD88) is known to have multiple effects on carcinogenesis, the role of MyD88 in hepatocarcinogenesis remains unknown. In this study, MyD88 expression was examined in 105 samples of human hepatocellular carcinoma (HCC) tissue by immunohistochemistry, Western blot, and quantitative reverse-transcriptase polymerase chain reaction methods. The relationships between MyD88 expression and clinical and pathological parameters were analyzed. The results showed that attenuated expression of MyD88 in HCC tissue tumor cells was significantly related to hepatitis B virus infection, large tumor size, positive vascular invasion, and intrahepatic metastasis (P < 0.05). Western blot analysis of MyD88 protein in nine normal livers and 28 HCCs showed gender disparity (P < 0.01, P < 0.05), and attenuated expression in cirrhotic livers (P < 0.05). Low expression of MyD88 mRNA was evident in HCCs with vascular invasion (P < 0.01). In contrast to previous reports, these results suggest that attenuated expression of MyD88 in HCC is associated with tumor progression.

Overexpression of Tim-3 reduces Helicobacter pylori-associated inflammation through TLR4/NFκB signaling invitro.

The present study aimed to investigate the interaction between T-cell immunoglobulin and mucin-domain-containing molecule-3 (Tim-3) and Toll-like receptor 4 (TLR4)/nuclear factor κB (NF‑κB) signaling in Helicobacterpylori-infected RAW264.7 macrophage cells. RAW264.7 cells were co‑cultured with H. pylori SS1 at different bacteria/cell ratios, and subsequently the mRNA expression of Tim‑3, TLR4, and myeloid differentiation factor 88 (MyD88) was measured by reverse transcription-quantitative polymerase chain reaction (RT‑qPCR). Furthermore, the effect of Tim‑3 overexpression was examined by transfection of RAW264.7 with pLVX-IRES-ZsGreen-Tim-3 and co‑culturing with H. pylori. mRNA and protein expression levels were then analyzed for Tim‑3, TLR4, MyD88, and phosphorylated (p‑) NF‑κB by RT‑qPCR and western blot analysis respectively. The concentrations of pro‑inflammatory cytokines [tumor necrosis factor‑α (TNF‑α), interleukin 6 (IL-6), interferon‑γ (IFN‑γ) and interleukin 10 (IL‑10)] released in the culture supernatants were measured by ELISA. H. pylori stimulation resulted in a significant increase of Tim‑3, TLR4, and MyD88 mRNA expression in RAW264.7 cells. H. pylori stimulation upregulated Tim‑3 expression even in the Tim‑3‑overexpressing RAW264.7 cells compared with unstimulated cells. TLR4, MyD88, and pNF‑κB protein expression and pro‑inflammatory cytokines (TNF‑α, IL‑6, and IFN‑γ) release levels were increased in the control RAW264.7 cells following H. pylori infection, but not in the Tim-3-overexpressing RAW264.7 cells. By contrast, IL‑10 levels were decreased following H. pylori infection in both control and Tim‑3‑overexpressing RAW264.7 cells. Overexpression of Tim-3 reduced H. pylori-associated inflammation in RAW264.7 macrophages, by downregulating expression of proteins in the TLR4 pathway and release of pro‑inflammatory cytokines. These findings suggest that Tim‑3 serves a crucial role in the negative regulation of H. pylori-associated inflammation and may be a novel therapeutic target for H. pylori infection.

Effects of dietary oxidized konjac glucomannan sulfates (OKGMS) and acidolysis-oxidized konjac glucomannan (A-OKGM) on the immunity and expression of immune-related genes of Schizothorax prenanti.

In the present study, konjac glucomannan (KGM) was degraded by H2O2, and then used trisulfonated sodium amine and HCl, individually, to obtain two kinds of derivatives: oxidized konjac glucomannan sulfates (OKGMS) and acidolysis-oxidized konjac glucomannan (A-OKGM). The effects of two OKGM modified products on the immune parameters and expressions of toll-like receptor 22 (TLR22), myeloid differentiation factor 88 (MyD88) and interferon regulatory factors 7 (IRF7) genes in Schizothorax prenanti were determined. The alternative haemolytic complement (ACH50) activity was found to be significantly increased by the OKGMS diets. The immunoglobulin M (IgM) level was significantly enhanced by the OKGMS diets. The lysozyme activity was significantly increased by both OKGMS and A-OKGM diets. The superoxide dismutase (T-SOD) activity in fish fed with all doses of OKGMS diets was significantly higher than that in fish fed with basal diet. The glutathione peroxidase (GSH-PX) activity in fish fed with 0.8% and 1.6% A-OKGM diets was significantly higher than control group. The malondialdehyde (MDA) level was significantly decreased by both OKGMS and A-OKGM diets. The 0.8% A-OKGM diet significantly up-regulated TLR22 gene expression in the head kidney and spleen. TLR22 gene expression was significantly promoted by all OKGMS diets in the mesonephros and liver. The MyD88 mRNA level in 1.6% A-OKGM group significantly increased in the head kidney. The low dose of OKGMS significantly induced the MyD88 gene expression in the mesonephros, gut and liver, while 0.8% A-OKGM group also showed a significantly enhanced MyD88 mRNA expression in the gut. High dose of OKGMS significantly increased the IRF7 mRNA expression in the mesonephros and spleen. Fish fed with low dose of A-OKGM showed significantly higher expression of IRF7 in the gut and liver. Present study suggested that OKGMS and A-OKGM can act as immunostimulant to improve the immune indexes and up-regulate the immune-related gene expressions.

Decreased toll-like receptor-4/myeloid differentiation factor 88 response leads to defective interleukin-1β production in term low birth weight newborns.

Morbidity and mortality rates are very high in low birth weight (LBW) newborns because of their increased susceptibility to infections compared with normal birth weight (NBW) newborns. A case and control study was designed to identify the status of toll-like receptor-4 (TLR-4) signaling and maternally derived immunoglobulin-G (IgG) subclasses in term LBW newborns compared with NBW newborns. To understand the basis of increased susceptibility to infections in LBW newborns, the levels of pro- and antiinflammatory cytokines interleukin-1β (IL-1β) and interleukin-10 (IL-10), respectively, released in response to lipopolysaccharide (LPS) stimulation of cord blood cells of LBW (n = 20) and NBW (n = 18) newborns, were quantified by enzyme-linked immunosorbent assay. Further, LPS-induced expression of TLR-4 and basal and LPS-induced expression of myeloid differentiation factor 88 (MyD88) were examined at mRNA levels in both groups. The levels of IgG subclasses in LBW (n = 20) and NBW (n = 18) newborns were quantified by enzyme-linked immunosorbent assay to explore the role of maternally derived immunity in LBW newborns. LPS-mediated release of IL-1β was significantly diminished in LBW newborns when compared with NBW newborns, whereas there was no significant difference in IL-10. Decreased production of IL-1β in LBW newborns was correlated with reduced expression of TLR-4 and MyD88 mRNA. No significant differences were observed in the levels of all 4 IgG subclasses between LBW and NBW newborns. Decreased production of IL-1β in LBW newborns was correlated with reduced expression of TLR-4 and MyD88 mRNA. This raises the possibility of increased susceptibility to infections in LBW when compared with the NBW newborns at term. Comparable levels of IgG subclasses in the 2 groups of newborns indicate that IgG is not a limiting factor in defense against infection in LBW newborns.

The impacts of regulating Toll-like receptor 2/nuclear factor-ΚB signal pathway on rats with ventilator-induced lung injury

To evaluate the role of Toll-like receptor 2/nuclear factor-ΚB (TLR2/NF-ΚB) signaling pathway pretreatment in ventilator-induced lung injury (VILI). Thirty male Sprague-Dawley (SD) rats were randomly divided into three groups by using random number scale, with 10 rats in each group. Group A: rats were given 200 μL of TLR2 monoclonal antibodies (TLR2mAb, 10 μg/kg) by slow instillation through tracheal catheter, and then ventilated with a high tidal volume (VT) of 40 mL/kg. Group B: ventilated with a normal VT of 8 mL/kg. Group C: rats were tracheally instilled with 10 μg/kg of TLR2mAb devoid of biologic activity, and then ventilated with a high VT of 40 mL/kg. The rats were mechanically ventilated for 4 hours, the lung wet to dry weight ratio (W/D) was calculated. The changes in pathology and ultrastructure in lung tissue were observed with microscope. Enzyme linked immunosorbent assay (ELISA) was performed to determine the concentration of interleukins (IL-1β, IL-6) and tumor necrosis factor-α (TNF-α) in serum and brconchoalveolar lavage fluid (BALF). Real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the mRNA expressions of TLR2, NF-ΚB and myeloid differentiation factor 88 (MyD88) in lung tissue. No obvious pathological changes in lungs were found in group A and group B, and no obvious damages to ultra-microstructure were found in lung macrophages, typeI epithelial cell and typeII epithelial cell. In group C, pathological changes were observed, including pulmonary alveoli fusion, alveoli septum thickening, inflammatory cells infiltration, and damages to ultrastructure of lung macrophage, damage to cell membrane of typeI epithelial cells and type II epithelial cells, vacuoles in cytoplasm, damage to organelle, and even pyknosis and perinuclear cistern thickening. Compared with group C, W/D ratio and mean concentration of inflammatory cytokines in serum and BALF showed a significant decrease in group A and B [W/D ratio: 1.151 ± 0.026, 1.128 ± 0.048 vs. 1.403 ± 0.062; concentration of IL-1β in serum (ng/L): 37.05 ± 5.61, 34.52 ± 4.31 vs. 51.45 ± 8.18; concentration of IL-6 in serum (ng/L): 53.65 ± 5.16, 55.77 ± 5.62 vs. 89.96 ± 7.08; concentration of TNF-α in serum (ng/L): 71.93 ± 13.29, 67.36 ± 11.42 vs. 96.20 ± 11.60; concentration of IL-1β in BALF (ng/L): 56.48 ± 6.16, 54.44 ± 7.26 vs. 99.77 ± 8.41; concentration of IL-6 in BALF (ng/L): 172.44 ± 21.26, 163.47±18.70 vs. 216.22 ± 23.90; concentration of TNF-α in BALF (ng/L): 235.81 ± 42.75, 231.72 ± 40.38 vs. 374.85 ± 69.61, all P<0.01], but there were no significant differences between group A and group B (all P>0.05). The mRNA expressions of TLR2, MyD88, and NF-ΚB were significantly decreased in group A and group B compared with those in group C [TLR2 mRNA(2(-Δ ΔCt)):1.021 ± 0.287, 0.938 ± 0.196 vs. 3.862 ± 0.871; MyD88 mRNA(2(-Δ ΔCt)):1.235 ± 0.277, 1.300 ± 0.306 vs. 3.618 ± 1.107; NF-ΚB mRNA(2(-Δ ΔCt)):0.519 ± 0.036, 1.043 ± 0.170 vs. 20.280 ± 9.466, P<0.05 or P<0.01], but there was no significant difference among the parameters mentioned above between group A and B (all P>0.05). To some extent, pre-intervention with TLR2mAb to block the TLR2/NF-ΚB signal pathway can inhibit the release of pro-inflammatory factors, and regulate the VILI.

Effect of hypothermia on TLR2/MyD88 signal pathway in lung tissue in rats with acute lung injury induced by lipopolysaccharide

To investigate the effect of hypothermia on the expression Toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88), nuclear factor-ΚBp65 (NF-ΚBp65), plasminogen activator inhibitor-1 (PAI-1) in the TLR2/MyD88 pathway in rats with acute lung injury (ALI) induced by lipopolysaccharide (LPS) inhalation. Ninety male Sprague-Dawley (SD) rats were randomly divided into control group (n=18), hypothermia group (n=24), temperature controlled group (n=24), and temperature-uncontrolled group (n=24). The ALI model was reproduced by 0.5 mL/kg LPS intratracheal instillation, while only normal saline was instilled intratracheally for control group. Arterial blood was collected and physical cooling was started 1 hour after instillation. The body temperature was lowered to 32-34 centigradein hypothermia group and 36-37 centigradein temperature controlled group, and no intervention was used for temperature-uncontrolled group and control group. The arterial blood gas was determined in all the groups before and 1 hour after instillation of saline or LPS and 1, 6, 12 hours after intervention. Rats were sacrificed respectively at 1, 6 and 12 hours after temperature control therapy, the morphological changes in lung tissue were observed under light microscope. The protein expression of PAI-1 in bronchoalveolar lavage fluid (BALF) was determined by enzyme linked immunosorbent assay (ELISA). TLR2 mRNA and MyD88 mRNA transcriptional level were determined by reverse transcription-polymeras chain reaction (RT-PCR). NF-ΚBp65 protein level was determined by Western Blot. After instillation of LPS, the oxygenation index (PaO₂/FiO₂) of each group was decreased obviously, the damage of lung tissues was aggravating, the lung injury score was increased significantly, PAI-1 protein in BALF and the expressions of TLR2 mRNA, MyD88 mRNA, NF-ΚBp65 protein in lung tissues were increased obviously. Each index was improved by therapeutic Hypothermia, the effect of which was best in using a cooling period in the 1-6 hours, while might be benefit at 6-12 hours. Compared with temperature controlled group, PaO2/FiO2 (mmHg, 1 mmHg=0.133 kPa) at 1 hour and 6 hours of hypothermia group was improved(1 hour: 402.49 ± 38.61 vs. 324.36 ± 28.93, 6 hours: 349.72 ± 98.20 vs. 284.35 ± 13.68, both P<0.01), the lung injury score at 1, 6 and 12 hours were significantly decreased (1 hour: 6.04 ± 0.74 vs. 7.96 ± 0.65, 6 hours: 9.09 ± 0.80 vs. 13.13 ± 1.02, 12 hours: 10.79 ± 1.42 vs. 13.42 ± 0.68, all P<0.01), the PAI-1 protein (ng/L) in BALF at 1, 6 and 12 hours were significantly decreased(1 hour: 121.36 ± 4.62 vs. 197.74 ± 9.42, 6 hours: 230.53 ± 10.76 vs. 294.06 ± 16.60, 12 hours: 270.48 ± 13.20 vs. 319.40 ± 10.24, all P<0.01), TLR2 mRNA and MyD88 mRNA expressions(2 (-Δ ΔCt)) in the lung tissues at 1, 6 and 12 hours were significantly decreased(TLR2 mRNA 1 hour: 2.18 ± 0.26 vs. 3.04 ± 0.39, 6 hours: 4.09 ± 0.29 vs. 4.90 ± 0.35, 12 hours: 6.02 ± 0.43 vs. 7.10 ± 0.54; MyD88 mRNA 1 hour: 2.25 ± 0.41 vs. 3.04 ± 0.30, 6 hours: 5.67 ± 0.55 vs. 7.01 ± 0.76, 12 hours: 7.14 ± 0.60 vs. 8.87 ± 0.54, all P<0.01), NF-ΚBp65 protein expression (A value) at 6 hours and 12 hours was significantly decreased(6 hours: 0.31 ± 0.08 vs. 0.53 ± 0.12, 12 hours: 1.05 ± 0.17 vs. 1.76 ± 0.35, both P<0.01). There was no difference in each index between temperature controlled group and temperature-uncontrolled group. Hypothermia can down-regulate the expression of TLR2 mRNA, MyD88 mRNA, NF-ΚBp65 protein and PAI-1 in the TLR2/MyD88 pathway to protect lung tissue of rats with ALI induced by LPS inhalation from injury.

Effect of dexmedetomidine on lung ischemia-reperfusion injury

Dexmedetomidine, a specific selective α2-adrenergic agonist, does not only have the characteristics of being a sedative and analgesic, but also exhibits a protective role in brain ischemia-reperfusion injury and inhibits the inflammation in animals with sepsis. The objective of the present study was to investigate whether dexmedetomidine is capable of attenuating rat pulmonary damage induced by ischemia-reperfusion injury, which is a type of acute sterile lung injury. Sprague-Dawley rats were randomly assigned into six groups: The sham-operated (sham) group, the lung ischemia-reperfusion (I/R) group, intravenous injection of dexmedetomidine 2.5 μg/kg/h (Dex2.5) or 5 μg/kg/h (Dex5) for 1 h prior to ischemia, combination of α2-adrenergic antagonist yohimbine prior to dexmedetomidine pre-treatment (Dex+Yoh) and pre-administration of yohimbine alone (Yoh) prior to ischemia. Lung injury was assessed by the histopathological changes, arterial blood gas, wet/dry (w/d) weight ratio and myeloperoxidase (MPO) activity of the lung. The concentration of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in bronchoalveolar lavage fluid (BALF) was measured by an enzyme-linked immunosorbent assay. The expression of toll-like receptor-4 (TLR4) and myeloid differentiation factor 88 (MyD88) mRNA in the lung were determined by quantitative PCR, and phosphorylated levels of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2 were determined by western blotting. Pre-treatment with dexmedetomidine significantly reduced the lung injury, w/d weight ratio and MPO activity, and decreased the concentration of TNF-α, IL-6 and MCP-1 in BALF compared with the I/R group. The expression of TLR4 and MyD88 mRNA and the levels of phosphorylated JNK and ERK1/2 in the lung tissue were markedly downregulated by intravenous injection of dexmedetomidne for 1 h prior to lung I/R. The protective effects of dexmedetomidine on the lung were not completely reversed by the α2-adrenergic antagonist, yohimbine. Pre-treatment with dexmedetomidine is capable of reducing pulmonary damage and inhibiting sterile inflammation induced by lung I/R injury. TLR4/MyD88/mitogen-activated protein kinase (MAPK) signaling is involved in the protective mechanism of dexmedetomidine through α2-adrenoceptor independence.

Change of Toll-like receptors in children with acute Epstein-barr virus infection

Objective To investigate the possible role of Toll-like receptors (TLRs) in immunological pathogenesis of Epstein-barr virus(EBV) infection. Methods Fifteen children with acute EBV infection, 18 children with infectious mononucleosis (IM), and 25 age-matched healthy children were enrolled in the study.Reverse-transcription polymerase chain reaction and real-time PCR were used to evaluate the levels of TLR2, TLR3, TLR7, TLR9, myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), TGF-β activated kinase 1 (TAK1), TIR domain-containing adaptor protein inducing interferon β (TRIF), tumor necrosis factor receptor-associated factor 3 (TRAF3), TBK1, IL-1β, TNF-α, IFN-α and IFN-β mRNA expression in peripheral blood mononuclear cells(PBMC). The plasma concentrations of cytokines such as IL-12 and interferon gamma (IFN-γ) were determined by enzyme-linked immunosorbent assay. Results 1.Compared with healthy control group, the expression levels of TLR2, TLR3, TLR7, TLR9, MyD88, TRAF6, TAK1, TRIF, TRAF3 and TBK1 mRNA were up-regulated significantly from the children with acute EBV infection and IM (all P 0.05).2.The levels of cytokines expression such as IL-1β, TNF- α, IFN-α and IFN-β in children with acute EBV infection were higher than those of the healthy control group (all P<0.05).3.The plasma concentrations of IL-12 and IFN-γ were up-regulated (all P<0.05) in children with acute EBV infection. Conclusion Imbalance of antiviral /inflammatory response resulting from the aberrant activation of TLRs may be one of the factors causing disturbed immunological function in vivo infected by EBV. Key words: Epstein-barr virus; Infectious mononucleosis; Toll-like receptor