Toll-like Receptor 4 mRNA Expression Research Articles

An oligodeoxynucleotide with CCT repeats restrains CpG ODN-induced TLR9 trafficking.

Toll-like receptor 9 (TLR9) can sense pathogen DNA and CpG ODN or even self-DNA by trafficking assisted by Unc93B1, an endoplasmic reticulum (ER) transmembrane protein, from ER to endolysosomes or cell surface. In previous study, we found that an oligodeoxynucleotide with CCT repeats (SAT05f) could selectively inhibit TLR7/9 activation. However, the mechanism for the inhibitory activity of SAT05f is still unknown. In present research, it was found that SAT05f could inhibit CpG ODN-induced the intracellular trafficking of TLR9 and Unc93B1 with feedback the responses of decreased surface TLR9 and enhanced TLR9 mRNA expression but not influence TLR9 protein level by using human plasmacytoid dendritic cell line CAL-1 cells, suggesting that SAT05f inhibits TLR9 activation by restraining TLR9 trafficking. Since the mitochondrial DNA released from injured tissue can cause systemic inflammatory response syndrome (SIRS), this study may provide valuable data for prevention and treatment of SIRS and rescue severe trauma patients.

Differential Toll-Like Receptor (TLR) mRNA Expression Patterns during Chicken Embryological Development

Toll-like receptors (TLRs), important components of innate immune response, play a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, the expression profile of chicken TLRs (TLR2A, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21) in various chicken embryonic tissues during embryo development was examined by real-time PCR assay. All the TLR mRNAs were expressed in whole embryonic tissue as early as 3rd embryonic day (ED). Four of the seven TLRs (TLR2, TLR3, TLR4, and TLR7) mRNA expressions were significantly (P < 0.01) higher at 12ED relative to expression at 3 ED, whereas TLR15 mRNA expression was significantly (P < 0.01) higher on 7ED and TLR5 and 21 were highly expressed on 18 ED. Among all the TLRs investigated TLR4 mRNA was the highest expressed and TLR15 mRNA expression was the lowest in all tissues during chicken embryo development. Tissue wise analysis of mRNA expression of TLRs showed that liver expressed significantly (P < 0.01) higher levels of most of the genes (TLR2, TLR4, and TLR21). However no significant difference was found in TLR15 mRNA expression among the tissues during development. Our results suggest the innate preparedness of chicken embryos and also a possible role for TLRs in the regulation of chicken embryo development that needs to be further evaluated.

Endotoxin tolerance induction in human periodontal ligament fibroblasts stimulated with different bacterial lipopolysaccharides

ObjectiveHuman periodontal ligament fibroblasts (hPDLFs) may play an important role in immune responses in the periodontal microenvironment by secreting proinflammatory cytokines. The present study aimed to explore the reaction of hPDLFs to a secondary lipopolysaccharide (LPS) challenge by examining cytokines and innate sensors. MethodsPrimary cultures of hPDLFs were obtained and identified by immunochemistry. Cells were treated with a secondary LPS challenge followed by a primary LPS or no LPS as a control. The levels of cytokines were assayed using cytometric bead array (CBA) kits, and the protein levels of Toll-like receptors (TLRs) were determined using flow cytometry. The messenger RNA (mRNA) levels of TLRs were determined using real-time polymerase chain reaction (PCR). ResultsWe show that the initial LPS exposure significantly induced hPDLFs to produce cytokines interleukin-8 (IL-8) and IL-6, whereas the secondary LPS challenge dramatically diminished the levels of cytokines IL-8 and IL-6. The mRNA and protein levels of TLRs were increased by the initial LPS stimulus but decreased by the secondary LPS challenge. ConclusionAn LPS stimulus induces immune responses in hPDLFs, whereas an LPS challenge exerts endotoxin tolerance by downregulating proinflammatory cytokines and TLR mRNA and protein expression. This process may confer hPDLFs with their essential functions for maintaining oral mucosal immunity homeostasis.

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.

Plasminogen activator inhibitor-1 regulates LPS-induced TLR4/MD-2 pathway activation and inflammation in alveolar macrophages.

Toll-like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) are the main lipopolysaccharide (LPS) binding receptors that respond to inflammatory stimuli and mediate NF-kappa B (NF-κB) signaling pathway in macrophages. We have previously shown that plasminogen activator inhibitor-1 (PAI-1) deletion increased lung injury induced by intratracheal instillation of LPS through downregulation of TLR4 negative regulators. However, the mechanisms by which PAI-1 regulates lung inflammation are largely unknown. The aim of this study is to assess the relationship between PAI-1 and TLR4 signaling pathways in LPS-induced NR8383 cells inflammatory reaction. The results showed that the levels of PAI-1, TNF-α, and IL-1β protein were increased remarkably in NR8383 cell supernatants after LPS stimulation. PAI-1 gene knockdown reduced TNF-α and IL-1β levels in cell supernatants and inhibited the NF-κB p65 protein expression in NR8383 cells. The upregulated mRNA and protein expressions of TLR4, MD-2, and myeloid differentiation protein (MyD88) induced by LPS were attenuated after PAI-1 gene knockdown. Conversely, overexpression of PAI-1 in NR8383 cells not only resulted in additional mRNA and protein production of PAI-1, TLR4, MD-2, and MyD88, it also aggravated the inflammatory response induced by LPS. In conclusion, PAI-1 contributes to the regulation of LPS-induced inflammatory response in NR8383 cells, likely by affecting the TLR4-MD-2/NF-κB signaling transduction pathway.

Toll-like receptor 2 mediates ischemia-reperfusion injury of the small intestine in adult mice.

Toll-like receptor 2 (TLR2) recognizes conserved molecular patterns associated with both gram-negative and gram-positive bacteria, and detects some endogenous ligands. Previous studies demonstrated that in ischemia-reperfusion (I/R) injury of the small intestine, the TLR2-dependent signaling exerted preventive effects on the damage in young mice, but did not have a significant effect in neonatal mice. We investigated the role of TLR2 in adult ischemia-reperfusion injury in the small intestine. Wild-type and TLR2 knockout mice at 16 weeks of age were subjected to intestinal I/R injury. Some wild-type mice received anti-Ly-6G antibodies to deplete circulating neutrophils. In wild-type mice, I/R induced severe small intestinal injury characterized by infiltration by inflammatory cells, disruption of the mucosal epithelium, and mucosal bleeding. Compared to wild-type mice, TLR2 knockout mice exhibited less severe mucosal injury induced by I/R, with a 35%, 33%, and 43% reduction in histological grading score and luminal concentration of hemoglobin, and the numbers of apoptotic epithelial cells, respectively. The I/R increased the activity of myeloperoxidase (MPO), a marker of neutrophil infiltration, and the levels of mRNA expression of tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and cyclooxygenase-2 (COX-2) in the small intestine of the wild-type mice by 3.3-, 3.2-, and 13.0-fold, respectively. TLR2 deficiency significantly inhibited the I/R-induced increase in MPO activity and the expression of mRNAs for TNF-α and ICAM-1, but did not affect the expression of COX-2 mRNA. I/R also enhanced TLR2 mRNA expression by 2.9-fold. TLR2 proteins were found to be expressed in the epithelial cells, inflammatory cells, and endothelial cells. Neutrophil depletion prevented intestinal I/R injury in wild-type mice. These findings suggest that TLR2 may mediate I/R injury of the small intestine in adult mice via induction of inflammatory mediators such as TNF-α and ICAM-1.

Toll-Like Receptors 2 and 4 in Pigs' Kidneys Early After Autotransplantation Procedure

ObjectivesThe aim of this paper was to evaluate mRNA expression of Toll-like receptors 2 (TLR2) and 4 (TLR4) and the adaptor protein myeloid differentiation primary-response protein 88 (MyD88) in pigs' kidneys 14 and 30 days after autotransplantation. MethodsThe research was conducted on 12 animals that underwent left renal transplantation procedure with further standardized rinsing with Biolasol solution and 24 hours' storage in 4°C; subsequently the kidneys were implanted in the right retroperitoneal space after right-side nephrectomy. Six randomly chosen animals (group I) were under observation for 14 days, the other 6 (group II) for 30 days. After these observation periods, the animals were killed and 4-g samples were collected from the renal cortex and medulla. ResultsExpression of mRNA in homogenates of collected samples were determined with the use of reverse-transcription polymerase chain reaction analysis. Obtained results in both groups, presented in relation to GAPDH, were compared with the use of Mann-Whitney U test. Stable graft function was observed in all animals from the 2nd day after the procedure. TLR2 in group I reached the mean value of 3.64 and was statistically significantly higher than in group II (2.19). Inverse proportion was observed in case of mRNA for TLR4: group II presented 2 times higher value than group I (0.25 vs 0.11). Similarly, significant difference was observed in MyD88 (group I, 0.067; group II, 0.45). ConclusionsAt 14 days after autotransplantation of a pig kidney, mRNA expression for TLR2 is dominant; later, expression increases for TLR4 and MyD88.

Irsogladine maleate inhibits Porphyromonas gingivalis-mediated expression of toll-like receptor 2 and interleukin-8 in human gingival epithelial cells.

Periodontitis is an infectious disease caused by an interaction between the host and periodontopathogenic bacteria. Regulating the immune response in human gingival epithelial cells (HGEC) may contribute to the prevention of periodontitis. Irsogladine maleate (IM) has previously been shown to regulate inflammation and the cell-cell junctional barrier in HGEC. In addition to these functions, control of bacterial recognition is important for preventing inflammation in periodontal tissue. Innate immunity in gingival epithelium is the first line of defense and plays a crucial role against bacterial challenge. Therefore, the effect of IM on regulating toll-like receptor 2 (TLR2), which is part of the innate immunity, was determined in this study. OBA-9, an immortalized human gingival epithelial cell line, and primary cultured HGEC were used in this study. Real-time PCR and western blotting were performed in OBA-9 or HGEC stimulated with whole cells of Porphyromonas gingivalis or with lipopolysaccharide (LPS) derived from P. gingivalis (PgLPS) in the presence or absence of IM to determine expression of TLR2 mRNA and production of TLR2 protein. Small interfering RNA (siRNA) against TLR2 was transfected into OBA-9 to clarify the association between the induction of TLR2 and interleukin-8 (IL-8) production. The addition of IM into P. gingivalis or PgLPS-induced OBA-9 suppressed IL-8 production (p < 0.01). The addition of IM also abolished the induction of TLR2 by P. gingivalis or PgLPS in OBA-9 and primary cultured HGEC (p < 0.01). The suppressive effect of IM on the induction of TLR2 was also confirmed by immunohistostaining. Stimulation with peptidoglycan, a specific ligand for TLR2, suppressed the expression of toll-like receptor 4 (TLR4) mRNA in the presence of IM (p < 0.01). However, LPS derived from Escherichia coli, a ligand for TLR4, did not induce the expression of TLR2 mRNA. The PgLPS-induced expression of TLR4 mRNA was abolished by IM. Knockdown of TLR2 by siRNA transfection resulted in a weaker response of induction of IL8 mRNA in P. gingivalis or PgLPS-stimulated OBA-9. These results suggest that IM suppresses the induction of IL-8 production by regulating increased levels of TLR2.

Toll-like receptor (TLR) expression and TLR‑mediated interleukin-8 production by human submandibular gland epithelial cells.

Toll-like receptor (TLR) family members are pattern recognition receptors that are essential in the activation of innate and adaptive immune responses. Submandibular gland epithelial cells (SMGCs) may recognize microbial components through TLRs and be involved in the development of inflammatory reactions in the submandibular glands. Therefore, the functional expression of TLRs in SMGCs was investigated in the present study. The mRNA expression of TLRs in SMGC and whole submandibular tissues was determined by RT-PCR. Subsequently, the effects of various TLR agonists and tumor necrosis factor alpha (TNF-α) on IL-8 production were examined using an ELISA. SMGCs, as well as whole submandibular tissues, expressed TLR1-10 mRNA. Furthermore, interleukin (IL)-8 production in SMGCs was increased by Pam3CSK4 (TLR1/2 agonist), polyI:C (TLR3 agonist), E.coli lipopolysaccharide (LPS; TLR4 agonist), flagellin (TLR5 agonist) and macrophage‑activating lipopeptide (MALP)-2 (TLR2/6 agonist) treatments in a dose‑dependent manner, whereas administration of either imiquimod (TLR7 agonist) or CpG-oligodeoxynucletide (TLR9 agonist) exerted no evident effect. Pam3CSK4, polyI:C, LPS, flagellin and MALP-2 also enhanced TNF‑α‑induced IL-8 production in SMGCs. These findings suggest that innate immune responses against microbial components result in the development of TNF-α-mediated autoimmune inflammatory disease in the submandibular glands.

Inhibitory effect of polydatin on expression of toll-like receptor 4 in ischemia-reperfusion injured NRK-52E cells

Polydatin is a monocrystaline compound isolated from Polygonum cuspidatum Sieb. et Zucc. (Polygonaceae) with biological properties, such as anti-inflammation, anti-oxidative and nephroprotective effects. Increasing number of studies have demonstrated the protective effect of polydatin on renal ischemia reperfusion injury. However, the possible mechanisms of this protection are not fully elucidated. This study aimed to investigate the effect of polydatin on ischemia-reperfusion induced expression of toll-like receptor4 (TLR4) in rat renal tubular epithelia cells (NRK-52E), and analyze the mechanism of polydatin on TLR4 signal pathway. The cultured NRK-52E cells were incubated in three gas incubators for a period of 6 h at hypoxia and 24h at reoxygenation to simulate the ischemia-reperfusion injury in vitro. TLR4 mRNA level was analyzed by real-time-PCR, and the protein expression of TLR4 and NF-κB by Western blotting, while TNF-α and IL-1β proteins expressions were detected by ELISA. Polydatin downregulated I/R induced mRNA and protein expressions of TLR4, and decreased the protein expression of NF-κB, TNF-α and IL-1β. The TLR4 blocker partially antagonized the effect of I/R on NF-κB signaling, and such inhibitory effect was markedly enhanced by polydatin. In the present study, polydatin protects NRK-52E cells from I/R injury possibly by relieving the inflammatory response through regulation of TLR4/NF-κB signaling pathway.

Postconditioning improvement effects of ulinastatin on brain injury following cardiopulmonary resuscitation.

The aim of the present study was to determine the effects of ulinastatin (UTI) on brain injury in rats subjected to cardiopulmonary resuscitation (CPR) following asphyxial cardiac arrest (CA) and identify the underlying mechanisms. In total, 100 healthy male Wistar rats were randomly divided into control and treatment groups (n=50). After 4 min of asphyxial CA, all the rats were immediately subjected to CPR. The treatment group animals were administered 15 mg/kg UTI at the onset of resuscitation. The mortality rate in the two groups was recorded at 24 h post-resuscitation. In addition, neurological function was evaluated at 24, 48 and 72 h post-resuscitation using a neurological deficit scale (NDS). Furthermore, the effects of UTI on the Toll-like receptor 4 (TLR4) signaling pathway in brain tissues were determined by assessing TLR4 mRNA expression, nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels at 1, 3, 6, 12, 24, 48 and 72 h post-resuscitation. After 24 h, the mortality rate significantly decreased in the treatment group when compared with the control animals (10 vs. 30%; P<0.05). Additionally, an overt improvement was observed in the NDS score following UTI treatment when compared with the control (P<0.01). Finally, statistically significant decreases in the levels of TLR4 mRNA expression, NF-κB activity and TNF-α and IL-6 were observed in the treatment group at each time point (P<0.01). Therefore, UTI treatment at the onset of CPR significantly inhibits the TLR4 signaling pathway, thereby alleviating the inflammatory responses following resuscitation and improving neurological function.

Studies on the correlation between the expression of Toll-like receptor 4 and the synovitis of the temporomandibular joint in rats

To investigate the expression of the Toll-like receptor-4 (TLR-4) in temporo-mandibular joint synovitis in rats, and to discuss the correlation between the expression of TLR-4 and the synovitis. Sixty male wistar rats were randomly divided into five groups, 12 each. Group A was the control group in which the rats were given normal diet.In Group B, the rats' bilateral masseter muscles were cut off (masseter resection group). In Group C, An cast metal crown were bonded on the mandibular right first molar of each rat (occlusal interference group). In Group D, occlusal pad were bonded on maxillary molars of each rat (occlusal dimension increase group). In Group E, rats' bilateral masseter muscles were re-sected and occlusal pads were bonded on their maxillary molars (masseter resection and occlusal dimension increase group). Pathological changes of synovium were observed using hematoxylin and eosin (HE) stains and pathology scores were evaluated. The expression of TLR- 4 were determined by immunohistochemical stains, and the expression of TLR-4 mRNA were determined by real-time PCR. The correlation between the expression of TLR-4, TLR-4 mRNA and the pathological score were analyzed using Spearman analysis. The pathological scores of Group A-E were 0.5 ± 0.5, 2.5 ± 1.0, 2.7 ± 1.0, 3.0 ± 0.9, 5.3 ± 1.2 respectively. The expression of TLR-4 were (3.2 ± 1.5)%, (16.± 2.6)%, (15.8 ± 2.1)%, (17.5 ± 2.4)%, (38.2 ± 4.4) %. The expression of TLR-4 mRNA were 1.07 ± 0.09, 2.12 ± 0.33, 2.07 ± 0.29, 2.17 ± 0.34, 4.53 ± 0.46. Compared with group A, groups B- E showed significant higher pathology score (P < 0.05) and increased expression of both TLR-4 (P < 0.05) and TLR-4 mRNA (P < 0.05). An significant positive correlation was found between the expression of TLR- 4 and the pathology score (r = 0.785, P < 0.05), and between the expression of TLR- 4 mRNA and the pathology score (r = 0.720, P < 0.05). TLR-4 may be closely associated with the development of the synovitis of TMJ of rats.

Mechanism of mitigation of neuropathic pain by spinal cord stimulation in rats: the relationship with HMGB1/TLR4/NF-κB signaling pathway in the spinal cord

Objective To investigate the relationship between the mechanism of mitigation of neuropathic pain by spinal cord stimulation (SCS) and high mobility group protein box-1 (HMGB1)/Toll-like receptor 4 (TLR4)/NF-κB signaling pathway in the spinal cord of rats.Methods Forty-eight male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were randomly divided into 4 groups (n =12 each) using a random number table:sham operation group (S group),chronic constrictive injury (CCI) group,sham SCS group (S-SCS group),and SCS group.Neuropathic pain was induced by CCI in the animals anesthetized with intraperitoneal chloral hydrate.The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 silk thread.Electrodes were placed into the epidural space at 5 days after CCI in S-SCS and SCS groups,and in addition SCS was performed at 12-14 days after CCI in SCS group.Paw withdrawal threshold to mechanical stimulation (MWT) were measured at 1 day before operation and 1,4,7,14 days after CCI.After measurement of pain threshold at day 14 after CCI,the animals were sacrificed and the lumbar segments (L4-6) of the spinal cord were obtained for determination of mRNA expression of HMGB1,TLR4,and NF-κB p65 (in the nuclear protein,by real-time fluorescent quantitative PCR),protein expression of TLR4 and NF-κBp65 (by Western blot),and protein expression of HMGB1 (by immunohistochemistry).Results Compared with S group,MWT was significantly decreased after CCI,the mRNA and protein expression of HMGB1 and TLR4 was up-regulated,and the expression of NF-κB p65in the nuclear protein and mRNA was up-regulated in CCI,S-SCS and SCS groups.Compared with CCI group,MWT was significantly increased after spinal cord stimulation,the mRNA and protein expression of HMGB1 and TLR4 was down-regulated,and the expression of NF-κB p65 in the nuclear protein and mRNA was down-regulated in SCS group.Conclusion The mechanism by which SCS mitigates neuropathic pain may be related to inhibition of HMGB1/TLR4/NF-κB signaling pathway in the spinal cord of rats. Key words: Electric stimulation; Neuralgia; Spinal cord; High mobility group proteins; Toll-like receptor 4; NF-kappaB

Postconditioning improvement effects of ulinastatin on brain injury following cardiopulmonary resuscitation

The aim of the present study was to determine the effects of ulinastatin (UTI) on brain injury in rats subjected to cardiopulmonary resuscitation (CPR) following asphyxial cardiac arrest (CA) and identify the underlying mechanisms. In total, 100 healthy male Wistar rats were randomly divided into control and treatment groups (n=50). After 4 min of asphyxial CA, all the rats were immediately subjected to CPR. The treatment group animals were administered 15 mg/kg UTI at the onset of resuscitation. The mortality rate in the two groups was recorded at 24 h post‑resuscitation. In addition, neurological function was evaluated at 24, 48 and 72 h post‑resuscitation using a neurological deficit scale (NDS). Furthermore, the effects of UTI on the Toll‑like receptor 4 (TLR4) signaling pathway in brain tissues were determined by assessing TLR4 mRNA expression, nuclear factor (NF)‑κB activity and tumor necrosis factor (TNF)‑α and interleukin (IL)‑6 levels at 1, 3, 6, 12, 24, 48 and 72 h post‑resuscitation. After 24 h, the mortality rate significantly decreased in the treatment group when compared with the control animals (10 vs. 30%; P<0.05). Additionally, an overt improvement was observed in the NDS score following UTI treatment when compared with the control (P<0.01). Finally, statistically significant decreases in the levels of TLR4 mRNA expression, NF‑κB activity and TNF‑α and IL‑6 were observed in the treatment group at each time point (P<0.01). Therefore, UTI treatment at the onset of CPR significantly inhibits the TLR4 signaling pathway, thereby alleviating the inflammatory responses following resuscitation and improving neurological function.

TLR9 and BAFF: Their expression in patients with IgA nephropathy

Since it was first described in 1968, immunoglobulin (Ig)A nephropathy (IgAN) has become the most commonly diagnosed form of primary glomerular disease worldwide. A number of reports have shown that toll‑like receptor 9 (TLR9) and B‑cell activating factor (BAFF) may be associated with IgAN; however, sufficient evidence has not yet to be delivered. In the present study, serum levels of BAFF as well as TLR9 mRNA and protein levels in peripheral blood mononuclear cells (PBMCs) were assessed. Expression of TLR9 mRNA in PBMCs was examined by quantitative polymerase chain reaction and the TLR9 protein was determined by western blot analysis. The levels of serum BAFF and IgA1 were determined by specific ELISA. Serum levels of BAFF and IgA1 as well as levels of TLR9 mRNA and protein in PMBCs were significantly higher in patients with IgAN compared with patients with minimal glomerular abnormalities (P<0.05, P<0.01, P<0.01 and P<0.01, respectively) and normal controls (P<0.01, P<0.01, P<0.05 and P<0.01, respectively). A correlation and regression analysis was performed to determine the pathogenesis of IgAN. In patients with IgAN, serum levels of BAFF were positively correlated with IgA1 levels (rp, 0.515; P<0.01) and mesangial IgA deposition density (rp, 0.746; P<0.01). Expression levels of TLR9 protein in PBMCs of IgAN patients were positively correlated with levels of serum BAFF (rp, 0.444; P<0.05) and IgA1 (rp, 0.633; P<0.01). These results suggested that overexpression of TLR9 mRNA and protein in PBMCs and elevated levels of serum BAFF may be associated with overexpression of serum IgA1, and, furthermore, may have a role in the development of IgAN.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer.

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

Toll-like receptor 4 monoclonal antibody attenuates lipopolysaccharide-induced acute lung injury in mice.

Toll-like receptor 4 (TLR4) has an important role in the recognition of lipopolysaccharide (LPS) and in the activation of the inflammatory cascade. In the present study, the effect of TLR4 monoclonal antibody (mAb) on LPS-induced acute lung injury (ALI) was investigated in mice. A total of 45 male BALB/c mice were randomly divided into three groups, namely, the control (group C), sepsis (group S) and pretreatment groups (group P). Mice in group P were intraperitoneally treated with TLR4 mAb 1 h prior to the intraperitoneal administration of LPS. Following treatment with LPS for increasing times periods in groups S and P, the mRNA expression level of TLR4 in the lung tissue and the expression of inflammatory factors in the serum were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. The degree of pulmonary edema, expressed as (wet weight - dry weight)/wet weight, as well as the lung injury scores, observed using a light microscope, were also analyzed. The results demonstrated that intraperitoneal administration of LPS in mice increased the mRNA expression levels of TLR4, the secretion of inflammatory factors in the serum, the degree of pulmonary edema and the lung injury score in a time-dependent manner. However, pretreatment with TLR4 mAb effectively attenuated the increased mRNA expression of TLR4 and the overproduction of inflammatory factors to correct the pulmonary edema and the elevated lung injury score induced by LPS. Therefore, TLR4 plays a critical role in LPS-induced ALI, and the TLR4 mAb decreases the secretion of inflammatory factors and attenuates the degree of pulmonary edema, thereby protecting the lungs from LPS-induced ALI.

N-(2-hydroxy phenyl) acetamide: a novel suppressor of Toll-like receptors (TLR-2 and TLR-4) in adjuvant-induced arthritic rats.

Toll-like receptors (TLRs) are key recognition structures of immune system and recently emerged as potential contributors to the inflammation observed in human and rodent models of arthritis. Present study aims to investigate the effect of N-(2-hydroxy phenyl)-acetamide (NA-2) on modulation of TLRs in the development of adjuvant-induced arthritis. Arthritis was induced by intradermal administration of heat-killed Mycobacterium tuberculosis H37Ra. The treatment of NA-2 (5 mg/kg) and indomethacin (5 mg/kg) was started in their respective group on the day of arthritis induction. Body weights, paw volume measurements, and nociception sensation (Plantar test) were done on alternate days to monitor the progression of the disease until arthritis score of four was observed in arthritic control group. Along with the clinical signs, histopathology of knee joints was also performed. The splenocytes cultures were prepared from each group; TLR-2 and TLR-4 mRNAs were analyzed in 48-h cultured splenocytes using RT-PCR; and the supernatants were used to determine IL-1β and TNF-α by ELISA. A significant reversal of deficit seen in body weights of the arthritic control group was observed in NA-2-treated animals with a parallel decrease in paw edema and transmission of nociception. Remission of the clinical signs and nociception was associated with improved histology. Compared with arthritic control, NA-2 treatment significantly decreased the level of IL-1β (p < 0.003) and TNF-α (p < 0.001) in the supernatants of cultured splenocytes. Likewise, NA-2 also reduced the expression of TLRs mRNA. Our findings suggest that NA-2 affects AIA in a pleiotropic manner, suppressing TLRs-mediated joint inflammation and related symptoms.

Bacteria and Toll-like receptor and cytokine mRNA expression profiles associated with canine arthritis

The major forms of inflammatory canine arthritis are immune-mediated arthritis (IMA) and septic arthritis (SA), although some cases of cruciate disease (CD) are associated with significant levels of synovitis. In this study, the bacteria associated with canine arthritis were identified and mRNA expression levels of Toll-like receptors (TLRs) and pro-inflammatory cytokines determined. Of the 40 synovial fluid samples analysed, bacteria were isolated from 12 samples by culture (2 CD, 10 SA) and detected in 4 samples (3 CD, 1 SA) using culture-independent methods. Statistically significant increases in TLR2, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12 mRNA expression were seen in all disease groups compared to normal controls. All disease groups had decreased mRNA expression of other TLRs compared to normal controls, but this did not reach statistical significance. Synovial fluid cell counts revealed that the highest number and proportion of mononuclear cells and neutrophils were found in the IMA and SA samples, respectively. Age had an effect on the TLR and cytokine mRNA expression profiles: TNF-α (p=0.043) and IL-12 (p=0.025) mRNA expression was increased and TLR4 mRNA expression was reduced (p=0.033) in dogs up to 4 years of age compared to older animals. In the 10 SA samples from which bacteria were isolated, statistically significant increases in TLR2, TLR7, TNF-α and IL-6 mRNA expression were observed. It is concluded that canine arthritis is associated with increased mRNA levels of pro-inflammatory cytokines, which could in some cases be mediated by bacteria through activation of TLR2.

Influences and mechanisms of somatostatin on inflammation in endotoxin-induced acute lung injury mice

To investigate the effects of somatostatin on inflammation in endotoxin-induced acute lung injury mice and its underlying mechanisms. 72 ICR male mice of specific pathogen free (SPF) were randomly divided into four groups: control group, SST control group, model group and SST intervention group, each group included 18 mice. The ALI model was reproduced by intraperitoneal injection of 40 mL/kg endotoxin, and the mice in control group was given intraperitoneal injection of equivalent normal saline. The mice in SST intervention group was given the subcutaneous injection of SST (20 μg, 20 mL/kg) at 0.5, 2, 6 and 12 hours after model reproduction. Six mice were sacrificed at 3, 8 and 16 hours after the first injection of LPS or NS. The lung wet/dry ratio (W/D) was calculated with oven drying method. Pathological changes in lung tissues were observed by light microscope. Serum SST, tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-10) levels were determined by enzyme linked immunosorbent assay (ELISA). The mRNA and protein expressions of Toll like receptor 4 (TLR4) and nuclear factor-ΚBp65 (NF-ΚBp65) were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot. There were no significant differences in above indexes between control group and SST control group. Compared with control group, lung W/D ratio, serum SST, TNF-α, IL-6, IL-10, and the mRNA and protein expressions of TLR4 and NF-ΚBp65 in model group were significantly increased. The W/D ratio, IL-6, IL-10, and the mRNA and protein expressions of TLR4 all peaked at 16 hours (W/D ratio: 6.32±0.18 vs. 4.14±0.14, IL-6: 673.78±56.13 ng/L vs. 50.17±7.06 ng/L, IL-10: 481.13±40.78 ng/L vs. 61.71±10.05 ng/L, TLR4 mRNA: 0.740±0.099 vs. 0.180±0.028, TLR4 protein: 0.935±0.067 vs. 0.222±0.019, all P<0.05), and SST, TNF-α, the mRNA and protein expressions of NF-ΚBp65 peaked at 8 hours (SST: 254.97±38.75 ng/L vs. 95.87±13.95 ng/L, TNF-α: 139.69±19.06 ng/L vs. 21.90±4.52 ng/L, NF-ΚBp65 mRNA: 0.753±0.065 vs. 0.190±0.026, NF-ΚBp65 protein: 1.214±0.079 vs. 0.303±0.067, all P<0.05). Compared with model group, the lung injury in SST intervention group was significantly improved, and the indexes were significantly decreased except for serum SST which was gradually increased (16-hour W/D ratio: 5.21±0.14 vs. 6.32±0.18, 8-hour TNF-α: 80.48±8.52 ng/L vs. 139.69±19.06 ng/L, 16-hour IL-6: 394.99±37.17 ng/L vs. 673.78±56.13 ng/L, 16-h IL-10: 326.95±36.41 ng/L vs. 481.13±40.78 ng/L, 16-hour TLR4 mRNA: 0.240±0.028 vs. 0.740±0.099, 16-hour TLR4 protein: 0.618±0.066 vs. 0.935±0.067, 8-hour NF-ΚBp65 mRNA: 0.240±0.045 vs. 0.753±0.065, 8-hour NF-ΚBp65 protein: 0.784±0.041 vs. 1.214±0.079, all P<0.05). SST has significant protective effects on endotoxin-induced ALI via direct suppression of the TLR4-NF-ΚB cytokine pathway, thus alleviate lung tissue inflammation.