Toll-like Receptor 4 mRNA Expression Research Articles

Surfactant protein-D attenuates the lipopolysaccharide-induced inflammation in human intestinal cells overexpressing toll-like receptor 4.

Necrotizing enterocolitis (NEC) is a devastating inflammatory disease of preterm infants that may depend on overexpression of toll-like receptor-4 (TLR4) in the immature intestine. Surfactant protein (SP)-D is a member of the collectin family and plays an important role in innate immunity, particularly in the airways. Although SP-D also exists in the intestines, little is known about its function. This study investigated whether SP-D can attenuate the inflammatory response of TLR4-overexpressing embryonal intestinal cells. All experimental procedures were performed using the human intestinal cell line INT407 originally derived from human embryonal intestines. Platelet-activating factor (PAF), reported to be elevated in NEC patients, was used to induce TLR4 overexpression in the human embryonal intestinal cell line INT407. TLR4 expression was measured using quantitative real-time PCR. Inflammatory responses to PAF (5µM), the TLR4 agonist lipopolysaccharide (LPS, 100ng/ml), PAF+LPS, and PAF+LPS following SP-D pretreatment (20µg/ml) were assessed by enzyme-linked immunosorbent assay (ELISA) of interleukin-8 (IL-8) release (in pg/ml). Expression of TLR4 mRNA (mean±SD) was upregulated by PAF (369%±28%, p<0.001). Stimulation with PAF+LPS resulted in higher IL-8 release (1959.3±52.3) than control (141.2±12.4), LPS (167.3±65.8), or PAF (1527.2±129.4) treatment (p<0.05). Release in response to PAF+LPS (1590.1±319.3) was attenuated by SP-D pretreatment (1161.6±131.6; p<0.05). SP-D attenuates LPS-induced IL-8 production in TLR4-overexpressing intestinal cells, suggesting that SP-D may have a protective effect in the development of NEC in preterm infants.

Vitamin D inhibits lipopolysaccharide-induced inflammatory response potentially through the Toll-like receptor 4 signalling pathway in the intestine and enterocytes of juvenile Jian carp (Cyprinus carpio var. Jian).

The present study was conducted to investigate the anti-inflammatory effect of vitamin D both in juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and in enterocytes in vitro. In primary enterocytes, exposure to 10 mg lipopolysaccharide (LPS)/l increased lactate dehydrogenase activity in the culture medium (P<0·05) and resulted in a significant loss of cell viability (P<0·05). LPS exposure increased (P<0·05) the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8), which was decreased by pre-treatment with 1,25-dihydroxyvitamin D (1,25D3) in a dose-dependent manner (P<0·05). Further results showed that pre-treatment with 1,25D3 down-regulated Toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (Myd88) and NF-κB p65 mRNA expression (P<0·05), suggesting potential mechanisms against LPS-induced inflammatory response. In vivo, intraperitoneal injection of LPS significantly increased TNF-α, IL-1β, IL-6 and IL-8 mRNA expression in the intestine of carp (P<0·05). Pre-treatment of fish with vitamin D3 protected the fish intestine from the LPS-induced increase of TNF-α, IL-1β, IL-6 and IL-8 mainly by downregulating TLR4, Myd88 and NF-κB p65 mRNA expression (P<0·05). These observations suggest that vitamin D could inhibit LPS-induced inflammatory response in juvenile Jian carp in vivo and in enterocytes in vitro. The anti-inflammatory effect of vitamin D is mediated at least in part by TLR4-Myd88 signalling pathways in the intestine and enterocytes of juvenile Jian carp.

The Toll-like receptors mRNA expression profile in the pineal gland of sheep during long and short days

The Toll-like receptors mRNA expression profile in the pineal gland of sheep during long and short days

Carnosic acid disrupts toll-like receptor 2 signaling pathway in Pam3CSK4-stimulated macrophages

Carnosic acid (CA) is a major bioactive component of rosemary (Rosmarinus officinalis) leaves and is known to exhibit anti-inflammatory activities. However, its molecular action in the inflammatory process has not been fully elucidated. In the present study, the anti-inflammatory effect of CA on Toll-like receptor 2 (TLR2) pathways in the Pam3CSK4-stimulated macrophage was investigated. Bone marrowderived macrophages (BMDM) were treated with CA (0-30 μM) for 1 h followed by Pam3CSK4 for 1 h, and then the mRNA expression levels and the production levels of cytokines were measured. Next, TLR2-related molecules were measured in Pam3CSK4-stimulated RAW264.7 macrophages. The results showed that CA significantly suppressed the secretion and mRNA expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in Pam3CSK4-stimulated BMDM. TLR2 mRNA expression, nuclear factorkappa B (NF-κB) activation, and mitogen-activated protein kinase (MAPK) phosphorylation were markedly diminished with CA (10 μM and 30 μM) in Pam3CSK4-stimulated macrophages. In conclusion, the anti-inflammatory activity of CA is partly due to modulation of the TLR2 and MAPK/NF-κB signaling pathway in macrophages.

Activin suppresses LPS-induced Toll-like receptor, cytokine and inducible nitric oxide synthase expression in normal human melanocytes by inhibiting NF-κB and MAPK pathway activation.

Activins are dimeric growth and differentiation factors that belong to the transforming growth factor(TGF)-β superfamily of structurally related signaling proteins. In the present study, we examined the mechanisms through which activin regulates the lipopolysaccharide(LPS)-induced transcription of Toll-like receptors(TLRs), cytokines and inducible nitric oxide synthase(iNOS) in human melanocytes, as well as the involvement of nuclear factor(NF)-κB and mitogen-activated protein kinase(MAPK) signaling. Cell proliferation was analyzed by cell viability assay, mRNA expression was detected by RT-qPCR, and protein expression was measured by western blot analysis. LPS increased the mRNA expression of TLRs(TLR1-10) and cytokines [interleukin(IL)-1β, IL-6, IL-8 and TNF-α], as well as the mRNA and protein expression of iNOS. Activin decreased the LPS-induced TLR and cytokine mRNA expression, as well as the LPS-induced iNOS mRNA and protein expression. In addition, activin suppressed NF-κBp65 activation and blocked inhibitor of NF-κB(IκBα) degradation in LPS-stimulated melanocytes, and reduced LPS-induced p38MAPK and MEK/ERK activation. On the whole, our results demonstrated that activin inhibited TLR and cytokine expression in LPS-activated normal human melanocytes and suppressed LPS-induced iNOS gene expression. Moreover, the anti-inflammatory effects of activin were shown to be mediated through the suppression of NF-κB and MAPK signaling, resulting in reduced TLR and iNOS expression, and in the inhibition of inflammatory cytokine expression.

Expression significance of Toll-like receptor 4 and myeloid derived suppressor cells in children with acute myeloid leukemia

Objective To investigate the expression of Toll-like receptor 4(TLR4) and myeloid derived suppressor cells(MDSC) in bone marrow cells in children with acute myeloid leukemia (AML), and to detect its relationship with the clinical features, the effect of chemotherapy and prognosis. Methods Twenty-nine cases of children with AML were collected from June 2013 to March 2014 in People's Hospital of Wuhan University, in which 11 cases of low-risk group, 10 cases of middle-risk group, 8 cases of high-risk group; and 17 cases of non blood disease was as the control group.The expressions of TLR4 and MDSC were detected by using reverse transcription-polymerase chain reaction(RT-PCR), Western blot methods, immunohistochemical staining, and flow cytometry, respectively, in the bone marrow cells of 29 children with AML. Results The mRNA and protein expression of TLR4 in the initial treatment group was higher than those in the complete remission group(t=3.092, 3.393, all P<0.05). The mRNA and protein expression of TLR4 in the relapse group was higher than those in the complete remission group(t=4.013, 4.279, all P<0.05). The positive expression rates of MDSC in the above 3 groups were(29.77±1.39)%, (5.19±0.65)%, (38.62±3.54)%, respectively, compared with the control group [(1.32±0.27)%] and there was significant difference(all P<0.05). The positive expression rates of TLR4 and MDSC in the initial treatment group, relapse group and complete remission group were significantly higher than those in the control group, with significant differences (initial treatment group TLR4: t=3.559, P<0.05; MDSC: t=3.727, P<0.05; relapse group TLR4: t=4.043, P<0.05; MDSC: t=4.125, P<0.05; complete remission group TLR4: t=2.798, P<0.05; MDSC: t=3.469, P<0.05). Pearson rank correlation analysis showed that there was a positive correlation between the expression of TLR4 and MDSC (r=0.673, P<0.01). Conclusions The expressions of both TLR4 and MDSC play an important role in onset, progression, curative effect and prognosis in children with AML, and the two may play an importment role in synergistic effect. Key words: Acute myeloid leukemia; Toll-like receptor 4; Myeloid derived suppressor cells

Effects of penehychdine hydrochloride on TLR4 mRNA expression during endotoxin-induced acute lung injury in neonatal rats

Objective To evaluate the effects of penehychdine hydrochloride on Toll-like receptor 4 (TLR4) mRNA expression during endotoxin-induced acute lung injury (ALI) in neonatal rats. Methods Thirty pathogen-free male Wistar rats, aged 7 days, weighing 12-18 g, were randomly divided into 3 groups (n=10 each) using a random number table: normal saline group (group NS), ALI group, and penehychdine hydrochloride group (group PH). ALI was induced with intraperitoneal endotoxin 5 mg/kg. In group PH, penehychdine hydrochloride 0.5 mg/kg was injected intraperitoneally at 30 min before ALI, while the equal volume of normal saline was given instead in group NS.At 4 h after endotoxin injection, the rats were sacrificed, and lungs were removed for determination of TLR4 mRNA expression (using real-time reverse transcriptase polymerase chain reaction), and contents of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 in the lung tissues (by enzyme-linked immunosorbent assay), and for examination of the pathologic changes of lung (with electron microscope). Results Compared with group NS, TLR4 mRNA expression was significantly up-regulated, and contents of TNF-α, IL-1β, and IL-10 were increased in ALI and PH groups.Compared with group ALI, TLR4 mRNA expression was significantly down-regulated, and contents of TNF-α, IL-1β, and IL-10 were decreased in group PH.The pathologic changes of lung were significantly mitigated in group PH compared with group ALI. Conclusion Penehychdine hydrochloride inhibits inflammatory responses through down-regulating TRL4 mRNA, thus reducing endotoxin-induced ALI in neonatal rats. Key words: Cholinergic antagonists; Infant, newborn; Endotoxemia; Respiratory distress syndrome, adult; Toll-like receptor 4

LPS-RS attenuation of lipopolysaccharide-induced acute lung injury involves NF-κB inhibition.

In this study, we studied the effect of lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS), an inhibitor of Toll-like receptor 4 (TLR4), in LPS-induced acute lung injury (ALI). Male Sprague-Dawley rats were treated with LPS-RS (0.1 mg/kg body mass, by intraperitoneal (i.p.) injection) 1 h before LPS injection (10 mg/kg, i.p.). Bronchoalveolar lavage fluid (BALF) and lung tissues were collected 24 h later to determine total and differential cell count, total protein content, levels of lactate dehydrogenase (LDH), histopathological changes, markers of oxidative stress, and mRNA expression of the inhibitory protein nuclear factor kappaB-α (NFκBIA) and TLR4. Additionally, rings of pulmonary artery were isolated for measuring vascular reactivity. LPS-induced ALI was indicated by increases in total and differential cell count, total protein, and LDH in BALF, and increased lung levels of malondialdehyde (MDA), as well as decreased activity of reduced glutathione (GSH) and superoxide dismutase (SOD). Moreover, LPS increased pulmonary artery contraction in response to phenylephrine (PE). Additionally, LPS downregulated mRNA expression of NFκBIA and upregulated mRNA expression of TLR4. LPS caused a marked inflammation in the lung tissue, with tubercular granuloma and numerous neutrophils. Pretreatment with LPS-RS protected against LPS-induced ALI by decreasing total and differential cell count, total protein, and LDH in BALF, and increased pulmonary GSH content and SOD activity without affecting MDA content. Additionally, it decreased the elevated PE-induced pulmonary artery contraction. LPS-RS upregulated mRNA expression of NFκBIA and downregulated mRNA expression of TLR4. Moreover, LPS-RS prevented inflammation in lung tissues. In conclusion, pretreatment with LPS-RS protects against LPS-induced ALI in rats through its anti-inflammatory effects, possibly by decreasing the mRNA expression of TLR4 and increasing that of NFκBIA.

Functional activity but not gene expression of toll-like receptors is decreased in the preterm versus term human placenta

Toll-like receptor (TLR) activity within gestation-associated tissues might have a role in normal pregnancy progression as well as adverse obstetric outcomes such as preterm birth (PTB). The expression and activity of TLRs 1-9 in placentas collected following preterm vaginal delivery after infection-associated preterm labour (IA-PTL) at 25-36 weeks of gestation (preterm-svd, n=10) were compared with those obtained after normal vaginal delivery at term (term-laboured; n=17). Placental explants were cultured in the presence of agonists for TLR2, 3, 4, 5, 7, 8 and 9 and cytokine production after 24h examined. Expression of TLR transcripts was determined using real time quantitative PCR. Reactivity to all agonists except CpG oligonucleotides was observed indicating that other than TLR9 all of the receptors studied yielded functional responses both term and preterm. Significantly less TNFα and IL-6, but not IL-10, were produced by preterm than term samples in response to all TLR agonists. Changes in TLR mRNA expression did not underlie functional differences in the preterm and term groups; nor does a pre-exposure/tolerance model mimic this finding. While glucocorticoids suppressed cytokine production in an invitro model using term tissue the association between lower gestational age and decreased cytokine outputs suggests a temporally regulated response. Pro-inflammatory cytokine output in response to multiple TLR ligands was decreased in the preterm compared to the term placenta but gene expression for each TLR tended to be similar. Reduced cytokine production by the preterm placenta in response to stimulation of TLRs therefore must be regulated at the post-transcriptional level in a gestational age dependent manner.

Lipopolysaccharide and toll-like receptor 4 in dogs with congenital portosystemic shunts

Surgical attenuation of a congenital portosystemic shunt (CPSS) results in increased portal vein perfusion, liver growth and clinical improvement. Portal lipopolysaccharide (LPS) is implicated in liver regeneration via toll-like receptor (TLR) 4 mediated cytokine activation. The aim of this study was to investigate factors associated with LPS in dogs with CPSS. Plasma LPS concentrations were measured in the peripheral and portal blood using a limulus amoebocyte lysate (LAL) assay.LPS concentration was significantly greater in the portal blood compared to peripheral blood in dogs with CPSS (P = 0.046) and control dogs (P = 0.002). LPS concentrations in the peripheral (P = 0.012) and portal (P = 0.005) blood of dogs with CPSS were significantly greater than those of control dogs. The relative mRNA expression of cytokines and TLRs was measured in liver biopsies from dogs with CPSS using quantitative PCR. TLR4 expression significantly increased following partial CPSS attenuation (P = 0.020). TLR4 expression was significantly greater in dogs that tolerated complete CPSS attenuation (P = 0.011) and those with good portal blood flow on pre-attenuation (P = 0.004) and post-attenuation (P = 0.015) portovenography. Serum interleukin (IL)-6 concentration was measured using a canine specific ELISA and significantly increased 24 h following CPSS attenuation (P < 0.001). Portal LPS was increased in dogs with CPSS, consistent with decreased hepatic clearance. TLR4 mRNA expression was significantly associated with portal blood flow and increased following surgery. These findings support the concept that portal LPS delivery is important in the hepatic response to surgical attenuation. Serum IL-6 significantly increased following surgery, consistent with LPS stimulation via TLR4, although this increase might be non-specific.

Ligustilide Ameliorates Inflammatory Pain and Inhibits TLR4 Upregulation in Spinal Astrocytes Following Complete Freund's Adjuvant Peripheral Injection.

Ligustilide is a major component of Radix Angelica Sinensis and reported to have anti-inflammatory and anti-nociceptive effects. Toll-like receptor 4 (TLR4) has been shown to be expressed in the spinal cord and be involved in inflammatory pain and neuropathic pain. Whether ligustilide can inhibit spinal TLR4 expression in inflammatory pain is still unknown. In the present study, we intravenously injected ligustilide daily for 4 days, with the first injection given at 1 h before complete Freund's adjuvant (CFA) injection. We tested the analgesic effect of ligustilide by behavioral test and checked the expression and distribution of TLR4 in the spinal cord by real-time quantitative PCR, Western blot, and immunofluorescence. Our data showed that repeated daily intravenous treatment with ligustilide alleviated CFA-induced heat hyperalgesia and mechanical allodynia. The same treatment also inhibited CFA-induced TLR4 mRNA and protein increase in the spinal cord. Immunofluorescence double staining showed that TLR4 was predominantly expressed in spinal astrocytes. In primary cultured astrocytes, ligustilide dose-dependently reduced lipopolysaccharide-induced upregulation of TLR4 mRNA expression. These data indicate that ligustilide treatment reduces TLR4 expression in spinal astrocytes and is an effective therapy for inflammatory pain.

Effect of remifentanil on Toll-like receptor 2 mRNA expression during renal ischemia-reperfusion in rats

Objective To evaluate the effect of remifentanil on Toll-like receptor 2 (TLR2) mRNA expression during renal ischemia-reperfusion (I/R) in rats. Methods Fifty-four male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 3 groups (n= 18 each) using a random number table: sham operation group (group S), I/R group and remifentanil group (group R). Renal I/R injury was produced by clamping the bilateral renal arteries for 45 min followed by reperfusion in I/R and R groups.Bilateral renal arteries were only exposed but not clamped in group S. Remifentanil 1.0 μg·kg-1·min-1 was infused via the tail vein starting from 15 min before ischemia until 30 min of reperfusion in group R, while the equal volume of normal saline was given instead in S and I/R groups.The animals were sacrificed at 15 min before ischemia and 6 and 24 h of reperfusion, and the renal specimens were obtained for examination of the pathological changes (with light microscope) and for determination of the contents of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) (by ELISA) and expression of TLR2 mRNA (by RT-PCR) and cell apoptosis (by double staining and flow cytometry). The apoptotic rate was calculated. Results Compared with group S, TLR2 mRNA expression was significantly up-regulated, and the contents of TNF-α and IL-6 and apoptotic rate were increased at 6 and 24 h of reperfusion in I/R and R groups.Compared with group I/R, TLR2 mRNA expression was significantly down-regulated, and the contents of TNF-α and IL-6 and apoptotic rate were decreased at 6 and 24 h of reperfusion in group R. The pathological changes were significantly attenuated in group R as compared with group I/R. Conclusion The mechanism by which remifentanil reduces renal I/R injury is related to down-regulation of TLR2 expression and decrease in TLR2 activity and inhibition of inflammatory responses in renal tissues and cell apoptosis in rats. Key words: Piperidines; Reperfusion injury; Kidney; Toll-like receptor-2

Expression of Toll-like receptors in human retinal and choroidal vascular endothelial cells

Toll-like receptors (TLRs) are a family of proteins that initiate the innate immune response in reaction to invading microbes. Studies confirm the expression of TLRs in a variety of ocular tissues and cells, and it has also been suggested that selected TLRs may be associated with geographic atrophy and neovascularisation in age-related macular degeneration, diabetic retinopathy and other vascular and inflammatory diseases of the ocular posterior segment. However, TLR expression and localisation in the retinal and choroidal vasculature has not been defined. A better understanding of differential TLR expression in the choroid and retina, particularly in endothelial cells would improve our knowledge of vascular and inflammatory diseases in the posterior segment of the eye. In this study the gene (mRNA) expression of TLRs 1–10 was investigated using RT-PCR and comparative qPCR and the protein expression and localisation of selected TLRs (3, 4, 6 and 9) were examined using western blotting, flow cytometry and immunofluorescent staining. PCR showed gene expression of TLR1-6 and 9 in human choroidal endothelial cells (hCEC) and TLR2-6, 9 and 10 in human retinal endothelial cells (hREC). Western blotting detected TLR3, 4 and 9 proteins in both hCEC and hREC with higher levels in hCEC, whilst TLR6 protein was not detectable in either endothelial cell type. Flow cytometry detected all four TLRs (3, 4, 6 and 9) on the cell surface and intracellularly, TLR6 expression was detectable but low. The expression and localisation of TLR3, 4 and 9 were confirmed by immunofluorescent staining in endothelial cells and whole tissue sections and their functionality tested by expression of IL-6 (ELISA) in response to stimulation with specific TLR ligands. This study has, for the first time, identified the differential expression and localisation of TLRs in intraocular endothelial cells. This profiling will help inform our understanding of different retinal and choroidal vascular diseases, as well as the development of future treatments for intraocular vascular diseases.

Asparagine attenuates hepatic injury caused by lipopolysaccharide in weaned piglets associated with modulation of Toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling and their negative regulators

Pro-inflammatory cytokines play a key role in many models of hepatic damage. In addition, asparagine (Asn) plays an important role in immune function. We aimed to investigate whether Asn could attenuate lipopolysaccharide (LPS)-induced liver damage. Forty-eight castrated barrows were allotted to four groups including: (1) non-challenged control; (2) LPS-challenged control; (3) LPS + 0.5% Asn; and (4) LPS + 1.0% Asn. After 19 d feeding with control, 0.5 or 1.0% Asn diets, pigs were injected with LPS or saline. Blood and liver samples were obtained at 4 h (early stage) and 24 h (late stage) post-injection. Asn alleviated liver injury, indicated by reduced serum aspartate aminotransferase and alkaline phosphatase activities linearly and quadratically; it increased claudin-1 protein expression linearly and quadratically at 24 h, and less severe liver morphological impairment at 4 or 24 h. In addition, Asn decreased mRNA expression of TNF-α and heat shock protein 70 (HSP70) linearly and quadratically at 4 h; it increased TNF-α mRNA expression, and HSP70 protein expression linearly and quadratically at 24 h. Moreover, Asn increased inducible NO synthase activity linearly and quadratically. Finally, Asn down-regulated the mRNA expression of Toll-like receptor 4 (TLR4) signalling molecules (TLR4, IL-1 receptor-associated kinase 1 (IRAK1), TNF-α receptor-associated factor 6), nucleotide-binding oligomerisation domain protein (NOD) signalling molecules (NOD1, NOD2 and their adaptor molecule receptor-interacting serine/threonine-protein kinase 2 (RIPK2)), and NF-κB p65 linearly or quadratically at 4 h. Oppositely, Asn up-regulated mRNA expressions of TLR4 and NOD signalling molecules (TLR4, myeloid differentiation factor 88, IRAK1, NOD2 and RIPK2), and their negative regulators (radioprotective 105, single Ig IL-1R-related molecule, Erbb2 interacting protein and centaurin β1) linearly or quadratically at 24 h. These results indicate that, in early and late stages of LPS challenge, Asn improves liver integrity and exerts different regulatory effects on mRNA expression of TLR4 and NOD signalling molecules.

Postnatal nutritional restriction affects growth and immune function of piglets with intra-uterine growth restriction.

Postnatal rapid growth by excess intake of nutrients has been associated with an increased susceptibility to diseases in neonates with intra-uterine growth restricted (IUGR). The aim of the present study was to determine whether postnatal nutritional restriction could improve intestinal development and immune function of neonates with IUGR using piglets as model. A total of twelve pairs of normal-birth weight (NBW) and IUGR piglets (7d old) were randomly assigned to receive adequate nutrient intake or restricted nutrient intake (RNI) by artificially liquid feeding for a period of 21d. Blood samples and intestinal tissues were collected at necropsy and were analysed for morphology, digestive enzyme activities, immune cells and expression of innate immunity-related genes. The results indicated that both IUGR and postnatal nutritional restriction delayed the growth rate during the sucking period. Irrespective of nutrient intake, piglets with IUGR had a significantly lower villous height and crypt depth in the ileum than the NBW piglets. Moreover, IUGR decreased alkaline phosphatase activity while enhanced lactase activity in the jejunum and mRNA expressions of Toll-like receptor 9 (TLR-9) and DNA methyltransferase 1 (DNMT1) in the ileum of piglets. Irrespective of body weight, RNI significantly decreased the number and/or percentage of peripheral leucocytes, lymphocytes and monocytes of piglets, whereas the percentage of neutrophils and the ratio of CD4+ to CD8+ were increased. Furthermore, RNI markedly enhanced the mRNA expression of TLR-9 and DNMT1, but decreased the expression of NOD2 and TRAF-6 in the ileum of piglets. In summary, postnatal nutritional restriction led to abnormal cellular and innate immune response, as well as delayed the growth and intestinal development of IUGR piglets.

Effects of simvastatin on airway inflammation and airway mucus hypersecretion in rats with chronic obstructive pulmonary disease

To explore the preventive and therapeutic effects of simvastatin on rats with chronic obstructive pulmonary disease (COPD) and examine the mechanism of airway inflammation and airway mucus hypersecretion. The rat model of COPD was established by cigarette smoking and an intratracheal injection of lipopolysaccharide (LPS). A total of 18 male Sprague-Dawley rats were randomly divided into control, COPD and simvastatin groups (n=6 each). The control and COPD groups received normal saline once daily via intragastric administration (i.g.) while the simvastatin group had simvastatin (0.5 g/L) 1 ml/100 g once daily via i.g. Pulmonary function was tested and pathological changes in bronchus and lung were observed. The bronchoalveolar lavage fluid (BALF) levels of interleukin-8 (IL-8), interleukin-17 (IL-17) and tumor necrosis factor (TNF)-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expressions of intercellular adhesion molecule 1 (ICAM-1), nuclear factor κB (NF-κB), mucin 5AC (MUC5AC) and Toll-like receptor 4 (TLR4) in rat airway were detected by immunohistochemical staining. The mRNA and protein expressions of TLR4 and MUC5AC in bronchi and lung tissue were detected by fluorescent real time quantitative polymerase chain reaction (RT-PCR) and Western blot. The changes of bronchi and lung tissues in COPD group were consistent with typical pathological manifestations of COPD. As compared with the COPD group, the degree of pulmonary pathological damage and the decline of pulmonary function in the simvastatin group were significantly lessened, but still remarkable as compared with the control group. The BALF levels of IL-8, IL-17 and TNF-α in the smvastatin group [(484.4 ± 11.1), (78.9 ± 2.0), (192.7 ± 2.0) ng/L] were significantly lower than those in the COPD group [(605 ± 48.7), (89.9 ± 6.9), (212.6 ± 10.7) ng/L], but still higher than those in the control group [(341.2 ± 21.4), (56.0 ± 2.9), (127.5 ± 9.0) ng/L respectively] (all P <0.01). Compared with COPD group, the expression levels of ICAM-1, NF-κB, MUC5AC and TLR4 protein were significantly lower in the simvastatin group, but still higher than the control group (all P <0.01). Furthermore, the expression levels of mRNA and protein of MUC5AC and TLR4 were significantly lower in the simvastatin group than those in the COPD group (all P <0.05). In COPD model rats, simvastatin can decrease the levels of IL-8, IL-17 and TNF-α in BALF and inhibit the expression levels of ICAM-1, NF-κB, MUC5AC and TLR4 protein in airway and lung tissue. Thus it plays preventive and therapeutic roles by reducing airway inflammation and airway mucus hypersecretion.

Expression of Toll-like receptors in nasal epithelium in allergic rhinitis.

Toll‐like receptors (TLRs) are important in barrier homeostasis, but their role in airborne allergies is not fully understood. The aim was to evaluate baseline and allergen‐induced expression of TLR proteins in nasal epithelium during allergic rhinitis. Nineteen otherwise healthy non‐smoking volunteers both allergic to birch pollen and non‐allergic controls were enrolled. We took nasal biopsies before and after off‐seasonal intranasal birch pollen or diluent challenge. The expression of epithelial TLR1‐7, TLR9‐10, and MyD88 proteins was immunohistochemically evaluated from the nasal biopsies. The TLR1‐3 and TLR5‐10 mRNAs were observed by RNA‐microarray. Baseline epithelial expression of TLR proteins was wide and identical in controls and atopics. After off‐seasonal intranasal birch pollen challenge, a negative change in the expression score of TLR1 and TLR6 proteins was detected in the atopic group. TLR mRNA expression was not affected by birch pollen challenge. Nasal epithelium seems to express all known TLRs. The mechanisms by which TLR1, and TLR6 proteins could affect pollen allergen transport need further studies.

Role of endothelial progenitor cell transplantation in rats with sepsis

To investigate the role of endothelial progenitor cells (EPCs) transplantation in rats with sepsis induced by endotoxin (lipopolysaccharides, LPS). Sixty clean grade Sprague-Dawley (SD) rats with genetic background were divided into three groups according to random number table method: control group, model group, and EPCs transplantation group, with 20 rats in each group. The sepsis model was reproduced by intravenous delivery of LPS 5 mg/kg. Rats in control group were injected with the same amount of normal saline. EPCs were isolated, and cultured and identified were fluorescently labeled with the green fluorescent protein (GFP) adenoviral transfection method. The EPC transplantation group was injected with LPS, then a fluorescently labeled EPCs suspension was injected via the tail vein 1 hour later. The expression of fluorescent markers of EPCs was detected with both small animal in vivo imaging instrument and frozen section. Seven days after transplantation, abdominal aorta blood was collected to determine interleukins (IL-6 and IL-10) in peripheral blood with enzyme linked immunosorbent assay (ELISA), and the lung, liver, and kidney tissues were harvested, the wet/dry ratio of the lung (W/D) was calculated, and hematoxylin and eosin (HE) staining was performed to observe, the change in histopathology. Toll-like receptor 4 (TLR4) mRNA expression in lung, liver, and kidney tissues was determined with real-time reverse transcription-polymerase chain reaction (RT-PCR). The positive rate of EPCs cells with double marking of CD133 and CD34 was 99.0% at the 5th generation of subculture by using flow cytometry. After the transplantation of EPCs labeled with the green fluorescent protein, the appearance of fluorescence indicated that EPCs were mainly localized in the chest, and a stronger fluorescence was observed near the blood vessels. EPCs transplantation could significantly reduce the inflammatory cell infiltration and cell damage in lung, liver, and kidney tissue in septic rats. Compared with control group, the expression of IL-6 and IL-10 in the peripheral blood, W/D ratio, and TLR4 mRNA in lung, liver, and kidney were increased significantly in the model group. Compared with model group, the expressions of IL-6 and IL-10 in the peripheral blood were significantly reduced after EPCs transplantation [IL-6 (μg/L): 2.127±0.118 vs. 2.664±0.438, IL-10 (ng/L): 24.5±3.9 vs. 31.5±3.8, both P<0.01]. EPCs transplantation reduced the W/D ratio of lung, liver and kidney tissues (lung: 4.68±0.24 vs. 5.48±0.15, liver: 3.33±0.11 vs. 3.94±0.09, kidney: 4.08±0.20 vs. 4.84±0.21, all P<0.05], and down-regulated the expression of TLR4 mRNA (×10(3), lung: 782±131 vs. 1 136±126, liver: 39.1±14.0 vs. 69.2±8.7, kidney: 52.2±15.2 vs. 83.5±17.1, all P<0.01). EPCs can enter the lung, liver and kidney tissues of the rat successfully after transplantation of EPCs via vein. EPCs transplantation can down-regulate pro-inflammatory process, help to recover the balance of pro- and anti-inflammatory processes, alleviate the damage to the lung, liver, and kidney tissue significantly.

Expression of Toll-like receptor signaling-related genes in pigs co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Pigs co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) have been shown to develop more severe diseases than pigs infected with PRRSV or PCV2 only. The underlying interaction mechanisms between the two viruses in developing the disease are unclear. The present study investigates the mRNA expression of Toll-like receptor (TLR) signaling-related molecules in peripheral blood mononuclear cells from pigs infected with PRRSV or PCV2 or both. The mRNA expression levels were determined by quantitative real-time RT-PCR. Co-infection of pigs with PRRSV and PCV2 resulted in a negatively synergistic effect on the mRNA expression of the negative regulators of TLR, including A20, Bcl-3, IRAK-M, MKP-1, SARM1 and SIGIRR, as well as the TLR downstream transcription factors IRF-1 and IRF-3. A positively synergistic effect of a combined infection of PRRSV and PCV2 on the CD14 mRNA expression was also observed.

A new mode of probiotic therapy: Specific targeting

Luminal bacteria are the most probable inducers of inflammatory bowel disease (IBD). The intestinal microbiota can be modified by probiotics, which reduce symptoms in IBD, via stimulation of the intestinal immune system. We aimed to evaluate the effects of oral and rectal administration of probiotics, on the morphology, gene expression, and microbial ecology of the colon, in a rat trinitrobenzenesulfonic acid (TNBS) model. Colitis was induced with simultaneous rectal or oral administration of probiotics. Both routes of introduction significantly increased the relative amounts of Lactobacillus spp. Rectal introduction almost eradicated damage to the tissue, while enteral introduction only improved it. Only rectal probiotics significantly decreased myeloperoxidase activity, and altered mucin and Toll-like receptor mRNA expression in the colon, to values close to control. We suggest that targeted introduction should be considered not only for probiotics but also for nutrients such as amino acids and vitamins into the colon and intestine.