This paper aims to explore the activity of Codonopsis canescens extract against rheumatoid arthritis(RA) based on the Toll-like receptors(TLRs)/mitogen-activated protein kinases(MAPKs)/nuclear factor kappa B(NF-κB) signaling pathways and its mechanism. The ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) was used to identify the components of C. canescens extract. Forty-eight male SD rats were randomly divided into six groups, namely the normal group, the model group, the methotrexate(MTX) tablet group, and the low, medium, and high-dose C. canescens extract(ZDS-L, ZDS-M, and ZDS-H) groups, with 8 rats in each group. The model of collagen-induced arthritis in rats was induced by injection of bovine type Ⅱ collagen emulsion. MTX(2.5 mg·kg~(-1)), ZDS-L, ZDS-M, and ZDS-H(0.3 g·kg~(-1), 0.6 g·kg~(-1), and 1.2 g·kg~(-1)) were administrated by gavage. Rats in the normal group and the model group received distilled water. MTX was given once every three days for 28 days, and the rest medicines were given once daily for 28 days. Body weight, degree of foot swelling, arthritis index, immune organ index, synovial histopathological changes, and serum levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6) were observed. Protein expressions of TLR2, TLR4, NF-κB p65, p38 MAPK, and p-p38 MAPK in rats were determined by Western blot. Thirty-four main components were identified by UPLC-Q-TOF-MS, including 15 flavonoids, 7 phenylpropanoids, 4 terpenoids, 4 organic acids, 2 esters, and 2 polyalkynes. As compared with the normal group, the body weight of the model group was significantly decreased(P<0.01), and foot swelling(P<0.05, P<0.01), arthritis index(P<0.01), and the immune organ index(P<0.01) were significantly increased. The synovial histopathological injury was obviously observed in the model group. The serum levels of inflammatory factors TNF-α, IL-1β, and IL-6 were significantly increased(P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK in the synovial tissue were significantly increased(P<0.01) in the model group. As compared with the model group, the body weights of the ZDS dose groups were increased(P<0.01), and the degree of foot swelling(P<0.01) and the arthritis index were decreased(P<0.05, P<0.01). The immune organ index was decreased(P<0.01) in the ZDS dose groups, and the synovial tissue hyperplasia and inflammatory cell infiltration were alleviated. The serum levels of TNF-α, IL-1β, and IL-6 were significantly decreased(P<0.05, P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK were decreased(P<0.05, P<0.01) in the ZDS dose groups. C. canescens extract containing apigenin, tricin, chlorogenic acid, aesculin, ferulic acid, caffeic acid, and oleanolic acid has a good anti-RA effect, and the mechanism may be related to the inhibition of TLRs/MAPKs/NF-κB signaling pathways.
Animals ; Cattle ; Male ; Rats ; Arthritis, Experimental/drug therapy* ; Arthritis, Rheumatoid/drug therapy* ; Body Weight ; Codonopsis/chemistry* ; Interleukin-6/blood* ; NF-kappa B/genetics* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; Plant Extracts/therapeutic use* ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism* ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/pharmacology*

OBJECTIVE: To characterize the expression of Toll-like receptor 4 (TLR4) in monocytes of diabetic nephropathy (DN) patients and the response of TLR4 to lipopolysaccharide (LPS), and, further, to explore the potential effects of inflammatory immune response in DN. METHODS: Thirty DN patients with uremia, ten early-type 2 DN patients, and twenty healthy volunteers were enrolled for the determination of TLR4 expression in monocytes by using peripheral blood flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated and subjected to 1 μg/mL LPS for 24 h. Monocytes were collected to assay NF-κB p65 and Notch1 expression by Western blot, with immuneofluorescence detection. Serum and supernatants were sampled for the determination of interleukin-6 (IL-6) concentration by using ELISA. Serum C-reactive protein (CRP) level was determined by using the immunoturbidimetry. RESULTS: Compared with the normal control, type 2 DN uremic patients had a significantly higher TLR4 fluorescence-blot intensities (FI), and serum CRP and IL-6 levels [TLR4 FI: DN uremia patients 2.8±0.9; early type 2 DN patients 3.4 ±0.7; healthy subjects 1.6±0.7. IL-6 concentration: DN uremia patients (84.8±20.7) pg/mL; early type 2 DN patients (63.20±14.4) pg/mL; healthy subjects (11.0±2.0) pg/mL. CRP concentraton: DN uremia patients (5.4±2.8) mg/L; early type 2 DN patients (3.7±1.7) mg/L; healthy subjects (1.7±0.7) mg/L. P<0.01 for any DN-group vs control]. In early type 2 DN patients, following exposure to LPS, PBMCs showed a significant upregulation in TLR4 and NF-κB p65 expression and a remarked increase in serum IL-6 level (all P<0.05), and NF-κB p65 transfer to the nucleus is enhanced. Notch1 protein expression was not significantly altered in any group. CONCLUSION A disturbance in proinflammatory CD14(+)CD16(+) monocytes occurs in type 2 DN patients. Such immunological dysfunction may be related to activation in NF-κB/TLR4 signaling pathways, and have nothing to do with the Notch1 signaling pathway.
Adult ; Aged ; Diabetes Mellitus, Type 2 ; blood ; Diabetic Nephropathies ; blood ; Female ; Humans ; Lipopolysaccharides ; pharmacology ; Male ; Middle Aged ; Monocytes ; metabolism ; Receptor, Notch1 ; genetics ; metabolism ; Signal Transduction ; Toll-Like Receptor 4 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; metabolism

To systematically clarify the effects of apolipoprotein E (aopE) and low-density lipoprotein receptor (LDLR) gene mutant on hyperlipidemia, vascular inflammation impairment and pathogenesis of atherosclerosis (AS), total RNA was isolated from fresh aortas of young apoE/LDLR double knockout (apoE(-/-)/LDLR(-/-)) and wild type (WT) mice using TRIzol reagent. Then RNA was reversely transcribed to first-strand cDNA by reverse transcriptase for reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Primer pairs were designed using primer design software according to the gene sequences available in GenBank. β-actin was used as an internal control. Then RT-PCR assay was used to analyze the expression patterns of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD36, endothelin-1 (ET-1), toll-like receptor 2 (TLR2), monocyte chemoattractant protein-1 (MCP-1), vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and platelet-derived growth factor-α (PDGF-α). SYBR Green quantitative real-time RT-PCR was used to validate gene expressions identified by RT-PCR. Blood samples were taken from the retro-orbital venous plexus, and serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured by using biochemical techniques. Serum concentrations of circulating TNF-α, IL-1β and oxidized LDL (ox-LDL) were determined by ELISA. Frozen sections of aortic sinus were stained with Sudan IV to visualize intimal fatty lesions. The results showed that the relative expressions of IL-1β, GM-CSF, ET-1, TLR2, CD36, MCP-1, ICAM-1 and VCAM-1 in apoE(-/-)/LDLR(-/-) mice at the age of 1 month were higher than those in age-matched WT mice (P<0.05, P<0.01), respectively. The expressions of PDGF-α and TNF-α in apoE(-/-)/LDLR(-/-) mice at the age of 2 months were up-regulated compared to those in age-matched WT mice (P<0.05). All the expressions of target genes continued to be up-regulated (P<0.05, P<0.01) except that ET-1 expression at the age of 2 months, TLR2, VCAM-1 and ICAM-1 expressions at the age of 3 months were down-regulated to that in WT mice. NF-κB expression had no significant changes between two genotype mice at different ages. All the gene expressions kept unchanged in WT mice at different ages, except that IL-1b expressions were slightly up-regulated at the ages of 2 and 3 months. Serum levels of TC, TG, LDL, HDL, TNF-α, IL-1β and ox-LDL in apoE(-/-)/LDLR(-/-) mice at different ages were higher than those in age-matched WT mice (P<0.05, P<0.01), and were increasing with age. Primary atherosclerotic lesions were observed in 1-month old apoE(-/-)/LDLR(-/-) mice and were progressing with age. There were no lesions observed in all WT mice at different ages. The data suggest that hyperlipidemia due to apoE and LDLR gene mutant may stimulate the temporal expressions of AS-related genes and contribute to primary atherogenetic lesions and vascular inflammation impairment.
Animals ; Aorta ; metabolism ; Apolipoproteins E ; genetics ; Atherosclerosis ; genetics ; CD36 Antigens ; metabolism ; Chemokine CCL2 ; metabolism ; Endothelin-1 ; metabolism ; Gene Expression ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Hyperlipidemias ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-1beta ; blood ; metabolism ; Lipoproteins, LDL ; blood ; Mice ; Mice, Knockout ; NF-kappa B ; metabolism ; Platelet-Derived Growth Factor ; Receptors, LDL ; genetics ; Toll-Like Receptor 2 ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo investigate the expression of TLR4/2 mRNA in neonatal cord blood mononuclear cells (MNC).

METHODSForty-six neonates without asphyxia and 40 neonates with asphyxia were divided into groups depending on the gestational age. In the neonates without asphyxia, there were 18 full term infants (the gestational age > or = 37 weeks), 16 preterm infants whose gestational age was > or = 32 weeks but < 37 weeks, and 12 preterm infants whose gestational age was < 32 weeks. In the neonates with asphyxia, 11 were full term infants, 15 were preterm infants whose gestational age was > or = 32 weeks but < 37 weeks and 14 were preterm infants at gestational age < 32 weeks. MNCs were separated and cultured with LPS (1 microg/ml) for 3 h. Cells were collected for analysis of gene expression of TLR4/2 by RT-PCR technique. Cell supernatants were taken to measure TNF-alpha production following the ELISA protocol. Fifteen healthy adults were enrolled into the control group. In addition, the Pearson correlation analyses were carried out between the levels of TLR4, TLR2 mRNA and the levels of TNF-alpha.

RESULTSIn the neonates without asphyxia, TLR4, TLR2 mRNA and TNF-alpha levels were 0.75 +/- 0.12, 0.63 +/- 0.08, 2502.6 +/- 273.1 ng/L, separately, in the full term infants, 0.37 +/- 0.04, 0.32 +/- 0.03, 1218.8 +/- 145.7 ng/L, separately, in the preterm infants whose gestational ages were > or = 32 weeks but < 37 weeks, and 0.26 +/- 0.03, 0.20 +/- 0.03, 811.8 +/- 105.2 ng/L separately, in the preterm infants whose gestational ages were < 32 weeks. In the neonates with asphyxia, TLR4, TLR2 mRNA and TNF-alpha levels were 0.58 +/- 0.07, 0.50 +/- 0.06, 1946.4 +/- 244.2 ng/L, separately, in the full term infants, 0.29 +/- 0.03, 0.26 +/- 0.03, 970.0 +/- 94.3 ng/L, separately, in the preterm infants whose gestational age was > or = 32 weeks but < 37 weeks, and 0.17 +/- 0.02, 0.14 +/- 0.02, 652.6 +/- 60.3 ng/L, separately, in the preterm infants whose gestational age was < 32 weeks. The levels of TLR4, TLR2 mRNA and TNF-alpha in the adults were 2.71 +/- 0.75, 2.61 +/- 0.33, 9270.1 +/- 1098.3 ng/L, separately. In the preterm infants and full term infants, the levels of TLR4, TLR2 mRNA and TNF-alpha were lower in comparison to the adults. The lower the gestational age, the lower the levels of TLR4, TLR2 mRNA and TNF-alpha. There were significant differences between the levels of TLR4, TLR2 mRNA and TNF-alpha of the neonates without asphyxia and those of the neonates with asphyxia. In the neonates with asphyxia, the levels of TLR4, TLR2 mRNA and TNF-alpha were lower than those in the neonates without asphyxia (P < 0.01). Whether the neonates were asphyxic or not, the levels of TLR4, TLR2 were paralleled with the levels of TNF-alpha.

CONCLUSIONSThe expression of TLRs in the neonates, especially in the preterm infants was lower than that in the adults, which probably contributes to the susceptibility of neonates to infections.

Blood Cells ; metabolism ; Gene Expression ; Humans ; Infant ; Infant, Newborn ; RNA, Messenger ; genetics ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptors ; metabolism ; Tumor Necrosis Factor-alpha ; immunology

Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Fetal Blood ; Humans ; Infant, Newborn ; Lipopolysaccharides ; Lymphocytes ; metabolism ; Male ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; Time Factors ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Toll-like receptors (TLRs) are pattern-recognition receptors that are important in innate immune responses to bacterial infection. The purpose of this study is to describe the prevalence of TLRs genetic variations in the bacteremic patients in Korea. A total of 154 patients with bacteremia and 179 healthy volunteers were included. The Asp299Gly and Thr399Ile allele of the TLR4 gene and Arg753Gln and Arg677Trp allele of the TLR2 gene were tested by PCR-RFLP. The DNA sequences were determined to confirm the PCR-RFLP results. Contrary to the expectation, no genetic polymorphisms were detected in both groups of this study, suggesting that it is very rare in Korean.
Toll-Like Receptor 4/blood/*genetics ; Toll-Like Receptor 2/blood/*genetics ; Risk Factors ; Risk Assessment/*methods ; Prevalence ; Polymorphism, Single Nucleotide/genetics ; Middle Aged ; Male ; Korea/epidemiology ; Humans ; Genetic Screening/methods ; Genetic Predisposition to Disease/epidemiology/genetics ; Female ; DNA Mutational Analysis ; Biological Markers/blood ; Bacteremia/blood/*epidemiology/*genetics

Toll-like receptors (TLRs) are pattern-recognition receptors that are important in innate immune responses to bacterial infection. The purpose of this study is to describe the prevalence of TLRs genetic variations in the bacteremic patients in Korea. A total of 154 patients with bacteremia and 179 healthy volunteers were included. The Asp299Gly and Thr399Ile allele of the TLR4 gene and Arg753Gln and Arg677Trp allele of the TLR2 gene were tested by PCR-RFLP. The DNA sequences were determined to confirm the PCR-RFLP results. Contrary to the expectation, no genetic polymorphisms were detected in both groups of this study, suggesting that it is very rare in Korean.
Toll-Like Receptor 4/blood/*genetics ; Toll-Like Receptor 2/blood/*genetics ; Risk Factors ; Risk Assessment/*methods ; Prevalence ; Polymorphism, Single Nucleotide/genetics ; Middle Aged ; Male ; Korea/epidemiology ; Humans ; Genetic Screening/methods ; Genetic Predisposition to Disease/epidemiology/genetics ; Female ; DNA Mutational Analysis ; Biological Markers/blood ; Bacteremia/blood/*epidemiology/*genetics

OBJECTIVETo study changes of TLR2 signaling pathway expression in Kupffer cells during the process of hepatic ischemia/reperfusion in a mice model and the mechanism of TLR2 signaling pathway participating in hepatic ischemia/reperfusion injury.

METHODSBALB/c mice were divided into 3 groups: sham operation (SH), ischemia/reperfusion (I/R) and GdCl3 treatment (Gd) groups. After 4 h of reperfusion, the expression of TLR2 mRNA and membrane TLR2 protein were analyzed in ischemic lobes of the livers, and in Kupffer cells isolated from ischemic lobes. The expression of NF-kappaB in ischemic lobes was also examined. Levels of endotoxin, ALT and TNFalpha were measured at the same time point.

RESULTSThe expressions of TLR2 mRNA and protein in both ischemic hepatic lobes and Kupffer cells isolated from ischemic lobes were increased in the I/R group compared to those in the SH group, as well as the expression of NF-kappaB in ischemic lobes, which was down regulated by intravenous GdCl3 treatment. Levels of ALT and TNFalpha in the portal vein were higher in the I/R group than in the SH group, which also were decreased with treatment of GdCl3. The level of endotoxin in the three groups remained constant.

CONCLUSIONTLR2 signaling pathway in Kupffer cells is activated during the process of hepatic ischemic/reperfusion injury. The activation of TLR2 signaling pathway in Kupffer cells may play a role in this process.

Animals ; Kupffer Cells ; metabolism ; Liver ; blood supply ; Male ; Mice ; Mice, Inbred BALB C ; Reperfusion Injury ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; biosynthesis ; genetics

To investigate TLR2 (Toll-like receptor 2) mRNA expression in ischemic hepatic lobes under the condition of partial hepatic ischemia/reperfusion injury in BALB/c mice and its relationship with liver function impairment. A partial ischemia/reperfusion injury model was established. The portal vein and hepatic artery supply to the median and left lobes of the liver were obstructed by an atraumatic artery micro-clip, with the obstruction lasting for about 60 min. Then reperfusion was fulfilled by removal of the clip. The liver samples were collected at the 4th h after the restoration of blood inflow. Total RNA was extracted from the liver samples and analyzed quantitatively by method of real-time PCR. At the same time, portal vein serum and plasma were taken respectively for further detection of the level of endotoxin, tumor necrosis factor alpha (TNF-alpha) and plasmic alanine aminotransferase (pALT). The results indicated that TLR2 mRNA in ischemic lobe was up-regulated markedly in mice partial liver ischemia/reperfusion injury model compared to that in sham operation group (deltaCt: 1.05 +/- 1.02 vs 5.08 +/- 1.36, P<0.001). The level of portal vein pALT and TNF-alpha increased significantly (112.32 +/- 17.56 pg/ml vs 6.07 +/- 5.33 pg/ml, P<0.01; 890 +/- 127 microm/L vs 30 +/- 5 microm/L, P<0.001) . However, the level of portal vein endotoxin remained below the normal line, suggesting a state of non-endotoxemia. TLR2 mRNA expression in ischemic lobe, as well as portal vein pALT and TNF-alpha, was up-regulated in the model of mice partial ischemia/reperfusion injury, suggesting the involvement of TLR2 in ischemia/reperfusion pathological process.
Animals ; Liver ; blood supply ; metabolism ; Male ; Mice ; RNA, Messenger ; biosynthesis ; genetics ; physiology ; Reperfusion Injury ; etiology ; metabolism ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; physiology ; Up-Regulation

OBJECTIVEIn order to explore the role of toll-like receptors 2 (TLR2) in initiating inflammatory response, the expression of TLR2 of the liver and IL-18, TNF-alpha and IFN-gamma of plasma in fulminant hepatic failure was analysed.

METHODSD-galactosamine (D-Gal, 900 mg/kg) and lipopolysaccharide (LPS, 10 microg/kg) were administered intraperitoneally into the BALB/C mice. To evaluate the hepatic injury, serum transaminase (ALT and AST) and plasma IL-18, TNF-alpha and IFN-gamma were determined and the mortality was observed at various time points following the intraperitoneal injection. The level of TLR2 mRNA was measured by semiquantitative RT-PCR. The protein expression of TLR2 in the liver was detected by immunohistochemistry. The data was analyzed by SAS software.

RESULTSAfter 4 hours of intraperitoneal injection of D-Gal/LPS, the serum transaminase and plasma IL-18, TNF-alpha and IFN-gamma levels were elevated. The treated mice began to die at 7 hours. The mortality reached up to 80% at 10 h. TLR2 mRNA was expressed at a low level in liver tissues of normal mice, while it was significantly increased and maintained at a higher level following intraperitoneal injection with D-Gal/LPS. The expression of TLR2 protein was similar to that of the TLR2 mRNA, and the expression of TLR2 mRNA was positively correlated with the concentration of plasma IL-18, TNF-alpha and IFN-gamma (r=0.36, P=0.02; r = 0.48, P 0.003; r = 0.72, P<0.001) at different time points.

CONCLUSIONSOur results showed that TLR2 was involved in initiating and inducing the expression of proinflammation cytokines in this model of fulminant hepatic failure. The results suggest that adjusting the expression of TLR2 might be a new strategy in preventing the development of infectious diseases

Animals ; Galactosamine ; Interferon-gamma ; blood ; Interleukin-18 ; blood ; Lipopolysaccharides ; Liver ; metabolism ; Liver Failure, Acute ; chemically induced ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; biosynthesis ; genetics ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; metabolism