OBJECTIVE: To investigate the role of cathepsin B in hepatic Kupffer cells (KCs) in activating Toll-like receptor 4(TLR- 4)-independent inflammatory pathways in mice with lipopolysaccharide (LPS)-induced sepsis. METHODS: Eighteen wild-type (WT) mice and 18 TLR4-knockout (TLR4) mice were both divided into 3 groups for intraperitoneal injections of a lethal dose (54 mg/kg) of LPS, LPS and CA-074(a cathepsin B inhibitor), or normal saline, and the survival of the mice were observed. Another 36 WT mice and 36 TLR4mice were also divided into 3 groups and subjected to intraperitoneal injections of normal saline, 20 mg/kg LPS, or LPS with CA-074 pretreatment.After the treatments, KCs were collected from the mice for assessing the protein level and activity of cathepsin B.The histopathological changes of the liver were observed with HE staining, and the serum levels of IL-1α, IL-1β, TNF-α and IL-18 were detected. RESULTS: Compared with the WT mice,TLR4mice receiving the lethal dose of LPS had significantly longer survival time (up to 84 h) after the injection,but were still unable to fully resist LPS challenge.CA-074 pretreatment prolonged the survival time of WT mice and TLR4mice to 60 h and 132 h,respectively.In the mouse models of sepsis,20 mg/kg LPS induced significantly enhanced activity of cathepsin B without affecting its expression level in the KCs (<0.05) and increased the serum levels of the inflammatory cytokines.CA-074 pretreatment of the mice obviously lessened the detrimental effects of LPS in TLR4mice by significantly lowering cathepsin B activity in the KCs,alleviating hepatocyte apoptosis and reducing the serum levels of inflammatory cytokines. CONCLUSIONS Cathepsin B plays an important role in activating TLR4-independent inflammatory pathways in mice with LPS-induced sepsis.
Animals ; Cathepsin B ; antagonists & inhibitors ; physiology ; Dipeptides ; pharmacology ; Gene Knockout Techniques ; Hepatocytes ; Inflammation ; metabolism ; Interleukin-18 ; blood ; Interleukin-1alpha ; blood ; Interleukin-1beta ; blood ; Kupffer Cells ; metabolism ; Lipopolysaccharides ; Liver ; pathology ; Mice ; Sepsis ; etiology ; metabolism ; Toll-Like Receptor 4 ; genetics ; Tumor Necrosis Factor-alpha ; blood

BACKGROUNDSurfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.

METHODSTetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.

RESULTSHK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.

CONCLUSIONSThe present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.

Cell Line ; Cell Survival ; drug effects ; physiology ; Colorimetry ; Humans ; Kidney ; cytology ; metabolism ; Lipopolysaccharides ; toxicity ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; NF-kappa B ; metabolism ; Pulmonary Surfactant-Associated Protein A ; metabolism ; Sulfonamides ; pharmacology ; Tetrazolium Salts ; chemistry ; Toll-Like Receptor 4 ; antagonists & inhibitors ; metabolism

OBJECTIVECelastrol has been established as a nuclear factor-κB (NF-κB) activation inhibitor; however, the exact mechanism behind this action is still unknown. Using text-mining technology, the authors predicted that interleukin-1 receptor-associated kinases (IRAKs) are potential celastrol targets, and hypothesized that targeting IRAKs might be one way that celastrol inhibits NF-κB. This is because IRAKs are key molecules for some crucial pathways to activate NF-κB (e.g., the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily).

METHODSThe human hepatocellular cell line (HepG2) treated with palmitic acid (PA) was used as a model for stimulating TLR4/NF-κB activation, in order to observe the potential effects of celastrol in IRAK regulation and NF-κB inhibition. The transfection of small interfering RNA was used for down-regulating TLR4, IRAK1 and IRAK4, and the Western blot method was used to detect changes in the protein expressions.

RESULTSThe results showed that celastrol could effectively inhibit PA-caused TLR4-dependent NF-κB activation in the HepG2 cells; PA also activated IRAKs, which were inhibited by celastrol. Knocking down IRAKs abolished PA-caused NF-κB activation.

CONCLUSIONThe results for the first time show that targeting IRAKs is one way in which celastrol inhibits NF-κB activation.

Hep G2 Cells ; Humans ; Interleukin-1 Receptor-Associated Kinases ; antagonists & inhibitors ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Phosphorylation ; Toll-Like Receptor 4 ; antagonists & inhibitors ; physiology ; Triterpenes ; pharmacology

To study the preventive effect of sophocarpine (Soc) on dextran sulfate sodium (DSS)-induced colitis in mice, in order to analyze the influence of Soc on toll like receptor 4 (TLR4)/mitogen-activated protein kinases (MAPKs) and janus tyrosine kinase 2 signal transducer and activator of transcription 3 (JAK2/STAT3) signal pathways in mice intestinal tissues. The mice was given 2.5% DSS for 6 days to induce the acute colitis model. The Soc-treated group was intraperitoneally injected with sophocarpine 30 mg · kg(-1) · d(-1) since the day before the experiment to the end. The disease activity index (DAI) was assessed everyday, and the colonic morphology and histological damage were observed with HE staining. The mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were detected by real-time RT-PCR. The changes in key protein kinase p38 mitogen-activated protein kinase (p38MAPK), c-Jun NH2-terminal protein kinase1/2 (JNK1/2), extracellular signal-regulated kinase1/2 (ERK1/2), JAK2, STAT3 in TLR4/MAPKs and JAK2/STAT3 signaling pathways were detected by western blot. The result showed that the model group showed statistical significance in body weight, DAI, colon length and histopathological changes compared with the normal group (P <0.05); however, the Soc-treated group showed significant improvements in the above indexes compared with the model group (P <0.05). TNF-α, IL-1β and IL-6 in the model group was significantly higher than that in the normal group (P <0.05), but lowered in the Soc-treated group to varying degrees (P <0.05). In the normal group, the expressions of TLR4 and the phosphorylation of P38, JNK1/2, JAK2, STAT3 were at low levels; in the model group, the phosphorylation of P38, JNK1/2, JAK2, STAT3 increased; the Soc-treated group showed a decrease in TLR4 expression compared with the model group, with notable declines in the phosphorylation of TLR4, P38, JNK1/2, JAK2, STAT3. These findings indicate that Soc can inhibit TLR4/MAPKs, K2/STAT3 signaling pathway activation, reduce the expression of proinflammatory cytokines TNF-α, IL-1β and IL-6 and relieve inflammatory reactions, so as to effectively prevent experimental colitis.
Alkaloids ; pharmacology ; therapeutic use ; Animals ; Colitis ; drug therapy ; immunology ; pathology ; Cytokines ; genetics ; Janus Kinase 2 ; antagonists & inhibitors ; physiology ; Male ; Mice ; Mice, Inbred BALB C ; Phosphorylation ; STAT3 Transcription Factor ; antagonists & inhibitors ; physiology ; Toll-Like Receptor 4 ; antagonists & inhibitors ; physiology

To study the protective effect and the mechanism of asiatic acid (AA) from Potentilla chinensis on alcohol hepatic injury in rats. Male Wistar rats were randomly divided into six groups: the normal control group, the AA control group (8 mg · kg(-1) AA), the model group (5.0-9.0 g · kg(-1) alcohol) and high, medium and low-dose AA-treated groups (alcohol + 8, 4, 2 mg · kg(-1) AA). Each group was orally administered with the corresponding drugs once a day for 24 weeks. Approximately 1. 5 hours after the final administration, all rats were killed, and their blood samples and hepatic tissues were collected. The AST and ALT in rat serum and the contents of MPO, TNF-α, IL-1β, SOD, GSH-Px, GSH-Rd and MDA in hepatic tissues were detected. The expressions of NF-κB, TLR4, CD14, MyD88, TRIF and protein expression in hepatic tissues were measured by western blot. The pathological changes in liver tissues were observed by histological examination. The results showed that compared with the model group, the AA-treated groups showed significant decreases in serum ALT, AST and MDA and increases in the activities of SOD, GSH-Px, GSH-Rd and MPO. Moreover, AA markedly inhibited the expressions of TNF-α, IL-1β, TLR4, CD14, MyD88 and NF-κB. The histological examination showed alleviated hepatic issue ijury to varying degrees. In short, asiatic acid (AA) from P. chinensis could protect alcohol-induced hepatic injury in rats. Its mechanism may be related to the inhibition of NF-κB inactivation and the reduction of inflammatory response.
Animals ; Liver ; drug effects ; pathology ; Liver Diseases, Alcoholic ; prevention & control ; Male ; NF-kappa B ; physiology ; Pentacyclic Triterpenes ; pharmacology ; Potentilla ; chemistry ; Protective Agents ; pharmacology ; Rats ; Rats, Wistar ; Toll-Like Receptor 4 ; antagonists & inhibitors

The present study was to investigate whether Toll-like receptor 4 (TLR4)-mediated Akt/FoxO3a/Bim signaling pathway participated in lipopolysaccharide (LPS)-induced apoptosis in hippocampal neurons. The primarily cultured rat hippocampal neurons were treated with LPS, TLR4 antibody+LPS, and LY294002+LPS, respectively. Cell vitality was assayed by CCK-8. Expressions of p-Akt, Akt, p-FoxO3a, FoxO3a, Bim and active-Caspase-3 of each group were detected by Western blot analysis; the mRNA expression of Bim was detected by real-time quantitative PCR; FoxO3a nuclear translocation was detected by fluorescence microscope. The rate of cell apoptosis was assayed by flow cytometry. The results showed that cell vitality of hippocampal neurons decreased after being treated with LPS in a time-dependent way. Compared with the control group, the expressions of p-Akt and p-FoxO3a decreased significantly, FoxO3a translocated into the nucleus, meanwhile, the expression of Bim and active-Caspase-3, and the apoptotic ratio of hippocampal neurons increased in LPS treated neurons. Pretreatment with TLR4 antibody significantly blocked, while PI3K antagonist LY294002 further strengthened these changes induced by LPS. In conclusion, the present study suggests that Akt/FoxO3a/Bim signaling pathways mediated by TLR4 participate in the apoptotic processes of primarily cultured hippocampal neurons treated with LPS, and the activation of TLR4 causes neuronal apoptosis.
Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Bcl-2-Like Protein 11 ; Caspase 3 ; metabolism ; Chromones ; pharmacology ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; metabolism ; Hippocampus ; cytology ; Lipopolysaccharides ; Membrane Proteins ; metabolism ; Morpholines ; pharmacology ; Neurons ; cytology ; metabolism ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Signal Transduction ; Toll-Like Receptor 4 ; metabolism

OBJECTIVE: To observe the expression of progesterone receptor (PR), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2) induced by lipopolysaccharide (LPS) or Toll-like receptor 4 antagonist (TLR4 mAb) in decidual cells in vitro, and then to explore the effect of LPS and its antagonist on PR of decidual cells and the relation between PR and inflammatory cytokines. METHODS: We isolated and cultured human decidua of early abortion in the sterile state. When the cells passaged to the 4th generation, the cells were randomly divided into 6 pore plates: A control group was added the culture medium alone; experimental group I was added 100 ng/mL of LPS; experimental group II was add 1 μg/mL of TLR4 mAb; experimental group III was added 3 μg/ mL of TLR4 mAb; experimental group IV was added 1 μg/mL of TLR4 mAb pretreatment for 24 h, and then 100 ng/mL LPS; and experimental group V was added 3 μg/mL of TLR4 mAb pretreatment for 24 h, and then 100 ng/mL LPS for 24 h culture. Subsequently, HE staining and immunofluorescence were used to observe the morphology and identify the purity of decidual cells in the 6 groups. The levels of mRNA expression of PR, IL-1β, and COX-2 were detected by reverse transcription PCR (RT-PCR). RESULTS: LPS reduced the mRNA expression of PR (P<0.05), increased the mRNA expression of IL-1β and COX-2 (P<0.05). TLR4 mAb increased the mRNA expression of PR (P<0.05) and reduced the mRNA expression of IL-1β (P<0.05) after LPS-stimulated decidual cells. High concentrations of TLR4 mAb reduced the mRNA expression of COX-2 (P<0.05) after LPS stimulated decidual cells. CONCLUSION The mRNA expression of PR is reduced, and the mRNA expressions of IL-1β and COX-2 are increased after LPS-stimulated decidual cells in vitro. TLR4 mAb antagonize the role of LPS on PR, IL-1β, and COX-2.
Adult ; Cells, Cultured ; Cyclooxygenase 2 ; genetics ; metabolism ; Decidua ; cytology ; metabolism ; Female ; Humans ; Interleukin-1beta ; genetics ; metabolism ; Lipopolysaccharides ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Receptors, Progesterone ; genetics ; metabolism ; Toll-Like Receptor 4 ; antagonists & inhibitors ; Young Adult

Type 1 diabetes mellitus is caused by the autoimmune destruction of beta cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were beta cells; few alpha cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on beta cells and that HMGB1 may signal via TLR4 to selectively damage beta cells rather than alpha cells during the development of type 1 diabetes mellitus.
Animals ; Diabetes Mellitus, Type 1/immunology/*metabolism/pathology ; Female ; Gene Expression Regulation ; Glucagon-Secreting Cells/immunology/metabolism/pathology ; HMGB1 Protein/*genetics/metabolism ; Humans ; Immunity, Innate ; Insulin-Secreting Cells/immunology/metabolism/*pathology ; Macrophages/immunology/pathology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Necrosis ; Protein Binding ; Signal Transduction ; Toll-Like Receptor 4/*antagonists & inhibitors/genetics/immunology

OBJECTIVETo study the effect of total coumarins (TC) from Urtica dentata on dextran sulfate sodium (DSS)-induced colitis in mice.

METHODThe colitis model was established by administering DSS. Having been treated with TC, their body weight was determined. Concentrations of IL-6, IL-10, TGF-beta1 and IFN-gamma were monitored by ELISA. Colon samples were collected for the histopathological examination. Western blot was used to detect TLR4 and NF-kappaB protein expression in colonic tissues.

RESULTTCs from U. dentata effectively controlled the body weight loss of mice with colitis, down-regulated the concentration of IL-6 and IFN-gamma and increased the suppressive cytokines IL-10 and TGF-beta1 in the serum. Additionally, TC alleviated the mucosal damage and decreased the expressions of TLR4 and NF-kappaB in colonic tissues.

CONCLUSIONTCs from U. dentata shows the anti-inflammatory effect on colitis in mice by reducing the expressions of TLR4 and NF-kappaB in colonic tissues and regulating pro-and anti-inflammatory cytokines.

Animals ; Colitis ; drug therapy ; metabolism ; Coumarins ; therapeutic use ; Cytokines ; blood ; Dextran Sulfate ; toxicity ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; antagonists & inhibitors ; Toll-Like Receptor 4 ; antagonists & inhibitors ; Urticaceae ; chemistry

OBJECTIVETo determine the role of toll like receptor-4 signal pathways activation in ischemia-reperfusion injury of island skin flap.

METHODSA totol of 50 adult male SD rats were randomized into 3 groups: sham-operated group (n=10), ischemia/reperfusion group (n=20) and TLR4 inhibitor-eritoran tetrasodium (E5564)-treated group (n=20). The inguinal island skin flaps models were set up. A bolus of E5564 (5 mg/kg) was infused intravenously 60 min before reper fusionm. TLR4 binding activity in flap tissue was analyzed at 1, 2, 4 and 6 h of reperfusion by immunohistochemical technique and flaps were assessed histologically at 6 h of reperfusion. The viability of flaps was assessed 7 days postoperatively.

RESULTSExprerssion TLR4 in skin flap tissue was significantly increased in I/R group, compared with E5564-treated group. Immunohistochemical exam showed TLR4 mainly expressed in skin flap vessel wall and PMN membrane. Marked neutrophil infiltration and edema was observed in I/R group, while less neutrophil infiltration was observed in E5564-treated group. In the E5564-treated group, the survival of flaps was (80.31 +/- 11.63)%, which was significantly greater than that in the I/R group (51.70 +/- 7.62)% (P < 0.01).

CONCLUSIONAfter ischemia-reperfusion injury in rats, the expression of TLR4 increased in the skin flap tissue with excessive neutrophil infiltration. Administration of E5564 can significantly improve flap survival by regulating the early activation of TLR4 and suppressing neutrophil infiltration within the flap.

Animals ; Groin ; Ischemia ; metabolism ; Lipid A ; analogs & derivatives ; pharmacology ; Male ; Random Allocation ; Rats ; Reperfusion Injury ; metabolism ; Signal Transduction ; Surgical Flaps ; blood supply ; Toll-Like Receptor 4 ; antagonists & inhibitors ; metabolism