The salmonella effector Hcp modulates infection response, and affects salmonella adhesion and egg contamination incidences in ducks.

Salmonella Entertidis (SE) often causes persistent infections and egg contamination in laying ducks. Hcp, the core structural and effector proteins of the Type VI Secretion System (T6SS) in SE, contributes to bacterial invasion, adhesion and virulence. However, little is known about the effect of Hcp on the host's infection responses and egg contamination incidences in duck. Herein, we generated an hcp deletion mutant SE MY1△hcp and detected its ability to invade duck granulosa cells (dGCs) and contaminate eggs. In comparison with MY1-infected group, the SE adhesion decreased by 15.96% in MY1△hcp-infected dGCs, and the apoptosis in MY1△hcp-infected dGCs decreased by 26.58% and 30.99% at 3 and 6 hours postinfection, respectively. However, the expression levels of immunogenic genes TLR4, NOD1, TNFα, IL-1ß and proinflammatory cytokines IL-6, IL-1ß, TNF-α release were markedly lower in the dGCs inoculated with MY1△hcp than that of the wild type. Besides, the laying ducks were challenged with MY1 or MY1△hcp in vivo, respectively. The lower egg production and higher egg contamination were observed in MY1-infected ducks in comparison with MY1△hcp-infected birds. Furthermore, the host's infection response of differentially abundant proteins (DAPs) to Salmonella effector Hcp was identified using quantitative proteomics. A total of 164 DAPs were identified between the MY1- and MY1△hcp-infected cells, which were mainly engaged in the immune, hormone synthesis, cell proliferation and cell apoptotic process. Among them, STAT3, AKT1, MAPK9, MAPK14, and CREBBP were the center of the regulatory network, which might serve as key host response regulators to bacterial Hcp. In conclusion, we demonstrated that effector Hcp contributed to not only SE invasion, induction of dGCs apoptosis, and trigger of immune responses, but also enhanced contamination incidences. Also, the STAT3, AKT1, MAPK9, MAPK14, and CREBBP were identified as host's infection response regulators of bacterial Hcp in duck. Overall, these results not only offered a novel evidence of SE ovarian transmission but also identified some promising candidate regulators during SE infection.

The role and difference of TLR2 and TLR4 in rhabdomyolysis induced acute kidney injury in mice.

To investigate the role of toll like receptors (TLRs) 2 and 4 in rhabdomyolysis (RM)-related acute kidney injury (AKI). Wild-type (WT) mice and TLR2 knockout (TLR2-/-) or TLR4 knockout (TLR4-/-) mice were injected with either saline (sham) or glycerin (to induce RM-related AKI). Samples were collected for detection of 0 h 24 h (Cr) creatinine, urea nitrogen (BUN), creatine kinase (CK), and PAS staining of renal tissues. Serum Cr and BUN level was significantly increased in TLR2-/- and TLR4-/-AKI groups more than those in the control group and the WT mice in AKI group. TLR4-/-AKI group Cr, BUN level, and the pathological damage was lightest. The expression levels of signal transduction proteins in TLR2-/- and TLR4-/-AKI group were higher than in the control group, but was lower than that in the wild AKI group, with the TLR4-/-AKI group having the lowest levels. The expression level of inflammatory factor mRNA in TLR2-/- and TLR4-/-AKI groups was higher than that in control group, but was lower than that in wild AKI group, with TLR4-/-AKI group displaying lowest levels. Knockout of TLRs 2 and 4 decreased kidney inflammation and improved RM-related AKI.

The renal level of a novel cytokine IL-35 is related to sepsis-associated acute kidney injury in mice.

Interleukin-35 (IL-35) is a novel immunosuppressive and anti-inflammatory cytokine. IL-35 is mainly secreted by regulatory T cells (Tregs), and exerts its effects through inducing proliferation of Tregs and reducing activity of helper T cells Th17. However, the effect of IL-35 on sepsis-associated acute kidney injury (SA-AKI) remains unclear. This study is aimed to examine the expression and role of IL-35 in an animal model of SA-AKI induced by cecal ligation and puncture (CLP). Eleven C57 male mice with SA-AKI and eight controls were used, and blood and kidney tissues were collected. Blood creatinine (Cr), urea nitrogen (BUN), alanine transaminase (ALT) and aspartate transaminase (AST) were measured to assess kidney and liver injury. The renal morphology and cell apoptosis were examined. The mRNA and protein expression levels of IL-35 in kidney tissues were tested by qRT-PCR, IHC-P, IF, and ELISA. Biochemical and histological examinations indicated CLP induced SA-AKI in mice. TUNEL assay showed apoptosis of renal tubular epithelial cells in SA-AKI mice. The mRNA and protein expression of IL-12α and EBI3 in kidney tissues decreased significantly in SA-AKI mice compared with those in sham controls. IL-35 levels in kidney tissues displayed a significantly negative correlation with the levels of Cr (r = -0.584, P = 0.009), ALT (r = -0.549, P = 0.015), AST (r = -0.475, P = 0.04), but not with BUN (r = -0.437, P = 0.061). These results demonstrated that IL-35 is associated with the pathological process of SA-AKI, and might represent a potential therapeutic agent for SA-AKI treatment.

Adenovirus-expressing miR-153-3p alleviates aortic calcification in a rat model with chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease (CKD) have abnormal calcification in vascular tissue that is a risk factor for cardiovascular disease. However, the specific molecular mechanisms for vascular calcification remain largely unknown. The present study aimed to determine the differentially expressed miRs and the underlying molecular mechanisms of miR-153-3p in vascular calcification induced by adenine. METHODS: Differentially expressed miRs were screened using a microarray chip in the thoracic aorta. miRs and mRNA expression were measured by RT-qPCR. Protein expression was performed by western blotting analysis. Aortic calcification was confirmed by Von Kossa staining. The targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. RESULTS: Our results revealed that the expression of miR-153-3p was significantly down-regulated in the thoracic aorta from adenine-fed rats compared with that of the control group. Transfection of miR-153-3p into the thoracic aorta markedly suppressed adenine-induced aortic calcification and significantly decreased the mRNA expression of ALP, OC, OSX, SOST and Runx2. Further studies indicated that Runx2 was a direct target gene of miR-153-3p, which was verified by dual luciferase reporter assay. CONCLUSION: These results suggest that increased vascular miR-153-3p expression attenuates adenine-induced aortic calcification via inhibiting osteogenic trans-differentiation in the thoracic aorta.

Xuebijing Injection Promotes M2 Polarization of Macrophages and Improves Survival Rate in Septic Mice.

Xuebijing (XBJ) injection, a concoction of several Chinese herbs, has been widely used as an immunomodulator for the treatment of severe sepsis in China. However, the precise mechanisms responsible for its efficacy have not been fully elucidated. In our study, we determined the flow cytometry markers (F4/80, CD11c, and CD206), the levels of secreted cytokines (TNF-α, IL-6, and IL-10), and the expression of specific proteins of M2 (Ym1, Fizz1, and Arg1) to assess macrophage polarization. Treatment with XBJ lowered M1 associated cytokine levels and increased the level of M2 associated cytokine level. The percentage of M2 phenotype cells of XBJ group was much higher than that of the control group. Expressions of phosphorylated Janus kinase 1 (JAK1) and signal transducer and activator of transcription 6 (STAT6) were markedly enhanced after the administration of XBJ; on the other hand, the M2 associated cytokines and proteins were decreased following treatment with JAK1 or STAT6 inhibitor. In addition, the treatment of XBJ significantly improved the survival rate of septic mice. These studies demonstrate that XBJ can markedly promote M2 polarization and improve the survival rate of septic mice, thereby contributing to therapeutic effect in the treatment of septic complications.

Flotillin-1 expression in human clear-cell renal cell carcinoma is associated with cancer progression and poor patient survival.

The present study was designed to elucidate the expression levels and the proliferative effect of flotillin-1, an integral membrane protein encoded by the FLOT1 gene, in human clear-cell renal cell carcinoma (RCC). Flotillin has been implicated in other types of cancer, but the role of flotillin in RCC has not been established. Immunohistochemistry and western blotting were used to determine FLOT1 protein expression levels in RCC samples from 182 patients who underwent nephrectomy. FLOT1 mRNA expression levels were analyzed using reverse-transcription (RT) and RT-quantitative polymerase chain reaction (PCR). The association between FLOT1 expression levels in the tumor samples and patient survival time was examined using Kaplan­Meier analysis. To demonstrate the proliferative effect of FLOT1 on RCC cells, a FLOT1 vector was transfected into four RCC cell lines and FLOT1 expression was inhibited using small interfering RNA. The proliferative ability of the RCC cells was investigated using a WST-1 assay and xenograft experiments with BALB/C nude mice. The results demonstrated that FLOT1 expression levels were significantly higher in RCC cell samples from patients than in healthy renal tissue, and the expression levels were associated with tumor stage, size and histological grade. In addition, FLOT1 significantly enhanced the proliferation of RCC cell lines in vitro and in vivo. These findings suggest that FLOT1, which is upregulated in RCC, is involved in RCC cell proliferation, tumorigenesis and progression. Therefore, FLOT1 is an independent prognostic marker and therapeutic target for patients with clear-cell RCC.

Quorum-sensing gene luxS regulates flagella expression and Shiga-like toxin production in F18ab Escherichia coli.

To investigate the effect of the luxS gene on the expression of virulence factors in Shiga-like toxin producing and verotoxin-producing Escherichia coli, the luxS gene from E. coli 107/86 (wild type, O139:H1:F18ab, Stx2e) was deleted. The successful deletion of luxS was confirmed by bioluminescence assays. The luxS deletion mutant exhibited changed flagella-related phenotypes, like impaired expression of flagella, decreased flagella motility, reduced biofilm formation, and reduced ability to induce pro-immunity response in host cells, which were restored after complementation with the intact luxS gene. The mutant strain also displayed attenuated production of Stx2e. This study provides new information to the crucial function of luxS in regulating Shiga-like toxin producing E. coli virulence.

Flagellin and F4 fimbriae have opposite effects on biofilm formation and quorum sensing in F4ac+ enterotoxigenic Escherichia coli.

Bacteria that form biofilms are often highly resistant to antibiotics and are capable of evading the host immune system. To evaluate the role of flagellin and F4 fimbriae on biofilm formation by enterotoxigenic Escherichia coli (ETEC), we deleted the fliC (encoding the major flagellin protein) and/or the faeG (encoding the major subunit of F4 fimbriae) genes from ETEC C83902. Biofilm formation was reduced in the fliC mutant but increased in the faeG mutant, as compared with the wild-type strain. The expression of AI-2 quorum sensing associated genes was regulated in the fliC and faeG mutants, consistent with the biofilm formation of these strains. But, deleting fliC and/or faeG also inhibited AI-2 quorum sensing activity.

Ischemia-reperfusion induces renal tubule pyroptosis via the CHOP-caspase-11 pathway.

The apoptotic or necrotic death of renal tubule epithelial cells is the main pathogenesis of renal ischemia-reperfusion-induced acute kidney injury (AKI). Pyroptosis is a programmed cell death pathway that depends on the activation of the caspase cascade and IL-1 cytokine family members. However, the role of pyroptosis in AKI induced by ischemia-reperfusion remains unclear. In this study, we found that the levels of the pyroptosis-related proteins, including caspase-1, caspase-11, and IL-1ß, were significantly increased after 6 h of renal ischemia-reperfusion injury (IRI) and peaked at 12 h after IRI. Enhanced pyroptosis was accompanied by elevated renal structural and functional injury. Similarly, hypoxia-reoxygenation injury (HRI) also induced pyroptosis in renal tubule epithelial NRK-52E cells, which was characterized by increased pore formation and elevated lactate dehydrogenase release. In addition, obvious upregulation of the endoplasmic reticulum (ER) stress biomarkers glucose-regulated protein 78 and C/EBP homologous protein (CHOP) preceded the incidence of pyroptosis in cells treated with IRI or HRI. Pretreatment with a low dose of tunicamycin, an inducer of ER stress, relieved IRI-induced pyroptosis and renal tissue injury. Silencing of CHOP by small interfering RNA significantly decreased HRI-induced pyroptosis of NRK-52E cells, as evidenced by reduced caspase-11 activity and IL-1ß generation. Therefore, we conclude that pyroptosis of renal tubule epithelial cells is a key event during IRI and that CHOP-caspase-11 triggered by overactivated ER stress may be an essential pathway involved in pyroptosis.

F18ab Escherichia coli flagella expression is regulated by acyl-homoserine lactone and contributes to bacterial virulence.

To investigate the effect of the Quorum Sensing (QS)-I system on the expression of virulence factors in Shiga toxin producing and verotoxin-producing Escherichia coli (STEC and VTEC), the yenI gene from Yersinia enterocolitica was cloned into E. coli F18ab 107/86. Recombinant E. coli transformed with yenI produced acyl-homoserine lactone synthase (AHL), as measured using cross-streaking assays with the reporter biosensor strain Chromobacterium violaceum CV026. The AI-1 positive recombinant F18ab E. coli exhibited impaired expression of flagella, decreased motility, reduced biofilm formation and AI-2 production, as well as attenuated adherence and invasion on IPEC-J2 cells. This study provides new insights to the crucial function of AI-1 in regulating STEC virulence.

Endoplasmic reticulum stress and its regulator XBP-1 contributes to dendritic cell maturation and activation induced by high mobility group box-1 protein.

High mobility group box-1 protein (HMGB1) had been proved to induce maturation and activation of dendritic cell (DC), however, the endogenous changes and mechanisms underlying are unknown. Since endoplasmic reticulum stress (ERS) activates an adaptive unfolded protein response (UPR) that facilitates cellular survival and repair, we hypothesized that HMGB1 may regulate the function of DC by modulating ERS. In our study, HMGB1 stimulation induced significant ERS responses in DCs in a time- and dose-dependent manner, demonstrated by the up-regulation of a number of ERS markers. Gene silence of XBP-1 in splenic DCs decreased the levels of CD80, CD86 as well as major histocompatibility complex (MHC)-II expression and cytokine secretion after HMGB1 treatment, when compared with untransfected or nontargeting-transfected DCs (all P<0.05). Moreover, XBP-1 silenced DCs after treatment with HMGB1 failed to stimulate notable proliferation and differentiation of T cells, unlike normal DCs or nontargeting-transfected DCs (all P<0.05). Gene silence of XBP-1 resulted in down-regulation of the receptor for advanced glycation end products (RAGE) expression on the surface of splenic DCs induced by HMGB1 stimulation (P<0.05). These findings demonstrate an important role for ERS and its regulator XBP-1 in HMGB1-induced maturation and activation of DCs.

[Effect of high mobility group box-1 protein on immune cells and its regulatory mechanism].

High mobility group box-1 protein (HMGB1), which is a nuclear protein, participates in chromatin architecture and transcriptional regulation. When released from cells, HMGB1 also plays a well-established role as a pro-inflammatory mediator during innate immune responses to injury. In the initial stage of injury, there is a release of large quantities of early pro-inflammatory mediators to initiate or perpetuate immune responses against pathogens, but this pro-inflammatory period is transient, and it is followed by a prolonged period of immune suppression. At present, several lines of evidences have suggested that HMGB1 is a late cytokine provoking delayed endotoxin morbidity, which may enhance the production of early proinflammatory mediators, and it can contribute potently to the activation of different immune cells and play a role in the development of host cell-mediated immunity. The biology of HMGB1 has been extensively studied as a pro-inflammatory cytokine of systemic inflammation, however, this review will attempt to provide a summary of the effects of HMGB1 on different immune cells and its regulatory mechanism in acute insults.

[Influence of splenic high mobility group box-1 protein on immune function of regulatory T lymphocytes in scald rats].

OBJECTIVE: To observe the influence of high mobility group box-1 protein (HMGB1) derived from spleen on the phenotype of regulatory T lymphocytes (Treg) and HMGB1-mediated immune function in severely scalded rats after delayed resuscitation. METHODS: One hundred and four Wistar rats were divided into normal control group (NC, n = 8), sham scald group (SS, n = 32), scald group (S, n = 32), and ethyl pyruvate (EP) treatment group (EPT, n = 32) according to the random comparison table. Rats in the latter 2 groups were subjected to 30%TBSA full-thickness scald, which were intraperitoneally injected with Ringer solution or EP solution at post scald hour (PSH) 6 (delayed antishock treatment) and administered with 4 mL Ringer solution or EP solution per 12 hours after PSH 12 till PSH 48. Rats in SS group were treated the same as that of S group except for sham scald with 37 degrees C water. Injured rats were sacrificed at post scald day (PSD) 1, 3, 5, 7 (rats in NC group were also sacrificed), and CD4(+)CD25(+)Treg were isolated from spleen with magnetic-activated cell sorting method. The content of HMGB1 in spleen and IL-2 level in supernatant were determined with ELISA. The expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) on Treg was determined with flow cytometry, and the proliferation activity of T lymphocytes was also detected (recorded as absorbance value). Data were processed with analysis of variance among groups and independent samples t test. RESULTS: (1) Compared with that of rats in SS group and EPT group, the expression of splenic HMGB1 in S group increased significantly on PSD 1 through PSD 7 [peaked on PSD 1: (46.7 +/- 8.3) ng/mg protein]. (2) Compared with that in SS group, the expression of CTLA-4 in S group was enhanced significantly on PSD 1 through PSD 5 (with t value respectively 10.459, 12.051, 4.029, P < 0.05 or P < 0.01); while that in EPT group decreased significantly on PSD 1 through PSD 7 as compared with that from S group (with t value respectively 2.796, 9.913, 9.581, 10.022, P < 0.05 or P < 0.01). (3) Compared with that of rats in SS group, the proliferation activity of T lymphocytes in S group was markedly suppressed on PSD 1 through PSD 7 (nadir on PSD1: 0.167 +/- 0.059), and release of IL-2 was decreased significantly [nadir on PSD 5: (44 +/- 24) pg/mL]. T lymphocytes proliferation activity was restored and excretion of IL-2 increased in EPT group as compared respectively with that of S group at each time point. CONCLUSIONS: The release of HMGB1 may stimulate splenic Treg to mature, thereby induce suppression of proliferation activity of T lymphocytes and immune function. EP can ameliorate immune dysfunction in animals with delayed resuscitation through inhibiting the synthesis and release of HMGB1.

Association between high-mobility group box-1 protein release and immune function of dendritic cells in thermal injury.

The present study was performed to investigate in vivo the effect of high-mobility group box-1 protein (HMGB1) on the maturation of dendritic cell (DC) and the influence on T-cell-mediated immunity after thermal injury. Rats were randomly divided into 3 groups as follows: sham burn group, burn group, and burn with ethyl pyruvate (EP) treatment group, and they were sacrificed on post burn days (PBD) 1, 3, 5, and 7 respectively. MACS microbeads were used to isolate splenic DCs and column of nylon wool to obtain T cells. Phenotypes were analyzed by flow cytometry and cytokines were determined with ELISA kits. The expression levels of splenic HMGB1 were significantly elevated during PBD 1-7. DC expressed similar CD80 levels, strongly enhanced CD86, and slightly elevated MHC class II levels in comparison to DC from sham-injured rats, and protein levels of IL-12 were not increased after thermal injury. Administration of EP to inhibit HMGB1 could significantly enhance expression levels of CD80, MHC class II on DC surface, and IL-12 production after burns. Simultaneously, proliferative activity and expression levels of IL-2 as well as IL-2R alpha of T cell were restored. These results suggested that the excessively released HMGB1 might stimulate splenic DC to mature abnormally and down-regulate the IL-12 production, and further shifting of Th1 to Th2 with suppression of T-lymphocyte immune function following burn injury.

[The pattern of nuclear factor-kappaB activation in rats with endotoxin shock and its role in biopterin-mediated nitric oxide induction].

OBJECTIVE: To investigate the pattern of nuclear factor-kappaB (NF-kappaB) activation in rats with lipopolysaccharide( LPS) shock, and to explore the mechanism of NF-kappaB signal pathway in the biopterin-mediated nitric oxide(NO) induction, as well as its role in the development of multiple organ dysfunction syndrome ( MODS) secondary to endotoxin challenge. METHODS: Fourty-seven male Wistar rats were randomly divided into control group ( C, n = 8) , LPS group ( n = 24, with 8 rats at each time-points, and shock model was made by injection of same dosage of LPS) , and pyrrolidine dithiocarbamate (PDTC) treatment group ( PDTC, n = 15, with 5 rats at each time-points, and the rats were injected with LPS and PDTC). The rats were sacrificed at 2,6,12 post-injection hour( PIH) , and the blood and tissue samples from liver, lungs and kidneys were harvested for the determination of NF-KB activity, GTP cyclohydrolase I (GTP-CH I ) , and inducible nitric oxide synthase (iNOS) mRNA expression in the liver, lungs and kidneys, plasma and tissue content of biopterin and NO, as well as hepatic and renal function, and pulmonary myeloperoxidase activity. RESULTS: NF-kappaB DNA binding activity in LPS group was rapidly enhanced in liver, lungs and kidneys after endotoxin challenge when compared with that in controls (e. g. in pulmonary tissue it was 26+/-6) , and it reached the peak at 2 PIH, which was 291 +/-44 in pulmonary tissue( P <0. 01). GTP-CH I mRNA expression and biopterin levels in the liver, lung and kidney of each group were obviously higher than those in control group( P <0.05 or 0.01) , and it maintained at high levels at 12 PIH. Additionally, different degrees of dysfunction of the above mentioned organs was observed. Treatment with PDTC, an inhibitor of NF-KB signal transduction pathway, could reduce NF-kappaB DNA binding activity, inhibit GTP-CH I and iNOS/NO mRNA expression, as well as BH4, and NO levels in various tissues. Meanwhile the multiple organ damage was significantly ameliorated by PDTC pretreatment. CONCLUSION: Endotoxin challenge can rapidly lead to activation of NF-kappaB in various tissues, and NF-KB pathway might markedly up-regulate the production of biopterin/NO following endotoxic shock. Inhibition of NF-kappaB pathway attenuates inflammatory response and ameliorates multiple organ dysfunction, which might be associated with its down-regulation of the excessive activation of iNOS mediated by biopterin.

[The mechanisms of extracellular-signal regulated protein kinase pathway in biopterin induction in rats with endotoxic shock].

OBJECTIVE: To observe the influence of treatment with the inhibitor of extracellular-signal regulated protein kinase (ERK) signal transduction pathway on the expression of biopterin/nitric oxide (NO) as well as the activation of nuclear factor-kappaB (NF-kappaB), and to clarify the potential cross-talk regulation mechanisms between ERK and NF-kappaB pathway in biopterin-mediated NO induction in rats with endotoxic shock. METHODS: Using an endotoxic shock model, 60 male Wistar rats were randomly divided into normal controls (n = 8), endotoxic shock group (n = 32) and PD98059 treatment group (n = 20). At serial time points animals in each group were sacrificed, and tissue samples from liver, lungs as well as kidneys were harvested to detect NF-kappaB activity, guanosine triphosphate-cyclohydrolase (GTP-CHI) and inducible nitric oxide synthase (iNOS) mRNA expression. Biopterin and NO levels in plasma and tissues were also assayed. RESULTS: It was found that after lipopolysaccharide (LPS) challenge, GTP-CHI mRNA expression and biopterin levels significantly elevated in liver, lungs and kidneys, keeping at high values up to 24 h, so did the values of iNOS mRNA expression and NO levels. NF-kappaB DNA binding activity was enhanced rapidly in various tissues, peaking at 2 h after LPS challenge. Treatment with PD98059, an inhibitor of ERK signal transduction pathway, could significantly inhibit GTP-CHI mRNA expression in kidneys, and GTP-CHI mRNA expression in liver and lungs showed certain down-regulation tendency. At the same time, biopterin level was significantly decreased in plasma, liver and kidneys at 12 h. Similarly, iNOS/NO induction at early stage markedly decreased in various tissues. In addition, treatment with PD98059 reduced NF-kappaB DNA binding activity in liver, lungs, as well as kidneys at 2-6 h, 2 h, 24 h and 24 h after LPS challenge, respectively. CONCLUSIONS: Inhibition of ERK pathway could partially inhibit the production of biopterin/NO as well as the activation of NF-kappaB pathway, which indicated that cross-talk regulation seems to be existed between ERK and NF-kappaB pathway, and they might be involved in the regulatory process of biopterin-mediated nitric oxide induction in rats with endotoxic shock.

[Relationship between nuclear factor-kappaB activity and kidney injury in rats with postburn sepsis].

OBJECTIVE: To investigate the relationship between nuclear factor-KappaB (NF-KappaB) activity and kidney injury in rats with postburn sepsis. METHODS: Rats subjected to 30% full-thickness scald injury, followed by intraperitoneal injection of lipopolysaccharide (LPS), were used in the present study. Fifty-four Wistar rats were randomly divided into normal control group, postburn sepsis 1, 2, 6, 12, 24 hours groups, and sepsis with NF-KappaB inhibitor pyrrolidine dithiocarbamate (PDTC) treatment 1, 2, and 6 hour groups. NF-KappaB activity, tumor necrosis factor-alpha (TNF-alpha) protein expression, and renal function were determined with electrophoretic mobility shift assay (EMSA), enzyme linked immunoadsorbent assay (ELISA), and automatic biochemistry analyzer, respectively. RESULTS: NF-KappaB activity in kidney was markedly enhanced and reached its peak 1 hour after scalding and injection of LPS (all P<0.01), and was decreased remarkably after the administration of PDTC. PDTC could suppress the elevated plasma TNF-alpha protein expression (both P<0.01), but not renal TNF-alpha levels. PDTC could not reduce blood urea nitrogen and blood creatinine contents, which were increased after scalding followed by LPS challenge. CONCLUSION: The results suggest that the treatment with NF-KappaB inhibitor PDTC could down-regulate the NF-KappaB activity in kidney, but it could not protect the renal function in rats with postburn sepsis.

[Changes in Toll-like receptor 2 and 4 gene expression in vital organs in septic rats and their regulation mechanisms].

OBJECTIVE: To investigate the changes in Toll-like receptor (TLR) 2 and 4 gene expression in vital organs in septic rats and their potential regulation mechanism. METHODS: One hundred Wistar rats were randomly divided into normal controls (n=10), sham-operated group (n=10), septic group (n=60), and recombinant bactericidal/permeability increasing protein (BPI)-treated group (n=20). Severe sepsis was replicated by cecal ligation and puncture (CLP). Animals were sacrificed at different time points after CLP, tissue TLR2/4 mRNA expression in the liver, lungs, kidneys as well as intestine were measured by reverse transcription-polymerase chain reaction (RT-PCR). Plasma levels of tumor necrosis factor-alpha(TNF-alpha) and interleukin-10 (IL-10) were also determined by enzyme linked immunoadsorbent assay (ELISA). RESULTS: TLR2/4 mRNA could be detected in various tissues with low values both in normal controls and sham-operated group, but they were markedly up-regulated at 2 hours after CLP, peaking at 6-12 hours. Tissue TLR4 mRNA was gradually down-regulated 24 hours later, while TLR2 mRNA levels maintained high values up to 72 hours. In comparison with the CLP group, treatment with BPI significantly decreased TLR2 mRNA in various tissues at 12 and 24 hours (P<0.05 or P<0.01), also tissue TLR4 mRNA at 12 hours (P<0.05 or P<0.01), without marked influence on TLR4 mRNA expression at 24 hours in liver, lungs and small intestine (P>0.05). In addition, treatment with BPI could significantly lower plasma TNF-alpha levels at 12 hours post-CLP, on the other hand markedly elevate plasma IL -10 levels 24 hours later (P<0.01). CONCLUSION: These data suggest that severe peritoneal infection could result in up-regulation of TLR2/4 mRNA expression in vital organs, which might play important roles in mediating proinflammatory cytokine synthesis and release. Endotoxemia appears to be involved in the activation of tissue TLR2/4 expression associated with CLP-induced sepsis.