Inhibitory effect of receptor for advanced glycation end product­specific small interfering RNAs on the development of hepatic fibrosis in primary rat hepatic stellate cells.

Specific small interfering RNAs (siRNAs) targeting receptor for advanced glycation end products (RAGE) inhibit the expression of RAGE, α-smooth muscle actin and type I collagen in the T6 hepatic stellate cells (HSCs), indicating that RAGE is important for the activation of HSCs and the expression of collagen. The present study aimed to investigate the effect of specific siRNAs targeting RAGE on the development of hepatic fibrosis (HF), using primary rat HSCs, which were isolated and cultured in vitro. The expression vectors for specific siRNAs targeting RAGE were constructed and transfected into primary rat HSCs. Untreated and nonspecific siRNA-transfected primary rat HSCs served as controls. The expression levels of RAGE, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), laminin (LN), hyaluronic acid (HA) and N-terminal procollagen III propeptide (PIIINP) in primary HSCs were detected by reverse transcription quantitative polymerase chain reaction and western blotting. The mRNA and 42 kD protein expression of RAGE in the pAKD-GR126-transfected primary HSCs were significantly downregulated compared with those in the untreated and the pAKD-negative control (NC)-transfected controls. The mRNA and protein expression levels of IL-6, TNF-α, TGF-ß1, CTGF, LN, HA and PIIINP in the pAKD-GR126-transfected primary HSCs were also markedly downregulated compared with those in the untreated and pAKD-NC-transfected controls. Therefore, RAGE-specific siRNAs inhibited the expression of RAGE in primary rat HSCs and inhibited the development of HF.

Anti-fibrotic effects of specific-siRNA targeting of the receptor for advanced glycation end products in a rat model of experimental hepatic fibrosis.

Since the receptor for advanced glycation end products (RAGE)-ligand axis has been demonstrated to be important in fibrogenesis, rat models may be used to assess whether specific small interfering RNAs (siRNAs) that target RAGE are able to reduce the progression of hepatic fibrosis. However, the effect of RAGE-targeted siRNA on established hepatic fibrosis remains to be elucidated. In the present study, RAGE-specific siRNA expression vectors were constructed prior to the animal experiment. Sprague-Dawley rats were treated initially with olive oil (2 ml/kg) or 50% CCl4 (2 ml/kg; CCl4/olive oil=1:1) twice per week for six weeks to generate the fibrosis model. The rats were then treated with phosphate­buffered saline, a RAGE-specific siRNA expression vector, at different doses or a non-specific siRNA expression vector twice weekly via tail vein injection for up to six weeks, and were sacrificed at week two, four or six. Compared with the control groups, RAGE­specific siRNA therapy significantly decreased RAGE mRNA and protein expression in rat livers (P<0.01). Following six weeks of RAGE gene-silencing treatment, the liver function, which was assessed by analyzing serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL), improved to varying degrees (P<0.01). The expression of nuclear factor-κB (NF-κB) significantly decreased following RAGE gene­silencing therapy (P<0.01). In addition, the serum levels of inflammatory cytokines, including tumor necrosis factor­α (TNF-α) and interleukin-6 (IL-6), and extracellular matrix (ECM) components, including hyaluronic acid (HA), laminin (LN) and procollagen type III (PCIII) also decreased (P<0.01). Furthermore, the expression of α-smooth muscle actin (α-SMA) and collagen I, which indicate the activation of hepatic stellate cells (HSCs), were downregulated following RAGE gene-silencing therapy (P<0.01). Furthermore, the inflammatory activity grade and fibrosis stage of rat livers also significantly improved compared with the control groups following RAGE gene-silencing therapy. Specific targeting of RAGE using siRNA may inhibit RAGE gene expression effectively in the rat hepatic fibrosis model and attenuate the progression of established hepatic fibrosis. This therapeutic effect may be mediated via inhibition of the expression of NF-κB. These findings suggest that RAGE may be a new target to prevent hepatic fibrosis.

In vivo and in vitro expression of the RASAL1 gene in human gastric adenocarcinoma and its clinicopathological significance.

Recent studies have suggested that the RAS protein activator like-1 (RASAL1) is a potential tumor suppressor, which is found to be reduced in certain human cancers. Its downregulation is involved in the progression of malignancies. However, whether or not RASAL1 plays a role in the development of gastric cancer remains to be determined. Our study aimed to clarify the role of RASAL1 in the progression of gastric adenocarcinoma. The expression of RASAL1 in primary gastric adenocarcinoma tissue specimens was determined by immunohistochemistry. The expression of RASAL1 mRNA and protein was detected by RT-PCR and western blotting in gastric adenocarcinoma cell lines with varying differentiation statuses, including well-differentiated MKN-28, moderately differentiated SGC-7901 and poorly differentiated BGC-823, respectively. A normal gastric epithelial cell line, GES-l, was used as the control line. The immunohistochemical results revealed that the expression of the RASAL1 protein was mainly observed in the cytoplasm. Among 50 cases of gastric adenocarcinoma tissues, 12 cases were identified as (-), 23 cases (+), 13 cases (++) and 2 cases (+++). Among 50 cases of normal gastric tissues, 16 cases were (++) and 34 cases (+++). The expression of the RASAL1 protein was found to be decreased in the gastric adenocarcinoma tissue compared with normal gastric tissue (p<0.01). Moreover, in the gastric carcinoma tissues, the expression of RASAL1 was correlated with carcinoma diameter, differentiation grades, invasive depth, lymph node metastasis and TNM. Additionally, the RASAL1 mRNA and proteins were decreased in the three gastric adenocarcinoma cell lines compared with the normal gastric epithelial cell line GES-l. In addition, the downregulation of RASAL1 correlated with the differentiation status of cancer cell lines. Based on the above investigation, we conclude that expression of the RASAL1 gene is decreased in gastric carcinoma tissues and cell lines. The results indicate that RASAL1 may be important in the tumorigenesis and development of gastric carcinoma.

HMBG1 mediates ischemia-reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling.

High-mobility group protein box-1 (HMGB1) has recently been recognized as a novel candidate in a specific upstream pathway promoting inflammation after brain ischemia. However, its downstream pathway and underlying mechanism have yet to be elucidated. The HMGB1 level in the acute cerebral infarct (ACI) group was significantly increased compared with that of control group, and correlated with the severity of neurologic impairment of ACI patients. Further, recombinant human HMGB1 (rhHMGB1) had no effect on microglia derived from mice lacking the Toll-like receptor 4 (TLR4(-/-)). Intracerebroventricular injection of rhHMGB1 in TLR4(+/+) mice cause significantly more injury after cerebral ischemia-reperfusion than control group. But, TLR4(-/-) mice administered with rhHMGB1 showed moderate impairment after ischemia-reperfusion than TLR4(+/+) mice. To determine the potential downstream signaling of HMGB1/TLR4 in cerebral ischemic injury, we used the ischemic-reperfusion model with Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-ß knockout mice (TRIF(-/-)) and evaluated the activity and expression of TRIF pathway-related kinases. The results suggest that the TRIF pathway is not likely to be involved in TLR4-mediated ischemia brain injury. Finally, we found that TLR4 expressed by immigrant macrophages was involved in the development of ischemic brain damage. These results suggest that HMBG1 mediates ischemia-reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling. The TLR4 expressed by immigrant macrophages may be involved in the development of ischemic brain damage.

Elevation of high-mobility group protein box-1 in serum correlates with severity of acute intracerebral hemorrhage.

High-mobility group protein box-1 (HMGB1) is a proinflammatory involved in many inflammatory diseases. However, its roles in intracerebral hemorrhage (ICH) remain unknown. The purpose of this study was to examine the correlation between changes in serum levels of HMGB1 following acute ICH and the severity of stroke as well as the underlying mechanism. Changes in serum levels of HMGB1 in 60 consecutive patients with primary hemispheric ICH within 12 hours of onset of symptoms were determined. The correlation of HMGB1 with disease severity, IL-6, and TNF-α was analyzed. Changes in HMGB1 levels were detected with ELISA and Western blot. Compared with normal controls, patients with ICH had markedly elevated levels of HMGB1, which was significantly correlated with the levels of IL-6 and TNF-α, NIHSS score at the 10th day, and mRS score at 3 months. In comparison with the control group, the levels of HMGB1 in the perihematomal tissue in mice with ICH increased dramatically, peaked at 72 hours, and decreased at 5 days. Meanwhile, heme could stimulate cultured microglia to release large amounts of HMGB1 whereas Fe(2+/3+) ions failed to stimulate HMGB1 production from microglia. Our findings suggest that HMGB1 may play an essential role in the ICH-caused inflammatory injury.

High-mobility group protein box-1 and its relevance to cerebral ischemia.

High-mobility group box-1 (HMGB1) was originally identified as a ubiquitously expressed, abundant, nonhistone DNA-binding protein. It has well-established functions in the maintenance of nuclear homeostasis. The HMGB1 can either be passively released into the extracellular milieu in response to necrotic signals or actively secreted in response to inflammatory signals. Extracellular HMGB1 interacts with receptors, including those for advanced glycation endproducts (RAGEs) as well as Toll-like receptor 2 (TLR2) and TLR4. The HMGB1 functions in a synergistic manner with other proinflammatory mediators and acts as a potent proinflammatory cytokine-like factor that contributes to the pathogenesis of diverse inflammatory and infectious disorders. Numerous reports point to HMGB1 as a novel player in the ischemic brain. This review provides an appraisal of the emerging roles of HMGB1 in cerebral ischemia injury, highlighting the relevance of HMGB1-blocking agents as potent therapeutic tools for neuroprotection.

Efficacy and safety of placing nasoenteral feeding tube with transnasal ultrathin endoscope in critically ill patients.

BACKGROUND: The placement of an enteral feeding tube is the foundation for providing enteral nutrition. But due to the anatomic complexity of the stomach and the duodenum, to a certain degree, there are some technical difficulties in the placement of postpyloric feeding tube, especially in critically ill patients. This study aimed to evaluate the efficacy and safety of placing nasoenteral feeding tube with a transnasal ultrathin endoscope. METHODS: Totally 49 patients, involving 46 (93.9%) being American Society of Anesthesiologists Physical Status (ASA-PS) grade III (n = 3) and grade IV (n = 43), in whom a nasoenteral feeding tube was placed with a transnasal ultrathin endoscope by using over-the-wire technique. The related clinic information during the procedure including success rate, time required, complications and monitoring results of vital signs was analyzed. RESULTS: The tube was placed at or beyond the Treitz's ligament in all of the 49 cases and the total tube-placement success rate was 100% including the one-time tube-placement success rate 95.9%. The tube placement was successful in 46 (93.9%) cases by transnasal method and 3 (6.1%) cases by transoral method. In the 47 cases whose one-time tube-placement success was obtained, the average procedure time was (6.2 +/- 5.6) minutes. For the 3 patients the endoscope inserted transorally due to the failure of transnasal insertion, the total procedure time was (12.3 +/- 2.1) minutes. In the period of nasoenteral tube placement, the average systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR) and average pulse oxygen saturation (SpO(2)) did not show any significant change. Apart from 3 patients in whom nausea occurred in the procedure and 2 nasal bleeding, no any other acute complications arose. CONCLUSION: The method of placing nasoenteral feeding tube with the transnasal ultrathin endoscope is not only efficient, time-saving, technically simple, and painless to patients, but also safe especially in critically ill patients.

Upregulated expression of toll-like receptor 4 in monocytes correlates with severity of acute cerebral infarction.

In the present study, we observed the expression of toll-like receptor 4 (TLR4) and its downstream signal pathway in peripheral blood monocytes (PBMs) from patients with acute cerebral infarct (ACI). The expression of TLR4 and MyD88 by PBMs was determined by flow cytometry and reverse transcriptase-polymerase chain reaction, and nuclear factor-kappaB (NF-kappaB) activity was detected by electrophoretic mobility shift assay. Ischemia/reperfusion injury-induced cerebral edema, infarction area, and neurologic impairment scores were determined in MyD88 gene knockout mice. The results indicated a significant increase in circulating TLR4(+) monocytes in ACI patients as compared with the control group and the transient ischemia attack (TIA) group. This change paralleled an elevation in TLR4mRNA transcription and serum tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 in the ACI and TIA groups. Correlation analysis showed TLR4 expression to significantly correlate with cytokine levels and stroke severity. MyD88mRNA differed insignificantly among the three groups. Compared with wild-type mice, 6 h of cerebral ischemia followed by 24 h of reperfusion did not significantly change cerebral edema, cerebral infarction area, and neurologic impairment scores in MyD88 gene knockout mice. Compared with the control group, serum heat shock protein (HSP) 60 increased significantly in the ACI and TIA groups, leading to NF-kappaB activation in TLR4/CD14-transfected HEK293 cells. It is suggested that upregulated TLR4 expression on PMBs may act as one of the peripheral mechanisms of inflammatory injury after ACI. Moreover, circulating HSP60 may be a ligand for TLR4, which is involved in the peripheral mechanism of inflammatory injury after ACI, possibly through an MyD88-independent signal pathway.

[Research progress in human artificial chromosomes(HACs) and the potentials in application].

Since the first report of the establishment of human artificial chromosome(HAC) was published in 1997, several types of HAC have been created by different strategies. Compared to other artificial chromosomes, such as yeast artificial chromosome (YAC) and bacterial artificial chromosome(BAC), HAC exists in a cell independently, in other words, HAC does not integrated into the cellular genome, and can undergo normal mitosis and meiosis from generation to generation in vitro and in vivo. Recent results proved that HAC, as a DNA carrier, is able to host a large fragment of DNA or mini-chromosome, thus it could be a very important tool in the study of human gene expression and regulation, human chromosome function and minimum functional elements and animal models for human diseases. In the near future, HAC can also be used in gene therapy for human genetic diseases.

[Cloning and identification of NF-kappaB p50 Rel homology domain and detection of its autonomous reporter gene activity].

AIM: To clone NF-kappaB p50 Rel homology domain (RHD) gene and construct the "bait" vector in yeast two-hybrid system, and detect the yeast cell toxicity and autonomous reporter gene activity of target gene. METHODS: Total RNA was extracted from human peripheral blood mononuclear cells and NF-kappaB p50 RHD gene was amplified by RT-PCR, and cloned into pGBKT7. The recombinant plasmid was transformed into yeast AH109. The growth condition of the transformants was observed in the selected medium SD/-Trp. The reporter gene activity of target gene in the yeast cells was verified by filter blotting. RESULTS: Using restriction enzyme digestion analysis and PCR, the length of inserted gene was confirmed correct. Sequencing result indicated that the sequence of the inserted gene and its open reading frame were completely correct, and then the recombinant plasmid was named pGBKT7-p50. p50 RHD gene had neither autonomous reporter gene activity nor yeast cytotoxicity. CONCLUSION: As a bait plasmid, pGBKT7-p50 could be used in yeast two-hybrid system to screen and capture the polypeptides which interact with p50 RHD.

[Changes in human leukocyte antigen-DR expression on monocytes and its value of prediction on infection complication in trauma patients].

OBJECTIVE: To observe the changes of human leukocyte antigen DR (HLA-DR) expression in monocytes of trauma patients and its value of prediction on infection complications. METHODS: Fifty-four trauma patients were divided into three groups according to severity of injury: severe trauma group injury severity score (ISS) >or=25, moderate trauma group (16or=16) were divided into three groups according to infection or not: no infection group, localized infection group and systemic infection group. Blood samples were collected immediately after admission and serially at 8:30 to 9:00 a.m. on days 1, 2, 4, 6, 8, 14 after admission, and monocyte HLA-DR expression was determined with monoclonal staining and flow cytometry. RESULTS: The HLA-DR expression in monocytes was reduced in the trauma patients. The lowest levels of HLA-DR were recorded on day 2 after trauma. Subsequently HLA-DR expression in monocytes increased gradually. During the whole observation, the HLA-DR expression was significantly decreased in both severe trauma group and moderate trauma group versus control group, but no significant differences were found between severe trauma group and moderate trauma group, mild trauma group and control group, Immediately after trauma, HLA-DR expression in monocytes was significantly lower in the localized infection group than that in the patients without infection, and lasted until day 4 after trauma. The mean fluorescence intensity of HLA-DR expression in monocytes on the day 2 to 14 after trauma, the percentage of HLA-DR monocytes on the day 1 to 14 after trauma were significantly lower in the systemic infection group than those in the localized infection group. The level of HLA-DR expression in monocyte in the 2 died trauma patients was lowered till died. CONCLUSION: In severe trauma patients, the HLA-DR expression in monocytes is significantly decreased, and decreased levels of HLA-DR expression in monocytes might be the early indicators of an immune deviation associated with the development of infection complications and prognosis.

Effect of decoy-oligodeoxynucleotides on expression of inflammation mediators in pMPhi cells from rats.

OBJECTIVE: To study the effect of decoy-oligodeoxynucleotides (decoy-ODNs) in dumbbell shape with the oligodeoxynucleotide sequence similar to nuclear factor kappa B (NF-kappaB) cis-elements on expression of inflammation mediators in pMPhi cells from rats. METHODS: With carriers of cationic liposomes, decoy-ODNs were transfected into pMPhi cells of rats. Then the inhibiting effects of the decoy-ODNs on tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6) and IL-10 were analyzed. RESULTS: Decoy-ODNs could decrease the expression of TNFalpha and IL-6 in dose-dependent fashion but had weaker inhibiting effect on IL-10. CONCLUSIONS: Decoy-ODNs targeting NF-kappaB can decrease the expression of inflammatory mediators in pMPhi cells from rats.