A synthetic cyclic peptide derived from Limulus anti-lipopolysaccharide factor neutralizes endotoxin in vitro and in vivo.

Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infections. Recently, much attention has been focused on cationic peptides which possess the potential to detoxify LPS. Limulus anti-LPS factor (LALF), a protein found in the horseshoe crab (Limulus polyphemus), has been proved with striking anti-LPS effects. We synthesized a cyclic peptide (CLP-19), and then investigated its bioactivity both in vitro and in vivo. The ability of CLP-19 to neutralize LPS in vitro was tested using a Limulus amebocyte lysate (LAL) assay and the LPS-binding affinity was measured with an affinity biosensor method. The synthetic peptide LALF31-52 (residues 31 to 52 of LALF) was used as the positive control peptide in this study. It was found that CLP-19 exhibited the significant activity to antagonize LPS without observable cytotoxicity effect on mouse macrophages. CLP-19 directly bound to LPS, and neutralized it in a dose-dependent manner in the LAL assay. Moreover, CLP-19 also showed the remarkable ability to protect mice from lethal LPS attack and to inhibit the LPS-induced tumor necrosis factor alpha (TNF-alpha) release by decreasing serum LPS in vivo. Our work suggests that this peptide is worthy of further investigation as a potential anti-LPS agent in the treatment of septic shock.

Polymyxin B antagonizing biological activity of lipopolysaccharide.

OBJECTIVE: To investigate the mechanism of polymyxin B (PMB) antagonizing the biological activity of lipopolysaccharide (LPS). METHODS: The affinity of PMB for LPS and lipid A was assayed by biosensor, and the neutralization of PMB for LPS (2 ng/ml) was detected by kinetic turbidimetric limulus test. The releases of TNF-alpha and IL-6 in murine peritoneal macrophages a (PMphi) after exposure to LPS (100 ng/ml) were detected, and the expression levels of TLR4, TNF-alpha and IL-6 mRNA in PMphi induced by LPS (100 ng/ml) were measured by RT-PCR. RESULTS: PMB had high-affinity to LPS and lipid A with dissociation equilibrium constants of 18.9 nmol/L and 11.1 nmol/L, respectively, and neutralized LPS in a dose-dependent manner. Furthermore, PMB could markedly inhibit the expressions of TLR4, TNF-alpha and IL-6 mRNA and the release of cycokines in LPS-stimulated murine peritoneal macrophages. CONCLUSIONS: PMB neutralizes LPS and inhibites the expression and release of cycokines in macrophages, in which the affinity of PMB for lipid A plays an important role.

[Release of DNA from Pseudomonas aeruginosa in vitro during spontaneous growth and treatment with ciprofloxacin].

OBJECTIVE: To observe the DNA release from Pseudomonas aeruginosa (P. aeruginosa) during spontaneous growth and exposure to different concentrations of ciprofloxacin (Cipro) in vitro. METHODS: The P. aeruginosa 1244 strain (ATCC 27317) was selected because it was sensitive to Cipro in vitro. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Cipro against this strain were determined, respectively. Different concentrations of Cipro were cultured with this strain at 37 degrees C for 4 h and 24 h. The samples of culture supernatant were filtered and electrophoresed in 1.8% agarose with SYBR Gold stain. Then the images of the gel sheets were photographed. RESULTS: The MIC and MBC of Cipro were 0.25 - 0.5 mg/L. The free bacterial DNA in 4 h culture samples with or without Cipro could not be detected by this method. The certain amount of free bacterial DNA molecules in 24 h culture samples without antibiotic appeared at the two zones whose molecular weights were more than 2000 bp and less than 100 bp. The large amount of free bacterial DNA molecules showed at three zones in 24 h culture samples with Cipro when its concentrations were much lower than its MIC. In terms of DNA molecular weight, the first two zones were above 2000 bp, and the third zone was below 100 bp. There was no detectable DNA release from bacteria in 24 h culture samples when Cipro was at or above its MIC. CONCLUSIONS: The certain amount of bacterial DNA were released from P. aeruginosa in the spontaneous growth. Different concentrations of Cipro had quite differential effects on the DNA release from P. aeruginosa in quantities and molecular weights in vitro.

Role of mitochondrial dysfunction in hydrogen peroxide-induced apoptosis of intestinal epithelial cells.

AIM: To study the role of mitochondrial dysfunction in hydrogen peroxide-induced apoptosis of intestinal epithelial cells. METHODS: Hydrogen peroxide-induced apoptosis of human intestinal epithelial cell line SW-480 was established. Cell apoptosis was determined by Annexin-V and PI double-stained flow cytometry and DNA gel electrophoresis. Morphological changes were examined with light and electron microscopy. For other observations, mitochondrial function, cytochrome c release, mitochondrial translocation and membrane potential were determined simultaneously. RESULTS: Percentage of apoptotic cells induced with 400 micro mol/L hydrogen peroxide increased significantly at l h or 3 h after stimulation and recovered rapidly. Meanwhile percentage of apoptotic cells induced with 4 mmol/L hydrogen peroxide increased with time. In accordance with these changes, we observed decreased mitochondrial function in 400 micro mol/L H2O2-stimualted cells at 1 h or 3 h and in 4 mmol/L H2O2-stimualted cells at times examined. Correspondingly, swelling cristae and vacuole-like mitochondria were noted. Release of cytochrome c, decreased mitochondrial membrane potential and mitochondrial translocation were also found to be the early signs of apoptosis. CONCLUSION: Dysfunctional mitochondria play a role in the apoptosis of SW-480 cell line induced by hydrogen peroxide.

Effects of hydrogen peroxide on mitochondrial gene expression of intestinal epithelial cells.

AIM: To study the effects of hydrogen peroxide on mitochondrial gene expression of intestinal epithelial cells in in vitro model of hydrogen peroxide-stimulated SW-480 cells. METHODS: RNA of hydrogen peroxide-induced SW-480 cells was isolated, and reverse-transcriptional polymerase chain reaction was performed to study gene expression of ATPase subunit 6, ATPase subunit 8, cytochrome c oxidase subunit I (COI), cytochrome coxidase subuit II (COII) and cytochrome c oxidase subunit III (COIII). Mitochondria were isolated and activities of mitochondrial cytochrome c oxidase and ATPase were also measured simultaneously. RESULTS: Hydrogen peroxide led to differential expression of mitochondrial genes with some genes up-regulated or down-regulated in a dose dependent manner. Differences were very obvious in expressions of mitochondrial genes of cells treated with hydrogen peroxide in a concentration of 400 micromol/L or 4 mmol/L. In general, differential expression of mitochondrial genes was characterized by up-regulation of mitochondrial genes in the concentration of 400 micromol/L and down-regulation in the concentration of 4 mmol/L. In consistence with changes in mitochondrial gene expressions, hydrogen peroxide resulted in decreased activities of cytochrome c oxidase and ATPase. CONCLUSIONS: The differential expression of mitochondrial genes encoding cytochrome c oxidase and ATPase is involved in apoptosis of intestinal epithelial cells by affecting activities of cytochorme c oxidase and ATPase.

Risk factors of development of gut-derived bacterial translocation in thermally injured rats.

AIM: Studies have demonstrated that gut-derived bacterial translocation (BT) might play a role in the occurrence of sepsis and multiple organ dysfunction syndrome (MODS). Yet, no convincing overall analysis of risk factors for BT has been reported. The purpose of this study was to evaluate the related factors for the development of BT in burned rats. METHODS: Wistar rats were subjected to 30% third-degree burns. Then samples were taken on postburn d 1, 3, and 5. Incidence of BT and counts of mucosal bifidobacteria, fungi and E. coli, mucus sIgA, degree of injury to ileal mucosa, and plasma interleukin-6 were observed. Univariate analysis and multivariate logistic regression analysis were performed. RESULTS: The overall BT rate was 53.9% (69 in 128). The result of univariate analysis showed that the levels of plasma endotoxin and interleukin-6, the counts of mucosal fungi and E. coli, and the scores of ileum lesion were markedly increased in animals with BT compared with those without (P=0.000-0.005), while the levels of mucus sIgA and the counts of mucosal bifidobacteria were significantly reduced in animals with translocation compared with those without (P=0.000). There was a significant positive correlation between mucus sIgA and the counts of mucosal bifidobacteria (r=0.74, P=0.001). Moreover, there were strong negative correlations between scores of ileum-lesion and counts of bifidobacteria (r=-0.67, P=0.001). Multivariate logistic regression revealed that ileum lesion score (odds ratio [OR] 45.52, 95% confidence interval [CI] 5.25-394.80), and counts of mucosal bifidobacteria (OR 0.039, 95% CI 0.0032-0.48) were independent predictors of BT secondary to severe burns. CONCLUSION: Ileal lesion score and counts of mucosal bifidobacteria can be chosen as independent prognosis factors of the development of BT. Specific interventions targeting these high-risk factors might be implemented to attenuate BT, including strategies for repair of damaged intestinal mucosae and restoration of the balance of gastrointestinal flora.

[Improvement of bifidobacterial supplement on the barrier function of intestinal mucosa and microbe flora induced by thermal injury in rats].

OBJECTIVE: To investigate the potential effect of bifidobacterial supplement on intestinal mucosal and biological barrier following severe burns. METHODS: Seventy Wistar rats were randomly divided into burn control group (BC, n=30), burn + treatment group (BT, n=30), and sham-burn group (NC, n=10). Animals in BT group were fed bifidobacterial preparation (5x10(12) CFU/L) after 30% total body surface area (TBSA) full-thickness thermal injury, 1.5 ml, twice daily. Samples were taken on postburn days 1,3,and 5. The incidence of bacteria/endotoxin translocation and the counts of bifidobacteria, E.coli, and fungi in ileo-cecum mucosa were determined with standard methods. Ileum mucosal injuries were evaluated light and electron microscopically by blinded examiners. RESULTS: (1) The incidence of bacterial translocation were 42% and 16% in BC and BT groups on postburn day 3 (P=0.004 0), 30% and 6% on day 5 (P=0.002 0), respectively. Plasma endotoxin levels were markedly higher in BC and BT groups than that in NC group at the early stage postburn, while there was a significant decrease in BT group compared with BC group on postburn day 1 (P=0.04). (2) The bifidobacteria counts in ileo-cecum mucosa were reduced by 10- to 60-fold after thermal injury, while there was a remarkable increase in animals fed with bifidobacteria. The E.coli counts in ileo -cecum mucosa were increased by 20- to 30-fold on postburn days 1 and 3, whereas those markedly reduced after 3-day bifidobacteria-supplemented formula, tending to normal range.(3)There were obviously ileum mucosal lesions on postburn days 1 and 3,the ileum lesion scores remained significant higher in B C group than that in NC group on postburn 5 day (P<0.05).However,the damage d ileum mucosa was markedly repaired after 3-day bifidobacteria-supplemented formula, and almost restored 5 days later. CONCLUSION: The intestinal mucosal and biological barrier appear to be markedly damaged after severe burns. The supplement of exogenous bifidobacteria can facilitate the improvement of mucosal as well as biological barrier function, thereby reducing the incidence of bacteria/endotoxin translocation secondary to major burns.

[Bifidobacterial supplement enhances the expression and excretion of intestinal sIgA in severely burned rats].

OBJECTIVE: To investigate the potential effect of bifidobacterial supplement on intestinal mucosal immunity associated with severe burns. METHODS: Wistar rats were randomly divided into burn control group (BC group, n = 30), treatment group (BT group, n = 30), and sham-burn group (NC group, n = 10). Rats in BT group were fed bifidobacterial preparation (5 x 10(9) CFU/ml) after 30% total body surface area full-thickness burns, 1.5 ml, twice daily. Rats in BC group and NC group were fed normal saline, 1.5 ml, twice daily. Samples were taken on post-burn 1-, 3-, and 5-day. The incidence of bacterial translocation and bifidobacteria counts in the cecum mucosa were determined with standard methods. The sIgA levels in the mucus of the small intestine were measured by RIA. The positive sIgA expression in the lamina propria was detected by immunohistochemical staining. RESULTS: The incidence of bacterial translocation was 42% and 16% in BC and BT groups on post-burn day 3 (P = 0.004), 30% and 8% on day 5 (P = 0.002), respectively. Plasma endotoxin levels were markedly higher in BC and BT groups than in NC group at the early stage post-burn. There was a significant decrease between BT group and BC group on post-burn day 1 (P = 0.0412). Bifidobacteria counts in cecum mucosa were reduced by 10- to 60-fold after thermal injury, but there was a remarkable increase in bifidobacteria counts in animals fed with bifidobacteria. sIgA levels in the intestinal mucus were significantly decreased in group BC, but they returned to normal range in BT group on post-burn day 5. Similarly, sIgA expression in the lamina propria was also weakened after burns, and had a tendency to recover after prescription of a 5-day bifidobacteria-supplemented formula. A strong positive correlation was observed between the counts of bifidobacteria in the cecal mucosa and the levels of sIgA in the intestinal mucus (r = 0.7534, P = 0.0000). CONCLUSIONS: The expression and excretion of sIgA in the intestine appear to be markedly inhibited following a severe thermal injury. The supplement of exogenous bifidobacteria could improve sIgA formation in the small intestine, thereby reducing the incidence of bacterial/endotoxin translocation secondary to major burns.

[Influence of haemoxygenase 1 (HO-1) gene expression on intestinal mucosa injury induced by intra-abdominal hypertension in rats].

OBJECTIVE: To observe the effects of up- or down-regulation of haemoxygenase 1 (HO-1) gene expression on intestinal mucosa injury induced by intra-abdominal hypertension (IAH). METHODS: (1) Reproduction of rat model of up- or down-regulation of HO-1 gene expression. Twenty-four healthy adult Wistar rats were divided into Co-PP (HO-1 specific revulsive) 2.5 mg, Co-PP 5.0 mg, Sn-PP (HO-1 specific inhibitor) 2.5 mg, and control groups according to the random number table, with six rats in each group. Rats in groups Co-PP 2.5 mg and Sn-PP 2.5 mg were respectively given Co-PP 2.5 mg/kg and Sn-PP 2.5 mg/kg by intraperitoneal injection, once every 12 hours for 3 days. The rats in group Co-PP 5.0 mg were intraperitoneally injected with Co-PP 5.0 mg/kg, once a day for 3 days. The rats in control group were treated with equal volume of normal saline by intraperitoneal injection. All rats were sacrificed on post injection day (PID) 4, and intestinal mucosa tissues were collected for determination of HO-1 mRNA expression. Optimal dose of Co-PP was chosen for the following experiment. (2) The influence of up- or down-regulation of HO-1 gene expression on intestinal mucosa injury under IAH condition. Another 24 healthy adult Wistar rats were divided into control, IAH, Co-PP+IAH, and Sn-PP+IAH groups according to the random number table, with six rats in each group. The rats in groups Co-PP+IAH and Sn-PP+IAH were intraperitoneally injected with 2.5 mg/kg Co-PP and 2.5 mg/kg Sn-PP, once every 12 hours for 3 days. Equal volume of normal saline was intraperitoneally injected into the rats in control group, once every 12 hours for 3 days. Then, nitrogen gas pneumoperitoneum was used to establish the model of IAH in rats of the latter three groups on PID 4, with IAP at 20 mm Hg (1 mm Hg = 0.133 kPa) , and it was maintained for 2 hours. Puncture and intubation were performed in rats of control group without inflating nitrogen gas. Jejunal segment in the length of 10-15 cm was harvested for collecting intestinal mucosa tissues to determine the HO-1 mRNA expression and diamine oxidase (DAO) content. Serum obtained from portal vein blood was collected to determine the D-lactate, TNF-α, and IL-6 contents. Another jejunal segment in the length of 1-2 cm was harvested for histopathological examination. Data were processed with one-way analysis of variance and t test. RESULTS: (1) The HO-1 mRNA expression in group Co-PP 2.5 mg was significantly higher than that in control and Co-PP 5.0 mg groups (with t values respectively 4.756, 3.175, P < 0.05 or P < 0.01). The HO-1 mRNA expression in group Sn-PP 2.5 mg was significantly lower than that in control group (t = 4.880, P < 0.01). The optimal dose of Co-PP for the following experiment was 2.5 mg/kg. (2) HO-1 mRNA expression in group Co-PP+IAH was 60 ± 5, and it was obviously higher than that of group IAH (49 ± 5, t = 3.811, P < 0.01) and control group (39 ± 4, t = 8.034, P < .001) . HO-1 mRNA expression was higher in group IAH than in control group (t = 3.826, P < 0.01). HO-1 mRNA expression in group Sn-PP+IAH was 29 ± 4, which was obviously lower than that of control group (t = 4.330, P < 0.01). The contents of DAO and D-lactate in group Co-PP+IAH were (0.52 ± 0.05) U/mL and (1.9 ± 0.6) mg/L, which were significantly lower than those in group IAH [(0.88 ± 0.06) U/mL and (4.3 ± 0.7) mg/L, with t values respectively 11.291, 6.376, P values all below 0.01], but still higher than those in control group [(0.34 ± 0.04) U/mL, (1.2 ± 0.5) mg/L, with t values respectively 6.886, 2.295, P < 0.05 or P < 0.01]. The contents of TNF-α and IL-6 were much lower in group Co-PP+IAH than in group IAH, but still higher than in control group (with t values from 3.781 to 18.557, P values all below 0.01). The contents of DAO, D-lactate, TNF-α, and IL-6 in group Sn-PP+IAH were all higher than those in the other 3 groups (with t values from 4.181 to 32.938, P values all below 0.01). Structure of epithelial cells from intestinal mucosa was intact and regularly arranged in rats of control group. Intestinal mucosal tissue was edematous, and the top of villi was anabrotic and necrotic in rats of group IAH. Compared with that of group IAH, the degree of intestinal mucosa injury was alleviated in rats of group Co-PP+IAH, while the pathology was aggravated in rats of group Sn-PP+IAH. CONCLUSIONS: Up-regulation of HO-1 gene expression can ameliorate intestinal mucosa injury caused by IAH, thus protecting intestinal mucosa tissues.

[Effect of mastoparan-1 on lipopolysaccharide-induced acute hepatic injury in mice].

OBJECTIVE: To observe the effect of mastoparan-1 (MP-1) on lipopolysaccharide (LPS)-induced acute hepatic injury in mice and probe into its possible mechanism. METHODS: One hundred and four BALB/c mice were randomly divided into healthy control group (n = 8, without treatment, HC), LPS group (n = 48, with injection of LPS 5 mg/kg via tail vein), and MP-1 group (n = 48, with injection of LPS 5 mg/kg and MP-1 3 mg/kg via tail vein). Mice in LPS group and MP-1 group were sacrificed at 2nd, 6th, 12th, 24th, 48th and 72nd post injection hour (PIH), 8 mice at each time point in each group. Blood samples were collected for determination of plasma levels of LPS by kinetic turbidimetric limulus test, TNF-alpha and IL-6 by ELISA, serum levels of ALT and AST by automatic biochemistry analyzer respectively. Hepatic tissue samples were collected for determination of TLR4, TNF-alpha and IL-6 mRNA by real-time fluorescent quantitation reverse transcription polymerase chain reaction, along with the observation of pathological changes in hepatic tissue at each time point. Above-mentioned examinations were also performed in HC group. RESULTS: Compared with those of HC group, plasma levels of LPS and TNF-alpha in LPS group significantly increased at 2nd PIH (18,320.50 +/- 2782.50 EU/mL and 988 +/- 130 ng/L, respectively), then decreased gradually to 1.80 +/- 0.80 EU/mL and 150 +/- 44 ng/L at 72nd PIH, which was close to those of HC group. The values of IL-6, ALT and AST peaked at 12th PIH, which declined to the levels close to those of HC group at 72nd PIH. Meanwhile, the expressions of TLR4, TNF-alpha and IL-6 mRNA in liver were remarkably up-regulated after injection, and the pathological changes in hepatic tissue pronounced significantly at 12th, 24th and 48th PIH. Compared with those of LPS group, the levels of LPS, cytokines, ALT and AST decreased in MP-1 group in different degrees after injection (P < 0.05 or P < 0.01), genes expression (P < 0.05 or P < 0.01) and pathological changes was respectively suppressed and alleviated in hepatic tissue. CONCLUSIONS: MP-1 can alleviate LPS-induced acute hepatic injury in mice, which may be associated with its neutralization of LPS and attenuation of synthesis and release of inflammatory mediators.

[The gut-origin infection in severe burns].

Early in 1962, after an extensive review including 312 cases of bacteremia in burn patients, we were surprised to find that there was about 30% of bacteremia in the patients who had no detectable microorganisms from repeated wound cultures, but blood cultures were usually positive for gut flora. From that time on the idea of gut-origin infection emerged. In following twenty years, a series of experiments were carried on in Wistar rats with 30% TBSA full-thickness burn. The results showed that the fluorescein labeled enteric microbes (Pseudomonas aeruginosa, Bacteroid fragilis and Candida albicans) could translocate through the stress injured intestinal wall and were recovered in visceral organs. The radioisotope 125I labeled endotoxin began to ascend in concentration in portal vein since 15 minutes postburn. Radioautography of liver sections demonstrated the labeled endotoxin granules. With the creation of minute mesenteric lymph fistulas, the clearance of endotoxin and TNFalpha was found to be significantly high in lymph fluid exited from the intestine. All above evidences indicated that the gut is a potential route of endogenous infection, and it also explained how did the patients manifest sepsis early after burn injury without a definite infectious focus. Now the concepts of gut-origin infection are commonly accepted, the measures like early enteral feeding for the protection of intestinal barrier has been established.

[Achievements in burn surgery over the past 50 years in China].

This paper reflects briefly the main advancements of clinical and scientific research in the field of burn surgery over the past 50 years in China. It includes emergency care of massive burns, resuscitation, anti-infection, prevention and treatment of internal organ injury, metabolic and nutritional support, repair of wound and rehabilitation, and special types of burns. The article also covers the researches in pathology, microbiology, immunology, cell biology, molecular biology, and tissue engineering pertaining to burn injury.

[Study of in vitro endotoxin neutralization effect and antibacterial activity of limulus anti-lipopolysaccharide factor simulated peptide REMP2].

OBJECTIVE: To investigate the role of REMP2 derived from limulus anti-lipopolysaccharide factor in neutralizing endotoxin in vitro and its antibacterial activity. METHODS: (1) REMP2 and PMB in the concentrations of 100.00, 10.00, 1.00, 0.10, 0.01 micromol/L were respectively mixed with LPS (lEU/mL), with PMB as positive control. The LPS concentrations in different specimens were determined by routine method, and the neutralizing percentage was respectively calculated. (2) After adding isotonic saline (NS), the final concentrations of REMP2 and PMB were 10, 20, 40, 80 micromol/L, and the concentration of LPS was 100 microg/L. The murine monocytic macrophages were stimulated with LPS, then cultured with REMP2 and PMB, with NS in culture as negative control. The content of tumor necrosis factor (TNF)-alpha was determined by ELISA kit. (3) The morphologic changes of Escherichia coli. was observed under electron microscope at 10, 20 and 40 minutes after addition of REMP2 to Escherichia coli suspension (with terminal concentration of REMP2 at 40 micromol/L). RESULTS: There were no significant difference in endotoxin-neutralizing percentages between PMB and REMP2 in concentrations of 0.10, 10.00, 100.00 micromol/L (P > 0.05). The contents of TNF-alpha were 1175 +/- 162, 859 +/- 122, 645 +/- 142, 489 +/- 102 ng/L, respectively,after treatment of 10, 20, 40, 80 micromol/L REMP2, which were obviously lower than that of NS (3463 +/- 218 ng/L, P < 0.01). Under transmission electron microscope, the outer and interior membranes of Escherichia coli were obscure and rough, bacterial bodies were swollen with vacuoles in cytoplasm after treatment with REMP2. CONCLUSION: REMP2 has ability of neutralizing endotoxin and also antibacterial activity.

[The present status, counter-measures and new trends on burn infection].

In recent fifty years, Pseudomonas aeruginosa and Staphylococcus aureus were continuously the predominant in burn infections, the only change seen was a rapid increase in their drug-resistance. Under the pressure of antibiotics, Some opportunistic bacteria that were resistant to all available antibiotics emerged, such as Acinetobacter baumanii and Maltophilia stenotrophomonas. For critically burn patients, basing on early surgical intervention, early and short-term use of broad-spectrum antibiotic is advisable, and it may control the infection promptly, prevent further inflammatory reaction, as well as minimize the emergence of antibacterial resistance. To control infections due to pandrug-resistant bacteria, cyclic use of some old antibiotics may be helpful. In dealing with severe infection, a combination of anti-pathogen and anti-inflammatory reaction measures should be considered.

[Study of lipopolysaccharide antagonizing effect of DPR-2 in vitro].

OBJECTIVE: To investigate the lipopolysaccharide (LPS) antagonizing biological activity of densefruit pattany root-bark extract (DPR-2) in vitro. METHODS: The effect of DPR-2 in neutralizing LPS (0.1 microg/L) was detected by kinetic turbidimetric limulus test. The effect of different concentrations of DPR-2 (0,8.0,16.0,32.0,64.0 mg/L) on binding of FITC-conjugated LPS (FITC-LPS,100.0 microg/L) to murine RAW264.7 cells was analyzed with laser scanning confocal microscopy. The expression of TNF-alpha and IL-6 mRNA in RAW264.7 cells after exposure to LPS (100.0 microg/L) were determined by real-time RT-PCR. RESULTS: DPR-2 could neutralize LPS (P < 0.05 or P < 0.01), and inhibit the binding of FITC-LPS to RAW264.7 cells in a dose-dependent manner when the concentration of DPR-2 was above 16.0 mg/L. Furthermore, DPR-2 could markedly inhibit the expression of TNF-alpha and IL-6 mRNA in LPS-stimulated murine RAW264.7 cells. CONCLUSION: DPR-2 exhibit an anti-LPS effect in vitro, which may be related to its capacity to neutralize LPS and inhibit binding of LPS for its receptors.

[Reproduction of the murine endotoxin shock model in D-galactosamine-sensitized KunMing mice].

OBJECTIVE: To reproduce a Kunming murine endotoxin shock model suitable for the anti-endotoxin pharmaceutical research. METHODS: Kunming mice were challenged with an intraperitoneal (i. p.) injection of different doses of D-galactosamine (D-Gal) and endotoxin (LPS) and divided into 10 groups: i.e, group 1 [with injection of isotonic saline solution (NS) and LPS]; group 2 (with injection of NS and 90mg/kg LPS), group 3 (with injection of NS and 500mg/kg D-Gal), group 4 (with injection of 500mg/kgD-Gal and 25 microg /kg LPS), group 5 (with injection of 500mg/kg D-Gal and 50 microg/kg LPS), group 6(with injection of 500mg/kg D-Gal and 250 microg/kg LPS), group 7( with injection of NS and 600mg/ kg D-Gal), group 8 (with injection of 600mg/kg D-Gal and 10 microg/kg LPS), group 9( with injection of 600mg/kg D-Gal and 25 microg/kg LPS), group 10 (with injection of 600mg/kg D-Gal and 50 microg/kg LPS). The death of the mice were observed and the mortality rate was recorded at 48 post-injection hour (PIH). The dose of D-Gal and LPS which caused 100% lethality was chosen for the subsequent experiment to serve as control group (with injection of NS and 600mg/kg D-Gal), LPS group (with injection of 600mg/kg D-Gal and 580mg/kg LPS for later experiment). The venous blood of the mice were collected for the detection of serum content of TNF-alpha with ELISA method at 30, 75 and 120 post-injection minutes (PIM). The tissues of lung, liver, intestine were also harvested at 5 PIH for the pathological examination. RESULTS: The lethality of mice was 100% in the groups 2, 6 and 10 (P < 0.01). The serum content of TNF-alpha was maintained in a low level in control group, but it increased remarkably in LPS group, and it reached peak at 75 PIM (6365 +/- 2087ng/L, P < 0.01). Obvious inflammatory reaction was observed in the lung, liver and intestine in LPS group, while only mild inflammatory reaction was observed in liver in control group. CONCLUSION: The Kunming mice showed signs of endotoxin shock after D-galactosamine presensitizing and endotoxin challenge, and it is suitable for anti-endotoxin pharmaceutical research.

[Protective effects of bactericidal/permeability increasing protein simulated peptide on murine acute lung injury induced by lipopolysaccharide].

OBJECTIVE: To investigate the protective effects of bactericidal/permeability increasing protein simulated peptide (bactericidal neutralizing endotoxin protein, BNEP) on murine acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: A murine model of ALI was reproduced by lipopolysaccharide via intranasal instillation. The Balb/c mice were randomly divided into control (n = 20, with nasal instillation of isotonic saline), LPS instillation (n = 20, with nasal instillation of isotonic saline and LPS) and BNEP treatment (n = 20, with nasal instillation of isotonic saline plus LPS and BNEP) groups. The ratio of lung wet weight to dry weight, the permeability of pulmonary capillary vessels and the histopathology of pulmonary tissue were determined in all groups. The change in the expression of Toll-like receptor 2 and 4 (TLR2/4) in the pulmonary tissue was detected by immunohistochemistry. RESULTS: Compared with LPS instillation group, the ratio of lung wet weight to dry weight and the permeability of pulmonary capillary vessel was decreased significantly in the BNEP group, and the inflammatory infiltration in the pulmonary tissue induced by neutrophil influx was alleviated markedly with BNEP treatment. The expression of TLR2 and TLR4 in pulmonary vascular endothelial cells, macrophages and alveolar type II epithelial cells in BNEP group were lower than those in LPS group (TLR2: 128 +/- 10 vs 214 +/- 12, P < 0.01). CONCLUSION: BNEP, as a simulated peptide of BPI, exerted a remarkable protective effect on ALI induced by LPS.

[Study of the anti-endotoxin effect of beta-1, 2, 3, 4, 6-penta-O-galloyl-D-glucopyranose in vitro].

OBJECTIVE: To study the anti-endotoxin effect of beta-1, 2, 3, 4, 6-penta-O-galloyl-D-glucopyranose (PGG) in vitro. METHODS: The affinity of PGG with lipid A was determined with biosensor technology, and the endotoxin-neutralizing effect was assayed with LAL. Human peripheral blood mononuclear cells (hPBMC) were separated from healthy donors and cultured in vitro. The effect of different concentrations of PGG on the release of TNF-alpha and hIL-6 from LPS-stimulated hPBMC were measured by ELISA method. RESULTS: Lipid A was combined with different concentrations of PGG. The combination speed was shortened with the increase in PGG concentration. The KD value between PGG and Lipid A was 5.2 x 10(-7) mol/L. The release of TNF-alpha and IL-6 of hPBMC under LPS stimulation (958 +/- 234 ng/L vs 1 351 +/- 99 ng/L) was obviously inhibited by PGG in the concentration of higher than 20 mg/L compared with that without PGG treatment (1 788 +/- 171 ng/L vs 1 965 +/- 232 ng/L, P < 0.05). CONCLUSION: PGG show an anti-endotoxin effect in vitro, which may be associated with its ability to combine and neutralize endotoxin.

[Preliminary study on the release of DNA from Pseudomona aeruginosa induced by piperacillin/tazobactam in vitro].

OBJECTIVE: To observe the release of DNA from Pseudomonas aeruginosa (P. aeruginosa) induced by different concentrations of piperacillin/tazobactam (Piper) in vitro. METHODS: The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Piper against 1244 strain (ATCC 27317) of P. aeruginosa were determined, respectively. This strain of P. aeruginosa was separately cultured with Piper in different concentrations at 37 degrees C for 4 h and 24 h. The samples of cultural supernatant were filtered and electrophoresis was conducted in 1.8% agarose with SYBR Gold stain. Then the images of the gel sheets were photographed. RESULTS: This strain of P. aeruginosa was sensitive to Piper. The bacterial DNA was not detected in 4-h cultured P. aeruginosa either with or without Piper by this method. The bacterial DNA molecules could be detected in 24 h samples in cultures without Piper, and they were displayed in two zones of molecular weight over 2000 base pairs (bp) and lower than 100 bp. Similar results were observed when the MIC of piper (0.002, 0.004 g/L) were under the MIC measured at the 3rd time (0.008 g/L), but there was much more bacterial DNA with molecular weight lower than 100 bp. When Piper concentration was higher than its MIC, only smaller quantities of bacterial DNA in the area with molecular weight lower than 400 bp could be detected in 24-h culture samples. CONCLUSION: A certain amount of bacterial DNA was released from P. aeruginosa under its natural growth circumstance. Different concentrations of Piper showed different effects on DNA release, in regard to its quantity and molecular weight, from P. aeruginosa cultures.