[The changes in secretory function of pulmonary intravascular macrophages after challenge of lipopolysaccharide].

OBJECTIVE: To investigate the role of pulmonary intravascular macrophages (PIMs) in the pathogenesis of acute lung injury (ALI) due to infection. METHODS: Porcine pulmonary blood vessels were flushed by modified Morton method, and PIMs were isolated and cultured. The adhered PIMs were collected with adhesion method and incubated in RPMI 1640 medium. They were challenged with lipopolysaccharide (LPS, 10 mg/L). The activity of interleukin-1ß (IL-1ß), and contents of IL 6 and IL 8 in the culture supernatant were measured by method of thymocyte proliferation and enzyme linked immunoadsorbent assay (ELISA). RESULTS: The released contents of IL-1ß, IL-6 and IL-8 from PIMs were increased significantly compared with those before LPS challenge , and they peaked at 2 hours [IL-1ß activity: (10 400 ± 2 389) scintillant count/min], 4 hours [IL-6 content: (0.80 ± 0.36) µg/L], and 6 hours [IL-8 content: (4.94 ± 1.19) µg/L ] after LPS challenge , and the differences were significant compared with hose before LPS challenge [IL-1ß activity: (213 ± 85) scintillant count/min, IL-6 content: (0.27 ± 0.12) µg/L, IL-8 content: (1.84 ± 0.53) µg/L, all P <0.01]. CONCLUSION: Among the cytokines released from PIMs after LPS challenge , the increase in IL-1ß occurred earlier in comparison with that of IL-6 and IL-8, suggesting that the former might play an important role at the early stage of ALI; on the other hand, though the increase in IL-6 and IL-8 contents occurred later than that of IL- 1ß but it lasted for a longer duration, suggesting that they might be associated with the advancement of ALI. The Results also suggested that interaction of these cytokines played a more important role in the pathogenesis of ALI.

[Changes in inflammatory cytokines released by pulmonary intravascular macrophages after stimulation with lipopolysaccharide].

OBJECTIVE: To investigate the roles of pulmonary intravascular macrophages (PIMs) in the pathogenesis of infective acute lung injury (ALI). METHODS: Porcine pulmonary blood vessels were flushed by modified Morton's method, PIMs were isolated with adhesion method and incubated in RPMI 1640 medium. They were stimulated with lipopolysaccharide (LPS, 10 mg/L). The contents of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatants were respectively measured by enzyme linked immunoadsorbent assay (EILSA). RESULTS: The release of TNF-alpha, IL-6 and IL-8 by PIMs was increased significantly as compared with the levels before stimulation by LPS, peaking at 1, 4, and 6 hours after LPS stimulation, respectively. The differences were significant (all P<0.01). CONCLUSION: Among the cytokines released by PIMs after LPS challenge, the increase in TNF-alpha content occurs earlier in comparison with that of IL-6 and IL-8, suggesting that the former may play an important role at the early stage of ALI. On the other hand, the increase in IL-6 and IL-8 contents is later than that of TNF-alpha and lasts for a longer time, suggesting that they may be associated with the development of ALI. The results also suggest that interaction of these cytokines is more important in the pathogenesis of ALI.

[Study on extraction process of polysaccharide from Sargassum fusiforme by enzymatic treatment].

OBJECTIVE: To investigate the optimal extraction process of polysaccharides from S. fusiforme by enzymatic treatment. METHOD: The optimum extraction conditions were obtained by the experiment with the orthogonal design. The content of polysaccharides of S. fusiforme was determined by spectraphotometry. RESULT: The amount of enzyme and temperature significantly affected total polysaccharides of S. fusiforme. CONCLUSION: The optimum extraction conditions include the addition of 1. 2 x 10 (4) U x 100 g(-1) enzyme into water at pH 4. 5, and the subsequent treatment for 10 min while the temperature is maintained at 45 degrees C.

Morphological and serum hyaluronic acid, laminin and type IV collagen changes in dimethylnitrosamine-induced hepatic fibrosis of rats.

AIM: To study the morphological and serum hyaluronic acid (HA), laminin (LN), and type IV collagen changes in hepatic fibrosis of rats induced by dimethylnitrosamine (DMN). METHODS: The rat model of liver fibrosis was induced by DMN. Serum HA, type IV collagen, and LN were measured by ELISA. The liver/weight index and morphological changes were examined under electron microscope on d 7, 14, 21, and 28 by immunohistochemical alpha smooth muscle actin alpha-SMA staining as well as Sirius-red and HE staining. RESULTS: The levels of serum HA, type IV collagen and LN significantly increased from d 7 to d 28 (P = 0.043). The liver/weight index increased on d 7 and decreased on d 28. In the model group, the rat liver stained with HE and Sirius-red showed evident hemorrhage and necrosis in the central vein of hepatic 10 lobules on d 7. Thin fibrotic septa were formed joining central areas of the liver on d 14. The number of alpha-SMA positive cells was markedly increased in the model group. Transitional hepatic stellate cells were observed under electron microscope. All rats in the model group showed micronodular fibrosis in the hepatic parenchyma and a network of alpha-SMA positive cells. Typical myofibroblasts were embedded in the core of a fibrous septum. Compared to the control group, the area-density percentage of collagen fibrosis and pathologic grading were significantly different in the model group (P<0.05) on different d (7, 14, and 28). The area-density percentage of collagen fibrosis in hepatic tissue had a positive correlation with the levels of serum HA, LN, and type IV collagen. CONCLUSION: The morphological and serum HA, type IV collagen, and LN are changed in DMN-induced liver fibrosis in rats.

The Effects of κ-Opioid Receptor Stimulation on Electrical Coupling during Ischemia in the Perfused Isolated Rat Heart.

Two series of experiments were performed in the perfused isolated rat heart to determine whether stimulation of κ-opioid receptor with U50,488H, a selective κ-opioid receptor agonist, produces any changes in electrical coupling during prolonged ischemia and whether these changes in electrical coupling is associated with the cardioprotection induced by U50,488H. It was found that U50,488H concentration dependently increased formazan content and reduced lactate dehydrogenase (LDH) release induced by 30 min of ischemia and 120 min of reperfusion, and the ameliorating effect of 10-5mol/L U50,488H was abolished by 5x10-6mol/L nor-binaltorphimine (nor-BNI), a selective Κ-opioid receptor antagonist, or 10-4mol/L 5-hydroxydecanoate (5-HD), a selective mitochondrial ATP-sensitive K+(KATP) channels blocker. The onset of electrical uncoupling during prolonged ischemia was delayed by U50,488H, and delaying effect was not only abolished, but also advanced by nor-BNI or 5-HD compared with control group. These results demonstrate that delayed electrical uncoupling is associated with the cardioprotection induced by U50,488H. These effects of U50,488H are mediated by mitochondrial KATPchannels.

The Effects of Heptanol on Electrical Coupling during Ischemia in the Perfused Isolated Rat Heart.

The aims of the present study were to examine the effect of heptanol on electrical coupling during ischemia, and to assess whether changes in electrical coupling by heptanol is associated with its cardiac protection. Perfused isolated rat hearts were subjected to a 24 min infusion of heptanol (0.05, 0.1, 0.5 or 1.0 mmol/L) followed by 70 min of global no-flow ischemia or by 20 min of regional ischemia and 60 min of reperfusion. Heptanol markedly decreased arrhythmia scores during ischemia and reperfusion as well as reduced infarct size to a degree similar to that induced by ischemic preconditioning. In the prolonged ischemia model, heptanol delayed the onset of uncoupling, increased time to plateau, and decreased the maximal rate of uncoupling during ischemia. Ischemic preconditioning had similar effects on these parameters. These results demonstrate that treatment with the gap junction uncoupler heptanol confers cardioprotection against ischemia, and this effect is related to delayed electrical uncoupling during prolonged ischemia.

A DNA replication-related element downstream from the initiation site of Drosophila selenophosphate synthetase 2 gene is essential for its transcription.

Selenophosphate synthetase catalyzes the synthesis of selenophosphate which is a selenium donor for Sec biosynthesis. In Drosophila melanogaster, there are two types of selenophosphate synthetases designated dSPS1 and dSPS2, where dSPS2 is a selenoprotein. The mechanism of gene expression of dSPS2 as well as other selenoproteins in Drosophila has not been elucidated. Herein, we report an essential regulator system that regulates the transcription of the dSPS2 gene (dsps2). Through deletion/substitution mutagenesis, the downstream DNA replication-related element (DRE) located at +71 has been identified as an essential element for dsps2 promoter activity. Furthermore, double-stranded RNA interference (dsRNAi) experiments were performed to ablate transcription factors such as TBP, TRF1, TRF2 and DREF in Schneider cells. The dsRNAi experiments showed that dsps2 promoter activities in DREF- and TRF2-depleted cells were significantly decreased by 90% and 50%, respectively. However, the depletion of TBP or TRF1 did not affect the expression level of dsps2 even though there is a putative TATA box at -20. These results strongly suggest that the DRE/DREF system controls the basal level of transcription of dsps2 by interacting with TRF2.