Carbon monoxide alleviates lipopolysaccharide-induced oxidative stress injury through suppressing the expression of Fis1 in NR8383 cells.

Acute respiratory distress syndrome (ARDS) is one of the most devastating complications of sepsis lacking of effective therapy. Mitochondrial dynamics undergoing continuous fusion and fission play a crucial role in mitochondrial structure and function. Fis1, as a small protein located on the outer membrane of mitochondria, has been thought to be an important protein mediated mitochondrial fission. During ARDS, alveolar macrophages suffer from increased oxidative stress and apoptosis, and also accompanied by disrupted mitochondrial dynamics. In addition, as one of the products of heme degradation catalyzed by heme oxygenase, carbon monoxide (CO) possesses powerful protective properties in vivo or in vitro models, such as anti-inflammatory, antioxidant and anti-apoptosis function. However, there is little evidence that CO alleviates oxidative stress damage through altering mitochondrial fission in alveolar macrophages. In the present study, our results showed that CO increased cell vitality, improved mitochondrial SOD activity, reduced reactive oxygen species (ROS) production and inhibited cell apoptosis in NR8383 exposed to LPS. Meanwhile, CO decreased the expression of Fis1, increased mitochondrial membrane potential and sustained elongation of mitochondria in LPS-incubated NR8383. Overall, our study underscored a critical role of CO in suppressing the expression of Fis1 and alleviating LPS- induced oxidative stress damage in alveolar macrophages.

Heme oxygenase-1 mediates the anti-inflammatory effect of molecular hydrogen in LPS-stimulated RAW 264.7 macrophages.

BACKGROUND: Molecular hydrogen (H2) as a new medical gas has an anti-inflammatory effect. In the present study, we investigated whether heme oxygenase-1 (HO-1) contributes to the anti-inflammatory effect of H2 in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. METHODS: RAW 264.7 macrophages were stimulated by LPS (1 µg/mL) with presence or absence of different concentrations of H2. Cell viability and injury were tested by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and lactate dehydrogenase (LDH) release, respectively. The cell culture supernatants were collected to measure inflammatory cytokines [TNF-α, IL-1ß, HMGB1 (high mobility group box-1) and IL-10] at different time points. Moreover, HO-1 protein expression and activity were tested at different time points. In addition, to further identify the role of HO-1 in this process, zinc protoporphyrin (ZnPP)-IX, an HO-1 inhibitor, was used. RESULTS: H2 treatment had no significant influence on cell viability and injury in normally cultured RAW 264.7 macrophages. Moreover, H2 treatment dose-dependently attenuated the increased levels of pro-inflammatory cytokines (TNF-α, IL-1ß, HMGB1), but further increased the level of anti-inflammatory cytokine IL-10 at 3 h, 6 h, 12 h and 24 h after LPS stimulation. Furthermore, H2 treatment could also dose-dependently increase the HO-1 protein expression and activity at 3 h, 6 h, 12 h and 24 h in LPS-activated macrophages. In addition, blockade of HO-1 activity with ZnPP-IX partly reversed the anti-inflammatory effect of H2 in LPS-stimulated macrophages. CONCLUSIONS: Molecular hydrogen exerts a regulating role in the release of pro- and anti-inflammatory cytokines in LPS-stimulated macrophages, and this effect is at least partly mediated by HO-1 expression and activation.

Role of HO-1 in protective effect of electro-acupuncture against endotoxin shock-induced acute lung injury in rabbits.

Heme oxygenase (HO)-1 has been reported to play a great role in attenuating lung injury during endotoxic shock in our previous research. Although electro-acupuncture has been explored to reduce oxidative stress and decrease inflammatory reaction in animals with endotoxic shock, the mechanism of this effect is still unclear. The aim of this study was to determine whether HO-1 is involved in the effect of electro-acupuncture on the injured lung during endotoxic shock in rabbits. Sixty New England white rabbits were randomly divided into groups C, Z, ES, EA, AP, and EAZ. Before inducing endotoxic shock, group ES received no electro-acupuncture, while group EA received electro-acupuncture at ST36 (zusanli) and BL13 (feishu) acupoints on both sides for five days and group AP received electro-acupuncture (EA) stimulation at a non-acupoint. Groups ES, AP, EA, and EAZ received LPS to replicate the experimental model of injured lung induced by endotoxic shock, and electro-acupuncture was performed throughout the procedure with the same parameter. Groups EAZ and Z received the HO-1 inhibitor, ZnPP-IX, intraperitoneally. The animals were sacrificed by blood-letting at 6 h after LPS administration. The blood samples were collected for serum examination, and the lungs were removed for pathology examination, detection of alveolaer epithelial cell apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL assay), determination of wet to dry ratio, measurement of Evans blue (EB) contents, and determination of HO-1protein and mRNA expression. According to the results, EA at ST36 and BL13 could increase the expression of HO-1. At the same time, index of quantitative assessment (IQA) score and the number of TUNEL-positive cells decreased, while electro-acupuncture at the other points did not exert this effect, and pretreatment with ZnPP-IX in group EAZ suppressed the efficacy of electro-acupuncture preconditioning. In summary, electro-acupuncture stimulation at ST36 and BL13, while not the non-acupoint, could attenuate the lung injury during the endotoxic shock, and this effect was due to increased expression of HO-1.

Probiotics improve survival of septic rats by suppressing conditioned pathogens in ascites.

AIM: To investigate the benefits of probiotics treatment in septic rats. METHODS: The septic rats were induced by cecal ligation and puncture. The animals of control, septic model and probiotics treated groups were treated with vehicle and mixed probiotics, respectively. The mixture of probiotics included Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus. We observed the survival of septic rats using different amounts of mixed probiotics. We also detected the bacterial population in ascites and blood of experimental sepsis using cultivation and real-time polymerase chain reaction. The severity of mucosal inflammation in colonic tissues was determined. RESULTS: Probiotics treatment improved survival of the rats significantly and this effect was dose dependent. The survival rate was 30% for vehicle-treated septic model group. However, 1 and 1/4 doses of probiotics treatment increased survival rate significantly compared with septic model group (80% and 55% vs 30%, P < 0.05). The total viable counts of bacteria in ascites decreased significantly in probiotics treated group compared with septic model group (5.20 ± 0.57 vs 9.81 ± 0.67, P < 0.05). The total positive rate of hemoculture decreased significantly in probiotics treated group compared with septic model group (33.3% vs 100.0%, P < 0.05). The population of Escherichia coli and Staphylococcus aureus in ascites of probiotics treated group were decreased significantly compared with that of septic model group (3.93 ± 0.73 vs 8.80 ± 0.83, P < 0.05; 2.80 ± 1.04 vs 5.39 ± 1.21, P < 0.05). With probiotics treatment, there was a decrease in the scores of inflammatory cell infiltration into the intestinal mucosa in septic animals (1.50 ± 0.25 vs 2.88 ± 0.14, P < 0.01). CONCLUSION: Escherichia coli and Staphylococcus aureus may be primary pathogens in septic rats. Probiotics improve survival of septic rats by suppressing these conditioned pathogens.

miR-200a modulate HUVECs viability and migration.

The posttranscriptional regulation of miRNAs is important for organism development. To investigate the role of miRNAs in angiogenesis, we performed a loss-of-function screening assay in human umbilical vein endothelial cells (HUVECs) and found that knockdown of 7 miRNAs (miR-95a, miR-126, miR-129, miR-137, miR-139, miR-200a, and miR-335) significantly suppressed cell viability. As miR-200a was highly expressed in HUVECs, blocking endogenous miR-200a using 2'-OMe antisense oligonucleotide (ASOs) resulted in a decrease of cell viability and migration. Bioinformatics analysis indicates the 3' untranslated region (UTR) of thrombospondin-1 (THBS1) has a putative binding site for miR-200a. MiR-200a can directly bind to THBS1 3'UTR and negatively regulate THBS1 expression. The identification of endothelial cells (ECs) related miRNA and its target gene may gain new insight into the mechanism of angiogenesis.

[Microwave coagulation at different temperatures for hepatocellular carcinoma management: efficacy evaluation by enzyme histochemical staining].

OBJECTIVE: To compare the application of HE and enzyme histochemical staining in assessing the viability of hepatocellular carcinoma (HCC) cells coagulated by microwave ablation at different temperatures. METHODS: Two groups of mice (n=6) with transplanted homogenic HCC were treated by microwave ablation at 60 degrees C and 50 degrees C for 3 min, respectively. Before and after microwave ablation, paraffin sections and frozen sections of the tumors were prepared for routine HE staining and enzyme histochemical staining with nicotinamide adenine dinucleotide diaphorase (NADH-diaphorase), respectively, and observed under microscope. RESULTS: Shortly after microwave ablation, the morphology and arrangements of the nucleus of the ablated tumor cells in the two groups showed no obvious alteration in HE stained sections, but in sections with enzyme histochemical staining, the activity of NADH-diaphorase in ablated tumor tissue at 60 degrees C disappeared, suggesting the death of HCC cells; sporadic activity of the enzyme was detected in the coagulated tumor at 50 degrees C, indicating tumor cells surviving the ablation. The ablation effect was markedly different between the two groups (P<0.01). CONCLUSION: HE staining is not suitable for evaluation of HCC destruction immediately after microwave ablation, and detection of NADH-diaphorase activity with the enzyme histochemical method better suits this purpose.