Objective To investigate the protective effect of hyperbaric oxygen (HBO) combined with ω-3 polyunsaturated fatty acid (PUFA ω-3) on lipopolysaccharide-induced acute lung injury in the rats and its related mechanism.Methods Forty-eight SD rats were randomly divided into the control group,the model group,the experimental group and the combined group,each consisting of 12 rats.With exception of the rats in the control group,acute lung injury (ALI) model was established by LPS tracheal instillation in the rats of the model and experimental groups.The experimental group received PUFA ω-3 (150 mg/kg) by gavage,the combined group was treated with HBO on the basis of PUFA omega-3,and equal volume of distilled water was given to the control and model groups.The course of treatment lasted for 14 days.After last medication,pulmonary tissues of the rats were collected and wet/dry ratio was calculated,and the inflammatory factors,TNF-α and IL-1β in the lung tissue homogenate were detected by ELISA test kit.HE staining was used to observe the pathological damage of the lung tissue,and the expression levels of TLR4 and NF-κB proteins in the lung tissue were detected by Western-blotting.Results Compared with the control group,pulmonary edema in the model group could clearly be seen,and the wet/dry ratio of the lung tissue increased markedly (5.57 ± 0.57 vs.4.16 ±0.19) (P ＜0.05).Compared with the model group,the wet/dry ratio of the lung tissue in the experimental and the combined groups decreased (4.78 ±0.25,4.42 ±0.18 vs.5.57 ±0.37) (P ＜0.01 or P ＜ 0.05),and the wet/dry ratio of the combined group was obviously lower than that of the experimental group (4.42 ±0.18 vs.4.78 ±0.25) (P ＜0.05).As compared with the control group,the inflammatory factor levels of TNF-α and IL-1β in the model group all significantly increased(P ＜ 0.01 or P ＜ 0.05).Following treatment with simple PUFA ω-3 or coupled with HBO,the inflammatory factor levels of TNF-α and IL-1β in lung tissues of the experimental and the combined groups all decreased markedly,as compared with those of the model group (P ＜ 0.05),with those of the combined group decreased more significantly (P ＜ 0.05).As compared with those of the control group,the expression levels of TLR4 and NF-κB proteins in the model group were significantly up-regulated (P ＜ 0.01).Compared with the model group,the expression levels of TLR4 and NF-κB proteins in the experimental and the combined groups all decreased(P ＜0.01 or P ＜0.05),with the down-regualtion of TLR4 in the experimental group displaying more markedly (P ＜ 0.01).Compared with the experimental group,the down-regulation of NF-κB protein in the combined group was more significant (P ＜ 0.05).Conclusion HBO could obviously enhance the protective effect of PUFA ω-3 on the lung tissue of the rats with ALI and reduce the level of inflammatory factors,which might be associated with the regulation of TLR4/NF-κB signal pathway.

Objective To investigate the protective effect of hyperbaric oxygen (HBO) combined with ω-3 polyunsaturated fatty acid (PUFA ω-3) on lipopolysaccharide-induced acute lung injury in the rats and its related mechanism.Methods Forty-eight SD rats were randomly divided into the control group,the model group,the experimental group and the combined group,each consisting of 12 rats.With exception of the rats in the control group,acute lung injury (ALI) model was established by LPS tracheal instillation in the rats of the model and experimental groups.The experimental group received PUFA ω-3 (150 mg/kg) by gavage,the combined group was treated with HBO on the basis of PUFA omega-3,and equal volume of distilled water was given to the control and model groups.The course of treatment lasted for 14 days.After last medication,pulmonary tissues of the rats were collected and wet/dry ratio was calculated,and the inflammatory factors,TNF-α and IL-1β in the lung tissue homogenate were detected by ELISA test kit.HE staining was used to observe the pathological damage of the lung tissue,and the expression levels of TLR4 and NF-κB proteins in the lung tissue were detected by Western-blotting.Results Compared with the control group,pulmonary edema in the model group could clearly be seen,and the wet/dry ratio of the lung tissue increased markedly (5.57 ± 0.57 vs.4.16 ±0.19) (P ＜0.05).Compared with the model group,the wet/dry ratio of the lung tissue in the experimental and the combined groups decreased (4.78 ±0.25,4.42 ±0.18 vs.5.57 ±0.37) (P ＜0.01 or P ＜ 0.05),and the wet/dry ratio of the combined group was obviously lower than that of the experimental group (4.42 ±0.18 vs.4.78 ±0.25) (P ＜0.05).As compared with the control group,the inflammatory factor levels of TNF-α and IL-1β in the model group all significantly increased(P ＜ 0.01 or P ＜ 0.05).Following treatment with simple PUFA ω-3 or coupled with HBO,the inflammatory factor levels of TNF-α and IL-1β in lung tissues of the experimental and the combined groups all decreased markedly,as compared with those of the model group (P ＜ 0.05),with those of the combined group decreased more significantly (P ＜ 0.05).As compared with those of the control group,the expression levels of TLR4 and NF-κB proteins in the model group were significantly up-regulated (P ＜ 0.01).Compared with the model group,the expression levels of TLR4 and NF-κB proteins in the experimental and the combined groups all decreased(P ＜0.01 or P ＜0.05),with the down-regualtion of TLR4 in the experimental group displaying more markedly (P ＜ 0.01).Compared with the experimental group,the down-regulation of NF-κB protein in the combined group was more significant (P ＜ 0.05).Conclusion HBO could obviously enhance the protective effect of PUFA ω-3 on the lung tissue of the rats with ALI and reduce the level of inflammatory factors,which might be associated with the regulation of TLR4/NF-κB signal pathway.

Objective To compare the immune effects of Coxsackievirus B3 (CVB3) capsid protein VP1 expressed bacterially, recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1which express VP1 protein in mice. Methods After expressed in prokaryotic cells, VP1 protein was purified. Recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1 were amplified and extracted. Six to 8-week-old, male BALB/c mice were divided into four groups randomly. Each group contained 18 mice. The mice of pcDNA3/VP1 group or VP1 protein group were immunized intramuscularly with three injections at three weeks apart, of recombinant plasmid pcDNA3/VP1 at a dose of 100 μg/mouse or recombinant protein VP1 at a dose of 50 μg/mouse. The mice of rAd/VP1 group were immunized intramuscularly twice at two weeks interval with rAd/VP1 at a dose of 1.2 × 107 PFU. The control group was mock-immunized with 100 μl of PBS intramuscularly. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. ELISA and micro-neutralization test were used to detect levels of CVB3-specific IgG antibody and neutralizing antibody titers in the sera of immunized mice. Three weeks after the last immunization, the cytotoxic T lymphocyte(CTL) killing activity of spleen lymphocytes was detected with CCK-8 assay. Subsequently, virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose( CCID50 ) assay on HeLa cell monolayers and percentage of animals surviving were observed after lethal CVB3 attack over a period of 21 days. Results The titers of specific IgG antibody and neutralizing antibody in sera of VP1 protein immunized mice were higher than other groups( P ＜0.05 ). While CTL killing activity of spleen lymphocytes of VP1 protein immunized mice was lower than mice in rAd/VP1 group( P ＜0. 05). Virus titers in sera of VP1 protein immunized mice were lower than the mice in pcDNA3/VP1 or rAd/VP1 groups ( P ＜ 0.05 ), while survival rate was significantly higher than these two groups ( P ＜ 0.05 ).Conclusion VP1 protein induced higher level of humoral immune response and acquired obvious immune protection effects in mice. The immunizing potency of VP1 protein vaccine surpassed plasmid pcDNA3/VP1or recombinant adenovirus rAd/VP1. It appeared to be a promising candidate among the three different vaccines.

Objective To construct recombinant adenovirus Ad/MDC-VP1 and investigate its im-muno-boosting effect of the mice primed with the experimental DNA vaccine against Coxsackievirus infection. Methods The recombinant adenovirus Ad/MDC-VP1 was constructed and packaged. The Western blot analysis was used to verify the target protein. BALB/c mice were divided into four groups: Ad/MDC-VP1 group, pcDNA3/MDC-VP1 group, pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group and PBS group. The mice in each group were immunized intramuscularly. The titers of serum IgG and neutralizing antibody were tested by ELISA and trace neutralization assay, respectively. The lymphocytes proliferation activity and specific CTL cytotoxic activity were tested by CCK-8 assay. The mice in each group were challenged with le-thal dose of Coxsackievirus, and the assay of the serum virus titers and the observation of protection efficacy against Coxsackievirus infection were carried out. Results The recombinant adenovirus Ad/MDC-VP1 was successfully constructed and the target protein was expressed. It was observed that the titers of CVB3 VP1 specific antibody, lymphocyte stimulation index, CTL cytotoxicity activities and protection rate of the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group were much higher than those of the rest groups( P < 0.05), and the titer of serum virus was lower after CVB3 challenged ( P < 0.05 ). Conclusion Both the cellular and humoral immune responses in mice could been significantly enhanced by the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost strategy.