Kallikrein-binding protein (KBP) was previously identified as a serpin family member with specific inhibitory effect on tissue kallikrein and angiogenesis, while there is little knowledge about the effects on inflammation. The aim of this study is to investigate whether KBP can suppress LPS-induced inflammatory process. Our results showed that both recombinant KBP and KBP overexpression inhibited LPS-stimulated TNF-α transcription and translation in macrophage cell line RAW264.7 and primary macrophages. Furthermore, KBP treatment protected mice from endotoxin shock and repressed serum TNF-α production, increasing survival rate of mice from 10% to 50% when compared to LPS alone. Moreover, qPCR and Western blot analysis demonstrated that both suppressor of cytokine signaling 3 (SOCS3) transcription and translation were induced by KBP treatment in the present of LPS. RNA interference assay and luciferase assay showed that SOCS3 was responsible for the down-regulation of TNF-α by KBP, rather than NF-κB subunit p65 and β-catenin. Therefore, we demonstrated that KBP suppressed LPS-induced TNF-α production via upregulating SOCS3 expression. These results present the protective effects of KBP on LPS-induced inflammation and provide novel information for the anti-inflammation mechanism.

Objective To provide an efficient, practicable and high specificity method for culture of bovine retinal capillary endothelial cells(BRCEC). Methods Fresh bovine eyes were obtained from a local market. The retinas were removed and washed several times in DMEM. Subsequently retinas were homogenized and the resultant pellet was resuspended in an isolation medium. Microvesseles were trapped on an 75 μm nylon mesh and transferred to a 50 ml centrifuge tube containing 10 ml enzyme cocktail which includes collagenase Ⅰ, DNase Ⅰand pronase and treated for 20 min. The resultant vessel fragments were trapped on a 46 μm nylon mesh,washed with the isolation medium and centrifuged for 5 min. For selective culture of BRCECs, the resultant pellet was resuspended in 2 ml of the BRCEC growth medium and transferred into 25 cm2 gelatin-coated plastic tissue culture flasks. BRCECs were identified by immunohistochemical method of factor Ⅷ. Results The purity of selectively cultured BRCECs was more than 95%. Cells seeded onto the surface of the polymerized EC Matrix could develop the cellular network structures by 12-18 h. Conclusion BRCECs can be attained easily and effectively by this method. This culture method would provide a useful model for studying disease related to retinal vessels in vitro.

Objective To determine the effects of pyrrolidine dithiocarbamate(PDTC),a nuclear factor-κB (NF-κB) inhibitor,on paraquat-induced early-stage acute lung injury.Methods Following establishment of paraquat-induced lung injury mice model,64 adult male SD rats were randomly divided into PQ+PBS gioup (32 SD rats were treated with phosphate buffer solution) and PQ+PDTC group (32 SD rats were treatecl with 100 mg/kg PDTC).The survival rate of two groups was assessed at hours 24,48 and 72 respectively.Haematoxylin-eosin staining was employed to assess the acute lung injury for scoring,and immunochemical assay was used to detect inflammatory cell infiltration in the lungs.The level of TNF-α and IL-6 was measured by enzyme-linked immunosorbent assay and activation of NF-κB in the lungs was assessed by using Western blotting.Results PDTC improved the survival rate at 72 hours as compared with group PQ+PBS[24/32(75％) in group PQ+PDTC vs 13/32(40.6%) in group PQ+PBS,P=0.015] and acute lung injury score at 24 hours (7.5±1.0 vs 9.8±1.5),48 hours (9.7±0.8 vs 12.0±0.9) and 72 hours (11.5±1.0 vs 14.5±1.0) (all P＜0.05).PDTC resulted in reduced cell count with myeloperoxidase expression,the levels of TNF-α and IL-6 in the lungs and the protein levels of NF-κB in the nucleus and cytoplasm (all P＜0.05).Conclusion PDTC reduces early-stage mortality of acute lung injury by suppressing infiltration of myeloperoxidase-positive inflammatory cells and inhibiting NF-κB activation leading to decreased TNF-α and IL-6 expression in the lungs.

Objective To explore the effects of pyrrolidine dithiocarbamate (PDTC) on the acute lung injury and the activation of Nrf2 pathway after Paraquat (PQ) induced lung injury.Methods Fortyeight adult male SD rats with lung injury induced by PQ were randomly (random number) divided into control group and PDTC group.Three animals were sacrificed at every 1-week interval,7d,14d and 21 days after PQ intoxication,and the lungs of rats were removed for acute lung injury score after HE staining,and for lung fibrosis assessment after Masson staining,and the levels of reduced glutathione (GSH) and malondialdehyde (MDA) in the lung tissue homogenate were assayed and the phosphorylation of Nrf2 (nuclear-E2-related factor 2) was detected by Weston blot.The mean values of detected variables between two groups were compared by t test,and survival curve was tested by Wilcoxon (Gehan) test.Results The intoxication symptoms of rats were obvious,and 4 rats in control group and 9 rats in PDTC group survived until 21days.The survival time of animals in PDTC group was longer than that in control group (Wilcoxon (Gehan) =10.17023,P =0.001).The levels of MDA in control group were higher than those in PDTC group,while the levels of GSH in control group were lower than those in PDTC group.The levels of phosphorylation of Nrf2 in PDTC group were higher than those in control group at 1-week intervals,1-week:(0.32±0.04) vs.(0.23±0.05),P=0.003; 2-week:(0.62±0.06) vs.(0.33±0.03),P＜0.001; 3-week:(0.61 ±0.04) vs.(0.33±0.05),P＜0.001.The acute lung injury (ALI) scores in PDTC group were lower than those in control group,1-week:(5 ± 0.95) vs.(8 ± 1.23),P =0.002 ; 2-week:(9±1.18) vs.(11±1.02),P=0.019; 3-week:(11±1.33) vs.(12±1.42),P=0.002.The percentages of lung fibrosis at 1-week intervals after PQ intoxication were (40.87 ± 7.25) ％,(43.38 ±5.71)％ and (45.91 ± 3.97)％ in control group,and they were higher than those in PDTC group (32.92±2.34)％,(33.45 ±3.04)％ and (35.27 ±3.81)％ in PDTC group,P=0.017,0.001 and 0.001 respectively.Conclusions Attenuation of acute lung injury and lung fibrosis,and prolongation of survival time of SD rats by PDTC were associated with activation of Nrf2 pathway.