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AIM:To investigate the effect of trans-activator of transcription-Rab guanine nucleotide exchange factor 1 (Tat-RabGEF1) fusion protein by intravenous injection on hepatic ischemia/reperfusion (IR) -induced lung injury, and its underlying mechanism.METHODS:Adult male C57BL/6 rats (n=24) were randomized into 3 groups with 8 rats each:sham group, IR group, and Tat-RabGEF1 group.IR was induced by occlusion of the vessels in the hepatic pedicle for 90 min followed by 4 h of reperfusion.The Tat-RabGEF1 protein was intravenously administered right after reperfusion through the caudal vein.The levels of tumor necrosis factor-α (TNF-α) , interleukin-6 (IL-6) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid (BALF) were assessed by ELISA.The lung histopathological injury score and lung wet/dry weight ratio were evaluated, and the levels ofβ-hexosaminidase A (β-Hex A) and myeloperoxidase (MPO) in the lung tissues were also measured.The expression of tryptase in the lung was determined by Western blot.RESULTS:The pathological changes after ischemia for 90 min followed by reperfusion for 4 h were significant, along with increased lung wet/dry weight ratio and lung injury score.Intravenous injection of Tat-RabGEF1 protein effectively alleviated the lung pathological injury (P<0.05).Compared with IR group, lower levels of TNF-α, IL-6 and IL-1βin BALF, and lower expression ofβ-Hex A, MPO and tryptase in the lung tissues in Tat-RabGEF1 group were observed (P<0.05).CONCLUSION:Intravenous injection of Tat-RabGEF1 protein attenuates the lung injury after IR, and its mechanism may be related with inhibition of mast cell activation, reduced releases ofβ-Hex A and tryptase, and decreased levels of TNF-α, IL-6, IL-1βand MPO.
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[Objective]To construct recombinant expression vector PET28a-TAT-RabGEF1,express,purify fusion pr-otein effectively in E.coli Rosetta(DE3),and investigate its transmembrane effect in vitro on P815 cells.[Methods]With cDNA in rats′tissues as the template,two primers containing the TAT sequence and two designed enzyme restriction cutting sites were designed. TAT-RabGEF1 fragment was amplified by PCR,and its product was inserted into PET-28a vector to construct recombinant plasmid PET28a-TAT-RabGEF1 prokaryotic expression vector. The recombinant vector was trans-formed into E.coli Rosetta(DE3)and express fusion proteins.The protein products were purified by affinity chromatography. The efficiency of the transduction of fusion protein into P815 cells were detected by immunofluorescence and analyzed by fluo-rescence spectro-photometer,with using methods of CCK-8 to analyze the cells viability after transduction of different con-centrations of fusion protein.[Results]The recombinant vector PET28a-TAT-RabGEF1 was constructed and the fusion pro-tein TAT-RabGEF1 was successfully expressed in E.coli Rosetta(DE3). By western blotting and SDS-PAGE we can see that the protein products′ relative molecular mass was about 57 ku,which was consistent with the target one,TAT-Rab-GEF1.The immunofluorescence results suggested that the fusion protein had the ability to transduct into P815 cells,and sat-uration of fusion proteins to be transducted into cells was 1 μmol/L.[Conclusion]Constructed recombinant vector PET28a-TAT-RabGEF1 and expressed fusion protein,TAT-RabGEF1,which verified the TAT′s ability of transduction. And it would build a solid technical foundation for the following research on the effect of RabGEF1′s on the activation of mast cells.
ABSTRACT
<p><b>BACKGROUND</b>The aim of this study was to investigate the potential relationship between the dynamic expression of Toll-like receptor 2 and 4 (TLR2/4) in peripheral blood mononuclear cells as well as changes in serum concentration of inflammatory factors and acute lung injury (ALI) in patients after orthotopic liver transplantation (OLT).</p><p><b>METHODS</b>The peripheral blood samples of 27 patients (23 men and 4 women with ASA III to IV) who received OLT were collected for measurement of TLR2/4 at T1 (after induction of anesthesia), T2 (25 minutes after anhepatic phase), T3 (3 hours after graft reperfusion) and T4 (24 hours after graft reperfusion). The expression of TLR2/4 in mononuclear cells was measured by flow cytometry. The serum concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA). Twenty-seven patients were assigned to ALI group (n = 9) and non-ALI group (n = 18) according to the diagnostic criteria of ALI. The expression of TLR2/4 in the ALI group or non-ALI group was analyzed.</p><p><b>RESULTS</b>Compared to the non-ALI group, the volumes of blood loss, ascites, total output and transfused red blood cells were higher in the ALI group, and the anhepatic phase lasted longer (P < 0.05, P < 0.01). The expression of TLR2/4 in mononuclear cells increased significantly at T3 and T4, and serum concentrations of TNF-alpha, IL-1beta and IL-8 increased significantly too. There was no significant difference in Child-Turcotte-Pugh (CTP) scores between the ALI group and non-ALI group (P > 0.05). The expression of TLR2/4 in mononuclear cells increased significantly at T3 and T4 in the ALI group (P < 0.05, P < 0.01). A positive correlation was noted between the expression of TLR4 in mononuclear cells and the serum concentrations of TNF-alpha, IL-1beta (P = 0.041, P = 0.046) in the ALI group. In the non-ALI group, statistical results showed that the expression level of TLR2/4 in mononuclear cells was not significantly different during the peri-operative period of OLT (besides TLR4 expression at T4). Compared to the non-ALI group, the increasing amplitude of TLR2/4 expression in mononuclear cells was more significant in the ALI group. The patients whose TLR2/4 expression in mononuclear cells exceeded that at T1 by one time were more likely to suffer from ALI (P = 0.013), with a relative risk of 16.</p><p><b>CONCLUSION</b>The expression level of TLR2/4 in mononuclear cells increases significantly in the peri-operative period of OLT, and it may be a high risk factor for occurrence of postoperative ALI.</p>