The Effects And Mechanisms Of Microgravity On Expression Of TF In Monocytes

Background and objectiveWith the establishment of space stations and the implementation of the Apollo Project, the effects of space environment on the body have become a new research focus in scientific field. The space environment with microgravity (10-2-10-5G) is completely different from the earth environment. The exprements can be carried out in space flight, unsuprisingly, space flight oportunity is fewer, with rigorous conditions, and huge cost, and it can not meet the requirements of study the effects of microgravity in-depth either. However, the development of ground simulator is compensated for this defect, Rotary cell culture system (RCCS) developed by National Aeronautics and Space Administration (NASA) is used to simulate microgravity conditions.Gravity is an essential force that affects many life functions and has played a key role in the evolution of all terrestrial life. While human organism is exposed in microgravity environment, which has been shown to lead to a series of changes. In the cardiovascular system, the reditribution of fluides of the body and the elimination of normal head-to-foot hydrostatic pressure gradients maybe cause the occurrence of "space anemia". Therefor, in recent years, the research focused on the blood disease which happened in the microgravity conditions. However, microgravity affects the coagulation function of the body is still unclear.Tissue factor, one of the coagulation factors, plays a vital role in the process of homeostasis and coagulation. It starts the blood clotting cascade by combination with the factorⅦ/Ⅶa. When TF is exposed to plasma proteins, TF will be mutual adhesion with a high affinity FⅦ. TF-FⅦcomplex can be activated to TF-Ⅶa complex by free factorⅦ(FⅦa) and formed TF-Ⅶcomplex. Free FⅦcan be further activated by TF-Ⅶa complex. These mechanisms are called TF mediated own activation of F VII. TF-Ⅶa complexes can rapidly catalyze the activation of factorⅩ. In addition, TF-Ⅶcan activate factor IX with a lower rate, in the presence of cofactorsⅧ, and the activated factorⅨcan activate factor X and transform into factor X a. these processes lead to generation thrombin ultimately. Thus fibrinogens catalyzed by thrombin transform into fibrin and poly synthetic protein clots. That TF can activate the coagulation factor IX and X simultaneously, and start the intrinsic and extrinsic joint amplification reaction of thrombin, which plays a crucial role in the process of thrombus formation.Monocytes in the blood system are important immune cells. But in recent years, the study found that it was the main source of blood-borne TF and related to thrombosis closely in atherosclerosis and coronary heart disease. Under physiological conditions, the monocytes of body does not express TF, but when it is stimulated by inflammatory mediators such as the lipopolysaccharide (LPS) or endotoxin, tumor necrosis factor, IL-l,then it expresses a large number of TF and activate the way of coagulation. LPS combines with LPS-binding protein (LBP), and then passes it to CD14 molecules, formation LPS-LBP-CD14 complex, and this complex interaction with TLR4-MD2, which activates signal transduction pathways and transcription factors and induces gene expression. Recently, it was shown that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex and others demonstrated that TLR4 plays a key role in the signaling pathway of LPS stimulation of monocytes and then expression of TF. Thus, CD 14 and TLR4 play key roles in the signaling pathway of LPS induced TF expression in monocytes.For monocytes has been adapted to the 1-G gravity, the simulated microgravity is a stimulating factor. With this stimulating factor, will the expression of monocyte TF increase? If the medium is supplemented with LPS, whether or not will it combines simulated microgravity and enhance monocyte TF expression? What is Specific mechanism? There are no relevant reports, so it is the main purpose of this research.The research includes three parts:(1) To simulate microgravity environment using RCCS, we analysed the effects of microgravity, LPS, alone or in combination together on the expressions of TF mRNA and TF protein.(2) To explain the change of TF expression in monocytes under microgravity environment, we detected the effects of microgravity on expression of CD 14 in monocytes.(3) To further explore the possible mechanism of variation of TF expression while monocytes are cultured in microgravity conditions, we detected the expression of TLR4 mRNA and TLR4 positive rate in THP-1 cells.Methods(1) Effects on expression of TF in monocyte by simulated microgravity.Human monocyte lines THP-1 cells were divided into four groups:1) the 1G control group (C group),2) the simulated with microgravity alone (M group),3) the treatment with LPS alone (L group), and 4) the treatment with simulated microgravity and LPS (ML group). Simulate microgravity environment using RCCS. 10ml culture medium for gene analysis by real-time quantitative PCR after cultured for 0h,2h,4h,8h,12h, respectively And 10ml culture medium for protein analysis by Western blotting assays were done respectively after cultured for 0h,4h,8h,12h, 24h.(2) The effects of simulated microgravity on the expression of CD14.Stimulated THP-1 cells with microgravity, taked out 10ml of the culture medium after cultured for 0h,2h,4h,8h,12h for flow cytometry assays, and detected the change of CD 14 expression.(3) The effects of simulated microgravity on the expression of TLR4.After THP-1 cells exposed to microgravity and LPS alone or in combination for 0h,2h,4h,8h,12h, the cells were harvested, counted, and analyzed for the expression of TLR4 mRNA were quantified by real-time quantitative PCR and the TLR4 positive rate were detected by flow cytometry.(4) Statistical analysisData are showed as means±SD, using independent samples T test to compare the positive rate of CD 14 and TLR4 at the same time between two groups. The relative gene expression and protein expression between different time and different groups were compared using two-way ANOVA, Fisher method is used with homogeneity of variance Bonferroni method multiple comparisons, the Welch approximation Heterogeneity of variance, and Dunnett T3 method multiple comparisons. It is considered significant difference when P<0.05 and calculated statistical software SPSS13.0 is used. Results(1) The effects on expression of TF in monocyte by simulated microgravity.The results showed that there was no significant difference between M group and C group at the five time points of TF mRNA and protein expression (P> 0.05). While added with LPS, there was significant difference of the expression of TF mRNA between ML group and L group after cultured for 2h,4h, (t=13.037 and 12.118, P= 0.000). Moreover, after cultured for 12h and 24h, TF protein expression in L group was significantly higher than that of ML group (t=9.369 and 38.622, P=0.001 and 0.000). Compared L group with the C group, the expression of TF mRNA increased significantly while THP-1 cells were cultured for 2h,4h,8h(t=-20.151,-19.465 and-23.925, P=0.002,0.000 and 0.000).(2) The effects of simulated microgravity on the expression of CD14.Without any stimulation in THP-1 cells, the positive rate of CD 14 is in basic level. Flow cytometry analysis showed that there was no significant difference of the positive rate of CD 14 between M group and C group after cultured for Oh,2h,4h,8h, 12h.(3) The effects of simulated microgravity on the expression of TLR4.Compared with C group, the expression of TLR4 mRNA of THP-1 cells in M group was down-regulated after cultured for 8h and 12h (t=5.843 and 8.198, P=0.004 and 0.001). while our study shown that the TLR4 mRNA expression was suppressed by simulated microgravity in ML group compared with L group after cultured for 2h,4h, 8h (t=3.560,14.962 and 4.260, P=0.024,0.000 and 0.013). There was no significant difference in positive rate of TLR4 between C group and M group (P>0.05). However, stimulated with LPS for 4h and 8h, up-regulation of positive rate of TLR4 in ML group was lower than L group (t=7.257,8.915,P=0.002,0.001) Conclusion(1) In M group and C group, there were little expression of TF mRNA and protein and exhibited no significant difference in the expression of TF between two groups. In contrast, TF expression was strongly induced by 20μg/ml LPS. Up-regulation of TF expression in ML group was significantly lower than that of L group.(2) CD 14 palys a key role in the expression of LPS-induced TF. In the present study, there was no significant difference of the expression of CD14 between M group and C group.(3) TLR4 is the vital element in the signal pathway of LPS-induced TF expression in monocytes. Our data suggested that microgravity suppressed the TLR4 expression, and this result was consistent with the effect of microgravity on expression of LPS-induced TF in monocytes. It suggests that simulated microgravity may inhibit the LPS-induced TF expression, through supressed the expression of TLR4.Innovations(1) Effects of simulate microgravity on expression of TF in monocytes was first detected and analysed. The results suggested that microgravity significantly inhibited the expression of TF induced by LPS in monocytes.(2)In this study, we observed the LPS-induced TF expression was partially suppressed by microgravity. This inhibition involved the suppression of TLR4 expression in monocytes.