| BackgroundVarious acute or chronic lung diseases commonly exist diversity infections, endotoxin, inflammatory media and inflammatory factors, ischemia, acidosis. The factors can result in the vascular endothelium damage and tissue factor releasing, activating various coagulation factors, making blood in the condition of high coagulation or thrombogenesis. Thrombin is one of the key coagulation factors. In addition, it is confirmed that thrombin has important non-coagulation functions, including promoting cells growth, proliferation, secretion, adhere, and so on through binding to protease activated receptor (PAR) in cell membrane and activating a series of downstream signal pathways. The concentration of thrombin was high significantly in lung alveoli in acute lung injury and chronic lung diseases such as COPD, lung interstitial diseases. Thrombin binding to PAR1 results in many kinds of effects including promoting various kinds inflammation factors such as IL-6,IL-8,PGE2,GM-CSF releasing, regulating the lung inflammation reaction and recovery. Another reseach indicates that thrombin stimulates various cells such as vascular smooth muscle, vascular endothelia cells, hippocampus neuron to produce ROS through activating NADPH oxidase subunit p47(phox) and p67(phox). ROS have two sides effects, one is injury effect via participating cell apoptosis and necrosis at the middle to high concentrations of ROS. Another side is participating cells signal transduction, activating MAPK pathway mainly at the lower concentration of ROS, finally to activate transcription factor and impact gene expression, resulting in cells proliferation and differentiation. NF kappa B is an important transcription factor and has the extensive biological effects and has the important roles in the pathological process such as oxidative stress, infection, inflammation reaction, cell apoptosis and abnormal proliferation.As we know lung fibroblast is the important structure cell in lung tissue and more than 95 percent of all lung interstitial cells in quantity. It is the main cell which participates the injury recovery process and manifestes higher proliferation activation. A great deal of damage factors make the capillary permeability increase and the plasm fractions leak into the outer of blood capillary. The leakage substances can touch fibroblast and then make cell proliferate. We presume that thrombin as one of the leakage substances could play a certain role in the recovery process.ObjectiveThrombin stimulated lung fibroblast and cell were detected to generate reactive oxygen series(ROS) and the produce pathway. ROS were investigated to activate MAPK signal pathway as the second messager, even more it has the effect on NF kappa B, regulating growth gene expression.MethodsHLF cells were chosen as the research object in vitro, a certain concentration of thrombin was added to cells. MTT method and Brdu incorporation assay were used to observe thrombin promoting HLF proliferation. Chemiluminescence method, flow cytometry and GSH detection kit were used to detect ROS in HLF. Western blot method was used to detect PAR1, NADPH oxidase, ERK1/2 and P38MAPK protein expression. Laser copolymerization technology was used to detect the p65 translation from kytoplasm to nucleus.Results1. Thrombin had reproductive activity with concentration dependence during the concentration of 0.1U/ml-20U/ml.Compared with control group, thrombin could significantly facilitate HLF to proliferate at the concentration of 10 U/ml or above (P<0.05), but there was no more proliferation effect when thrombin concentration was more than 20U/ml. Thrombin at the concentration of 20U/ml for 24h,36h,48h,60h,72h,respectively, compared with the control group, had significant enhance in proliferation from 36h to 60h (P<0.05).Brdu incorporation assay showed that thrombin enhanced cells to proliferate significantly above the concentration of lOU/ml, compared with control group (P<0.05), and it was a dose-response relationship. But there was no more proliferation ability when thrombin's concentration was above 20U/ml.2. Thrombin significantly increased the amount of ROS at the concentration of 5U/ml compared with control group (P<0.05), showing a dose-response relationship. ROS did not increase more when the concentration was more than 40U/ml. Thrombin at the concentration of 20 U/ml for 30 minutes increased intracellular ROS (mean value 298.0) by 64.7% compared with the control (mean value 180.6). The same concentration of thrombin for 1 hour caused the increase ROS (mean value 431.2) by 138.8% compared with the control. ROS generation in HLF pretreated with hirudin (20 U/ml) for 30 minutes had no significant change (mean value 171.2) compared with the control.Ratio of GSH/GSSG had a significant decrease in HLF treated with thrombin at the concentration of 10U/ml,20U/ml(2.1,1.7 respectively) compared with the control group(5.2) (P<0.05). The ratio was no significant change (5.0,4.9) compared with the control group (5.2) when pretreated with hirudin or NAC (P> 0.05)3.Subunits p47phox and Rac2 specific bands markedly decreased in cytosol following treatment with thrombin(20 U/ml) at 10 minutes and 30 minutes time points(P<0.05). There was little protein expression at 1h time point (P<0.05). But there was no significant change between the control group and hirudin+thrombin group (P> 0.05)4.p-ERK1/2 protein expressing increased significantly from 10 minutes to 2 hours time points with thrombin treatment compared with control (P<0.05). There was little protein expression in the cells pretreated with NAC and hirudin. Thrombin did not affect the change of p-p38 expression at all from 10 minutes to 2 hours time point (P >0.05)5. Thrombin stimulated HLF at 5min,10min,30min,1h and 2h respectively. The fluorescence contents representing p65 at different time points in nucleus had no statistical difference compared with control group (P>0.05). The fluorescence contents in cells stimulated with TPA had the significantly increase and there was a statistical difference (P<0.05)6. The protein expression of PAR1 was found in both bronchial epithelial cell and human lung fibroblast and there was more in the later (P<0.05)Conclusion1. Thrombin promotes human lung fibroblast to proliferation.2. The mechanism of promoting proliferation is as follows.2.1 PAR1 is expressed higher in HLF than that in bronchial epithelial cell.2.2 Thrombin induced HLF generate ROS via activating NADPH oxidase.2.3 Thrombin activated growth signal pathway via phosphorylating ERK1/2 but not p38MAPK.2.4 NF kappa B was not activated in thrombin promoting proliferation process.SignificanceThrombin activates protease activated receptor-PAR1, inducing NADPH oxidase generate ROS and ROS activate ERK1/2 signal pathway, resulting to HLF proliferation. Our study explore primarily the mechanism of thrombin on human lung fibroblast, which provides the theory evidence for blood coagulation disorder relative respiratory diseases revention and therapy. |
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