The Effects Of Tar And Nicotine In Tobacco On The Expression Of Thrombomodulin And The Interaction Between Thrombomodulin And Thrombin And The Intervention By Rosuvastatin

Cigarette smoking is an independent risk factor of cardiovascular disease. Numerous studies and clinical practice have shown that smoking can lead to thrombosis in the blood vessels, increase rates of acute myocardial infarction. But the mechanism by which cigarette smoking cause thrombosis is not clear. Endothelia cells play a key role in maintaining the blood flow, resisting vascular thrombosis. Thrombomodulin(TM) is a glycoprotein which is expressed on the surface of endothelial cells and plays a critical role in anticoagulation. It is the formation of a high affinity 1:1 complex with thrombin to activate protein C which plays a role in anticoagulation. The anticoagulation effect of TM not only depends on the amount of TM expression but also on the singe-molecule interactional between TM and thrombin. Currently the effect of smoking on the expression of TM in vitro observation research is less. Tar and nicotine, as two important harmful substances of tobacco, are widely studied. But the effects of tar and nicotine on the expression of TM in endothelial cells are less reported. Rosuvastatin, as a new type of lipid-lowering drugs, not only has the function of adjusting blood fat, but also can resist to oxidative stress, decrease the inflammation state, stabilize the plaques, improve endothelial functions. But there has not been reported if it can improve the expression of TM induced by tar and nicotine. Moreover, about the single-molecule interactional between TM and thrombin, Atomic Force Microscopy(AFM) single molecule force spectroscopy( SMFS) has been developed to measure the biomolecular interaction force at the single molecule level under physiologically relevant conditions with high force resolution. Although in recent years the study of AFM on biomolecular interaction is more and more, but there has not been reported the effects of tar and nicotine on the interaction between TM and thrombin and the intervention by rosuvastatin. Part Ⅰ: The effects of tar and nicotine on the expression of TMObjective: To observe the proliferation of Human Umbilical Vein Endothelial Cells(HUVECs) and the expression of TM in HUVECs under different tar and nicotine conditions.Methods: HUVECs were isolated by 0.1% type I collagenase digestion for 15-20 min at 37℃, and cultured in low serum endothelial cell medium. After passage cultivation, we used the factor VIII related antibody to identify endothelial cell. The 2-3 generation of HUVECs were used in experiment.1 CCK-8 test detected the effects of tar and nicotine on the proliferation of HUVECsHUVECs seeded in 96-well plates, incubated respectively with 50mg/L, 20 mg/L, 10 mg/L, 1 mg/L tar and 10-3mol/L, 10-5mol/L, 10-7mol/L, 10-9mol/L nicotine for 6h and 24 h. Microplate Reader was used for detecting A value.2 Flow cytometric analysis technique detected the effects of tar and nicotine on the protein expression of TM in HUVECsHUVECs seeded in 6-well plates, incubated respectively with 50mg/L, 20 mg/L, 10 mg/L, 1 mg/L tar and 10-3mol/L, 10-5mol/L, 10-7mol/L, 10-9mol/L nicotine for 6hours or exposed to 20mg/L tar and 10-5mol/L nicotine for 0, 1, 6, 12, 24 h. Flow cytometric analysis technique was used for detecting TM protein.3 RT-PCR detected the effects of tar and nicotine on the m RNA expression of TM in HUVECsHUVECs seeded in 6-well plates, incubated respectively with 50mg/L, 20 mg/L, 10 mg/L, 1 mg/L tar and 10-3mol/L, 10-5mol/L, 10-7mol/L, 10-9mol/L nicotine for 6hours or exposed to 20mg/L tar and 10-5mol/L nicotine for 0, 1, 6, 12, 24 h. RT-PCR was used for detecting TM m RNA.Results:1 CCK-8 test detected the effects of tar and nicotine on the proliferation of HUVECsCompared to control group, different concentration of 6h, 24 h tar groups were observed to gradually reduce A value with the increasing of concentration. But only 6h and 24 h 50mg/L tar groups were significant difference(P<0.05). Different concentration of 6h, 24 h nicotine groups compared to control group were observed to gradually increase A value with the increasing of concentration. But there were no significant difference.2 Flow cytometric analysis technique detected the effects of tar and nicotine on the protein expression of TM in HUVECsCompared to control group, different concentration of tar groups were observed to gradually increase the TM protein expression with the increasing of concentration, in a concentration dependent manner. And 10mg/L tar group was significant difference(P<0.05). Compared to 0h group, different time of tar groups were observed to gradually increase the TM protein expression with the increasing of time, in a time dependent manner. And 6h tar group was significant difference(P<0.05). It was also observed that the TM protein expression was increased fastest within 6h. With the extension of time, the increasing rate of TM protein expression was slow down. However nicotine groups were no effect on the TM protein expression in HUVECs.3 RT-PCR detected the effects of tar and nicotine on the m RNA expression of TM in HUVECsCompared to control group, different concentration of tar groups were observed to gradually increase the TM m RNA expression with the increasing of concentration, in a concentration dependent manner. And 1mg/L tar group was significant difference(P<0.05) Compared to 0h group, different time of tar groups were observed to gradually increase the TM m RNA expression with the increasing of time, in a time dependent manner. And 1h tar group was significant difference(P<0.05). It was also observed that the TM m RNA expression was increased fastest within 6h. With the extension of time, the increasing rate of TM m RNA expression was slow down. However nicotine groups were no effect on the TM m RNA expression in HUVECs.Conclusion:1 Tar inhibited the proliferation of HUVECs, however nicotine increased the proliferation of HUVECs.2 Tar increased the expression of TM on HUVECs in concentration andtime dependent manner. Nicotine was no effect on the expression of TM inHUVECs. Part II: The effect of Rosuvastatin on the expression and activity of TMinduced by tar and nicotineObjective: To observe the effect of rosuvastatin on the proliferation of HUVECs and the expression and activity of TM in HUVECs induced by tar and nicotine. So as to explore the mechanism by which statins improve smoking-induced intravascular thrombosis.Methods: HUVECs were isolated by 0.1% type I collagenase digestion for 15-20 min at 37℃, and cultured in low serum endothelial cell medium. The 2-3 generation of HUVECs were used in experiment.1 CCK-8 test detected the effect of rosuvastatin on the proliferation of HUVECs induced by tar and nicotineHUVECs seeded in 96-well plates, incubated respectively with 50mg/L tar, 10-3mol/L nicotine, 50umol/L ROS+50mg/L tar, 50umol/L ROS+10-3mol/L nicotine, 50umol/L ROS for 6h and 24 h. Microplate Reader was used for detecting A value.2 Flow cytometric analysis technique detected the effect of rosuvastatin on the protein expression of TM in HUVECs induced by tar and nicotineHUVECs seeded in 6-well plates, incubated respectively with 20mg/L tar, 10-5mol/L nicotine, 50umol/L ROS+20mg/L tar, 50umol/L ROS+10-5mol/L nicotine, 50umol/L ROS for 6h. Flow cytometric analysis technique was used for detecting TM protein.3 RT-PCR detected the effect of rosuvastatin on the m RNA expression of TM in HUVECs induced by tar and nicotine.HUVECs seeded in 6-well plates, incubated respectively with 20mg/L tar, 10-5mol/L nicotine, 50umol/L ROS+20mg/L tar, 50umol/L ROS+10-5mol/L nicotine, 50umol/L ROS for 6h. RT-PCR was used for detecting TM m RNA.4 TM activity test detected the effects of tar and nicotine on the TM activity in HUVECs and the intervention by rosuvastatinHUVECs seeded in 96-well plates, incubated respectively with 20mg/L tar, 10-5mol/L nicotine, 50umol/L ROS+20mg/L tar, 50umol/L ROS+10-5mol/L nicotine, 50umol/L ROS for 6h and 24 h. Microplate Reader was used for detecting TM activity.Results:1 CCK-8 test detected the effect of rosuvastatin on the proliferation of HUVECs induced by tar and nicotineCompared to tar group, ROS+tar group significant increased A value(P<0.05), but ROS group was no significant difference compared to control group. Also ROS+nico group was no significant difference compared to nico group.2 Flow cytometric analysis technique detected the effect of rosuvastatin on the protein expression of TM in HUVECs induced by tar and nicotineCompared to control group, ROS group significantly increased the TM protein expression in HUVECs(P<0.05). Compared to tar group, ROS+tar group significantly increased the TM protein expression in HUVECs(P<0.05). Compared to nico group, ROS+nico group significantly increased the TM protein expression in HUVECs(P<0.05).3 RT-PCR detected the effect of rosuvastatin on the m RNA expression of TM in HUVECs induced by tar and nicotineCompared to control group, ROS group significantly increased the TM m RNA expression in HUVECs(P<0.05). Compared to tar group, ROS+tar group significantly increased the TM m RNA expression in HUVECs(P<0.05). Compared to nico group, ROS+nico group significantly increased the TM m RNA expression in HUVECs(P<0.05).4 TM activity test detected the effects of tar and nicotine on the TM activity in HUVECs and the intervention by rosuvastatinCompared to contral group, tar group significant decreased A value(P<0.05), but ROS+tar group significant increased A value compared to tar group(P<0.05). nico group was no significant difference compared to control group, but ROS+nico group and ROS group increased A value(P<0.05).Conclusion:1 Rosuvastatin could improve the proliferation of HUVECs induced by tar, however rosuvastatin was no effect on the proliferation of HUVECs.2 Rosuvastatin increased the expression of TM in HUVECs. And it could increase the expression of TM on the basis of the increasing TM expression induced by tar.3. Tar significantly decreased the TM activity, rosuvastatin could improve this effect. And rosuvastatin itself also increased the TM activity. Nicotine was no effect on the TM activity. Part III: The effects of tar and nicotine on the interaction between TMand thrombin by live-cell single-molecule force spectroscopy andthe intervention by rosuvastatinObjective: To study the effects of tar and nicotine on the single-molecule interactional force between TM and thrombin by live-cell single-molecule force spectroscopy and the intervention by rosuvastatin.Methods:1 COS-7 cells were transfected with the recombinant plasmid TM-GFP. The transfected COS-7 cells were grouped(1) blank control group(2) control group(3) tar group(4) nico group(5) ROS+tar group(6) ROS+nico group(7) ROS group. Force measurements with the thrombin modified AFM tips on the living cell surface were carried out on Pico SPM II with a Pico-Scan 5500 controller and a larger scanner. The force curves measured in living cells were recorded by Pico Scan software and analyzed by MATLAB R2009 a Metlab. Drawn affinity between TM and thrombin of the Gaussian probability distribution and binding probability were analyzed by Origin8.0 software.2 Flow cytometric analysis technique was used to detect tar, nicotine and rosuvastatin on the effect of the expression TM-GFP in COS-7 cellsTransfected COS-7 cells seeded in 6-well plates, incubated respectively with 50mg/L tar, 10-3mol/L nicotine, 50umol/L ROS+50mg/L tar, 50umol/L ROS+10-3mol/L nicotine, 50umol/L ROS for 1h. Flow cytometric analysis technique was used for detecting TM-GFP protein.Results:1 AFM detected the effects of tar and nicotine on the interaction between TM and thrombin and the intervention by rosuvastatinCompared to control group, tar group significantly decreased the binding probability between TM and thrombin(P<0.05). Compared to tar group, ROS+tar group significantly increased the binding probability(P<0.05). Also ROS+tar group significantly increased the binding force compared to control group(P<0.05). However nico group was no significant difference of the binding probability and the binding force compared to control group. ROS+nico group was no significant difference of the binding probability compared to nico group, but increased the binding force. And ROS group was also no significant difference of the binding probability compared to control group, but increased the binding force.2 Flow cytometric analysis technique detected the effects of tar, nicotine and rosuvastatin on the protein expression of TM-GFP in COS-7 cells.Compared to control group, tar group and ROS+tar group significantly increased the TM-GFP protein expression in COS-7 cells(P<0.05), other groups were no significant difference.Conclusion:1 Tar significantly decreased the binding probability between TM and thrombin, rosuvastatin could improve this effect. But rosuvastatin itself was no effect on the binding probability. Nicotine was no effect on the binding probability and the binding force. After joining rosuvastatin, all could increase the binding force between TM and thrombin.2 Tar significantly increased the TM-GFP protein expression in COS-7 cells. Nicotine and rosuvastatin were no effect on the TM-GFP protein expression in COS-7 cells.