Perivascular Adipose Tissue In Role Of Nicotine Induced Endothelial Cell Inflammatory Response

Objective:Smoking is one of the most important factor causes atherosclerosis, and the nicotine in the smoke is considered the main reason that lead to Cardiovascular damage. Past research mainly focused on the causing of atherosclerosis through direct damage to endothelium by smoking. In recent years, many experiments prove that PVAT can secrete many types of cells thus participate in the internal environment adjustment between PVAT and vascular. Therefore hypothesis arises: other than the direct damage to vascular endothelium caused by nicotine, there may be changes happens on the endocrine and paracrine of PVAT after stimulated by nicotine that lead to the abnormal secretion of cytokines and inflammatory cytokine, which influences the structure and function of blood vessel wall itself and ultimately causes the intimal hyperplasia. For this reason, we adopted experiment in vitro and use nicotine to stimulate adipocyte and endothelial cells, and construct co-culture system of adipocyte and endothelial cells, to discover the influences of nicotine has on adipocyte, endothelial cells, and the cell and proinflammatory cytokinein the co-culture system, and study the possibility of whether nicotine can stimulate NF-k B to support the expression of the co-developing.Method:we foster the adipocyte individually, diluting nicotine to 10-6 mol/l,10-7 mol/l,10-8 mol/l, using these three kinds of nicotine to dispose the cell for 24 hours and extract supernatant, and testing the secretion of IL-1β, IL-6, TNF-α and adiponectin by ELISA. Use the same kind of nicotine to dispose the cell for 24 hours and extract supernatant, and testing the secretion of IL-1β, IL-6, TNF-α and adiponectin by ELISA. Finally,after using transwell to contruct the co-culture system, we adopt 10-6 mol/l nicotine to dispose co-culture system.Testing the phosphorylation of NF-k B-p65 and the expression level of VCAM-1,ICAM-1 by western-blot method. Then, after 1 hour of dispose of PDTC by NF-k B, we test the secretion of IL-1β, IL-6, TNF-α and adiponectin by ELISA and VCAM-1,ICAM-1 expression level by western-blot.Result:After the foster of 3T3-L1 as well as it induced differentiation to mature adipocyte is succeed, and the adipocyte was stimulated by nicotine alone, the expression of adiponectin is significantly lowered(P<0.05)with the increasing of nicotine. The expression of IL-1β and IL-6 increased obviously(P<0.05) with 10-6、10-7mol/l nicotine, and with no changes occurs compare to the control group(P>0.05) with 10-8 mol/l nicotine. With 10-7、10-8 mol/l nicotine, the expression of TNF-α remains the same with control group(P>0.05), while it increases significately compare with control groupwith 10-6mol/l nicotine. When the endothelial cells is stimulated by nicotine alone, the expression of IL-1β and IL-6 increased(P<0.05)with the increasing of nicotine, while the expression of TNF-α remain unchanged. When the co-culture system is stimulated by nicotine, the expression of IL-1β and IL-6 has a major increase(P<0.05)compare to the individually fostered group. Analysis result of western blot shows the expression of NF-k B p65 in the co-culture system is higher than individually fostered endothelial cells(P<0.01), VCAM-1、ICAM-1 is also higher in the co-developing group(P<0.01). After 1 hour dispose of co-culture system by PDTC, the expression of IL-1β、IL-6 and TNF-α increased significantly IL-1β、IL-6 and TNF-α, on the other hand, these numbers decreased in the PDTC co-culture system( P<0.05).The expression of adiponectin rises in the PDTC co-developing group, and it was statistically meaningful. Western blot shows that after the stimulation of nicotine, VCAM-1、ICAM-1 in PDTC co-culture system is lower than pure co-culture system(P<0.01).Conclusion:Nicotine can lead to the abnormal secretion ofadiponectin、IL-1β、IL-6 and TNF-α of mature adipocyte. Nicotine can also cause the abnormal secretion of IL-1β、IL-6 of endothelial cells, while it of TNF-α is unchanged. Nicotine can activate NF-k B to support the abnormal secretion of IL-1β、IL-6 and TNF-α of co-culture system, and the expression of VCAM-1 and ICAM-1.