Effect Of TFPIct32 On Progress Of Atherosclerotic Lesions In ApoE^-/-Mice And Its Mechanism

Experimental background:Atherosclerosis is a chronic inflammatory disease that occurs mainly in large and medium-sized arteries.It is the main pathogenesis of cardiovascular and cerebrovascular diseases,which seriously affects human health and quality of life.A variety of cells involves in the progresses of atherosclerosis,among which macrophages are one of the key factors.Monocytes were accumulated under the damaged blood vessel wall and activated into macrophages which secrete a series of inflammatory factors,chemokines,to promote inflammation and accumulation of inflammatory cells.A number of studies have reported that tissue factor pathway inhibitor?TFPI?is a physiological inhibitor of tissue factor?TF?,which not only inhibits TF activity,but also prevents the infiltration of inflammatory cells in atherosclerosis lesions,thereby inhibiting the development of atherothrombosis.In addition,TFPI has a unique antithrombotic effect.Therefore,TFPI may be an ideal factor to promote atherogenesis regression.The C-terminus of TFPI has a small fragment consisting of 32 amino acid sequences?TFPIct32?with TFPI-like attenuation of atherogenesis,but the attenuate effects of atherosclerosis and the involving mechanisms remains to be further studied.Purpose:By constructing an atherosclerosis model,the effect of TFPI on ApoE^-/-mice was observed,and its mechanism of plaque regression was preliminarily explored.Method:Forty ApoE^-/-mice were randomly divided into 4 groups?n=10?:10 weeks group,control group,negative control peptide group,TFPIct32group.The mice in the model group were sacrificed after 10 weeks of high-fat diet.The mice in the negative control peptide group received a high-fat diet for 10 weeks.The TFPIs group received a 4-week TFPI scrambled amino acid sequence peptide?TFPIs?at a dose of 2.5nmol/g/week after 10 weeks of high-fat diet administration.After 10weeks of high fat diet in TFPIct32 group,TFPIct32 was injected through the tail vein for 4 weeks at a dose of 2.5 nmol/g/week.The control group was given a high-fat diet for 10 weeks and then injected with a 4-week equivalent volume of normal saline via the tail vein.The mice were fed on a high-fat diet while injecting the drug.At the end of the experiment,the tail bleeding time?BT?was measured and sacrificed.Blood was taken from the eyeball and serum was collected after centrifugation.Serum TC,TG,LDL-C and HDL-C were detected by automatic biochemical analyzer.The concentrations of serum TAT,D-D,TNF?,IL-1?and IL-6were measured by enzyme-linked immunosorbent assay.Oil red O staining was performed on the mouse aorta to observe atherosclerotic lesions of the aortic intima.The frozen heart sections of the aortic sinus tissue were made in the mouse heart,and atherosclerosis lesions were observed by HE,Masson,and oil red O staining.The expression of macrophage markers?CD68?in aortic sinus was detected by immunofluorescence staining.The expressions of UNC5B,CCR7,ICAM-1 and VCAM-1 were detected by immunofluorescence staining.Western blot was used to detect the expression of UNC5B,CCR7,ICAM-1 and VCAM-1 proteins in mouse aortic tissues.Experimental results:1.Compared with the model group,the TC levels in the control group,TFPIs group and TFPIct32 group increased by 27.07%?P<0.05?,17.18%?P<0.05?,and 6.14%,respectively.The TG levels increased by 24.03%?P<0.05?,28.02%?P<0.05?and 17.37%,respectively;HDL-C levels decreased by 24.75%?P<0.05?,16.79%?P<0.05?and 19.20%?P<0.01?,respectively.Compared with the model group,The LDL-C level in the TFPIct32 group decreased by 5.92%.The level of LDL-C in the TFPIct32 group was significantly decreased about 15.6%compared with the control group?P<0.05?.Compared with the TFPIs group,the LDL-C level in the TFPIct32 group decreased by 18.14%?P<0.05?.2.There were no significant changes in BT,TAT and D-D in each group of mice.3.Compared with the model group,the area of aortic plaque in the control group and the TFPIs group increased by 118.53%?P<0.01?and126.28%?P<0.01?,respectively,the area of aortic sinus plaque increased by 36.84%?P<0.01?,33.38%?P<0.05?,and the area of aortic plaque in the TFPIct32 group increased by 25.34%?P<0.05?.Compared with the control group and TFPIs group,the area of aortic plaque in the TFPIct32group was decreased by 42.65%?P<0.01?and 44.61%?P<0.01?,respectively,and the area of aortic sinus plaque decreased by 28.83%?P<0.01?and 26.98%?P<0.01?.Compared with the model group,These indicate that TFPIct32 can inhibit ApoE^-/-mouse aortic sinus atherogenesis plaque.4.The immunofluorescence results of frozen sections of mouse aortic sinus suggested that the expression of macrophage marker CD68 was visibly decreased and the number of macrophages in plaques was decreased in TFPIct32 group compared with the control group.5.The TFPIct32 group was compared to the other three groups,the expression of CCR7 was evidently increased and the expression of UNC5B was significantly decreased in TFPIct32 mice,indicating that TFPIct32 can promote macrophage emigration in plaque.6.Compared with the model group,the levels of TNF?,IL-1?and IL-6 in the TFPIct32 group ApoE^-/-mice decreased by 35.13%?P<0.01?,35.30%?P<0.05?,and 55.29%?P<0.01?,respectively.Compared with the control group,the levels of TNF?,IL-1?and IL-6 in the TFPIct32 group ApoE^-/-mice decreased by 30.34%?P<0.01?,29.43%?P<0.05?,57.41?P<0.01?,respectively.compared with the TFPIs group,the levels of TNF?,IL-1?and IL-6 in the TFPIct32 group ApoE^-/-mice decreased by 27.74%?P<0.01?and 34.36%?P<0.01?,62.51%?P<0.01?.These suggest that TFPIct32 can inhibit inflammation in ApoE^-/-mice.Compared with the model group,the expression of ICAM-1 and VCAM-1 in the aortic sinus and aorta of the control group increased.Compared with the other three groups,the expression of ICAM-1 and VCAM-1 in the aortic sinus and aorta decreased significantly in the TFPIct32 group,indicating that TFPIct32 inhibited monocyte aggregation.Conclusion:TFPIct32 has a prevention effect on ApoE^-/-mouse atherosclerosis plaques,and its mechanism may be related to the increase of macrophage emigration and inhibition of monocyte recruitment.