Effects Of Endurance Training On Inflammatory Factor Expression And Therapeutic Mechanism Of Atherosclerosis In ApoE^-/- Mice

Atherosclerosis is one of the serious diseases.At present,Atherosclerosis is a major cause of cardiovascular and cerebrovascular diseases and is responsible for a large proportion of incidence and mortality.Many researches focus on the mechanism and therapy of atherosclerosis.Atherosclerosis is associated with inheritance and environment.The recent studies indicated that the injury of endothelial cells is one of the key pathomechanisms of atherosclerosis.Exercise is a deterrent of atherosclerosis,and its antiatherogenic effects have been described in different studies.However,the studies focused on apparatus and body.Data from large population-based studies indicated that regular physical exercise reduced the incidence of atherosclerosis.However,the mechanism(s)by which exercise might benefit atherosclerosis remains unknown.At present,there were no reports on the mechanism of inflammatory response by which exercise prevents/treated atherosclerosis.Objective:Our experiment was designed to study the role of the expression of the transcription factor nuclear factor(NF-κB)and its proinflammatory genes linked to NF-κB in atherosclerosis during exercise.We used the apolipoprotein E-knockout(ApoE^-/-)mice,a particularly useful model for analyzing the pathomechanisms of atherosclerosis in vivo,to study the pathomechanisms of atherosclersis associated with the major atherosclerotic factors regulated by NF-κB and how they may affect different steps in the atherosclerotic process during exercise training by the methods of pathology,biochemistry and molecular biology.Methods:Eight-week-old ApoE-knockout mice(n=48)were fed western food,and were randomly divided into precautionary groups and therapeutic groups which include fourteen-week western food diet group(H),fourteen-week exercised group(HE), twenty-six-week western diet group(HH),fourteen-week western food diet and twelve-week exercised group(H+E).Every group has twelve mice,all males.Eight-week-old C₅₇BL/6J(n=24)were randomly divided two groups which include fourteen-week sedentary group(N₁,male:n=12)and twenty-six-week sedentary group(N₂,male:n=12).HE and H+E groups were trained on a treadmill for 60 minutes per day,5 days per week(at 13 m/min,). Fourteen-week group was used to observe the preventing effect of endurance training on atherosclerosis.Twenty-six-week group was used to observe the therapeutic effect of endurance training on formed atherosclerosis.The end of every periods,the mouse blood was drawn from the eye for assay of the levels of serum lipids and OX-LDL.The aortic trunk was perfused with formalin,then removed heart and 1-1.5cm aortic trunk closed heart for frozen section.The aortic trunk was cut consecutively from aortic valve(cross-section thickness,7μm).Aortic sections were saved for oil red O and H.E dyeing and hybridizing in vivo and immunohistochemical analysis.Blood sampling assays for the levels of serum lipids.OX-LDL level was measured by ELISA.The plaque areas of aortas which were dyed by oil red O were quantified by using computor imaging system.The aortic sections which were dyed by H.E were analysised pathologic change by microscope,and measured the expression of the transcription factor nuclear factor(NF-κB)by hybridizing in vivo,and measured the protein content of NF-κB and its proinflammatory proteins linked to NF-κB by Immunohistochemistry.Results:1,The ApoE^-/-mouse growth and weight change.The weight of all the mice increased gradually.The exercised groups adapted exercise load well.2,Effect of endurance training on the formation of atherosclerosis and the pathologic change of aortic vessel wall in ApoE^-/-mice.1)Effect of endurance training on the formation of the aortic atherosclerosis in ApoE^-/- miceA lot of atheromatous plaques of aortas appeared in H group(no lesions were observed in control).The degree of atheromatous plaque progression in HE group was significantly lower than H group(the area of proportion of atheromatous plaques:25.07±7.04%vs41.79±6.93%, P＜0.01).There were a lot of fatty streaks-fibrous plaques and a few atheromatous plaques appeared on aortas(dyed by H.E)in H group,it showed that ApoE^-/-mice had been typical atheromatous models.Compared with H group,HE group mainly presented period of fatty streak,partly fibrous plaques appeared.The results indicated that fourteen endurance training prevented/decreased formation of atherosclerosis.2)Effect of endurance training on the formed atherosclerosis in ApoE^-/-mice.Compared with H group,the larger size of the lesions observed in the HH group(the area of proportion of atheromatous plaques:58.80±6.40%),characterized by the atheromatous plaques or damageable plaques.Compared with H+E group,the area of proportion of atheromatous plaques of H group was significantly lower(41.79±6.93%vs 50.35±3.52%, P＜0.05),while HH group was significantly higer(P＜0.05).The results showed that twelve endurance training prevented/decreased the development of formed atherosclerosis in ApoE^-/- mice.3,Effect of endurance training on the levels of serum lipids in ApoE^-/-mice.The levels of TC,TG,LDL-c increased significantly in ApoE^-/-mice,while HDL-c significantly decreased.There was no effect on serum lipids and lipoproteins in ApoE^-/-mice by endurance training.4,Effect of endurance training on the level of serum OX-LDL in ApoE^-/-mice.The level of serum OX-LDL was higher in ApoE^-/-mice than control.Compared with H group,the level of OX-LDL in HE group decreased significantly(P＜0.05).Compared with HH group,the level of OX-LDL in H+E group decreased significantly(P＜0.05).It showed that there was higher the level of OX-LDL,while endurance training effectively decreased the level of OX-LDL.5,Effect of endurance training on NF-κBmRNA and protein aortic level in ApoE^-/-mice.Compared with control,NF-κBmRNA and protein aortic levels increased significantly in ApoE^-/-mice.Compared with H group,NF-κBmRNA and NF-κBp65 protein aortic levels decreased significantly in HE group(P＜0.01).NF-κBmRNA and NF-κBp65 protein aortic levels in H+E group were lower than in HH group.It showed that NF-κBmRNA and NF-κBp65 protein aortic levels increased in ApoE^-/-mice,endurance training effectively inhibited or decreased them expression.6,Effect of endurance training on the expression of inflammatory factors in aortas in ApoE^-/-mice.Compared with control,the expression of TNF-α,ICAM-1 and MMP-9 in aortas were higher significantly in all ApoE^-/-mice groups,it indicated that there was tight relationship between formation of atherosclerosis and inflammation response.Endurance training effectively decreased the expression of inflammatory factors in aortas in ApoE^-/-mice.7,The correlation between the areas of atheromatous plaques and the level of NF-κBp65 and OX-LDL in ApoE^-/-mice.The experiment showed that the areas of atheromatous plaques positively with the level of NF-κBp65 and OX-LDL in ApoE^-/-mice.Conclusions:1,Fourteen-week endurance training effectively prevented the formation of atherosclerosis.2,Twelve-week endurance training can control/stay the development of the formed atherosclerosis in ApoE^-/-mice.3,The effect of endurance training on potential therapeutic mechanism of atherosclerosis: endurance training decreased the level of OX-LDL and the expression of NF-κB,accordingly down-regulating the expression of inflammatory factors,ICAM-1 and MMP-9 associated with the formation and development of atherosclerosis,thereby decreasing inflammation in atheromatous plaques,probably it was one of the therapeutic mechanisms of endurance training on inhibiting or impairing the formation of atherosclerosis in ApoE^-/-mice.