The Regulation Of Toll-like Receptor 4 Expression Mediate The Effect Of Erythropoietin On Antiinflammatory And Attenuating Myocardial In Rat Heart

Background and Objective: Cardiovascular disease is the first cause of death in the world.Myocardial fibrosis is the pathological basis of a variety of cardiovascular diseases,such as myocardial infarction and hypertension.It is the key pathological features and reasons of myocardial remodeling.Previous studies have confirmed that myocardial protection of erythropoiesis(EPO)were through the inhibition of myocardial fibrosis.However,the specific mechanism is still unclear.The objective of this study is to explore the specific mechanism of EPO against myocardial fibrosis in rat for providing new ideas,new targets and new directions for the prevention and treatment of myocardial remodeling and myocardial fibrosis and providing more theoretical basis for the application of EPO in cardiovascular disease.Methods: Thirty of 250-300 g healthy adult male SD rats were divided into five groups at random: control,abdominal aorta constriction(AAC),AAC+EPO,AAC+EPO+TLR4 and AAC+EPO+LY294002.Rats in group AAC underwent abdominal aortic banding.AAC+EPO group rats underwent abdominal aortic constriction surgery by intramuscular injection of EPO slow virus infection.In group AAC+EPO+TLR4 rats,the EPO and TLR4 were injected into the abdominal aorta after abdominal aortic banding.In group AAC+EPO+LY294002,EPO was injected into the myocardium after abdominal aortic banding,and the LY294002 was injected into the abdominal cavity for a month.In group Control,the abdominal aorta was not too narrow,but the same incision was performed in the same incision,and the same volume of normal saline was injected into the myocardium.Rats in each group were sacrificed after 8 weeks.Hematoxylin eosin(HE)and masson staining was used to observe the pathological changes of myocardial tissue in rats.QPCR was used to detect the expression level of PI3 K,Akt1 and TLR4 in rat myocardium.Western blotting was used to detect the expression levels of PI3 K,p-Akt,TLR4,TGF-beta 1,MMP-2,MMP-9 in rat myocardium.Immunofluorescence was used to observe the expression levels of,TGF-beta 1,TNF-alpha,MMP-2,MMP-9 and TLR4 in myocardium of rats.Enzyme linked immunosorbent assay was used to detect the changes of biochemical indexes of TGF-beta 1,TNF-alpha,IL-1 beta,IL-6 and IL-17 A in rat serum.Results:(1)HE staining showed that,compared with control group,the diameter of myocardial cells of the AAC group was incresed and myocardial fiber beam was wider,the muscle fiber loose edema,the gap widened.Myocardial injury of AAC+EPO group was lighter when compared with AAC group.Myocardial injury of AAC+EPO+TLR4 AAC+EPO+LY294002 were significantly aggravated when compared with AAC+EPO group.Masson staining showed that,compared with control group,cardiac muscle cell hypertrophy,myocardial fibers arranged in disorder,collagen deposition in AAC groups,and AAC+EPO group of myocardial cells with respect to the AAC group,the myocardial fibers were arranged in order,collagen deposition than the light.AAC+EPO+TLR4 group and AAC+EPO+LY294002 group myocardial fibers arranged disorder obviously,collagen deposition was significantly increased when compared to AAC+EPO group.(2)qPCR results show that compared with the control group,TLR4 protein expression in AAC group was up-regulated,PI3 K,Akt1 expression level was significantly decreased(P < 0.05);AAC+EPO group than in the AAC group,TLR4 protein expression was significantly down regulated,PI3 K,Akt1 expression levels were significantly increased(P<0.05);AAC+EPO+ TLR4 group than in the AAC+EPO group,TLR4 protein expression was increased(P<0.05).AAC+EPO+LY294002 group than in the AAC+EPO group,TLR4 protein expression was up-regulated,PI3 K and Akt1 expression level was significantly decreased(P< 0.05).(3)Western blotting results show that compared with the control group,TLR4 TGF-beta 1,MMP-2,MMP-9 protein expression in AAC group was up-regulated,PI3 K,p-Akt expression level was significantly decreased(P<0.05);AAC+EPO group than in the AAC group,TLR4,TGF-beta 1,MMP-2 and MMP-9 protein expression was significantly down regulated,PI3 K,p-Akt expression levels were significantly increased(P<0.05);AAC+EPO+TLR4 group than in the AAC+EPO group,TLR4,TGF-beta 1,MMP-2 and MMP-9 protein expression was increased(P<0.05).AAC+ EPO+LY294002 group than in the AAC+EPO group,TLR4,TGF-beta 1,MMP-2 and MMP-9 protein expression was up-regulated,PI3 K,p-Akt expression level was significantly decreased(P<0.05).(4)Immunofluorescence staining showed that,compared with control group,TLR4 in AAC group,TGF-beta 1,TNF-alpha,MMP-2,MMP-9 protein expression was significantly increased(P<0.05);AAC+EPO group than in the AAC group,TLR4,TGF-beta 1,TNF-alpha,MMP-2 and MMP-9 protein expression significantly decreased(P<0.05),AAC+ EPO+TLR4 group and AAC+ EPO+LY294002 group compared with the AAC+EPO group TLR4,TGF-beta 1 and TNF-alpha,MMP-2 and MMP-9 protein expression was significantly increased(P<0.05).(5)ELISA results showed that compared with the control group,AAC rats serum TGF-beta 1,TNF-alpha,IL-1,IL-6 and IL-17 and concentration was significantly increased(P<0.05),AAC+EPO group than in the AAC group significantly reduced(P<0.05),and compared with group AAC+EPO,AAC+EPO+ TLR4 group and AAC+EPO+ LY294002 group significantly increased with(P< 0.05).Conclusion:(1)EPO can inhibit myocardial fibrosis and improve myocardial remodeling,which closely related to its function of inhibition the expression of inflammatory cytokines.(2)The role of EPO in anti myocardial fibrosis may be mediated by TLR4.(3)The protective of against myocardial fibrosis of EPO may be through PI3K/Akt signaling pathway down-regulating the expression of TLR4,and then reducing the expression of TGF-beta 1,IL-1,IL-6,IL-17 A and TNF-alpha.