| Osteoprotegerin(OPG) is a kind of member in tumor necrosis factor (TNF) receptor superfamily. The competitive bingding of OPG and receptor activator of nuclear factor-κB ligand(RANKL) blocks the association of RANK with receptor activator of nuclear factor-κB(RANK). As a soluble induced receptor, OPG break the combination of RANKL and RANK and ihibit the physiological effects therefore. OPG/RANKL/RANK system is a major breakthrough in the field of orthopedics in recent years. The studies find that many hormones,cytokines and so on adjust the expression of OPG, RANKL and RANK directly or indirectly. The regulation of the ratio between OPG, RANKL and RANK affects bone metabolism by mediating differentiation and maturation osteoclast (OC). More and more research show that the system of OPG/RANKL/RANK is revolved in the development of cardiovascular disease. It plays an important role in vascular calcification, atherosclerosis and heart failure. The system may provide certain clinical guides of diagnosis, treatment and prognosis in cardiovascular diseases. At present the exact molecular mechanism is not clear. The pathways may include the regulation of vascular calcification and inflammatory factors of endothelial cells and the influence of gene polymorphism,etc. Now the system has been a new hotspot in the research of cardiovascular disease.Chronic heart failure is a set of comprehensive clinical manifestations in the terminal stages of various heart diseases. A large number of research results confirm that myocardial cell apoptosis and ventricular remodeling are the main mechanism of the development of chronic heart failure. Erkol’s research showed that serum OPG levels were closely related to the area of recurrent myocardial infarction after percutaneous coronary intervention, which suggest that the level of OPG may affect the left ventricular remodeling. Another study showed that the OPG levels were higher in patients with acute coronary syndromes and heart failure than those with acute coronary syndromes only because heart failure may induce vascular cells to secrete OPG. It has been found that serum OPG/RANKL ratio is closely related to severity of heart failure, which can be used as a new predictive factor in patients with heart failure. Omland found that high levels of serum OPG were positive related to high quality of ventricle, high thickness of left ventricular wall and low left ventricular ejection fraction(LVEF). After adjusting some common factors of coronary artery disease, the levels of serum OPG were still closely associated with the severity of heart failure. OPG/RANKL/RANK may be involved in the pathological process of heart failure.The cardiovascular research of OPG/RANKL/RANK system are mostly focused on vascular calcification, atherosclerosis and coronary heart disease. The domestic research about the effects of OPG/RANKL/RANK on the mechanism of chronic heart failure is relatively rare. The study is aimed to explore the expression of OPG/RANKL/RANK systemin in myocardial tissues of rats with chronic heart failure and the effect of OPG/RANKL/RANK on myocardial fibrosis and its mechanism, which provide a new approach for clinical evaluation and treatment of chronic heart failure.Chapter one The effects of RANKL with different concentrations on signal mechanism of cardiomyocytes fibrosis induced by LPS in newborn ratsObjective:Using the stimulation of lipopolysaccharide(LPS) to upregulate fibrosis factors in myocardial cells of newborn rats and to study the effects of RANKL with different concentrations on cytokines of Interleukins(ILs), matrix metalloproteinases (MMPs)/tissue inhibitor of matrix metalloproteinases(TIMPs) and transforming growth factor-β1(TGF-β1)/Smad signaling pathways to partly clarify the mechanism of myocardial fibrosis.Methods:Rat myocardial cells in vitro are cultured and identified. After 72 h,the cells were divided into six groups according to different drugs:the control group, LPS group, LPS+RANKL100ng/ml group, LPS+RANKL200ng/ml group and RANKL 200ng/ml group. IL-1β, IL-6, IL-10 and TNF-a are detected by enzyme-linked immunosorbent method. MMP-2, MMP-9 and TIMP-1 are tested by immunohistochemical methods. The mRNA expressions of TGF-β1, Smad2/3 and Smad7 in myocardial tissues were detected by real-time fluorescence quantitative polymerase chain reaction. The protein expressions were tested by western blot.Results:(1) The expressions of IL-1β, IL-6, IL-10 and TNF-a Compared with the control group, the levels of IL-1β, IL-6, IL-10 and TNF-a were increased significantly in LPS group(P<0.05). The levels of IL-1β, IL-6, IL-10 and TNF-a were higher in all groups induced by LPS and RANKL than in LPS group(P<0.05). The levels of IL-1β, IL-6, IL-10 and TNF-a were increased gradually in groups induced by LPS and RANKL with the concentrations of RANKL(P<0.05). There was no statistically significant difference of IL-10 between groups induced by LPS combined with RANKL and LPS group(P>0.05). And there was no statistically significant difference of the levels of IL-1β, IL-6, IL-10 and TNF-a between the control group and RANKL200ng/ml group(P>0.05).(2) The expressions of MMP-2, MMP-9 and TIMP-1 Compared with the control group, the levels of MMP-2, MMP-9 and TIMP-1 were increased significantly in LPS group(P<0.05). The levels of MMP-2, MMP-9 were higher and TIMP-1 level was lower in all groups induced by LPS and RANKL than in LPS group(P<0.05). The levels of MMP-2 and MMP-9 were increased and TIMP-1 level was decreased gradually in groups induced by LPS and RANKL with the concentrations of RANKL(P<0.05). There was no statistically significant difference of the levels of MMP-2, MMP-9 and TIMP-1 between the control group and RANKL200ng/ml group(P>0.05).(3) The mRNA results of TGF-β1, Smad2/3 and Smad7 Compared with the control group, the mRNA expression of TGF-β1,Smad2/3 and Smad7 were increased significantly in LPS group(P<0.05). The mRNA expression of TGF-β1, Smad2/3 were higher in all groups induced by LPS and RANKL with different concentrations than in LPS group(P<0.05). The mRNA expression of TGF-β1 and Smad2/3 were increased gradually in groups induced by LPS and RANKL with the concentrations of RANKL(P<0.05). The mRNA expression of Smad7 were lower in LPS group than in LPS+RANKL200ng/ml group(P<0.05). There was no statistically significant difference of the mRNA expression of TGF-β1 and Smad2/3 between the control group and RANKL 200ng/ml group(P>0.05).(4) The protein expression of TGF-β1, Smad2/3 and Smad7 The protein expression of TGF-β1, Smad2/3 and Smad7 were increased significantly in LPS group(P<0.05). The protein expression of TGF-β1 and Smad2/3 were higher in all groups induced by LPS and RANKL with different concentrations than in LPS group(P<0.05). The protein expression of TGF-β1 and Smad2/3 were increased gradually in groups induced by LPS and RANKL with the concentrations of RANKL(P<0.05). There was no statistically significant difference of Smad7 expression between groups induced by LPS combined with RANKL and LPS group(P>0.05).Conclusions:(1) Lipopolysaccharide LPS (1mg/L) can induce the expression of fibrosis factors in myocardial cells of rats.(2) RANKL could enhance the levels of IL-1β, IL-6 and TNF-a of cardiomyocytes cells induced by LPS in rats, which suggest that RANKL can promote myocardial fibrosis by up-regulating inflammatory cytokines and activating inflammation,(3) RANKL could enhance the levels of MMP-2 and MMP-9 and inhibit TIMP-1 level induced by LPS in cardiomyocytes cells of rats, which suggest that RANKL can promote myocardial fibrosis by up-regulating MMPs/TIMPs which can regulate the extracellular matrix(ECM) and unbalance the prodution and degradation of collagen.(4) RANKL could enhance the expression of TGF-β1 and Smad2/3 induced by LPS in cardiomyocytes cells of rats, which suggest that RANKL can promote cardiomyocytes to produce fibrosis related factors by enhancing the activity of TGF-β1/Smad signaling pathways.Chapter two Effects of metformin on the expression of OPG/RANK/RANKL in myocardial tissues of rats with chronic heart failure and the relevant mechanismObjective:To study the effects of metformin on the expression of OPG/RANK/RANKL in myocardial tissues of rats with chronic heart failure and the relevant mechanism.Methods:Seventy-five male SD rats were randomly divided into sham operation group(S group) and model group. Establish the heart failure models by abdominal aorta coarctation. The rats were randomly divided to S group, untreated group, low-dose metformin group, high-dose metformin group, metformin combined inhibitor group and inhibitor group according to different drugs. Blood sugar, blood lactic and nitric oxide(NO) in blood and cardiac tissues were tested after treatment for 8 weeks. All rats performed echocardiography examination and hemodynamic detection. Myocardial collagen fibers were evaluated through Masson staining and CVF were measured. The contents of HYP and the total collagen in myocardium were tested. The mRNA expressions of OPG, RANKL and RANK in myocardial tissues were detected by real-time fluorescence quantitative polymerase chain reaction. The protein expressions of adenosine monophosphate activated protein kinase(AMPK), P-adenosine monophosphate activated protein kinase(P-AMPK), endothelial nitric oxide synthase(eNOS), p-endothelial nitric oxide synthase(p-eNOS), OPG, RANKL and RANK were tested by western blot.Results:(1) Body weight, heart rate, blood sugar, and blood lactic acid Compared with S group, heart rate were increased in all model groups(P<0.05). There was no statistically significant difference of body weight, blood sugar and blood lactic acid in all model groups(P>0.05).(2) The echocardiogram indexes Compared with S group, LVEDD and LVESD were decreased and LVEF, LVFS were increased significantly in all model groups(P<0.05). LVEDD and LVESD were significantly lower and LVEF, LVFS were higher in groups incubated by metformin with different concentrations than in untreated group(P<0.05). Compared with high-dose metformin group, LVEDD and LVESD were decreased and LVEF, LVFS were increased significantly in metformin combined inhibitor group (P<0.05). There were no statistically significant difference of LVEDD, LVESD, LVEF and LVFS between inhibitor group and untreated group(P>0.05).(3) The hemodynamics indexes Compared with S group, LVEDP were increased and LVESP,+dp/dt and -dp/dt were decreased significantly in all model groups (P<0.05). LVEDP were significantly lower and LVESP,+dp/dt and -dp/dt were higher in groups incubated by metformin with different concentrations than in untreated group(P<0.05). Compared with high-dose metformin group, LVESP, +dp/dt and -dp/dt were decreased and LVEDP were increased significantly in metformin combined inhibitor group(P<0.05). There were no statistically significant difference of LVEDP, LVESP,+dp/dt and -dp/dt between inhibitor group and untreated group(P>0.05).(4) The collagen fiber of myocardial tissue Compared with S group, the degree of myocardial fibrosis was deteriorated in all model groups. Myocardial fibrosis were improved in groups with the treatment of metformin,especially in high-dose metformin group. CVF was higher in all model groups than in S group(P<0.05). CVF were significantly lower in groups incubated by metformin with different concentrations than in untreated group(P<0.05). Compared with high-dose metformin group, CVF were increased significantly in metformin combined inhibitor group (P<0.05). There were no statistically significant difference of CVF between inhibitor group and untreated group(P>0.05).(5) The protein of AMPKa, p-AMPKa, eNOS and p-eNOS There were no statistically significant difference of the protein expression of AMPKa and eNOS between S group and all the model groups(P>0.05). The protein expression of AMPKa and eNOS have no significant difference in S group and untreated group(P >0.05). The protein expression of AMPKa and eNOS were significantly upregulated in groups incubated by metformin with different concentrations than in untreated group(P<0.05). Compared with high-dose metformin group, the protein expression of AMPKa and eNOS were decreased significantly in metformin combined inhibitor group(P< 0.05). The protein expression of AMPKa and eNOS were lower in inhibitor group than in untreated group(P<0.05).(6) NO in blood and cardiac tissues There were no statistically significant difference of NO in blood and cardiac tissues between S group and untreated group(P >0.05). NO in blood and cardiac tissues were significantly increased in groups incubated by metformin with different concentrations than in untreated group(P< 0.05). Compared with high-dose metformin group, NO in blood and cardiac tissues were decreased significantly in metformin combined inhibitor group(P<0.05). NO in blood and cardiac tissues were lower in inhibitor group than in untreated group(P <0.05).(7) The mRNA expression of OPG, RANKL and RANK Compared with S group, the mRNA expression of OPG, RANKL and RANK were increased significantly in all model groups (P<0.05). The mRNA expression of RANKL was significantly lower in groups incubated by metformin with low concentrations than in untreated group(P<0.05). But there were no statistically significant difference of the mRNA expression of OPG and RANK between the two groups(P>0.05). The mRNA expression of RANKL was significantly down-regulated and OPG expression was up-regulated in groups incubated by metformin with high concentrations than in untreated group(P<0.05). Compared with high-dose metformin group, the mRNA expression of RANK and RANKL were significantly increased and OPG expression was decreased in metformin combined inhibitor group(P<0.05). The mRNA expression of OPG was lower and the expression of RANK and RANK was higher in inhibitor group than in untreated group(P<0.05).(8) The protein expression of OPG, RANKL and RANK Compared with S group, the protein expression of OPG, RANKL and RANK were increased significantly in all model groups(P<0.05). The protein expression of RANKL was significantly lower in groups incubated by metformin with low concentrations than in untreated group(P<0.05). There were no statistically significant difference of the protein expression of OPG and RANK between the two groups(P>0.05). But the protein expression of RANKL was significantly down-regulated and OPG expression was up-regulated in groups incubated by metformin with high concentrations than in untreated group(P<0.05). Compared with high-dose metformin group, the protein expression of RANK and RANKL were significantly increased and OPG expression was decreased in metformin combined inhibitor group(P<0.05). The protein expression of OPG was lower and the expression of RANK and RANK were higher in inhibitor group than in untreated group(P<0.05).Conclusions:(1) Metformin could improve cardiac function, reduce myocardial fibrosis and delay the ventricular remodeling.(2) Metformin could improve myocardial fibrosis by up-regulating OPG expression and down-regulating expressions of RANKL and RANK.(3) Metformin could regulate OPG/RANKL/RANK axis by activating the signaling process of AMPK-eNOS-NO. |
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