| Myocardial fibrosis?MF?referred to as diverse pathological factors lead to collagen deposition significant,such as collagen concentration and collagen volume increased and disorders of the normal ratio and arrangement of different types of collagen.The proliferation of myocardial fibroblasts and over deposition of extracellular matrix are the most cause of MF which would lead to heart failure at last.So inhibition of the proliferation of fibroblast and collagen synthesis is the crucial therapy to reduce MF.Cardiac fibroblasts accounted for 25%of the cardiac volume and 70%of the whole cells,and as the most non-cardiomocytes in cardiac which involved in the myocardial fibrosis and ventricular remodeling.As we know,myocardial fibrosis was closely related with ischemic cardiomyopathy,hypertensiveheartdisease,dilatedcardiomyopathy,hypertrophic cardiomyopathy,etc,which is also account for pathology of chronic heart failure?CHF?.According to epidemiological investigation,CHF was one of the most common causes of death and CHF has became a hot point in medical research recently.Nowadays,we've known that the molecular mechanisms of myocardial fibrosis mainly included:1)Extracellular factors;2)Signal transduction pathway of intracellular;3)Materials that caused extracellular matrix deposition or fibroblast proliferation.Adipose tissue,was more than energy storage,but a highly active endocrine organ,which secreted diverse active factors,related with metabolism,differentiation and inflammation,neuroendocrine and cardiovascular diseases.These factors are called as adipose factor,and are rich in blood plasma and they can regulate the organs in the whole-body through different types of secretion,more and more members of adipose are involved in the progress of myocardial remodeling.Chemotaxis,which was found by Goralski in 2007,mainly from white adipose tissue and liver,and had a less expression in lungs,heart,ovaries,kidneys and other organs,played an important role in adipocytes,metabolism and inflammation.As we known the important function of chemotaxis effects on the cells are based on chemokine receptor-CMKLR1 untill now,such as on immune cells,vascular endothelial cells,adipocytes and skeletal muscle cells,to induce immune response,inflammatory response,oxidative stress,autophagy,lipid metabolism and insulin resistance.It was shown that epicardial adipose synthesis of coronary heart disease?CHD?was related with high expression of chemotaxis,which was not only reduced the myocardial contractility,but also lead to remodel by blocking insulin signal pathway to inhibit the metabolism,as well as a variety of inflammatory factors.There were variety pathogenesis of MF and renin-angiotensin-aldosterone system?RAAS?activation was the most important one;further more,aldosterone was an independent risk factor of cardiovascular disease,also more and more evidences showed that cardiac fibroblasts are the most respose cell of aldosterone,and high expression level of aldosterone also an independent predictor of mortality of CHF,which would cause proliferation of cardiac fibroblasts.Rho/ROCK?Rho-associated coiled-coil protein kinase?was the upstream of multiple signal pathway,as a"molecular switch",it could transfer the signal from cytoplasmic into the nucleus,and regulated the gene and protein expression.Some investigations have shown that the expression of Rho/ROCK mRNA increased in CHF and it suggests that Rho/ROCK signal pathway has a crucial role during the progress of CHF.Meanwhile,Mmitogen activated protine kinase?MAPKs?was the common pathway for a variety of extracellular signals to cell proliferation,differentiation and apoptosis,which would also lead to fibroblast proliferation in CHF.Since there was a lack of evidence about chemokines expression and its role in aldosterone induced cardiac fibroblasts in cardiac.In this work we would investigate the change of chemerin during the progress of aldosterone induced cardiac fibroblast and the role of Rho?ROCK?MAPK signal way during this progress.Part one Expression of chemokines in cardiac fibroblasts of ratObjective:To investigate the expression of chemokines and CMKLR1 in cardiac fibroblasts of SD neonatal rat.Methods:1 Using the acute enzyme digestion method to get the single cell of SD rats born within 2-3 days and cultured,cells grew after 23 times of passages was used for experiments.2 The expression of chemokines and CMKLR1 was examinated by RT-PCR,Western-Blot and immunocytochemistry.Results:1 Real-Time PCR results show that positive results of chemokine mRNA in cardiac fibroblasts of SD neonatal rat.2 Western-Blot results show that there is also the protein expression of chemokine and CMKLR1 cardiac fibroblasts of SD neonatal rats.Conclusion:Chemokine and CMKLR1 expressed in cardiac fibroblasts of SD neonatal rats.Conclusion:There is the MRNA and protein expression of chemerin and its receptor CMKLR1 in cardiac fibroblasts of SD neonatal rats.Part two Effect of aldosteron on chemokines in cardiac fibroblasts ofneonatal ratsObjective:To investigate the effect of aldosterone on the expression of chemokines in cardiac fibroblasts of neonatal rats.Methods:1 Using the acute enzyme digestion method to get the single cell of SD rats born within 2-3 days and cultured,cells grew after 23 times of passages was used for experiments.The cells were treated with different concentration of aldosterone for different durations,then the mRNA and protein expression of chemokines was examinated by RT-PCR and Western-Blot.2 The cells were treated with different concentration of aldosterone for different durations,the protein expression of TGF-?,Collagen?and Collagen?were also detected by Western-Blot.Results:1 The protein expression of chemokines,TGF-?,collagen?and collagen?in cardiac fibroblasts increased significantly along with the increasing of aldosterone concentration and durations?P<0.05?.2 The peak value of the mRNA and protein of chemokines was the cells treated with aldosterone 10-7 mo1/L for 24 h.3 The peak value of the protein of TGF-?was the cells treated with aldosterone 10-7 mo1/L for 24 h.4 The protein expression of collagen?and collagen?increased markedly along with the increasing of aldosterone concentration and durations,and the peak value of collagen?/collagen?reached when the cells treated with aldosterone 10-7 mo1/L for 24 h.Conclusion:1 During the progress of aldosteron induced cardiac fibroblast,the mRNA and protein expression of chemokines and TGF-?increased significly with a concentration-and time-dependent manner.2 During the progress of aldosteron induced cardiac fibroblast,aldosteron induced the protein expression of collagen?,collagen?and the ratio of collagen?/collagen?increased significantly.Part 3 Role of Ros/ROCK and MAPK signal pathway in regulating theexpression of chemokines undergoing cardiac fibroblasts induced by aldosteronObjective:To investigate the mechanism of aldosterone induced the expression of chemokines in cardiac fibroblasts through Ros/ROCK and MAPK signal pathway.Methods:1 Using the acute enzyme digestion method to get the single cell of SD rats born within 2-3 days and cultured,cells grew after 23 times of passages was used for experiments.The cells were treated with 10-7 mmol/L aldosteron for 24 h,to investigate the effect of Y27632?the inhibitor of Rho/ROCK signal pathway?on chemerin in control group,aldosterone group(treated with aldosterone 10-7 mmol/L,24h),aldosterone+Y27632 group and Y27632group.2 The cells were treated with 10-7 mmol/L aldosteron for 24 h,o investigate the effect of spironolactone?the inhibitor of aldosteron receptor?on the expression of p-MYPT1 and total MYPT1 in cardiac fibroblasts of control group,aldosterone group(treated with aldosterone 10-7 mmol/L,24h),aldosterone+spironolactone?aldosterone receptor blocker?group and spironolactone group.3 The cells were treated with 10-7 mmol/L aldosteron for 24 h,to investigate the effect of SB203580?p38 MAPK blocker?,PD98059?ERK1/2blocker?and SP600125?JNK blocker?on chemerin in cardiac fibroblast.4 The cells were treated with 10-7 mmol/L aldosteron for 24 h,o investigate the effect of Y27632?the inhibitor of Rho/ROCK signal pathway?on p-JNK/total JNK in control group,aldosterone group(treated with aldosterone 10-7 mmol/L,24h),aldosterone+Y27632 group and Y27632group.Results:1 The expression of chemokine mRNA and protein in cardiac fibroblasts was significantly lower than that in aldosterone group?P<0.05?after intervention of Y27632.Total MYPT1 and total JNK was the same in aldosterone group and control group,while the p-MYPT1 and p-JNK levels were significantly higher,so the ratio of p-MYPT1/total MYPT1,p-JNK/total JNK increased.2 The levels of p-MYPT1/total MYPT1 were significantly decreased after24 hours of treatment with aldosterone receptor antagonist spironolactone?P<0.05?.The expression of p-JNK/total JNK was significantly decreased after Y27632 intervention on cardiac fibroblasts?P<0.05?.3 There was no differences of expression of chemokines between aldosterone+SB203580?p38 MAPK blocker?and aldosterone group;aldosterone+PD98059?ERK1/2 blocker?and aldosterone group,but significant lower in aldosterone+SP600125?JNK blocker?group than aldosterone group?P<0.05?.Conclusion:Rho/ROCK pathway was a key pathway in aldosterone-inducing chemokine increase,which interacted with MAPK pathway,and JNK pathway was the downstream of Rho/ROCK in chemokine expression. |
PDF Full Text Request