| BackgroundIn recent years,with the continuous increase of the prevalence rate of cardiovascular disease,the influence of coronary disease,pulmonary heart disease,heart failure and other diseases on the residents can not be ignored,myocardial fibrosis and ventricular remodeling have been paid more and more attention.and not only in heart disease,glucose-related myocardial fibrosis will occur in the diabetes.The pathophysiological character of myocardial fibrosis is excessive deposition of extracellular matrix components in the myocardial interstitium,resulting the weak ventricular wall elasticity,impairment of systolic and diastolic function,and finally heart failure.Extracellular matrix deposition includes collagen proliferation and fibroblast proliferation.In the end stage of cardiovascular disease,abnormal collagen fibers cross-link each other,aggravating myocardial fibrosis.Therefore,preventing and treating the progress of myocardial fibrosis has important clinical significance.The mechanism of myocardial fibrosis is complex.In the study of myocardial fibrosis signal pathway,it is found that TGF-? is the most important molecule in the process of promoting myocardial fibrosis.It is considered to be the switch of tissue transformation from inflammation to scar tissue after myocardial infarction.It stimulates cytokines and signal pathways through Smad protein-dependent pathway and non-Smad protein-dependent pathway.In the TGF-? family,TGF-?1 is found almost ubiquitously.It is a pleiotropic cytokines,and its effection may be different or even opposite even in the same cell.At present,it has been found that there are three informs of TGF-? in mammals.Three informs of TGF-?1,TGF-?2 and TGF-?3 have different functions in vivo.After the binding of TGF-? and TGF?R,it participates in the process of differentiation,apoptosis and inflammation through the cascade of downstream signal molecules.The TGF-? signaling pathway is mainly based on the classical approach of Smad dependence.The activated TGF?R binding to R-Smad and finally translocates to the nucleus to regulate the transcriptional process of targeted genes.In addition,Smad-independent signaling pathways also play a key role in TGF-?response.Non-canonical pathways signaling pathways contain ERK-MAPK pathway,TAK1-mediated JNK/p38MAPK pathway,PI3K/AKT/mTOR pathway and so on.Through cascade regulation after binding to activated TGF-? receptors,these non-canonical signaling pathways participate in T GF-?-mediated epithelial-mesenchymal transition,apoptosis,fibrosis.In addition to mediating the signal pathway,TGF-? also participates in the process of promoting fibrosis by interacting with other cytokines.Therefore,anti-TGF-? treatment has undoubtedly become an important target to prevent myocardial fibrosis.The p38MAPK signal pathway mediated by mitogen-activated protein kinase MAPK has been found to promote the synthesis of collagen fibers in the early stage.Classical MAPK is a three-stage phosphorylation process.After activated TGF-?R activates TAK1,also named MAP3K,downstream MKKs and p38 are activated by a single phosphorylation to regulate the expression of type ? and type ? collagen.Granulocyte colony-stimulating factor(G-CSF)is an effective stem cell mobilization agent.By recruiting bone marrow stem cells from blood and mobilizing to the site of myocardial necrosis,G-CSF can differentiate into vascular endothelial cells,promote angiogenesis,reduce the expression of apoptotic cells,and play a role in protecting the heart in acute myocardial infarction and heart failure.At present,the mechanism of G-CSF mobilization of stem cells in the treatment of myocardial fibrosis after myocardial infarction is mainly focused on Smad-dependent signaling pathways,but it is not clear whether be regulated by Smad-independent pathway.ObjectiveOur Experiment is intended to use Wistar rats as experimental subjects.After the myocardial fibrosis model was established by isoproterenol Iso,G-CSF was injected subcutaneously to observe the effect of mobilization in rats.Detect the expression of TGF-?,TAK1,MKK3 and p38MAPK in myocardial tissue to explore the possible mechanism of G-CSF against myocardial fibrosis.Methods1.22 Wistar rats were subcutaneously injected with Iso 4mg/kg-1d-1 for 10 days to establish the model of myocardial fibrosis.2.20 rats were randomly divided into control group and GT group,G-CSF diluted with normal saline.The mobilization group was subcutaneously injected with G-CSF 50 ?g/kg-1.d-1,for 5 days,and the control group was subcutaneously injected with normal saline.After the last administration,the rats were fed normally for 4 weeks.3.Before mobilization and on the 5th day after mobilization,the number of WBC in peripheral blood was detected after tail vein blood was collected and diluted.4.On the fifth day of mobilization,after collecting blood from tail vein,the blood were centrifuged,and the mononuclear cell layer was absorbed for cell culture and flow identification.5.The rats were performed cardiac ultrasound before they were sacrificed.6.After blood collection from the abdominal aorta,the upper serum was collected by standing and packed for Elisa to detect the content of BNP.7.The rat heart was transected at about 2mm below the maximum transverse diameter,the upper part was fixed in paraformaldehyde,and the lower part was frozen in liquid nitrogen.8.After 24 hours of fixation,myocardial tissue was used for HE/Masson and immune tissue detection.9.Western Blot was used to detect the expression of Col I,TGF-?,TAK1,MKK3 and p38MAPK protein in myocardial tissue.Results1.Iso induced myocardial fibrosis in rats.After continuous subcutaneous injection of Iso for 10 days,myocardial HE staining shows that the myocardial structure of normal rats was normal without collagen hyperplasia,while the myocardial structure of rats injected with Iso was disordered,interstitial collagen fibers proliferated.2.Detection and identification of granulocytes in peripheral blood.G-CSF can mobilize hematopoietic stem cells and bone marrow stem cells,so we can know the situation of mobilization by counting the number of WBC in peripheral blood leukocytes.According to the results of WBC count,the number of peripheral blood granulocytes in the GT group increased significantly before and after mobilization treatment,and there was significant difference between the two groups(P<0.01).On the 5th day after mobilization,the granulocyte count in the GT group was also significantly higher than that in the control group(P<0.01),while there was no significant change in the control group before and after mobilization.After the peripheral blood was isolated and cultured on the 5th day after mobilization,the results of flow cytometry showed that the surface markers of granulocytes were positive for CD29/CD90 and negative for CD45,which proved that after G-CSF the mononuclear cells in peripheral blood were mainly bone marrow stem cells.3.Detection of BNP content in serum by Elisa.After mobilization treatment,the content of serum BNP in the GT group was lower than that in the control group,and the difference between the twogroups was statistically significant(P<0.05).4.Detection of myocardial pathological morphology.Masson staining showed that the collagen deposition of myocardial interstitium in GT group was significantly lower than that in control group,and the CVF value of collagen volume fraction measured by Image-pro software in GT group was lower than that in control group,and there wassignificant difference between the two groups(P<0.05).5.Expression of type ? collagen fibers in myocardium.The immunohistochemical results of myocardial tissue showed that there was obvious positive expression under the endocardium and diffuse proliferation of type ? collagen in the interstitium in the control group,while the positive expression and collagen deposition decreased in the GT group.6.Western Blot was used to detect the expression of Col I,TGF-?,TAK1,MKK3 and p38MAPK protein in myocardial tissue.Compared with the control group,the expression of type ? collagen and fibrosis in GT group decreased,and the expression of TGF-?,TAK1,MKK3 and p38MAPK decreased.The protein bands of the two groups were measured by Image-pro software,and the difference of protein expression between the two groups was statistically significant(P<0.05).Couclusion1.Wistar rats were subcutaneously injected with Iso 4mg/kg-1d-1 for 10 days,Obvious fibrotic changes were observed in rat myocardial tissue.The modeling method was stable and the animal survival rate was high.2.After G-CSF mobilization therapy,the number of stem cells in peripheral blood of rats with myocardial fibrosis increased significantly,and the increased cells were mainly bone marrow stem cells.3.G-CSF mobilization therapy can improve cardiac function,reduce interstitial collagen deposition and reduce the degree of myocardial fibrosis in rats with myocardial fibrosis.4.By detecting the expression of TGF-?,TAK1,MKK3 and p38MAPK in myocardium,it was found that the expression of TGF-?,TAK1,MKK3 and p38MAPK decreased in GT group.It was preliminarily confirmed that the mechanism of G-CSF mobilization of stem cells against myocardial fibrosis was through inhibition of TGF-?-mediated TAKl-MKK3-p38MAPK pathway. |
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