| BackgroundIn recent years,with the acceleration of urbanization and population aging in China,the incidence of cardiovascular disease continues to increase,and the incidence and mortality of acute myocardial infarction are much higher than those of 10 years ago.Myocardial fibrosis after myocardial infarction is very important for the prognosis of patients.After the attack of acute myocardial infarction,cardiac fibroblasts proliferate and secrete extracellular matrix under the stimulation of cytokines,replacing necrotic myocardial tissue to maintain the integrity of cardiac structure.the interstitial reactive remodeling occurs in the non-infarcted area due to the change of ventricular wall tension,that is,the deposition of extracellular matrix occurs in the myocardial stroma.However,excessive deposition of extracellular matrix can cause myocardial fibrosis,affect cardiac function,and eventually lead to refractory heart failure.In addition,myocardial fibrosis will also interfere with the normal electrical conduction function of the heart,increase the incidence of arrhythmia,seriously endanger the life safety of patients,and affect the quality of life of patients.Therefore,it is of extraordinary significance to inhibit or reverse myocardial fibers.One of the pathological features of myocardial fibrosis is the net accumulation of extracellular matrix in myocardial interstitium.MMPs/TIMPs plays an important role in extracellular matrix metabolism.MMPs is the main regulator of extracellular matrix,which can preferentially degrade denatured collagen and participate in ECM remodeling.Overexpression of MMPs can also promote the deposition of collagen lacking junction structure in normal cardiac interstitium and aggravate myocardial fibrosis.TIMPs is an endogenous inhibitor of MMPs,which can inhibit the activation of MMPs and bind to the substrate.Usually,both of them are in dynamic equilibrium and play a key role in regulating the metabolism of ECM.Studies have shown that MMP-2 and MMP-9 are the main enzymes for degrading extracellular matrix in myocardial tissue.TIMP-1 and TIMP-2 are endogenous specific inhibitors of MMP-9 and MMP-2 respectively.Therefore,MMP-9/TIMP-1 and MMP-2/TIMP-2 can be used as important indexes to evaluate myocardial fibrosis.As the upstream stimulating factor of MMPs,the expression of CD 147 is significantly up-regulated after myocardial infarction,and induces the secretion of MMP-2 and MMP-9,participates in extracellular matrix remodeling and aggravates myocardial fibrosis.G-CSF is a powerful stem cell mobilization agent,which can mobilize bone marrow mesenchymal stem cells to the ischemic area of myocardial infarction in the early stage of myocardial infarction,inhibit apoptosis,promote angiogenesis,regulate extracellular matrix metabolism and improve myocardial fibrosis.However,there are few studies on whether the inhibitory effect of G-CSF on myocardial fibrosis after myocardial infarction is related to CD 147.Purpose1.To study the effect of G-CSF on myocardial fibrosis after myocardial infarction in rats.2.To study the effect of G-CSF on extracellular matrix metabolism after myocardial infarction in rats.3.To study the effect of G-CSF on CD 147 after myocardial infarction in rats.Cymbals.Methods1.The model of myocardial fibrosis was established by myocardial ischemia induced by coronary artery ligation in SPF male Wistar rats.2.The successfully established rats were randomly divided into model group and treatment group(n=7),and another 7 rats were randomly selected as blank control group.3.24 hours after the establishment of the model,the treatment group was subcutaneously injected with G-CSF(50μg/kg/day).The model group and the blank control group were injected with corresponding volume of normal saline once a day for 5 days.4.Four weeks after the last dose,blood was collected from the abdominal aorta,and serum was collected after centrifugation for Elisa detection;myocardial tissue was cut at the maximum transverse diameter of the heart,fixed in 4%paraformaldehyde,left ventricle was left on the apical side and frozen in a cryopreservation tube,and liquid nitrogen was cryopreserved.5.After 24 hours of myocardial tissue fixation,paraffin sections were prepared for HE and Masson staining.6.The distribution of CD147,MMP-2,MMP-9,TIMP-1 and TIMP-2 was observed by immunohistochemical staining and the average optical density was calculated.7.Determination of P Ⅰ CP and P Ⅲ NP in serum and HYP in myocardial tissue by enzyme linked immunosorbent assay.8.Western Blot was used to detect the expression of type I collagen,III collagen,CD 147,MMP-2,MMP-9,TIMP-1 and TIMP-2 in myocardial tissue.Results1.Morphological changes of myocardial tissue in rats of each group.Compared with the blank control group,the cardiomyocytes in the model group arranged disorderly and contained a large number of inflammatory cells;compared with the model group,the cardiomyocytes in the treatment group arranged relatively regularly and scattered a small number of inflammatory cells.2.Distribution of collagen in rats of each group.Compared with the blank control group,the collagen fiber and CVF increased significantly in the model group,while the collagen fiber and CVF decreased notably in the treatment group compared with the model group(P<0.001).3.Determination of P Ⅰ CP and P Ⅲ NP in serum by Elisa.The serum P Ⅰ CP and P Ⅲ NP of the model group were increased remarkably(P<0.001),while the P Ⅰ CP and P Ⅲ NP of the treatment group were significantly lower than those of the model group(P<0.001).4.Determination of HYP content in myocardial tissue by Elisa.The HYP of myocardial tissue in the model group increased significantly(P<0.001),while the HYP in the treatment group was notably lower than that in the model group(P<0.001).5.Detection of CD 147,MMP-2,MMP-9,TIMP-1 and TIMP-2 protein expression in myocardial tissue by immunohistochemistry and Western Blot.Compared with the blank control group,the protein expression of CD 147,MMP2 and MMP-9 in the model group increased significantly,while the expression of TIMP1 and TIMP-2 decreased significantly(P<0.001).Compared with the model group,the protein expression of CD 147,MMP-2 and MMP-9 in the treatment group decreased significantly,while the expression of TIMP-1 and TIMP-2 increased significantly(P<0.001).6.Detection of type Ⅰ collagen and Ⅲ collagen in myocardial tissue by Western Blot.The expression of collagen type Ⅰ and collagen Ⅲ in the model group was significantly higher than that in the blank control group(P<0.001),and decreased significantly after G-CSF treatment(P<0.001).Conclusion1.G-CSF can inhibit collagen metabolism and effectively improve myocardial fibrosis.2.G-CSF can improve the imbalance of MMP-2/TIMP-2 and MMP-9/TIMP-1,inhibit collagen deposition and improve myocardial fibrosis by down-regulating MMP2 and MMP-9,up-regulating TIMP-1 and TIMP-2.3.The effect of G-CSF on myocardial fibrosis may be related to CD147/MMPs signal pathway. |
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