Study On The Effect And Mechanism Of CCL7’s Regulation Of Myocardial Fibrosis

Background:CCL7 is widely expressed in mononuclear cells, T lymphocytes, dendritic cells and other immune cells. It was researched as inflammatory factor at first, with further study we realized that CCL7 may play significant roles in immune regulation, tissue regeneration, and promoting fibrosis. Our previous study found that CCL7 plays an important role in the process of cardiac fibrosis which is induced by aldosterone, but the intracellular signal pathway is unclear. Therefore study the mechanism and signal pathway of CCL7 in myocardial fibrosis will be very useful to understand the molecular mechanism and treatment of myocardial fibrosis.Objective:To investigate the signal pathway and mechanism of CCL7 induced myocardial fibrosis.Methods:Cardiac fibroblasts were separated from the hearts of 1-3 day’s neonatal SD rats.Cells were divided into control group, CCL7 group, TGF-β₁ group and the combined group. The cells were given 200ng/ml CCL7 and / or 4ng/ml TGF-β₁ factors,respectively. The expressing of COL1A1 gene, COL3A1 gene and CCL7 gene was detected by RT-PCR. The variation of type Ⅰ collagen and type Ⅲ collagen was detected by Western blot. Then the virus carried ERK2 shRNA or Smad3 shRNA were used to knock down ERK2 protein and Smad3 protein in cardiac fibroblasts. Then the cells were treated with CCL7 and / or TGF-β₁ factors before the observation of COL1A1 gene,COL3A1 gene and CCL7 gene expression and the expression of type Ⅰcollagen and type Ⅲ collagen. Then the virus carried ERK1 shRNA and ERK2 shRNA were used to knock down ERK1 protein and ERK2 protein at the same time. Then observe the phosphorylation of Smad2 and Smad3.We use the virus carried Smad2 shRNA and Smad3 shRNA to knock down Smad2 protein and Smad3 protein at the same time. Then observe the phosphorylation of ERK1/2. All date were analyzed by SPSS 20.0.Student’s t-test was used to compare the difference between different groups.P<0.05 was accepted as statistical difference.Results:（1）The COL1A1 gene and COLDA1 gene of cardiac fibroblasts was increased（P<0.05） after using CCL7 factors and / or TGF-β₁ factors, type I and III collagen expression was increased（P<0.05） too,CCL7 gene expression was increased（P<0.05）after using TGF-β₁ factors.（2） The expression of COL1A1 gene and COL3A1 gene of the cells which transfected by virus carried ERK2 shRNA before using CCL7 and / or TGF-β₁ factors was decreased（P<0.05）,but the expression of Type I and III collagen was not increased, the expression of CCL7 was decreased（P<0.05） after use TGF-β₁.（3）The expression of COL1A1 gene and COL3A1 gene of the cells which transfected by virus carried Smad3 shRNA before using CCL7 and / or TGF-β₁ factors was decreased（P< 0.05）,but the expression of Type I and III collagen was not increased, the expression of CCL7 was decreased（P< 0.05） after use TGF-β₁. （4）When knock down ERK1/2 at the same time, the phosphorylation of Smad2 and Smad3 was decreased（P<0.05）.If we knock down Smad2 and Smad3 at the same time, the phosphorylation of ERK1/2 was decreased（P<0.05）,too.Conclusion:ERK2 and Smad3 play important role in the process of CCL7 induced myocardial fibrosis. TGF-β₁ factors induced CCL7 gene increasing though ERK2 and Smad3.There is cross-talk between ERK signal pathway and Smad signal pathway in the process of myocardial fibrosis.CCL7 is an important molecules in the process of TGF-β₁ induced myocardial fibrosis...