Effect And Mechanism Of Tranilast On Cardiac Fibroblasts Proliferation And Phenotype Transformation Incubated With High Concentration Of Glucose

ObjectiveTo investigate the effect of tranilast on cardiac fibroblasts proliferation and phenotype transformation incubated with high concentration of glucose and the possible mechanism.MethodsThe heart of 1³ day-old Sprague-Dawley（SD）rats were collected under sterile conditions,then cut into pieces,digested it.Primary CFs were cultured in DMEM with 10% fetal bovine serum（FBS）,after 3 day in culture,the CFs were digested and passage with 1:2,use the 2³ generation in the experiment.Then the cardiac fibroblasts were divided into seven groups in accordance with different nutrient solutions: Normal control group（5.5 mmol·L-1 glucose）,hypertonic group（5.5 mmol·L-1 glucose+mannitol 25 mmol·L-1）,high glucose group（25 mmol·L-1 glucose）,tranilast intervention groups（25 mmol·L-1 glucose + tranilast 50 μmol·L-1,100 μmol·L-1,200 μmol·L-1）,ALK7 inhibitor group（25 mmol·L-1 glucose+10 μmol·L-1 SB431542）.The cell morphology was observed under inverted microscope combined with HE staining,then identify the cell by immunofluorescence staining.The cell proliferation was detected by MTT method.The transformation of cardiac fibroblasts was determined by immunfluorescence staining.The expression of FSP-1,α-SMA,TGF-β₁ and ALK7 were assessed by western blotting.Results 1、 Morphological observation and immunofluorescence staining showed: spindle-shaped cells,the nucleus is relatively large,each cell within 1³ nucleus,CFs with HE staining showed clearly visible fibroblast morphology;FSP-1 immune response was positive and α-SMA was negative,confirmed it was CFs.2、 MTT assay showed: Compared with normal control group,the cell proliferation in high glucose group was significantly facilitated（P<0.01）;Compared with high glucose group,the cell proliferation were decreased（P<0.05）in tranilast or SB431542 intervention groups（P<0.05）,then concentration-dependent.There were no difference between normal control group and hypertonic group.3、 Immunfluorescence staining showed: Compared with normal control group,the expression of a-SMA in high glucose group were significantly increased,but FSP-1 protein was significantly decreased;Compared with high glucose group,the expression of a-SMA protein was significantly decreased,but FSP-1 protein was increased in tranilast or SB431542 intervention groups.There were no difference between normal control group and hypertonic group.4、 Western results showed: Compared with normal control group,the expression of a-SMA,TGF-β₁ and ALK7 protein in high glucose group were significantly increased（P<0.01）,but FSP-1 protein was significantly decreased（P<0.01）.There were no difference between normal control group and hypertonic group.Compared with high glucose group,the expression of a-SMA,TGF-β₁ and ALK7 protein were significantly decreased（P<0.05）,but FSP-1 protein was increased（P<0.05）in tranilast or SB431542 intervention groups.ConclusionsThe cardiac fibroblasts proliferation and phenotype transformation can be induced by high glucose,in which TGF-β₁、ALK7 may participate in.Tranilast can inhibit the proliferation and phenotype transformation of cardiac fibroblasts induced by high glucose,which may be related to down-regulating the expression of TGF-β₁、ALK7.