Protective Effects Of Sodium Tanshinone ?A Sulfonate On X-ray-induced Cardiac Fibroblasts Injury And Its Mechanism In Vivo

Objective The purpose of this study is to explore the effects of X-ray in cardiac fibroblasts(CFs),and whether sodium tanshinone ?A sulfonate(STS)has a role in the above process and its possible molecular mechanism.Methods CFs were extracted from Sprague-Dawley(SD)rat(1~3 days old)using differential centrifugation and identified by immunofluorescence.The cells were cultured in DMEM medium containing 10% fetal bovine serum.After the cells were randomly grouped,they were respectively subjected to X-ray irradiation treatment of 0.5Gy,1Gy,2Gy,and 4Gy.The clonal formation experiment was used to determine the number of cell clones and determine the radiation dose of cell damage model caused by X-rays.The cells were pretreated with different STS concentrations(2.5,5,10,20?g/mL).The proliferation effect of STS on CFs was analyzed by MTT assay.The leakage of lactate dehydrogenase(LDH)determined the damage of X-rays to CFs and the protective of STS to CFs.Determine cell grouping: control group(0+0);4+0 group;4+2.5 group;4+5 group;4+10 group;4+20 group.The reactive oxygen species(ROS)was determined by flow cytometry.And the activity of superoxide dismutase(SOD)and the level of malondialdehyde(MDA)were determined by microplate method to determine the oxidation state of cells.The enzyme linked immunosorbent assay(ELISA)was used to measure the levels of B-type natriuretic peptide(BNP),angiotensin II(Ang II)and vascular endothelial growth factor(VEGF)in cell supernatant.Cell cycle and apoptosis were detected by flow cytometry.Western blot was used to detect the expression levels of apoptosis-related proteins and p38 mitogen-activated protein kinase(MAPK)signal pathway-related proteins.Results1.Identification of cell purity: CFs was identified by immunofluorescence and vimentin antigen expression was positive with cell purity of 97.5%±1.3%.2.Cell colony formation assay: Compared with the control group,the clone formation rate after irradiation was significantly reduced in a dose-dependent manner(P < 0.05);The number of cell-forming clones was the lowest after 4Gy irradiation(P < 0.05).3.Cell viability detection: MTT assay results showed that compared with the control group,the cell viability of STS pretreatment 24 h,48h(2.5,5,10,20?g/mL)is increased(P < 0.05),and the cell viability increased most significantly at 48 h,20?g/mL after STS pretreatment(P < 0.05).The measurement results of LDH showed that with the increase of radiation dose,the leakage of LDH increased in a dose-dependent manner(P < 0.05).Compared with 4+0 radiation group,LDH content in STS pretreatment group(2.5,5,10,20?g/mL)was significantly reduced(P < 0.05).Compared with the 0+0 group,there was no statistically significant difference in LDH content after STS pretreatment(P > 0.05).4.Detection of oxidative damage: Compared with 0+0 group,SOD content in 4+0 group decreased significantly and ROS positive rate increased significantly(P < 0.05).Compared with 4+0 group,SOD increased and ROS positive rate decreased in STS pretreatment group(4+5,4+10,4+20)(P < 0.05).Compared with the 0+0 group,MDA content decreased after 4Gy X-ray irradiation and MDA content decreased after STS pretreatment,but the difference is not statistically significant(P > 0.05).5.Detection of cell secretion levels: Compared with 0+0 group,the levels of Ang II and BNP in group 4+0 increased(P < 0.05).After STS pretreatment,the levels of Ang II decreased in a dose-dependent manner(P < 0.05).The levels of BNP decreased from group 4+5 to group 4+20(P < 0.05).There was no significant difference in VEGF level between X-ray and STS intervention(P > 0.05).6.Detection of apoptosis and cell cycle by flow cytometry: Compared with 0+0 group,4+0 group had S phase cell cycle arrest.Compared with 4+0 group,the proportion of S-phase cells in STS pretreatment group decreased,with significant differences among 4+5 group,4+0 group and 4+0 group(P < 0.05).Compared with 0+0 group,the apoptosis in 4+0 group increased,and the apoptosis rate decreased significantly after STS pretreatment.There were significant differences among 4+5 group,4+10 group and 4+20 group(P < 0.05),while there was no significant difference between 4+0 group and 0+20 group(P > 0.05).7.Western blot was used to detect the expression of related proteins: Compared with 0+0 group,X-rays induced p38,B cell lymphoma/leukemia-2 associated X protein(Bax),caspase-3,expression increased and B cell lymphoma/leukmia-2(Bcl-2)expression decreased,and phosphorylated extracellular signal-regulated kinase 1/2(p-ERK 1/2)protein expression was significantly down-regulated(P < 0.05);Compared with 4+0 group,the relative expression of caspase-3,Bax/Bcl-2,p-p38 protein was significantly decreased and the expression of pERK1/2 protein was significantly increased after STS pretreatment(P < 0.05).Conclusion X-ray can induce CFs damage,while STS can protect cells from X-ray damage.X-ray increased oxidative stress and apoptosis of CFs,and changed cell cycle.The potential mechanism may be related to the levels of Ang II and BNP,activation of p38 MAPK signaling pathway and increased expression of apoptosis-related proteins.STS alleviated X-ray damage to CFs by inhibiting the above molecular changes,and CFs may be an important target of cardiovascular drug polymorphism,the specific mechanism of which needs further study.