| Objective Over-production of radicals lead to increased cardiomyocyte apoptosis,which is the main mechanism of radiation induced heart disease(RIHD).This ultimately results in symptoms of congestive heart failure.The treatment for RIHD is currently mainly based on symptomatic and supportive treatment or through surgical approaches(cardiac transplantation).However,all these methods exhibit little ideal effect,developing new methods is of great significance.Accordingly, the inhibition of oxidative stress is of importance for preventing or suppressing apoptosis in order to protect the heart against radiation injury. In this paper, we therefore characterize the regulatory effects of Sodium tanshinon ⅡA sulfonate(STS) which has anti-oxidant effects against oxidative stress induced by radiation injury and the possible mechanisms in H9c2 cardiomyocytes in vitro,which provides a new insight of STS serving as an anti-RIHD reagent.Methods(1)X-ray induced H9c2 cardiomyocyte apoptosis H9c2 cardiomyocytes were cultured in Dulbecco’s modified Eagle’s medium F12 containing10%fetal bovine serum.They were exposed to different dose of X-ray and divided into five groups:1Control group22 Gy group34Gy group46 Gy group58Gy group.Cytotoxicity of X-ray was assessed by MTT and the activity of lactate dehydrogenase(LDH), superoxide dismutase(SOD) activity and malondialdehyde(MDA) content were determined by colorimetric assay.Hochest33258 staining, clonogenic survival assay and Annexin V/propidium iodide(PI) double-staining assays were used to detect cell apoptosis.Cell cycle distribution was employed to determined cell cycle arrest.Western blot was used to detect the expression of relative protein in the cell.(2)Protective effect of STS on radiation induced myocardial cell injury The cells were cultured with 10%FBS DMEM F12 culture medium, and the proper concentration of STS was determined by MTT method, and then the cells were divided into 5groups: 1 control group(Con): no X- ray irradiation and no STS. 2 2Gy+STS(2STS) group:The cells were pretreated with 10μg/m L STS and then irradiated with 2Gy X- rays.34Gy+STS(4STS) group:The cells were pretreated with 10 μ g/m L STS and then irradiated with 4Gy X- rays.46Gy+STS(6STS)group:The cells were pretreated with 10μg/m L STS and then irradiated with 6Gy X- rays. 5 8Gy+STS(8STS)group:The cells were pretreated with 10μg/m L STS and then irradiated with 8Gy X- rays.Cytotoxicity of X-ray was assessed by MTT and the activity of lactate dehydrogenase(LDH), superoxide dismutase(SOD)activity and malondialdehyde(MDA) content were determined by colorimetric assay.Hochest33258 staining,clonogenic survival assay and Annexin V/propidium iodide(PI)double-staining assays were used to detect cell apoptosis.Cell cycle distribution was employed to determined cell cycle arrest.Western blot was used to detect the expression of relative protein in the cell.Results(1)Observation of cell morphology by inverted phase contrast microscope:Compared with the control group, the cells were changed into round or malformation,and the number of H9C2 cells exposed to X-ray radiation was decreased in a dose-dependent manner. Compared with the relative single irradiation group, the number of cells in each group was significantly increased by STS pretreatment.Compared with the control group, the number of cells in STS pretreatment group were decreased, among which the number of 6STS and 8STS cells decreased significantly.(2)MTT, LDH cell viability assay:Compared with Con, the cell survival rate of simple irradiation group(4Gy, 6Gy, 8Gy) was significantly lower(P < 0.05), and the content of LDH was higher(P < 0.05). Compared with the relative single irradiation group, in STS pretreatment group(4STS, 6STS, 8STS) cell survival rate increased(P < 0.05),and LDH content was lower(P < 0.05).Compared with con group,in STS groups cell survival rate were decreased and LDH levels were increased,among which 6STS and 8STS group were changed statistically(P < 0.05).(3)Antioxidant and lipid peroxide assay:Compared with Con, the activities of SOD in single radiation groups were significantly decreased, and the contents of MDA were significantly increased(P < 0.05).Compared with the single irradiation group, in STS pretreatment groups SOD were increased,and the levels of MDA were decreased(P< 0.05).Compared with Con, in STS group(4STS,6STS, 8STS) SOD contents were decreased,and MDA levels were increased(P < 0.05).(4)The clonogenic survival assay :Compared with Con, preincubation without STS exposed to various dose(2,4,6,8Gy)of radiation significantly reduces the number of colonies by61%,25%,17%, 6% respectively.Compared with the single irradiation group,the results show that preincubation with STS attenuates the cell apoptic effects of IR and especially the number of clonies in the 2STS group nearly restored to the control level. Compared with Con,in STS groups except 2STS group,the number of clonies were decreased statistically(P< 0.05).(5)Morphological detection of apoptosis in H9c2 cells by Hochest33258:Compared with Con,chromatin condensation, indicative of apoptosis, was examined using the Hochest staining method in single radiation groups. Typical apoptotic cells with fragmented chromatin,chromatin condensation or apoptotic bodies in 8Gy group were observed after X-ray radiation by Hochest 33258 staining.Hochest 33258 staining demonstrated that the number of chromatin condensation and apoptotic bodies pretreatment with STS exposed to radiation decreased.Compared with Con, the number of chromatin condensation and apoptotic bodies pretreatment with STS were increased.(6)Cell apoptosis and cell cycle were detected by flow cytometry:Compared with the Con group, the number of apoptotic cells(4Gy, 6Gy, 8Gy) increased gradually, the proportion of G1 phase cells increased significantly, and the proportion of S cells was significantly decreased(P < 0.05).Compared with the simple irradiation group, the number of apoptosis in STS pretreatment group was decreased, the proportion of G1 phase cells were decreased, and the proportion of S phase cells were increased(P < 0.05).Compared with Con, the apoptosis of STS pretreatment group(6STS, 8STS) increased, the proportion of G1 phase cells increased, and the proportion of S phase cells decreased(P < 0.05).(7)The expression of apoptosis-related proteins and P38-MAPK signal transduction pathway related protein :Bax/ Bcl-2 ratio increased markedly,and expressions of Caspase-3 also increased in the single radiation group when compared with the control group; however, it was reversed by pretreatment with STS:expressions of Caspase-3 decreased markedly and ratio of Bax / Bcl-2 also decreased.To further assess the signal pathways potentially involved in the cardiomyocyte apoptosis process induced by radiation, we examined key components of the MAPK pathways in the single radiation groups.There was marked decrease of P-ERK and P-P38 MAPK in the single radiation group compared to control group;however,STS pretreatment significantly mitigated p38 MAPK activation.Conclusions: X-ray could inhibit H9c2 growth by oxidative stress and induced apoptosis in H9c2 cell. STS could protect the cells from inhibition induced by X-ray via regulating cell cycle distribution, depressing oxidative stress level and apoptosis related gene products.Therefore, we suggested the STS could be useful for the preventment and treatment of radiation induced cardiovascular disease in future. |
PDF Full Text Request