| Objective Ischemic Heart Disease is one of the most devastating diseases in our moden lives. Myocardial anoxia is the basic pathophysiological manifestation in acute stage of the disease. So, improving ischemic and anoxic condition , lessening the damage of myocardial cells may play a very important role in the treatment. Hydroxysafflor yellow A, abstracted from a traditional Chinese drug safflower, is a kind of monomeric compound. Chinese indigenous medicine considered that Safflower might promote blood flow, remove blood stasis and had a function of detumescence. It was used to treating with coronary artery disease, pneumocardial disease and cerebral thrombosis etc. Safflor yellow is abstracted from safflower and it is the water-soluble and effectual composition of safflower. Many researches indicated that Safflor yellow also had a lot of pharmacologic actions, including dilating vascular, improving myocardial blood-supply, inhibiting platelet aggregation and thrombosis and anti-oxidation. HSYA was lately found to be the effectual content of SY. A lot of experiments showed that HSYA resisted free radicals and PAF and protected nerves. But the direct protective study of HSYA to the heart is very scarce. In present studies hypoxia model of cultural myocardial cells was set up ,and then myocardial protections of HSYA and the mechanisms was investigated.Methods Primary cultures of cardiac myocytes in vitro: Cardiac myocytes cultures were prepared from the ventricles of 1-3 d -day-old Wistar rats. The cardiac ventricle were firstly obtained and broken, then Trypsinization was performed with 0.125% trypsin by 4-6 times at 37℃ . Supernatant was took out and DMEM medium supplemented with 20% calf serum was added in. Then they were centrifuged at 1000 r/min for 10 min and cells obtained at the bottom of the centrifuge tube. Cells were then suspended in medium with 20% calf serum .Culture was enriched for myocardial cells by preplating two times to deplete the population of non-myocardial cells. Nonattached cells were then plated at a concentration of 1×105 cells/mL and cultured at 37℃ in 5% CO2. The culture medium was changed after 24h and 0.5μg/ml mitomycin was added and maintained for 24h. The culture medium was changed every 24h.(l)The protection of HSYA to anoxic Cardiac myocytes: Cardiac myocytes were divided into nine groups xontrol ,model,HSYA1×10-6 mol/L-1×10-10mol/L groups and Diltiazem hydrochloride group, 1×10-6 mol/L HSYA on normal cells group. After grouping, the model was established by changing for sugar-free and serum-free medium after 2 hours of normal medium seeding and cells of the model group were transferred to a cuvette for 3 hours, which was filled with high purity Nitrogen gas .In other groups, medium were changed for sugar-free and serum-free medicated medium after 2 hours of medicated normal medium seeding, then cells were also put into the anoxic cuvette for 3 hours. Cells of 1×10-6 mol/L HSYA on normal cells group were treated by normal medium contained HSYA for 5 hours. After this, the morphological changes were observed and the beating frequency was counted. The survival rate of the cells ,contents of lactate dehydrogenase in the culture medium, and malondialdehyde ATPase in the cells were determined too. (2)Mechanisms of the protection: Cardiac myocytes were divided into five groups: control,model,HSYA1×10-7 mol/L group ,1×10-8 mol/L group and Diltiazem hydrochloride group. After grouping, cells of different groups were treated according to (1). Concentration of intracellular Ca2+was determined by fluorescent probeFURA2/AM.The expression of sarcoplasmic reticulum Ca2+-ATPase( calcium pump) and membrane Ca2+-ATPase were also be investigated by RT-PCR method. Results Compared with the model group, cells of 1 × 10-7 mol/L and 1×10-8mol/L HSYA groups still had a rhythmical beat. HSYA at the dose of 1×10-7mol/L and 1×10-8 mol/L caused a significant increase of survival rate, had a notable decrease of lactate dehydrogenase in the culture medium (p<0.05) and a notable increase of ATPase contents in the cells(p<0.01). Contents of malondialdehyde in the cells were significantly decreased (p<0.01). Concentration of intracellular Ca2+ of 1 × 10-7 mol/L and 1 × 10-8mol/L HSYA groups was significantly lowered compared with the model group(p<0.01),and they had no statistical significance with Diltiazem hydrochloride group. The results of RT-PCR showed that HSYA at the dose of 1×10-8mol/L can boost the expression of sarcoplasmic reticulum Ca2+-ATPase (calcium pump) (p<0.01)and membrane Ca2+-ATPase (p<0.05) . Conclusions HSYA at dose of 1×10-7 mol/L and 1×10-8 mol/L significantly protect the anoxic cardiac myocytes: HSYA lightened the morphologic injury, decreased the leak of LDH, inhibited the production of MDA and raised the activity of membrane ATPase. Then they caused an advance of survival rate. The protection may be related to the lowered expression of sarcoplasmic reticulum and membrane Ca2+-ATPase which would lessen calcium overloading. |
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