| Background: Association between iron and cardiovascular diseases was first proposed by Sullivan in 1980 s, whereasnow it becomes a hot research topic.Ironoverloadisafrequentcause of cardiovasculardamage, andstronglyassociatewith the occurrence and progress of cardiovascular diseases. Significant increase of reactive oxygen species(ROS) occurs when iron overload takes place, ROS can induce apoptosis of cardiomyocyte. Iron chelators are thought to be effective in alleviating these pathological changes.Objectives & Significance: In this research, ROS levels of Meriones unguiculatus iron overload models administrated with iron chelators were determined to evaluate the conditions of oxidative / anti-oxidative system.Expression levels of apoptic proteins(Bax, Bcl-2) were determined by Western blotting and Bax/Bcl-2 ratio was employed for investigating the association between iron overload and cardiomyocyte apoptosis, and the efficacy of iron chelators. This research is expected to provide more specific laboratory basis for clinical prevention and therapy of iron overload induced cardiovascular diseases.Methods: Three-month old healthy Meriones unguiculatus(n=30, male &female, weight 200-500g) were bred at 22±2℃, 12 h / 12 h day-night cycle was maintained, food and drink were not restricted. Before the test, the rodents were kept for 2 weeks to adapt the environment, body weight and status were strictly monitored, weight-losing and inadaptive subjects were excluded. Included subjects were randomly assigned to 3 groups including 3 months control(C-3m, no treatment, comparable to iron overload group), ten-week iron overload group(IO, iron dextran was subcutaneously injected at 100 mg/kg each 5 days for 3 months), iron overload treatment group(IO-DFR, after 10 weeks’ iron overload, the subjects were administrated with iron chelator deferasirox 100mg/kg/d for 3 months). iron overload treatment group(IO-DFR): iron chelator(deferasirox) was administrated by a 1 m L synringe at 100 mg/kg/d orally for 3months after 10-week iron overload. The model was validated by a former study1. MDA levels and SOD activity of all groups were determined to evaluate the conditions of oxidative / anti-oxidative system. ROS levels of the rodents were determined by specific histochemical staining, immunohistochemistry,fluorescence staining. Expression of apoptic proteins in cardiomyocyte was determined by Western blotting. The data were analyzed to elucidate the association between cardiomyocyte hyperplasia in iron overload induced oxidative stress and cardiomyocyte apoptosis and to evaluate the efficacy of iron chelators.Results:(1) MDA and SOD determination indicated that MDA levels of the IO-3 m and C-3 m groups elevated for 83.71%, SOD activity decreased for 49.41%,whereas MDA levels dropped for 28.14% and SOD activity increased for65.12% in the IO-DFR group comparing to IO-3 m group. All differences were statistically significant(P<0.05).(2) In HE staining, fluorescence was enhanced in the IO-3m group comparing to the control group, indicating increase in ROS. Oppositely the fluorescence was weakened in the IO-DFR group thus the ROS level of IO-DFR group decreased. Nitrotyrosine and 4-HNE immunohistochemical staining showed increased green fluorescene in the IO-3m group and weakened green fluorescene in the IO-DFR group comparing to the control, which indicated ROS increased in the IO-3m group and decreased in the IO-DFR group. The differences were statistically significant(P<0.05).(3) Western blotting showed that no significant difference was observed in Bcl-2 among the groups(P>0.05). Bax level of IO-3m group increased for31.28% comparing to the control group, whereas Bax level of IO-DF group was18.76% lower than IO-3m group(P<0.05). Bax/Bcl-2 ratio of the IO-3m group was higher than the control group, and Bax/Bcl-2 ratio of the IO-DFR group was lower than IO-3m group(P<0.05). These results indicated that iron overloade drove the expression of apoptic proteins higher and iron chelators can significantly down regulate the expression of apoptic proteins.Conclusions:(1) Iron overload can trigger oxidative stress in rodents, ROS increased,anti-oxidative function was undermined. Balance between oxidation and anti-oxidation was broken.(2) Iron overload can induce up-regulation of apoptic proteins, Bax/Bcl-2ratio elevated. Thus, iron overload can promote apoptosis of cardiomyocyte.(3) Iron chelator agents can reverse the increasing trend of apoptic proteins in cardiomyocyte and lower the ratio of Bax/Bcl-2... |
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