PostGraduation Student: Name: Sudeep K.C.

Faculty (major): Internal Medicine (Cardiology)

Professior name ： Lu JunKun

Thesis defense date ： June 2015

The study of Slc25a3 gene effect in apoptosis of cardiomyocytes in SD Rats by doxorubicin and the protective mechanism using by curcumin.

The study of Slc25a3 gene effect in apoptosis of cardiomyocytes in SD Rats by doxorubicin and the

protective mechanism using by curcumin.

1. Preface2. Aims & Objectives3. Methods4. Result5. Discussion6. Conclusion

PREFACEDoxorubicin causes acute myocardial injury, leading to myocardial interstitial edema, myocardial fibers derangement, swelling and heart volume to be increased significantly .

Doxorubicin cardiomyopathy mechanism:Existing relevant ultrastructural studies have found that anthracycline-induced myocardial cell injury two main forms, namely myocardial fiber necrosis and degeneration of the myocardium, these changes can lead to progressive myocardial necrosis, electron microscopy shows myocardial muscle fibers disorder, muscle dearray, mitochondrial hyperplasia, swelling, deformity, and reduce cristae.

Studies have also indicated that doxorubicin may lead to non-species-dependent myocardial injury . Although the exact mechanism of doxorubicin-induced cardiotoxicity still not clear, but most studies have shown that oxidative stress may play the most critical role, which is due to the chemical structure of doxorubicin in the metabolic process to produce reactive oxygen species

The purpose of this experiment is to study the Protective effect of curcumin against doxorubicin induced Myocardial cell injury on SD rats and to explore the expression of Pic Protein and Slc25a3 gene inhibition on Doxorubicin induced Myocardial injury under Curcumin intervention, depending upon the different concentration and duaration of use of curcumin.

To study the protective effect of Curcumin against Doxorubicin induced myocardial injury on SD rats by inhibiting expression of Slc25a3 gene.

Aims&Objectives

Methods Normal healthy adult SD rats (male and female,

180-220g) were used to establish doxorubicin-induced myocardial injury animal models, randomly divided into three groups: control group, doxorubicin group, curcumin with doxorubicin group.

Control group : was injected 0.9% sodium chloride injection (2.5ml/kg).

Doxorubicin group :was injected diluents of doxorubicin i.e. 2.5mg/kg, by 0.9% sodium chloride injection .

Curcumin with doxorubicin group was injected diluents of doxorubicin i.e. 2.5mg/kg, by 0.9% sodium chloride injection with curcumin (30mg/kg).

All groups were injected via rat tail vein once every week following for 6 weeks .

After eleven weeks, all rats were sacrificed using spinal cord transection method and heart tissues were taken for the next experiment.

Flow cytometry is used to test the SD rat cardiomyocytes transferred level; Application of Real-time PCR technology is used to detect Slc25a3 gene expression.

Using SPSS 16.0 software, the results were expressed as mean ± standard deviation (Mean± SD). Data between the two groups were compared using t test, among groups were compared using ANOVA. With P <0.05 was considered statistically significant

ResultDetection of cardiomyocytes apoptosis in SD rats by flow cytometry

In this study, flow cytometry is used in animal model to detect myocardial apoptosis.

The results showed that apoptotic rate was significantly higher in doxorubicin injury group than curcumin intervention group (Figure), the differences were statistically significant (p <0.05).

Figure - Animal models of myocardial apoptosis

Result Level of Expression of PiC after curcumin on adriamycin-induced myocardial cell injury on SD Rats:

The expression of Slc25a3 mRNA was significantly higher in doxorubicin injury group than that in control group and curcumin intervention group, while the expression of Slc25a3 mRNA is significantly lower in curcumin intervention group than that in control group (p <0.01, p <0.05).

Figure 16 expression of PiC after curcumin intervention in adriamycin-induced myocardial cell injury in SD rats

Discussion

Doxorubicin is broadly used antitumor drug. The study shows patients using long duration of doxorubicin cause cardiac toxicity, till now the exact mechanism is not clear but many research and experiments have done and shown that oxidative stress plays a critical role due to chemical structure of doxorubicin to produce reactive oxygen species and cause myocardial injury and mitochondrial sub cellular injury.

Curcumin a family of Zingiberacae, is a plant used for spice, coloring and a major source of curry powder in country like India and has a long traditional value in medicine in country like China.

Many studies shown curcumin to be inhibitor of tumor initiation in vivo. It also showed cardioprotective effect by suppression of myocardial protein expression of inducible nitric oxide synthase and the catalytic subunit of nicotinamide adenine dinucleotide phosphate reduced oxidate and significantly decrease myocardial endoplasmic reticulum stress signaling proteins.

Discussion

Mitochondrial is a protein that in humans encoded by Slc25a3gene. The protein encoded by this gene catalyzes the transport of phosphate into the mitochondrial matrix, either by protein co-transport or in exchange for hydroxyl ions.

The experiment here is to study the Protective effect of curcumin against doxorubicin induced Myocardial cell injury on SD rats and to explore the expression of Pic Protein and Slc25a3 gene inhibition on Doxorubicin induced Myocardial injury under Curcumin intervention, depending upon the different concentration and duaration of use of curcumin.

We studies the Slc25a3 gene effect in apoptosis of cardiac injury in SD rats by establishing protective mechanism of curcumin against doxorubicin injuced myocardial injury.

Discussion

结论（ Conclusion）• 1. Doxorubicin intervention in SD rats myocardial

cells can induce myocardial cells apoptosis, curcumin can significantly reduce the doxorubicin-induced myocardial cells apoptosis.

• 2. The expression of PiC and Slc25a3 gene on doxorubicin-induced myocardial injury in rats have an important role. The Slc25a3 gene up-regulation is closely related to doxorubicin myocardial injury. Curcumin down regulates Slc25a3 gene expression, thus reducing doxorubicin induced myocardial cell damage in rats.

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