Abstract:

Objective To discuss the ameliorative effect of empagliflozin （EMPA） on the doxorubicin （DOX）-induced heart injury （HI） model in the rats， and to clarify its mechanism of action. Methods Twenty-four 6-7-week-old male Wistar rats were randomly divided into control group （normal healthy rats were maintained）， HI group （the DOX-induced rat HI model was established）， and HI+EMPA group （after 6 weeks of establishing the DOX-induced rat HI model， the rats were given 10 mg·kg?1 EMPA daily by gavage for 14 consecutive days）， with 8 rats in each group. Echocardiography was used to detect the left ventricular internal diameter at end-systole （LVIDs）， left ventricular ejection fraction （LVEF）， and left ventricular fractional shortening （LVFS） of the rats in various groups； HE staining and Masson staining were used to detect the pathomorphology of myocaridium tissue and collagen fibers in myocardium tissue of the rats in various groups； terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） method was used to analyze the apoptosis in the left ventricular myocardium cells of the rats in various groups； enzyme-linked immunosorbent assay （ELISA） was used to detect the serum levels of lactate dehydrogenase （LDH） and creatine kinase （CK） of the rats in various groups. Rat myocardial H9c2 cells were used for in vitro experiments. A DOX-induced rat cardiomyocyte injury model was established in vitro （DOX group）. Cell experiment grouping-1 was divided into control group （H9c2 cells were cultured normally without any treatment）， DOX group （0.1 μmol·L^-1 DOX was added to the H9c2 cell culture medium for 48 h to induce cardiomyocyte injury）， DOX+EMPA group （0.1 μmol·L^-1 DOX and 500 nmol·L^-1 EMPA were added to the H9c2 cell culture medium for 48 h）， and DOX+EMPA+sirtuin 1 （SIRT1） inhibitor （EX527） group （H9c2 cells were pretreated with 10 μmol·L?1 EX527 for 1 h， then 0.1 μmol·L^-1 DOX and 500 nmol·L^-1 EMPA were added for 48 h）. Cell experiment grouping-2 was divided into control group （H9c2 cells were cultured normally without any treatment）， DOX group （0.1 μmol·L^-1 DOX was added to the H9c2 cell culture medium for 48 h to induce cardiomyocyte injury）， and DOX+dynamin-related protein 1 （Drp-1） inhibitor group （H9c2 cells were pretreated with 75 μmol·L^-1 Drp-1 inhibitor Mdivi-1 for 2 h， then treated with 0.1 μmol·L^-1 DOX for 48 h）. Western blotting method was used to determine the expression levels of autophagy-related proteins microtubule-associated protein 1 light chain 3 （LC3）， autophagy key molecule yeast Atg6 homolog BECLIN1， and ubiquitin-binding protein P62， SIRT1， peroxisome proliferator-activated receptor gamma coactivator 1-alpha （PGC-1α）， and Drp-1； mitochondrial fission 1 protein （Fis-1）， and mitochondrial fission factor （MFF） proteins in the cells in various groups. Results In control group， the myocardial fibers of the rat hearts were arranged neatly， and no obvious inflammatory cell infiltration or collagen fiber deposition was seen in the interstitium； compared with control group， in HI group， the myocardial fibers of the rats were disordered， the myocardial interstitium was enlarged， and inflammatory cell infiltration and collagen fiber deposition were observed； compared with HI group， in HI+EMPA group， the myocardial fibers of the rats were more regular， no obvious inflammatory cell infiltration was seen in the interstitium， and a small amount of collagen fiber deposition was observed. The TUNEL staining results showed that compared with control group， the number of TUNEL-positive cells in the myocardium tissue of the rats in HI group was increased （P<0.05）. The echocardiography results showed that compared with control group， the LVIDs of the rats in HI group was increased （P<0.05）， and the LVEF and LVFS were decreased （P<0.05）； compared with HI group， the LVIDs of the rats in HI+EMPA group was decreased （P<0.05）， and the LVEF and LVFS were increased （P<0.05）. The ELISA results showed that compared with control group， the serum levels of LDH and CK of the rats in HI group were increased （P<0.05）； compared with HI group， the serum levels of LDH and CK of the rats in HI+EMPA group were decreased （P<0.05）. The Western blotting results showed that compared with control group， the BECLIN1 protein expression level and LC3-Ⅰ/LC3-Ⅱ ratio in the H9c2 cells in DOX group were increased （P<0.05）， the expression levels of P62， SIRT1， and PGC-1α proteins were decreased （P<0.05）， and the expression level of Drp-1 protein was increased （P<0.05）； compared with DOX group， the BECLIN1 protein expression level and LC3-Ⅰ/LC3-Ⅱ ratio in the H9c2 cells in DOX+EMPA group were decreased （P<0.05）， the expression levels of P62， SIRT1， and PGC-1α proteins were increased （P<0.05）， and the expression level of Drp-1 protein was decreased （P<0.05）； compared with DOX+EMPA group， the BECLIN1 protein expression level and LC3-Ⅰ/LC3-Ⅱ ratio in the H9c2 cells in DOX+EMPA+EX527 group were increased （P<0.05）， and the expression levels of P62 and Drp-1 proteins were decreased （P<0.05）. The Western blotting results showed that compared with control group， the expression levels of Drp-1， Fis-1， and MFF proteins in the H9c2 cells in DOX group were increased （P<0.05）； compared with DOX group， the expression levels of Fis-1 and MFF proteins in the H9c2 cells in DOX+Drp-1 inhibitor group were decreased （P<0.05）. Conclusion EEMPA can alleviate DOX-induced myocardial lesions and cardiac dysfunction in rats， and reduce cardiomyocyte apoptosis and autophagy， and its mechanism may be related to the up-regulation of SIRT1 and PGC-1α protein expressions and the down-regulation of Drp1 protein expression by EMPA.

Key words: Doxorubicin, Cardiotoxicity, Empagliflozin, Mitochondrial fission, Sirtuin 1, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha, Dynamin-related protein 1