Yellow Wine Polyphenolic Compounds Regulate SIRT3 Expression to Alleviate Doxorubicin-induced Myocardial Injury

Background Doxorubicin (DOX) , a common and powerful anthracycline antitumor agent, has been widely used in the treatment of various tumors, especially solid tumors. However, its strong cardiotoxicity-induced irreversible cardiomyopathy and congestive heart failure limit its clinical application. Yellow Wine Polyphenols Compounds (YWPC) are polyphenols extracted from Shaoxing yellow rice wine which have anti-inflammatory and antioxidant activities, and can alleviate DOX-induced myocardial injury, but the mechanism of actions is still unclear. Objective To explore the mechanism of YWPC reducing DOX-induced myocardial injury via in vitro and in vivo experiments. Methods SD rats were used for an in vivo experiment and divided into four groups: control group (intervened with normal saline) , YWPC group (intervened with YWPC and normal saline) , DOX group (intervened with DOX and normal saline for establishing a DOX-induced myocardial injury model) and DOX+YWPC group (intervened with DOX, YWPC and normal saline for observing the myocardial protective effect of YWPC in a DOX-induced myocardial injury model) . When the experiment ended, all rats were sacrificed and cardiac tissues were taken out for examining myocardial fiber morphology using Masson's trichrome staining, myocardial cell apoptosis using TUNEL assay, pathological changes using immunohistochemical assay, and levels of proteins (Bcl-2 and Bax) involved in apoptosis as well as expression level of SIRT3 using Western blotting. And the serum lactate dehydrogenase (LDH) was also measured. Cardiomyocyte H9C2 cells of rats were used for an in vitro experiment and divided into five groups: control group, YWPC group (intervened with YWPC and normal saline) , DOX group (intervened with DOX for establishing a DOX-induced myocardial injury model within 24 hours) and three DOX+YWPC (1 mg/L, 10 mg/L, 100 mg/L) groups〔first intervened with DOX for establishing a DOX-induced myocardial injury model within 24 hours, then with YWPC for observing the myocardial protective effect of three concentrations of YWPC (1 mg/L, 10 mg/L, 100 mg/L) , respectively〕. Viability of H9C2 cells was measured by CCK-8 assay. Apoptosis, and SIRT3 expressed in H9C2 cells were measured by Western blotting. Then another batch of the same H9C2 cells were took and divided into control group, DOX group, DOX+YWPC group, DOX+3-TYP group and DOX+YWPC+3-TYP group, and SIRT3 inhibitor 3-TYP was used to inhibit the activity of SIRT3 protein in the latter two groups. Then apoptosis level and SIRT3 protein expressed in H9C2 cells were detected by Western blotting for further assessing the effect of SIRT3 in reducing DOX-induced myocardial injury. Results In vivo experiment: (1) Under the microscope, Masson's trichrome-stained myocardial fibers of rats in DOX group were disordered and intersected with a large number of blue collagen fibers. The myocardial texture of DOX+YWPC group was partially restored with decreased blue collagen fibers. DOX group had higher proportion of collagen fibers distributed in cardiovascular tissues, and higher serum LDH than control and DOX+YWPC groups (P<0.05) . DOX group had lower ratio of Bcl-2/Bax and level of expression of SIRT3 than control and DOX+YWPC groups (P<0.05) . The apoptotic myocardial cells with a patch distribution appearing as bright green dots were significantly increased in DOX group at first, but were decreased after YWPC treatment. In vitro experiment: (1) The absorbance value, Bcl-2/Bax ratio and expression level of SIRT3 in DOX group were lower than those of control group, DOX+YWPC (1 mg/L) group, DOX+YWPC (10 mg/L) group and DOX+YWPC (100 mg/L) group (P<0.05) . (2) The Bcl-2 /Bax ratio and expression level of SIRT3 in DOX or DOX+3-TYP group were lower than those in control group (P<0.05) . (3) The Bcl-2/Bax ratio in DOX+YWPC+3-TYP group was lower than that in DOX+YWPC group (P<0.05) . (4) The expression level of SIRT3 was similar in DOX+YWPC and DOX+YWPC+3-TYP groups (P>0.05) . Conclusion DOX-induced myocardial injury in in vitro and in vivo experiments with rats may be alleviated by YWPC via improving the expression level of SIRT3, and the effect may be reduced if the expression level of SIRT3 protein is inhibited