Effects And Mechanism Of Naringenin On Angiotensinâ…¡-induced Myocardial Hypertrophy

Objective: To observe the protective effects and mechanism of naringenin onangiotensinⅡ-induced myocardial hypertrophy in C57BL/6J mice or H9C2cells.Methods: The model of angiotensin-induced hypertrophy of H9c2cells wasestablished. Cultured H9C2cells were randomly divided into four groups: controlgroup, angiotensinⅡgroup (AngⅡgroup,10-7mol/L), Naringenin (75μM)+AngⅡgroup (Nar+AngⅡgroup) and Naringenin (75μM) group (Nar group). During thegroups, naringenin was added into the medium for60min pretreatment in Nar+AngⅡgroup and Nar group, then angiotensinⅡwas added into the medium and the cellswere maintained for another24h. CCK-8was used to assay the cell viability;immunofluorescent staining of F-actin were adopted to observe and calculate the cellsize. Expression of ANP mRNA and BNP mRNA was detected by real-time PCR;mitochondrial ROS production was measured by H2DCFH-DA staining, and theexpression of p-AMPK, Nox-4and p-Erk was detected by western blot.32C57BL/6J male mice were randomly divided into four group: control group,AngⅡgroup, Nar (50mg/kg/d)+AngⅡgroup and Nar (50mg/kg/d) group. The modelof myocardial hypertrophy was induced by consecutive injection of angiotansinⅡ(1.6mg/kg/d, ip) for3weeks in mice. At the end of experiment, the mice were sacrificatedand the hearts were taken photos and weighed to calculate the heart mass index(HW/BW). HE staining was used to observe the myocardial morphology; Xanthineoxidase method was used to measure the activity of superoxide dismutase (SOD);thiobarbituric acid method was to assay the content of malondialdehyde (MDA);electrochemiluminescence immunoassay was adopted to assay the activities of lactatedehydrogenase (LDH) and creatine kinase (CK). Results: Naringenin (12.5,25,50,75μmol/L) had no effect on viability of H9C2cells. Compared with control group, angiotensinⅡsignificantly increased the size ofH9C2cells, promoted the expression of ANP mRNA and BNP mRNA, the differencewas significant between the two groups (P<0.05). H2DCFH-DA staining showed thatmitochondrial ROS production in AngⅡgroup was higher than that of control group.Compared with AngⅡgroup, naringenin treatment could obviously decrease the sizeof H9c2cells and the expression of ANP mRNA and BNP mRNA (P<0.05), inhibitthe ROS production, reduce the expression of Nox-4, and p-Erk, and elevate thephosphorylation of AMPK.Data in vivo showed that the heart mass index (HW/BW) and the trans diameterof myocardial cells were obviously increased in AngⅡ-treated mice as compared tocontrol mice (P<0.05). Compared with control group, the activities of LDH and ASTthe content of MDA were increased significant myocardial tissues of Ang Ⅱgroup(P<0.01), the level of SOD was markedly decreased (P<0.01). In addition,morphological staining also showed disorder of myocardial fibers in AngⅡgroup.Compared with AngⅡgroup, naringenin treatment for3weeks could obviouslyimprove the disorder of myocardial structure, decrease the trans diameter ofmyocardial cells and the heart mass index (P<0.05), increase SOD level in hearttissues, and reduce the release of LDH, CK and MDA content (P<0.05).Conclusion: These results suggest that naringenin could defend cardiachypertrophy induced by angiotensinⅡin vitro and in vivo by activating AMPKphosphorylation and subsequently negative regulating of Nox-4expression andmitochondrial ROS production.