Cardioprotective Effect Of Berberine Against Ischemia/Reperfusion Injury Via AMPK And PI3K/Akt Signaling Pathway In Diabetic Rats

BackgroundDiabetes mellitus (DM) has reached epidemic proportions in the general adultpopulation in China with high prevalence and increasing incidence. Diabetic patients havehigher risk of developing heart disease and more severe and fatal myocardial infarctionsthan nondiabetic population. Cardiovascular complications are the leading cause ofmorbidity and mortality in diabetic patients. Therefore, treatment strategies for patientswith diabetes require not only metabolic control but also alleviation of cardiovascularmorbidity and mortality. Evidence from experimental models and human cardiac diseaseshave shown that myocardial apoptosis is significant in various heart diseases andinevitably leads to heart failure. Cardiomyocyte apoptosis is one of the major pathogenicmechanisms underlying myocardial ischemia/reperfusion (MI/R) injury. Blocking theapoptosis process could prevent the loss of contractile cells, minimize ischemia/reperfusion (I/R)-induced cardiac injury and therefore slow down or evenprevent heart failure. Berberine, an isoquinoline alkaloid, derived from medicinal herbsincluding Berberis, Hydrastis canadensis, Coptis chinensis Franch and CortexPhellodendri Chinensis, has antibacterial activities in addition to pharmacologic actionssuch as antiarrhythmic activities. Recent studies have also demonstrated the anti-diabeticeffect of berberine in type1or type2diabetic animal models through lowering bloodglucose and regulating lipid metabolism. In both humans and rodents with diabetes,berberine decreases hyperglycemia, alleviates insulin resistance and inhibits lipidsynthesis possibly via the activation of adenosine monophosphate-activated protein kinase(AMPK). Some other studies showed that berberine lowered high blood glucose inalloxan-induced diabetic mice by upregulating Akt activity. It has also been reported thatberberine decreases mortality in patients with chronic congestive heart failure. However,the underlying mechanisms of berberine beneficial effects on acute myocardial injury,especially in the diabetic state, remain incompletely understood. Our previous studies havedemonstrated that the survival signaling, i.e., PI3-kinase-Akt-dependent pathway, plays akey role in antiapoptotic action and protection of cardiomyocyte fromischemia/reperfusion injury. Therefore, we aimed to investigate whether berberine canattenuate MI/R injury in diabetic rat hearts via activating PI3-kinase-Akt-dependentpathway.ObjectivesThe present study aims1) to investigate whether berberine has cardioprotectiveeffects against ischemia/reperfusion injury in diabetic rats, and2) to elucidate theunderlying mechanisms of berberine cardioprotective effects.Methods1. Male Sprague-Dawley rats were injected with low dose streptozotocin and fed withhigh-fat diet for8weeks to induce type2diabetes. Diabetic rats were randomized toreceive saline or berberine by oral gavage of different doses (100mg/kg/d,200mg/kg/d and400mg/kg/d, respectively) for another4weeks.2. Four weeks after berberine treatment, oral glucose tolerance test (OGTT) wasperformed. Glucose (5g/kg body weight) was administered to rats. A one-drop bloodsample was obtained at0,30,60,120and180min respectively through the tip of thetail for the determination of blood glucose using a glucose meter.3. Anesthetized rats were subjected to MI/R. Myocardial ischemia was produced by exteriorizing the heart with a left thoracic incision followed by making a slipknotaround left anterior descending coronary artery. After30min of ischemia, the slipknotwas released and the myocardium was reperfused for3h. Myocardial function wascontinuously monitored before and during the entire I/R procedure. The arterial bloodpressure and LV pressure were sampled and digitally processed via a hemodynamicanalyzing system. Heart rate (HR), left ventricular developed pressure (LVDP) andthe instantaneous first derivation of LV pressure (+dP/dtmaxand–dP/dtmax) werederived by computer algorithms. Cardiomyocyte apoptosis was determined byTUNEL staining at the end of reperfusion.4. Primary cultures of neonatal rat cardiomyocyte from1-to3-day-old Sprague-Dawleyrats were prepared, cultured and randomly allocated into the following groups:(1) HG(high glucose,25mmol/L);(2) HG+H/R group;(3) HG+H/R+BBR0.5μmol/L group;(4) HG+H/R+BBR5μmol/L group;(5) HG+H/R+BBR50μmol/L group.5. Hypoxia/reoxygenation (H/R) model: the oxygen-glucose deprivation injury occurredby placing cells in a hypoxic environment (1%O2,5%CO2,94%N2) maintained byincubator in the presence of glucose-free DMEM medium for2h, during which themedium was exchanged with oxygenated and normal glucose DMEM medium withdifferent concentrations of berberine (0.5,5and50μmol/L) in an incubator (95%O2,5%CO2) at37°C to simulate the reoxygenation condition for2h.6. Cell viability was determined by MTT assay. Culture medium was collected formeasurement of total nitric oxide (NOx) production. TUNEL, hoechst33342stainingand fluorescein isothiocyanate-conjugated (FITC)-Annexin V and propidium iodide(PI) were performed to identify apoptotic cells. AMPK, Akt and eNOS expression andphosphorylation, PI3K, Bcl-2, Bax and Caspase-3were determined by Western Blot.Results1. Berberine lowered blood glucose levels in diabetic rats. OGTT was performed beforeMI/R. Compared with the diabetic control, rats treated with200mg/kg/d and400mg/kg/d berberine showed a significant improvement in oral glucose tolerance, asevidenced by lower blood glucose levels (4.0±0.2mmol/L, P<0.01) throughoutOGTT. Berberine treatment (at dose of200mg/kg/d) significantly improved+LVdP/dtmax(6619±249mmHg/s vs.4601±812mmHg/s, P<0.01) and–LV dP/dtmax(4801±285mmHg/s vs.2950±474mmHg/s, P<0.01) following MI/R when comparedwith untreated diabetic animals. Moreover, MI/R resulted in a significant increase of TUNEL positive nuclei in diabetic rat hearts, which was significantly attenuated byberberine treatment (P<0.05). These results showed that berberine improved cardiacfunctional recovery and prevented cardiomyocyte apoptosis in diabetic rats followingMI/R.2. Berberine protected cell viability of neonatal rat cardiomyocyte treated with HG+H/Rin vitro. Neonatal rat cardiomyocyte survival following HG+H/R was assessed byMTT assay. HG+H/R induced significant decrease in cell viability to60.7±2.5%ascompared to control cells (P<0.01), while berberine treatment (at concentration of50μmol/L) resulted in a significant increase in cell survival, restoring cell survival to89.3±3.5%(P<0.01) at the end of reoxygenation. Berberine decreased apoptosis inneonatal rat cardiomyocyte treated with HG+H/R (P<0.01). Berberine at theconcentration of50μmol/L produced the best effect.3. Berberine at the concentration of50μmol/L activated PI3K/Akt and AMPK andincreased eNOS phosphorylation in cultured neonatal rat cardiomyocyte underHG+H/R (1.6-fold,2.6-fold,1.5-fold and1.7-fold, respectively, P<0.01). Berberinesignificantly increased NO release in neonatal rat cardiomyocyte treated withHG+H/R. Berberine-induced PI3K/Akt, AMPK and eNOS activation were completelyabolished by PI3K/Akt inhibitor wortmannin (100nmol/L) or AMPK inhibitorCompound C (1μmol/L) respectively. Berberine-induced NO increase wascompletely abolished by PI3K/Akt inhibitor wortmannin (100nmol/L) or AMPKinhibitor Compound C (1μmol/L) respectively.4. Berberine increased Bcl-2/Bax ratio and decreased Caspase-3expression in neonatalrat cardiomyocyte cultured in H/R+HG. H/R+HG resulted in a noticeable increase inCaspase-3expression compared with that of control group (P<0.01), while50μmol/Lberberine reduced Caspase-3expression compared with that of H/R+HG group(P<0.05). Treatment with berberine plus wortmannin or Compound C markedlydecreased Bcl-2/Bax ratio and increased active Caspase-3expression (both P<0.05).These results demonstrated that berberine exerted anti-apoptotic effects throughactivating PI3K/Akt and AMPK signaling pathways.ConclusionsBerberine exerts anti-apoptotic action against MI/R injury and improves cardiacfunction recovery following MI/R via activating AMPK and PI3K-Akt-eNOS signaling in diabetic rats. The findings suggest the potential therapeutic value of berberine in theprevention and rescue for diabetic cardiovascular complications.