Autophagy and its therapeutic potential in diabetic nephropathy.

Diabetic nephropathy (DN), the leading cause of end-stage renal disease, is the most significant microvascular complication of diabetes and poses a severe public health concern due to a lack of effective clinical treatments. Autophagy is a lysosomal process that degrades damaged proteins and organelles to preserve cellular homeostasis. Emerging studies have shown that disorder in autophagy results in the accumulation of damaged proteins and organelles in diabetic renal cells and promotes the development of DN. Autophagy is regulated by nutrient-sensing pathways including AMPK, mTOR, and Sirt1, and several intracellular stress signaling pathways such as oxidative stress and endoplasmic reticulum stress. An abnormal nutritional status and excess cellular stresses caused by diabetes-related metabolic disorders disturb the autophagic flux, leading to cellular dysfunction and DN. Here, we summarized the role of autophagy in DN focusing on signaling pathways to modulate autophagy and therapeutic interferences of autophagy in DN.

The protection of luteolin against diabetic cardiomyopathy in rats is related to reversing JNK-suppressed autophagy.

Increasing evidence has shown that impaired autophagy dramatically causes myocardial hypertrophy and fibrosis in the diabetic heart, ultimately leading to diabetic cardiomyopathy (DCM). Luteolin has been reported to effectively attenuate diabetic cardiovascular injury by inhibiting oxidative stress and alleviate sepsis-induced myocardial injury by enhancing autophagy. However, whether luteolin can reduce DCM through activating autophagy and the underlying mechanism remain unclear. Here, reversing the c-Jun N-terminal kinase (JNK)-suppressed autophagy pathway by which luteolin attenuates DCM was explored. Male Sprague-Dawley rats were injected with streptozotocin to induce diabetes. After 6 weeks of diabetes, rats were treated with luteolin (50, 100 and 200 mg kg-1, i.g.) for 4 weeks. Histological and functional alterations in the diabetic heart were determined using HE staining, Masson staining and echocardiography. The expressions of myocardial miR-221, JNK, and c-Jun and autophagic vesicles in diabetes were evaluated by quantitative PCR, Western blotting and electron microscopy. Luteolin significantly improved cardiac function and attenuated myocardial disorganization and fibrosis in the diabetic rat accompanying the dose-dependent down-regulation of JNK, c-Jun, miR-221 and p62, increase of LC3-II/I and autophagic vesicles, and decrease of mitochondrial swelling in the diabetic heart. These data suggest that the protection of luteolin against DCM, at least, is related to suppressing JNK/c-Jun-regulated miR-221 and the subsequent blockage of autophagy.

Profile of crosstalk between glucose and lipid metabolic disturbance and diabetic cardiomyopathy: Inflammation and oxidative stress.

In recent years, the risk, such as hypertension, obesity and diabetes mellitus, of cardiovascular diseases has been increasing explosively with the development of living conditions and the expansion of social psychological pressure. The disturbance of glucose and lipid metabolism contributes to both collapse of myocardial structure and cardiac dysfunction, which ultimately leads to diabetic cardiomyopathy. The pathogenesis of diabetic cardiomyopathy is multifactorial, including inflammatory cascade activation, oxidative/nitrative stress, and the following impaired Ca2+ handling induced by insulin resistance/hyperinsulinemia, hyperglycemia, hyperlipidemia in diabetes. Some key alterations of cellular signaling network, such as translocation of CD36 to sarcolemma, activation of NLRP3 inflammasome, up-regulation of AGE/RAGE system, and disequilibrium of micro-RNA, mediate diabetic oxidative stress/inflammation related myocardial remodeling and ventricular dysfunction in the context of glucose and lipid metabolic disturbance. Here, we summarized the detailed oxidative stress/inflammation network by which the abnormality of glucose and lipid metabolism facilitates diabetic cardiomyopathy.

Sestrin2 is involved in the Nrf2-regulated antioxidative signaling pathway in luteolin-induced prevention of the diabetic rat heart from ischemia/reperfusion injury.

Luteolin attenuates myocardial ischemia/reperfusion (I/R) injury in diabetes through activating the nuclear factor erythroid 2-related factor 2 (Nrf2)-related antioxidative response. Though sestrin2, a highly conserved stress-inducible protein, is regarded as a modulator of Nrf2 and reduces I/R injury, the effect of sestrin2 on luteolin-induced prevention of the diabetic heart from I/R injury remains unclear. We hypothesized that luteolin could relieve myocardial I/R injury in diabetes by activating the sestrin2-modulated Nrf2 antioxidative response. Diabetes was induced in rats using a single dose of streptozotocin (65 mg kg-1, i.p.) for 6 weeks, and then luteolin (100 mg kg-1 d-1, i.g.), Nrf2 inhibitor brusatol, or sestrin2 blocker leucine was administered for 2 consecutive weeks. After that, the hearts were isolated and exposed to global I/R (30 min/120 min). Luteolin markedly improved cardiac function, myocardial viability and expressions of Nrf2-regulated antioxidative genes, and reduced lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R hearts. Ca2+-induced mitochondrial permeability transition and membrane potential disruption were markedly inhibited in luteolin-treated diabetic ventricular myocytes. All these effects of luteolin were significantly reversed by Nrf2 inhibitor brusatol or sestrin2 inhibitor leucine. Luteolin-induced diminished Keap1 and augmented nuclear translocation and ARE binding activity of Nrf2 were hampered by leucine in the diabetic I/R heart. In addition, luteolin-induced augmented transcription of sestrin2 was markedly blocked by brusatol in the diabetic I/R heart. These data suggest that sestrin2 and Nrf2 positively interact to promote antioxidative actions and attenuate mitochondrial damage, by which luteolin relieves diabetic myocardial I/R injury.

Betulinic acid attenuates lipopolysaccharide-induced vascular hyporeactivity in the rat aorta by modulating Nrf2 antioxidative function.

Betulinic acid (BA), a pentacyclic triterpenoid, has been reported to inhibit cardiovascular dysfunction under sepsis-induced oxidative stress. Nuclear factor erythroid-2 related factor-2 (Nrf2) is regarded as a key transcription factor regulating expression of endogenous antioxidative genes. To explore the preventive effects of BA against vascular hyporeactivity and the related antioxidative mechanism in sepsis, contraction and relaxation in aortas isolated from lipopolysaccharide (LPS)-challenged rats were performed. Male Sprague-Dawley rats were pretreated with brusatol (Bru, 0.4 mg/kg/2 days, i.p.), an inhibitor of Nrf2, and BA (10, 25, 50 mg/kg/day, i.g.) for 3 days and injected with LPS (10 mg/kg, i.p.) at the 4th day. Rats were anesthetized and killed by cervical dislocation after they were treated with LPS for 4 h. Thoracic aortas were immediately dissected out to determine contraction and relaxation using the organ bath system. Pro-inflammatory factors interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) and oxidative stress were measured in aortic tissues and plasma. mRNA expression of Nrf2-regulated antioxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and heme oxygenase-1 (HO-1), in rat aortas was determined. Increases of IL-1ß, TNF-α, nitric oxide, and malondialdehyde and the decrease of glutathione induced by LPS were significantly attenuated by pretreatment with different doses of BA in plasma and aortas (p < 0.05 versus LPS), all of which were blocked by Bru (p < 0.01). Inhibition of phenylephrine (PE)- and KCl-induced contractions and acetylcholine (ACh)-induced vasodilatation in aortas from LPS-challenged rats was dose-dependently reduced by BA (p < 0.05; percentage improvements by BA in PE-induced contraction were 55.38%, 96.41%, and 104.33%; those in KCl-induced contraction were 15.11%, 23.96%, and 22.96%; and those in ACh-induced vasodilatation were 16.08%, 42.99%, and 47.97%), all of which were reversed by Bru (p < 0.01). Improvements of SOD, GPx, and HO-1 mRNA expression conferred by BA in LPS-challenged rat aortas were inhibited by Bru (p < 0.01; 145.45% versus 17.42%, 160.69% versus 22.76%, and 166.88% versus 23.57%). These findings suggest that BA attenuates impairments of aortic contraction and relaxation in LPS-challenged rats by activating Nrf2-regulated antioxidative pathways.

Luteolin Attenuates Cardiac Ischemia/Reperfusion Injury in Diabetic Rats by Modulating Nrf2 Antioxidative Function.

Luteolin has been reported to attenuate ischemia/reperfusion (I/R) injury in the diabetic heart through endothelial nitric oxide synthase- (eNOS-) related antioxidative response. Though the nuclear factor erythroid 2-related factor 2 (Nrf2) is regarded as a key endogenous factor to reduce diabetic oxidative stress, whether luteolin reduces cardiac I/R injury in the diabetic heart via enhancing Nrf2 function needs to be clarified. We hypothesized that pretreatment with luteolin could alleviate cardiac I/R injury in the diabetic heart by affecting the eNOS/Nrf2 signaling pathway. The diabetic rat was produced by a single injection of streptozotocin (65 mg/kg, i.p.) for 6 weeks, and then, luteolin (100 mg/kg/day, i.g.), eNOS inhibitor L-NAME, or Nrf2 inhibitor brusatol was administered for the succedent 2 weeks. After that, the isolated rat heart was exposed to 30 min of global ischemia and 120 min of reperfusion to establish I/R injury. Luteolin markedly ameliorated cardiac function and myocardial viability; upregulated expressions of heme oxygenase-1, superoxide dismutase, glutathione peroxidase, and catalase; and reduced myocardial lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R heart. All these ameliorating effects of luteolin were significantly reversed by L-NAME or brusatol. Luteolin also markedly reduced S-nitrosylation of Kelch-like ECH-associated protein 1 (Keap1) and upregulated Nrf2 and its transcriptional activity. This effect of luteolin on Keap1/Nrf2 signaling was attenuated by L-NAME. These data reveal that luteolin protects the diabetic heart against I/R injury by enhancing eNOS-mediated S-nitrosylation of Keap1, with subsequent upregulation of Nrf2 and the Nrf2-related antioxidative signaling pathway.

Luteolin alleviates cardiac ischemia/reperfusion injury in the hypercholesterolemic rat via activating Akt/Nrf2 signaling.

Myocardial ischemia/reperfusion (I/R) injury in hypercholesterolemia is associated with oxidative stress, while luteolin is known to reduce oxidative stress by activating Akt/nuclear factor erythroid-2-related factor 2 (Nrf2) signaling and alleviate cardiac I/R injury. Here, we investigated whether luteolin pretreatment diminishes myocardial I/R injury in hypercholesterolemic rats by activating Akt/Nrf2 signaling. Hypercholesterolemic rats were produced by 2% cholesterol diet for 8 weeks. Luteolin (100 mg/kg/day, i.g.) or LY294002 was administered for the last 2 weeks. The hearts were then isolated and subjected to 30 min of global ischemia followed by 120 min of reperfusion. Pretreatment with luteolin significantly improved left ventricular function throughout reperfusion, increased cardiac tissue viability, reduced coronary lactate dehydrogenase release and the myocardial malondialdehyde level, upregulated p-Akt and p-GSK3ß expressions, inhibited nuclear translocation of Fyn, and activated Nrf2 function in hypercholesterolemic I/R rat hearts. All these improving effects of luteolin were significantly attenuated by LY294002. Ca2+-induced mitochondrial permeability transition pore (mPTP) opening and mitochondrial inner membrane potential reduction were significantly inhibited in ventricular myocytes isolated from luteolin-treated hypercholesterolemic rats, which were attenuated by LY294002. These results indicate that luteolin protects the hypercholesterolemic heart against I/R injury due to upregulation of Akt-mediated Nrf2 antioxidative function and inhibition of mPTP.

Inhibition of HDAC3 prevents diabetic cardiomyopathy in OVE26 mice via epigenetic regulation of DUSP5-ERK1/2 pathway.

Inhibition of total histone deacetylases (HDACs) was phenomenally associated with the prevention of diabetic cardiomyopathy (DCM). However, which specific HDAC plays the key role in DCM remains unclear. The present study was designed to determine whether DCM can be prevented by specific inhibition of HDAC3 and to elucidate the mechanisms by which inhibition of HDAC3 prevents DCM. Type 1 diabetes OVE26 and age-matched wild-type (WT) mice were given the selective HDAC3 inhibitor RGFP966 or vehicle for 3 months. These mice were then killed immediately or 3 months later for cardiac function and pathological examination. HDAC3 activity was significantly increased in the heart of diabetic mice. Administration of RGFP966 significantly prevented DCM, as evidenced by improved diabetes-induced cardiac dysfunction, hypertrophy, and fibrosis, along with diminished cardiac oxidative stress, inflammation, and insulin resistance, not only in the mice killed immediately or 3 months later following the 3-month treatment. Furthermore, phosphorylated extracellular signal-regulated kinases (ERK) 1/2, a well-known initiator of cardiac hypertrophy, was significantly increased, while dual specificity phosphatase 5 (DUSP5), an ERK1/2 nuclear phosphatase, was substantially decreased in diabetic hearts. Both of these changes were prevented by RGFP966. Chromatin immunoprecipitation (ChIP) assay showed that HDAC3 inhibition elevated histone H3 acetylation on the DUSP5 gene promoter at both two time points. These findings suggest that diabetes-activated HDAC3 inhibits DUSP5 expression through deacetylating histone H3 on the primer region of DUSP5 gene, leading to the derepression of ERK1/2 and the initiation of DCM. The present study indicates the potential application of HDAC3 inhibitor for the prevention of DCM.

Exacerbation of diabetic cardiac hypertrophy in OVE26 mice by angiotensin II is associated with JNK/c-Jun/miR-221-mediated autophagy inhibition.

Both diabetes and angiotensin II (Ang II) excess trigger cardiac remodeling and dysfunction, and diabetic cardiomyopathy. We hypothesized that cardiac hypertrophy associated with the development of diabetic cardiomyopathy is worsened by increased Ang II. Male type 1 diabetic OVE26 and wild-type mice were given Ang II (sc., 1.15 mg/kg, twice a day) for 14 days. Diabetes-induced cardiac dysfunction and hypertrophy was exacerbated by Ang II treatment as determined by echocardiography, wheat germ agglutinin staining and atrial natriuretic peptide. Ang II treatment dramatically exacerbated diabetes-caused decreased LC3-II, a marker of autophagy, and increased p62, an indicator of cytosolic protein clearance. Ang II treatment also augmented diabetes-associated increased phosphorylated levels of c-Jun, JNK, mTOR, and miR-221, and decreased of p27 expression, a direct target of miR-221. Chromatin immunoprecipitation assay showed that Ang II elevated c-Jun binding to the promoter of miR-221 in diabetic mice. These results suggest that Ang II accelerates cardiac hypertrophy in the early stage of murine diabetes, probably through activation of the JKN/c-Jun/miR-221 axis and inhibition of downstream autophagy. Therefore, inhibition of Ang II or miR-221 in diabetic individuals may be a potential approach for delaying the onset and/or reducing the severity of diabetic cardiomyopathy.

Cardioprotective effects of luteolin on ischemia/reperfusion injury in diabetic rats are modulated by eNOS and the mitochondrial permeability transition pathway.

Myocardial ischemia/reperfusion (I/R) injury in diabetes is associated with oxidative stress, endothelial nitric oxide synthase (eNOS) dysfunction, and mitochondrial collapse, whereas luteolin is known to protect the cardiovascular system against diabetes and I/R injury. Here, we investigated whether luteolin pretreatment diminishes myocardial I/R injury in diabetic rats by affecting eNOS and the mitochondrial permeability transition pore (mPTP). After diabetic rats were produced by streptozotocin treatment (65 mg/kg) for 3 weeks, luteolin (100 mg·kg·d) or L-NAME (25 mg·kg·d) was administered intragastrically for 2 weeks. Hearts were then isolated and subjected to 30 minutes of global ischemia followed by 120 minutes of reperfusion. Pretreatment with luteolin significantly improved left ventricular function and coronary flow throughout reperfusion, increased cardiac tissue viability and manganese superoxide dismutase (MnSOD) activity, and reduced coronary lactate dehydrogenase release, and the myocardial malonaldehyde level in diabetic I/R rat hearts. All these improving effects of luteolin were significantly attenuated by L-NAME. Luteolin also significantly upregulated eNOS expression in diabetic rat hearts after I/R. Ca-induced mPTP opening and mitochondrial inner membrane potential reduction were significantly inhibited in ventricular myocytes isolated from luteolin-treated diabetic rats, and this effect was attenuated by L-NAME. These findings indicate that luteolin protects the diabetic heart against I/R injury by upregulating the myocardial eNOS pathway, and downstream effects include the enhancement of MnSOD and inhibition of mPTP.

Endothelium-dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas.

Elsholtzia splendens (ES) is, rich in flavonoids, used to repair copper contaminated soil in China, which has been reported to benefit cardiovascular systems as folk medicine. However, few direct evidences have been found to clarify the vasorelaxation effect of total flavonoids of ES (TFES). The vasoactive effect of TFES and its underlying mechanisms in rat thoracic aortas were investigated using the organ bath system. TFES (5-200mg/L) caused a concentration-dependent vasorelaxation in endothelium-intact rings, which was not abolished but significantly reduced by the removal of endothelium. The nitric oxide synthase (NOS) inhibitor N(ω)-nitro-l-arginine methyl ester (100µM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (30µM) significantly blocked the endothelium-dependent vasorelaxation of TFES. Meanwhile, NOS activity in endothelium-intact aortas was concentration-dependently elevated by TFES. However, indomethacin (10µM) did not affect TFES-induced vasorelaxation. Endothelium-independent vasorelaxation of TFES was significantly attenuated by KATP channel blocker glibenclamide. The accumulative Ca(2+)-induced contraction in endothelium-denuded aortic rings primed with KCl or phenylephrine was markedly weakened by TFES. These results revealed that the NOS/NO/cGMP pathway is likely involved in the endothelium-dependent vasorelaxation induced by TFES, while activating KATP channel, inhibiting intracellular Ca(2+) release, blocking Ca(2+) channels and decreasing Ca(2+) influx into vascular smooth muscle cells might contribute to the endothelium-independent vasorelaxation conferred by TFES.

[Effects of total flavones of Elsholtzia splendens in isolated ischemia/reperfusion rat hearts].

OBJECTIVE: To investigate the influence of total flavonoids of Elsholtzia splendens (TFES) on isolated ischemia/reperfusion rat hearts and its underlying mechanisms. METHODS: Hearts isolated from male SD rats were perfused on the Langendorff apparatus and subjected to global ischemia for 30 min followed by 120 min of reperfusion. The cardiac infarct size was measured by TTC staining. Hemodynamic parameters and the level of lactate dehydrogenase (LDH) in the coronary effluent were measured. Absorbance at 520 nm was determined in isolated cardiac mitochondria exposed to 200 micromol/L CaCl2 to detect the opening of the mitochondrial permeability transition pore. RESULTS: Pretreatment with TFES (1, 10, 100 microg/ml) for 5 min decreased infarct size and LDH release and improved the recovery of the left ventricular developed pressure. In mitochondria, the decrease of absorbance at 520 nm evoked by CaCl2 was greatly inhibited by TFES. CONCLUSION: TFES prevents myocardial ischemia/reperfusion injury, and this cardioprotective effect is probably via inhibiting mitochondrial permeability transition pore opening.

Ventricular hypertrophy abrogates intralipid-induced cardioprotection by alteration of reperfusion injury salvage kinase/glycogen synthase kinase 3ß signal.

Recent studies have demonstrated that intralipid (ILP) conferred myocardial protection against ischemia-reperfusion (IR) injury through activation of reperfusion injury salvage kinase (RISK) pathway. As RISK signal has been shown to be impaired in hypertrophied myocardium, we investigated whether ILP-induced cardiac protection was maintained in hypertrophied rat hearts. Transverse aortic constriction was performed on male Sprague-Dawley rats to induce left ventricular hypertrophy, then sham-operated or hypertrophied rat hearts were isolated and perfused retrogradely by the Langendorff for 30 min (equilibration) followed by 40 min of ischemia and then 120 min of reperfusion. The isolated hearts received 15-min episode of 1% ILP separated by 15 min of washout or three episodes of 5-min ischemia followed by 5-min reperfusion before ischemia. The hemodynamics, infarct size, apoptosis, phosphorylated protein kinase B (p-Akt), phosphorylated extracellular regulated protein kinase 1/2 (ERK1/2), phosphorylated glycogen synthase kinase 3ß (GSK3ß), Bcl-2, phosphorylated Bad, and Bax were determined. We found that ILP significantly improved left ventricular hemodynamics and reduced infarct size and the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cells in the sham-operated rat hearts exposed to IR. However, such myocardial infarct-sparing effect of ILP was completely blocked by phosphatidylinositol-3-kinase inhibitor wortmannin, but only partially by mitogen-activated protein kinase kinase inhibitor PD98059 in sham-operated hearts. Intralipd upregulated the phosphorylation of Akt, extracellular regulated protein kinase 1/2 (ERK1/2), and their downstream target of GSK3ß and antiapoptotic Bcl-2 expression in healthy rat hearts. Nonetheless, ILP failed to improve left ventricular hemodynamics and reduced infarct size and apoptosis and increase the phosphorylated Akt, ERK1/2, GSK3ß, and antiapoptotic Bcl-2 in hypertrophied myocardium. In contrast, ischemic preconditioning increased the phosphorylation of Akt, ERK1/2 and GSK3ß, improved heart pump function, and reduced myocardial necrosis in sham-operated hearts, a phenomenon partially attenuated by ventricular hypertrophy. Interestingly, GSK inhibitor SB216763 conferred cardioprotection against IR injury in sham-operated hearts, but failed to exert cardioprotection in hypertrophied myocardium. Our results indicated that ventricular hypertrophy abrogated ILP-induced cardioprotection against IR injury by alteration of RISK/GSK3ß signal.

Hypercholesterolemic myocardium is vulnerable to ischemia-reperfusion injury and refractory to sevoflurane-induced protection.

Recent studies have demonstrated that volatile anesthetic postconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia. Therefore, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. In the present study, normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane for 5 min or 3 cycles of 10-s ischemia/10-s reperfusion. The hemodynamic parameters, including left ventricular developed pressure, left ventricular end-diastolic pressure and heart rate, were continuously monitored. The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3ß were determined. We found that both sevoflurane and ischemic postconditioning significantly improved heart pump function, reduced infarct size and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3ß in the healthy rats. In the hypercholesterolemic rats, neither sevoflurane nor ischemic postconditioning improved left ventricular hemodynamics, reduced infarct size and increased the phosphorylated Akt, ERK1/2 and GSK3ß. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. In conclusions, hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3ß and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.

Hypercholesterolemia blocked sevoflurane-induced cardioprotection against ischemia-reperfusion injury by alteration of the MG53/RISK/GSK3ß signaling.

BACKGROUND: Recent studies have demonstrated that volatile anesthetic preconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. METHODS: Normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane during three 5 min periods with and without PI3K antagonist wortmannin (10 µg/kg, Wort) or the ERK inhibitor PD 98059 (1 mg/kg, PD). The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3ß were determined. RESULTS: Two hundred and six rats were analyzed in the study. In the healthy rats, sevoflurane significantly reduced infarct size by 42%, a phenomenon completely reversed by wortmannin and PD98059 and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3ß. In the hypercholesterolemic rats, sevoflurane failed to reduce infarct size and increase the phosphorylated Akt, ERK1/2 and GSK3ß. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. CONCLUSIONS: Hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3ß and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.

Hypertrophied myocardium is refractory to sevoflurane-induced protection with alteration of reperfusion injury salvage kinase/glycogen synthase kinase 3ß signals.

Recent studies have demonstrated that volatile anesthetic postconditioning confers myocardial protection against ischemia-reperfusion injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired by ventricular hypertrophy, we investigate whether anesthetic-induced cardiac protection was maintained in rat hearts with ventricular hypertrophy. Transverse aortic constriction operation was performed on male Sprague-Dawley rats to induce left ventricular (LV) hypertrophy, then sham-operated or hypertrophied rat hearts were subjected to 40 min of global ischemia and 2 h of reperfusion. The isolated hearts received 3% sevoflurane for 10 min or six cycles of 10-s ischemia/10-s reperfusion after reperfusion. The hemodynamics, infarct size, PTEN (phosphatase and tensin homolog deleted on chromosome ten), phosphorylated Akt, phosphorylated extracellular regulated protein kinase (ERK) 1/2, and phosphorylated glycogen synthase kinase 3ß (GSK3ß) were determined. We found the myocardial expression of PTEN, phosphorylated Akt, ERK1/2, and phosphorylated GSK3ß did not significantly differ between sham-operated and transverse aortic constriction-control groups. Both sevoflurane and ischemic postconditioning significantly improved LV hemodynamics, reduced infarct size, and increased the phosphorylation of Akt, ERK1/2, and their downstream target of GSK3ß in the sham-operated rat hearts. In contrast, neither sevoflurane nor ischemic postconditioning improved LV hemodynamic, reduced infarct size, and increased the phosphorylated Akt, ERK1/2, and GSK3ß in hypertrophied myocardium. All the results above indicate that ventricular hypertrophy abrogated sevoflurane-induced cardioprotection against ischemia-reperfusion injury by alteration of RISK/GSK3ß signals.

Reducing the oxidative stress mediates the cardioprotection of bicyclol against ischemia-reperfusion injury in rats.

OBJECTIVE: To investigate the beneficial effect of bicyclol on rat hearts subjected to ischemia-reperfusion (IR) injuries and its possible mechanism. METHODS: Male Sprague-Dawley rats were intragastrically administered with bicyclol (25, 50 or 100 mg/(kg∙d)) for 3 d. Myocardial IR was produced by occlusion of the coronary artery for 1 h and reperfusion for 3 h. Left ventricular hemodynamics was continuously monitored. At the end of reperfusion, myocardial infarct was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and serum lactate dehydrogenase (LDH) level and myocardial superoxide dismutase (SOD) activity were determined by spectrophotometry. Isolated ventricular myocytes from adult rats were exposed to 60 min anoxia and 30 min reoxygenation to simulate IR injuries. After reperfusion, cell viability was determined with trypan blue; reactive oxygen species (ROS) and mitochondrial membrane potential of the cardiomyocytes were measured with the fluorescent probe. The mitochondrial permeability transition pore (mPTP) opening induced by Ca(2+) (200 µmol/L) was measured with the absorbance at 520 nm in the isolated myocardial mitochondria. RESULTS: Low dose of bicyclol (25 mg/(kg∙d)) had no significant improving effect on all cardiac parameters, whereas pretreatment with high bicyclol markedly reduced the myocardial infarct and improved the left ventricular contractility in the myocardium exposed to IR (P<0.05). Medium dose of bicyclol (50 mg/(kg∙d)) markedly improved the myocardial contractility, left ventricular myocyte viability, and SOD activity, as well decreased infarct size, serum LDH level, ROS production, and mitochondrial membrane potential in rat myocardium exposed to IR. The reduction of ventricular myocyte viability in IR group was inhibited by pretreatment with 50 and 100 mg/(kg∙d) bicyclol (P<0.05 vs. IR), but not by 25 mg/(kg∙d) bicyclol. The opening of mPTP evoked by Ca(2+) was significantly inhibited by medium bicyclol. CONCLUSIONS: Bicyclol exerts cardioprotection against IR injury, at least, via reducing oxidative stress and its subsequent mPTP opening.

Activation of Akt and cardioprotection against reperfusion injury are maximal with only five minutes of sevoflurane postconditioning in isolated rat hearts.

It had been proved that administration of sevoflurane for the first two minutes of reperfusion effectively protects the heart against reperfusion injury in rats in vivo. Our aim was to investigate the duration of effective sevoflurane administration and its underlying mechanism in isolated rat hearts exposed to global ischemia/reperfusion (I/R) injury. Adult male Sprague-Dawley rats were randomly divided into six groups (n=12): a sham-operation group, an I/R group, and four sevoflurane postconditioning groups (S2, S5, S10, and S15). In the S2, S5, S10, and S15 groups, the duration times of sevoflurane administration were 2, 5, 10, and 15 min after the onset of reperfusion, respectively. The isolated rat hearts were mounted on the Langendorff system, and after a period of equilibrium were subjected to 40 min global ischemia and 120 min reperfusion. Left ventricular (LV) hemodynamic parameters were monitored throughout each experiment and the data at 30 min of equilibrium and 30, 60, 90, and 120 min of reperfusion were analyzed. Myocardial infarct size at the end of reperfusion (n=7 in each group) and the expression of myocardial phosphorylated Akt (p-Akt) after 15-min reperfusion were determined in a duplicate set of six groups of rat hearts (n=5 in each group). Compared with the I/R group, the S5, S10, and S15 groups had significantly improved left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), and the maximal rate of rise or fall of the LV pressure (±dP/dtmax), and decreased myocardial infarct size (P<0.05), but not the S2 group. After 15 min of reperfusion, the expression of p-Akt was markedly up-regulated in the S5, S10, and S15 groups compared with that in the I/R group (P<0.05), but not in the S2 group. Sevoflurane postconditioning for 5 min was sufficient to activate Akt and exert maximal cardioprotection against I/R injury in isolated rat hearts.

Both PI3K/Akt and ERK1/2 pathways participate in the protection by dexmedetomidine against transient focal cerebral ischemia/reperfusion injury in rats.

Dexmedetomidine (Dex) has been demonstrated to provide neuroprotection against ischemia/reperfusion (I/R) injury. However, the exact mechanism of this protection remains unknown. Here, we explored the neuroprotective effect of Dex in rats exposed to cerebral I/R-induced by middle cerebral artery occlusion (MCAO) and the role of phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and glycogen synthase kinase-3ß (GSK-3ß) in this protective action. Adult male Sprague-Dawley rats were subjected to MCAO for 90 min followed by reperfusion for 24h and Dex (15 µg/kg, i.v.) was administered immediately after the onset of MCAO. The neurological deficit score, cerebral infarct volume, brain edema, and neuron survival were evaluated at 24h of reperfusion. The effect of Dex on p-Akt, p-ERK1/2 and p-GSK-3ß expression in the ischemic hemisphere was assayed by Western blot. Treatment of rats exposed to I/R with Dex caused not only marked reduction in the neurological deficit score, cerebral infarct volume, and brain edema (P <0.01 vs. I/R alone), but also a decrease in neuron death in hippocampal CA1 and cortex (P<0.01 vs. I/R alone). The Dex-induced increment of neuron survival in the ischemic CA1 and cortex was diminished by the PI3K inhibitor LY294002 and the MEK inhibitor U0126. The increasing expressions of p-Akt and p-ERK1/2 induced by Dex in the ischemic hemisphere were markedly inhibited by LY294002 (or wortmannin) and U0126 (or PD98059), respectively. The up-regulation of p-GSK-3ß by Dex in the ischemic hemisphere was significantly decreased by both LY294002 (or wortmannin) and U0126 (or PD98059). Our data demonstrated that treatment with Dex reduced cerebral injury in rats exposed to transient focal I/R, and this was mediated by the activation of the PI3K/Akt and ERK1/2 pathways as well the phosphorylation of downstream GSK-3ß.

Betulinic acid inhibits superoxide anion-mediated impairment of endothelium-dependent relaxation in rat aortas.

OBJECTIVES: To investigate the protective effect of betulinic acid (BA) on endothelium-dependent relaxation (EDR) in rat aortas exposed to pyrogallol-produced superoxide anion and its underlying mechanism. MATERIALS AND METHODS: The thoracic aorta of male Sprague-Dawley rats was isolated to mount in the organ bath system and the effect of BA on acetylcholine (ACh)-induced EDR, nitric oxide (NO) level, reactive oxygen species (ROS) level, nitric oxide synthase (NOS) activity, and superoxide dismutase (SOD) activity of aortic rings exposed to pyrogallol (500 µM) for 15 min were measured. RESULTS: BA evoked a concentration-dependent EDR in aortas, and pretreatment with EC(50) (2.0 µM) concentration of BA markedly enhanced ACh-induced EDR of aortas exposed to pyrogallol-produced superoxide anion (E(max) rose from 23.91 ± 5.41% to 42.45 ± 9.99%), which was markedly reversed by both N(w) -nitro-L-arginine methyl ester hydrochloride (L-NAME) and methylene blue, but not by indomethacin. Moreover, BA significantly inhibited the increase of ROS level, as well as the decrease of NO level, the endothelial NOS (eNOS) activity, and the SOD activity in aortas induced by pyrogallol-derived superoxide anion. CONCLUSION: These results indicate that BA reduces the impairment of EDR in rat aortas exposed to exogenous superoxide anion, which may closely relate to the reduction of oxidative stress and activation of eNOS-NO pathway.