Minocycline has been shown to protect against myocardial ischemia-reperfusion injury. This study investigated the effects of minocycline on ischemia-induced ventricular arrhythmias in rats. Anesthetized male rats were once treated with minocycline (45mg/kg, i.p.) 1h before ischemia in the absence and/or presence of 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002, 0.3mg/kg, i.v., a PI3K inhibitor) and 5-hydroxydecanoic acid [5-HD, 10mg/kg, i.v., a specific inhibitor of mitochondrial ATP-sensitive potassium (K(ATP)) channels] which were once injected 10min before ischemia and then subjected to ischemia for 30min. Ventricular arrhythmias were assessed. L-type Ca(2+) current was measured by the patch-clamp technique. During the 30-minute ischemia, minocycline significantly reduced the incidence of ventricular fibrillation (VF) (P<0.05). The duration of VT+VF, the number of VT+VF episodes and the severity of arrhythmias were all significantly reduced by minocycline compared to those in myocardial ischemia group (P<0.05 for all). Administration of LY294002 or 5-HD abolished the protective effects of minocycline on VF incidence, the duration of VT+VF, the number of VT+VF episodes and the severity of arrhythmias (P<0.05 for all). In addition, minocycline inhibited L-type Ca(2+) currents of normal myocardial cell membrane in a dose-dependent manner. This study suggested that minocycline could attenuate ischemia-induced ventricular arrhythmias in rats in which PI3K/Akt signaling pathway, mitochondrial K(ATP) channels and L-type Ca(2+) channels may be involved.

In this study, we investigated whether hydralazine could reduce renal ischemia and reperfusion (I/R) injury in rats. Renal I/R was induced by a 70-min occlusion of the bilateral renal arteries and a 24-h reperfusion, which was confirmed by the increased the mortality, the levels of blood urea nitrogen (BUN), blood creatinine (Cr), renal tissue NO and the visible histological damage of the kidneys. Apoptosis was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) staining. Furthermore, the serum levels of malonaldehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly elevated in renal I/R group, while the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) levels were suppressed. However, intragastric pretreatment with hydralazine at doses of 7.5-30 mg/kg before renal I/R significantly limited the increase in mortality, BUN, Cr, oxidative stress, inflammatory factors, histological damage and apoptosis in the kidneys. In addition, hydralazine also increased p-AKT, Bcl-2 expression and decreased iNOS, Bax, cleaved caspase-3 expression in the kidneys. In conclusion, hydralazine reduced renal I/R injury probably via inhibiting NO production by iNOS/NO pathway, inhibiting oxidative stress, inflammatory response and apoptosis by a mitochondrial-dependent pathway.

Myocardial ischemia/reperfusion (IR) injury is caused by the restoration of the coronary blood flow following an ischemic episode. Accumulating evidence suggests that galectin-3, a β-galactoside-binding lectin, acts as a biomarker in heart disease. However, it remains unclear whether manipulating galectin-3 affects the susceptibility of the heart to IR injury. In this study, RNA sequencing (RNA-seq) analysis identified that Lgals3 (galecin-3) plays an indispensable role in IR-induced cardiac damage. Immunostaining and immunoblot assays confirmed that the expression of galectin-3 was markedly increased in myocardial IR injury both in vivo and in vitro. Echocardiographic analysis showed that cardiac dysfunction in experimental IR injury was significantly attenuated by galectin-3 inhibitors including pectin (1%, i.p.) from citrus and binding peptide G3-C12 (5.0 mg/kg, i.p.). Galectin-3 inhibitor-treated mice exhibited smaller infarct sizes and decreased tissue injury. Furthermore, TUNEL staining showed that galectin-3 inhibition suppressed IR-mediated cardiomyocyte apoptosis. Mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) levels were well-preserved and IR-induced changes of mitochondrial cyto c, cytosol cyto c, caspase-9, caspase-3, Bcl-2 and Bax in the galectin-3 inhibitor-treated groups were observed. Our findings indicate that the pathological upregulation of galectin-3 contributes to IR-induced cardiac dysfunction and that galectin-3 inhibition ameliorates myocardial injury, highlighting its therapeutic potential.

This study aimed to investigate whether hydralazine could reduce cardiac ischemia/reperfusion (I/R) injury in rats. Anesthetized male Sprague-Dawley rats underwent myocardial I/R injury. Saline, hydralazine (HYD, 10-30 mg/kg) was administered intraperitoneally 10 min before reperfusion. After 30 min of ischemia and 24 h of reperfusion, the myocardial infarct size was determined using TTC staining. Heart function and oxidative stress were determined through biochemical assay and DHE staining. HE staining was used for histopathological evaluation. Additionally, the cardiomyocytes apoptosis and protein expression of PI3K-Akt-eNOS pathway marker were detected by TUNEL and Western blotting. The serum levels of malonaldehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and reactive oxygen species were significantly elevated in cardiac I/R group, but the superoxide dismutase (SOD) level was suppressed. However, intraperitoneal pretreatment with hydralazine at a dose of 10-30 mg/kg before cardiac I/R significantly limited the increase in CK-MB, LDH, oxidative stress, inflammatory factors, histological damage and apoptosis in the hearts. In addition, hydralazine also increased p-PI3K, p-AKT, p-eNOS expression and decreased Cleaved Caspase-3, Cleaved Caspase-9 expression in the hearts. Our results suggest that the cardioprotective effect of hydralazine against I/R injury might be a cooperation of the inhibition of oxidative stress, inflammatory response, apoptosis with the motivation of eNOS phosphorylation via activating the PI3K/AKT signal pathway.

Acute kidney injury caused by ischemia-reperfusion injury (IRI) is a major risk factor for chronic kidney disease, which is characterized by renal interstitial fibrosis. However, the molecular mechanisms underlying renal fibrosis induced by IRI are not fully understood. Our results showed that interleukin (IL)-33 was induced markedly after IRI insult, and the kidneys of mice following IRI plus IL-33 treatment presented more severe renal fibrosis compared with mice treated with IRI alone. Therefore, we investigated whether inhibition of IL-33 protects against IRI-induced renal fibrosis. Mice were administrated with soluble ST2 (sST2), a decoy receptor that neutralizes IL-33 activity, or vehicle by intraperitoneal injection for 14 days after IRI challenge. We revealed that mice treated with sST2 exhibited less severe renal dysfunction and fibrosis in response to IRI compared with vehicle-treated mice. Inhibition of IL-33 suppressed bone marrow-derived fibroblast accumulation and myofibroblast formation in the kidneys after IRI stress, which was associated with less expression of extracellular matrix proteins. Furthermore, inhibition of IL-33 also showed a significant reduction of F4/80+ macrophages and CD3+ T cells in the kidneys of mice after IRI treatment. Finally, Treatment with IL-33 inhibitor reduced proinflammatory cytokine and chemokine levels in the kidneys of mice following IRI insult. Taken together, our findings indicate that IL-33 signaling plays a critical role in the pathogenesis of IRI-induced renal fibrosis through regulating myeloid fibroblast accumulation, inflammation cell infiltration, and the expression of proinflammatory cytokines and chemokines.

Reperfusion causes undesirable damage to the ischemic myocardium while restoring the blood flow. In this study, we evaluated the effects of dexpramipexole (DPX) on myocardial injury induced by ischemia/reperfusion (I/R) in-vivo and the hypoxia/reoxygenation (HR) in-vitro and examined the functional mechanisms of DPX. DPX protected cells against H/R-induced mitochondrial dysfunction and prevented H/R damage. Both myocardial infarct size and tissue damage due to I/R was reduced upon DPX treatment. We discovered that DPX enhanced mitophagy in-vivo and in-vitro, which was accompanied by enhanced expression of PINK1 and Parkin. Knock-down of PINK1 and Parkin by specific siRNAs reversed DPX-induced inhibition of myocardial I/R injury. These findings suggest that DPX might protect against myocardial injury via PINK1 and Parkin.

Hepatic ischemia/reperfusion (I/R) injury is a common complication in the clinical setting. Our previous study has shown that connexin 32 (Cx32) plays a major role in renal I/R injury; however, the role of Cx32 in hepatic I/R injury remains unknown. Liver tissue and serum samples from patients undergoing orthotopic liver transplantation (OLT) were used to evaluate the function of Cx32 in OLT post-reperfusion injury. Then, partial hepatic ischemia was established in global Cx32 knockout mice and wild-type mice followed by reperfusion. Hepatic injury markers were examined. Cx32 small interfering RNA and the p53 inhibitor, pifithrin-α, tenovin-1 were used to examine the relationship between Cx32 and the p53/puma pathways in the BRL-3A and murine primary hepatocytes hypoxia/reoxygenation (H/R) model. Corresponding to liver damage, Cx32 was significantly induced both during OLT in human patients and partial hepatic I/R in mice. Cx32 KO mice exhibited less liver injury than controls. Cx32 deficiency significantly suppressed the p53/puma pathways and hepatocyte apoptosis. Similar results were observed in the BRL-3A and murine primary hepatocytes H/R model. Propofol protected against OLT post-reperfusion injury and hepatocyte apoptosis by inhibiting Cx32. In conclusion Cx32 is a novel regulator of hepatic I/R injury through the modulation of hepatocyte apoptosis and damage, largely via the p53/puma signaling pathway.

During the processes of myocardial ischemia reperfusion (I/R) injury, inflammation and apoptosis play an important role. I/R and its induced acute myocardial infarction (AMI) with high morbidity and mortality, and there is no effective treatment for it so far. TRAF5 has been shown to regulate inflammation and apoptosis in atherosclerosis, steatosis and melanoma cells, but its function in myocardial I/R injury is still unclear. This study demonstrates that the expression of TRAF5 is significant up-regulation in heart tissues of I/R injury mice and hypoxia/reoxygenation (H/R)-stimulated cardiomyocytes. TRAF5 knockout mice exhibites heavier heart damage, inflammatory response and cell death after myocardial I/R injury. Further, TRAF5 overexpression inhibites inflammation and apoptosis of H/R-stimulated cardiomyocytes. Mechanistically, we prove that TRAF5 promotes the activation of AKT. Overall, our study indicates that TRAF5 can regulate the processes of myocardial I/R injury. TRAF5 can be a new therapy target for myocardial I/R injury.

Disruption of the blood-brain barrier (BBB) after cerebral ischemia is considered to be the initial step in the development of brain injuries, and an increase in the tyrosine phosphorylation of the tight junctional protein occludin has been shown to cause an increase in BBB permeability. Prostaglandin E2 (PGE2) appears to be associated with both toxic and protective effects on neuronal survival in vitro. However, it remains to be determined whether the prostanoid EP1 receptor is involved in the disruption of the BBB after cerebral ischemia. So we examined the effect of a prostanoid EP1 receptor antagonist, SC51089, on BBB leakage and tyrosine phosphorylation of occludin after cerebral ischemia. We demonstrated that SC51089 attenuated the increase in the tyrosine phosphorylation of occludin in isolated brain capillaries, which was coincident with a decrease in BBB leakage. These results suggest that the prostanoid EP1 receptor is involved in the tyrosine phosphorylation of occludin at tight junction, which may lead to disruption of the BBB and be linked to the development of cerebral infarctions.

We determined effects of the nitric oxide (NO) precursor L-arginine, on isolated guinea pig ventricular myocytes under normoxic conditions and simulated ischemia and reperfusion. Currents and contractions were recorded with voltage clamp and a video edge detector, respectively. In normoxia, L-arginine (50-200 microM) had little effect on Ca2+ current, but significantly decreased contraction. Ischemia in the absence of L-arginine reduced Ca2+ current and abolished contractions. In reperfusion, the arrhythmogenic transient inward current was induced and cells exhibited sustained contractile depression (stunning). With L-arginine (100 microM) in ischemia, Ca2+ current did not decline and recovery of contraction was potentiated in reperfusion. L-Arginine had no effect on transient inward current. Inhibition of nitric oxide synthase reversed effects of L-arginine on contractions but not Ca2+ current. Thus, NO contributes to beneficial effects of L-arginine in reperfusion, although effects on I(Ca-L) are independent of NO. Further, L-arginine effects differ under normoxic and ischemic conditions.

Ischemia/reperfusion (I/R)-induced autophagy increases the severity of cardiomyocyte injury. The aim of this study was to investigate the effects of berberine, a natural extract from Rhizoma coptidis, on the I/R-induced excessive autophagy in in vitro and in vivo models. Autophagy was increased both in H9c2 myocytes during hypoxia/reoxygenation (H/R) injury and in mouse hearts exposed to I/R. And the expression level of p-AMPK and p-mTORC2 (Ser2481) were increased during H/R period. In addition, the increased autophagy level was correlated with reduced cell survival in H9c2 myocytes and increased infarct size in mouse hearts. However, berberine treatment significantly enhanced the H/R-induced cell viability and reduced I/R-induced myocardial infarct size, which was accompanied by improved cardiac function. The beneficial effect of berberine is associated with inhibiting the cellular autophagy level, due to decreasing the expression level of autophagy-related proteins such as SIRT1, BNIP3, and Beclin-1. Furthermore, both the level of p-AMPK and p-mTORC2 (Ser2481) in H9c2 myocytes exposed to H/R were decreased by berberine. In summary, berberine protects myocytes during I/R injury through suppressing autophagy activation. Therefore, berberine may be a promising agent for treating I/R-induced cardiac myocyte injury.

Although arterial limb tourniquet is one of the first-line treatments to prevent exsanguinating hemorrhage in both civilian pre-hospital and battlefield casualty care, prolonged application of a limb tourniquet can lead to serious ischemia-reperfusion injury. However, the underlying pathomechanisms of tourniquet-induced ischemia-reperfusion injury are still poorly understood. Using a murine model of acute limb ischemia-reperfusion, we investigated if acute limb ischemia-reperfusion injury is mediated by superoxide overproduction and mitochondrial dysfunction. Hind limbs of C57/BL6 mice were subjected to 3h ischemia and 4h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Approximately 40% of the gastrocnemius muscle suffered infarction in this model. Activities of mitochondrial electron transport chain complexes including complex I, II, III, and IV in the gastrocnemius muscle were decreased in the ischemia-reperfusion group compared to sham. Superoxide production was increased while activity of manganese superoxide dismutase (MnSOD, the mitochondria-targeted SOD isoform) was decreased in the ischemia-reperfusion group compared to the sham group. Pretreatment with tempol (a SOD mimetic, 50mg/kg) or co-enzyme Q(10) (50mg/kg) not only decreased the superoxide production, but also reduced the infarct size and normalized mitochondrial dysfunction in the gastrocnemius muscle. Our results suggest that tourniquet-induced skeletal muscle ischemia-reperfusion injuries including infarct size and mitochondrial dysfunction may be mediated via superoxide overproduction and reduced antioxidant activity. In the future, this murine ischemia-reperfusion model can be adapted to mechanistically evaluate anti-ischemic molecules in tourniquet-induced skeletal muscle injury.

Inflammatory reactions play an important role in ischemia/reperfusion injury in various organs. Since histamine is closely related to inflammatory reactions and immune responses, effects of postischemic administration of histaminergic ligands on ischemia-induced liver injury were examined in rats. Animals were subjected to warm ischemia for 30 min by occlusion of the left portal vein and hepatic artery under halothane anesthesia, and liver damage was evaluated by assessing plasma concentrations of transaminases after 24 h. Warm ischemia for 30 min provoked severe liver damage after 24 h, and the plasma concentrations of alanine transaminase (ALT) and aspartate transaminase (AST) were 8600 I.U./l and 13100 I.U./l, respectively. Subcutaneous injections of histamine twice, immediately and 6 h after reperfusion (20 mg/kg, each), alleviated liver damage. The plasma concentrations of ALT and AST in the histamine group were 35% and 24% of those in the control group, respectively. Neither mepyramine (3 mg/kg x 2), an H1 antagonist, nor cimetidine (15 mg/kg x 2), an H2 antagonist, affected the outcome in histamine-treated rats. However, thioperamide (5 mg/kg x 2), an H3/H4 antagonist, completely abolished the alleviation caused by histamine. Administration of dimaprit (1-10 mg/kg x 2), an H2/H4 agonist, mimicked the protective effect of histamine, and the effect of dimaprit is reversed by thioperamide, whereas neither H1 nor H2 antagonists altered the outcome caused by dimaprit. Clozapine (15 mg/kg x 2), an H4 agonist, also mimicked the protective effect of histamine. These findings indicate that stimulation of histamine H4 receptors after ischemic events prevents development of reperfusion injury in the liver.

Poncirin, a flavonoid glycoside derivative extracted from the fruits of Poncirus trifoliata (trifoliate orange or Chinese bitter orange), has a variety of documented bioactivities, including anti-tumor, anti-inflammatory, and antioxidant effects. Oxidative stress is a major underlying factor in the pathogenesis of cardiac ischemia-reperfusion (I/R) injury. Therefore, we investigated the protective efficacy of poncirin on primary cardiomyocytes subjected to anoxia-reoxygenation (A/R) injury in vitro, and on rat hearts subjected to ischemia-reperfusion (I/R) injury in vivo. Poncirin pretreatment enhanced cardiomyocyte survival, inhibited A/R-induced oxidative stress by upregulating cellular antioxidant capacity, suppressed mitochondrial depolarization, and ultimately inhibited apoptosis. Similarly, systemic poncirin treatment significantly reduced cardiomyocyte apoptosis and infarct size in rat hearts. In addition, activity of the PI3K/AKT/PGC-1α pathway was significantly increased by poncirin pretreatment in both A/R and I/R injury models, while PI3K and PGC-1α inhibitors abolished all poncirin related effects, suggesting that this pathway is essential for the cardioprotective effects of poncirin. Pretreatment with the PGC-1α inhibitor reversed effects of poncirin without affecting p-AKT expression, indicating that PGC-1α is downstream of AKT. In conclusion, both in vitro and in vivo studies suggested that poncirin alleviates cardiac ischemia-reperfusion injury by mitigating oxidative stress, which is dependent on activation of the PI3K/AKT/PGC-1α signaling pathway.

Myocardial ischemia is the malperfusion of cardiac tissue due to a blockage in a coronary artery. Subsequent return of blood flow to the ischemic area of the heart, results in ischemia/reperfusion (I/R) injury in the heart and other organs, including the brain. Besides the cardioprotective effects of metformin on the heart against cardiac I/R injury, metformin also reduced neuronal injury in a stroke model. However, the effects of metformin on the brain following cardiac I/R injury has not yet been investigated. Therefore, we hypothesize that metformin reduces brain damage via decreasing brain mitochondrial dysfunction, microglial hyperactivity, and Alzheimer's proteins in rats after cardiac I/R injury. Rats (n = 50) received either a sham operation (n = 10) or cardiac I/R (n = 40). Cardiac I/R was induced by 30 min of cardiac ischemia, followed by 120 min of reperfusion. Rats in cardiac I/R group were divided into 4 groups (n = 10/group); vehicle, metformin 100 mg/kg, metformin 200 mg/kg, and metformin 400 mg/kg. Metformin was given via femoral vein at 15 min prior to cardiac ischemia. At the end of reperfusion, brains were removed to determine dendritic spine density, brain mitochondrial function, microglial morphology, and amyloid beta formation. Cardiac I/R injury led to brain mitochondrial dysfunction, microglial hyperactivation, amyloid beta formation, Tau hyperphosphorylation, and reduced dendritic spine density with an increase in AMPK activation. All doses of metformin improved brain pathologies in rats with cardiac I/R injury possibly via activating cerebral AMPK. In summary, pre-treatment with metformin offers neuroprotection against the brain damages caused by cardiac I/R injury.

The aim of our study was to investigate the neuroprotective properties of shikonin, a naphthoquinone pigment isolated from the roots of the traditional Chinese herb Lithospermum erythrorhizon. In the present study, mice were divided randomly into sham, model, shikonin and edaravone-treated groups. Shikonin (50, 25, and 12.5mg/kg, i.g.) or maize oil was administered three times before ischemia and once at 2h after the onset of ischemia. Mice were anesthetized with chloral hydrate and subjected to middle cerebral artery 2h of occlusion and then 22h of reperfusion. Different antioxidant assays were employed in order to evaluate the antioxidant activities of shikonin in vitro. Neurological deficit, infarct size, histopathology changes and oxidative stress markers were evaluated after 22h of reperfusion. In comparison with the model group, treatment with shikonin significantly decreased neurological deficit scores, infarct size, the levels of malondialdehyde(MDA), carbonyl and reactive oxygen species, and attenuated neuronal damage, up-regulated superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) activities and reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio. Taken together, these results suggested that the neuroprotective effects of shikonin against cerebral ischemia/reperfusion injury may be attributed to its antioxidant effects.

Acute kidney injury (AKI) is one of the major complications with increased oxidative stress and inflammation in diabetic patients. Hyperglycemia stimulates the formation of advanced glycation end products (AGEs). However, hyperglycemia directly triggers the interaction between AGEs and transmembrane AGEs receptors (RAGE), which enhances oxidative stress and increases the production of inflammatory substances. Therefore, diabetes plays a pivotal role in kidney injury. Hydralazine, a vasodilator and antihypertensive drug, was found to have the ability to reduce ROS, oxidative stress, and inflammation. We applied Hydralazine co-culture with AGEs in rat mesangial cells (RMC) and to renal ischemia/reperfusion(I/R) injury models in streptozotocin-induced diabetic rats. Hydralazine significantly decreased AGEs-induced RAGE, iNOS, and COX-2 expressions in RMC. Compared to the diabetic with AKI group, hydralazine decreased inflammation-related protein, and JAK2, STAT3 signaling in rat kidney tissue. Our studies indicate that Hydralazine has the potential to become a beneficial drug in the treatment of diabetic acute kidney injury.

Myocardial infarction (MI) refers to the loss of cardiomyocytes due to inadequate coronary blood flow and subsequently a reduced oxygen supply. Activation of N-methyl-D-aspartate (NMDA) receptors has been linked to myocardial infarction. The aim of the present study was to determine the cardioprotective effects of memantine, in myocardial infarction both in ex vivo and in vivo models. Effects of memantine on the electrocardiogram (ECG) pattern, cardiodynamic parameters, infarct size and lipid peroxidation were evaluated in the isolated perfused rat heart. Moreover, in in vivo studies in rats, the protective effects of memantine on isoproterenol-induced myocardial infarction model (administration of 100 mg/kg isoproterenol subcutaneously for 2 consecutive days) was evaluated by measuring ECG pattern, mean arterial pressure, malondialdehyde (MDA) levels, myeloperoxidase (MPO) activity, cardiac tumor necrosis factor-alpha (TNF-α) level and cardiac remodeling. The results from the ex vivo isolated perfused heart showed that memantine treatment increased heart rate, left ventricular systolic pressure and left ventricular maximal rate of pressure increase, and decreased cardiac arrhythmia, MDA level and infarct size in comparison to ischemia/reperfusion (IR) group. The isoproterenol-induced MI (Iso) as used in the in vivo model demonstrated that MDA levels and MPO activity were decreased in memantine groups. Memantine treatment reduced the expression of cardiac TNF-α in comparison to Iso group. Cardiac fibrosis and hypertrophy were lower in memantine groups. In conclusion, memantine exerts cardioprotective effects in models of myocardial infarction, which may be attributed to reduction of pro-inflammatory and oxidative stress factors and subsequently a decrease in cardiac remodeling.

Cerebral ischemia-reperfusion injury is a thorny issue in the treatment of stroke. Energy depletion and oxidative stress are the core mechanisms underlying cerebral ischemia-reperfusion injury. Mitochondrial function is involved in energy production and oxidative stress. It has been reported that mitochondrial uncoupling protein 2 (UCP2) may be involved in the regulation of cerebral ischemia-reperfusion injury. We hypothesized that UCP2 can regulate cerebral ischemia-reperfusion injury by regulating energy supply and oxidative stress. To test this hypothesis, we used a middle cerebral artery occlusion model in male C57BL/6 mice with/without genipin--an UCP2-specific inhibitor. We measured the expression and/or activity of UCP2, SIRT3, the level of ATP, and antioxidant-related molecules in the cerebral cortex and the LDH in serum after ischemia-reperfusion, the level of apoptosis was reflected by the level of cleaved-caspase3 and tunel staining. The results showed an increase in the expression of UCP2, coinciding with an increase in the level of apoptosis, NAD+/NADH ratio, SIRT3 activity, LDH release and a decrease in the level of ATP and antioxidant-related molecules after 1 h of ischemia and 24 h of reperfusion. These findings suggest that UCP2 may regulate energy supply and oxidative stress in ischemia-reperfusion injury. Interestinly, above changes can be reserved by administration of genipin with the brain damage level going down. In conclusion, the UCP2-SIRT3 signaling pathway is involved in the regulation of cerebral ischemia-reperfusion injury as a bridge between energy metabolism and oxidative stress. Genipin protects against cerebral ischemia-reperfusion injury by inhibiting UCP2.

Pharmacological strategies aimed at co-activating peroxisome proliferator-activated receptor-gamma (PPAR-γ)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway have shown promising results in alleviating myocardial injury. The aim of the study was to evaluate the role of chrysin, a PPAR-γ agonist, in ischemia-reperfusion (IR)-induced myocardial infarction (MI) in rats and to explore the molecular mechanism driving this activity. To evaluate this hypothesis, chrysin (60 mg/kg, orally), PPAR-γ antagonist (GW9662, 1 mg/kg, intraperitoneally), or both were administered to rats for 28 days. On the 29th day, one-stage ligation of left anterior descending coronary artery for 45 min followed by 60 min of reperfusion was performed. Chrysin significantly decreased infarct size and improved cardiac functions following IR-induced MI. This improvement was corroborated by augmented PPAR-γ/Nrf2 expression as confirmed by immunohistochemistry and western blotting analysis. Chrysin exhibited strong anti-oxidant property as demonstrated by increased GSH and CAT levels and decreased 8-OHdG and TBARS levels. Our findings also imply that chrysin significantly inhibited inflammatory response as validated by decreased NF-κB, IKK-β, CRP, TNF-α and MPO levels. In addition, chrysin decreased TUNEL/DAPI positivity, a marker of apoptotic response and normalized cardiac injury markers. The histopathological and ultrastructural analysis further supported the functional and biochemical outcomes, showing preserved myocardial architecture. Intriguingly, co-administration with GW9662 significantly diminished the cardioprotective effect of chrysin as demonstrated by depressed myocardial function, decreased PPAR-γ/Nrf2 expression and increased oxidative stress. In conclusion, the present study demonstrates that co-activation of PPAR-γ/Nrf2 by chrysin may be crucial for its cardioprotective effect.